Program of the 2004 East Coast Worm Meeting - Caenorhabditis ...

Program of the 2004 East Coast Worm Meeting 

A full-text search is available at http://elegans.swmed/edu/ECWM/2004/. 

Session 1. Friday June 11, 7-8:48 PM Chair: Mónica Colaiácovo 

1. Candidate EGO-1 interactors that function in germline development 

Xiang Yu, Valarie Vought, Jamie Wasilenko, Tom Ratliff, Bill Kelly, Eleanor Maine 

2. Two controls of FBF expression in the C. elegans germ line 

Liana B. Lamont, Sarah L. Crittenden, David S. Bernstein, Marvin Wickens, Judith Kimble 

3. Robust germline amplification and the precise timing of initial meiosis are dependent upon 

interactions with specific cells of the developing gonadal sheath 

Darrell J. Killian, E. Jane Albert Hubbard 

4. The NR4A nuclear receptor is required for spermatheca morphogenesis during somatic 

gonad development 

Chris R. Gissendanner, Tri Q. Nguyen, Marius Hoener, Ann E. Sluder, Claude V. Maina 

5. The spe-38 gene encodes a novel tetraspan integral membrane protein and is required for 

sperm function at fertilization 

Indrani Chatterjee, Andrew W Singson 

6. Sperm-oocyte interactions in C. elegans 

Alissa Richmond, Diane C. Shakes 

7. SPE-42 is required for sperm-egg interaction during C. elegans fertilization 

Tim L. Kroft, Steven W. L’Hernault 

8. A Vesicle-Budding Model for the Release of MSP from C. elegans Sperm 

Mary Kosinski, Kent McDonald, Jay Jerome, David Greenstein 

9. EFL-1 and DPL-1 Activate Genes Required for Oogenesis and Proper Embryonic 

Specification in the Maternal Germline 

Woo Chi, Valerie Reinke 

Session 2. Friday June 11, 9:10-10:46 PM Chair: Martha Soto 

10. RGS-7 Completes a Receptor-independent Heterotrimeric G Protein Signaling Cycle to 

Regulate Mitotic Spindle Positioning in C. elegans 

Heather A. Hess, Michael R. Koelle 

11. Three conserved protein kinases, DYRK, CDC2 and GSK3 promote OMA-1 degradation to 

establish proper cell fate and cell division polarity in early C. elegans embryos 

Masaki Shirayama, Takao Ishidate, Kuniaki Nakamura, Craig C. Mello 

12. Automated production of standardized, easy-to-share, easy-to-compare 4D embryonic image 

data 

Ariel B. Isaacson, William A. Mohler 

13. The germ granule protein PGL-1 is required for efficient RNA interference 

Darryl Conte Jr., Yingdee Unhavathaya, Craig C. Mello 

14. Enhancers of ksr-1 lethality define new potential regulators of small regulatory RNAs 

Christian E. Rocheleau, Yelena Bernstein, Meera V. Sundaram 

15. Multiple, dynamic microRNA ribonucleoprotein complexes with selective microRNA cargos in 

C. elegans 

Gopalakrishna Ramaswamy, Eun-Young Choi, Frank J. Slack

16. eak (enhancer-of-akt-1) genes encode membrane-associated proteins that potentiate AKT-1 

signaling in the C. elegans XXX cells 

Patrick J. Hu, Jinling Xu, Gary Ruvkun 

17. Notch function in differentiated neurons is required to maintain dauer 

Jimmy Ouellet, Richard Roy 

Session 3. Saturday June 12, 8:30-10:06 AM Chair: Siu Sylvia Lee 

18. Control of aging and developmental arrest by TGFbeta and insulin pathways during C. 

elegans diapause 

Manjing Pan, Li Sun da Graca, Tao Liu, Garth I. Patterson 

19. Life span regulation by JNK MAP kinase in C. elegans: a novel input into daf-16 

Seung Wook Oh, Nenad Svrzikapa, Heidi A. Tissenbaum 

20. Regulation of chemoreceptor gene expression by MEF-2 and class II HDACs in C. elegans 

Alexander M van der Linden, Katie Nolan, Piali Sengupta 

21. mig-10 functions downstream of unc-6 and slt-1 to mediate axon guidance 

Christopher C. Quinn, Elizabeth Stovall, Elizabeth F. Ryder, William G. Wadsworth 

22. Genes Involved In Serotonergic Neurotransmission 

Megan Higginbotham, Bob Horvitz 

23. Ryanodine Receptors Regulate Neurotransmitter Release at the C. elegans Neuromuscular 

Junction 

Qiang Liu, Michael Nonet, Lawrence Salkoff, Zhao-Wen Wang 

24. KEL-8, a novel Kelch-like protein, is required for glutamate receptor degradation 

Henry Schaefer, Christopher Rongo 

25. Knockout of GLT-3 C. elegans Glutamate Transporter: A Genetic Approach to Study 

Excitotoxic Neurodegeneration 

Itzhak Mano, Sarah Straud, Monica Driscoll 

Session 4. Saturday June 12, 10:40 AM-12:16 PM Chair: Peter Roy 

26. A non-developmental role for lin-12 Notch signaling in the C. elegans adult nervous system 

Michael Y. Chao, Jonah Larkins-Ford, Anne C. Hart 

27. Calcium permeability of death-inducing DEG/ENaC ion channel MEC-4(d) 

Laura Bianchi, Wei-Hsiang Lee, Gargi Mukherjee, Beate Gerstbrein, Dewey Royal, 

Maryanne Royal, Jian Xue, Monica Driscoll 

28. UNC-55, a Nuclear Receptor, is Essential for Male Mating 

Ge Shan, Bill Walthall 

29. Genes Controlling Sensory Axon Patterning in the C. elegans Male Tail 

Lingyun Jia, Scott W. Emmons 

30. A genomic approach to the development and function of the C. elegans male tail rays 

Douglas S. Portman, Daryl D. Hurd, Nicole Juskiw, Kwi Yeon Lee, William R. Mowrey, 

Carolyn Tyler, Hai Wu 

31. WormBase: What’s New and What’s Next? 

Nansheng Chen, Lincoln D. Stein, WormBase Consortium

32. Frogs and snails and puppy dog tails? Wormatlas launches a guide to what boy worms are 

made of 

Robyn Lints, Zeynep F. Altun, Huawei Weng, Gloria Stephney, Maurice Volaski, David H. 

Hall 

33. A New Phylogeny Reveals Frequent Loss of Introns During Nematode Evolution 

Ronald E Ellis, Soochin Cho 

Session 5. Saturday June 12, 5:30-7:06 PM Chair: Landon Moore 

34. Caenorhabditis phylogeny predicts convergence of hermaphroditism and extensive intron 

loss 

Karin C. Kiontke, Nicholas P. Gavin, Yevgeniy Raynes, Casey Roehrig, Fabio Piano, David 

H. A. Fitch 

35. Evolutionary innovation of excretory system in Caenorhabditis elegans 

Xiaodong Wang, Helen M. Chamberlin 

36. cdc-14 regulates cki-1 to control cell-cycle arrest 

R. Mako Saito, Audrey Perreault, Bethan Peach, John S. Satterlee, Sander van den Heuvel 

37. Transcriptional regulation of Hox gene lin-39 during vulval cell fate specification 

Javier A. Wagmaister, Julie E. Gleason, Corey A. Morris, Leilani M. Miller, Ginger R. Miley, 

Kerry Kornfeld, David M. Eisenmann 

38. EPS-8 regulates LET-23/EGFR localization during C. elegans vulval development 

Attila Stetak, Assunta Croce, Giuseppe Cassata, Pier P. DiFiore, Erika Fröhli Hoier, Alex 

Hajnal 

39. The C-terminal sequence of C. elegans smad/SMA-3 has multiple roles 

Jianjun Wang, Cathy Savage-Dunn 

40. PKC2 A Calcium-Diacylglcerol Kinase that Runs Hot and Cold 

Marianne Land, Charles S. Rubin 

41. Regulation of a Conserved Oxidative Stress Defense by GSK-3 and p38 signaling in C. 

elegans 

Jae Hyung An, Riva Oliveira, Rosanna Baker, Kelly Vranas, Hideki Inoue, Naoki Hisamoto, 

Yanxia Bei, Craig C. Mello, Kunihiro Matsumoto, T. Keith Blackwell 

Session 6. Sunday June 13, 8:30-9:42 AM Chair: Laura Mathies 

42. LIN-28 and LIN-46 converge at a branchpoint in the heterochronic pathway 

Eric G. Moss, Keven Kemper 

43. The role of daf-6 and cell-cell interactions in amphid morphogenesis 

Elliot A. Perens, Shai Shaham 

44. An FGF Signaling Pathway Regulates Membrane Extensions from the Body Wall Muscles in 

C. elegans 

Scott Dixon, Raynah Fernandes, Peter J. Roy 

45. EGL-15 FGF Receptor Isoforms Play Different Roles in SM Migration 

Te-Wen Lo, Catherine S. Branda, Peng Huang, Isaac E. Sasson, S. Jay Goodman, Michael 

J. Stern 

46. MLS-2, an HMX class homeodomain protein essential for mesodermal patterning and cell 

fate specification 

Yuan Jiang, Jun Kelly Liu

47. Regulation of TRA-1 by sex specific proteolytic processing and localization 

Mara Schvarzstein, Laura Mathies, Andrew Spence 

Session 7. Sunday June 13, 10:10 AM-12:10 PM Chair: Barth Grant 

48. Mutations in him-8 suppress developmental defects of egl-13 mutants 

Brian L. Nelms, Wendy Hanna-Rose 

49. Generating a more comprehensive picture of apoptosis using multiple functional genomic 

techniques 

Stuart Milstein, Pierre-Olivier Vidalain, Siming Li, David Hill, Marc Vidal 

50. Characterization and cloning of a novel component in the cell-corpse engulfment pathway 

Xiaomeng Yu, Xiaohong Leng, Chin-Hua Chuang, Sampeter Odera, H. Robert Horvitz, 

Zheng Zhou 

51. Characterization of the Cell Deaths Caused by Mutations in lin-24 and lin-33 

Brendan Galvin, Saechin Kim, Erika Hartwieg, Bob Horvitz 

52. RME-6 is a new regulator of Rab5-mediated endocytosis 

Miyuki Sato, Ken Sato, Paul Andre Fonarev, Barth Grant 

53. Septins function in morphogenesis of the C. elegans pharynx 

Fern P. Finger 

54. An Essential Role for HTP-3, a HIM-3 Paralog, in Mediating Meiotic Chromosome Behaviour 

and Structure 

William Goodyer, Monique Zetka 

55. HDA-1 regulates C. elegans embryogenesis: a potential role for a ubiquitous chromatin 

modifier in regulating tissue-specific gene expression and patterning 

Johnathan R. Whetstine, Julian Ceron, Valerie Reinke, Yang Shi 

56. Genetics of telomere replication in C. elegans 

Bettina Meier, Sarah Mense, Yan Zhao, Shawn Ahmed 

57. Centromere resolution is inhibited by cohesin proteins and requires condensin II 

components, HCP-6 and Mix-1 

Landon L. Moore, Matt Stankiewicz, David Rosen, Tovah Day 

Poster session: Saturday June 12, 1:45-5:30 PM 

58. Identification and Characterization of C. elegans Amine-gated Chloride Channels 

Namiko Abe, Niels Ringstad, Bob Horvitz 

59. Linker cell death may be caspase-independent 

Mary C. Abraham, Shai Shaham 

60. Octopamine Inhibits Pharyngeal Pumping and Egg Laying and Stimulates Locomotion 

Mark Alkema, Niels Ringstad, Bob Horvitz 

61. Establishment of anteroposterior neuronal polarity 

Eleanor Allen, Anastasia Bakoulis, Dave Hunt, Scott Clark 

62. The let-7 and mir-35 Families of MicroRNAs Each Act Redundantly in C. elegans 

Ezequiel Alvarez-Saavedra, Eric A Miska, Allison L Abbott, Nelson C Lau, David P Bartel, 

Victor Ambros, Bob Horvitz

63. Characterization of the Synthetic Multivulva Suppressor Gene isw-1, a Homolog of the 

Drosophila Chromatin-Remodeling ATPase ISWI 

Erik Andersen, Xiaowei Lu, Scott Clark, Bob Horvitz 

64. met-1 and met-2, Two Putative Histone Methyltransferases, May Act as Synthetic Multivulva 

Genes 

Erik Andersen, Bob Horvitz 

65. Generation of left/right asymmetry in the nervous system of C. elegans 

Celia Antonio, Oliver Hobert 

66. Alterations of the C. elegans Excretory Duct in Dauer Larvae 

Kristin R. Armstrong*, Helen M. Chamberlin* 

67. Sheath cell-dependent maintenance of AWC dendritic morphology 

Taulant Bacaj, Shai Shaham 

68. Association of Oscheius species with millipedes in the Wright State University Woods 

Scott Everet Baird, Christine M. Spice 

69. Females, Hermaphrodites and Developmental Bias 

Chris Baldi, Soochin Cho, Ronald E Ellis 

70. Genome-wide RNAi screen to identify novel regulators of membrane trafficking 

Zita Balklava, Barth D. Grant 

71. Molecular connections between developmental timing and circadian timing: The C. elegans 

homolog of the circadian gene doubletime regulates post-embryonic developmental timing 

Diya Banerjee, Alvin Kwok, Shin-Yi Lin, Frank Slack 

72. Towards cloning mutations isolated in a genetic screen for hyperactive egg-laying mutants 

I. Amy Bany, Michael R. Koelle 

73. C. elegans HDACs, CBP, and CREB play roles in polyglutamine neurotoxicity 

Emily A. Bates, Cindy Voisine, Martin Victor, Yang Shi, Anne Hart 

74. Study of the genetic and cellular bases of ventral nerve cord maintenance 

Claire Benard, Oliver Hobert 

75. A Genome-Wide RNAi Screen for Components Affecting Sex Muscle Differentiation 

Daniel C. Bennett, Isaac E. Sasson, Michael J. Stern 

76. A Genetic Screen for New Genes Involved in Aging 

Ala Berdichevsky, Leonard Guarente, Bob Horvitz 

77. Towards understanding the role of the stomatin-like protein UNC-24 in the process of gentle 

touch sensation: evidence for functional interaction with the mechanosensory ion channel 

complex MEC-4/MEC-10 

Laura Bianchi, Wei-Hsiang Lee, Dan Slone, Julie Y. Koh, David M. Miller III, Monica Driscoll 

78. A germline-specific RNA-binding protein required for germ cell survival and cytokinesis 

Peter R, Boag, T. Keith Blackwell 

79. Identification of C. elegans spindle assembly checkpoint components 

Mike Boxem, Marc Vidal 

80. Development of high-throughput sublehtal toxicity tests using Caenorhabditis elegans 

Windy A. Boyd, Sandra J. McBride, Julie R. Rice, Jonathan H. Freedman

81. Using enriched populations of single neuron types and microarrays to identify sensory 

neuron-specific genes 

Marc Colosimo, Adam Brown, Anne Lanjuin, Saikat Mukhopadhyay, Piali Sengupta 

82. Modulations of thermotactic behavior by food 

Adam Brown, Damon Clark, Chris Gabel, Steven Lin, Ares Perides, Piali Sengupta, Aravi 

Samuel 

83. A Systematic Genome-Wide Genetic Interaction Analysis of Signaling Pathways in C. 

elegans 

Alexandra Byrne, Scott J. Dixon, Jason Moffat, Peter J. Roy 

84. Temporal regulation of postmitotic neural differentiation by heterochronic genes including the 

microRNA lin-4 

Katherine O. Carter, Kristy Reinert, Shin-Yi Lin, Frank Slack 

85. Identification of genes acting redundantly with lin-35 Rb 

Julian Ceron, Abha Chandra, Khursheed Wani, Jean-Francois Rual, Marc Vidal, Sander van 

den Heuvel 

86. Functional analysis of AMPA-type glutamate receptor tail sequences in C. elegans. 

Howard Chang, Chris Rongo 

87. Feeding status and serotonin modulate a chemosensory circuit in C. elegans 

Michael Y. Chao, Hidetoshi Komatsu, Hana S. Fukuto, Heather M. Dionne, Anne C. Hart 

88. D1- and D2-like dopamine receptors antagonistically modulate C. elegans behavior through 

Galphaq and Galphao signaling 

Daniel Chase, Judy Pepper, Michael Koelle 

89. Germline establishment and maintenance in the early embryo 

Paula M. Checchi, Christine E. Schaner, William G. Kelly 

90. GUM-1, a protein affecting the subcellular localization of RME-1, is required for endocytic 

recycling in the C. elegans intestine 

Carlos Chih-Hsiung, Chen, Peter Schweinsberg, Eric Lambie, Barth Grant 

91. Translational control in the germ plasm: nos-2 RNA regulation. 

Pei-Lung Chen, Ingrid D’Agostino, Geraldine Seydoux 

92. Translational control in the germ plasm: nos-2 RNA regulation 


93. Age-associated feeding decline in C. elegans can be modulated by genetic and 

environmental inputs 

David K. Chow, Catherine A. Wolkow 

94. An Investigation into the potential regulatory relationships between components of a network 

specified by pal-1 

Julia M. Claggett, L. Ryan Baugh, Craig P. Hunter 

95. Genome-wide RNAi analysis of Caenorhabditis elegans distal tip cell migration 

Erin J. Cram, Jean E. Schwarzbauer 

96. Characterization of the DTC niche and germline stem cells in C. elegans 

Sarah L. Crittenden, Dana Byrd, Kim Leonhard, Judith Kimble 

97. Structure/function studies of the polarity regulator PAR-1 in C. elegans 

Adrian Cuenca, Geraldine Seydoux

98. Experimental Design for C. elegans Microarray 

Yuxia Cui, Sandra J. McBride, Jonathan H. Freedman 

99. Tracking the mid-life crisis of C. elegans 

Diana David-Rus, Peter J. Schmeissner, Beate Hartmann, Christophe Grundschober, Uri 

Einav, Eytan Domany, Patrick Nef, Garth Patterson, Monica Driscoll 

100. An HCP-6 Suppression Screen for Genes Involved in Centromere Resolution 

Tovah A. Day, Landon Moore 

101. Analysis of sel-2, an enhancer of lin-12 activity in vulval precursor cells 

Natalie de Souza, Laura G. Vallier, Hanna Fares, Iva Greenwald 

102. Function of a novel protein EFF-1 in cell fusion 

Jacob J del Campo, Ariel B Issacson, Morgan Tucker, Min Han, William A Mohler 

103. Degenerate binding sites for the FAX-1 nuclear receptor predict potential downstream target 

genes 

Stephen DeMeo, Rebecca Lombel, Danielle Snowflack, Aaron Wagner, Eric Smith, Sheila 

Clever, Bruce Wightman 

104. Mapping transcription regulatory networks in C. elegans 

B. Deplancke, D. Dupuy, M. Vidal, A.J. Marian Walhout 

105. SYP-3, a coiled-coil protein required for chromosome synapsis and chiasma formation in C. 

elegans 

Andreas Eizinger, Allison Hurlburt, JoAnne Engebrecht, Kirthi Reddy, Anne Villeneuve, 

Mónica Colaiácovo 

106. A genetic screen for mutants defective in male leaving, a mate-searching behavior of C. 

elegans 

Chunhui Fang, Rajarshi Ghosh, Scott W. Emmons 

107. DKF-2 is a Novel Target-Effector of Protein Kinase C 

Hui Feng, Min Ren, Charles S. Rubin 

108. A novel mutant that partially suppresses the daf-2 (e1370) Daf-c phenotype 

Manuel A. Fidalgo, Manuel J. Munoz 

109. Specificity of the C. elegans Putative Transmembrane Channel SID-1 

Michael C. Fitzgerald, Craig P. Hunter 

110. SMA-9, a Protein Involved in Patterning of the C.elegans M Lineage 

Marisa L Foehr, Ming Xu, Jun Liu 

111. Cloning and characterization of the C. elegans post-embryonic cytokinesis gene unc-85 

Iwen Fu, Jason W. Reuter, Fern P. Finger 

112. CeMyoD(hlh-1) in embryonic muscle fate determination 

Tetsunari Fukushige, Joan McDermott, Thomas Brodigan, Michael Krause 

113. Quantification of electrotaxis 

Chris Gabel, Albert Kao, Dmitri Pavlichin, Aravi Samuel 

114. Barotaxis 

Chris Gabel, Alex Dahlen, Aravi Samuel 

115. Genetic analysis of mutations that suppress dauer arrest in age-1/PI3 kinase mutants 

Minaxi S. Gami, Keaton Hanselman, Catherine A. Wolkow

116. spe-19, a Gene Affecting Spermiogenesis 

Brian Geldziler, Andy Singson 

117. Death defying acts: RNAi screen for genes influencing neuronal necrosis 

Beate Gerstbrein, Vienna Lo, Monica Driscoll 

118. An in vivo analysis of age-related biomarkers in C. elegans 

Beate Gerstbrein, Georgios Stamatas, Nikiforos Kollias, Monica Driscoll 

119. Serotonin and octopamine modulate thrashing behavior of C elegans 

Rajarshi Ghosh, Scott W. Emmons 

120. Functional Characterization of the Vertebrate Homologs of LIN-10: Mint 1, 2, and 3 

Doreen R. Glodowski, Bonnie L. Firestein, Christopher Rongo 

121. The Myt1 ortholog in C. elegans is essential for oocyte maturation 

Andy Golden 

122. RecQ Helicases, Genomic Stability and Lifespan in C. elegans 

Melissa M. Grabowski, Nenad Svrzikapa, Heidi Tissenbaum 

123. The temporal patterning microRNA let-7 controls multiple transcription factors including the 

nuclear hormone receptor DAF-12 

Helge Grosshans, Ted Johnson, Mark Gerstein, Frank J. Slack 

124. The genes tra-4 and mog-7 are necessary to ensure hermaphrodite development in the 

soma and in the germline 

Phillip Grote, Claudia Huber, Barbara Conradt 

125. SMA-10 is a novel extracellular regulator of the Sma/Mab TGF-beta pathway in C. elegans 

Tina L. Gumienny, Cole M. Zimmerman, Andrew F. Roberts, Huang Wang, Lena Chin, 

Richard W. Padgett 

126. LON-2 is a glypican heparan sulfate proteoglycan that regulates the Sma/Mab TGF-beta 

pathway in C. elegans 

Tina L. Gumienny, Huang Wang, Richard W. Padgett 

127. Reproductive isolation of C. briggsae haplotypes 

Rachael M. Hampton, Scott E. Baird 

128. High Throughput TILLING, Ecotilling, Genotyping, and Sequencing Instrumentation 

Jeff Harford 

129. Analysis of synMuv Protein Complexes in vivo and Characterization of the Class B synMuv 

Gene lin-61 

Melissa M Harrison, Xiaowei Lu, Bob Horvitz 

130. Visualizing activity of C. elegans interneurons 

Gal Haspel, Anne C. Hart 

131. Multiple factors act in concert to initiate the cell death of the NSM sister cells 

Julia Hatzold, Barbara Conradt 

132. An RNAi-based suppressor screen for components of the Aurora B kinase pathway 

Todd R. Heallen, Jill M. Schumacher 

133. Characterization of tissue-specific suppressors of cdc-25.1(gf) 

Michael Hebeisen, Roshni Basu, Richard Roy

134. Form and function of glia-neuron interactions 

Maxwell G. Heiman, Shai Shaham 

135. Vulval and uterine development are not temporally coordinated in ku212 mutants 

Li Huang, Wendy Hanna-Rose 

136. COMPUTER MODELING, SIMULATION AND ANALYSIS OF C. elegans VULVAL 

INDUCTION 

Na’aman Kam, Jasmin Fisher, David Harel, Amir Pnueli, Michael J. Stern, E. Jane Albert 

Hubbard 

137. A Screen for Genes Synthetically Lethal with lin-35 Rb 

Mike Hurwitz, Bob Horvitz 

138. Genetic variation reveals differences among C. elegans isolates for ASH mediated behaviors 

Rhonda Hyde, Anne C. Hart 

139. Study of the Effects of Oxidative Damage Repair on Aging and Muscle HealthSpan 

Carolina Ibanez-Ventoso, Samuel Bassous, Peter J. Schmeisser, Suzhen Guo, Monica 

Driscoll 

140. Guidance and cell-matching of a migrating epidermal sheet during ventral enclosure by 

MAB-20 and PLX-2 

Richard Ikegami, Kristin Simokat, Louise Dixon, Jeff Hardin, Joseph Culotti 

141. Identification of Wnt Pathway Target Genes in C. elegans by Microarray Analysis 

Belinda M. Jackson, David M. Eisenmann 

142. Combinatorial control of lin-48 expression in the C. elegans excretory duct cell is mediated 

through Pax and bZip transcription factors 

Hongtao Jia, Xiaodong Wang, Helen M. Chamberlin 

143. Structural and functional studies of the C.elegans Hsp90 ortholog DAF21 

Dayadevi Jirage, Harold Smith 

144. GNA-2, Chitin and the Functionally-Redundant CEJ-1/B0280.5 are Required for the 

Synthesis of a Lipophobic Extraembryonic Matrix (EEM) and are Essential for Development and 

Polarity in the One-cell Embryo 

Wendy L. Johnston, Aldis Krizus, James W. Dennis 

145. Biochemical and structure/function analysis of DGK-1, a neuronal diacylglycerol kinase and 

putative downstream effector of Gapha o signaling 

Antony Jose, Michael Koelle 

146. Cytological screening of the germ lines of sterile mutants for meiotic defects 

Malek Jundi, Monique C. Zetka 

147. EGL-26 belongs to the NlpC/P60 superfamily of putative enzymes and is closely related to a 

mammalian acyl transferase 

Rasika Kalamegham, Wendy Hanna-Rose 

148. Genes controlling the developmental response to nutrients in L1 larvae 

Gautam Kao, Peter Naredi, Simon Tuck 

149. Characterization of F11A10.3, a RING domain protein that interacts with the transcription 

factor UNC-3 

Ozgur Karakuzu, Brinda Prasad, Scott Cameron

150. Characterization of sel-6, a suppressor of lin-12 gain-of-function mutants 

Iskra Katic, Iva Greenwald 

151. How Does Ivermectin Induce Cell Death? 

Aamna Kaul, Joseph Dent 

152. Expression, function and regulation of gon-2 

Ben Kemp, Rachel West, Diane Church, Samantha Schilling, Janet Lee, Robert Bruce III, 

Matthew Ambros, Eric Lambie 

153. Centrosome Maturation and Duplication In C. elegans Require the Coiled-Coil Protein SPD-2 

Catherine A Kemp, Kevin R. Kopish, Peder Zipperlen, Julie Ahringer, Kevin F. O’Connell 

154. nhr-67 and nhr-111, two NR2E nuclear receptors that may function in nervous system 

development 

Ryan Kennedy, Kristy Reinert, Genna Albert, Chris Gissendanner, Ann Sluder, Bruce 

Wightman 

155. The lin-4 homologue, mir-237, directs proper vulva and gonad development in C. elegans 

Aurora Esquela Kerscher, Lei Bai, Frank J. Slack 

156. Sex-specific centrosome inheritance requires cki-2 in C. elegans 

Dae Young Kim, Richard Roy 

157. Roles of PAR-3 and PKC-3 in establishment and maintenance of epithelial cell polarity in C. 

elegans 

Heon S. Kim 

158. The Regulation of the CDC-6 Replication Licensing by CUL-4 

Jihyun Kim, Hui Feng, Edward T. Kipreos 

159. The O/E transcription factor UNC-3 specifies the identities of the ASI chemosensory neurons 

via cell-specific repression and activation mechanisms 

Kyuhyung Kim, Marc Colosimo, Piali Sengupta 

160. Identification of CUL-4 complex components 

Youngjo Kim, Edward T. Kipreos 

161. Genetic pathways that affect C. elegans leaving, a mate searching behavior 

Gunnar A. Kleemann, Ling yun Jia, Johnathan O .Lipton, Scott W. Emmons 

162. A screen for suppressors of cyclin-D1 in C. elegans 

John Koreth, Mike Boxem, Huihong Xu, Stuart H. Orkin, Sander van den Heuvel 

163. Attempts to develop molecular genetic tools to study parasitic nematodes 

Kelly Kraus, Meera Sundaram 

164. Reiteration of a lineage branch generating right-sided amphid neurons in ref-1 bHLH mutants 

Anne Lanjuin, Julia K. Thompson, Piali Sengupta 

165. Identification and Characterization of Suppressors of him-3 

Ka-Lun Law, Monique Zetka 

166. A him-8 mutation suppresses the PIE-1-induced synMuv defect 

Jungsoon Lee, Prashant Raghavan, Byung-Jae Park, Tae Ho Shin 

167. Interaction between the SEK-1-PMK-1 p38 MAPK and DAF-2/DAF-16 insulin signaling 

pathways mediating pathogen resistance and longevity in C. elegans 

Dennis H. Kim, Valerie Reinke, Danielle A. Garsin, Gary Ruvkun, Siu Sylvia Lee, Frederick 

M. Ausubel

168. Global transcriptional changes caused by cognition enhancing compounds in C. elegans N2 

French A. Lewis, III, Brian A. Dougherty 

169. Study of par-3 function in C. elegans 

Bingsi Li 

170. sma-9, A Gene that Regulates Body Size Development in C. elegans 

Jun Liang, Ling Yu, Cathy Savage-Dunn 

171. Toward Identifying Targets of MAP Kinase During C. elegans germline Development Using 

Functional Proteomic Approaches 

Baiqing Lin, Valerie Reinke 

172. CaM KII Regulates Neurotransmitter Release at the C. elegans Neuromuscular Junction 

Qiang Liu, Zhao-Wen Wang 

173. Control of aging and developmental arrest by TGF and insulin pathways during C. 

elegansdiapause 

Tao Liu, Manjing Pan, Garth Patterson 

174. End-to-end chromosome fusions in C elegans 

Mia R. Lowden, Bettina Meier, Shawn C. Ahmed 

175. An intracellular serpin, srp-6, is required for survival from hypoosmotic shock in 

Caenorhabditis elegans 

Cliff J. Luke, Stephen C. Pak, Vasantha Kumar, Sule Çataltepe, Carmen M. Knebel, 

Anthony Clark, Deiter Brömme, Gary A. Silverman 

176. Genetic Analysis of the Putative SUP-9/SUP-10/UNC-93 Two-Pore Domain K + Channel 

Complex 

Long Ma, Bob Horvitz 

177. Modeling and simulation of the behavior of proliferating C. elegans germ cells 

John Maciejowski, Nadia Ugel, Marco Isopi, Bud Mishra, E. Jane Albert Hubbard 

178. Nog mutants and early germline proliferation in C. elegans 

John Maciejowski, Giselle Cipriani, Ji-Inn Lee, James Ahn, Kerry Donny-Clark, E. Jane 

Albert Hubbard 

179. DNA replication and the proliferation versus meiotic development decision 

Valarie Vought, Min-Ho Lee, Larissa Wirlo, Katy Michalak, Deborah Springer, Ying Liu, 

Valerie Reinke, Tim Schedl, Eleanor Maine 

180. The molecular analysis of ego-2 

Ying Liu, Deborah Swenton, Dave Hansen, Eleanor Maine 

181. Control of lipid accumulation by ciliated neurons in C. elegans 

Ho Yi Mak, Gary Ruvkun 

182. n3263 is a mutant with persistent cell corpses that defines a candidate new engulfment gene 

Paolo M. Mangahas, H. Robert Horvitz, Zheng Zhou 

183. Localization of APH-1 Protein in Embryos 

David McGaughey, Valerie Hale, Caroline Goutte 

184. EGL-32 Functions in Sperm to Regulate Egg-Laying through the TGF-beta Pathway in 

C.elegans 

Marie McGovern, Ling Yu, Cathy Savage-Dunn

185. sns-10, the C. elegans ortholog of Aristaless/ARX, regulates sensory and motor neuron 

development 

Tal J. Melkman, Piali Sengupta 

186. him genes and X chromosome meiosis 

Philip M. Meneely, Joshua Havassy, Kathryn Crozier 

187. Characterization of an hlh-8 mutant 

Stephany G. Meyers, Ann K. Corsi 

188. Functional Analysis of the MicroRNA Genes of C. elegans 

Eric A Miska, Ezequiel Alvarez-Saavedra, Allison L Abbott, Andrew B Hellman, Nelson C 

Lau, David P Bartel, Victor Ambros, Bob Horvitz 

189. Genetic and molecular analysis of the C2H2 zinc-finger gene ehn-3 

Kristin M. Morphy, Judith Kimble, Laura D. Mathies 

190. Analysis of an UNC-13 protein expressed from an internal promoter 

Theresa Moser, Bethany Stitt, Kristin Servent, Brooke Swalm, Lydia Sanchez, Monique 

Spencer, Rebecca Kohn 

191. cwp-4, a novel male-specific C. elegans gene with a potential role in mating behavior 

William R. Mowrey, Douglas S. Portman 

192. Identification of genes regulating chemosensory neuron-specific morphologies 

Saikat Mukhopadhyay, Anne Lanjuin, Piali Sengupta 

193. The Role of PDZ Domain Proteins in GLR-1 Localization 

Vidhya Munnamalai, Christopher Rongo 

194. Uncovering the role for sperm contributed SCU-1 in regulating meiotic exit and axis formation 

in the early Caenorhabditis elegans embryo 

MaryAnn Murrow, Anna Mazor, Rebecca Lyczak 

195. Characterization of the identity and specificty of RGS protein targets in C. elegans 

Edith M. Myers, Michael R. Koelle 

196. Evidence that lin-35 Rb functions in hyp7 to inhibit vulval fates 

Toshia R Myers, Iva Greenwald 

197. RNAi-mediated screen for meiotic genes in C. elegans 

Sandra Nagl, Allison Hurlburt, Mónica Colaiácovo 

198. A germline-specific cell cycle inhibitor involved in dauer and adult lifespan 

Patrick Narbonne, Richard Roy 

199. The C. elegans pumilio ortholog, puf-9, controls the timing of development 

Mona J. Nolde, Kristy Reinert, Nazli Saka, Frank J. Slack 

200. High throughput genetic screen for suppressors of necrotic cell death 

Yury O. Nunez, Dewey Royal, MaryAnn Royal, Michael Lizzio Jr., Monica Driscoll 

201. Activation of SKN-1 stress response by a chemoprotective antioxidant 

Riva P. Oliveira, Jae Hyung An, Rosana P. Baker, Hideki Inoue, Kunihiro Matsumoto, T. 

Keith Blackwell 

202. Some Dauer Formation, Social Feeding, and Chemotaxis Mutants are Abnormal in the 

Enhanced Slowing Response 

Daniel Omura, Bob Horvitz

203. Transcriptional regulation and stochasticity of eff-1 in fusing cell types 

Eugene Opoku-Serebuoh, Victoria L. Scranton, William A. Mohler 

204. Characterization of mutants defective in intestinal nuclear division 


205. SRP-2 is an intracellular serpin that participates in postembryonic development 

Stephen C. Pak, Vasantha Kumar, Christopher Tsu, Cliff J. Luke, Yuko S. Askew, David J. 

Askew, David R. Mills, Anthony C. Clark, Gary Silverman 

206. The Identification of Factors Mediating the Downregulation of MEP-1 Function by PIE-1 

Byung-Jae Park, Prashant Raghavan, Seung-Il Kim, Jungsoon Lee, Keunhee Park, Tae Ho 

Shin 

207. A Screen for Mutants Resistant to Serotonin: a Search for Genes Involved in 

Neurotransmitter Signaling and Centrosome Movement 

Judy S. Pepper, Michael R. Koelle 

208. Investigating interacting partners of CeTwist 

Mary C. Philogene, Ann Corsi 

209. Genome wide RNAi screen for new synMuv genes 

Gino Poulin, Yan Dong, Andrew Fraser, Neil Hopper, Julie Ahringer 

210. A screen for axon branching and guidance mutants 

Brinda Prasad, Scott Clark 

211. Identification of Genetic Pathways Dependent on Protein N-glycosylation by GlcNAc-TV 

Justin M. Prien, Justin M. Crocker, Aldis Krizus, James W. Dennis, Charles E. Warren 

212. Components of the dosage compensation machinery antagonize the MEP-1 complex 

function 

Prashant Raghavan, Jungsoon Lee, Byun-Jae Park, Seun-il Kim, Keunhee Park, Tae Ho 

Shin 

213. Behavioral quiescence during the L1 stage and its alteration in eat-7 mutants 

David M. Raizen, Meera Sundaram, Allan I. Pack 

214. Characterization of UNC-43/CaMKII transport and activity in neurons 

Paris Rapp, Toru Umemura, Christopher Rongo 

215. Identification of factors required for germline silencing 

Tom Ratliff, Karissa McClinic, David Han, Bill Kelly 

216. Histone variant H2A.Z is essential for development in C.elegans 

Brianne J. Ray, William G. Kelly, Adam Raymond 

217. A Mos1 transposon mutagenesis screen for suppressors of the let-7 microRNA 

K. Reinert, F. J. Slack 

218. DKF-1 is A Diacylglycerol-regulated Kinase Involved in C.elegans Motility 

Min Ren, Hui Feng, Charles S. Rubin 

219. Modulation of C. elegans Egg-laying Behavior by the Environment and Experience 

Niels Ringstad, Bob Horvitz 

220. Dissecting the role of CNK-1 in LIN-45 Raf activation 

Christian E. Rocheleau, Meera V. Sundaram

221. Progress Towards the Cloning of lin-38 and Identification of Novel Class A SynMuv Genes 

Adam Saffer, Ewa Davison, Bob Horvitz 

222. C. elegans rme-3 encodes a clathrin heavy chain required for embryogenesis and 

neuro-muscular function 

Ken Sato, Chih-Hsiung Chen, Miyuki Sato, Barth D. Grant 

223. Characterization and mapping of sig-1, a new gene involved in germline-specific silencing of 

extrachromosomal transgenes 

Christine E. Schaner, William G. Kelly 

224. The mutation bc202 blocks physiological as well as non-physiological germ cell death in the 

adult hermaphrodite gonad 

Claus Schertel, Barbara Conradt 

225. Thrashing in liquid as a quantifiable measurement of aging 

Peter J. Schmeissner, Suzhen Guo, Shih-Hung Yu, Monica Driscoll 

226. The BarH Class Homeodomain Gene ceh-30 is Directly Regulated by tra-1 to Specify the 

Sexually Dimorphic Survival of the CEM Neurons 

Hillel Schwartz, Bob Horvitz 

227. The "Green Pharynx" Phenotype of Transgene Misexpression Yields New Insight into the 

synMuv Genes 

Hillel Schwartz, Dawn Wendell, Bob Horvitz 

228. Toward expression and biochemical characterization of EFF-1 

Victoria L. Scranton, William A. Mohler 

229. atx-2 Promotes Germline Proliferation and the Female Fate 

Xingyu She, Valarie E. Vought, Dave Hansen, Deborah Springer, Eleanor M. Maine 

230. The Unfolded Protein Response Regulates Glutamate Receptor Export from the ER 

Jaegal Shim, Toru Umemura, Erika Nothstein, Christopher Rongo 

231. Microarrays analysis of two essential factors required for RNAi, RDE-1 and RDE-4 

Martin J. Simard, Darryl Conte Jr, Jennifer A. Keys, Juerg Straubhaar, Danila Ulyanov, 

Craig C. Mello 

232. Regulation of gene expression by the Pax factor EGL-38 

Sama F. Sleiman, Helen M. Chamberlin 

233. C.elegans recognizes protons as a nociceptive stimulus through the DEG/EnaC and TRP 

channel 

Alfonso J. Apicella, Robert D. Slone, Monica Driscoll, William R. Schafer 

234. Move or Die: Epidermal Migration in the Caenorhabditis elegans Embryo 

Esteban Chen*, Michael M. S. Huang*, Veronica Zappi, Martha C. Soto 

235. Phenotypic characterization of egg-1, a molecule involved in fertilization 

Pavan Kadandale, Allison Stewart, Richard Klancer, Barth Grant, Andrew Singson 

236. How are cytoplasmic asymmetries achieved? In vivo studies of germ plasm localization 

dynamics during early embryogenesis 

Michael L. Stitzel, Denis Wirtz, Geraldine Seydoux 

237. Screen for Enhancers of ksr-2 Lethality 

Craig Stone, Meera Sundaram

238. EGL-26 controls vulF morphogenesis 

Hongliu Sun, Rita Sharma, Wendy Hanna-Rose 

239. Development of Caenorhabditis elegans in CeHR Axenic Medium 

Maria Szilagyi, Hugh F. LaPenotiere, Eric D. Clegg 

240. The Wnt genes egl-20 and cwn-1 are redundantly required for proper vulval cell fate 

specification 

Elizabeth Szyleyko, Julie E. Gleason, David M. Eisenmann 

241. The G proteins GOA-1 and EGL-30 function antagonistically in the HSN neurons that 

regulate egg-laying behavior in C. elegans 

Jessica E. Tanis, James J. Moresco, Robert A. Lindquist, Michael R. Koelle 

242. SRY-box containing protein SOX-2 directly binds to the promoter of Hox gene egl-5 and 

negatively regulates its expression 

Yingqi Teng, Scott W. Emmons 

243. Identification of genes involved in the specification of the sexually dimorphic CEMs 

Tatiana Tomasi, Stefanie Löser, Phillip Grote, Barbara Conradt 

244. Genetic screen for factors functioning with EGL-38 Pax to regulate lin-48 expression in 


Rong-Jeng Tseng, Helen M. Chamberlin 

245. Transcriptional specification of neural subtype in the C. elegans male tail 

Carolyn Tyler, Nicole Juskiw, Douglas S. Portman 

246. Suppressors of pha-4/FoxA loss of function mutations define potential pha-4 regulators 

Dustin L. Updike, Susan E. Mango 

247. Identification of genes involved in cell fate specification of the gonadal sheath cells in 


Laura G. Vallier, Helaina Skop, Lindsay Eisemann 

248. Genetic Screens for Suppressors of the ceh-30(n3714gf) Phenotype of Inappropriate 

Survival of the Male-Specific CEM Neurons in Hermaphrodites 

Johanna Varner, Hillel Schwartz, Bob Horvitz 

249. Screens for suppressors of cul-2 and zyg-11 

Srividya Vasudevan, Edward T. Kipreos 

250. Half-molecule ATP-binding cassette transporter, CeHMT1, is required for PC-dependent 

heavy metal detoxification in Caenorhabditis elegans 

Olena K. Vatamaniuk, Elizabeth A. Bucher, Meera V. Sundaram, Philip A. Rea 

251. How are apoptotic cells recognized by their phagocytes? 

Victor Venegas, Zheng Zhou 

252. Modifiers of polyglutamine-mediated neurodegeneration 

Cindy, Voisine, Adriana, K., Jones, Anne, C., Hart 

253. Disruption of germline pattern by forward and reverse genetics 

Roumen V. Voutev, E. Jane Albert Hubbard 

254. Regulation of Cell Death in the C. elegans Tail Spike Cell 

Carine Waase, Shai Shaham 

255. A suppressor screen for genes involved in UNC-6 mediated guidance 

Gauri Kulkarni, Chaunte Cannon, William G. Wadsworth

256. Regulation of RNA Polymerase II in C. elegans embryos and germline 

Amy K. Walker, T. Keith Blackwell 

257. Identification and characterization of the downstream target genes of CeTwist and its partner 

CeE/DA 

Peng Wang, Ann Corsi 

258. Identification of regulatory sequences necessary for egl-1 function in the ventral nerve cord 

Erin Webster, Tamara Strauss, Kelly Liu, Scott Cameron 

259. Identification and Characterization of MAPK Signaling Targets in the C. elegans Germline 

Stefanie West, Valerie Reinke 

260. The nuclear receptor gene fax-1 and homeobox gene unc-42 coordinate interneuron identity 

by regulating the expression of glutamate receptor subunits and other neuron-specific genes 

Bruce Wightman, Sheila Clever 

261. Characterization of loci that control or depend upon N-glycosylation in C. elegans 

William C. Wiswall Jr, Kristin M.D. Shaw, Weston B. Struwe, Charles E. Warren 

262. sid-5 is required for robust environmental RNAi 

Amanda J. Wright, Craig P. Hunter 

263. LIN-10 inhibits the synaptic delivery of GLR-1 

Tricia Wright, Henry Schaefer, Keri Martinowich, Howard Chang, Douglas Beach, 

Christopher Rongo 

264. MSP signals microtubule reorganization in C. elegans oocytes prior to fertilization 

Jana E. Harris, Ikuko Yamamoto, David Greenstein 

265. The conserved DEAD-box helicase CGH-1 negatively regulates MAP kinase activation in C. 

elegans oocytes 

Ikuko Yamamoto, David I. Greenstein 

266. Functional genomic characterization of germline stem cells in C. elegans 

Zhang Yang, Michelle Banfill, Valerie Reinke 

267. The Function of rde-1 homologs in C. elegans 

Erbay Yigit, Martin Simard, Ka Ming Pang, Ngan K. Vo, Shih-Chang Tsai, Shohei Mitani, 

Craig C. Mello 

268. Alteration of Pax protein DNA binding properties affects tissue-specific activities of the C. 

elegans gene egl-38 

Guojuan Zhang, Rong-Jeng Tseng, Helen M. Chamberlin 

269. The regulation of egl-5 expression in C. elegans by sop-2 

Hongjie Zhang, Yingqi Teng, Scott Emmons 

270. Characterizing the Cell Biology of mec-4(d)-induced Necrotic Cell Death in C. elegans 

Wenying Zhang, Monica Driscoll 

271. The Roles of Chitin and Chitin Synthases in C. elegans and Parasitic Nematodes 

Yinhua Zhang, Jeremy Foster, Laura Nelson, Dong Ma, Clotilde Carlow 

272. Identification and characterization of a mutation which causes arg-1 to be expressed 

ectopically in C. elegans 

Jie Zhao, Ann Corsi 

273. Neuropeptide modulation of C. elegans male mating behavior 

Tiewen Liu, Maureen M. Barr


Diana David-Rus, Peter J. Schmeissner, Beate Hartmann, Christophe Grundschober, Uri 

Einav, Eytan Domany, Patrick Nef, Garth Patterson, Monica Driscoll 

275. Isolation of Mutations that Cause Mini-Chromosome Loss 

Christine Barbishs, Sandi-Jo Galati, Stephanie Keller, Stacey Eggert, Mary Howe 

276. Abstract for Leica Microsystems Inc. 

Lon Nelson 

The author index follows the abstracts.

1. Candidate EGO-1 interactors that function in germline development. 

Xiang Yu 1 , Valarie Vought 1 , Jamie Wasilenko 1 , Tom Ratliff 2 , Bill Kelly 2 , Eleanor Maine 1 

1Department of Biology, Syracuse University, Syracuse, NY 

2Biology Department, Emory University, Atlanta, GA 

ego-1 was identified in a genetic screen for enhancers of glp-1 (Qiao et al.1995). Later studies 

showed that ego-1 mutants have defects in germline proliferation, meiotic progression, 

gametogenesis and RNA interference (RNAi) (Smardon et al. 2000). EGO-1 belongs to the 

RNA-dependent RNA Polymerase (RdRP) family. RdRP family members have been shown to 

function in RNA silencing in many organisms, although their specific role is unclear. Our working 

model is that the ego-1 developmental defects result from defects in RNA metabolism, perhaps 

specifically from the failure to produce or amplify small, non-coding RNA important for various 

cellular processes. Two approaches that we have taken to understanding the role of EGO-1 in 

germline development are to screen for interaction partners using the yeast two-hybrid system 

and to investigate chromatin structure in ego-1 mutant germ lines. 

EGO-1 is predicted to be a 1632 amino acid (aa) protein with a conserved RdRP domain (813 

aa) located between N-terminal (491 aa) and C-terminal (328 aa) domains. We used both 

N-terminal and C-terminal domains as "bait" in two-hybrid screens. We then tested candidate 

interactors by RNAi to identify those that function in the germ line, and particularly those whose 

germline function might be related to ego-1. We identified three genes that bind the EGO-1 

N-terminal domain and have severe proliferation defects in RNAi injection experiments. We then 

used tempered RNAi to produce milder proliferation defects, and checked for genetic interactions 

with glp-1. We find that R08C7.12 RNAi causes a meiotic progression defect similar to ego-1 

mutants, but does not enhance glp-1(ts). In contrast, ZC518.2 RNAi causes a masculinized germ 

line (a Mog defect) in wildtype animals and enhances glp-1(ts). Finally, the Y54F10BM.2 RNAi 

proliferation defect is more severe in a glp-1(ts) background than in a wildtype background, 

suggesting that it is enhanced by glp-1(ts). We are now extending these RNAi studies to include 

other relevant genes, and are collaborating with the Conradt lab (Dartmouth College) to recover 

deletion alleles of all three genes. To complement these studies, we are using a directed yeast 

two-hybrid approach to better define the physical interactions between EGO-1 and these three 

proteins. 

Studies in S. pombe have shown that chromatin silencing modifications depend, at least in part, 

on components of the RNAi machinery, including the pombe RdRP, RdP1 (Volpe et al., 2002). 

For example, accumulation of a silencing modification on centromeres, methylation of histone H3 

on the lysine 9 residue (H3meK9), is defective in RdP1 mutants. In the C. elegans germ line, 

H3meK9 silencing modifications accumulate on the X chromosome during male meiosis and on 

other unpaired DNA (Bean et al., 2004). We are investigating whether EGO-1 function is 

important for this aspect of germline chromatin assembly. Preliminary data suggest that silencing 

modifications on the male X are dependent on EGO-1 function. 

Bean et al. (2004) Nature Genetics 36, 100-105 

Qiao et al. (1995) Genetics 141, 551-569 

Smardon et al. (2000) Current Biology 10, 169-178 

Volpe et al. (2002) Science 297, 1833-1837.

2. Two controls of FBF expression in the C. elegans germ line 

Liana B. Lamont 1 , Sarah L. Crittenden 2 , David S. Bernstein 1 , Marvin Wickens 1 , Judith 

Kimble 1,2 

1Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706 

2Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin 53706 

The GLP-1 (Notch) signaling pathway and FBF RNA-binding proteins are crucial regulators of 

germline mitoses and germline stem cell (GSC) proliferation. The FBF proteins are Puf 

translational repressors and homologs of Drosophila Pumilio. FBF is encoded by two nearly 

identical genes, fbf-1 and fbf-2. An fbf-1 fbf-2 double mutant is sterile and lacks GSC, but fbf-1 or 

fbf-2 single deletion mutants are similar to wild-type. Therefore, the nearly identical FBF-1 and 

FBF-2 proteins can promote mitosis interchangeably. 

Although most fbf-1 and fbf-2 single mutants are fertile, rare animals are sterile. Surprisingly, 

these rare sterile mutants have opposite defects: fbf-1 single mutants can have a masculinized 

Mog germ line and make only sperm, and fbf-2 single mutants can have a feminized Fog germ 

line and make only oocytes. Therefore, fbf-1 and fbf-2 have distinct, albeit subtle, roles in 

controlling germ line fates. 

While analyzing the individual characteristics of fbf-1 and fbf-2, we uncovered two types of FBF 

control. First, FBF-1 protein increases in an fbf-2 deletion mutant, and FBF-2 protein increases in 

an fbf-1 deletion mutant. One simple model is that FBF directly controls its own expression. 

Indeed, FBF binding sites are present in both fbf-1 and fbf-2 3’UTRs, as assessed by both yeast 

three-hybrid and gel shift experiments. Therefore, in the wild-type germ line, FBF levels appear to 

be restricted by negative auto-regulation. 

We also noticed that the fbf-2 5’flanking region possesses four consensus LAG-1 binding sites, 

while fbf-1 has none. This suggested to us that fbf-2 might be a direct target of GLP-1 signaling. 

We completed several experiments to test this idea. Gel shift assays demonstrate that predicted 

LAG-1 sites upstream of fbf-2 can bind purified LAG-1. In contrast, an fbf-1 region that differs by 

only a single base pair does not bind LAG-1. Both fbf-2 mRNA and FBF-2 protein localize to the 

distal-most germ line, whereas fbf-1 products are more broadly distributed. Moreover, the level of 

FBF-2, but not FBF-1, responds to changes in GLP-1 signaling. We attempted to generate 

multiple fbf-2 transcriptional reporters without success. Nonetheless, our cumulative data 

supports the idea that GLP-1 signaling controls fbf-2 transcription directly. 

We will propose a model in which GLP-1 signaling and cross-regulation between the fbf genes 

work together to establish the normal pattern of GSC proliferation and differentiation.

3. Robust germline amplification and the precise timing of initial meiosis are dependent 

upon interactions with specific cells of the developing gonadal sheath 

Darrell J. Killian, E. Jane Albert Hubbard 

New York University, Department of Biology 

An early step in animal development is the establishment of the germ line, an "immortal" cell 

lineage that produces gametes as a means to contribute genetic information to the next 

generation. In animals as diverse as flies, nematodes, and mammals, the adult germ line is a 

spatial gradient of differentiation from germline stem cells progressing through meiosis to gamete 

formation. However, germline development begins from a pool of presumably equivalent 

primordial germ cells (PGCs). The development of the germ line from PGCs to a patterned adult 

tissue is dependent upon interactions with the developing somatic gonad. We are interested in 

somatic gonad/germline interactions that influence proliferation and differentiation of the 

developing germ line. 

In C. elegans, soma/germline interactions occur throughout germline development and can be 

studied through genetic and anatomical perturbations. The PGCs, Z2 and Z3, associate with the 

somatic gonad precursor cells Z1 and Z4. This interaction is essential for initial germ cell divisions 

and their competence to differentiate. Later in development, germline proliferation is dependent 

on the somatic distal tip cell (DTC). Ablation of the DTC or disruption of the underlying 

LAG-2/GLP-1 signaling pathway results in a loss of germline stem cells to meiosis (Kimble and 

White 1981; Austin and Kimble 1987). Conversely, constitutive activation of GLP-1 leads to 

germline hyper-proliferation at the expense of differentiation (Berry et al., 1997). Yet another 

germline pattern defect is proximal proliferation (Pro phenotype) characterized by ectopic 

germline proliferation in the proximal region of the germ line. Pro mutant germ lines contain, from 

distal to proximal, germline stem cells, meiotic cells, gametes, and a proximal germline tumor. In 

several Pro mutants the germline tumor is derived of a subset of the pre-meiotic germ line that 

failed to differentiate (Seydoux et al., 1990; Pepper et al., 2003; Killian and Hubbard 2004). 

We have shown that a reduction-of-function mutation in pro-1 results in weak distal germline 

proliferation, delayed meiosis, and a highly penetrant Pro phenotype. These phenotypes are due 

to loss of pro-1 activity in the sheath/spermatheca (SS) lineage of the somatic gonad, not the 

germ line. Consistent with this finding, the earliest germline defects in pro-1 mutants are detected 

just following the division of the SS cells. Furthermore, a stronger reduction of pro-1 function by 

RNAi deletes the SS lineage, further impairs germline proliferation, but does not cause a Pro 

phenotype. pro-1 encodes a member of a highly conserved but poorly characterized sub-family of 

the WD-repeat containing proteins (Killian and Hubbard 2004). 

Our studies with pro-1 prompted an investigation of early SS lineage/germline interactions with 

respect to germline proliferation, the timing of initial meiosis, and the molecular function of 

PRO-1. Our time-course analysis of coordinate somatic gonad/germline development and our 

cell-killing experiments complement and extend earlier findings by McCarter et al. (1997). We find 

that the distal SS daughter, sheath 1, is in direct contact with mitotic germ cells in the L3 and L4 

and this contact is crucial for robust proliferation. Sheath 1 does not contact the mitotic germ line 

in adults (Hall et al., 1999) when the germline is in a steady state. In addition, we find that 

ablation of the proximal daughter of the SS cell, the precursor to sheath 2-5 and spermathecal 

cells, delays the onset of meiosis in the germ line. Together, our results support important and 

antagonistic roles of the SS daughter cells in early germline pattern formation. We are also 

investigating the role of PRO-1 with respect to the function of the SS lineage cells. The S. 

cerevisiae ortholog of pro-1, Ipi3, has been implicated in the rRNA processing step of ribosome. 

Our genetic analysis supports an analogous role for PRO-1. Up-regulation of ribosome 

biogenesis via mutation in either ncl-1 or lin-35/Rb significantly rescues pro-1(na48) germline 

pattern defects. Defects in ribosome biogenesis have been linked to both inhibition of cell growth 

and cell cycle arrest. We are currently investigating a potential developmental control of ribosome 

biogenesis related to the functions of the SS lineage cells and germline patterning.

4. The NR4A nuclear receptor is required for spermatheca morphogenesis during somatic 

gonad development 

Chris R. Gissendanner 1 , Tri Q. Nguyen 2 , Marius Hoener 3 , Ann E. Sluder 4 , Claude V. Maina 1 

1New England Biolabs, Beverly, MA 

2Develogen AG, Goettingen, Germany 

3F. Hoffmann-La Roche Ltd., Basel, Switzerland 

4Cambria Biosciences, Woburn, MA 

The NR4A group of nuclear receptor (NR) transcription factors regulates a wide variety of 

biological processes in the metazoa. The vertebrate NR4A paralogs (NGFI-Balpha, beta, and 

gamma) predominately have neuronal functions but also regulate other processes such as 

embryonic development, organogenesis, and apoptosis. The insect NR4A NR, DHR38, has been 

proposed to have a complex function in ecdysone signaling during metamorphosis. The C. 

elegans genome also encodes an NR4A NR gene, nhr-6. We previously reported that nhr-6 is 

required for C. elegans reproduction (IWM 2003). Animals homozygous for a null allele of nhr-6, 

as well as animals subjected to nhr-6 RNAi, have ovulation defects and lay abnormally shaped 

eggs. Further analysis has demonstrated that the ovulation defects are due to abnormal 

spermatheca development in nhr-6 mutant animals. The spermathecae of nhr-6 mutants are 

severely malformed and exhibit cytoskeletal defects. In particular, the spermathecal valves 

display the most severe morphological phenotype. nhr-6 is expressed in the somatic gonad 

beginning in L3 and becomes restricted to the spermathecal region in L4 animals. The highest 

levels of nhr-6 expression are in cells that likely form the spermatheca-uterine junction. We are 

currently investigating the molecular mechanisms of NHR-6 function including the identification of 

potential target genes, characterization of the NHR-6 binding sites, and the regulation of nhr-6 

expression. Our analysis suggests that NHR-6 can be utilized as a useful model system for 

dissecting the complex developmental functions of nuclear receptors.

5. The spe-38 gene encodes a novel tetraspan integral membrane protein and is required 

for sperm function at fertilization. 

Indrani Chatterjee, Andrew W Singson 

Waksman Instiute, Rutgers University, Piscataway, NJ 08854 

Fertilization involves the union of haploid gametes; a sperm and an oocyte to give rise to a 

diploid zygote. C.elegans serves as an excellent model system to identify and study genes that 

are required for the process of fertilization. Mutations in the spe-38 gene result in both male and 

hermaphrodite sterility due to a sperm specific defect. Sperm produced by spe-38 mutants are 

morphologically identical to wild type sperm. They have normal motility and can make contact 

with oocytes without fertilizing them. spe-38 sperm can also stimulate ovulation and can indulge 

in sperm competition. We cloned the spe-38 gene in order to determine its molecular function in 

fertilization. The spe-38 gene encodes a novel tetraspan integral membrane protein. Other 

structurally similar tetraspan molecules have been implicated in processes such as gamete 

adhesion/fusion in mammals, membrane adhesion/fusion during yeast mating, and the formation 

of tight-junctions in metazoa. This suggests that SPE-38 is required for gamete adhesion and/or 

fusion at fertilization. The ongoing characterization of SPE-38 will lead to a better understanding 

of C. elegans fertilization and complement work in other organism.

6. Sperm-oocyte interactions in C. elegans 

Alissa Richmond, Diane C. Shakes 

Department of Biology, College of William and Mary 

Fertilization is the key event that initiates the developmental program of any organism. Gamete 

interactions have been studied extensively in organisms such as sea urchin, Xenopus, and 

mammals. In C. elegans, much is known about gametogenesiss and early embryonic 

development but fertilization itself remains poorly understood. For many organisms, fertilization 

occurs through the fusion of the oocyte and sperm plasma membranes. In contrast, TEM studies 

in the filarial nematode, Dirofilaria immitis, suggest that nematode sperm is engulfed by the 

oocyte (Sacchi et al., 2002). To determine the mechanism (fusion or endocytosis) of fertilization in 

C. elegans, we have analyzed the localization of various sperm markers immediately after 

fertilization. Our analysis of two classes of sperm membrane proteins strongly suggests that in C. 

elegans, the oocyte and sperm membranes fuse. Most convincingly, staining of newly fertilized 

embryos using the 1CB4 monoclonal antibody revealed a tight patch of staining at the point of 

sperm entry.

7. SPE-42 is required for sperm-egg interaction during C. elegans fertilization 

Tim L. Kroft, Steven W. L’Hernault 

Department of Biology, Emory University, Atlanta, GA 30322 

We exploit the unusual reproductive biology of C. elegans to discover mutants that are 

defective in fertilization. We generate spermatogenesis defective (Spe) mutants by mutagenizing 

hermaphrodites and selecting self-sterile worms that lay oocytes. Self-sterile hermaphrodites can 

be mated to wild type males, and mutants that produce cross progeny are usually defective only 

in spermatogenesis. Spe mutant sperm are examined and only mutants with cytologically normal 

spermatozoa are chosen for further study. Identification, cloning and analysis of genes whose 

protein products function during fertilization will lead to a better understanding of sperm-egg 

interactions necessary for fertilization and cell-cell communication in general. Here we report our 

analyses of spe-42, a late-acting fertilization defective mutant. Spermatids from spe-42 mutant 

animals activate normally when exposed to the protease pronase and form spermatozoa that are 

morphologically indistinguishable from wild type by light microscopy. A sensitive mating assay 

showed that spe-42 males can mate normally and produce apparently wild type seminal fluid that 

is competent to activate spermatids. spe-42 was placed on the genetic map by three-point, 

deficiency and single nucleotide polymorphism mapping techniques. A cosmid within the genetic 

interval defined by these mapping techniques restores self-fertility to spe-42 mutants. Both alleles 

(eb5 and tn1231) have been sequenced and the underlying genetic lesions have been 

determined. eb5is an opal nonsense mutation within exon 10 and tn1231 is a splice donor mutant 

following exon 12. spe-42 consists of 15 exons and encodes a predicted transmembrane protein. 

We have identified SPE-42 homologs in a number of species including mosquitoes, fruit flies, 

mice, rats and humans.

8. A Vesicle-Budding Model for the Release of MSP from C. elegans Sperm 

Mary Kosinski 1 , Kent McDonald 2 , Jay Jerome 1 , David Greenstein 1 

1Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN USA 

2The University of California-Berkely, CA USA 

Oocyte meiotic maturation is essential to prepare the oocyte for fertilization and embryonic 

development. C. elegans sperm stimulate oocyte meiotic maturation and gonadal sheath cell 

contraction using major sperm protein (MSP) as a signaling molecule. MSP promotes meiotic 

maturation and activates MAP kinase in oocytes in part by binding the VAB-1 Eph receptor 

protein-tyrosine kinase. The discovery of MSP’s signaling role raised the question of how sperm 

release MSP to signal oocytes and sheath cells at a distance. MSP is a cytoskeletal protein that 

nematode sperm utilize for motility and it does not possess a hydrophobic leader sequence. In 

addition, C. eleganssperm lack many standard secretory components, such as ribosomes, ER, or 

Golgi. Thus, the release of MSP may depend on a novel mechanism. 

Using specific antibodies, we detect MSP as far as 90 microns outside of spermatids and 

spermatozoa in vivo,consistent with its function as an extracellular signal. Labeling with vital dyes 

and sperm specific antibodies rules out sperm lysis as a potential mechanism. Wide-field and 

confocal microscopy shows extracellular MSP to be punctate with large (

9. EFL-1 and DPL-1 Activate Genes Required for Oogenesis and Proper Embryonic 

Specification in the Maternal Germline 

Woo Chi, Valerie Reinke 

Department of Genetics Yale University School of Medicine 333 Cedar Street New Haven, CT 

06520 

E2F, a heterodimer consisting of an E2F and a DP subunit, is a sequence-specific transcription 

factor that regulates genes required for DNA synthesis and cell proliferation during the G1-S 

transition in the mammalian cell cycle. In C. elegans, dpl-1(DP-like) and efl-1(E2F-like) have been 

shown to function in both embryonic development and vulval development (Page et al., 2001; 

Coel and Horvitz, 2001). A null mutation in dpl-1 results in embryonic lethality at (or prior to) the 

one-cell stage, while a strong loss-of-function or null mutation of efl-1 causes embryonic lethality 

with apparent defects in DNA replication and cell division. We wished to identify transcriptional 

target genes of dpl-1 and efl-1 in the maternal germline and to investigate their roles in germline 

and embryonic development. 

Microarray analysis comparing dissected young adult gonads from dpl-1(n3316) or efl-1(n3639) 

to wild type show a highly significant target gene overlap (p

10. RGS-7 Completes a Receptor-independent Heterotrimeric G Protein Signaling Cycle to 

Regulate Mitotic Spindle Positioning in C. elegans 

Heather A. Hess, Michael R. Koelle 

Yale University 

Heterotrimeric G proteins promote astral microtubule forces on centrosomes to position mitotic 

spindles properly during asymmetric cell division in C. elegans embryos. While all previously 

studied G protein functions require activation by seven-transmembrane receptors, this function 

appears to be receptor-independent, and the active form of the G proteins remains unclear. We 

obtained mutants for all 13 C. elegans regulators of G protein signaling and found that one, 

RGS-7, decreases the speed and magnitude of centrosome movements. The effects of RGS-7 

require two redundant Gao-related G proteins and their non-receptor activators. Using 

recombinant proteins, we found that the non-receptor activator RIC-8 stimulates GTP binding by 

Gao, and the RGS domain of RGS-7 stimulates GTP hydrolysis by Gao. These results 

demonstrate that the active species in the receptor-independent G protein cycle is Gao·GTP, and 

that RGS-7 completes the cycle by driving Gao to its inactive, GDP-bound form.

11. Three conserved protein kinases, DYRK, CDC2 and GSK3 promote OMA-1 degradation 

to establish proper cell fate and cell division polarity in early C. elegans embryos 

Masaki Shirayama, Takao Ishidate, Kuniaki Nakamura, Craig C. Mello 

UMass Medical School, Worcester, MA 01605 

The proper establishment of cell polarity and identity underlies development in all multicellular 

animals. In C. elegans, the 4-cell stage blastomere, EMS receives inductive signals that orient its 

division axis and specify the endoderm fate. GSK-3 is positively required for Wnt-mediated EMS 

cell fate determination and mitotic spindle alignment in EMS. Paradoxically, despite lacking 

E-derived endoderm, gsk-3 mutants frequently produce ectopic endoderm from the C blastomere. 

Thus GSK-3 functions to promote endoderm development in E and simultaneously functions to 

repress endoderm development in C. In our screens for temperature-sensitive mutants, we 

isolated 5 mutants that (like gsk-3 mutants) produce ectopic endoderm derived from the C 

blastomere and are defective in EMS cell polarity. These mutations include one allele of cdk-1, 

one allele of its binding partner, cks-1, one allele of the dyrk-family kinase member mbk-2, and 

two alleles of the zinc finger gene oma-1. Previous work has linked OMA-1 destruction to C-cell 

fate specification and EMS polarity1), therefore, we tested whether cdk-1, mbk-2 and gsk-3 

mutants exhibit defects in OMA-1 degradation. We found that OMA-1 protein persists ectopically 

in all of these mutants. We have also found that MBK-2 directly phosphorylates OMA-1 protein 

and this phosphorylation is abolished in the ne411and zu405 OMA-1 mutant protein due to a 

mutation in a consensus MBK-2 site. We are currently testing the model that sequential 

phosphorylation of OMA-1 by MBK-2, CDK-1 and GSK-3 kinases is required for the timely 

degradation of OMA-1 protein. 

1) Lin, R. Dev. Biol. 258 (2003) 226

12. Automated production of standardized, easy-to-share, easy-to-compare 4D embryonic 

image data. 

Ariel B. Isaacson, William A. Mohler 

Dept. of Genetics and Developmental Biology, UConn Health Center, Farmington, CT 

06030-3301 

We are developing software tools for production of "publishable" 4D movie data from 

microscope recordings of GFP expression in embryos. Raw image stacks comprising a field of 

several randomly-oriented embryos are typically processed to produce three useful standardized 

data types for each embryo: sliced-4D QuickTimeVR (QTVR) movies, stereo-4D QTVR movies, 

and embryonic developmental expression chronograms (EDECs). A finished sliced-4D QTVR 

allows free animation through focus and over time for an individual embryo, re-aligned into the 

canonical anterior (left), posterior (right), dorsal(up), ventral(down) orientation for display of C. 

elegans embryos. A stereo-4D QTVR allows free rotational animation of 3D volume 

reconstructions (maximum point projection) around the X and Y-axes, as well as animation 

through time. By keying on the onset of embryonic movement, each movie isolated from an 

asynchronous field of embryos can be properly annotated as to developmental time. Both 

sliced-4D and stereo-4D QTVRs benefit from several aspects of the established QuickTimeVR 

data format: facile navigation of the movie using the computer mouse or keyboard, availability of 

free viewing software and web browser plug-ins, streaming-download web access to large 

movies, and flexible choices for data compression. The third data type, an EDEC, represents the 

full 4D data set in a 2-dimensional summary image - averaged fluorescence intensity along the 

one dimension of the anterior-posterior axis is plotted against time. Each of these formats has 

unique advantages in analysis and comparison among expression patterns for different gene 

products. All three should be applicable to the "super-imposable" comparison and correlation of 

the expression/localization patterns of different gene products.

13. The germ granule protein PGL-1 is required for efficient RNA interference 

Darryl Conte Jr. 1 , Yingdee Unhavathaya 1 , Craig C. Mello 1,2 

1Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 

01605 

2Howard Hughes Medical Institute 

In C. elegans, very small quantities of dsRNA are sufficient to generate RNA interference not 

only in the animal that is exposed to dsRNA but also in subsequent generations (1, 2). These 

findings indicate that amplification and inheritance of a silencing agent(s) (such as siRNA) are 

important to the mechanism of RNAi. P-granules, the worm equivalent of germ granules, are 

expressed in the maternal germline, deposited into early embryos and are implicated in 

translational regulation of maternal mRNAs. Therefore, we hypothesized that the germline 

P-granules of C. elegans may be important for the transmission of a silencing factor and hence 

for RNAi. 

An examination of mutations in known P-granule components revealed that the PGL-1 protein 

is required for RNAi. siRNAs failed to accumulate in pgl-1 mutant worms exposed to dsRNA 

targeting a maternal mRNA, while siRNAs appeared to accumulate in the embryos of wild-type 

worms. Remarkably, animals challenged with dsRNA targeting zygotic mRNAs expressed in 

somatic tissues produced progeny with a reduced penetrance of RNAi phenotypes. These data 

are consistent with the hypothesis that PGL-1 may be important for efficient transmission of a 

silencing agent to the progeny of animals exposed to dsRNA. 

PGL-1 encodes a protein with an RGG-box found in RNA-binding proteins and is required for 

fertility in C. elegans (3). An attractive scenario is that PGL-1 interacts with and transmits siRNA 

to the progeny of animals exposed to dsRNA. Alternatively, PGL-1 may regulate the activity of the 

RNAi pathway by modulating the accessibility, localization or expression of RNAi pathway 

components or intermediates. We will present our recent efforts to elucidate the function of PGL-1 

in RNAi and the relationship to the role of PGL-1 in development. 

1) Fire et al. (1998) Nature 391:806-811 

2) Grishok, Tabara and Mello (2000) Science 287:2494-2497 

3) Kawasaki et al. (1998) Cell 94:635-645 

This work is funded by National Institutes of Health grants GM058800 (to C.C.M.) and 

GM063348 (to D.C.)

14. Enhancers of ksr-1 lethality define new potential regulators of small regulatory RNAs 

Christian E. Rocheleau, Yelena Bernstein, Meera V. Sundaram 

Department of Genetics, University of Pennsylvania, Philadelphia, PA 

Ras signaling is required for multiple cell fate decisions during C. elegans development 

including specification of the excretory duct cell. Strong mutations in let-60 ras result in distinct 

rod-like larval lethal phenotype owing to loss of the excretory duct cell. Mutations in positive 

regulators ksr-1 and cnk-1 display little or no rod-like lethality (90% rod-like larval phenotype (1). To identify additional positive regulators of 

Ras signaling, we performed a RNAi screen for enhancers of ksr-1 rod-like lethality. From a 

screen of chromosome I we identified eleven ekl genes (enhancers of ksr-1 lethality). 

Several observations suggest that at least a subset of the ekl genes may function together in 

the generation or function of small regulatory RNAs. First, the molecular identities of three strong 

enhancers ego-1 (a putative RNA-dependent RNA polymerase), ekl-1 (a tudor domain protein), 

and ekl-3 (a Dicer-related helicase), are consistent with an RNA based mechanism. Second, two 

additional ekl genes may be co-regulated with one or more of the above genes based on the 

microarray topology map (2), and Gene Recommender algorhythm (3). Third, RNAi of ekl-4 (a 

conserved SANT domain protein) has embryonic lethal and heterochronic related phenotypes 

similar to those described for alg-1/alg-2 and dcr-1 (4), suggestive of a role in microRNA 

processing. These cumulative observations suggest that small regulatory RNAs may be 

cooperating with Ras signaling in specification of the excretory duct cell fate. 

We are in the process of confirming that ekl-4(RNAi) phenotypes are due to heterochronic 

defects and if ekl-4 is required for miRNA processing. In addition, we are determining if the other 

ekl genes have heterochronic phenotypes and if loss of alg-1/alg-2 can enhance the ksr-1 ras-like 

lethal phenotype. The results of these experiments will be presented at the meeting. 

1. see poster by Rocheleau and Sundaram 2. Kim et al., Science (2001) 3. Owen et al., 

Genome Biology (2003) 4. Grishok et al., Cell (2001)

15. Multiple, dynamic microRNA ribonucleoprotein complexes with selective microRNA 

cargos in C. elegans 

Gopalakrishna Ramaswamy, Eun-Young Choi, Frank J. Slack 

Department of Molecular, Cellular and Developmental Biology, Yale University, P.O. Box 208103, 

New Haven, CT 06520 

Hundreds of small, non-translated regulatory RNAs, known as microRNAs (miRNAs) are 

expressed from the genomes of animals and plants. miRNAs as well as small interfering RNAs 

(siRNAs) are associated with protein components of the RNA induced silencing complex (RISC), 

including Argonaute and VIG proteins, but little is known about miRNA mechanism of action or 

miRNA ribonucleoprotein (miRNP) assembly. Here we demonstrate the presence of multiple, 

distinct miRNPs in C. elegans extracts. These complexes form in a temporal sequence, which 

suggests that a series of smaller intermediates form into a larger, functional miRNP. All of these 

complexes require Argonaute-like-2 (ALG-2) to form, and are thus, likely to be related to RISC. 

We detected two large complexes (~ 500 kDa and > 669 kDa), which are able to bind both let-7 

and lin-4 miRNAs, while two smaller complexes of ~160 kDa and ~250 kDa, show selectivity and 

bind let-7, but not lin-4. We identified two proteins, p41 and p14 that bind directly to the let-7 

miRNA in all complexes, suggesting that these might be core miRNP factors. p41 is similar in size 

to C. elegans VIG-1 and we demonstrate that VIG-1 can bind directly to let-7 miRNA. We 

conclude that multiple miRNP complexes exist in C. elegans, and that these may have different 

miRNA cargos and roles.

16. eak (enhancer-of-akt-1) genes encode membrane-associated proteins that potentiate 

AKT-1 signaling in the C. elegans XXX cells. 

Patrick J. Hu 1,2 , Jinling Xu 2 , Gary Ruvkun 2 

1Division of Hematology/Oncology, Massachusetts General Hospital 

2Department of Molecular Biology, Massachusetts General Hospital 

Akt/PKB proteins transduce signals that regulate growth and apoptosis in mammals. 

Dysregulation of Akt/PKB signaling plays prominent roles in the pathogenesis of cancer and 

diabetes in humans. In C. elegans, AKT-1 prevents dauer formation by transducing insulin-like 

signals from the DAF-2 insulin/IGF-1 receptor to inhibit the FoxO transcription factor DAF-16. 

In order to identify novel components of DAF-2 signaling, we performed an Eak 

(enhancer-of-akt-1) screen. We have isolated 21 independent Eak mutants defining 7 

complementation groups from a screen of approximately 20,000 haploid genomes. eak mutants 

have weak dauer-constitutive phenotypes and strongly enhance the weak dauer-constitutive 

phenotype of akt-1 loss-of-function mutants. eak phenotypes are suppressed by daf-16 null 

mutants, suggesting that EAK proteins function in DAF-2 signaling. Interestingly, all eak alleles 

assayed thus far have normal lifespans. 

4 eak genes have been cloned. eak-3 and eak-4 encode novel proteins; eak-5 and eak-6 

encode proteins with similarity to protein tyrosine phosphatases. Analysis of transgenic animals 

expressing transcriptional and translational fluorescent protein reporter constructs indicates that 

all 4 EAK proteins are predominantly expressed in and localize to the plasma membrane of the 

XXX cells. Expression of AKT-1 in the XXX cells fully rescues the dauer phenotype of eak;akt-1 

double mutants, suggesting that the XXX cells are a major site of AKT-1 function in dauer 

regulation. Taken together, the data support a model whereby AKT-1 and EAK proteins function 

in parallel in the XXX cells to prevent dauer formation during larval development.

17. Notch function in differentiated neurons is required to maintain dauer. 


Department of Biology, McGill University, Montréal, Québec 

Appropriate cell fate specification is highly dependent on cell-cell communication and the 

interplay between different pathways provides the developmental complexity typical of higher 

animals. In the case of dauer formation in C. elegans, three conserved signaling pathways 

(TGF-β, Insulin-like, and cGMP signaling pathways) regulate the complex morphological and 

metabolic changes typical of this stage, and many of these changes must occur globally in the 

animal. Evidence suggests that these signaling pathways are required in a small subset of 

neurons, implicating that a second signal (probably neuroendocrine) is produced in these neurons 

to cause these global changes. Although the Notch signaling pathway has not been involved in 

dauer formation, we have shown that when the Delta-like ligand (LAG-2) or the Notch receptors 

themselves (LIN-12 and GLP-1) are mutated, dauer larvae are unable to maintain this stage and 

therefore recover prematurely. When we tested the downstream effector of Notch signaling, the 

transcription factor lag-1, for recovery in the daf-7 mutant background we noticed that the animals 

maintained the dauer stage better than the single mutant daf-7. This suggests that the 

downstream genes activated by Notch, which are required for dauer maintenance, are 

’’derepressed’’ in the lag-1mutant background and are rendered independent of Notch signaling. 

We also found, through the analysis of the different double mutants, that the Insulin-like pathway 

was absolutely required for the premature dauer recovery caused by mutation in components of 

the Notch signaling pathway. This suggests that downstream Notch targets may repress 

Insulin-like signaling in order to maintain the dauer stage. More interestingly, all these interactions 

occur in a small collection of neurons in the head of the animal. We are currently trying to 

determine which neurons are involved in Notch signaling during dauer. 

Animals harboring a lag-2::GFP transgene express GFP in the IL1 head neurons at the onset 

and throughout the dauer stage. Although these neurons were never shown to be required for 

dauer development, they are nonetheless among the subset of neurons remodeled during the 

dauer stage. To determine if these neurons play a role in dauer development, we used the mutant 

background deg-1(u38), which causes the degeneration of the IL1 neurons as well as the 

motorneurons AVD, AVG and PVC. We found that these dauer animals are incapable of 

maintaining dauer, as was observed in daf-7; lag-2 mutants, suggesting that these neurons are 

required for dauer maintenance. Our analysis of the lag-2 promoter indicated that 3 forkhead 

binding sites are sufficient for the dauer-specific expression in these head neurons. We are 

currently testing the 15 predicted forkhead transcription factors (Fkh) identified from the C. 

elegans genome database to determine which Fkh binds to these sites to initiate the Notch 

cascade at the onset of dauer. Based on our genetic studies, it appears that the Fkh DAF-16 is 

not involved. 

We propose that the pathways required for dauer formation (TGF-β and/or the Insulin-like 

pathway) activate the Notch ligand in head neurons specifically during dauer through a Fkh. 

Ligand binding to the nearby Notch receptors leads to the expression of genes required for the 

inhibition of the Insulin-like pathway, probably through a neuroendocrine mechanism since these 

changes are rapid and must occur in all cells throughout the animal. Therefore, our results 

provide a link between three important signaling pathways, as well as implicating Notch signaling 

in a novel function, perhaps different than its more canonical role in binary cell fate specification.

18. Control of aging and developmental arrest by TGFbeta and insulin pathways during C. 

elegans diapause 

Manjing Pan, Li Sun da Graca, Tao Liu, Garth I. Patterson 

Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 

When resources are scant, C. elegans larvae arrest as non-aging dauers under the control of 

insulin- and TGFbeta-related signaling pathways. Recent experiments suggest that insulin- and 

TGFbeta-related pathways function in neurons to control the dauer decision (Wolkow et al., 2000 

Science. 290:147; Inoue & Thomas 2000 Dev Biol 217:192; da Graca et al. 2003 Dev 131:435; 

Libina et al, 2003 Cell 115:489). Many genes in the TGFbeta pathway are broadly expressed in 

the nervous system, but daf-5, a putative transcriptional coregulator that is negatively regulated 

by the TGFbeta pathway, is strongly expressed in only about a dozen neurons. These neurons 

include four pairs of neurons that innervate the amphid sensilla, which makes them very good 

candidates for being the initial source of dauer signal in the nervous system. 

We have used microarray analysis to identify many genes that are regulated by the TGFbeta 

pathway (see abstract by Liu et al). We find that many insulin-related ligands show 

TGFbeta-pathway dependent regulation. C. elegans has over thirty insulin-related ligands with 

diverse structures, but only one known insulin receptor kinase. The abundance of ligands is 

puzzling, and genetic analysis of these diverse ligands has shed light on their function. RNAi 

analysis has shown that four of the ligands promote dauer and five promote reproductive growth. 

Of these, only ins-7 has been previously shown to play this role. Another clue to the puzzle of the 

seemingly excessive number of ligands is the presence of a large family of genes with a small 

degree of sequence similarity to the extracellular, ligand-binding domain of insulin and other 

tyrosine-kinase receptors. Unlike daf-2, which is the only known insulin receptor in C. elegans, 

these novel genes have no kinase domain. We have performed RNAi on a subset of this large 

family of genes, and find that one promotes dauer formation, and three promote reproductive 

growth. We will present our functional analysis of these and other TGFbeta-regulated genes.

19. Life span regulation by JNK MAP kinase in C. elegans: a novel input into daf-16 

Seung Wook Oh, Nenad Svrzikapa, Heidi A. Tissenbaum 

Program in Molecular Medicine, University of Massachusetts Medical School, Aaron Lazare 

Research Building, 364 Plantation Street, Worcester, MA 01605 

The insulin-like signaling plays a pivotal role in life span regulation in many diverse organisms. 

In C. elegans, mutations attenuating daf-2, an ortholog of the mammalian insulin and insulin-like 

growth factor-1 (IGF-1) receptor, or age-1, the PI 3-kinase catalytic subunit (p110), extend life 

span and confer stress resistance. These phenotypes require the FOXO transcription factor 

ortholog, DAF-16, which is down regulated by this pathway. Here we show the C. elegans JNK 

MAP kinase as a novel input into daf-16 for life span regulation. Overexpression of jnk-1 extends 

life span by up to 45%, which requires two upstream kinases, jkk-1 and mek-1. Genetic analysis 

indicates that life span extension by jnk-1 overexpression is suppressed by daf-16 RNAi and 

synergizes with a mutation in daf-2. In addition, overexpression of jnk-1 promotes resistance to 

oxidative and heat stress. Taken together, these findings define a new role for the JNK MAP 

kinase signaling pathway in life span regulation and identify a new pathway that targets daf-16.

20. Regulation of chemoreceptor gene expression by MEF-2 and class II HDACs in C. 

elegans 

Alexander M van der Linden, Katie Nolan, Piali Sengupta 

Brandeis University, Department of Biology, MS008, 405 South Street, MA02454, Waltham 

C. elegans is able to regulate the expression of chemoreceptors in response to environmental 

cues. This provides a simple mechanism by which C. elegans can rapidly respond to changing 

environmental conditions. How is the expression of chemoreceptor genes regulated, both during 

development and in response to environmental signals? Previously, we identified a role for the 

Ser/Thr-kinase KIN-29 in regulating the efficient expression of a subset of candidate olfactory 

receptors in the chemosensory system [1]. kin-29 mutants exhibit reduced expression of the str-1 

receptor in the AWB olfactory neurons and the sra-6 receptor in the ASH sensory neurons. In 

addition, kin-29 mutants exhibit a reduced body-size and are hypersensitive to dauer pheromone, 

indicative of alterations in the ability to correctly sense environmental conditions. All phenotypes 

can be rescued by kin-29 expression in chemosensory neurons, suggesting that modulation of 

receptor expression may be crucial for regulating body-size and pheromone responses. However, 

the mechanisms through which KIN-29 functions to regulate gene expression are unknown. 

In order to identify targets of KIN-29, we identified suppressor mutations in the MADS box 

transcription factor mef-2 (MEF2) and the class II histone deacetylase hda-4. All phenotypes of 

kin-29 mutants are suppressed by mutations in mef-2 and hda-4. MEF2 and class II HDACs have 

previously been shown to regulate gene expression in response to intracellular signaling and 

electrical activity in muscles and neurons. Cell-specific expression indicates that mef-2 functions 

in the nervous system to regulate both str-1::gfp expression and body-size. Promoter deletion 

experiments suggest that sequences required for developmentally regulated str-1 expression in 

the AWB neurons are distinct from sequences required for KIN-29-regulated modulation of 

expression levels. There are several potential MEF2 binding sequences within the str-1 proximal 

promoter, indicating that KIN-29 may function via MEF-2 to modify the direct transcription of str-1. 

All together, our findings suggest an intriguing model for a remodeling of chromatin structure in 

regulating chemoreceptor gene expression in response to environmental signals in C. elegans. 

[1] Lanjuin & Sengupta (2002), Regulation of chemosensory receptor expression and sensory 

signaling by the KIN-29 Ser/Thr kinase, Neuron, Vol. 33, 369-381.

21. mig-10 functions downstream of unc-6 and slt-1 to mediate axon guidance. 

Christopher C. Quinn 1 , Elizabeth Stovall 2 , Elizabeth F. Ryder 2 , William G. Wadsworth 1 

1Dept. of Pathology, Robert Wood Johnson Medical School, Piscataway, NJ 

2Biology and Biotechnology, Worcester Polytechnic Institutute, Worcester, MA 

Migrating axons are guided through the developing nervous system by extracellular guidance 

cues. These cues interact with transmembrane receptors, causing an attractive or repulsive 

response to the guidance cue. While many extracellular guidance cues and their receptors have 

been identified, the intracellular mechanisms that mediate the response to the guidance cues are 

largely unknown. 

The ventral migrations of the AVM and PVM axons are guided by attraction to netrin/UNC-6 

and repulsion from slit/SLT-1 (Gitai et al, 2002, Neuron 37,53-65; C.C.Q, unpublished 

observations). We have found that MIG-10 is expressed in the AVM and PVM axons where it 

functions cell-autonomously to mediate the axon’s response to both netrin/UNC-6 and slit/SLT-1. 

This function occurs in parallel to UNC-34/enabled, a regulator of actin polymerization that acts 

downstream of netrin/UNC-6 and slit/SLT-1 (Yu et al, 2002, Nat. Neurosci. 5, 1147-1154). MIG-10 

is a cytoplasmic protein that includes several proline rich domains as well as a Ras Association 

domain and a Plekstrin Homology domain. We propose that MIG-10 serves as a 

signaling/adaptor protein that can integrate the repulsive and attractive guidance signals received 

by these axons.

22. Genes Involved In Serotonergic Neurotransmission 

Megan Higginbotham, Bob Horvitz 

MIT Biology, 77 Massachusetts Ave., Cambridge MA 02139 

Wild-type animals that have been acutely food deprived slow their locomotory rate upon 

encountering bacteria more than do well-fed animals. This behavior, called the enhanced slowing 

response, is serotonin (5-HT) dependent. Animals mutant for the 5-HT reuptake transporter 

mod-5 slow even more than wild-type animals because endogenous serotonin activity is 

potentiated. We call this behavior the hyperenhanced slowing response. mod-5 animals are 

hypersensitive to immobilization by exogenous 5-HT. To identify additional genes involved in 

5-HT signaling and possibly in the enhanced slowing response, we screened for suppressors of 

the 5-HT hypersensitivity of mod-5 animals. We also used a candidate gene approach, testing for 

5-HT resistance of strains containing deletions in genes that encode proteins similar to 

metabotropic serotonin receptors. 

Using Mos1 transposon mutagenesis (Bessereau et al. Nature, 413: 70-74, 2001), we 

screened worms corresponding to 46,200 haploid genomes and identified three suppressors. 

Two contain insertions in genes that when mutated are known to suppress mod-5 for both the 

exogenous 5-HT hypersensitivity and the hyperenhanced slowing response. One of these 

suppressors is an allele of mod-1, which encodes a 5-HT-gated chloride channel, and the other is 

an allele of goa-1, a predicted alpha subunit of a heterotrimeric G-protein. The third suppressor, 

n4094, partially suppresses the 5-HT hypersensitivity of mod-5 animals but does not suppress the 

hyperenhanced slowing response of mod-5 animals. n4094 animals contain an insertion in a 

gene with similarity to bicarbonate transporters. A deletion allele of this gene phenocopies 

n4094. Experiments are currently underway to determine whether this deletion mutant displays 

other defects and to determine the expression pattern of this gene. 

Using our candidate gene approach, we found a deletion, ser-4(ok512),that confers resistance 

to 5-HT and defects in the enhanced slowing response. ser-4 likely encodes a metabotropic 

serotonin receptor (Olde and McCombie, J. Mol. Neurosci., 8:53-62, 1997). ser-4(ok512) 

suppressed both the 5-HT hypersensitivity and the hyperenhanced slowing response of mod-5 

animals. We will place ser-4 in a genetic pathway with other genes known to function in the 

hyperenhanced slowing response, including mod-1, goa-1, and dgk-1 (diacylglycerol kinase). 

We also hope to define the neural circuit(s) through which mod-5 and suppressors of mod-5 act 

to affect the enhanced slowing response. Using antibodies raised against MOD-5,we have 

identified two pairs of head neurons in which MOD-5 is expressed: the NSMs and either the 

AIMs or the AIYs. Additionally, a translational mod-1::rfp reporter has been constructed and 

studied (by Eric Miska), and the expression pattern of SER-4 has been reported (Tsalik et al., 

Dev. Biol., 263(1): 81-102, 2003) using a translational GFP reporter. We will confirm these 

expression patterns by raising antibodies against these proteins. We will then express these 

genes in subsets of the neurons in which they are expressed to determine where these genes are 

required to function for a normal enhanced slowing response.

23. Ryanodine Receptors Regulate Neurotransmitter Release at the C. elegans 

Neuromuscular Junction 

Qiang Liu 1 , Michael Nonet 2 , Lawrence Salkoff 2 , Zhao-Wen Wang 1 

1Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 06030 

2Department of Anatomy and Neurobiology, Washington University School of Medicine, St. 

Louis, MO 63110 

Traditionally, calcium influx through voltage-gated calcium channels was thought as the sole 

source of calcium for synaptic release. Emerging evidence suggests that calcium release from 

intracellular stores may also play a role. We investigated a potential role of presynaptic ryanodine 

receptors (RYRs), which are calcium release channels in the endoplasmic reticulum membrane, 

in spontaneous and evoked synaptic exocytosis using C. elegans as a model system. The 

existence of a single RYR gene (ryr-1) and the availability of ryr-1 mutants in C. elegans make 

the analysis convenient. The potential role of RYRs was investigated by analyzing miniature and 

evoked postsynaptic currents (mPSCs and ePSCs) at the neuromuscular junction. In ryr-1 

loss-of-function mutants, both the frequency and mean amplitude of mPSCs were significantly 

reduced compared with the wild-type. These changes appeared due to presynaptic RYR-1 defect 

since the sensitivity of body-wall muscle cells to exogenously applied neurotransmitters was 

similar to that of the wild-type. Acute pharmacological blockade of RYRs produced similar 

changes in mPSCs as the ryr-1 mutations, suggesting that a secondary developmental defect 

was not involved. In wild-type preparations, elimination of extracellular calcium reduced mPSC 

frequency by ~70%. By contrast, similar treatment essentially abolished mPSCs in the ryr-1 

mutant. Thus, calcium influx and RYR-mediated calcium release are likely the exclusive sources 

of calcium for spontaneous synaptic release. The ryr-1 mutant also showed a significant reduction 

in ePSC amplitude. However, the quantal content (ePSC current integral divided by the mean 

mPSC current integral) did not change, suggesting that RYRs regulate ePSCs by controlling 

quantal size but not quantal number. Quantal number appeared to be solely determined by 

depolarization-mediated calcium influx. Given their large effects on mPSCs and ePSCs, RYRs 

may play an important role in synaptic plasticity.

24. KEL-8, a novel Kelch-like protein, is required for glutamate receptor degradation 

Henry Schaefer, Christopher Rongo 

The Waksman Institute, Department of Genetics, Rutgers University, Piscataway, NJ 08854. 

In the central nervous system, glutamate receptor insertion into and removal from the 

postsynaptic membrane is an important mechanism for changes in synaptic strength. Glutamate 

receptor localization is a stepwise process that includes transport from the cell body to 

postsynaptic specializations in the neurite, exocytosis into the membrane, endocytosis, and 

eventual degradation. Using a visual-based, forward genetic screen with animals expressing a 

fluorescent-tagged receptor subunit, we isolated mutants with mislocalized glutamate receptors. 

One of the genes found to be required for receptor turnover produces a Kelch-like protein we are 

calling KEL-8. In kel-8 mutants, unlocalized receptor fills the ventral nerve cord. Other synaptic 

proteins are properly localized in kel-8 mutants, suggesting that KEL-8 is not required for general 

cell polarity or synapse formation per se. A possible actin-binding protein, KEL-8 has previously 

been found to bind proteasome subunits(1). We find that KEL-8 is expressed in neurons and is 

localized to punctate structures in the neurite. It has been shown that ubiquitination of glutamate 

receptors causes their degradation, and overexpression of monoubiquitin reduces the abundance 

of glutamate receptor in the neurite(2). However, the accumulation of receptor in kel-8 mutants is 

resistant to this ubiquitin-mediated turnover. This evidence suggests a novel function for a 

Kelch-like protein in the degradation of glutamate receptors by the proteasome. 

1. A. Davy et al., EMBO Rep 2, 821-8 (Sep, 2001). 

2. M. Burbea, L. Dreier, J. S. Dittman, M. E. Grunwald, J. M. Kaplan, Neuron 35, 107-20 (Jul 3, 

2002).

25. Knockout of GLT-3 C. elegans Glutamate Transporter: A Genetic Approach to Study 

Excitotoxic Neurodegeneration 

Itzhak Mano 1 , Sarah Straud 2 , Monica Driscoll 1 

1Mol Biol & Biochem, Rutgers University, Piscataway, NJ 

2Mol Biol, University of Texas Southwestern Medical Center, Dallas, TX 

In stroke, injury, and several neurodegenerative diseases such as ALS, over-stimulation of 

neurons by glutamate (Glu) hyperactivates postsynaptic Glu receptors (GluRs) and triggers a 

process of necrotic neuronal death termed excitotoxicity. Malfunction of surface glutamate 

transporters (sGluTs), which normally remove glutamate from the synapse, makes critical 

contributions to this process. To model sGluT malfunction in an in vivo physiological context and 

apply the power of genetics to the study of excitotoxicity we generated sGluT knockout strains in 

C. elegans. After establishing that sGluT knockout causes Glu overstimulation, we document the 

first definitive case of excitotoxic-like condition in C. elegans. We show that disruption of the 

sGluT gene glt-3 acts synergistically with activated GalphaS* signaling to promote 

neurodegeneration. We demonstrate that the Glu-dependent effect is mediated through GluRs of 

the AMPA subtype and a Type 9 adenylyl cyclase, assigning the latter a novel role in an 

excitotoxic-like condition. Our observations suggest that in addition to the other well-appreciated 

mechanisms, AMPA receptor-cAMP synergism may be critical to excitotoxicity, a hypothesis with 

significant clinical implications.

26. A non-developmental role for lin-12 Notch signaling in the C. elegans adult nervous 

system 

Michael Y. Chao 1,2 , Jonah Larkins-Ford 1 , Anne C. Hart 1,2 

1Massachusetts General Hospital Center for Cancer Research, 149-7202 13th Street, 

Charlestown, MA 02129 

2Dept. of Pathlogy, Harvard Medical School, Boston, MA 

The Notch signaling pathway is conserved between species and plays important roles in cell 

fate determination during development of many tissue types, including the nervous system. Here 

we describe a non-developmental role for the C. elegans Notch homolog lin-12 in the adult 

nervous system. C. elegans predominantly moves forward but intermittently initiates short 

spontaneous reversals. lin-12 gain and loss of function mutant animals both had increased 

spontaneous reversal rates compared to wild type. Overexpressing lin-12 and knocking down 

lin-12 function by RNAi in otherwise wild type animals led to similar results. To rule out cell fate 

changes, we observed the cellular expression patterns of several reporter genes and saw no 

significant differences between lin-12 and wild type animals in AVA, AVB, AVD, AVC, or AIY, 

interneurons important for locomotion and initiation of reversals. These results suggested that the 

behavioral defects we observed in lin-12 mutants were not due to developmental changes in 

neurons. 

Instead, using a conditional, cold-sensitive gain of function lin-12 allele, we found that 

increasing lin-12 activity in adults (i.e., after nervous system development is complete) was 

sufficient to confer increased reversal rates. Cellular ablations, cell type specific expression 

experiments, and epistatic analysis suggested that both gain and loss of function lin-12 activity in 

a subset of neurons (AVA, AVB, and AVG) was sufficient to confer increased reversals. 

Previous studies have implicated AMPA-class glutamate gated cation channels in regulating 

reversals. We therefore examined the role of the C. elegans AMPA receptor homolog glr-1. 

Reversals caused by lin-12 gain or loss of function were strongly suppressed in a glr-1 mutant 

background. We also observed nose touch defects in lin-12 gain of function mutants that were 

consistent with defects in glr-1 function. However, it is as yet unclear if lin-12 regulates the 

function and/or expression of glr-1 or other glutamate-gated ion channels. Our results 

demonstrate that lin-12 Notch signaling regulates behavior and thus neuronal activity in the adult 

nervous system of C. elegans.

27. Calcium permeability of death-inducing DEG/ENaC ion channel MEC-4(d) 

Laura Bianchi, Wei-Hsiang Lee, Gargi Mukherjee, Beate Gerstbrein, Dewey Royal, Maryanne 

Royal, Jian Xue, Monica Driscoll 

Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ. 

The C. elegans ion channel complex formed by the DEG/ENaC MEC-4 and MEC-10 subunits, 

exclusively assembled in six specialized touch sensing neurons, is thought to constitute the core 

of a voltage-independent Na + -selective mechanosensory ion channel. In a screen for touch 

insensitive nematodes, a very interesting mec-4 mutant was isolated. This mec-4(d) mutation 

specified a large side-chain amino acid substitution near the channel pore and induces 

degeneration of touch neurons through a mechanism that appears to involve channel 

hyperactivation. Analysis of mec-4(d)-induced neurodegeneration, however, indicated that large 

increase of [Ca 2+ ] i is one of the molecular requirements for cell death. While Ca 2+ release from 

ER-based stores is certainly occurring in mec-4(d)-induced cell-death, the triggering factor 

remains unknown. We speculated that Ca 2+ could permeate through MEC-4(d) channels to 

perhaps induce Ca 2+ -induced Ca 2+ release from stores. 

P> P> 

We analyzed Ca 2+ permeability of MEC-4(d) channels reconstituted in Xenopus oocytes and 

found that they are permeable to Ca 2+ (P Ca/P Na ~ 0.22). Interestingly, Ca 2+ currents through 

MEC-4(d) channels display the same sensitivity to amiloride that Na + currents do, with Ki for the 

drug being reduced by co-expression with MEC-10(d). We also found that Ca 2+ permeability is 

independent of co-expression with MEC-10(d), MEC-2 or MEC-6, and appears to be determined 

by MEC-4(d) residues. We are currently searching for residues involved in Ca 2+ permeability, by 

introducing second site mutations in and near the pore, identified in our screen for suppressors of 

mec-4(d)-induced cell death. In addition we are evaluating the intracellular pathway activated by 

entry of Ca 2+ through MEC-4(d) that leads to massive release of Ca 2+ from the stores that 

ultimately results in cell death.

28. UNC-55, a Nuclear Receptor, is Essential for Male Mating 

Ge Shan, Bill Walthall 

Department of Biology, Georgia State University, Atlanta, GA 30303. 

unc-55has a sexually dimorphic expression pattern: it is necessary for 

organizing the synaptic pattern of the VD motor neurons, which are 

involved in locomotion in both males and hermaphrodites; and is 

necessary for successful male copulation. unc-55 is a nuclear receptor 

gene in the coup-tf gene family. Transgenic males express unc-55::gfp 

reporters in a male specific sensory neuron and structural cells 

associated with the spicules. Developmentally staged RNA interference 

experiments show that UNC-55 in the male tail cells is required for 

successful copulation but expression in the motor neurons is not. There 

are two mRNA isoforms (unc-55a and unc-55b) detected throughout 

postembryonic development in males, whereas only unc-55a is detected in 

hermaphrodites. The difference between the two isoforms is 12 amino 

acids that link the DNA binding domain to the ligand-binding domain. Two 

fusion genes were constructed one from each of the isoforms, both under 

the control of the unc-55 promoter and both with the gfp cassette on the 

3’ end. In an unc-55 mutant background the gfp expression pattern in the 

motor neurons was the same for both, however, locomotion defects were 

rescued only by UNC-55A, the isoform presnt in both males and 

hermaphrodites. Although there were subtle differences in the gfp 

expression pattern; both isoforms were capable of rescuing male mating 

in unc-55 mutants. unc-55b appears to sustain male mating ability as 

they age. Additional experiments will address the genetic program to 

which unc-55 contributes in male mating.

29. Genes Controlling Sensory Axon Patterning in the C. elegans Male Tail 

Lingyun Jia, Scott W. Emmons 

Albert Einstein College of Medicine, Bronx, 10461, USA 

The C. elegans male exhibits sex-specific behaviors like mate searching and copulation. The 

generation and modification of such behaviors depends on the precise connections between 

male-specific neurons. However, how the male-specific circuits are established during 

development is not well known. We initiated a genetic analysis by focusing on sensory neurons of 

the rays to identify genes involved in this process. We first demonstrated by using fluorescent 

protein that each ray axon takes a distinct but stereotyped pathway into the preanal ganglion and 

is highly branched there, identical with previous study by Sulston(1980). In a genetic screen for 

mutations with abnormal ray axon morphology, we isolated mutations in nine genes that cause a 

diversity of ray axon morphology defects. A subset of these mutations define two non-sex-specific 

genes: unc-27 and sax-2, and two potentially new genes: egl-35 and rax-2 with sex-specific 

functions. 

unc-27 encodes troponin I, a subunit of Troponin Complex. Our results show that UNC-27/TnI 

controls axon pathway by ordering muscle position and the substrate for the axons to migrate 

along, indicating that nerves and muscles are spatially coincidently regulated during 

development. unc-27 loss of function results in axon wandering. Polarized microscope analysis 

demonstrated that mutations in unc-27 distort the evenly spaced dense bodies, detaching muscle 

from basement membrane and epidermis. Mutations in most genes encoding myofilament 

proteins do not affect axon guidance except for mup-2/TnT, another subunit of the Troponin 

Complex that is also required for muscle cell positioning during development. We showed by 

mosaic analysis that UNC-27 expression in muscles but not in neurons or hypodermis rescued 

the axon wandering; suggesting muscles provide a scaffold for axon pathfinding. 

sax-2 encodes the C.elegans homologue of the drosophila furry, a conserved protein without 

any known functional domains. sax-2 plays an important role in maintaining axon morphology. 

The mutation sax-2(bx130) causes ray neurons to send supernumerary processes in the adult 

stage and reduces the density of the presyanptic vesicles in the PAG. It also affects the stability 

of amphid sensory neurons, as do other sax-2 alleles (Zallen et al, 1999). Other studies have 

shown that furry is required for cellular morphogenesis and the polarity of cell division in the fly 

and yeast. The disruption of neuronal morphology indicates furry is also required for maintaining 

integrity of the mature nervous system. Consistently, the reduced density of presynaptic vesicles 

and the low mating efficiency of males suggest that sax-2 may play a role in establishing neural 

circuits important for copulation. 

Finally, two genes, egl-35 and rax-1 (ray axon defect), specifically control ray axon guidance 

and also appear to affect sex behaviors. Mutations in egl-35 and rax-1 cause ray axons to fail to 

project into the PAG, but do not affect the non-sex- specific axons, consistent with normal 

non-sex-specific behaviors like locomotion. However, egl-35(bx129) males loose attraction to 

hermaphrodites and fail to initiate any steps of the mating program. Our assay for mate searching 

(sexual motivation) suggests that bx129 males may have reduced sex drive. Males of 

rax-1(bx132) also exhibit reduced mating frequency. Currently, we are cloning them and 

analyzing the genetic interaction with known guidance factors and other rax genes.

30. A genomic approach to the development and function of the C. elegans male tail rays 

Douglas S. Portman 1,2 , Daryl D. Hurd 1,3 , Nicole Juskiw 4 , Kwi Yeon Lee 1 , William R. Mowrey 5 , 

Carolyn Tyler 5 , Hai Wu 6 

1 Center for Aging and Developmental Biology, University of Rochester Medical Center, 

Rochester, NY 14642 

2 Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, NY 

14642 

3 Biology Dept., St. John Fisher College, Rochester, NY 14618 

4 GEBS Summer Scholar Program, University of Rochester Medical Center, Rochester, NY 

14642 

5 Neuroscience Graduate Cluster, University of Rochester Medical Center, Rochester, NY 14642 

6 Genetics, Genomics and Development Graduate Cluster, University of Rochester Medical 

Center, Rochester, NY 14642 

The rays of the C. elegans male tail present an ideal opportunity to understand the genetic 

mechanisms by which distinct neural subtypes arise from neuroblast precursors. As sensory 

organs, the rays are also a model to address the means by which chemo- and mechanosensation 

regulate behavior. Each of the eighteen rays develops during L3 and L4 from a single ray 

precursor cell (Rn). Triggered by the action of the proneural bHLH gene lin-32, each ray 

precursor executes the ray sublineage to generate two neurons of distinct subtype (RnA and 

RnB), the ray structural cell (Rnst), and a cell that undergoes programmed cell death. To better 

understand how the ray sublineage generates these distinct cell fates, we carried out microarray 

experiments designed to identify new ray-expressed genes. In these studies, we compared total 

patterns of gene expression between hlh-2; lin-32 and lin-22 adult males, which respectively lack 

and have supernumerary rays. By testing candidate ray genes with reporter constructs, we 

identified 17 genes whose ray expression was not previously known. 

Among these new ray-expressed genes are several transcription factors, including egl-46, 

hlh-10, lim-7 and an uncharacterized DM domain gene. These represent excellent candidates to 

act downstream of or in parallel with lin-32 to specify individual ray cell fates. We have found that 

mutations in egl-46 and hlh-10 do not seem to have an effect on ray development; lim-7 and the 

DM gene are currently being characterized. We also found a beta-tubulin isoform, tbb-4, that is 

expressed in all ray neurons. Preliminary results indicate that this tubulin may be present in all 

ciliated sensory neurons and could contribute to the structure of the cilium itself. Finally, we have 

identified five novel genes, cwp-1 through -5, that share the highly-specific expression pattern of 

lov-1 and pkd-2, two genes known to be essential for male mating behavior. lov-1, pkd-2 and the 

five cwp genes are expressed in all RnB neurons (except the R6Bs), the hook sensory neuron 

HOB and the male-specific CEM head sensory neurons. We are currently testing the possibility 

that the cwp genes function in a sensory pathway with lov-1 and pkd-2. In addition, by defining 

minimal elements from these genes that are sufficient to drive RnB expression, we hope to 

characterize the regulatory mechanisms downstream of lin-32 that implement specific gene 

expression in the RnB neural subtype.

31. WormBase: What’s New and What’s Next? 

Nansheng Chen 1 , Lincoln D. Stein 1 , WormBase Consortium 2 

1 Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724 

2 Californica Institute of Technology, Wellcome Trust Sanger Center, Washingtown University at 

St. Louis, Cold Spring Harbor Laboratory 

As a data and software resource for nematode biology and genomics, WormBase 

(http://www.wormbase.org) continues to grow and improve. In this presentation, I will highlight the 

growth and improvement in the past year from three major aspects: the database, user interface, 

and bioinformatics support. The growth of the database was highlighted by the incorporation, 

expansion and integration of new large-scale datasets including microarray, SAGE, interactome, 

3D protein structure, ORFeome and RNAi data. Significant changes also included our continued 

effort in curation of sequence data, literature, gene expression and antibodies, gene ontology, 

phenotype ontology, and anatomy ontology. On the user interface side, much effort was made to 

make various web pages uniform, informative and stable. Textpresso, a literature search tool, is a 

new and useful feature added last year. New bioinformatics support includes batch downloads 

(’Batch Genes’ and ’Batch Sequences’ pages), remote access to WormBase databases (ACeDB 

database and Bio::DB::GFF MySQL databases), and periodic genome freezes. Looking ahead, 

WormBase will continue to grow and improve in all these aspects. WormBase data models and 

software architecture are being reorganized and optimized to accommodate the genomics and 

biology data from more nematode species, including the three Caenorhabditis species whose 

genomes are currently sequenced.

32. Frogs and snails and puppy dog tails? Wormatlas launches a guide to what boy worms 

are made of. 

Robyn Lints, Zeynep F. Altun, Huawei Weng, Gloria Stephney, Maurice Volaski, David H. Hall 

Wormatlas consortium, Center for C. elegans Anatomy, Department of Neuroscience, Albert 

Einstein College of Medicine, 1410 Pelham Parkway S., Bronx, NY 10164, USA. 

C. elegans is sexually dimorphic, producing hermaphrodite and male sexes that differ from one 

another in both their morphology and behavior. The hermaphrodite has been studied extensively 

and over the past three years we have developed a web-based guide to its anatomy 

(www.wormatlas.org) designed to assist researchers with interests ranging from gene 

characterization to computational modeling. In addition, The Handbook, Glossary and selected 

Slideable Worm electron micrographs (EMs) from the site are to be published as a laboratory 

handbook in the coming year (Cold Spring Harbor Laboratory Press, NY). 

As part of our long-term objective to describe C. elegans developmental stages and sexes, we 

are now embarking on a description of the male anatomy. Males differ from hermaphrodites 

primarily in the reproductive tract and in the tail, which bears the male copulatory apparatus (1, 

2). The male has a single-armed gonad containing a germ line that produces only sperm. The 

tract opens to the exterior at the male anus (cloaca) via a modified rectal epithelial chamber 

called the proctodeum. The tail copulatory apparatus is organized around this opening and 

consists of the copulatory spicules, several types of male-specific external sensory organs, 

interneurons, motor neurons and muscles. Most male-specific cells arise post-embryonically 

through sex-specific division of precursors common to both sexes. Thus, establishing the adult 

male form involves the generation and organization of a large number of male-specific cells and 

their integration into an existing framework of non-sex-specific tissues. 

Several studies have assigned roles for male-specific cells in fascinating sex-specific behaviors 

such as mate-searching (3) and copulation (4). However, a full understanding of these behaviors 

requires a detailed knowledge of the cellular substrates underlying their expression, both at the 

level of individual cells and the functional units they form through their interconnection. In 

contrast to the hermaphrodite, the male anatomy is only partially described and, in particular, the 

connectivity of many male neurons is still unknown. To resolve this problem, we are currently 

collaborating with the Emmons lab to describe the fine structure and connectivity of individual 

neurons and other cell types in the male. This project resumes the effort initiated by the MRC in 

the 1970s to reconstruct the male posterior nervous system from the N2Y EM series. Emerging 

connectivity data will be incorporated into a web-based guide to the male anatomy. The male 

anatomy will be presented in a format similar to that used for the hermaphrodite. Additional 

features we hope to incorporate in the future include a Slideable Male Worm, 3D models of cell 

shapes generated from EM serial reconstructions and quick-time movies of those important 

events in a male’s life. We anticipate that the male web pages will provide a comparative basis for 

gene expression or developmental studies and represent a significant advancement on current 

descriptions of this sex. It is our hope that, through this multi-tiered web-based description and 

an accompanying handbook, the workings of at least this male sex will become less of a 

mystery. 

1. Horvitz and Sulston (1977) Dev. Biol. 56, 110-156. 

2. Sulston et al.(1980) Dev. Biol. 78, 542-576. 

3. Lipton and Emmons (2003) J. Neurobiol 54: 93-110. 

4. Sternberg and Emmons (1997). In C. elegans II (Eds. Riddle et al., CSHL Press, NY), pp. 

295-334.

33. A New Phylogeny Reveals Frequent Loss of Introns During Nematode Evolution 

Ronald E Ellis 1 , Soochin Cho 2 

1Department of Molecular Biology, UMDNJ - SOM, Stratford, NJ 08084 

2Department of EEB, University of Michigan, Ann Arbor, MI 48109 

Since introns were discovered 26 years ago, people have wondered how changes in 

intron/exon structure occur, and what role these changes play in evolution. To answer these 

questions, we have begun studying gene structure in nematodes related to C. elegans. As a first 

step, we cloned a set of five genes from six different Caenorhabditis species, and used their 

amino acid sequences to construct a detailed phylogeny of the genus. Our results show that C. 

briggsae and C. remanei are sister species, and imply that mating systems have changed 

frequently during recent nematode evolution. 

Using this phylogeny, and the species C. sp. PS1010 as an outgroup, we were able to 

determine which changes in intron/exon structure were caused by the deletion of ancestral 

introns, and which were caused by the insertion of new introns. Our results show that nematode 

introns are lost at a very high rate during evolution, almost 400-fold higher than in mammals. 

These losses do not occur randomly, but instead favor some introns and do not affect others. By 

contrast, intron gains are far less common than losses in these genes. 

For years, people have focused on recombination between a gene and a cDNA copy of its 

transcript as the primary cause of intron loss. However, we found that adjacent introns were not 

likely to be lost together, as one might expect from this model, which implies that other 

mechanisms are also important. Based on sequence data, we suggest that both simple deletions 

and mutations at splice donor sites might play a significant role in causing the loss of introns 

during evolution. Furthermore, each of these species has small introns, much like those in C. 

elegans. The small size of these introns should increase the rate at which each type of loss 

occurs, and could account for the dramatic difference in loss rate between nematodes and 

mammals. 

Because of the wealth of genomic data that will be generated for these species, we should 

soon be able to expand our studies and determine the relative importance of these mechanisms 

during evolution.

34. Caenorhabditis phylogeny predicts convergence of hermaphroditism and extensive 

intron loss 

Karin C. Kiontke, Nicholas P. Gavin, Yevgeniy Raynes, Casey Roehrig, Fabio Piano, David H. 

A. Fitch 

Department of Biology, New York University, New York, NY 10003 

Despite the prominence of Caenorhabditis elegans as a major developmental and genetic 

model system, its phylogenetic relationship to its closest relatives has not been resolved. 

Resolution of these relationships is necessary for studying the steps that underlie life history, 

genomic, and morphological evolution of this important system. Using nucleotide sequence data 

from five different nuclear genes [small subunit ribosomal RNA gene, large subunit ribosomal 

RNA gene, gene for the largest subunit of RNA polymerase II (RNAP2), par-6 and pkc-3] from 10 

Caenorhabditis species currently in culture, we find a well-resolved phylogeny: C. briggsae is the 

sister species of C. remanei, C. elegans is the sister species of a clade consisting of C. briggsae, 

C. remanei and C. sp. CB5161. These 4 species are representatives of the Elegans group of 

Caenorhabditis, the sister species of which is C. japonica. The next branches down the tree are 

formed by C. sp. PS1010, C. drosophilae plus C. sp. DF5070, C. plicata and C. sp. SB341. 

RNAP2 analyzed separately, however, supports a clade consisting of C. sp. PS1010 and the pair 

of sister species, C. drosophilae and C. sp. DF5070. 

Our phylogeny reveals three striking patterns in the evolution of Caenorhabditis: (1) 

Hermaphroditism has evolved independently in C. elegans and its close relative C. briggsae. (2) 

In the RNAP2 gene there is a large degree of intron turnover within Caenorhabditis and intron 

losses are much more frequent than intron gains. Together with data from 28 other species this 

also indicates that while in some lineages intron losses are more frequent than gains, in other 

lineages intron gains exceed the losses. (3) Despite the lack of marked morphological diversity in 

these nematodes, more genetic disparity is present within this one genus than has occurred 

within all vertebrates and possibly within all arthropods.

35. Evolutionary innovation of excretory system in Caenorhabditis elegans 

Xiaodong Wang, Helen M. Chamberlin 

Department of Molecular Genetics, Ohio State University, Columbus, OH 43210 

Changes in developmental gene regulation can result in physiological and morphological 

differences between species. To understand the molecular changes responsible for the evolution 

of gene expression patterns, our lab studies the excretory system from several Caenorhabditis 

species. We have found that there are unique features of the C. elegans excretory system, and 

that these changes result from the evolution of novel regulation of the zinc-finger transcription 

factor gene lin-48. 

Previously, we showed that the morphology of the C. elegans excretory system differed from 

that of C. briggsae, and that this difference resulted from the expression of lin-48 in the C. 

elegans excretory duct cell (Wang and Chamberlin, 2002). To investigate whether this difference 

resulted from the gain of lin-48 regulatory elements in C. elegans of loss from C. briggsae, we 

have studied excretory system structure and function in other Caenorhabditis species. We have 

found that the C. elegans excretory system exhibits unique structural and functional features. The 

morphology of the C. elegans excretory system differs from other Caenorhabditis species, and C. 

elegans animals show greater tolerance to a high-salt environment compared to other species. 

Evolution of the regulation of lin-48 expression is a key event in producing these unique features. 

For example, The C. elegans gene contains critical CES-2-responsive regulatory sequences, 

whereas these sequences are absent from the lin-48 gene of C. briggsae, C. remanei, and C. sp 

CB5161. Likewise, the C. elegans lin-48 mutant phenotype can be rescued by lin-48 cDNA from 

each species when it is expressed under control of the C. elegans lin-48 regulatory sequences. 

Finally, we have shown that ectopic expression of lin-48 in the C. briggsae excretory duct can 

confer C. elegans morphology on an otherwise normal C. briggsae animal. Taken together, our 

results illustrate how increased organism complexity can result from the evolution of novel gene 

regulatory features. 

To better understand other molecular changes responsible for species differences, we are 

conducting two genetic screens for increased salt tolerance in C. briggsae. The screens may 

allow us to identify LIN-48-responsive genes that mediate higher salt tolerance, as well as other 

genes that act independent of LIN-48. 

Wang, X. & Chamberlin, H. M. Genes Dev. 16, 2345-2349 (2002).

36. cdc-14 regulates cki-1 to control cell-cycle arrest 

R. Mako Saito, Audrey Perreault, Bethan Peach, John S. Satterlee, Sander van den Heuvel 

MGH Cancer Center, Charlestown, MA 02129 

The development of multicellular organisms requires proper temporal and spatial control of cell 

divisions. To reveal underlying mechanisms, we screened for cell-cycle mutants that disrupt the 

reproducible pattern of somatic divisions in the nematode C. elegans. The screen for mutations 

that allowed extra divisions of the vulva precursor cells (VPC) led to the identification of genes 

that are required for their developmental arrest. 

Surprisingly, we found a novel role for the cdc-14 phosphatase in establishing the temporary 

quiescence of the VPCs. While budding yeast Cdc14p is essential for mitotic exit, inactivation of 

C. elegans cdc-14 resulted in extra cell divisions within multiple lineages, with no apparent 

defects in mitosis or cell-fate determination. The localization of a functional CDC-14 reporter was 

dynamic and cell-cycle dependent. Several lines of genetic evidence suggest that loss of cdc-14 

function disrupts a cki-1 dependent pathway to arrest cell divisions during development. 

Moreover, we show that cdc-14 acts upstream of cki-1 and elevates CKI-1 nuclear accumulation. 

These data demonstrate that cdc-14 contributes to developmental regulation of cell-cycle arrest 

by promoting cki-1 activity. If conserved, a role for Cdc14 in Cip/Kip stabilization may have 

important implications for human cancer.

37. Transcriptional regulation of Hox gene lin-39 during vulval cell fate specification 

Javier A. Wagmaister 1 , Julie E. Gleason 1 , Corey A. Morris 2 , Leilani M. Miller 2 , Ginger R. 

Miley 3 , Kerry Kornfeld 3 , David M. Eisenmann 1 

1Biological Sciences, University of Maryland, Baltimore County, Baltimore, MD 

2Department of Biology, Santa Clara University, Santa Clara, CA 

3Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 

We are interested in understanding how extracellular signaling processes regulate Hox gene 

activity and cell fate specification during C. elegans vulval development. The vulva arises from six 

Vulval Precursor Cells (P3.p-P8.p) located in the ventral midline of the body. Vulval specification 

requires the activity of Wnt, Notch, and RTK/RAS signaling pathways and the expression of 

transcription factors including the Hox gene lin-39. LIN-39 protein levels increase specifically in 

P6.p at the time of vulval induction 1 and lin-39 levels are regulated by Wnt 2 and Ras 1 signaling 

pathways. However, it is not clear whether regulation is at the transcriptional or 

post-transcriptional level. Our goals are to understand how lin-39 integrates information from Wnt 

and Ras signaling pathways, and to study the interaction of lin-39 with the regulatory network 

participating in VPC fate specification. 

Using transcriptional and translational lin-39::GFP fusions containing the entire lin-39 genomic 

sequence, we expanded the previous analysis of lin-39expression throughout development in 

ventral cord neurons, sex myoblast descendants, and the VPCs. We found that at the time of 

vulval induction GFP levels increased in P6.p from both constructs, indicating that lin-39 is 

regulated at the transcriptional level. Transcriptional upregulation is dependent on Ras signaling 

and the zinc-finger transcription factor sem-4, but it can be independent of the Forkhead 

transcription factor lin-31. We are currently analyzing the interaction of lin-39 with other members 

of the transcriptional regulatory apparatus like eor-1, eor-2, and lin-25. 

In an effort to understand the details of lin-39 transcriptional regulation, we isolated from the 

lin-39 promoter one fragment responsive to Ras (TCF3) and another element required for the 

initiation and maintenance of lin-39 expression in a subset of VPCs (TCF4D). TCF3 is located 

approximately 6 kb upstream of the transcription starting site and is sufficient for the expression 

of GFP in P6.p at the time of vulval induction. Furthermore, GFP expression driven by TCF3 is 

expanded to the six VPCs in a let-60(gof) background. TCF3 contains putative LIN-31 and LIN-1 

binding sites and these two transcription factors are able to bind to several regions in TCF3. The 

fragment TCF4D drives GFP expression mainly before vulval induction in P5.p to P8.p, starting 

early during embryogenesis in the P cells. The pattern of GFP expression is stronger in P7-8.p 

than in P5-6.p and absent in P3-4.p suggesting that a gradient coming from the posterior might 

be implicated in regulating lin-39 expression through TCF4D. We identified a 300 bp element 

sufficient for the expression of GFP, and a 20 bp region conserved in C. briggsae that is 

necessary for GFP expression. Considering the specific pattern of TCF4D expression we propose 

that TCF4D might be regulated by the Wnt pathway. 

1 Maloof, J. N. and Kenyon C. (1998). ÒThe Hox gene lin-39 is required during C. 

elegansvulval induction to select the outcome of Ras signaling.Ó Development 125(2): 181-90. 

2 Eisenmann, D. M., et al. (1998). ÒThe beta-catenin homolog BAR-1 and LET-60 Ras 

coordinately regulate the Hox gene lin-39 during Caenorhabditis elegans vulval development.Ó 

Development 125(18): 3667-80.

38. EPS-8 regulates LET-23/EGFR localization during C. elegans vulval development 

Attila Stetak 1 , Assunta Croce 2 , Giuseppe Cassata 2 , Pier P. DiFiore 2 , Erika Fröhli Hoier 1 , Alex 

Hajnal 1 

1University of Zürich, Inst. of Zoology, Winterthurerstr. 190, 8057 Zürich, Switzerland 

2IFOM-FIRC Institute of Molecular Oncology, Via Adamello, 16, I-20139 Milano, Italy 

During the development of the hermaphrodite vulva, three out of six equivalent vulval precursor 

cells (P3.p to P8.p, theVPCs) are induced by the gonadal anchor cell (AC) to adopt vulval cell 

fates. The anchor cell signal activates in P6.p the conserved EGFR/RAS/MAPK pathway. For the 

proper signalling in P6.p, the EGF-receptor (LET-23) has to be localized at the baso-lateral 

surface of the cell. A ternary complex composed of LIN-7, LIN-2 and LIN-10 has been identified 

that anchors LET-23 on the baso-lateral cell surface. Mutations in lin-2, lin-7 or lin-10 cause 

receptor mislocalisation and a vulvaless phenotype. However, it is unknown how this ternary 

complex is anchored to the baso-lateral surface, and how receptor-endocytosis influences the 

status and composition of this complex. To address these questions, we looked for interactors of 

the LIN-7/LIN-2/LIN-10 complex in the ORFeome database and depleted the candidates using 

RNAi. We found that one of the putative interactors, the C. elegans homologue of the mammalian 

EGF-receptor substratre 8 (Y57G11C.24) interacts genetically with the EGFR/RAS/MAPK 

pathway and co-precipitates with LIN-2/CASK in a stimulation dependent manner. Using yeast 

two-hybrid and GST pull-down experiments we demonstrate that C. elegans EPS-8 binds 

exclusively to the C-terminal L27-domain of LIN-2, while LIN-7 binds to the N-terminal 

L27-domain. The eps-8 promoter is active in the VPCs, and it is positively regulated by the 

EGFR/RAS/MAPK pathway. Overexpression of EPS-8 in the VPCs transforms the fate of P5.p 

and P7.p from 2° to 1° fate. Genetic analysis revealed that eps-8 is a positive regulator of the 

EGFR/RAS/MAPK pathway that acts at the level of LIN-2. Furthermore, overexpression of EPS-8 

restores baso-lateral LET-23 localization in LIN-7 but not in LIN-2 mutant animals. In summary, 

we identified EPS-8 as a positive regulator of the EGFR/RAS/MAPK pathway that links receptor 

endocytosis to vulval cell fate specification.

39. The C-terminal sequence of C. elegans smad/SMA-3 has multiple roles 

Jianjun Wang, Cathy Savage-Dunn 

Department of Biology, Queens College and GSUC-CUNY 

TGF-beta pathways have been extensively studied in both vertebrate and invertebrate animals. 

The signaling begins when the ligand (TGF-beta/BMP) binds on the type I and type II receptors. 

The type I receptor is phosphorylated by type II receptor. And the activated type I receptor 

phosphorylates intracellular transducers, smads. The smads form a complex and go into the 

nucleus. They form special structure complexes with transcription factors and regulate target 

gene expression. 

In C. elegans, there are at least two TGF-beta related pathways, the Dauer pathway and the 

Sma/Mab pathway. Our lab focuses primarily on the Sma/Mab pathway. The mutants have small 

body size and male tail defects, fused rays and crumpled spicules. There are three smad 

proteins, SMA-2, SMA-3 and SMA-4. Using a GFP reporter, we find the sma-3 gene expresses in 

pharynx, intestine and hypodermis. We further demonstrated that hypodermal expression of 

sma-3 is necessary and sufficient for body size rescue. The results by other investigators 

studying DAF-4, SMA-6 and LON-1 are consistent with the conclusion that the hypodermis is the 

essential tissue to control body size. To study the role of phosphorylation in Smad activity in vivo, 

we designed several kinds of C-terminal mutations of SMA-3. The amino acids SMT were 

changed into DME, AMA, YMY or deleted. Instead of being constitutively active, the 

pseudophosphorylation mutation DME is dominant negative and nonfunctional in body size 

regulation. Except for YMY, the remaining mutations are also dominant negative in body size. 

Using a GFP-tagged SMA-3 reporter construct, we find that the dominant negative mutations 

could turn on a feedback loop leading to a reduction in SMA-3(+) protein accumulation. 

Interestingly, all of the mutations are still partially functional in rescuing male tail ray fusions 

except YMY. Finally, only DME could rescue the crumpled spicules, a result consistent with our 

original speculation. The results from yeast two hybridization show that SMA-3, but not SMA-2, 

interacts with the forkhead transcription factor, LIN-31, which is important for spicule 

formation.The results suggest that SMA-3 has multiple targets in different cell types and that they 

have differential requirements for SMA-3 phosphorylation.

40. PKC2 A Calcium-Diacylglcerol Kinase that Runs Hot and Cold 

Marianne Land, Charles S. Rubin 

Dept. Mol Pharmacology, Albert Einstein Col. Med., Bronx NY 10461 

PKC2/KIN11 is the only diacylglycerol (DAG) and Ca 2+ protein kinase C isoform in C.elegans. 

pkc-2 null worms wander randomly at temperatures higher and lower than their cultivation 

temperature when placed in a thermal gradient. These athermotactic worms were rescued with 

the 30 kbp kin-11 gene (containing 3 promoters previously characterized in this lab). Expression 

of PKC2 protein at the wild type level (in stable transgenic integrants) normalized the mutant 

phenotype. High level PKC2 expression caused worms to migrate to regions where temperatures 

are lower than their cultivation temperature (cryophilic phenotype). pkc-2 null animals are unable 

to transmit signals generated by binding of various receptors with classical model odorants. 

High-level expression of PKC2 in worms carrying an inactivating mutation [tax-6(p695)] in the 

catalytic domain of PP2B (calcineurin) converts thermophilic worms to cryophiles. 

Overexpression of PKC2 might cause chronic phosphorylation of a target-effector, thereby 

locking temperature perception circuitry in a one state and bypassing tax-6. Another possibility is 

that TAX-6 may be involved in up-regulation of a channel or receptor that elevates DAG and/or 

Ca 2+ . A high concentration of PKC2 could overcome inactive TAX-6 by enabling kinase activation 

at basal levels of DAG and Ca 2+ . 

tax-4 or tax-2, which are loss of function mutants that encode defective subunits of a cGMP 

gated Ca 2+ channel, exhibit an athermotactic phenotype (V Komatsu H, Mori I, Rhee J-S, Akaike 

N, Ohshima Y, Neuron 17: 707-718 1996) in the absence or presence of over expression of 

PKC-2. TAX-2 and/or TAX-4 may be substrates of PKC2 , regulated by a PKC2 effector or 

generate Ca 2+ that could activate PKC2. N and C terminal portions of TAX-4 and TAX-2, 

generated as His 6-fusion proteins in E.coli, were not phosphorylated by PKC2 in vitro. Thus it is 

more likely that the TAX-2/TAX-4 channel imports Ca 2+ that activates PKC2. 

Analysis of functions of PKC2 exons (and corresponding protein domains) is under 

investigation. Large segments of genomic DNA upstream from C and B promoters were deleted 

from the 30kbp kin-11 gene. The modified DNA was sufficient to drive normal PKC2 expression 

and rescue the athermotactic phenotype of null worms. Like mammalian PKC beta, PKC2 uses 

alternative splicing of the final and penultimate exons to generate potentially functionally 

divergent isoforms. Expression of PKC2 cDNA containing only the penultimate exon is capable of 

rescuing the athermotactic phenotype in kin-11 -/- worms. Its ability to rescue the chemotactic 

response of PKC2 null worms is being tested. We are currently utilizing genetic and proteomic 

analysis to identify PKC2 substrates.

41. Regulation of a Conserved Oxidative Stress Defense by GSK-3 and p38 signaling in C. 

elegans 

Jae Hyung An 1 , Riva Oliveira 1 , Rosanna Baker 1 , Kelly Vranas 1 , Hideki Inoue 2 , Naoki 

Hisamoto 2 , Yanxia Bei 3 , Craig C. Mello 3 , Kunihiro Matsumoto 2 , T. Keith Blackwell 1 

1 Joslin Diabetes Center, One Joslin Place, Boston, MA 02215 

2 Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, 

Nagoya 464-8602, JAPAN 

3 Program in Molecular Medicine, University of Massachusetts Medical School, 373 Plantation 

Street, Worcester, MA 01605 USA 

Oxidative stress is a central etiologic factor in diabetes, cardiovascular disease, reperfusion 

injury, and various other pathologies. In vertebrates a major defense against oxidative and 

xenobiotic stress is orchestrated by the two Nrf bZIP transcription factors, which induce 

expression of a battery of Phase II detoxification enzymes. These enzymes synthesize 

glutathione and scavenge free radicals directly. This stress response system is also activated by 

chemoprotective antioxidants that are produced by many plants, and can inhibit chemical 

carcinogenesis in mice. We have determined that in C. elegans, this oxidative stress defense is 

mediated by the transcription factor SKN-1 (An and Blackwell (2003) Genes Dev., 17, 1882). 

SKN-1 is distantly related to Nrf proteins but binds DNA through a unique monomeric mechanism, 

indicating that this detoxification system has been conserved despite a dramatic divergence in 

DNA recognition. Previous work showed that in the embryo, maternally expressed SKN-1 initiates 

formation of the entire digestive system and other mesendodermal tissues. We have found that 

during postembryonic stages, zygotically expressed SKN-1 accumulates in intestinal nuclei in 

response to oxidative stress and directly regulates a key Phase II gene through constitutive and 

stress-inducible mechanisms in different tissues. In contrast, SKN-1 is constitutively nuclear and 

active in the ASI neurons. skn-1 mutants are sensitive to oxidative stress, and age prematurely. 

We believe that the developmental function of SKN-1 arose from this ancient conserved stress 

response. 

We are now investigating how this detoxification response is regulated in the C. elegans 

intestine. The Matsumoto lab has shown that a p38-like stress-activated kinase (PMK-1) 

phosphorylates SKN-1, and p38 signaling is required for SKN-1 to accumulate in nuclei or 

activate target genes in response to oxidative stresses. We have found that the C. elegans 

glycogen synthase kinase-3 (GSK-3) alpha/beta ortholog gsk-3 prevents constitutive SKN-1 

activation. When gsk-3 is inhibited by RNAi, SKN-1 is localized to intestinal nuclei without 

oxidative stress. A direct GSK-3 phosphorylation site and putative priming kinase site that we 

have identified are each required to prevent constitutive localization of SKN-1 to intestinal nuclei. 

The p38 pathway regulates SKN-1 epistatically to GSK-3, indicating that it is required for SKN-1 

function independently of mechanisms that overcome effects of GSK-3 on SKN-1. Our 

experiments have identified novel components of this conserved oxidative stress response, and 

have shown that oxidative stress resistance is modulated by a kinase (GSK-3) that has many key 

functions in metabolism, growth control, and differentiation. We believe that in the intestine SKN-1 

integrates multiple redox and metabolic inputs. Mechanisms through which these and other 

signals may regulate SKN-1 are under investigation.

42. LIN-28 and LIN-46 converge at a branchpoint in the heterochronic pathway 

Eric G. Moss, Keven Kemper 

Dept. of Molecular Biology, UMDNJ, Stratford, NJ 

LIN-28 acts early in larval development to determine what developmental events happen in the 

L2 stage and later. LIN-46 acts antagonistically at a step immediately downstream of LIN-28. Our 

genetic analysis has revealed that LIN-46 independently affects cell fates at the L2/L3 transition 

and the larval-to-adult (L/A) switch. Thus, LIN-28 and LIN-46 converge at a branch-point in the 

heterochronic pathway. LIN-28 is a predominantly cytoplasmic protein that is present in 

complexes with non-polysomal mRNAs. LIN-28 is conserved in evolution to humans and its 

expression is governed by microRNAs. The genetic analysis of LIN-28 has indicated it is a highly 

specific regulator, and is therefore likely to bind to specific RNA targets. We have isolated 

LIN-28-RNA complexes using Tandem Affinity Purification for the purpose of identifying those 

targets. Our molecular analysis of LIN-46 has suggested that this protein acts as a scaffold for a 

multi-protein complex. Some of LIN-28’s targets may encode components of this complex. The 

major question is: Are the components of this developmental timing complex (and LIN-28’s 

targets) animal-wide regulators (as are LIN-28 and LIN-46) or do they act in specific tissues to 

control cell type developmental events? The identity of LIN-28’s targets, their expression patterns 

and phenotypes will answer this question. Our purification procedure and the characterization of 

the candidate targets will be presented.

43. The role of daf-6 and cell-cell interactions in amphid morphogenesis 

Elliot A. Perens, Shai Shaham 

Rockefeller University, 1230 York Ave., New York , NY 10021 

Sensory organs are composed of sensory neuron dendrites surrounded by ensheathing 

glial-like cells. We are interested in understanding sensory organ morphogenesis. The generation 

of sensory organ morphology is dependent on the formation of a tube by the ensheathing cells 

and the coordination of this lumen with the sensory neuron processes. To understand how tube 

morphogenesis is coupled to sensory neuron development, we are studying the morphogenesis 

of the C. elegans amphid sensory organ. 

To gain insight into the genetic basis of sensory organ morphogenesis, we characterized daf-6. 

daf-6 encodes a sterol-sensing domain containing protein related to Drosophila and vertebrate 

Patched. Microscopy analysis suggests that daf-6 sensory defects are due to a failure to generate 

a proper tube within the amphid sheath cells. Mosaic analysis demonstrated that daf-6 functions 

in the amphid sheath cells(1), and heat shock rescue experiments revealed that daf-6 is required 

in the amphid during a narrow window of embryogenesis when the amphid sheath lumen forms. 

DAF-6 is expressed in and lines the luminal surfaces of the amphid sheath and socket cells, as 

well as other tube cells, including the phasmid sheath and socket cells, excretory system cells, 

vulval cells, and rectal cells, suggesting that daf-6 may play a general role in lumen formation. In 

animals mutant for both daf-6 and che-14, a C. elegans Dispatched homolog, the excretory canal 

lumen is malformed, and embryos do not pass through the vulva opening. Thus, daf-6 functions 

with che-14 to regulate proper lumen formation. 

To address the coordination of amphid sheath lumen development with formation of the ciliated 

ending of the sensory neurons, we examined daf-19 mutants, which lack sensory neuron cilia. 

We have observed several sheath cell defects, most notably an alteration in the pattern of 

DAF-6::GFP localization in the amphid sheath tube. Currently, we are attempting to determine 

whether this defect is due to a defect in amphid lumen formation or a more selective defect in 

DAF-6 subcellular localization. 

We are also interested in identifying additional genes that function with daf-6 to regulate 

amphid morphogenesis. To do so, we are carrying out genetic screens to identify daf-6 

suppressors or enhancers. Our results will be presented at the meeting. 

(1) Herman R.K. (1987). Mosaic analysis of two genes that affect nervous system structure in 

Caenorhabditis elegans. Genetics 116, 377-388.

44. An FGF Signaling Pathway Regulates Membrane Extensions from the Body Wall 

Muscles in C. elegans 

Scott Dixon, Raynah Fernandes, Peter J. Roy 

Department of Molecular and Medical Genetics, University of Toronto, Toronto, Canada 

In C. elegans, membrane projections from the body wall muscles (BWMs), called arms, form 

the post-synaptic element of the neuromuscular junction. To characterize muscle arm 

development we generated transgenic worms expressing membrane-anchored yellow fluorescent 

protein in the BWMs and observed that individual muscles extend a stereotypical number of 

arms. Furthermore, we noted a burst of muscle arm extension late in the first larval stage that is 

coincident with, and at least partially dependent upon, motor neuron proliferation. In a screen for 

genes required for normal muscle arm development we isolated sem-5, which encodes a small 

adapter protein that functions in receptor tyrosine kinase signaling. We determined that the 

number of muscle arm projections to the nerve cord is decreased, and the number of aberrant 

membrane extensions (AMEs) from the BWMs increased, in a sem-5(RNAi) background 

compared to controls. Moreover, we show that BWM expression of sem-5 is both necessary and 

sufficient to prevent AME formation. We have determined that disruption in any component of a 

conserved FGF/MAPK signaling pathway, comprising the genes let-756, egl-15, sem-5, soc-2, 

sos-1, let-60, mek-2 and mpk-1 results in the AME phenotype. Finally, we show that expression 

of the LET-756 ligand from several distinct cell types can rescue the AME phenotype conferred 

by a let-756 null mutation. Our results suggest a non-instructive role for FGF signaling in the 

control of muscle membrane extensions in C. elegans.

45. EGL-15 FGF Receptor Isoforms Play Different Roles in SM Migration 

Te-Wen Lo 1 , Catherine S. Branda 1 , Peng Huang 1 , Isaac E. Sasson 1 , S. Jay Goodman 2 , 

Michael J. Stern 1 

1Department of Genetics, Yale University 

2Department of Cell Biology, Yale University 

Fibroblast growth factor receptors (FGF receptors) are transmembrane tyrosine kinases that 

can initiate many different cellular processes as a response to their activation. The C. elegans 

FGF receptor, EGL-15, is similarly involved in multiple biological processes. These processes 

include an essential function and a chemoattraction of the migrating sex myoblasts (SMs) to flank 

the center of the gonad. To carry out these functions, EGL-15 has two functionally distinct 

isoforms resulting from alternative splicing of two distinct fifth exons. These exons encode an 

alternative EGL-15-specific domain which lies N-terminal to the major ligand-binding domain in 

the extracellular region of the receptor. These isoforms, termed EGL-15(5A) and EGL-15(5B), 

mediate two major functions of EGL-15: 5A is necessary for SM chemoattraction, while 5B is 

required for viability. 

Structural as well as expression differences contribute to the different roles of the isoforms. 

Immunohistochemistry with EGL-15-specific antibodies have shown that these isoforms are 

expressed in a tissue-specific manner. 5A is predominantly expressed in the M lineage, which 

gives rise to the migrating SMs and their sex muscle descendants. By contrast, 5B is 

predominantly expressed in the hypodermis. Tissue-specific expression, however, explains only 

part of the functional differences between these two receptor isoforms. Ectopic expression 

experiments have demonstrated that 5A can carry out the reciprocal essential function of 5B 

when expressed appropriately, but that 5B is incapable of carrying out the SM chemoattraction 

function normally mediated by 5A. These data indicate that the structural differences between 

these two isoforms contribute to their functional differences. 

Although the 5B isoform does not function in SM chemoattraction, it does play a role in SM 

migration. In the absence of 5A-mediated chemoattraction, a chemorepulsion by the gonad is 

revealed, resulting in posteriorly displaced SMs. Three lines of evidence implicate the 

involvement of the 5B isoform in this chemorepulsion. These include a mosaic analysis of egl-15 

and two transgenic approaches that eliminate all EGL-15 expression in the migrating SMs. All 

three approaches resulted in centrally-distributed final SM positions. SMs in control animals that 

retained the 5B isoform remained repelled to posterior distributions. These data, therefore, 

indicate a role for 5B in SM repulsion, suggesting that structural differences in the extracellular 

domains of these two isoforms can help specify either attraction to or repulsion from the gonad.

46. MLS-2, an HMX class homeodomain protein essential for mesodermal patterning and 

cell fate specification 

Yuan Jiang, Jun Kelly Liu 

Biotech. Building Rm. 441, cornell University. Ithaca, NY 14853 

We are interested in understanding mesodermal patterning and fate specification by studying 

the C. elegans postembryonic mesodermal lineage, the M lineage. The M lineage is derived from 

a single precursor cell, the M mesoblast, and gives rise to six cell types: striated bodywall 

muscles (BWMs), nonmuscle coelomocytes (CCs), and four classes of non-striated sex muscles 

which are descendants of the sex myoblasts (SMs). We are studying the function of the mls-2 

(mesodermal lineage specification) gene in M lineage patterning and fate specification. 

The mls-2(cc615) mutation causes randomization of division planes in the M lineage, and 

subsequent fate transformation of CCs and BWMs to SMs. In addition, cc615mutants have 

defects in SM migration and show some larval and adult lethality. We have cloned the wild type 

mls-2 gene (C39E6.4). mls-2 encodes a homeodomain protein that belongs to the HMX family of 

homeodomain proteins that are also present in sea urchin, Drosophila and vertebrates., We 

examined the expression pattern of mls-2 using both functional mls-2::gfp fusion construct and 

affinity purified anti-MLS-2 antibodies. We found that the MLS-2 protein is localized in nuclei of 

early M lineage cells and a subset of head neurons. Furthermore, mls-2 expression in the M 

lineage and the head neurons appears to require distinct cis-acting elements. Overexpression of 

mls-2 in the early M lineage where mls-2 is normally expressed caused a variety of defects in the 

M lineage. Forced expression of mls-2 in the later M lineage such as in SMs where mls-2 is not 

normally expressed resulted in extra rounds of divisions of SMs. This suggests that mls-2 may 

have multiple roles in the M lineage and that MLS-2 protein level is critical for the correct 

patterning of the M lineage. The M lineage defects of mls-2(cc615) are very similar to those of 

mab-5, hlh-1, egl-27 and egl-20 mutants. We are currently carrying out molecular and genetic 

epistasis experiments to investigate the relationship between mls-2 and those factors.

47. Regulation of TRA-1 by sex specific proteolytic processing and localization. 

Mara Schvarzstein 1 , Laura Mathies 2 , Andrew Spence 1 

1Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, M5S 

1A8, Canada 

2Department of Genetics, North Carolina State University, Raleigh, NC 

TRA-1A is the terminal global regulator of sex determination in C. elegans. Its activity is 

required to specify all somatic female cell fates. In the germline, active TRA-1A plays a minor role 

in maintaining spermatogenesis. In addition, TRA-1A has a role in patterning the somatic gonad 

in both sexes. TRA-1A is a member of the Gli/Ci family of zinc finger transcription factors. 

TRA-1A regulation appears to be post-transcriptional because the tra-1 transcripts are present at 

equal levels in both males and hermaphrodites. To study TRA-1 regulation in detail, we 

generated an antibody against TRA-1A. Western blot analysis of nematode lysates revealed that 

while both males and hermaphrodites have similar levels of full length TRA-1A, hermaphrodites 

accumulate a set of smaller, more abundant TRA-1 isoforms. We suggest that these 

hermaphrodite-specific TRA-1 isoforms are generated by proteolytic processing of the full length 

protein. Others have shown that TRA-1 promotes certain female cell fates by repressing 

transcription of genes required for male cell fates. We hypothesize that the 

hermaphrodite-specific TRA-1 isoforms that we observe are transcriptional repressors 

responsible for promoting female differentiation. 

The observation that several feminizing tra-1 alleles encode truncated derivatives of TRA-1A 

provides genetic support for our hypothesis. To test it directly, I am mapping the endpoints of the 

hermaphrodite-specific isoforms of TRA-1A and using that information to produce transgenes that 

encode only these isoforms. I will then test these transgenes for feminizing activity. 

TRA-1 immunostaining revealed differences in the expression pattern of TRA-1 in males and 

hermaphrodites. In the embryo I detect TRA-1 in the somatic gonad precursor cells (Z1 and Z4) in 

both sexes, consistent with evidence that tra-1 is required in both sexes to establish the two-fold 

symmetry of the gonad primordium. Early in the first larval (L1) stage TRA-1 is nuclear in 

hermaphrodite Z1 and Z4, but it is evenly distributed in the nucleus and cytosol of these cells in 

the male. TRA-1 expression persists in the descendants of Z1 and Z4 in the hermaphrodite but 

not in the male. By adulthood TRA-1 is expressed in the nuclei of many different tissues in the 

hermaphrodite. In adult males, TRA-1 is detected only weakly in a few unidentified somatic cells 

and in the germline, consistent with its requirement for maintaining spermatogenesis. 

Our analysis suggests that TRA-1 activity is regulated by sex-specific proteolytic processing 

and by differential localization. Thus the regulation of TRA-1 may be analogous to that of Ci/Gli, 

which undergo regulated proteolysis to yield transcriptional repressors.

48. Mutations in him-8 suppress developmental defects of egl-13 mutants 

Brian L. Nelms, Wendy Hanna-Rose 

Penn State University, 406 Althouse Lab, University Park, PA 16802 

EGL-13 is a Sox domain transcription factor that is required for the maintenance of uterine 

seam cell fate. Mutant alleles of egl-13 cause connection-of-gonad and egg-laying defects in C. 

elegans hermaphrodites. By looking for genetic interactors of egl-13, we hope to gain insight into 

how Sox family members, which are important in many aspects of metazoan development, are 

regulated and function. We performed a screen for suppressors of the developmental defects of 

egl-13 mutants and isolated two such suppressors. 

Multiple recessive mutations in the gene him-8 partially suppress the defects caused by 

incompletely penetrant mutant alleles of egl-13. We tested several other him genes, and none of 

these mutants suppressed the defects of mutant egl-13, suggesting that suppression is not an 

indirect effect of the Him phenotype in general. The him-8 gene product is haploinsufficient for 

suppression of egl-13, and we conclude that normal HIM-8 protein acts antagonistically to 

EGL-13. Because null alleles of egl-13 cannot be suppressed, we conclude that this antagonistic 

interaction most likely occurs either upstream of EGL-13 or more directly on EGL-13 protein itself. 

RNAi of egl-13 in suppressed animals abrogates suppression, consistent with the conclusion that 

mutant HIM-8 does not bypass the requirement for EGL-13. Furthermore, the addition of multiple 

copies of the egl-13 promoter via an integrated transgene array can also abrogate the 

suppression of egg-laying defects by him-8 mutations. We are currently investigating the 

molecular mechanism of suppression and the role of the egl-13 promoter. 

In addition to him-8, we have isolated a second suppressor of egl-13 that shows genetic 

behavior similar to that of suppression by him-8, but does not exhibit a Him phenotype. We are 

currently in the process of mapping and cloning this second gene.

49. Generating a more comprehensive picture of apoptosis using multiple functional 

genomic techniques 

Stuart Milstein 1,2 , Pierre-Olivier Vidalain 1 , Siming Li 1 , David Hill 1 , Marc Vidal 1 

1Center for Cancer Systems Biology and Deparmtent of Cancer Biology Dana-Farber Cancer 

Institute Boston MA 

2stuart_milstein@dfic.harvard.edu 

In C. elegans 131 somatic cells and several hundred germ cells undergo programmed cell 

death, or apoptosis. Through a combination of forward and reverse genetics, a total of 21 genes 

have been shown to be involved in C. elegans apoptosis. In addition, 13 additional genes might 

have a role in apoptosis because their predicted products share significant homology to proteins 

already known to be involved in the process in other organisms. Our ultimate goal is to develop a 

predictive model of apoptosis in C. elegans by identifying most components involved and a nearly 

complete set of functional interactions between them. Our first step is the generation of a physical 

protein-protein interaction, or interactome map, starting with the product of each one of the 34 

known or suspected apoptosis genes. 

We performed yeast two-hybrid screens using 31 proteins known or suspected to act in C. 

elegans apoptosis and combined the resulting map with WI5 (worm interactome version 5), our 

recently published interactome dataset consisting of ~2,900 proteins connected by nearly 5,500 

potential interactions. After filtering WI5 for the highest quality interactions we generated an 

apoptosis interactome map consisting of 218 potential interactions involving 20known or 

suspected apopotosis proteins and 167 new potential components. We have been able to 

confirme the majority of the interactions that we have tested so far (14/17) by co-affinity 

purification using GST and Myc tagged proteins in a mammalian cell line. 

We are currently using this interactome map to derive a more comprehensive model of 

apoptosis in C. elegans. Our strategy is two-fold. First we perturb the system by systematically 

removing gene products using RNAi, either in wild-type or in apoptotic mutant animals. Second, 

we are beginning to develop reagents to test the consequences of removing individual 

interactions, rather than removing proteins entirely, by generating interaction defective alleles 

using the reverse yeast two-hybrid system. Such alleles will then be tested back in various 

backgrounds to determine phenotype of these interaction defective alleles. Using these methods 

we hope to develop a more complete and predictive model of apoptosis.

50. Characterization and cloning of a novel component in the cell-corpse engulfment 

pathway 

Xiaomeng Yu 1 , Xiaohong Leng 1 , Chin-Hua Chuang 1 , Sampeter Odera 1 , H. Robert Horvitz 2 , 

Zheng Zhou 1,3 

1 Verna and Marrs McLean Department of Biochemistry & Molecular Biology., Baylor College of 

Medicine, Houston, TX 77030 

2 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 

3 Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030 

In multicellular organisms, cells that undergo apoptosis are swiftly removed by other cells via 

the process of phagocytosis. This is an evolutionarily conserved process of which many 

components identified so far are shared throughout animal kingdom. During the development of 

C. elegans, 131 somatic cells undergo apoptosis and are removed by their neighboring cells. 

Genetic studies have identified at least seven genes that are required for efficient engulfment of 

these apoptotic cells. These seven genes define two parallel and partially redundant pathways. In 

one pathway, ced-1, -6 and -7 function together to promote the recognition of cell-corpse by the 

engulfing cell. CED-1 functions as a phagocytic receptor. It is majorly expressed in and presented 

onto the surface of engulfing cells and has been shown to cluster around cell-corpses during 

engulfment. CED-6, an adaptor protein that also contains PTB domain, potentially relays the 

CED-1 signaling downstream. CED-7, an ABC transporter protein, may function in presenting the 

CED-1 ligand onto the surface of dying cells, since CED-1 clustering around dying cell is greatly 

diminished in ced-7 mutant. In the other pathway, CED-2/CrkII, CED-5/DOCK180 and 

CED-12/ELMO1 form a protein complex that activates CED-10/Rac1 GTPase. Activation of 

CED-10/Rac-1 leads to the rearrangement of cytoskeleton, an event required for the extension of 

pseudopods surrounding cell-corpse. 

P> P> 

Defects in the removal of apoptotic cells have been associated with multiple human diseases. 

In worms, however, this process may not be essential, since mutants that are strongly defective 

in the engulfment of cell-corpses are still viable. From screens that we recently conducted, 

however, 17 new alleles were discovered that cause lethality in addition to the persistent 

cell-corpse phenotype, which suggests that genes defined by these mutants have other essential 

roles in worm development. These 17 mutants define at least three complementation groups. I 

am particularly interested in one group which includes 14 alleles. By studying one representative 

allele in this group, n4039, I found that the gene affected in this mutant is a novel component that 

has not been characterized in the engulfment process. This gene functions in the ced-1, -6 and -7 

pathway and may also be required for CED-1 function, since CED-1 clustering is diminished in 

n4039 mutant. However, it is unlikely that this gene functions in the presentation of CED-1 ligand 

as has been suggested for CED-7, since tissue-specific expression of this gene in engulfing but 

not dying cells is sufficient to rescue the engulfment defect in n4039 mutant. In this meeting, I will 

present the molecular characterization of the gene defined by n4039 and 13 other alleles 

regarding its function in cell-corpse engulfment.

51. Characterization of the Cell Deaths Caused by Mutations in lin-24 and lin-33 

Brendan Galvin, Saechin Kim, Erika Hartwieg, Bob Horvitz 

HHMI, Dept. of Biology, MIT, Cambridge, MA 02139, USA 

Mutations in the genes lin-24 and lin-33 can semidominantly cause the inappropriate deaths of 

Pn.p cells. Some Pn.p cells are vulval precursor cells, and lin-24 and lin-33 mutations can result 

in a vulvaless phenotype. The Pn.p cell deaths caused by lin-24 and lin-33 mutations are 

morphologically distinct from both programmed cell deaths and the necrotic deaths observed in 

strains containing certain mutant alleles of genes that encode degenerin ion channels (mec-4, 

deg-1, mec-10, and unc-8). Interestingly, the inappropriate deaths of the Pn.p cells in lin-24 and 

lin-33 mutants require a subset of the genes necessary for programmed cell death, including at 

least some of the genes required for corpse engulfment. 

We have cloned lin-24 and lin-33. lin-24 encodes a protein containing an Aerolysin toxin 

domain, while lin-33 encodes a novel protein. Aerolysin-like toxins are cytolytic toxins made 

predominantly by bacteria and act by destroying the membrane permeability barrier of eukaryotic 

cells and causing osmotic lysis. 

A deletion allele we isolated of lin-24 and an intragenic suppressor we isolated of lin-33(n1043) 

each result in a wild-type phenotype. Genetic analyses suggest that the deaths caused by a 

semidominant mutation in one gene require the function of the other gene. Additionally, the 

deletion allele of lin-24 can dominantly suppress the semidominant Pn.p death phenotypes of 

both lin-24 and lin-33 mutants. To characterize the lin-24 and lin-33 Pn.p cell deaths, we are 

analyzing these deaths by electron microscopy. We are also analyzing the expression of the 

lin-24 and lin-33 genes using gfp reporter constructs and an antibody raised against LIN-24. The 

results of ectopic expression studies, mosaic analysis, and dosage studies of these genes will be 

presented.

52. RME-6 is a new regulator of Rab5-mediated endocytosis 

Miyuki Sato, Ken Sato, Paul Andre Fonarev, Barth Grant 

Dept. of Molecular Biology and Biochemistry, Rutgers University, 604 Allison Road, Piscataway, 

NJ 08854 

Endocytosis is an essential process required for key cellular functions such as the uptake of 

nutrients, recycling of membranes and regulation of signaling pathways. Using a GFP fusion of 

YP170, a C. elegans yolk protein, we have isolated rme mutants that show defects in 

receptor-mediated endocytosis. This genetic screen was successful in identifying 11 new genes, 

including several that encode novel conserved proteins required for endocytic traffic. 

One such rme mutant, rme-6, shows a particularly strong yolk uptake defect in oocytes. In 

rme-6 mutants, yolk receptors accumulate to abnormally high levels on the cell surface, 

suggesting that receptor internalization is blocked. rme-6 also causes defect in fluid-phase 

endocytosis in coelomocytes, indicating that rme-6 is required for at least two classes of 

endocytosis in at least two cell types. The rme-6 gene encodes a novel protein that is conserved 

from worms to humans. RME-6 has a predicted Vps9 domain at its C-terminus and a 

RasGAP-like domain at its N-terminus. The Vps9 domain is a known motif conserved among 

guanine nucleotide exchange factors (GEFs) of the small GTPase Rab5. Rab5 is a key regulator 

of the early endocytic pathway in mammalian cells required for transport from the plasma 

membrane to the early endosome, and homotypic fusion of early endosomes with one-another. 

rme-6 mutants display several phenotypes that specifically indicate low Ce-RAB5 activity, as 

would be expected if RME-6 functions as a Ce-RAB5 exchange factor. These phenotypes include 

very small early endosomes and failure to recruit the Rab5 effector EEA1 to early endosomal 

membranes. Furthermore, double knockdown of rme-6 and the apparent C. elegans orthologue of 

the canonical Rab5 exchange factor rabex-5 (Y39A1A.5a) results in complete dispersal of 

Ce-RAB5 from endosomes to the cytoplasm and loss of embryo viability. A rescuing GFP::rme-6 

transgene is broadly expressed and the GFP::RME-6 fusion protein is enriched in cortical 

structures of oocytes and coleomocytes, showing significant spatial overlap with the coated-pit 

protein clathrin and markers of the early endosome. Finally we found that RME-6 interacts 

physically with the GDP-form of Ce-RAB5 in two-hybrid and co-immunoprecipitation assays. 

These results demonstrate that RME-6 is a new regulator of Rab5 and indicate that it is a key 

regulator of endocytosis in metazoans.

53. Septins function in morphogenesis of the C. elegans pharynx 

Fern P. Finger 

Biology Department, Rensselaer Polytechnic Institute, Troy, NY 12180 

Septins are a family of GTPases involved in cytokinesis in diverse organisms. They are also 

expressed in post-mitotic cells, suggestive of other cellular functions. C. elegans have two 

septins, encoded by the unc-59 and unc-61 genes. We have recently described novel roles for 

septins in cellular and axonal migration (1). Loss of septin function also results in up to 50% 

lethality in the first larval (L1) stage due to defective formation of the pharynx. Some pharynges 

appear to not have properly elongated and are not attached to the buccal cavity, while others 

appear morphologically normal, but are also unattached. The range of defects seen in the septin 

mutants suggests that some pharynges may not elongate properly, whereas others may attach to 

the buccal cavity and then snap back under the stress of embryonic elongation. Cuticle and 

basement membrane are produced, and the mutant pharynges contract, indicating epithelial and 

neuromuscular function, suggesting that all of the normal types of pharyngeal cells are present. 

The pharynges of septin mutants expressing a plasma membrane-localized GFP, 

pha-4::GFP::PM (2), do not contain any unusually large cells, suggesting that no early cytokinesis 

failures have occurred, as is suggested by the normal embryonic lineages in previous analysis of 

unc-59(e1005) (3). In indirect immunofluorescence studies, UNC-59 appears to be associated 

with junctional complexes containing AJM-1 in the embryonic pharynx, and UNC-59 is able to 

localize correctly in pharynges of the null mutant unc-61(e228). This suggests that UNC-59 

localization is independent of UNC-61. Expression of both septins is observed in the pharynx 

using previously made septin::GFP fusions. My working hypothesis for septin function in 

pharyngeal morphogenesis is that the septins may organize or stabilize the actomyosin-rich 

apical domain of the pharynx during the contraction phase of pharyngeal attachment (2), 

analogous to their potential role in organizing the contractile ring during cytokinesis. This 

potential function of the C. elegans septins may not be restricted to the pharynx, as we also 

detect septins at other epithelial junctions that are subject to considerable mechanical stress. 

1. Finger, F. P., Kopish, K. R., and White, J. G. A role for septins in cellular and axonal 

migration in C. elegans. Dev. Biol., 261: 220-234, 2003. 

2. Portereiko, M. F. and Mango, S. E. Early morphogenesis of the Caenorhabditis elegans 

pharynx. Dev. Biol., 233: 482-494, 2001. 

3. Sulston, J. and Horvitz, H. Abnormal cell lineages in mutants of the nematode C. 

elegans. Dev. Biol., 82: 41-55, 1981.

54. An Essential Role for HTP-3, a HIM-3 Paralog, in Mediating Meiotic Chromosome 

Behaviour and Structure 

William Goodyer, Monique Zetka 

Department of Biology, McGill University, Quebec, Canada, H3A 1B1 

Meiosis is essential for the creation of genetic variation and the accurate transmission of 

genetic material in sexually reproducing organisms. Throughout this process, chromosomes 

undergo complex structural rearrangements whose relationship to universally conserved meiotic 

processes is poorly understood. The him-3 gene encodes a meiosis-specific structural 

component of chromosome cores and is required for several crucial meiotic events including 

chromosome pairing, the nuclear reorganization that accompanies the onset of homolog 

alignment, synapsis, and recombination 1 ,2 . Three paralogs of him-3, known as the him-three 

paralogs (htp-1, 2, and 3), have been identified, raising the possibility that they constitute a family 

of genes involved in mediating chromosome structure and behaviour. 

We are investigating the localization and function of HTP-3, one of these paralogs, and 

its interaction with the him-3 family of proteins. Yeast two-hybrid screening data suggests that 

HTP-3 interacts directly with both HIM-3 3 and HTP-1 4 . Stainings with anti-HTP-3 antibodies 

have revealed that HTP-3 localizes to all germ line nuclei. HTP-3 is diffusely present within the 

nucleoplasm of mitotic nuclei, but like HIM-3, HTP-3 localizes to chromosome axes in meiotic 

nuclei. HTP-3 localization is independent of both HIM-3 and HTP-1. However, consistent with an 

interaction between HTP-3 and HIM-3, HTP-3 is required for the localization of HIM-3 to meiotic 

chromosome cores. Additionally, htp-3 (RNAi) hermaphrodites exhibit the most severe meiotic 

defects of him-3 mutants. Given these results, HTP-3 is hypothesized to have a primary meiotic 

function in direct recruitment of HIM-3 to chromosome axes. However, due to the mitotic 

localization of HTP-3 and other meiotic defects in the htp-3 (RNAi) background unique from him-3 

mutants, HTP-3 likely has additional roles. 

Further cytological and genetic analyses of HTP-3 are in progress and will be 

presented. Our goal is to ultimately understand how HTP-3, in conjunction with its paralogs, 

mediates these essential meiotic processes as well as other germ line events. 

Supported by NSERC and CIHR. 

1 Zetka et al. 1999. Genes and Dev. 12: 2258, 2 Couteau et al.. 2004. Curr Biol. 14: 585 

3 Zetka, unpublished results, 4 Walhout, et al. 2002. Curr Biol. 12: 1952

55. HDA-1 regulates C. elegans embryogenesis: a potential role for a ubiquitous chromatin 

modifier in regulating tissue-specific gene expression and patterning. 

Johnathan R. Whetstine 1 , Julian Ceron 1 , Valerie Reinke 2 , Yang Shi 1 

1Harvard Medical School, Department of Pathology 

2Yale School of Medicine, Department of Genetics 

In order for an organism to properly develop, a series of complex signals must be deciphered 

and processed into extra- and intracellular signals that will result in the temporal and spatial 

modulation of gene expression. The modification of the DNA environment is one such intracellular 

signal. DNA is organized and compartmentalized into a complex structure called chromatin. The 

chromatin contains both chromosomal and histone proteins (H2A, H2B, H3, and H4), and the 

post-translational modifications of these histone proteins (i.e. , phosphorylation, acetylation, 

ubiquitination, and methylation) serve as critical marks for a series of cellular processes (i.e. , 

transcription). Therefore, understanding the enzymes responsible for these modifications is 

critical in understanding how multi-cellular organisms develop. For this reason, a gene expression 

profile was generated for a highly conserved histone deacetylase (HDA-1) that results in 

embryonic lethality when depleted by RNAi. N2 worms were fed a potent hda-1 RNAi vector that 

resulted in a dramatic depletion of hda-1 transcripts and protein. The transcripts from RNAi 

treated animals were hybridized to a microarray containing ~94% of the C. elegans genome. 

Interestingly, the microarray data revealed that certain clusters of genes are enriched in these 

RNAi treated embryos (i.e. , intestine related genes). In fact, many of the targets were confirmed 

by RT-PCR. The confirmation was then followed up by assessing the occupancy of HDA-1 and 

acetylation status of several target promoters using chromatin immunoprecipitation assays. 

HDA-1 was found at the target promoters and was associated with decreased acetylation status. 

In addition to identifying key genes downstream of HDA-1, the clusters from the microarray 

suggested that certain target organs are highly influenced by HDA-1 during early embryogenesis. 

These observations were then confirmed by using a series of tissue-specific markers. Overall, our 

data demonstrated that HDA-1 directly regulated specific sub-sets of genes that are 

tissue-specifically expressed. Future studies will focus on the role of various gene families 

identified in this study and their contribution to the hda-1 RNAi treated embryos.

56. Genetics of telomere replication in C. elegans 

Bettina Meier 1 , Sarah Mense 2 , Yan Zhao 2 , Shawn Ahmed 1,2 

1Department of Genetics, University of North Carolina, Chapel Hill, NC 27599-3280, USA 

2Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280, USA 

Replication of chromosome ends by telomerase is pivotal to maintain immortality of the 

germline in many organisms. Telomerase, a reverse transcriptase, adds multiple repeats of a 6 

bp sequence to the end of the chromosomes, the telomeres, and thereby confers stable 

chromosome length. The telomerase catalytic subunit, TERT, and it’s RNA component have been 

shown to be sufficient for the addition of repetitive sequences to chromosome ends in vitro, 

however several additional proteins are required to ensure telomerase function in vivo. We are 

interested in studying telomerase function in C. elegans. 

We are currently characterizing strains with different mutant alleles of the catalytic subunit of 

telomerase, trt-1. These mutants become sterile after multiple generations and show variable 

germline phenotypes that might mirror a late proliferation defect. In addition, we are studying a 

telomere replication mutant, mrt-1, which maps nearby trt-1. mrt-1 mutants are not rescued by an 

extragenic trt-1 gene. Analysis of cosmids spanning the map position of mrt-1 identified no 

obvious candidate gene, suggesting that mrt-1 may be a novel factor required for telomere 

replication in C. elegans, and we are in the process of cloning this gene. 

To identify genes that synthetically interact with trt-1, transient gene knockdowns of all 

essential genes by RNA interference have been performed in a trt-1 mutant background. We 

have identified several essential genes whose RNAi phenotypes are more severe in the absence 

of trt-1. Among these candidates are genes required for cell-cycle control, chromatin structure, 

and nucleic acid metabolism. The specificity of these genetic interactions in the context of 

different trt-1 alleles or other telomere replication mutants, such as mrt-1, may help to define the 

nature of the telomere replication defects in these mutants.

57. Centromere resolution is inhibited by cohesin proteins and requires condensin II 

components, HCP-6 and Mix-1 

Landon L. Moore, Matt Stankiewicz, David Rosen, Tovah Day 

Department of Genetics and Genomics, Boston University School of Medicine, Boston, MA 02118 

Centromere maturation, the processes of organizing centromeric chromatin, resolving sister 

centromeres and assembling sister kinetochores, is essential for maintaining chromosome 

stability. The centromere protein HCP-4/CeCENP-C is required for centromere resolution and 

kinetochore assembly. Prior to microtubule capture, sister centromeres resolve from one another, 

coming to rest on opposite surfaces of the condensing chromosomes. Despite the importance of 

this process in chromosome segregation, centromere resolution remains poorly understood. To 

better understand the process of centromere resolution and CeCENP-C’s role, we took 

advantage of the holocentric chromosomes present in C. elegans. Because centromere 

resolution resembles the resolution of sister chromatids that occurs during prophase of 

monocentric chromosomes, we investigated the involvement of proteins required for cohesion in 

centromere resolution. While removal of individual cohesin proteins (CeSCC1, CeSCC3, 

CeSMC1) or a genetic loss-of-function mutation in CeSMC1 did not abrogate the close 

association of sister chromatids in mitosis, it did suppress the requirement for CeCENP-C in 

resolution. In addition, to the cohesin complex proteins, we observed that loss of CeSCC2, which 

in yeast is necessary to recruit the cohesin complex to DNA, and CeTRF4, an alternative DNA 

polymerase required for establishment of cohesion, were required to inhibit centromere 

resolution. Other proteins involved in cohesion, such as EVL-14/PDS-5 however, did not appear 

to be involved. Interestingly, the restored centromere resolution correlated with the recruitment of 

HCP-6, a component of the condensin II complex. We found that condensin II proteins 

Mix-1/CeSMC2 and HCP-6 together with the removal of cohesin-mediated inhibition are required 

for centromere resolution to occur prior to microtubule capture. Additionally, defects in 

centromere resolution timing led to aberrant kinetochore microtubule interactions. These results 

support the idea that centromere resolution involves an organized pathway that is regulated by 

the CeCENP-C mediated release of cohesion.

58. Identification and Characterization of C. elegans Amine-gated Chloride Channels 

Namiko Abe, Niels Ringstad, Bob Horvitz 

HHMI, Dept. Biology, MIT, Cambridge, MA 02139 USA 

Biogenic amines generally function as neuromodulators by activating metabotropic G 

protein-coupled receptors. In vertebrates, the biogenic amine serotonin (5-HT) also acts as a 

fast-acting excitatory neurotransmitter by activating the ionotropic 5-HT 3 receptor, a non-selective 

cation channel. In C. elegans, 5-HT may function as a fast-acting inhibitory neurotransmitter 

through MOD-1, a 5-HT-gated chloride channel (Ranganathan, Cannon, and Horvitz, Nature 408: 

470-475, 2000). 

To determine whether there are other MOD-1-like receptors in C. elegans, we searched the C. 

elegans genome for sequences similar to the mod-1 coding sequence and found 26 

uncharacterized genes predicted to encode ligand-gated chloride channels. We are expressing 

each of these genes in Xenopus oocytes and testing them for amine receptor activity. So far, 

this approach has identified three novel ionotropic receptors gated by biogenic amines. One of 

these receptors is activated most potently by dopamine, another by tyramine, and one receptor is 

weakly activated by 5-HT and not by other known amines. Ion-replacement studies suggest that, 

like MOD-1, these channels selectively pass chloride ions. We are characterizing the 

pharmacological properties of these channels. We have found that some dopamine receptor 

antagonists, including chloropromazine, haloperidol, spiperone, raclopride, and SCH 23390 

antagonize the current carried by the putative dopamine recepor. To understand how these 

receptors function in vivo, we have isolated deletion mutants in these three genes and are 

determining if any of these mutants are abnormal for any behaviors. The mutant with a deletion 

in the putative tyramine receptor gene has a phenotype consistent with behaviors known to be 

mediated by tyramine signaling (Alkema et al., IWM Abstract no. 14, 2003).

59. Linker cell death may be caspase-independent 

Mary C. Abraham, Shai Shaham 

The Rockefeller University, 1230 York Avenue, New York, NY 10021 

Programmed cell death is important for development and inappropriate programmed cell death 

can contribute to disease. An important breakthrough in the understanding of cell death in C. 

elegans was the identification of the caspase ced-3 as a key regulator of apoptosis. Caspases 

also play crucial roles in the execution of cell death across many species. Are caspases 

absolutely required for programmed cell death, or do caspase-independent mechanisms exist 

enabling cells to die in a programmed way? The C. elegans linker cell, which leads the migration 

of the male gonad, has been identified as a rare example of a C. elegans cell that can die in a 

ced-3 mutant background (1). We have studied linker cell death by microscopy studies of a GFP 

marked linker cell in a number of mutant animals including animals harboring mutations in core 

apoptotic pathway genes such as ced-3 and ced-4 and also in engulfment genes. None of the 

known cell death genes that we have tested appear to play roles in linker cell death, suggesting 

that the mechanism for linker cell death involves unknown cell death molecules. For example, 

within a 3-hour time window when the linker cell in a wild-type animal undergoes the 

morphological hallmarks of the final stages of cell death, 32/41 ced-4 animals and 43/57 ced-3 

animals undergo the same morphological changes. Apart from ced-3, there are three caspase 

related genes in C. elegans (2), and we have investigated the potential involvement of these 

other caspases in linker cell death by use of p35 (a broad spectrum caspase inhibitor) or RNAi 

techniques against the caspase related gene csp-3. To date, no evidence for caspase 

involvement in linker cell death has been observed. It had previously been reported that linker cell 

death requires a "murder" signal from a U cell descendant to die. This hypothesis was based on 

reports of linker cell survival either after U cell ablation or in a linker cell migration defective strain 

in which the linker cell failed to reach its normal position adjacent to the U cell descendants (3). 

Our investigation of U cell involvement in the death of the linker cell presented here suggests that 

the U cell descendants may not be required for the linker cell to die after all. To identify genes 

that are responsible for linker cell death, we are conducting a visual genetic screen for animals in 

which the linker cell fails to die. 

References: 

(1) Ellis H.M. and Horvitz H.R. (1986) Genetic Control of Programmed Cell Death in the 

Nematode C. elegans. Cell, 44:817-29. 

(2) Shaham S. (1998) Identification of multiple Caenorhabditis elegans caspases and their 

potential roles in proteolytic cascades. J. Cell Biol., 273:35109-35117. 

(3) Sulston J.E., Albertson D.G. and Thompson J.N. (1980) The Caenorhabditis elegans male: 

postembryonic development of nongonadal structures. Dev Biol.,78:542-76.

60. Octopamine Inhibits Pharyngeal Pumping and Egg Laying and Stimulates Locomotion. 

Mark Alkema, Niels Ringstad, Bob Horvitz 

HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA 

Octopamine is a biogenic amine implicated in several invertebrate behaviors. Octopamine 

biosynthesis requires a tyrosine decarboxylase (TDC) to convert tyrosine to tyramine and a 

tyramine beta-hydroxylase (TBH) to convert tyramine to octopamine. We characterized a C. 

elegans tyrosine decarboxylase gene (tdc-1) and a tyramine-beta-hydroxylase gene (tbh-1). We 

showed that tdc-1 deletion mutants lack tyramine and octopamine, whereas tbh-1 deletion 

mutants lack only octopamine. This observation indicates that tdc-1 is required for tyramine 

biosynthesis and that tdc-1 and tbh-1 are required for octopamine biosynthesis. tdc-1 and tbh-1 

expression overlap in the RIC interneurons and the gonadal sheath cells. tdc-1, but not tbh-1, is 

expressed in the RIM motor neurons and four uterine cells. These expression patterns suggest 

that the RIC interneurons and gonadal sheath cells are octopaminergic and the RIM motor 

neurons and UV cells are tyraminergic. 

tdc-1 mutants have several behavioral defects that are not shared by tbh-1mutants, suggesting 

a role for tyramine in the suppression of head oscillations upon anterior touch and in reversal 

behavior (Alkema et al. 14 IWM 2003). tbh-1 and tdc-1 mutants share several behavioral defects: 

they fail to properly inhibit egg laying and pharyngeal pumping in the absence of food and move 

more slowly than wild-type animals. tbh-1 and tdc-1 mutants are also hypersensitive to 

exogenous serotonin in assays of locomotion, pharyngeal pumping and egg laying. Our results 

indicate that endogenous octopamine inhibits pharyngeal pumping and egg laying and stimulates 

locomotion and thus acts antagonistically to serotonin in these behaviors. Mutations in tdc-1 and 

tbh-1 suppress the pharyngeal pumping defect but not the egg-laying defect of serotonin-deficient 

tph-1 mutants. Food-deprived tbh-1 and tdc-1 mutants, much like food-deprived animals mutant 

for the serotonin-reuptake transporter MOD-5, become almost immobilized when they encounter 

a bacterial lawn. tbh-1 deletions can suppress the resistance to exogenous serotonin in 

locomotion assays of animals mutant for the serotonin-gated chloride channel MOD-1. 

Furthermore, mod-1; tbh-1 double mutants display a hyperenhanced slowing response similar to 

that of the tbh-1 single mutants. These data suggest that tbh-1acts downstream of or in parallel to 

mod-1 in the modulation of locomotion. mod-1 is expressed in a small number of motor neurons 

and interneurons, including the octopaminergic RIC neurons (Eric Miska, unpublished 

observation). This finding suggests that serotonin release can hyperpolarize the RIC interneurons 

and thereby inhibit octopamine signaling.

61. Establishment of anteroposterior neuronal polarity 

Eleanor Allen, Anastasia Bakoulis, Dave Hunt, Scott Clark 

Skirball Institute, NYU School of Medicine, New York, New York 

Each neuron type in C. elegans projects axons with characteristic trajectories along the 

dorsoventral and/or anteroposterior body axes. Axons extend from the cell body at distinct 

orientations, revealing an intrinsic cellular polarity. Although evident in a mature neuron, when or 

how neuronal polarity is established is unclear. In addition, the processes that direct growth cone 

extensions along the anteroposterior body axis are poorly understood. To gain insight into the 

molecular mechanisms that determine neuronal polarity and anteroposterior axon guidance, we 

have begun screens for mutants with defects in the growth of the AVG axons. AVG is located in 

the retrovesicular ganglion and projects a very short anteriorly directed process and a long 

posteriorly directed process that extends to the tail. AVG pioneers the right ventral nerve bundle 

and is thought to provide cues that promote the proper assembly and organization of the ventral 

nerve cord. 

We isolated several mutations that alter AVG axonal outgrowth. zd101 caused the anteriorly 

directed AVG axon to over extend and enter the nerve ring; the posteriorly directed axon was 

unaffected. zd101 is an allele of klp-7, which encodes a kinesin-related protein involved in the 

regulation of microtubules. We had found previously that klp-7 mutations cause neurons that 

normally extend only a single axon to project a second axon. Together these observations 

indicate that the regulation of microtubules can influence both the initiation and termination of 

axon extension. 

Several mutations were recovered that cause the apparent reversal of AVG anteroposterior 

polarity, as defined by the lengths of anterior and posterior axons. These mutants exhibited a 

range of three AVG axonal phenotypes: animals with a wildtype anterior axon and a posterior 

axon shorter than wild type, animals with anterior and posterior processes about equal in length 

and animals with a very long anterior axon and a very short or no posterior axon. When the axons 

were about equal in length, the anterior process terminated in the nerve ring and the posterior 

process stopped before the vulva. However, when the posterior axon was absent and the anterior 

axon was very long, the long axon typically entered the nerve ring and the looped back to the tail 

via the dorsal cord or lateral fascicle. These observations suggest that if the neuronal polarity of 

AVG is completely reversed, the long axon can be guided to the tail and that the AVG guidance 

cues are not restricted to the ventral nerve cord. Furthermore, these cues might only influence the 

direction of growth and not extension per se. We also found several genes that influence the 

anteroposterior polarity of other neurons, suggesting that multiple processes are involved in the 

establishment of anteroposterior neuronal polarity.

62. The let-7 and mir-35 Families of MicroRNAs Each Act Redundantly in C. elegans 

Ezequiel Alvarez-Saavedra 1 , Eric A Miska 1 , Allison L Abbott 2 , Nelson C Lau 3 , David P 

Bartel 3 , Victor Ambros 2 , Bob Horvitz 1 

1HHMI, Dept Biology, MIT, Cambridge, MA 02139, USA 

2Dept. Genetics, Dartmouth Medical School, Hanover, NH 03755, USA 

33Whitehead Institute for Biomedical Research and Dept. Biology, MIT, Cambridge, MA 02139, 

USA 

From our efforts to obtain deletion alleles for all microRNA genes in C. elegans (see abstract by 

Miska et al.) we have identified and are characterizing two families of microRNAs the members of 

each of which appear to act redundantly. 

The let-7 microRNA regulates the larval-to-adult transition. mir-48, mir-84, and mir-241 encode 

microRNAs similar in sequence to let-7. To study their functions we obtained strains with 

deletions in these microRNA genes by screening a library of mutagenized worms. Strains with 

single mutations in mir-48, mir-84or mir-241 have a wild-type phenotype. However, mir-48; 

mir-84double mutants undergo an additional molt in the adult stage. Worms mutant for both 

mir-48 and mir-241 generate extra seam cells in the third and fourth larval stages, probably as a 

consequence of reiterations of the second larval stage developmental program. These findings 

suggest functional redundancy amonglet-7 family members and roles for the let-7 family in the 

control of the L2-to-L3 transition and the larval-to-adult transition. 

The mir-35 genomic cluster of microRNAs consists of seven genes, mir-35 through mir-41, that 

share closely related sequences. These microRNAs are expressed only during embryogenesis, 

as assayed by northern blot and reporter GFP constructs. A deletion that removes all seven of 

these microRNAs results in a temperature-sensitive late embryonic lethal phenotype, while a 

deletion that affects only mir-37 to mir-41 does not cause lethality. The embryonic lethality can be 

rescued by expression of mir-35and mir-36. We are conducting a screen for suppressors of the 

temperature-sensitive lethality to seek targets of this family of microRNAs.

63. Characterization of the Synthetic Multivulva Suppressor Gene isw-1, a Homolog of the 

Drosophila Chromatin-Remodeling ATPase ISWI 

Erik Andersen, Xiaowei Lu, Scott Clark, Bob Horvitz 


The synthetic Multivulva (synMuv) genes are grouped into at least three functionally redundant 

classes, A, B, and C, that negatively regulate the induction of vulval cell fates. Animals with a 

mutation in one or more genes within the same class are non-Muv. Animals with mutations in 

genes within any two classes are Muv. Among some of the identified class B gene products are 

counterparts of a transcriptional repression complex, and among the identified class C gene 

products are homologs of a putative transcriptional activation complex. 

To identify genes that interact genetically with the synMuv genes, we performed two screens 

for synMuv suppressors. From these screens, 166 suppressors of the synMuv phenotype of 

lin-15AB(n765ts) animals and 43 suppressors of the synMuv phenotype of lin-53(n833); 

lin-15A(n767) animals were isolated. We cloned one lin-53(n833); lin-15A(n767) synMuv 

suppressor. It encodes a homolog of the chromatin-remodeling ATPase ISWI. We named this 

gene isw-1. RNAi or a presumptive null allele of isw-1 can suppress the Muv phenotype of most, 

if not all, synMuv mutant combinations. By contrast, loss of isw-1 function did not suppress the 

Muv phenotype of a null mutant of lin-1, a RTK/Ras pathway effector. The loss-of-function 

Vulvaless phenotype of mutants in the RTK/Ras pathway is epistatic to the synMuv phenotype. 

We conclude that isw-1 is likely to act downstream of or in parallel to the synMuv genes and 

upstream of or in parallel to the RTK/Ras pathway. 

Immunohistochemistry using antisera specific to ISW-1 suggests that ISW-1 is in the nuclei of 

most if not all cells throughout development. Levels of ISW-1 are not significantly changed in 

synMuv mutants as compared to the wild type based on quantitative western blotting. We are 

currently trying to identify in which cells and during which developmental times isw-1 is required 

to allow the synMuv phenotype. 

ISWI has been biochemically purified from Drosophila embryo extracts as a component of 

multiple complexes, including the Nucleosome Remodeling Factor (NURF), the Chromatin 

Accessibility Complex (CHRAC), and the ATP-utilizing Chromatin assembly and remodeling 

Factor (ACF). RNAi of C. elegans homologs of NURF, CHRAC, and ACF complex members in a 

lin-15AB(n765ts) background was used to test for suppression of the synMuv phenotype. A 

NURF301 homolog and the NURF38 homolog dhp-2 were identified as synMuv suppressor 

genes likely to act with isw-1 to regulate vulval development. Additionally, a deletion allele of the 

NURF301 homolog can suppress the synMuv phenotype of lin-15AB(n765ts) animals. Thus, 

ISW-1 may be acting as a component of a C. elegans NURF-like complex to antagonize the 

synMuv phenotype. We are mapping and cloning additional mutationally-defined suppressors of 

the synMuv phenotype in an effort to identify other factors that may act with a putative C. elegans 

NURF-like complex or in parallel processes to antagonize the synMuv phenotype.

64. met-1 and met-2, Two Putative Histone Methyltransferases, May Act as Synthetic 

Multivulva Genes 

Erik Andersen, Bob Horvitz 


Repression of euchromatic gene transcription may occur through the specific covalent 

modification of histone tails leading to the recruitment of trans-acting repressive factors. In one 

model, through the actions of the Nucleosome Remodeling and Deacetylase (NuRD) complex, 

lysine 9 of histone H3 (H3K9) is deacetylated and methylated by a histone methyltransferase 

(HMTase), which ultimately leads to recruitment of heterochromatin protein 1 (HP1) and a 

transcriptionally silenced state. 

Among the proteins encoded by the class B synthetic Multivulva (synMuv) genes are homologs 

of some NuRD complex members, including LIN-53 p48, HDA-1 HDAC, and LET-418 

Mi-2. Within the Pn.p cells fated to adopt a non-vulval cell fate, a putative LIN-53/HDA-1/LET-418 

transcriptional repression complex may be recruited to genes to be repressed through interaction 

with LIN-35 Rb and the sequence-specific heterodimeric transcription factor DPL-1 DP/EFL-1 

E2F. Through the actions of the histone deacetylase HDA-1, histones may be deacetylated. 

After subsequent methylation of histones, the C. elegans HP1 homolog (and synMuv protein) 

HPL-2 may be recruited, leading to the repression of genes that would normally promote a vulval 

fate. Given this model, we decided to seek HMTase-encoding genes that interact genetically with 

the synMuv genes. 

All known HMTases specific for modifying histone tail residues have an enzymatic SET 

domain. Through BLAST, Pfam, and SMART database searches we identified and classified the 

31 genes predicted to encode SET-domain-containing proteins in C. elegans. The HMTases 

predicted to modify histone H3 lysines 4, 9, or 36 have conserved domains flanking the SET 

domain. There are nine genes in C. elegans predicted to encode such HMTases. Using existing 

mutant alleles or deletion alleles that we have generated, we tested all nine of these HMTases for 

a synMuv phenotype with mutations in synMuv class A or B genes. Additionally, we injected 

dsRNA to inactivate each of the 31 genes predicted to encode SET domain containing genes and 

tested for a synMuv phenotype with mutations in synMuv class A or B genes. 

Mutations in two genes, met-1 and met-2, cause a synMuv phenotype in combination with 

mutations in all synMuv class A genes but not with mutations in synMuv class B genes. Based 

upon sequence similarity, met-1 is predicted to encode a histone H3 lysine 36 (H3K36) HMTase. 

In S. cerevisiae, methylation of H3K36 may play a role in transcriptional elongation or repression. 

We are currently testing whether perturbations in transcriptional elongation can cause a synMuv 

phenotype. met-2 is predicted to encode a histone H3K9 HMTase. We are testing whether 

met-2 plays a role in the recruitment of HPL-2. Interestingly, strains doubly mutant for met-1 and 

met-2 are synthetically Muv. We will discuss our model for the redundant actions of these 

putative histone methyltransferases and their interactions with the products of the synMuv genes.

65. Generation of left/right asymmetry in the nervous system of C. elegans 

Celia Antonio, Oliver Hobert 

Department of biochemistry and molecular biophysics, Columbia University, 701W 168th street 

HHSC724, New York, NY-10032 

Although the nervous system of most animals is superficially bilaterally symmetric, there is 

some degree of left-right (L/R) asymmetry. The asymmetry could be at the level of anatomical 

size difference of particular structures, patterns of gene expression and/or functional output of 

those neurons. The main taste receptors in the nematode Caenorhabditis elegans, the ASE left 

(ASEL) and right (ASER) neurons, are an example of cells that although bilaterally symmetric in 

respect to all morphological criteria, show different patterns of gene expression. The putative 

guanyl cyclase receptors gcy-6 and gcy-7 are expressed only in ASEL while gcy-5 is only 

expressed in ASER leading to different sensitivities of the two neurons to soluble compounds 

such as sodium and potassium. 

Previously in the lab, a forward genetic screen was performed to find the genes involved in 

restricting the expression of the receptors to one side of the animal. Through this screen, several 

transcription factors and a microRNA were found and shown to act in a regulatory cascade to 

promote the asymmetric gene expression in the ASE neurons. This cascade has however already 

an asymmetric activity with its most genetically upstream component, the zinc-finger transcription 

factor die-1, only expressed in ASEL. Since the original screen was not saturated, I performed a 

new screen with the aim to identify the molecular nature of the initial process that leads to the 

asymmetries observed in the ASE neurons. Using the promotor region of gcy-5 fused to green 

fluorescent protein as a reporter I have screened 19,800 haploid genomes and retrieved 26 

mutants. These mutants were divided into three classes: class I, in which ASER now expresses 

the left cell markers (2 ASEL); class II, in which the right cell markers are now also expressed in 

the ASEL (2 ASER) and finally class III, in which the ASE cells form but fail to express most of the 

identity determining markers. The mutants retrieved were called, as previously, lsy (pronounced 

lousy) for lateral symmetry mutants. Complementation tests and sequencing analysis have shown 

that at least 1 class I gene is not allelic to any of the known class I genes and is therefore a new 

lsy mutant. Four of the class II mutants failed to complement with known genes and are therefore 

also new lsy genes. Three of the retrieved mutants are allelic to che-1, the only member of class 

III known until now. After grouping all the mutants into complementation groups the new lsy 

mutants will be mapped giving priority to the mutants that affect die-1 expression and their role in 

the L/R asymmetry of the ASE neurons will be examined.

66. Alterations of the C. elegans Excretory Duct in Dauer Larvae 

Kristin R. Armstrong*, Helen M. Chamberlin* 

*Department of Molecular Genetics, Ohio State University, Columbus, OH 43210 

We are interested in studying organ development and function in order to understand both the 

normal processes and how improper organ development and function can lead to birth defects or 

cancer. The research in this project focuses on how an organ’s structure, function, and response 

can be modified by external stimuli, such as environmental conditions. The C. elegans excretory 

system is a good model for organ development and function because of its simplicity. The 

excretory system is composed of only four cell types, yet it performs all of the necessary functions 

of osmotic regulation and waste removal, similar to mammalian kidneys. The simple structure of 

the excretory system makes it possible to study development and function at the cellular level. 

Dauer formation is an example of a change induced by environmental conditions in C. elegans. 

During dauer formation, the morphology of numerous cells and organs is altered, including the 

pharynx, hypodermis, and excretory system (Riddle, Blumenthal, Meyer, and Priess, 1997). It 

was previously shown that the excretory system might play a role in the C. elegans’ 

morphological transition into dauer state (Nelson and Riddle, 1984). We are following up on this 

research using GFP markers to help visualize each component of the excretory system. We 

currently have markers for the excretory cell and excretory duct and are working on markers for 

the excretory pore and gland cells. Using the markers, we are able to observe the morphology of 

the excretory system during its development. 

As an initial experiment, we compared the morphology of the excretory duct in normal L3 larvae 

and L3 larvae that had entered dauer. Our studies showed there is a difference in the excretory 

duct morphology between dauer and non-dauer larvae. In dauer larvae, the duct’s position had 

shifted, the duct cell body was elongated, and the appendages were hyper-extended as 

compared to normal L3 larvae. We plan to expand these studies to other excretory cell types, and 

to investigate how these changes alter the excretory system function. 

Riddle, D.L., Blumenthal, T., Meyer, B.J., and Priess, J.R., C. elegans II (1997): 739-768 

Nelson, F.K and Riddle, D.L., J. Exp. Zool. (1984) Jul; 231 (1): 45-56

67. Sheath cell-dependent maintenance of AWC dendritic morphology 

Taulant Bacaj, Shai Shaham 

Laboratory of Developmental Genetics, The Rockefeller University, New York, NY 10021 

A complex set of interactions takes place between neurons and their associated glial cells. 

Glia provide the tracks and cues along which axons migrate, they are responsible for axon 

myelination as well as neuronal synapse ensheathment. We have chosen the amphid sensory 

structures in C. elegans as a model for studying the pathways that underlie the glia-neuron 

dialog. Each of the two bilaterally symmetric amphid structures is composed of a glial-like sheath 

cell, a glial-like socket cell, and twelve neurons that endow C. elegans with many of its sensory 

modalities. 

To test whether neuronal structure is regulated by its associated glia we have focused on the 

AWC olfactory neuron. At the tip of its dendrite, AWC possesses an elaborate wing-like structure 

that is completely ensheathed by the sheath cell. 

We ablated sheath cells on one side of L1 larvae in a strain expressing the sheath cell marker 

vap-1::GFP and the AWC marker odr-1::RFP using a laser microbeam. Operated animals were 

then imaged at the L4 or adult stages using a spinning-disk confocal microscope. The shape of 

the AWC wing-like projection was dramatically different in laser operated amphids (as determined 

by loss of GFP expression) as compared to non-operated control amphids. Thus, in 12/15 L4 

larvae and 36/37 adults scored, the ablated amphids had lost the wing-like structure, and instead, 

a bright spherical structure or a finger-like protrusion was often observed. In contrast, 52/52 

non-ablated amphids displayed wild-type AWC morpholgy. 

These results suggest that sheath cell-dependent signals may be required for active 

maintenance of AWC dendritic morphology. Interestingly, odr-1::RFP is faintly expressed in the 

AWB neuron which ends in a V-like dendritic process. Our preliminary findings suggest that the 

morphology of this neuron remained unchanged in ablated amphids. Thus, it is possible that the 

maintenance of some dendritic structures within the amphid are not dependent on an intact 

sheath cell. 

Our goal is to determine if mutations exist that phenocopy the laser ablated amphids. We 

reason that mutant animals with abnormal AWC morphology will be defective in AWC olfaction. 

To identify mutants, we will first visualize the AWC dendritic processes in known mutants 

defective in AWC olfaction. In addition, we will perform a saturation screen for mutants that fail to 

chemotax toward AWC-mediated attractants. These chemotaxis-defective mutants will be 

screened for abnormal AWC morphology.

68. Association of Oscheius species with millipedes in the Wright State University Woods. 

Scott Everet Baird, Christine M. Spice 

Department of Biological Sciences, Wright State University, Dayton OH 45435 

Rhabditid nematodes commonly form phoretic or necromenic associations with other soil 

invertebrates. These associations typically are as dauers although although associations of other 

larval stages also have been reported. Two hermaphroditic species of Oscheius, O. myriophila 

and an isolate tenatively identified as O. necromena, have been reported as necromenic 

associates of millipedes. Both of these species have been found in association with the millipede 

Pseudopolydesmus serratus in the Biology Preserve on the campus of Wright State University. A 

third as yet unnamed hermaphroditic species of Oscheius was found in association with a second 

millipede, Oxidus gracilis. O. myriophila, O. necromena, and O. sp. were found at the same 

collection site, i.e. they were sympatric. O. myriophila never was found in association with Ox. 

gracilis despite this millipede being its type host. When first observed, all collected nematodes 

were L4s, consistant with a previous report of the association of O. myriophila L4s with 

millipedes. Relationships among these species were investigated through sequence comparisons 

of 18S rDNA. O. myriophila, O. necromena, and O. sp. clustered together as a monophyletic 

clade within the insectivora-group of Oscheius. Thus, association with millipedes and their 

hermaphroditic mode of reproduction may be ancestral characters of this group of species.

69. Females, Hermaphrodites and Developmental Bias 

Chris Baldi 1 , Soochin Cho 2 , Ronald E Ellis 1 

1Department of Molecular Biology, UMDNJ - SOM, Stratford, NJ 08084 

2Department of EEB, University of Michigan, Ann Arbor, MI 48109 

A developmental bias is a restriction in the direction of evolution caused by the nature of 

development itself. For example, which digits are lost during frog or salamander evolution is very 

predictable, and appears to be determined by the way these digits are patterned during 

development. Although such biases are likely to influence the direction of evolutionary change, 

they have been difficult to study, and the best models involve physical restrictions imposed by 

patterning mechanisms. 

Mating systems provide an excellent model for studying how the structure of genetic regulatory 

pathways might create developmental biases. Although self-fertilizing hermaphrodites are ideally 

suited for colonizing new environments, species that contain such hermaphrodites are common in 

nematodes, but absent from insects or vertebrates. To learn why, we are studying the regulation 

of female and hermaphroditic mating systems in C. elegans and related nematodes. 

We recently prepared a detailed phylogeny for Caenorhabditis. Our results show that the two 

male/hermaphrodite species are not sisters. Along with work by T. Schedl, this phylogeny 

indicates that hermaphroditism might have evolved independently in C. elegans and C. briggsae, 

which implies that underlying features of the sex-determination system are likely to favor such 

changes. 

What are these features? The key difference between hermaphrodites and females is that the 

former produce sperm and oocytes, but the latter make only ooctytes. Since fog-1 and fog-3 

control which cells produce sperm in C. elegans, we cloned their homologs from other 

nematodes. The sequences of both genes have been diverging rapidly, but RNA-mediated 

interference shows that their functions are completely conserved. Inactivating either fog-1 or fog-3 

causes germ cells to become oocytes, but has no other effects on development. 

Why, then, do fog-1 and fog-3 cause XX animals to make sperm in some species, but not in 

others? We observed significant levels of fog-1 expression at all times and in both sexes of C. 

briggsae and C. remanei. By contrast, we found that fog-3 expression peaks during larval 

development in C. briggsae hermaphrodites, disappears in adults, and is never observed in C. 

remanei females. Thus, expression of fog-3 is strongly correlated with spermatogenesis. 

Furthermore, in both species fog-3 expression is controlled by sex-determination genes, which 

may act through conserved TRA-1A binding sites in the fog-3 promoters. We suggest that 

changes in the regulation of fog-3 by upstream genes could explain why C. briggsae produces 

XX hermaphrodites, but C. remanei makes XX females. 

We are now studying these upstream regulators. In C. elegans,mutations in fog-2 transform 

XX animals into true females. We isolated a mutation with a similar phenotype, glf-1(v35), in C. 

briggsae, and are now characterizing the gene. Preliminary results suggest it is probably not 

related to known C. elegans sex-determination genes. In addition, we have begun the process of 

manipulating fog-3 levels in C. remanei by cloning and characterizing the tra-1 gene. 

Surprisingly, lowering tra-1 activity by RNAi causes a decrease in fog-3 transcript levels and 

prevents spermatogenesis. Thus, tra-1 plays a role in male spermatogenesis in C. remanei, just 

as it does in C. elegans. 

Our results suggest that several factors allow nematode mating systems to undergo rapid 

evolution. First, in nematodes, the decision to produce sperm or oocytes is controlled by two 

genes, fog-1 and fog-3, that lack pleiotropic effects. By contrast, mutations that alter germline 

sex in fruit flies also cause tumor formation or cell death. Second, mutations in any of a number 

of upstream regulators could change the activity of fog-3 during development, and thus switch 

mating systems. In fact, the nature of these regulatory mechanisms might differ between C. 

elegans and C. briggsae. Third, once nematodes develop a male/hermaphroditic mating system, 

it might be more stable than in other species, because males will always be produced by 

non-disjunction, and their presence should prevent inbreeding depression.

70. Genome-wide RNAi screen to identify novel regulators of membrane trafficking. 

Zita Balklava, Barth D. Grant 

Department of Molecular Biology and Biochemistry, Rutgers University, 604 Allison Road, Nelson 

Biological Laboratories, Piscataway, NJ 08854 

Endocytosis is the vesicle-mediated cellular process by which membranes and extracellular 

macromolecules are internalized and sorted in the cell. Endocytic trafficking is essential to many 

aspects of eukaryotic life, including nutrient uptake, recycling of membranes and membrane 

proteins, antigen processing by the immune system, synaptic vesicle recycling by the nervous 

system, and growth factor receptor regulation during development. This work is focused on 

identifying all of the components required for membrane traffic in multicellular organisms. To 

study the mechanisms of membrane transport we have utilized an in vivo endocytosis assay, 

where we follow the trafficking of a yolk protein-green fluorescent protein chimera (YP170::GFP) 

in transgenic C. elegans strains. We have shown before that C. elegans yolk is endocytosed by 

receptors of the LDL receptor superfamily on the surface of growing oocytes, sorted in the 

endosomal system, and stored in vesicles. Since the basic components and pathways of 

endocytic trafficking are conserved between C. elegans and vertebrates, this system can be used 

to identify completely novel proteins and to test the endocytic functions of any gene of interest. 

We have combined RNAi with the YP170::GFP trafficking assay to systematically screen the 

whole C.elegans genome for all regulators of this membrane trafficking pathway. 

We have completed screening five of the six C. elegans chromosomes (>13,000 genes out of 

18,000 total) and have identified >600 candidate regulators of membrane traffic. Significant part 

of candidate genes we have identified so far encode novel proteins, or they encode proteins that 

have been previously studied in other contexts such as actin dynamics but had not been 

previously connected to the regulation of membrane traffic. We are currently developing 

secondary assays in C. elegans that will allow us to determine the specific steps in membrane 

traffic regulated by each new protein.

71. Molecular connections between developmental timing and circadian timing: The C. 

elegans homolog of the circadian gene doubletime regulates post-embryonic 

developmental timing 

Diya Banerjee, Alvin Kwok, Shin-Yi Lin, Frank Slack 

Dept. of Molecular, Cellular and Developmental Biology, Yale University, PO Box 208103, New 

Haven, CT 06520 

Spatial patterning during animal development is genetically controlled by genes such as the 

Hoxcluster. Similarly, the temporal aspect of developmental patterning is under the genetic 

control of heterochronic genes. The most extensively investigated heterochronic pathway defines 

a somatic clock that controls the timing of cell fate determination during C. elegans 

post-embryonic development. Although a number of key heterochronic genes have been 

identified, the molecular mechanisms that underlie temporal control of cell division and 

proliferation remain elusive. The heterochronic gene lin-42 is the C. elegans homolog of 

Drosophila and mammalian period, key regulators of circadian rhythms, which specify behavior 

and physiology over a 24 hour day/night cycle. lin-42 thus defines a molecular link between two 

different types of biological clocks: developmetal timing and circadian timing. In addition to lin-42, 

the C. elegans genome contains homologs of a number of core circadian clock genes. We have 

investigated the possible function of these homologs in control of developmental timing by using 

RNA interference (RNAi) to knock-down gene expression. We describe the identification and 

characterization of two genes that interact with the developmental clock: kin-20 (casein kinase 

Iε/CKIε) and kin-19 (casein kinase Iα/CKΙα), which are C. eleganshomologs of Drosophila 

doubletime. kin-19/CKΙαand kin-20/CKIε antagonistically regulate cell fate decisions during late 

larval development. kin-19 is downstream of and negatively regulated by kin-20, lin-42, lin-41 

and hbl-1, which either work together or in parallel. We have also found that the homologs to the 

Drosophila clock genes timeless, casein kinase IIα, protein phosphatase 2A, vrille, and pdp1 

appear to function in the developmental clock. Our results, showing function of multiple circadian 

gene homologs in the developmental clock, suggest that circadian and developmental timing 

pathways may utilize conserved mechanisms of temporal regulation. Our work also suggests that 

one or more of the heterochronic genes already identified, including CKΙα and microRNAs, may 

play a role in circadian timing. Furthermore, our findings point to circadian genes being 

regulators of cell fate timing, and support reports that circadian genes can function as oncogenes 

in mammals.

72. Towards cloning mutations isolated in a genetic screen for hyperactive egg-laying 

mutants 

I. Amy Bany, Michael R. Koelle 

Yale University School of Medicine 

Genetic studies in C. elegans indicate that neurotransmitter(s) signal through the neural 

Galpha(o) protein GOA-1 to inhibit egg laying, and this inhibition is opposed by signaling through 

the neural Galpha(q) protein EGL-30. To gain a more complete understanding of the mechanisms 

by which Galpha(o)/Galpha(q) signaling regulates egg-laying behavior, we have screened 39,000 

mutagenized haploid genomes for mutations that confer a hyperactive egg-laying phenotype 

similar to that caused by loss-of-function mutations in goa-1. We isolated 17 mutants that display 

a strongly hyperactive egg-laying phenotype, including mutations in the known G protein signaling 

genes eat-16 and dgk-1. We also identified one allele of the homeodomain transcription factor 

unc-4. UNC-4 is necessary for acetylcholine expression in the VC neurons, and acetylcholine 

from the VC neurons can inhibit egg-laying behavior 1 . The remaining eleven mutants appear to 

identify eleven different genes. Eight of these isolates belong to a new class of mutants that are 

hyperactive for egg laying but, unlike goa-1 mutants, are not hyperactive for locomotion. We are 

pursuing two of the mutations isolated in this screen: vs25 and vs33. 

The strongest mutation isolated in the screen was vs25. vs25 is semi-dominant: the 

homozygous mutants lay virtually all of their eggs at an early developmental stage and the 

heterozygotes lay approximately 50% early-stage eggs. vs25 is wild-type for locomotion and 

gross body morphology. Mutants hyperactive for only their egg-laying behavior have never been 

identified before this study, and might represent elements of the GOA-1 pathway that specifically 

regulate egg-laying behavior. Alternatively, these mutants might identify a new pathway that 

inhibits egg-laying behavior. vs25 has been mapped to a 200kb interval on the left arm of the X 

chromosome between the visible markers unc-10 and dpy-7, and transformation rescue 

experiments are ongoing. 

Another mutant identified in the screen, vs33, closely phenocopies goa-1 mutants. In addition 

to being strongly hyperactive for egg laying, vs33 mutants are hyperactive for locomotion; they 

initiate body bends and back up more frequently, and their body bends are deeper than those of 

wild type. vs33 animals also appear thin and pale, a body morphology typical of the previously 

known hyperactive mutants, presumably due to defects in eating behavior. The pleiotropies vs33 

shares with goa-1 mutants suggest that it represents a new component of the GOA-1 signaling 

pathway. We mapped vs33 to a 1.3 cM interval on the X chromosome, between the visible 

markers dpy-8 and unc-97. SNP mapping has begun to narrow the interval containing vs33. 

Studying the genes identified by these mutations should help us gain a more complete cellular 

and molecular picture of how neurotransmitters, acting through G proteins, regulate behavior. 

1 Bany, I.A., Dong, M-Q., Koelle, M.R. J Neurosci. 23, 8060-8069 (2003).

73. C. elegans HDACs, CBP, and CREB play roles in polyglutamine neurotoxicity 

Emily A. Bates 1 , Cindy Voisine 1 , Martin Victor 1 , Yang Shi 2 , Anne Hart 1 

1Massachusetts General Hospital, 149 13th St., Charlestown, MA 02129 

2Harvard Medical School, New Research Building, 854, 77Louis Pasteur Ave, Boston, MA 02115 

The expansion of a polyglutamine (polyQ) tract in the huntingtin protein causes neuronal death 

in Huntington’s disease, but the molecular basis of cell death is unknown. Transcriptional 

regulation via histone acetylase (HAT) activity and histone deacetylase (HDAC) activity is critical 

to many cellular processes. Some results from Drosophila and vertebrate systems suggest that 

expanded polyQ tracts may alter transcription by sequestering glutamine rich transcriptional 

regulatory proteins. CREB binding protein (CBP), a glutamine rich histone acetyltransferase, has 

been found in expanded polyQ aggregates in patient tissue and cell culture models. HDAC 

inhibitors reduce polyQ induced death in cell culture and Drosophila. This transcriptional 

hypothesis for polyQ toxicity suggests that 1) overexpression of CBP and CREB or loss of HDAC 

function will protect cells from polyQ-induced degeneration and 2) CBP (cbp-1) or CREB (crh-1) 

loss of function mutations will enhance polyQ-induced neurodegeneration. 

We are testing this hypothesis in a C. elegans model of polyQ toxicity. N-terminal fragments of 

human huntingtin with varying lengths of glutamine tracts are expressed in C. elegans ASH 

sensory neurons. At least 30% of the ASH neurons expressing mutant huntingtin fragment 

degenerate in 8 day old animals. Putative roles of crh-1 (Alkema & Horvitz), CBP, and HDAC in 

polyQ-induced neurodegeneration were examined using RNA interference and loss of function 

alleles. Deletion of CREB enhances polyQ toxicity in ASH neurons. Similarly, deletion of one copy 

of cbp-1 enhances polyQ toxicity. Loss of function alleles and RNA interference were used to 

systematically reduce function of each C. elegans HDAC in our model of polyQ toxicity. 

Knockdown of most CeHDACs enhanced polyglutamine toxicity; only one CeHDAC knockdown 

resulted in suppressed polyQ toxicity. Additional experiments are underway to determine if 

overexpression of these proteins modulates polyQ toxicity. These studies will further define the 

role of the CREB/CBP pathway in polyQ expansion diseases.

74. Study of the genetic and cellular bases of ventral nerve cord maintenance 

Claire Benard, Oliver Hobert 

Dept of Biochemistry and Molecular Biophysics, Center for Neurobiology and Behavior, Columbia 

University, 701 W., 168th St, HHSC 724, New York, NY 10032 

The correct wiring of the nervous system requires not only that axons navigate to their targets, 

but also that they maintain their correct position in axon fascicles after termination of axon 

outgrowth. Previous work in the laboratory uncovered the existence of a novel mechanism that 

ensures that axons maintain their correct positioning in the axonal tracts of the ventral nerve cord 

of C. elegans. Aurelio et al. (2002) showed that microsurgical removal of the neuron PVT in the 

first larval stage leads to the inability of embryonically generated axons to maintain their proper 

position within the ventral nerve cord. Aurelio et al. (2002) further discovered that the PVT neuron 

expresses the zig genes, which encode a family of proteins containing two immunoglobulin 

domains. Six family members are expressed in PVT, five of which show a precisely timed onset 

of expression in PVT at the first larval stage. In addition, the knockout of zig-4 leads to defects in 

the maintenance of axon positioning in the VNC (Aurelio et al., 2002). Also, abrogating the proper 

expression of the zig genes in PVT in lim-6 ceh-14 or in lin-14 mutants leads to ventral nerve cord 

maintenance defects (Aurelio et al., 2003). Thus, the role of PVT in maintaining proper axon 

positioning in the ventral nerve cord appears to be mediated by the temporally controlled 

secretion of 2-immunoglobulin domain proteins during the first larval stage. One attractive 

possibility is that ZIG-4 functions as an anti-adhesive between contralateral neurons. Additional 

work in the lab has recently implicated egl-15, which encodes the fibroblast growth factor 

receptor, in the maintenance of axons in the ventral midline (Bulow et al., in press). 

To further our understanding of the cellular and genetic bases of maintenance of axon 

positioning in the ventral nerve cord we are carrying out laser ablations of the neurons that flip 

over the midline in the absence of the zig- and egl-15-mediated maintenance. We expect that this 

analysis will reveal the type of cellular interactions that exist between the neurons that fail to 

maintain their position in the ventral nerve cord. We are also undertaking a screen for mutants 

defective in maintenance in the ventral nerve cord. Progress will be discussed. 

Aurelio O, Hall D H, Hobert O (2002). Science 295, 686. 

Aurelio O, Hobert O (2002). Development 130, 599. 

Bulow H, Boulin T, Hobert O (2004). Neuron, in press.

75. A Genome-Wide RNAi Screen for Components Affecting Sex Muscle Differentiation 

Daniel C. Bennett, Isaac E. Sasson, Michael J. Stern 

Department of Genetics, Yale University, New Haven, CT, USA 

FGF receptor signaling plays a role in many varied biological processes including cell viability, 

cell migration, and cell differentiation. The C. elegans FGF receptor is encoded by a single gene, 

EGL-15. The functions of EGL-15, like mammalian FGF receptors, are biologically diverse. The 

5A isoform of EGL-15 functions in the M lineage of the developing hermaphrodite to regulate 

migration of the sex muscle precursors, and can also play a role in the subsequent differentiation 

of the sex muscles. 

Hyperactivation of the C. elegans FGFR inhibits muscle differentiation. Our lab has generated 

a hyperactivated EGL-15(5A) isoform bearing the transmembrane region of oncogenic 

NEU/Her-2. This construct is known as egl-15(5A*). The egl-15(5A*) construct causes 

constitutive ligand-independent dimerization of the receptor and stimulation of downstream 

signaling. The sex muscle precursors in egl-15(5A*) animals fail to differentiate to functional sex 

muscles, and the animals are severely egg-laying defective. This failure of the sex muscles to 

differentiate can be visualized by the lack of muscle actin filaments in these cells and by 

disorganized expression of the vulval muscle-specific marker 16Nde::RFP (modified from Harfe 

and Fire, 1998 1 ). The downstream effectors of EGL-15 in the M-lineage and the mechanism by 

which egl-15(5A*) inhibits sex muscle differentiation remain unknown. 

To identify additional factors which influence sex muscle differentiation, we are looking for 

genes which, when compromised, can restore sex muscle differentiation in the presence of 

egl-15(5A*). To do this, we are utilizing the recently available genome-wide RNA interference 

library 2 to perform a suppressor screen. To simplify the screening process, we have generated a 

strain bearing an integrated array containing the vulval muscle-specific marker 16Nde::RFP, the 

egl-15(5A*) construct, and the marker of differentiated body wall muscles P myo-3::GFP-NLS. This 

strain allows rapid visual scoring of vulval muscle and body wall muscle differentiation in live 

animals using a dissecting microscope and appropriate fluorescent light filters. 

Sex muscle differentiation in C. elegans acts as a model for the differentiation of mammalian 

muscle cells in vitro. In mammalian muscle precursors, activation of the FGF receptor can inhibit 

muscle differentiation, whereas the PI3-kinase pathway is thought to promote muscle 

differentiation. A candidate gene approach has already shown that activation of the C. elegans 

PI3-kinase signaling pathway can partially suppress muscle differentiation in the presence of 

egl-15(5A*). Thus, sex muscle differentiation in C. elegans is regulated by pathways similar to 

those that affect mammalian sex muscle differentiation. The suppressors of egl-15(5A*)are 

expected to include both the components of the egl-15(5A*) pathway responsible for inhibiting 

muscle differentiation, and signaling components in other myogenic pathways. Candidate 

pathways are those involved in muscle differentiation in mammalian systems, including the p38 

MAP kinase and p70S6-kinase pathways. 

Suppressors will be tested for potential roles in EGL-15 signaling with respect to sex myoblast 

migration, sex muscle differentiation, and other FGF signaling-dependent processes. 

Suppressors will also be tested for roles in regulating the differentiation of other muscle types in 

C. elegans. Thus, this project provides an opportunity to study both FGF signaling in the M 

lineage and the process of muscle differentiation in general. 

1. Harfe, BD., and Fire, A. 1998 Development 125: 421-429. 

2. Kamath RS., et al. 2003 Nature. 421: 231-237.

76. A Genetic Screen for New Genes Involved in Aging 

Ala Berdichevsky 1 , Leonard Guarente 2 , Bob Horvitz 1 

1HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA 

2Dept. Biology, MIT, Cambridge, MA 02139, USA 

Over the last decade, studies of C. elegans and other organisms have resulted in progress 

towards understanding molecular mechanisms of aging. Mutations in genes involved in the 

insulin-like pathway or in mitochondrial function significantly affect C. elegans lifespan. Research 

in the field has largely focused on genes that normally function to reduce longevity, that is, genes 

that when mutated extend lifespan. This focus is primarily a consequence of the difficulties in 

distinguishing accelerated aging from sickness in short-lived mutants. Recently, Garigan et al.(1) 

and Herndon et al.(2) described characteristics of aging C. elegans, including decline in 

locomotion, muscle deterioration, and accumulation of autofluorescence in the intestine of aging 

animals. Such observations help distinguish aged from sick worms and therefore facilitate the 

study of the aging process in C. elegans. 

We are interested in identifying and studying genes that function to prevent or delay the aging 

of C. elegans. Loss-of-function mutations of such genes should result in accelerated aging, 

similar to human progeria. We are performing a genetic screen for mutants that age prematurely, 

using accumulation of intestinal autofluorescence as a marker. 

The accumulation of a lipofuscin-like fluorescent pigment in the gut of old worms has long been 

known to be a marker of aging. The amount of pigment is consistent among wild-type animals of 

the same age and gradually increases throughout adulthood. We measured the spectrum of the 

autofluorescence of old worms and found that emission peaks at 420 nm upon excitation at 350 

nm. We are using a filter set with similar excitation and emission values to visualize lipofuscin and 

to screen for mutants showing early accumulation of this pigment. 

So far we have screened worms corresponding to 20,000 mutagenized haploid genomes and 

identified 12 isolates that exhibit premature accumulation of gut autofluorescence. Eight of these 

isolates have significantly shorter lifespans than the wild type. We are currently mapping the 

mutations and characterizing the mutants with respect to other aging-related characteristics, such 

as behavioral decline and tissue deterioration. 

1. Garigan, Hsu, Fraser, Kamath, Ahringer and Kenyon, Genetics 161: 1101-1112 (2002). 

2. Herndon, Schmeissner, Dudaronek, Brown, Listner, Sakano, Paupard, Hall and Driscoll, 

Nature 419: 808-814 (2002).

77. Towards understanding the role of the stomatin-like protein UNC-24 in the process of 

gentle touch sensation: evidence for functional interaction with the mechanosensory ion 

channel complex MEC-4/MEC-10 

Laura Bianchi 1 , Wei-Hsiang Lee 1 , Dan Slone 1 , Julie Y. Koh 2 , David M. Miller III 2 , Monica 

Driscoll 1 

1Dept. of Mol. Biol. and Biochem., Rutgers University, Piscataway, NJ 

2Dept. of Cell. and Developmental Biology, Vanderbilt University Med. Ctr., Nashville, TN 

unc-24 encodes a 415 aa long protein endowed with a stomatin-like domain and a lipid transfer 

domain. unc-24 is expressed in many C. elegans neurons including ventral cord neurons and 

touch neurons, and unc-24 mutants are forward Uncs, indicating that UNC-24 is required for 

normal function of the forward movement motor circuit. Recent work has shown that in ventral 

cord neurons UNC-24 is required for stability and postraslational processing of UNC-1, another 

stomatin-like protein (Sedensky et al., 2001 and 2004). In addition UNC-24 was shown to be 

required for localization of UNC-1 in specialized membrane microdomains called lipid rafts. Since 

genetic as well as biochemical experiments have detected interactions between UNC-1 and 

UNC-8, another DEG/ENaC ion channel subunit, UNC-24 may be important for the processing 

and/or stability of DEG/ENaC ion channel complexes. 

In touch neurons, the DEG/ENaC Na + channel subunits MEC-4 and MEC-10 form a 

heteromeric channel postulated to constitute the core of a mechanosensory complex. The 

membrane-anchored stomatin-like protein MEC-2 is required for MEC-4/MEC-10 channel 

function, but it does not influence MEC-4 cellular localization (Goodman et al., 2002). Based on 

previous work as well as our observation that UNC-24 is expressed in touch neurons, we 

postulated that it may be important for cellular processing and/or function of the mechanosensory 

ion channel complex. 

When we assayed unc-24(0) mutants sensitivity to gentle touch stimulation, we found that they 

are strikingly touch insensitive at the L1 stage, but recover to full touch sensitivity by L2/L3. In the 

attempt to determine the mechanism by which UNC-24 affects sensitivity to gentle touch, we 

assayed cellular localization of MEC::GFP proteins in unc-24(0) mutant nematodes as well as 

cellular localization of UNC-24::GFP in mec mutant worms, during development. We found that 

while MEC-10::GFP localization was defective in unc-24(0) adult worms but not in L1, 

UNC-24::GFP was mislocalized in L1 as well as in adult mec-2(0) worms, suggesting physical 

and/or functional interaction between UNC-24 and MEC-2 and implying a possible role of UNC-24 

in controlling the channel processing or function. To determine if UNC-24 affected channel 

function, we co-expressed it in Xenopusoocytes with the MEC channel complex and found an 

effect on current amplitude that was time dependent. Our results propose a developmentally 

regulated role of UNC-24 in controlling the function of the mechanosensory ion channel complex.

78. A germline-specific RNA-binding protein required for germ cell survival and 

cytokinesis 

Peter R Boag 1,2 , T. Keith Blackwell 1,2 

1Joslin Center for Diabetes, One Joslin Place, Boston, MA 02215 

2Department of Pathology, Harvard medical School 

During oogenesis in C. elegans approximately half of the developing germ cells undergo 

apoptosis at the pachytene stage of meiosis I. This process has been termed physiological 

apoptosis and is genetically distinct from both somatic and genotoxic programmed cell death, 

although the core apoptotic components ced-3, ced-4 and ced-9 are shared. The observation 

that the apoptotic germ cells ÒdonateÓ their cytoplasm to the germline syncytium suggest they 

may be acting as nurse cells (Gumienny, et al., Development 126, 1011-22,1999). The 

mechanism through which physiological cell death is initiated is unclear, however the requirement 

of the conserved germline helicase CGH-1 for oocyte survival suggests RNA metabolism could 

be involved (Navarro, et al., Development 128, 3221-32, 2001). In Drosophila, clam and 

Xenopus the CGH-1 orthologs are associated with maternal RNAs and their translational control, 

suggesting that a similar mechanism may function in C. elegans. 

To determine if other RNA interacting proteins are associated with physiological germ cell 

death, we used RNAi to test selected germline enriched RNA-binding proteins. We identified the 

C. elegans homolog of a small family of conserved RNA-binding proteins, which included 

oocyte-specific proteins from amphibians. The 340 amino acid germline-specific protein contains 

three conserved regions, including a RNA-binding domain (RGG box) at the carboxyl terminus. 

We have named this gene rsc-1 (RNA binding, germ cell survival and cytokinesis). 

Immunostaining localised RSC-1 to the germline syncytium and P granules, and to the early 

embryonic germline precursor cells. Low level staining was also detected in early somatic 

lineages and were lost in a pattern reminiscent of class II RNAs. Co-immunostaining with RSC-1and 

CGH-1-specific antibodies indicates that the two proteins have overlapping localisation in 

both the germline and embryo. RSC-1 and CGH-1 co-immunoprecipitated from adult 

hermaphrodite protein lysates, however if the lysate was pre-treated with RNAse A this was 

abolished. This indicates the two proteins are part of a germline ribonucleoprotein complex 

(RNP). 

An elevated level of ced-3 and ced-9(1950gf) dependent, but P53 independent death was 

evident in rsc-1(RNAi) hermaphrodites, indicating that germ cell death was regulated by the 

physiological apoptosis pathway. Interestingly, while cgh-1 RNAi resulted in increased 

physiological germ cell death and sterility, rsc-1(RNAi) hermaphrodites were fertile, although 

maternal affect lethal. rsc-1(RNAi) animals also displayed germline and embryonic cytokinesis 

defects. Small spherical bodies (SSB) accumulated in the proximal gonad in rsc-1(RNAi) 

hermaphrodites. These bodies first appear immediately after the loop region, contained the yolk 

receptor protein RME-2, but no DNA or GLD-1, the translational repressor of rme-2. Formation of 

the SSB was independent of apoptosis and required germ cell exit from pachytene. Taken 

together, this suggests that SSBs arise through defects in oocyte formation as the germ cell exit 

pachytene. Embryonic cytokinesis failed and resulted in multinucleated embryos. Embryos 

displayed defects in pronuclei fusion and failed cleavage furrow closure. 

Our data indicates rsc-1 is required for cytokinesis in the germline and embryo, and for normal 

regulation of physiological apoptosis. The RSC-1/CGH-1 RNP complex provides a link between 

germline RNA dynamics, celluarization and physiological cell death, and is consistent with the 

model that this apoptosis contributes to germline homeostasis.

79. Identification of C. elegans spindle assembly checkpoint components 

Mike Boxem, Marc Vidal 

Dana Farber Cancer Institute and Department of Genetics, Harvard Medical School, 44 Binney 

Street, Boston, MA 02115, USA. 

The spindle assembly checkpoint is an essential mechanism to prevent cells from exiting 

mitosis until all chromosomes have attached to both spindle poles and are properly aligned at the 

metaphase plate. We are combining high-throughput yeast two-hybrid (HT-Y2H) screens with 

reverse genetic approaches to study the spindle assembly checkpoint in the metazoan 

Caenorhabditis elegans. 

Our laboratory recently mapped a large fraction of C. elegans protein interactions by HT-Y2H * . 

The overall quality of this Y2H dataset was validated experimentally by independent coaffinity 

purification assays. Together with already described interactions and interactions from other 

species potentially conserved in C. elegans (interologs), the current Worm Interactome map 

(WI5) contains ~5,500 interactions. 

From WI5, smaller subnetworks, or "modules", can be derived which represent particular 

biological processes. The subnetwork surrounding the C. elegans homologs of the known spindle 

assembly checkpoint components Mad1-3p, Bub1-3p, Rod, and ZW10 consists of 62 interactions. 

Of these, 53 are novel interactions, and 9 are interologs or previously described interactions. 

Following the "guilt by association" reasoning, the 57 proteins involved in these interactions have 

a relatively high probability of being involved in some aspect of the spindle assembly checkpoint. 

We are using RNA interference in conjunction with a number of assays to identify checkpoint 

functions for the corresponding genes. By studying a limited set of candidate genes in detail, we 

may uncover gene functions that would be difficult to identify in genome wide genetic or RNAi 

screens, for example because they have a partially penetrant phenotype, or because they are 

partially redundant with another gene. 

* Li, S. et al. (2004). A map of the interactome network of the metazoan C. elegans. Science 

303, 540-3.

80. Development of high-throughput sublehtal toxicity tests using Caenorhabditis elegans 

Windy A. Boyd, Sandra J. McBride, Julie R. Rice, Jonathan H. Freedman 

Nicholas School of the Environment and Earth Sciences, Duke University, Durham, NC 27708 

The National Toxicology Program (NTP), a division of the Department of Health and Human 

Services, is responsible for the development of sound scientific tests designed to estimate the 

effects of chemicals on human health. Recently, the NTP and other regulatory agencies have 

recognized the need for alternative toxicological methods and models to decrease the time and 

expense associated with the current testing protocols. The numerous advantages of using 

Caenorhabditis elegans as a model organism have been well documented in the fields of 

developmental and molecular biology and genetics. Advantages such as short life cycles, easy 

and inexpensive maintenance and culture, and detailed biological knowledge have led to a rise in 

the use of C. elegans as a toxicity testing organism. This has allowed for the development of 

rapid, low-cost toxicity tests that readily lend themselves to mechanistic studies of toxicant 

actions in a multi-cellular organism. Through collaboration with the NTP, our lab is developing the 

infrastructure to examine at least 200 potential neurological and developmental toxicants using C. 

elegans. To this point, we have developed automated systems to measure sublethal toxicity 

endpoints including growth, reproduction, movement, and feeding. These automated systems 

include two robotic laboratory workstations for liquid handling and pipetting; a Complex Object 

Parametric Analyzer and Sorter (COPAS) BIOSORT, for dispensing and analyzing nematode 

length and fluorescence; and an imaging workstation, which includes a computer-controlled 

microscope interfaced with a CCD camera for motion tracking and multidimensional image 

analysis. To increase the rate and efficiency of chemical screens, a 96-well plate format is being 

used for dispensing of test organisms, and quantification of specific toxicological endpoints. As C. 

elegans are dispensed into wells, the time of flight (TOF), extinction (EXT), and green and red 

fluorescence are measured for each nematode, and data are automatically recorded for later 

analysis. In a typical toxicological test, gravid adult C. elegans are bleached, and then the eggs 

are either transferred directly to plates seeded with OP50 E. coli or allowed to hatch overnight in 

M9 buffer. C. elegans at specific developmental stages are used for each test: L1s for 72-h 

growth, L4s for 48-h reproduction, and 3-day-old adults for 4-h or 24-h movement and feeding 

assays. To determine the effects of various concentrations of toxicant on growth, reproduction, or 

feeding, the characteristics of the nematode population in each well are measured using the 

COPAS BIOSORT following toxicant exposure. To monitor feeding, immediately after toxicant 

exposure, red fluorescent microspheres (0.5 µm) are added to the wells. To assess the effects of 

toxicants on C. elegans movement, a series of images of toxicant-exposed nematodes are 

collected and then analyzed for changes in distance traveled and velocity, using commercially 

available motion tracking software. For each test, the effective concentration that results in a 50% 

reduction in response relative to controls (EC50) is then calculated. Four chemicals (cadmium 

chloride, acetaminophen, ferric citrate, and 1-methyl-3-nitro-1-nitroso-guanidine) are currently 

being used as preliminary test chemicals for development of the screens. All of the toxicity tests 

have been completed with cadmium chloride, and these data will be presented to illustrate the 

technology.

81. Using enriched populations of single neuron types and microarrays to identify sensory 


Marc Colosimo 1,2 , Adam Brown 1 , Anne Lanjuin 1 , Saikat Mukhopadhyay 1 , Piali Sengupta 1 

1Brandeis University, 415 South St., Waltham, MA 02454 

2Current address: MITRE, 202 Burlington Road, Bedford, MA 01730 

To gain a better understanding of the gene sets that are expressed in and required for the 

functions of specific sensory neuron types, our lab utilized a previously described C. elegans 

primary cell culture technique (1) coupled with FACS sorting to obtain enriched collections of 

single neuron types. We extracted RNA from these populations and hybridized to the C. elegans 

Affymetrix Genechips to compare the gene expression profiles of the aversive olfactory neuron 

type, AWB, and the primary thermosensory neuron type, AFD. We identified a list of 

approximately 250 genes with at least a 2 fold change in one neuron type as compared to the 

other. The expression predictions were further validated via real-time PCR and in vivo expression 

analyses using gfp fusion constructs. Expression patterns of several genes were confirmed by 

green fluorescent protein (gfp) fusion constructs. Genes analyzed represent a diverse array of 

gene families encoding sensory receptors, kinases, and transcription factors. 

We have further characterized a subset of genes identified in these experiments (also see 

abstract by Brown et al). In particular, we have shown that dac-1, the C. elegans dachshund 

homolog, is expressed in AFD, as well as weakly in the neurons ASK, ASE, and AWC. 

dachshund belongs to the SKI/SNO family of proteins, and encodes a nuclear factor involved in 

the development of several organ types, including the Drosophila eye. Interestingly, a dac-1 

knockout allele, dac-1(gk211), exhibits a cryophilic phenotype when placed on a linear 

temperature gradient, similar to ttx-1, an Otd/otx transcription factor required for AFD 

development. This implicates dac-1 in the development and/or function of the AFD 

thermosensory neurons. 

These experiments validate the use of cell culture and microarrays in C. elegans to identify new 

genes required for development and function of specific neuronal subtypes. Current experiments 

are aimed at further analyzing the functions of genes identified by these experiments, and 

application of this technology to identify genes expressed in additional sensory neuron types. 

1.) Christensen M, Estevez A, Yin X, Fox R, Morrison R, McDonnell M, Gleason C, Miller DM 

3rd, Strange K. (2002). A primary culture system for functional analysis of C. elegans neurons 

and muscle cells. Neuron 33, 503-14.

82. Modulations of thermotactic behavior by food 

Adam Brown 1 , Damon Clark 2 , Chris Gabel 2 , Steven Lin 2 , Ares Perides 2 , Piali Sengupta 1 , 

Aravi Samuel 2 

1Biology Department, Brandeis University 

2Physics Department, Harvard University 

When navigating at temperatures above their cultivation temperature, worms move down 

spatial thermal gradients. However, at or near their cultivation temperature, worms track 

isotherms. We have shown that while the mechanism for isothermal tracking works both in the 

presence and absence of food, migration down gradients is suppressed in the presence of food. 

Therefore, food appears to specifically inhibit only one of the two thermotactic behavioral modes. 

Since the positions of isothermal tracks reflect the memory of cultivation temperature, this 

provides us with a simple assay to measure thermal memory. In this assay, we impose a linear 

thermal gradient from 15C-25C on a plate containing a population of worms on a bacterial lawn, 

calculate the trajectories of crawling worms using video microscopy and particle tracking, and 

score the positions of isothermal tracks. We have used this memory assay to determine the rates 

and characteristics of learning in normal and mutant worms. N2 worms originally cultivated at 15C 

and shifted as adults to 25C will adjust the locations of their isothermal tracks to reflect the new 

temperature in ~3 hours. However, animals originally cultivated at 25C and shifted to 15C require 

~4-6 hours to ’learn’ the new temperature. Using enriched populations of AFD neurons and 

microarrays, we identified the diacylglycerol kinase gene dgk-3 which is expressed strongly in the 

AFD neuron and more weakly in additional sensory neuron types. dgk-3 mutants exhibit wild-type 

behaviors on spatial and temporal thermal gradients. Interestingly, while dgk-3 mutant adults 

shifted from 25C to 15C learn the new temperature at the same rate as wild-type animals (~4-6 

hours), they require ~10-12 hours to learn the new temperature when shifted from 15C to 25C. 

These data suggest that the pathways required for resetting thermal memory upon temperature 

upshifts and downshifts may be distinct, and that dgk-3 might play a specific role in memory 

formation or resetting upon upshifts. We are exploring the cellular basis of the memory formation 

and investigating the role of DGK-3 mediated pathways in thermotactic behavioral plasticity.

83. A Systematic Genome-Wide Genetic Interaction Analysis of Signaling Pathways in C. 

elegans 

Alexandra Byrne, Scott J. Dixon, Jason Moffat, Peter J. Roy 

Dept. of Molecular and Medical Genetics, University of Toronto, Toronto, Canada 

We are systematically creating a genome-wide network of genetic interactions in C. elegans 

focused on signaling pathways of universal importance to animal development and pathogenesis. 

In Saccharomyces cerevisiae, Synthetic Genetic Array (SGA) analysis relies on the principle that 

if two genes function redundantly in an essential process, disrupting both in the same cell will 

result in a new (synthetic) lethal phenotype 1 . While there is no comprehensive library of C. 

elegans deletion mutants, we can systematically disrupt gene function by RNA-interference 

(RNAi) in the background of hypomorphic mutations and search for synthetic genetic interactions. 

By screening viable hypomorphic mutants whose null phenotypes are lethal, we will identify 

interactions as a result of genetic enhancement as well as synthetic genetic interactions. In 

deference to yeast SGA analysis, we call our approach "worm Systematic Synthetic Genetic 

Analysis" (worm SSGA). We are focusing on seven conserved signaling pathways, namely the 

Insulin, EGF, FGF, Ros, Notch, Wingless and TGF-beta signaling pathways. We are also 

including two components of the DNA-damage response (DDR) pathway to serve as a global 

control, since this pathway is expected to have minimal links to the other pathways. We have 

begun to build the network of genetic interactions amongst genes that are known or predicted to 

function in the aforementioned signaling pathways. In doing so, we obtained variable success 

with positive controls for six of seven pathways: Insulin (100%), EGF (56%), FGF (63%), Ros 

(100%), Notch (30%), and Wingless (18%). No positive controls were available for the TGF-beta 

pathway since analyses of single and double mutants have not revealed any embryonic or 

essential functions 2 . We have also uncovered previously unknown links between well 

characterized pathways. Our hope is that SSGA will define new function for many 

uncharacterized genes and reveal several new interactions between classic signaling pathways 

important in animal development and pathogenesis. 

1 Tong, A. H. et al. Systematic genetic analysis with ordered arrays of yeast deletion mutants. 

Science 294, 2364-8 (2001). 2 Patterson, G. I. & Padgett, R. W. TGF beta-related pathways. 

Roles in Caenorhabditis elegans development. Trends Genet 16, 27-33 (2000).

84. Temporal regulation of postmitotic neural differentiation by heterochronic genes 

including the microRNA lin-4 

Katherine O. Carter 1 , Kristy Reinert 1 , Shin-Yi Lin 2 , Frank Slack 3 

1 Yale University, Department of Molecular, Cellular and Developmental Biology, KBT 938, PO 

Box 208103, New Haven CT 06520 

2 Princeton University, Department of Molecular Biology, Washington Road, Princeton, NJ 

08544-1014 

3 Yale University, Department of Molecular, Cellular and Developmental Biology, KBT 936, PO 

Box 208103, New Haven CT 06520 

In the two hermaphrodite specific neurons (HSNs) of C. elegans, axon outgrowth and serotonin 

synthesis occur at fixed times during larval development, but the mechanisms underlying their 

temporal regulation are largely unknown. Genes that control developmental timing in hypodermal 

tissues have been identified and ordered into a dedicated pathway for temporal control, the 

heterochronic pathway. One of these genes, lin-14, has been shown to regulate timing of 

postmitotic development in the DD neuron subtype, suggesting that heterochronic genes could 

also control temporal patterning in neural tissues. In order to test this possibility, timing of axon 

outgrowth and initiation of serotonin synthesis in the HSNs were scored using integrated 

unc-86::myr-GFP and tph-1::GFP markers in heterochronic mutant backgrounds. Of the nine 

heterochronic genes tested, four have caused significant defects in timing of HSN differentiation, 

including the microRNA lin-4 as well as hbl-1, lin-29, and lin-28. lin-4 mutants display retarded 

HSN differentiation, while hbl-1, lin-29, and lin-28 result in partially penetrant precocious 

phenotypes. The lin-4 defects are partially rescued by hbl-1 RNAi knockdown, suggesting that 

hbl-1 acts downstream of lin-4. Since both lin-4 and hbl-1 are expressed in the HSNs and normal 

hbl-1 down-regulation requires its 3’UTR, this interaction could be direct. Based on these data, it 

appears that the hypodermis and HSNs utilize some but not all of the same heterochronic genes 

to regulate timing of developmental events, suggesting that temporal patterning may be controlled 

by distinct tissue-specific mechanisms.

85. Identification of genes acting redundantly with lin-35 Rb 

Julian Ceron 1 , Abha Chandra 1 , Khursheed Wani 1 , Jean-Francois Rual 2 , Marc Vidal 2 , Sander 

van den Heuvel 1 

1MGH Cancer Center and Department of Pathology, Harvard Medical School, Charlestown, MA 

02129,USA 

2Dana-Faber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, MA 

02115, USA 

lin-35 encodes the single C. elegans representative of the Retinoblastoma (Rb) protein family, 

which consist of three members in mammals (pRb, p107, p130). Rb has been shown to act in the 

transcriptional repression of genes with roles in cell cycle control, differentiation and apoptosis. 

Importantly, components of the "Rb pathway" are mutated or deregulated in most human 

cancers. 

Although lin-35 loss of function results in viable animals that predominantly show a reduced 

brood size, there are additional defects that become apparent when other genes are inactivated. 

For instance, a multivulva phenotype is observed when lin-35 loss of function is combined with 

inactivation of genes of a second class. Therefore, lin-35 functions as a SynMuv (synthetic 

multivulva) class B gene to repress vulval cell fates redundantly with SynMuv class A genes. 

More recently, redundant functions of lin-35 in cell-cycle control and pharyngeal morphogenesis 

have also been discovered (Boxem M and van den Heuvel S, 2001; Fay DS et al, 2003). In order 

to find additional genes that genetically interact with lin-35, we are carrying out novel forward and 

reverse genetic screens for phenotypes that are synthetic, suppressed or enhanced in the context 

of lin-35 loss of function. In these screens, we may identify genes that are required for cell 

survival in the absence of lin-35 function, revealing potential targets for anti-cancer therapeutics. 

In addition, genes may be found that act to regulate lin-35 function, or that act in parallel to lin-35. 

In the forward genetic screen of ~2850 haploid genomes, we isolated 20 mutants that showed 

interaction with lin-35 Rb. These included 6 SynMuv class A mutants, which validates the screen. 

For the reverse genetic approach, we have screened more than 10,000 RNAi feeding clones of 

the ORFeome RNAi library. This RNAi screen has identified a large number of genes, most of 

which display a feeding RNAi phenotype in lin-35 mutants but not in wild-type animals. We are 

using different genetic backgrounds and other gene inactivation methods in secondary screens to 

further select true candidates. Encouragingly, cki-1 (known to interact genetically with lin-35) has 

already been identified in the reverse screen.

86. Functional analysis of AMPA-type glutamate receptor tail sequences in C. elegans. 

Howard Chang, Chris Rongo 

Waksman Institute, Rutgers University, Piscataway, NJ 

The modulation of AMPA-type glutamate receptor localization to central nervous system 

synapses is an important component of synaptic plasticity, and can be triggered and regulated by 

LTP (Long-Term Potentiation) and CaMKII (Type II calcium-calmodulin-dependent protein kinase) 

activity. In mammalian hippocampal neurons, the different AMPA receptor subunits GluR1, GluR2 

and GluR3 are proposed to form heterotetramers, and individual subunits can confer localization 

specificity to the tetrameric channels that they comprise. One likely explanation for such subunit 

specificity is that the targeting of AMPA receptors to the synapses is probably due to the 

interaction of PDZ domain-containing proteins and the receptor subunit cytosolic tail sequences. 

In C. elegans, there are four AMPA/kainate-like receptor subunits that are expressed in the 

command interneurons, including, GLR-1, GLR-2, GLR-4 and GLR-5. GLR-1 has C-terminal 

sequences that contributes to channel turnover. We have identified that the GLR-2 subunit 

cytosolic tail domain appears to be instructive for channel localization. We will perform a 

yeast-two-hybrid screen using GLR-2 tail as a bait. Hopefully, we can identify the direct binding 

partner of AMPA-type glutamate receptors in C. elegans. These results should increase our 

understanding of the localization and regulation of AMPA-type glutamate receptors in vivo.

87. Feeding status and serotonin modulate a chemosensory circuit in C. elegans 

Michael Y. Chao 1,2 , Hidetoshi Komatsu 1,3 , Hana S. Fukuto 1,2 , Heather M. Dionne 1 , Anne C. 

Hart 1,2 

1Massachusetts General Hospital Center for Cancer Research, 149-7202 13th Street, 

Charlestown, MA 02129 

2Dept. of Pathlogy, Harvard Medical School, Boston, MA 

3Current address: Takeda Chemical Industries Ltd., Ibaraki 300-4293, Japan 

C. elegans responds to the odorant octanol by initiating backward movement. This behavioral 

response is modulated by food. Response to octanol is concentration dependent, and animals 

are consistently more responsive on food than off food. The biogenic amine serotonin (5-HT) is 

thought to signal the presence of food in C. elegans, in part because many behaviors that are 

modulated by food are also modulated by 5-HT. We found that tph-1 animals, which lack 5-HT, 

are normal in their response to undiluted octanol but fail to respond to diluted octanol both on and 

off food. Response is restored by exogenous 5-HT. Consistent with a role for 5-HT in modulating 

the sensitivity of the behavioral response, mod-5 animals, which are defective for a 5-HT 

selective reuptake transporter and consequently have increased 5-HT signaling, are 

hypersensitive to octanol. 

We have previously found that three pairs of amphid neurons, ASH, ADL, and AWB, contribute 

to octanol detection. ASH neurons are primarily responsible for octanol detection on food, while 

all three neurons contribute off food. These experiments addressed the role of 5-HT on a neural 

circuit at large, but did not address whether the focus of 5-HT activity is pre- or post-synaptic. 

Behavioral (i.e., reversals) and physiological (i.e., Ca 2+ influx; Hilliard and Schafer, submitted) 

response to nose touch, an ASH mediated behavior, depended on the presence of food or 5-HT, 

suggesting that 5-HT may be acting pre-synaptically. However, mutants that are defective for 

postsynaptic glutamate gated cation channels (glr-1, glr-2, and nmr-1) all had varying degrees of 

octanol response defects, suggesting that the postsynaptic command interneurons may also be a 

target for 5-HT modulation. 

A genetic screen is planned in which mutants that are hypersensitive to octanol are isolated. 

This approach should lead to the identification of genes that play a role in 5-HT signal 

transduction (ala mod-5), as well as general negative regulators of sensory transduction such as 

tax-6.

88. D1- and D2-like dopamine receptors antagonistically modulate C. elegans behavior 

through Galphaq and Galphao signaling 

Daniel Chase, Judy Pepper, Michael Koelle 

Yale University School of Medicine 

Defects in signaling by the neurotransmitter dopamine underlie schizophrenia and drug 

addiction and loss of dopamine signaling causes Parkinson’s disease. Dopamine controls the 

activity of neurons by acting through two classes of G protein-coupled receptors known as D1-like 

and D2-like. Activation of D1- and D2-like receptors have opposing effects on behavior, but the 

signaling mechanisms underlying this antagonism remain debated. We analyzed the mechanisms 

of dopamine signaling genetically in C. elegans. Knocking out a D2-like receptor, DOP-3, caused 

behavioral defects similar to those observed in animals lacking dopamine. Knocking out a D1-like 

receptor, DOP-1, reversed the defects seen in the DOP-3 knockout. Thus in C. elegans, as in 

mammals, D1- and D2-like signaling antagonize each other to control behavior. We identified the 

physiological signaling pathways responsible for this antagonism in C. elegans using a genetic 

screen for mutants unable to respond to dopamine. The screen identified four genes that encode 

components of the antagonistic Gα o and Gα q signaling pathways, including Gα o itself and two 

subunits of the RGS complex that inhibits Gα q. Thus dopamine regulates behavior in C. elegans 

through D1- and D2-like receptors that activate the antagonistic Gα q and Gα o signaling 

pathways, respectively. While the antagonistic Gα q and Gα o signaling pathways have been 

well-characterized in C. elegans this mechanism has not been previously implicated in dopamine 

signaling. Each of the signaling components identified in C. elegansis conserved in mammals and 

expressed in the brain. If the mechanism of Gα q and Gα o signaling is also conserved from C. 

elegans to mammals, signaling by D1-like receptors through Gα q and D2-like receptors through 

Gα o could explain many of the observed antagonistic effects of these receptors in mammals.

89. Germline establishment and maintenance in the early embryo. 

Paula M. Checchi 1,2 , Christine E. Schaner 1,2 , William G. Kelly 1 

1Biology, Emory University, Atlanta, GA 30306 

2Program in Biochemistry, Cell and Developmental Biology 

In C. elegans, embryogenesis is orchestrated by a well-characterized series of cellular 

divisions, resulting in both a somatic lineage as well as the germline, which will later give rise to 

the eggs and sperm. In the early embryo, a number of protective mechanisms exist to shield 

germline blastomeres from inductive signals that specify the fates of somatic cells. Specifically, 

germ cells are kept transcriptionally quiescent by the activity of the maternally loaded CCCH 

protein PIE-1. However, upon the birth of the germ cell precursors Z2 and Z3, PIE-1 disappears. 

The mechanisms regulating the spatial and temporal expression of PIE-1 are incompletely 

understood. In the early embryo, the Seydoux lab has demonstrated that PIE-1 is degraded from 

the somatic blastomeres by the zinc finger protein ZIF-1. While the factors mediating PIE-1 

degradation in Z2/Z3 have not yet been identified, data from our lab suggest that ZIF-1 may also 

play a role in degrading PIE-1 from the germ cell precursors. In addition, we have previously 

demonstrated that there is a chromatin based repressive mechanism that succeeds PIE-1 

degradation, effectively marking and maintaining the germ lineage. The germ cell precursors 

Z2/Z3 are the only cells that lose the histone modification H3 K4 dimethylation, a conserved 

marker for transcriptionally competent chromatin. The loss of this modification temporally 

coincides with the disappearance of PIE-1 in Z2/Z3, but it is not yet known whether there is a 

mechanistic link between PIE-1 degradation and chromatin remodeling. In zif-1(RNAi) embryos, 

PIE-1 is abnormally stabilized in Z2/Z3 and H3 K4 dimethylation is inappropriately retained, 

suggesting that failure to degrade PIE-1 may prevent chromatin remodeling in the germ cell 

precursors. Furthermore, we have demonstrated that two temperature sensitive embryonic arrest 

mutants emb-4(hc60) and mal-2(qm31) have aberrant patterns of both PIE-1 expression and H3 

K4 dimethylation, both of which abnormally persist in Z2/Z3. Using polymorphisms, we have 

physically mapped these loci to a small interval on LGV, and we are currently determining the 

molecular identity of these mutants and their potential role in regulating loss of PIE-1 and/or the 

onset of chromatin remodeling at germline restriction.

90. GUM-1, a protein affecting the subcellular localization of RME-1, is required for 

endocytic recycling in the C. elegans intestine 

Carlos Chih-Hsiung Chen 1 , Peter Schweinsberg 1 , Eric Lambie 2 , Barth Grant 1 

1Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854 

2Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 

The endocytic pathway of eukaryotes is essential for the internalization and trafficking of 

macromolecules, fluid, membranes, and membrane proteins. One of the most enigmatic aspects 

of this process is endocytic recycling, the return of macromolecules (often receptors) and fluid 

from endosomes to the plasma membrane. A mechanistic understanding of this critical transport 

step will require the identification of the proteins that regulate it. C. elegans genetics has allowed 

us to identify several new factors that function in this portion of the endocytic pathway. 

We have shown that a new receptor-mediated endocytosis (rme) gene identified in our genetic 

screens, rme-1, is a critical regulator of endocytic recycling in worms and mammalian systems. 

RME-1 protein is normally localized to the limiting membrane of endosomes in many cell-types. 

rme-1 mutants display endocytic defects in several tissues including strongly reduced uptake of 

yolk proteins by oocytes, due to poor recycling of yolk receptors, and the formation of gigantic 

endosomes in the basolateral intestine, due to defective recycling of pseudocoelomic fluid. 

Recently we found another mutant, gum-1,that displays rme-1-like intestinal morphology. 

Furthermore in gum-1 mutants RME-1 protein appears diffuse in the cytoplasm and lacks its 

normal localization to endosomes, indicating that gum-1 and rme-1 function in the same pathway. 

Two likely scenarios are suggested by these findings. Either GUM-1 functions together with 

RME-1 in recycling endosome to plasma membrane transport, and in gum-1 mutants RME-1 fails 

to be recruited to recycling endosomes, or GUM-1 functions earlier in the pathway in early 

endosome to recycling endosome transport, and in gum-1 mutants recycling endosomes are 

missing altogether. We are currently addressing these possibilities by examining the localization 

of GUM-1 and RME-1 relative to one-another, and examining the affects of these mutants on the 

structure and function of each endosomal compartment using a set of GFP markers we have 

developed for worm intestinal endosomes. Preliminary analysis indicates that gum-1 mutants 

have abnormally abundant and abnormally large early endosomes, consistent with our second 

model where GUM-1 and RME-1 function in sequential steps of endosome transport. 

We have cloned GUM-1 and found that it encodes the C. elegans homologue of a small rab 

GTPase of the ras superfamily. A variety of small GTPases are known to be master regulators of 

membrane transport, but the function of GUM-1 and its orthologues was unknown until this work. 

Using a gum-1p::gfp::gum-1(+) reporter transgene, we found that gum-1 is broadly expressed 

and is localized to cytoplasmic puncta that likely represent endosomes. Colocalization 

experiments are underway that should identify the specific identity of these GUM-1 positive 

structures. Results will be presented at the meeting.



Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore MD 

Nanos is a conserved regulator of germ cell differentiation. In many organisms, nanos RNA is 

maternally inherited and is translated only in the germ plasm, a specialized cytoplasm that 

segregates with the germline during early embryogenesis. How nanos translation is activated 

specifically in the germ plasm is poorly understood. 

We are investigating this question using the C. elegans nanoshomolog nos-2. nos-2 encodes a 

maternal RNA that is enriched on P granules (germ plasm organelles) and is translated 

specifically in the germline founder cell, P 4, and its daughters the primordial germ cells Z2 and 

Z3. This expression depends on the nos-2 3’UTR. A GFP:Histone H2B:nos-2 3’UTR transgene 

(driven by the pie-1 promoter) is expressed only in P 4 and Z2 and Z3. 

To begin to identify the transacting factors that act on the nos-2 3’UTR, we have examined the 

expression of the GFP: H2B: nos-23’UTR transgene in 2 classes of mutants: mutants in P 

granule components and mutants in the RNAi pathway. Our initial results indicate that certain P 

granules components are needed to repress ectopic nos-2 translation, whereas others are 

needed to activate nos-2 translation in P 4.



Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore 

MD. 

Nanos is a conserved regulator of germ cell differentiation. In many organisms, nanos RNA is 

maternally inherited and is translated only in the germ plasm, a specialized cytoplasm that 

segregates with the germline during early embryogenesis. How nanos translation is activated 

specifically in the germ plasm is poorly understood. 

We are investigating this question using the C. elegans nanoshomolog nos-2. nos-2 encodes a 

maternal RNA that is enriched on P granules (germ plasm organelles) and is translated 

specifically in the germline founder cell, P 4, and its daughters the primordial germ cells Z2 and 

Z3. This expression depends on the nos-2 3’UTR. A GFP:Histone H2B:nos-2 3’UTR transgene 

(driven by the pie-1 promoter) is expressed only in P 4 and Z2 and Z3. 

To begin to identify the transacting factors that act on the nos-2 3’UTR, we have examined the 

expression of the GFP: H2B: nos-23’UTR transgene in 2 classes of mutants: mutants in P 

granule components and mutants in the RNAi pathway. Our initial results indicate that certain P 

granules components are needed to repress ectopic nos-2 translation, whereas others are 

needed to activate nos-2 translation in P 4.

93. Age-associated feeding decline in C. elegans can be modulated by genetic and 

environmental inputs. 

David K. Chow, Catherine A. Wolkow 

National Institute on Aging Gerontology Research Center, 5600 Nathan Shock Dr. Rm. 4-E-10 

Baltimore, MD 21224 

Our goal was to study organ aging using the C. elegans pharynx as a model. The pharynx is a 

neuromuscular organ that primarily functions to ingest and crush bacterial food. This process is 

marked by a pumping movement that can be observed within the pharynx. The rate at which the 

pharynx pumps is well-known to decline over lifespan. Our experiments explored genetic and 

environmental inputs that modulate the age-related decline of pharynx function. 

We considered two models of pharyngeal aging that could account for the decrease in pumping 

rate across lifespan. The first suggests that muscle usage over lifespan leads to mechanical 

damage, the accumulation of which impairs pumping. The second model proposes that efficiency 

of cellular function decreases as the animal ages, in turn, decreasing function. We found that 

dramatic reductions of pumping rate in youth appeared to protect from declines of pharynx 

function in old age, although mild pumping rate reductions in youth were not apparently 

protective. These results tentatively support our second model of pharyngeal aging where 

reduced cellular function during aging leads to age-related declines in pharynx function.

94. An Investigation into the potential regulatory relationships between components of a 

network specified by pal-1. 

Julia M. Claggett, L. Ryan Baugh, Craig P. Hunter 

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138. 

PAL-1 protein, contributed both maternally and zygotically, is necessary and sufficient to 

specify and maintain the C blastomere lineage in the C. elegans embryo (Hunter and Kenyon, 

1996). A number of this master regulator’s targets were identified by microarrays comparing the 

transcript abundance in wild-type and mutant embryos either lacking or containing extra C 

blastomeres. Furthermore, we collected these embryos at defined time points, thus additionally 

providing temporal information. Target genes could then be separated by their transcriptional 

initiation into four consecutive temporal phases defined by a singular cell cycle beginning with the 

2C-cell stage (Baugh et al, 2003). Using reporter YFP constructs for thirteen of the targets and a 

volume-rendering program, the 3D spatial expression pattern of each target gene was 

established. On the basis of this spatial information and knowledge of the temporal phase to 

which each target belongs, we have proposed a set of regulatory relationships between the 

components. We are currently testing these hypotheses by disrupting potential ÒupstreamÓ 

regulators via RNAi and/or mutation and either observing the effect on individual ÒdownstreamÓ 

reporters or analyzing the effect on transcript abundance using QPCR. We hope that such 

measurements will give us insight into how the genes within the pal-1 network regulate each 

other in order to establish and maintain the various cell fates within the C blastomere lineage. 

Hunter, C.P. and Kenyon, C. (1996). Spatial and temporal controls target pal-1 

blastomere-specification activity to a single blastomere lineage in C. elegans embryos. Cell 87, 

217-26. 

Baugh, L.R., Hill, A.A., Slonim, D.K., Brown, E.L. and Hunter, C.P. (2003). Composition and 

dynamics of the Caenorhabditis elegans early embryonic transcriptome. Development 130, 

889-900.

95. Genome-wide RNAi analysis of Caenorhabditis elegans distal tip cell migration 

Erin J. Cram, Jean E. Schwarzbauer 

Princeton University Department of Molecular Biology Princeton, NJ 08544 

Integrin receptors for extracellular matrix transmit mechanical and biochemical information 

through molecular connections to the actin cytoskeleton and to a number of intracellular signaling 

pathways. In C. elegans, integrins (pat-2, pat-3, and ina-1) are essential for embryonic 

development, muscle cell adhesion and contraction, and migration of nerve cell axons and 

gonadal distal tip cells. To identify key components involved in distal tip cell migration, I am using 

an RNA interference (RNAi)-based genetic screen for deformities in gonad morphogenesis. We 

have obtained a library of most C. elegans open reading frames, each contained in a vector 

optimized for bacterial mediated RNAi. I have surveyed 5540 clones or 32% of the RNAi library. 

So far, 42 clones (0.75%) cause highly penetrant DTC migration defects. Some of these genes 

include talin, various tubulins (eg tba-1, tba-2), Rac (ced-10), daughterless (hlh-2), and the plakin 

shortstop (vab-10). Information from these screens will identify a set of genes required for distal 

tip cell migration in vivo, and may advance our knowledge of the role of integrins in cell responses 

to the extracellular environment.

96. Characterization of the DTC niche and germline stem cells in C. elegans 

Sarah L. Crittenden 1 , Dana Byrd 1 , Kim Leonhard 2 , Judith Kimble 1,2 

1Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin 53706 

2Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706 

The mitotic region of the C. elegans germ line includes stem cells, as defined by its ability to 

maintain a proliferating population of germ cells as well as to generate gametes. [Note that all 

"cells" in the germ line are part of a syncytium; we call them "cells" for simplicity; all are partially 

enclosed by membranes and appear to behave as individuals within the mitotic region.] In young 

wild-type adults, the mitotic region is composed of about 225 germ cells that extend 18-20 germ 

cell diameters along the distal-proximal axis. The somatic distal tip cell (DTC) promotes 

proliferation by GLP-1/Notch signaling and provides the "stem cell niche". 

To ask whether contact between the DTC and the germ cells defines the extent of the mitotic 

region, we examined the DTC and its processes using a lag-2::GFP reporter. In animals of 

different ages and in mutants with longer and shorter mitotic regions we found that DTC process 

length does not correlate with the length of the mitotic region. These findings extend the work of 

Hall et al. (1999) and confirm the idea that the length of DTC processes does not define the 

extent of the mitotic region. 

In an attempt to identify classical germline stem cells, we are characterizing cell cycles and 

orientation of mitotic spindles within the germline mitotic region. Preliminary results using BrdU, 

Cy3-dUTP and anti-PH3 reveal no differences along the distal-proximal axis with respect to 

frequency of mitosis or time from BrdU incorporation to M phase. In addition, the orientation of 

mitotic spindles appears random throughout the region. We suggest that stem cells in the C. 

elegans germ line may be controlled at a population level rather than by asymmetric divisions. 

Although we see no difference in cell cycle timing or orientation of cell divisions along the 

distal-proximal axis, germ cells in the mitotic region are not uniform by other criteria. First, cells in 

the adult mitotic region do not respond uniformly to removal of GLP-1 signaling (shift of glp-1(ts) 

from permissive to restrictive temperature). Instead, entry into meiosis occurs in a wave that 

begins with the most proximal cells and progresses to the most distal cells. Second, cells within 

the mitotic region do not express regulators of the mitosis/meiosis decision uniformly (e.g., see 

Hansen et al., 2004). Our current model is that the mitotic region of the germ line includes distal 

cells that reside within the DTC niche and remain undifferentiated, while more proximal cells that 

have left the niche begin the transition into differentiation. Before the more proximal cells can 

leave the mitotic cell cycle and enter meiosis, they must achieve a critical level of regulatory 

activity that drives them forward into the meiotic cell cycle. 

Hall et al. (1999) Developmental Biology 212, 101-123 

Hansen et al. (2004), Developmental Biology 268, 342-357

97. Structure/function studies of the polarity regulator PAR-1 in C. elegans. 

Adrian Cuenca, Geraldine Seydoux 

Dept. of Molecular Biology and Genetics, Johns Hopkins U. School of Medicine, Baltimore MD 

21205 

Polarization of the C. elegans zygote along the anterior-posterior axis depends on a signal from 

the sperm asters, which causes the cortically-enriched PAR proteins to relocalize to distinct 

anterior and posterior domains. The sperm asters exclude the PAR-3/PAR-6/PKC3 complex from 

the nearby cortex, allowing PAR-1 and PAR-2 to accumulate there (Goldstein and Hird, 1996; 

O’Connell et al., 2000; Wallenfang and Seydoux, 2000; Cuenca et al., 2003). 

We are investigating how the serine-threonine kinase PAR-1 localizes to the cortex. A 

GFP:PAR-1 fusion accumulates around the sperm-derived centrosomes and on the nearby 

posterior cortex during polarization. We have identified a domain at the carboxy-terminal end of 

PAR-1 necessary and sufficient for both of these localizations. This domain is conserved in 

PAR-1 kinases from other organisms and includes a region that binds to the non-muscle myosin 

NMY-2 (Guo and Kemphues, 1996). Consistent with earlier results (Boyd et al., 1996), analysis of 

GFP fused to the localization domain in zygotes depleted for PAR-2 or PAR-3 suggest that 

PAR-1 can associate with the cortex in the absence of other PAR proteins, but requires PAR-2 

and PAR-3 to accumulate specifically on the posterior cortex. We are currently constructing 

PAR-1 transgenes lacking the localization domain to investigate its relevance to PAR-1 polarity 

function in vivo.

98. Experimental Design for C. elegans Microarray 

Yuxia Cui, Sandra J. McBride, Jonathan H. Freedman 

Nicholas School of the Environment and Earth Sciences, Duke University, Durham, NC 27708 

To determine the optimal conditions to be used in future C. elegans microarray analyses, the 

effects of different RNA isolation protocols on microarray data quality were examined. Total RNA 

was collected from cadmium-treated C. elegans (100uM CdCl2, 24-h exposure) and non-treated 

nematodes. Total RNA was labeled and then hybridized to a printed 96-oligonuclotide array. The 

96 genes are part of Qiagen C. elegans Array-Ready Oligo set. Three RNA isolation procedures 

were tested in this study. Data analysis showed gene specific dye effect on the relative 

expression levels of the gene. It was also found that the RNA isolation procedure affects 

microarray hybridization quality in terms of signal/background ratio (one-way ANOVA F-test alpha 

= 0.05). The highest signal/background ratio (mean signal/background ratio across arrays = 3.73, 

95% C.I. from 1.99 to 5.47 for green channel signal, and mean signal/background ratio across 

arrays = 4.21, 95% C.I. from 1.42 to 7.00 for red channel signal) was obtained when frozen 

nematodes were ground prior to Trizol extraction. In order to determine the major source of 

variance in C. elegans microarray, variance components of data were estimated by ANOVA 

analysis and variance caused by arrays contributes to the majority of data variance. To calculate 

the reasonable minimum sample size, the median standard deviation over all genes was 

estimated and the optimal subsampling method was determined.


Diana David-Rus 1 , Peter J. Schmeissner 1 , Beate Hartmann 2 , Christophe Grundschober 2 , Uri 

Einav 3 , Eytan Domany 3 , Patrick Nef 4 , Garth Patterson 1 , Monica Driscoll 1 

1Rutgers University, Piscataway, New Jersey 

2Hoffman-LaRoche, Basel, Switzerland 

3Weizmann Institute of Science, Rehovot, Israel 

4Hoffmann-LaRoche, Basel, Switzerland 

In an effort to better understand the biology of aging with an emphasis on mid-life changes that 

influence healthspan, we have undertaken a DNA microarray analysis of global gene expression 

profiles over time using Affymetrix gene chip arrays. Our experiment includes time points 

including the reproductive and post-reproductive periods, with a series of consecutive mid-life 

time points being covered. Studies in our lab and others have suggested that critical events 

during the mid-life of the nematode can influence the aging of this organism. For our analyses, we 

used supervised and unsupervised methods, including a clustering method developed in the 

Domany lab. Interestingly, we find a sharp change in the transcriptional levels of numerous genes 

at about 10 days post egg-lay. This abrupt change in gene expression on day 10 is consistent 

with a window of time identified previously by our lab as a time when mutations in the age-1 gene 

are able to delay the deterioration of muscle tissue (Herndon et al., 2002, Nature, 419: 808-14). 

The abrupt change also correlates with a transition point at which autofluorescent biomarkers 

accumulate (see abstract by Gerstbrein et al., this volume). Since two similar microarray 

experiments already have been performed (Lund et al., 2001, Curr Biol. 12(18): 1566-73; Murphy 

et al., 2003, Nature, 424(6946):277-83), we attempted a detailed cross-comparison between data 

from all three experiments, using non-parametric techniques. Again, a dramatic change in gene 

expression was observed for some genes at time points corresponding to our day 10 time point. 

Approximately 100 genes show significant changes in gene expression over adulthood in all three 

studies. These expression changes might be relevant to rapid end-stage deterioration in old 

nematodes.

100. An HCP-6 Suppression Screen for Genes Involved in Centromere Resolution 

Tovah A. Day, Landon Moore 

Department of Genetics and Genomics, Boston University School of Medicine, Boston, MA 02118 

Holocentric centromere protein (HCP)-6 was originally discovered in a screen for aberrant 

chromosome segregation. A temperature sensitive mutation, hcp-6(mr17), causes embryonic 

lethality as a result of defects in chromosome segregation. Although the mechanism of HCP-6 

action remains unclear, hcp-6(mr17) chromosomes are characterized by a failure to properly 

condense during prophase. When HCP-6 function is compromised, defects in both centromere 

resolution and twisting lead to merotelic orientation, attachment of a single kinetochore to 

microtubules from both centromeres. Seemingly, HCP-6 is pleiotropic, exhibiting additional 

phenotypes that range from everted vulva to genomic instability. In order to distinguish the 

requirements for HCP-6 in these different processes, we designed a suppressor screen that 

depended upon the temperature sensitive nature of allele mr17. Screening for growth at the 

non-permissive temperature allowed identification of mutations that suppressed embryonic 

lethality. We screened 2000 EMS-mutagenized haploid genomes for growth at 26 degrees and 

identified one putative suppressor, Suppressor of HCP-6 Embryonic Lethality (SHL) -1. Although 

the initial screen was performed at 26 degrees, suppression was only weakly penetrant at this 

temperature. Because HCP-6 retains a strong embryonic lethal phenotype at 23 degrees, we 

examined SHL-1 at 23 degrees and observed more robust suppression at the lower temperature. 

Interestingly, the suppressor strain maintains the phenotype of everted vulva and as such may be 

able to help us differentiate the various functions of HCP-6. Future work will include microscopic 

characterization of the suppressor’s chromosome phenotype and SNP mapping to determine its 

identity.

101. Analysis of sel-2, an enhancer of lin-12 activity in vulval precursor cells 

Natalie de Souza 1 , Laura G. Vallier 2 , Hanna Fares 3 , Iva Greenwald 1 

1 HHMI/Dept. of Biochemistry and Mol. Biophysics, Columbia University College of Physicians 

and Surgeons, 701 W. 168th St., New York, NY 10032 

2 Biology Dept., 114 Hofstra University, Hempstead, NY 11549 

3 University of Arizona, Dept. of Mol. and Cellular Biology, 1007 E. Lowell St., Tucson, AZ 85721 

LIN-12 mediates intercellular signaling in several developmental decisions in C. elegans, 

including lateral specification during vulval precursor cell (VPC) patterning. Elucidation of the 

function of sel genes (suppressor or enhancer of lin-12), has furthered our understanding of 

LIN-12/Notch signaling at the molecular level. An apparent null allele of sel-2 enhances lin-12 

activity in the VPCs: lin-12(n302) hermaphrodites lack an anchor cell and do not display any 

vulval induction, but sel-2(0)lin-12(n302) hermaphrodites are Multivulva, with all VPCs adopting 

the secondary fate (the fate associated with LIN-12 activation). sel-2 was cloned and found to 

encode a protein with a mammalian ortholog that has been implicated in intracellular traffic in 

various polarized cell types. As the VPCs are also polarized cells, studying the role of sel-2 in the 

VPCs offers the possibility to understand further the mechanisms of polarized traffic in C.elegans 

and in other organisms. In wild type hermaphrodites, LIN-12::GFP is visualized in the apical 

domain of the VPCs, but in a sel-2 mutant, LIN-12 can be visualized throughout the cells. The 

distribution of LET-23, which is normally basolateral, appears not to be affected. Experiments to 

investigate the role of sel-2 in LIN-12 subcellular localization and/or turnover in the VPCs are in 

progress.

102. Function of a novel protein EFF-1 in cell fusion. 

Jacob J del Campo 1 , Ariel B Issacson 1 , Morgan Tucker 2 , Min Han 2 , William A Mohler 1 

1University of Connecticut Health Center Farmington, CT 

2University of Colorado Boulder, CO 

The mechanism of cell membrane fusion remains a mystery in every organism in which it is 

observed. Forward genetics has identified a single gene (eff-1) required specifically for cell fusion 

in C. elegans. Mutations within the extracellular domain of EFF-1 do not seem to affect prefusion 

events (differentiation, adhesion, etc.). However, in embryos, eff-1 mutants cannot fuse dorsal or 

ventral hypodermis. eff-1 mutant larvae can also be phenotyped by a small ball at the end of the 

tail, presumably resulting from a failed embryonic fusion of two tail spike cells. As the worm 

matures and elongates, other morphological structures are subject to cell fusions (i.e. vulva, 

seam cells, pharynx). Although eff-1 mutant worms fail to fuse cells within these tissues, they are 

remarkably viable and are self-fertile. 

EFF-1 is a predicted type I integral membrane glycoprotein. BLAST searches indicate that 

EFF-1 is not a member of any known protein family. We are employing two approaches to 

deduce the functional domains and motifs of EFF-1. (1) We are changing putative structural and 

functional domains (phospholipase A2 active site, putative fusion peptide), via site directed 

mutagenesis and testing for rescue in eff-1 mutant worms. (2) We have isolated several new 

alleles with EMS, including several probable eff-1 null mutations. To observe EFF-1 localization in 

vivo, EFF-1 was tagged with GFP at the C-terminus. EFF-1::GFP expression precedes embryonic 

cell fusion events, and the labeled protein localizes dynamically and specifically to fusion 

competent cell contacts. Mutant EFF-1::GFP constructs show atypical localization. As ectopic 

expression of EFF-1 in worms produces extra cell fusions, we are currently testing whether EFF-1 

can induce cell fusion in cells of other species.

103. Degenerate binding sites for the FAX-1 nuclear receptor predict potential downstream 

target genes 

Stephen DeMeo, Rebecca Lombel, Danielle Snowflack, Aaron Wagner, Eric Smith, Sheila 

Clever, Bruce Wightman 

Biology Department, Muhlenberg College, Allentown, PA 18104 

The fax-1 gene encodes a nuclear hormone receptor that regulates neuron specification during 

embryogenesis by orchestrating the coordinated transcription of neuron-specific gene products. 

The human ortholog of fax-1, PNR, functions in the generation of photoreceptor cells in the retina. 

Mutations in human PNR are a cause of inherited retinal degeneration and blindness. The human 

diseases caused by PNR mutations include Enhanced S-cone Sensitivity, in which S-cones are 

over-represented in the retina at the expense of rods and other cone types. This defect in neuron 

specification is reminiscent of the incorrect specification of neurons in fax-1 mutants (see 

Wightman et al. abstract). We are studying the DNA-binding properties of FAX-1 towards the goal 

of predicting the candidate regulatory target genes that mediate neuron specification. Like other 

nuclear receptors, FAX-1 demonstrates selective binding to hexameric half-sites. Using yeast 

one-hybrid and gel-shift assay techniques, we have shown preferential binding of FAX-1 to two 

dimeric half sites, AAGTCA, a known target of PNR and also AGGTCA, a sequence not 

previously shown to be a target of the NR2E class of nuclear hormone receptors. Optimal binding 

occurs when the directly-repeated hexamers are separated by a single base-pair (e.g., 

AAGTCANAAGTCA). FAX-1 does not bind monomeric hexamer sites, but the existence of a 

strong binding site allows dimerization and binding to an adjacent weak binding site. FAX-1 

exhibits only marginal preference for purines at the second position of hexamers, suggesting that 

the protein is relatively indifferent to the bases at that position. There are approximately 130 sites 

in the C. elegans genome with the sequence ANGTCANANGTCA, the predicted FAX-1 binding 

site from our analysis. We are evaluating candidate downstream genes by creating 

promoter::GFP fusions and testing them for dependence on fax-1. 

P> P> 

Our studies also have implications for amino acid-base pair contacts in DNA-binding by FAX-1. 

In crystal structures, the second base-pair of each hexamer DNA site is contacted by amino acid 

19 of the nuclear receptor DNA-binding domain. Both FAX-1 and PNR have asparagines at this 

position, while Tailless and Tlx, two nuclear receptors that discriminate between A and G at 

hexamer second base positions have aspartates. Thus the asparagines at amino acid 19 may 

mediate relaxed DNA-binding specificity for FAX-1 and PNR. Because FAX-1 and PNR have 

identical P-boxes, the short α-helical region that directly contact the DNA base-pairs, we expect 

that core hexameric binding properties of both proteins will be very similar or identical. Therefore, 

the investigation of FAX-1 DNA-binding properties may be predictive for identifying candidate 

targets of FAX-1 in C. elegans and PNR in humans.

104. Mapping transcription regulatory networks in C. elegans 

B. Deplancke 1 , D. Dupuy 2 , M. Vidal 2 , A.J. Marian Walhout 1 

1Program in Gene Function and Expression, University of Massachusetts Medical School, 

Worcester, MA 

2Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 

Since the advent of microarrays, large amounts of gene expression data have become 

available for C. elegans. However, the transcription regulatory mechanisms that dictate the gene 

expression profiles observed frequently remain elusive. This is mainly because the identity of the 

regulatory transcription factors (TFs) that control differential gene expression are difficult to infer 

from such datasets. Regulatory TFs activate or repress transcription of their target genes by 

binding to cis-regulatory elements that are frequently located in or near a gene’s promoter. Thus, 

in order to experimentally "match" a regulatory TF with its target genes, assays are needed that 

demonstrate a physical interaction between regulatory TFs and the cis-regulatory elements or 

promoters they bind to. 

To facilitate the large-scale identification of TF-promoter interactions, we developed a 

Gateway-compatible version of the yeast one-hybrid (Y1H) system. This system enables the 

rapid identification of TF-DNA interactions using both small (i.e. repeats of DNA elements of 

interest) and large DNA fragments (i.e. gene promoters). The possibility to generate many Y1H 

DNA bait constructs simultaneously by Gateway cloning makes this system amenable to 

high-throughput settings. Here, we present Y1H data obtained using various DNA fragments as 

baits including four intact C. elegans promoters. These DNA baits were screened versus a 

Y1H-compatible worm cDNA library and a newly generated worm TF mini-library consisting of 

675 predicted TF-encoding full-length open reading frames in the right orientation and frame, and 

in roughly equimolar amounts. The TF mini-library has a highly reduced complexity compared to 

the cDNA library, which increases both the throughput of the Y1H assay and the probability to 

detect TF-DNA interactions with TFs that are relatively underrepresented in cDNA libraries. 

Because of the ability to associate C. elegans TFs with their respective target genes, the 

high-throughput Y1H system will be a valuable tool to decipher the transcription regulatory 

networks that control differential gene expression during worm development.

105. SYP-3, a coiled-coil protein required for chromosome synapsis and chiasma 

formation in C. elegans 

Andreas Eizinger 1 , Allison Hurlburt 1 , JoAnne Engebrecht 2 , Kirthi Reddy 3 , Anne Villeneuve 3 , 

Mónica Colaiácovo 1 

1Department of Genetics, Harvard Medical School, Boston, MA 02115 

2Department of Pharmacological Sciences, State University of New York, Stony Brook, NY 

11794-8651 

3Department of Developmental Biology, Stanford University, Stanford, CA 94305 

Meiosis is the form of cell division, essential for sexually reproducing organisms, that leads to 

the formation of haploid gametes. During prophase of the first meiotic division, a proteinaceous 

structure called the synaptonemal complex (SC) forms between paired and aligned homologous 

chromosomes. The SC is an evolutionarily conserved structure consisting, in C. elegans, of axial 

components such as HIM-3, and central region components, SYP-1 and SYP-2. Analysis of syp-1 

and syp-2 mutants revealed that initial homolog pairing unfolds independent of the presence of 

central region components, but that these are necessary for further stabilization of pairing 

interactions as well as for completion of crossover recombination. 

We now report on the analysis of syp-3, which encodes a 213 aa protein containing a 

coiled-coil domain. syp-3 was identified in an RNAi based screen for genes that affect meiosis 

and matched to me42, identified through a genetic screen for meiotic mutants. syp-3 (me42) 

mutants exhibit increased progeny lethality (96%) associated with a high incidence of male 

progeny (36%) indicative of problems with both autosomal and X chromosome segregation. 

Analysis of meiotic chromosome morphology in syp-3 (me42) indicates that chromosomes fail 

to fully synapse at pachytene and lack chiasmata at diakinesis. This lack of chiasmata does not 

reflect an inability to initiate meiotic double-strand breaks (DSB) given that RAD-51, a protein 

involved in strand exchange during DSB repair, associates with both wild type kinetics and levels 

in syp-3(me42). Furthermore, processing of DSBs is not impaired in syp-3(me42) as determined 

by lack of chromosome fragmentation at diakinesis, normal turnover of RAD-51 foci and the lack 

of activation of a late pachytene DNA damage checkpoint. Taken together, these results along 

with the lack of proximity to a homologous partner from which to repair DSBs and the absence of 

functional chiasmata, suggest repair might be unfolding between sister chromatids. 

Localization studies indicate that SYP-3 first associates with chromosomes during transition 

zone of meiotic prophase, and localizes in a continuous fashion between synapsed chromosomes 

at pachytene (a pattern reminiscent of SYP-1 and SYP-2 localization). However, unlike SYP-1 

and SYP-2, SYP-3 persists associated onto chromosomes throughout diakinesis. 

Preliminary data suggests that HIM-3 and the meiotic cohesin REC-8 do not require SYP-3 for 

proper chromosomal association and dissociation. However, SYP-1 and SYP-2 are delayed in 

localizing onto chromosomes upon entrance into meiosis in the syp-3 (me42) mutant, and localize 

continuously along unsynapsed chromosomes at pachytene. Furthermore, SYP-3 localization in 

syp-1 and syp-2 mutants is strongly reduced. Altogether, these results implicate SYP-3 as a 

possible regulator of the assembly of SYP-1 and SYP-2 on properly synapsed homologous 

chromosomes.

106. A genetic screen for mutants defective in male leaving, a mate-searching behavior of 

C. elegans 

Chunhui Fang, Rajarshi Ghosh, Scott W. Emmons 

Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY 10461 

When an individual adult of the nematode C. elegans is cultured on a small patch of bacteria, it 

will display a sexually dimorphic behavior we termed as leaving behavior: males will wander away 

from the food source as time goes by while hermaphrodites will usually stay. This is a putative 

male mate-searching behavior, since the presence of hermaphrodite can fully suppress this 

behavior. The successful execution of leaving requires sensory input from external environment 

(the presence of food and absence of hermaphrodite), integration of sensory information and 

locomotion output. Mutations that disrupt any of these steps can cause defective leaving 

behavior. While we have previously shown that mutations that disrupt sensory input or locomotion 

output will alter the leaving behavior, little is known about mutations that affect the process of 

information processing. 

To identify genes involved in the execution of this behavior, we performed a genetic screen for 

mutations that alter the leaving behavior of males. him-5 (e1490) hermaphrodites were 

mutagenized at L4 stage with PLACE>EMSPLACE> and the leaving behavior of male progeny of 

F2 animals was scored. Mutations that produce apparent morphological or other behavioral 

phenotypes such as dpy orunc were discarded. After screening about 1200 genomes, we 

recovered 20 Las (Leaving assay defective) mutations that render males to stay instead of leave. 

Four of them, bx121, bx137, bx138 and bx139, were chosen to characterize further due to their 

strong Las phenotype. These mutant males have normal ray morphology and can mate 

efficiently. One of these mutants, bx137, has a higher reversal frequency on food than wildtype 

animals, but it is currently unknown whether this is the underlying reason for the defective leaving 

behavior. No apparent phenotype has been identified for the other 3 mutants. In no case is the 

leaving behavior of hermaphrodites of these mutants altered. In order to further characterize the 

genes defined by these mutations, we are currently mapping them using SNP-based method.

107. DKF-2 is a Novel Target-Effector of Protein Kinase C 

Hui Feng, Min Ren, Charles S. Rubin 

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461 

Extensive knowledge of structures, modes of activation and properties of protein kinase C 

isoforms (PKCs) is available. In contrast, information about downstream substrate-effectors and 

the molecular and cellular basis for physiological roles of PKCs is limited. Recently-discovered 

protein kinase D (PKD) isoforms may contribute significantly to diversification and integration in 

DAG-controlled signal transduction. PKDs have PKC-like DAG binding sites coupled to novel 

catalytic domains. Moreover, PKDs are candidate target-effectors for PKCs in signaling 

pathways. Consequently, characterization of structure, function, regulatory properties, substrate 

specificities, intracellular locations, molecular genetics and physiological roles of PKDs will 

advance the quest to comprehend molecular principles underlying DAG-mediated signal 

transduction. 

A cDNA that encodes a novel C. elegans PKD (named D kinase family-2 or DKF-2) was 

isolated and characterized. DKF-2 (1068 amino acids, Mr ~ 120,000) protein contains tandem 

DAG-binding and PH regulatory domains. Incubation of transfected cells with TPA (a phorbol 

ester tumor promoter that mimics DAG) elicits translocation of DKF-2 from cytoplasm to plasma 

membrane, thereby enabling kinase activation via upstream activators. Subsequently, DKF-2 is 

internalized and degraded in a phosphorylation-dependent manner. PKC inhibitors (which do not 

diminish DKF-2 kinase activity in vitro) abolish DKF-2 activation in intact cells. Thus, PKCs control 

DKF2 in vivo. A centrally-located PH domain is not essential for targeting DKF-2 to plasma 

membrane. Mutation of several conserved residues within the PH module or PH domain deletion 

have little effect on DKF-2 kinase activity. However, substitution of W717 with Ala decreases total 

DKF-2 phosphotransferase activity ~ 70%. The data document a novel regulatory property for a 

PKD and suggest that the C-terminal region of the PH domain may co-operate with adjacent 

downstream amino acids to occlude the PKD catalytic cleft. Ser925 and Ser929 in the DKF-2 

activation loop regulate the activity and stability of the enzyme. Application of phospho-specific 

antibodies, PKC inhibitors and kinase assays demonstrated that phosphorylation of Ser925 by a 

PKC is essential for DKF-2 activation by growth factors. Phosphorylation of additional sites on 

DKF-2 is also required for expression of maximal enzymic activity. S925A substitution decreases 

phosphotransferase activity on a model substrate by 75%, while double Ala mutations (S925A, 

S929A) render the kinase inactive. The S929A mutant kinase is very stable because it is resistant 

to proteasome-mediated degradation. Down-regulation of DKF-2 is triggered by phosphorylation 

of Ser929. Reversal or suppression of Ser929 phosphorylation generates a stable, 

proteasome-resistant kinase. Cellular and temporal patterns of DKF-2 gene transcription were 

determined to provide insights into physiological functions of the kinase. A 1.4 kbp DNA fragment 

(promoter/enhancer) that flanks the 5’ end of the dkf-2 gene was inserted upstream from a 

nucleus-directed, GFP reporter gene in a C. elegans expression vector (designated dkf2P::GFP). 

Transgenic C. elegans that carry a chimeric dkf2P::GFP gene were created and assayed by 

immunofluorescence microscopy. High level dkf-2 promoter activity was evident principally in 

nuclei of intestinal cells. The dkf-2 gene promoter sequence contains a tandem pair of GATA 

regulatory elements (CTGATAA) that are separated by 25 nts. Thus, dkf-2 expression may be 

partly regulated via the gut-specific transcriptional activator, elt-2. RNAi and gene disruption 

experiments were used to elucidate in vivo functions for DKF-2. dkf-2 null mutants live 30% 

longer than wild type worms. In a population of dkf-2 null animals, the first occurrence of death 

and the day at which 50% survival occurs are increased by 4 days relative to N2 worms. The 

maximum life span of dfk-2 null worms is 5-6 days longer than the maximal lifetime of wild type C. 

elegans. Thus, DKF-2 may function in regulating feeding, energy metabolism, digestion or 

responses to toxic stress.

108. A novel mutant that partially suppresses the daf-2 (e1370) Daf-c phenotype 

Manuel A. Fidalgo, Manuel J. Munoz 

Centro Andaluz de Biologia del Desarrollo. Univ. Pablo de Olavide. Ctra. de Utrera, Km. 1. 

41013. SEVILLE (SPAIN) 

Mutations in the IGF/insulin like receptor daf-2 confer a daf-c phenotype together with an 

increase of longevity in the adult. 

Suppressors of the daf-c phenotype of daf-2(e1370) mutant have been isolated. Mutations in 

the daf-16 or daf-18 genes suppress daf-c phenotype of e1370, making the animal enter to adult. 

In the other hand, mutations in the daf-12 gene make the e1370 mutant arrest in L2d, a larval 

stage previous to dauer. We have found a novel mutant (named pv12) that suppresses the daf-c 

phenotype but they do not let the animal to develop to adult. Most of worms from the daf-2 

(e1370); pv12 double showed an L3-L4 arrest. Preliminary result suggest that pv12 works 

downstream of daf-16 because is enable to relocate the DAF-16 out of the nucleus in an insulin 

signaling defective mutant background. This new weak suppressor can help to understand how 

the insulin-signaling pathway regulates dauer formation. The mutant itself is short lived and also 

has a reduction in the brood size. Mapping and further characterization of this mutant is under 

progress.

109. Specificity of the C. elegans Putative Transmembrane Channel SID-1 

Michael C. Fitzgerald, Craig P. Hunter 

Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Ave, Cambridge, 

MA 02138 

SID-1 was identified in a genetic screen for mutants capable of cell autonomous RNAi but 

deficient for systemic RNAi (Winston et al., 2002). The systemic RNAi defect is likely due to the 

inability of cells lacking SID-1 to import double-stranded RNA (dsRNA) from neighboring cells. 

The nature of SID-1 activity was elucidated using a heterologous system whereby either wild-type 

SID-1 or, as a negative control, a missense mutant form of SID-1 was transiently expressed in 

Drosophila S2 cells (Feinberg & Hunter 2003). These investigations showed that SID-1 enables 

efficient RNAi by adding dsRNA to the media of transfected S2 cells (soaking RNAi) and uptake 

of labeled dsRNA into cells. Furthermore, long dsRNA was shown to be more effective than short 

dsRNA for SID-1 mediated soaking RNAi in S2 cells and for systemic RNAi in C. elegans. 

We are investigating the length dependence of silencing as well as substrate specificity of the 

SID-1 channel. We have shown that SID-1 is extremely efficient, enabling soaking RNAi with less 

than one molecule of dsRNA per transfected cell and will present evidence that transport is 

extremely rapid. We will also report the results of ongoing analyses of length-dependent 

transport and channel selectivity, which may have implications for the use of SID-1 as a tool for 

molecular biology. 

W. M. Winston, C. Molodowitch, C. P. Hunter, Science 295, 2456-59 (2002). Systemic RNAi in 

C. elegans requires the putative transmembrane protein SID-1. 

E. H. Feinberg, C. P. Hunter, Science 301, 1545-7 (2003). Transport of dsRNA into cells by 

the transmembrane protein SID-1.

110. SMA-9, a Protein Involved in Patterning of the C.elegans M Lineage 

Marisa L Foehr, Ming Xu, Jun Liu 

Department of Molecular Biology and Genetics. Cornell University, Ithaca, NY 14853 

We are interested in understanding mesodermal patterning and cell fate specification using the 

C. elegans postembryonic mesodermal lineage, the M lineage, as a model system. The M lineage 

is derived from a single precursor cell (M), which undergoes a series of asymmetric divisions to 

generate a number of differentiated cell types. In particular, the dorsal daughter of M produces 

two coelomocytes (CCs), while the ventral daughter produces two sex myoblasts (SMs). The 

mechanisms underlying this asymmetry are not clear. We are interested in a molecule, SMA-9, 

that functions in establishing this polarity and in regulating cell fate specification in the M lineage. 

sma-9 mutants generate a duplication of the ventral descendants of the M lineage. This results 

in an end phenotype in which the M derived CCs are missing and extra SMs are generated. In 

addition, sma-9 mutant animals are smaller than wild type and males have tail patterning defects 

(Liang et al, 2003 1 ). In order to understand how sma-9 functions in regulating the dorsal/ventral 

asymmetry of the M lineage, we set out to determine the expression pattern of sma-9. sma-9 is a 

highly complex molecule that has multiple splicing isoforms. We are interested in dissecting the 

expression patterns of the different isoforms, as well as understanding which isoforms are 

specifically functioning in the M lineage. To this end, we have generated isoform specific 

antibodies to two of the three SMA-9 C-terminal isoforms and examined the expression pattern of 

SMA-9 using these antibodies. Additionally, GFP fusion constructs linking the genomic N 

terminus to the different C terminal ends have been made. We are currently generating 

transgenic lines carrying these fusion constructs. 

sma-9 encodes the C. elegans homolog of the Drosophila protein Schnurri, a known regulator 

of the TGF-beta signaling pathway. While sma-9 functions in the TGF-beta signaling pathway in 

regulating body size and male tail patterning (Liang et al, 2003), it does not appear to function in 

the TGF-beta signaling pathway in patterning the M lineage. Instead, we have found some 

intriguing evidence that sma-9 genetically interacts with the lin-12/Notch signaling pathway in 

patterning of the M lineage. 

1. J.Liang et al. Development 130, 6453-6464. 2003.

111. Cloning and characterization of the C. elegans post-embryonic cytokinesis gene 

unc-85 

Iwen Fu 1 , Jason W. Reuter 2 , Fern P. Finger 1 

1Biology Department, Rensselaer Polytechnic Institute, Troy, NY 12180 

2Laboratory of Molecular Biology, University of Wisconsin, Madison, WI 53706 

Cytokinesis, the physical division of a mother cell into two daughter cells, is the final stage of 

the cell cycle.We are studying the unc-85 gene, which is required for post-embryonic cytokinesis 

in C. elegans. Consistent with its known role in post-embryonic cytokinesis (1, 2), we find little 

lethality in unc-85(e1414) mutants during embryogenesis and larval development. The 

uncoordinated phenotype of unc-85(e1414) mutants has been attributed to cell division failures of 

post-embryonic ventral cord neuronal precursors (1, 2). However, assays of locomotory behavior 

reveal that approximately 45% of newly hatched unc-85 mutants are uncoordinated. Because all 

of the embryonic ventral cord neurons are present, the uncoordination cannot be attributed to 

cytokinesis failures in the ventral cord neuronal precursors. This suggests an additional function 

for unc-85 in neuronal morphogenesis or function. We are cloning the unc-85 gene to determine 

its molecular identity. Its genetic location has previously been mapped to a 664 kb region on 

chromosome II between vab-1 and pho-1. Using a combination of deficiency and snip-SNP 

mapping, we have narrowed the position of unc-85 to a 225 kb region between F59A6.3 and 

pho-1, a region spanned by eight cosmids, and we are continuing to narrow the unc-85region by 

additional SNP mapping. We will attempt to rescue unc-85(e1414) mutants by injection of 

cosmids, and we will also use RNAi to test candidate genes in the unc-85 region for 

unc-85(e1414) phenocopy. 

1. Sulston, J. and Horvitz, H. Abnormal cell lineages in mutants of the nematode C. 

elegans. Dev. Biol., 82:41-55, 1981. 

2. White, J. G., Horvitz, H. R., and Sulston, J. E. Neurone differentiation in cell lineage 

mutants of Caenorhabditis elegans. Nature, 297: 584-587, 1982.

112. CeMyoD(hlh-1) in embryonic muscle fate determination. 

Tetsunari Fukushige, Joan McDermott, Thomas Brodigan, Michael Krause 

Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, Maryland, 20892 

The MyoD family of basic helix-loop-helix transcription factors regulates skeletal muscle 

formation and differentiation in mammals. The lone C. elegans member of this family, CeMyoD 

(encoded by hlh-1), is the earliest known factor identified in the body wall muscle cell lineage 

consistent with an important role in fate specification. However, mutant analysis has shown that 

CeMyoD activity is not required for body wall muscle formation in C. elegans. The correct number 

of embryonic muscle cells (81) are formed in hlh-1 loss-of-function mutants and many terminal 

muscle markers are expressed at apparently normal levels. These mutant animals do have a 

severe muscle phenotype; hlh-1 mutant muscle functions poorly and results in severe 

morphogenesis defects and larval lethality. 

To test if CeMyoD is sufficient for muscle cell fate determination, we generated an integrated 

transgenic worm carrying a heat-shock promoter::hlh-1 construct. Ectopically expressed hlh-1 in 

the early stages of embryogenesis results in widespread conversion of many embryonic cell 

lineages to muscle as assayed by terminal muscle markers. Markers for other cell fates, such as 

3ENB12 (pharyngeal cells), ELT-2 (gut cells) and LIN-26 (hypodermal cells), are not detected in 

embryos strongly expressing ectopic hlh-1. These results demonstrate that CeMyoD alone is 

sufficient to activate the striated muscle cell fate. We have begun to address how the hlh-1 gene 

is activated during embryogenesis. The early presence of CeMyoD protein in cells restricted to 

the body wall muscle fate suggests that maternal factors may be regulating hlh-1 expression. This 

is particularly true in the C and D lineages in which formation of the founder cell is only one or two 

divisions prior to the onset of CeMyoD accumulation. Using RNAi of maternal components 

important for early cell determination, we are trying to identify factors that are necessary and 

sufficient to activate hlh-1.

113. Quantification of electrotaxis 

Chris Gabel, Albert Kao, Dmitri Pavlichin, Aravi Samuel 

Physics Department, Harvard University 

In an electric field, the worm has preferred trajectories for forward crawling movement that 

depend on the direction and magnitude of the electric field. In weak fields (5 V/cm, worms crawl towards the negative 

pole in trajectories at an angle to the direction of the field. The angle of approach towards the 

negative pole increases with field strength, rising from 0 degrees (parallel to the field lines) 

towards 90 degrees (perpendicular to the field lines). We have quantified these electrotactic 

movements by tracking wild-type and mutant worms crawling over agar surfaces containing 

different types and concentrations of ions while responding to electric fields varied in amplitude, 

frequency, and direction.

114. Barotaxis 

Chris Gabel, Alex Dahlen, Aravi Samuel 

Physics Department, Harvard University 

Touching the worm on the nose or tail is transduced reflexively through a mechanosensory 

circuit into forward or backward movement. But what happens when a worm is touched all over at 

once? Can the mechanosensory circuit process a spatially isotropic stimulus that varies only in 

time, e.g., hydrostatic pressure? For these experiments, we use a computer-controlled hyperbaric 

chamber that allows us to deliver arbitrary waveforms of hydrostatic pressure, and we monitor 

and quantify worm movements using video microscopy and machine vision. Analyzing the 

movements of worms responding to sinusoidal, triangle waves, and step changes in pressure is 

revealing a sophisticated mechanism for the detection of pressure waveforms at least as small as 

0.01 psi/sec. For example, triangle waves stimulate redirections during upramps and stimulate 

forward movement during downramps with a behavioral hysteresis of ~8 seconds delaying the 

switch to the new pattern of movement after the change in stimulus direction. This hysteresis is 

symptomatic of an algorithm that performs temporal comparisons in a recently elapsed window of 

time to compute whether ambient pressure is rising or falling. By correlating response patterns to 

stimulus patterns, we are uncovering the quantitative structure of this algorithm. The actual 

detection of hydrostatic pressure also presents a puzzle. Since worms are essentially bags of 

water and water is nearly incompressible how is hydrostatic pressure converted into mechanical 

sensation? We are sensitive to pressure variations in our eardrums because gas is compressible, 

but worms lack ears. However, worms might be exploiting their resting internal pressurization: 

any curved interface can support a pressure difference proportional to surface tension divided by 

radius of curvature. A sensor associated with any curved interfaces between the inside and 

outside of the worm is thus a candidate pressure transducer. We hypothesized that overwhelming 

the transducer by pulling a sudden vacuum might disrupt barotactic signal transduction. We 

suddenly pulled a brief vacuum on worms, and while many behaviors are completely unaffected 

(including avoidances to osmolarity and body touch) the barotactic response is completely wiped 

out. After ~2 hours, vacuum-treated worms recover their barotactic response, so if we are 

damaging the transducer, it is capable of repair. We are now investigating the neuronal correlates 

of the barotactic response using mutant analysis and laser dissection.

115. Genetic analysis of mutations that suppress dauer arrest in age-1/PI3 kinase mutants. 

Minaxi S. Gami, Keaton Hanselman, Catherine A. Wolkow 

National Institute of Aging, Laboratory of Invertebrate Molecular Genetics Unit, Baltimore, 

Maryland. USA 

The DAF 2/insulin-like signaling pathway regulates lifespan and dauer arrest in C. elegans . 

Ligand activation of the insulin/IGF1-receptor like protein DAF-2, activates AGE-1, which is 

homologous to the catalytic subunit of phosphatidylinositol 3-kinase (PI-3 kinase). Activated 

AGE-1/PI3 kinase generates 3-phosphoinositides (PIPs), lipid secondary messengers that in turn 

activate serine threonine kinases AKT-1, AKT-1 and PDK-1. PIP levels are negatively regulated 

by the PTEN tumor suppressor homologue DAF-18. Activated AKT-1 and AKT-2 phosphorylate 

DAF-16, the ultimate output of the DAF-2 /insulin-like signaling. DAF-16 encodes a fork-head 

transcription factor that is required in dauer formation and longevity in age-1 mutants. In order to 

identify additional components of the age-1 pathway, a genetic screen was performed for 

suppressors of the constitutive dauer arrest phenotype in age-1 (mg44) null mutants. 40 mutants 

were identified from approximately 20, 000 haploid genomes screened, including alleles of daf-16 

and daf-18. In addition, several mutations appear to represent previously unknown components 

of the age-1 pathway. We are currently mapping and characterizing these mutations.

116. spe-19, a Gene Affecting Spermiogenesis 

Brian Geldziler, Andy Singson 

Waksman Institute, Rutgers University, Piscataway, New Jersey 

A primary goal of our lab is the characterization of genes required by sperm for fertilization. 

Toward that end, we have been studying spe-19, and will present our ongoing characterization. 

SNP mapping has allowed us to localize spe-19 between cosmids M162 and ZC15 on the far 

right arm of chromosome V. Hermaphrodites homozygous in either of our two alleles of spe-19 

ovulate at rates comparable to wild-type, but are self-sterile. Outcross progeny can be produced 

by crossing to wild-type males. Homozygous male spe-19 worms are fertile. Their sperm engage 

in sperm competition as do wild-type, and sire broods of comparable size to wild-type males. 

Our phenotypic analysis suggests that spe-19 functions in spermiogenesis (sperm activation). 

spe-19 mutant worms produce normal numbers of sperm but become sperm-depleted more 

quickly than wild type. Although they produce normal looking spermatids, these spermatids fail to 

complete spermiogenesis in vitro, with most arresting at the spiked intermediate stage. 

Unlike a number of well-characterized spermiogenesis-defective mutants in C. elegans, spe-19 

mutant hermaphrodites exhibit little or no spermatid activation when exposed to male seminal 

fluid, and thus spe-19 may encode a novel component of the spermiogenesis pathway. spe-19 

sterility is suppressed in spe-19 spe-6 double mutants, suggesting that spe-19 functions 

upstream of spe-6, in the same genetic pathway as spes 8, 12, 27 and 29.

117. Death defying acts: RNAi screen for genes influencing neuronal necrosis 

Beate Gerstbrein, Vienna Lo, Monica Driscoll 

Rutgers University, Dept. of Molecular Biology and Biochemistry, 604 Allison Road, Piscataway, 

NJ, 08855 

Understanding the molecular mechanisms of neuronal degeneration is a prerequisite for the 

development of successful pharmaceutical interventions to prevent or cure inappropriate cell 

death in conditions such as stroke or neuronal injury. C. elegans lends itself as a model for 

elaborating mechanisms of necrotic neuronal death because of the availibility of mutations that 

induce cell death with morphological characteristics of mammalian necrosis. Molecular 

requirements, such as the rise in intracellular calcium concentration and roles for calpains, are 

conserved from nematodes to humans. C. elegans necrosis is clearly distinct from apoptosis in 

both molecular mechanism and morphology. 

In the necrosis model we use most extensively, touch neurons undergo necrotic degeneration 

as a consequence of expression of a hyperactive ion channel, similar to excitotoxic cell death in 

mammals. Healthy touch receptor neurons sense external touch stimuli through a 

mechanosensory protein complex localized in the plasma membrane, which includes the ion 

channel subunit MEC-4. A specific amino acid substitution (MEC-4(d)) near the channel pore 

hyperactivates the channel and causes necrotic cell death, leaving worms insensitive to gentle 

touch. Besides mec-4(d) hyperactivating mutations affecting the G-protein subunit Galpha s, and 

the acetylcholine receptor subunit deg-3 also induce necrotic-like cell death. 

Our previous work has identified Ca 2+ -dependent proteins calreticulin and calpains as well as 

cathepsin proteases to be required for the efficient execution of mec-4(d)-induced cell death. We 

had not, however, conducted a saturation genetic screen for all mutations that could block 

mec-4(d) dependent death in the touch neurons (but see also abstract by Nunez et al., this 

volume). In order to identify additional genes needed for the efficient progression through 

necrosis, we knocked down the function of 2280 genes on chromosome I using the RNAi library 

constructed by Ahringer et al. We found that the reduction of function of about 40 genes 

decreases the extent of degeneration. We are testing the effect of RNAi-mediated knock down of 

the mec-4(d) suppressors on other cell death inducers, as well as their effect on oxidative stress 

and beta amyloid-mediated toxicity. This will reveal if the cell death suppressors identified in our 

screen are required for a "death pathway" common to different cell death inducers, and if they 

affect stress conditions other than necrotic injury. 

We have also tested specific genes/mutations affecing ion homeostasis for their involvement in 

mec-4(d) induced degeneration. We identified a K + - channel which, when mutated, functions as a 

strong cell death suppressor. The control of K + homeostasis plays an important role in the 

regulation of cell volume and is a key factor in apoptosis in higher organisms. We are 

investigating the effect of this mutation on different cell death inducers by itself and in 

combination with other cell death suppressors.

118. An in vivo analysis of age-related biomarkers in C. elegans 

Beate Gerstbrein 1 , Georgios Stamatas 2 , Nikiforos Kollias 2 , Monica Driscoll 1 

1Rutgers University, Dept. of Molecular Biology and Biochemistry, 604 Allison Road, Piscataway, 

NJ 

2Methods and Models Development, Johnson & Johnson, 199 Grandview Road, Skillman, NJ 

The rapidly growing elderly human population confronts an ever-increasing risk for several 

debilitating diseases, possible cognitive decline, and decline in muscle strength over time--all of 

which adds up to serious health and economic concerns for both the individual and society as a 

whole. A critical priority in aging research is thus to understand the basic biology of aging and to 

define strategies for the delay/prevention of components of age-related decline. Given the 

existence of several conserved mechanisms of aging, simple models such as C. elegans have 

become important tools for elaborating the basic biology of aging and might be exploited to 

provide insight into the problem of aging gracefully. 

Our recent focus is on defining the genetics of nematode healthspan, describing genetic 

interventions that enable animals to live healthier for longer, delaying the age-related decline 

phase. For this reason, we have been carefully analyzing biomarkers of aging. 

One "universal" feature of aging shared by nematodes and humans is the accumulation of 

autofluorescent compounds over time. In humans, a heterogenous group of such compounds, 

referred to as AGEs (advanced glycation endproducts), is also found in elevated concentrations 

in some neurodegenerative disease conditions and in diabetic patients, adding to complications 

associated with the disease. The chemistry of the generation of AGEs involves non-enzymatic 

reactions between sugar groups and proteins, resulting in modifications that impair protein 

function and turnover. 

We measured autofluorescence in live C. elegans using a fiber-optic probe coupled to a 

spectrofluorimetry. Living C. elegans displays a characteristic autofluorescence 

excitation/emission spectrum with 2 distinct major bands: The first one is the excitation/emission 

pair 290 nm/330 nm, which corresponds to the aromatic amino acid tryptophan (TRP peak). This 

peak value measures overall protein content. The second excitation/emission pair 340 nm/430 

nm can be attributed to lipofuscin and advanced glycation endproducts (AGE peak). 

Our data show that the TRP peak does not change significantly over time in adult worms, 

indicating that protein levels stay relatively constant in mature nematodes. In marked contrast 

however, AGE fluorescence increases over adult lifespan. The increase does not occur at a 

uniform rate, however. Rather, at a midlife timepoint of about 10 days of adulthood, there is a shift 

to an accelerated accumulation rate. 

We assayed how AGE fluorescence changes over time in the background of various 

lifespan-extending mutations. In age-1 mutants, the shift to accelerated accumulation is delayed 

for a few days whereas with daf-2, a generally low rate of accumulation is maintained. By 

contrast, daf-16 mutations markedly increase accumulation rates. However, not all short-lived 

nematodes accumulate high levels of AGE products--nematodes with shortened life span due to 

mutations in the respiratory chain did not show increased autofluorescence as compared to wild 

type. 

We also sorted same-age animals into groups that appear to move well and age well, to be in 

moderate decline, or to have aged very poorly (ABC locomotory classes described in Herndon et 

al, 2002). Unexpectedly, we found that class C animals have greatly enhanced levels of AGE 

products as compared to their same-age siblings that appear to have aged better. This indicates 

that AGE levels may report physiological aging rather than chronological age. In vivo AGE 

biomarker analysis may be used to screen for genetic influences on healthspan. 

We found the lowest AGE fluorescence in nematodes starved either by growth in liquid culture 

or by eat mutations. Interestingly, the AGE fluorescence maximum peak is shifted in starved 

worms, a result never observed in any of the numerous other experiments we performed. This 

suggests there may be a "signature" of the calorically restricted physiological state that could be 

used to genetically dissect controls on this important process.

119. Serotonin and octopamine modulate thrashing behavior of C elegans 

Rajarshi Ghosh, Scott W. Emmons 

Albert Einstein College of Medicine 1300, Morris Park Avenue Bronx New York 10461 

Certain mutants defective in male leaving behavior (these males, unlike wild type ones, do not 

leave a patch of food over a period of time) were also serotonin hypersensitive i.e. they got 

paralyzed in a 10mM solution of serotonin in M9 buffer. While studying the serotonin 

hypersensitivity at this concentration we found that ~70% of wild type worms were in a paralyzed 

state after 15 minutes of thrashing while 50% were in a paralyzed state after 20 minutes, implying 

that 20% of worms came out of paralysis state. We hypothesized that in liquid the worms might 

be going into a resting state and eventually coming out of it, and that this pattern is modulated by 

serotonin. To investigate such a phenomenon we studied thrashing in greater details. 

We found that in contrast to locomotion in agar plates, the body bends/20 seconds of wild type 

worms is ~ 4 times greater in M9. This is consistent with a previous observation that the worms 

exhibited a forward bias in their movement i.e. waves passed backwards ~4 times more 

frequently in M9 than when worms were moving on agar plates (Croll, 1975). Most interestingly, 

we observed that this thrashing pattern was interrupted by bouts of rest periods (~6-7 minutes), 

which for the wild type worm occurred after a period of ~ 50 minutes of thrashing from the time 

they were put in M9. The worms then come out of the resting state and continued thrashing. 

Given the striking differences in locomotion we wondered what the neural correlate of transition 

from slow (on plates) to fast (in M9) movement might be and what genes might be involved in 

such a process? In addition we also wanted to address the question of how such a behavior 

comes to a halt and then resumes again? 

To answer these questions we devised an assay where single worms were put into micro titer 

wells containing 200microliters of M9 buffer and watched every minute for 60 minutes and their 

thrash/rest pattern were recorded. We noted several interesting properties of thrashing. 

We found that the rearing temperature has no obvious detectable influence on the thrash/rest 

cycle but the assay temperature is critical for the wild type thrash/rest pattern. By using sensory 

pathway mutants like che-2(e1033), osm-5(p118) and unc-86(sm117) we found that sensory 

input is essential for exhibiting wild type thrashing behavior. These mutants show very slow 

thrashing and the wild-type thrash-rest pattern is remarkably disrupted. 

We also found that the wild type pattern of thrash/rest cycle is disrupted by exogenous 

serotonin and octopamine. Wild type worms go more frequently into paralysis with increasing 

concentration of exogenous serotonin. Octopamine has the opposite effect i.e. it increases the 

thrash periods but has a moderate effect on paralysis periods. We found that like the null allele, a 

novel, mis-sense allele of mod-5 (the serotonin reuptake transporter in C.elegans.) exhibits 

defective thrashing behavior. Interestingly the thrashing defect of the mis-sense mutation can be 

supressed by exogenous octopamine. We are characterizing the genetic complexity of mod-5 

locus with respect to thrashing behavior. Using mutants in the serotonergic pathway we also 

found that serotonin is required for wild-type thrash- rest cycle and excess serotonin tend to 

disrupt this pattern. We are characterizing the modulation of thrashing behavior by serotonin and 

octopamine in greater details.

120. Functional Characterization of the Vertebrate Homologs of LIN-10: Mint 1, 2, and 3 

Doreen R. Glodowski 1 , Bonnie L. Firestein 2 , Christopher Rongo 1 

1Waksman Institute, Rutgers University, 190 Frelinghuysen Rd, Piscataway, NJ 08854 

2Dept. of Cell Biology and Neuroscience, Rutgers University, 604 Allison Rd, Piscataway, NJ 

08854 

The C. elegans protein, LIN-10, functions in neurons to direct postsynaptic localization of 

AMPA-type glutamate receptors (AMPARs) and in epithelial cells to direct basolateral localization 

of the epidermal growth factor receptor (EGFR) protein, LET-23 1,2 . There are three vertebrate 

homologs of LIN-10: Mint 1, 2, and 3. Sequence comparison between LIN-10 and the Mint 

proteins, which revealed the presence of a highly divergent amino terminal region, led to the 

hypothesis that LIN-10 evolved in higher eukaryotes into a family of proteins with distinct cellular 

functions mediated by their amino terminal domains. To test this hypothesis, the activities of each 

Mint protein were assayed in lin-10 mutant strains of C. elegans, which display distinct 

phenotypes for mislocalization of AMPARs and EGFRs. Significantly, of the Mint proteins, only 

Mint 2 rescued mislocalization of AMPARs, demonstrating that the Mint proteins have distinct 

activities. Experiments testing the ability of each Mint protein to rescue mislocalization of EGFRs 

are ongoing and results will be presented. Further functional characterization of the Mint proteins 

is being performed in a vertebrate system by observing the effects of altered expression of Mint 1 

and/or Mint 2 in cultured hippocampal neurons. Preliminary experiments show that a 

snRNA-mediated approach 3 can be used to attenuate expression of Mint 1 and/or Mint 2 in 

cultured hippocampal neurons. 

1. Rongo, C., Whitfield, C.W., Rodal, A., Kim, S.K., and Kaplan, J.M. (1998) Cell: 94, 751-759. 

2. Whitfield, C.W., Benard, C., Barnes, T., Hekimi, S., and Kim, S.K. (1999) Mol Biol Cell: 10, 

2087-2100. 

3. Akum, B.F., Chen, M., Gunderson, S.I., Riefler, G.M., Scerri-Hansen, M.M., and Firestein, 

B.L. (2004) Nature Neurosci: 2, 145-152.

121. The Myt1 ortholog in C. elegans is essential for oocyte maturation. 

Andy Golden 

Laboratory of Biochemistry and Genetics, NIDDK, NIH, Bethesda, Maryland 20892 

Myt1, a member of the WEE1 kinase family, is an essential negative regulator of the 

CDK/cyclin complex that acts during the G2/M transition of the meiotic cell cycle in Xenopus 

oocytes. Using RNA interference (RNAi), we have shown C. elegans oocytes depleted of the 

Myt1 homolog, WEE-1.3, fail to develop and mature normally within the oviduct of the 

hermaphrodite. At the earliest stages of WEE-1.3 depletion, the oocytes appear to mature 

precociously. Despite this precocious maturation, oocytes are ovulated normally. However, such 

WEE-1.3-depleted oocytes are fertilization-incompetent in the presence of wild-type sperm and 

appear to remain in an extended M-phase state within the uterus. With longer exposure to 

wee-1.3 RNAi, the ability of the hermaphrodite germline to produce normal oocytes becomes 

greatly compromised. Our explanation is that CDK-1, the presumed WEE-1.3 target, is an 

abundant maternal protein that must to be kept inactive during critical phases of oocyte 

development. Presumably, in the absence of WEE-1.3, CDK-1 is precociously activated in 

immature oocytes. This model is strengthened by our observation that CDK-1 depletion 

suppresses the wee-1.3 RNAi maturation/fertilization defects. Our experimental results confirm 

that WEE-1.3 acts through CDK-1 in the proper regulation of the G2/M transition during C. 

elegans oocyte development and maturation.

122. RecQ Helicases, Genomic Stability and Lifespan in C. elegans 

Melissa M. Grabowski, Nenad Svrzikapa, Heidi Tissenbaum 

Program in Gene Function and Expression, University of Massachusetts Medical School, Aaron 

Lazare Research Building, 364 Plantation Street, Worcester MA 01605 

Bloom and Werner syndrome are heritable syndromes caused by mutations in the RecQ 

helicases BLM and WRN respectively. While humans have 5 RecQ helicases (three of which are 

responsible for disease phenotypes characterized by genomic instability), C. elegans has 4 RecQ 

helicase family memebers. To investigate the relationship between genomic stability and 

organismal lifespan we are studying the RecQ helicase family in C. elegans . 

The four members of the C. elegans RecQ family are T04A11.6 (him-6), F18C5.3 (wrn-1), 

E03A3.2 (rcq-5), and K02F3.1. HIM-6 is homologous to human BLM, and WRN-1 is homolgous to 

human WRN. The remaining two helicases RCQ-5 and K02F3.1 are homologous to human 

RecQL and RecQL5 respectively. We have obtained knock out strains of all four C. elegans 

RecQ helicases (him-6, wrn-1, rcq-5, K02F3.1) from the CGC. 

We are currently characterizing the phenotypes of the deletion strains. Preliminary results 

indicate wrn-1 and rcq-5 show no effect on brood size but him-6 shows a significant decrease in 

brood size. Interestingly, thus far single mutations of these genes seem to have no significant 

effect on lifespan. In the case of him-6 this may be due to the fact that at least 50% of the 

progeny die as embryos and we are only able to perform life spans on the surviving worms. We 

are currently examining the effects of X-ray exposure and the role of these genes during S-phase. 

These studies should provide further insight into the role of genomic instability and aging.

123. The temporal patterning microRNA let-7 controls multiple transcription factors 

including the nuclear hormone receptor DAF-12 

Helge Grosshans, Ted Johnson, Mark Gerstein, Frank J. Slack 

Yale University, New Haven, CT 06520-8103, USA. Email: helge.grosshans@yale.edu 

The C. elegans let-7 noncoding RNA is a member of the large family of microRNAs (miRNAs), 

which posttranscriptionally regulate gene expression in plants and animals. let-7 is 

phylogenetically conserved and its expression is temporally regulated in many animals. In C. 

elegans, let-7 controls terminal differentiation in the seam cells as a part of the heterochronic 

temporal patterning pathway. To understand the role of let-7 in temporal control of worm 

development, we used a combination of computational sequence analysis and reverse genetics 

to identify and validate new target genes of the let-7miRNA. Strikingly, the DAF-12 nuclear 

hormone receptor, which has previously been reported to act upstream of let-7 (A. Antebi et al., 

1998), is among the newly identified targets. We show that daf-12 is epistatic to let-7 and, 

importantly, that its expression is under posttranscriptional control, mediated by its 3’ untranslated 

region and let-7. A strong enrichment of transcription factors among the nine novel let-7 targets 

supports the notion that let-7 acts as master regulator of temporal patterning, allowing the cell to 

connect miRNA-mediated translational control to transcriptional control of a larger number of 

more down-stream genes.

124. The genes tra-4 and mog-7 are necessary to ensure hermaphrodite development in 

the soma and in the germline 

Phillip Grote, Claudia Huber, Barbara Conradt 

Department of Genetics, Dartmouth Medical School, 7400 Remsen, Hanover, NH USA 

During C. elegans development, the decision to develop into a hermaphrodite (XX) or a male 

(XO) is determined by the ratio of the number of X chromosomes to the number of sets of 

autosomes (X:A ratio). In the soma the signal induced by this ratio is transmitted through a 

cascade of interacting genes, which determines the activity of the most downstream factor of this 

cascade, the transcription factor TRA-1A (tra, transformer). The same genes are involved in 

determining sexual fate in the germline; however, additional genes are required for this process. 

The determination of the sexual fate in the germline is a complex regulatory process because 

self-fertilizing hermaphrodites produce sperm during the last larval stage (L4) but have to switch 

to the production of oocytes after becoming adults. 

Strong loss-of-function (lf) mutations in genes required for feminization (tra genes) can lead to 

the development of completely transformed animals: XX animals develop into phenotypic males. 

Weak lf mutations in tra genes can cause a weak masculinization: XX animals lack the 

hermaphrodite-specific neurons (HSNs), have the male-specific cephalic companion neurons 

(CEMs) and show additional defects indicative of masculinization. In a forward genetic screen 

originally designed to identify factors involved in the regulation of the sexually dimorphic death of 

the CEMs, we identified a gene, which when mutated causes a weak masculinization of XX 

animals, suggesting that it is required for feminization. We therefore named this gene tra-4. tra-4 

encodes a protein with seven C2H2 type zinc-finger domains, which are known to interact with 

DNA and/or RNA. Furthermore we identified a homologue of tra-4 in the C. elegans genome. 

Hermaphrodites homozygous for a lf mutation in the tra-4 homologue fail to switch from sperm 

production to oocyte production after the L4 stage. We therefore refer to this gene as mog-7 

(mog, male-only gonad). 

We are currently investigating the roles of tra-4 and mog-7 in the regulation of sex 

determination in the soma and in the germline, respectively. We propose that tra-4 is necessary 

for female development in the soma and that mog-7 fulfills a similar function in the germline. 

Furthermore, while tra4 and mog-7 seem to play similar but independent roles in sex 

determination, they appear to have a redundant function in an essential process, since tra-4(lf); 

mog-7(lf) double mutants are non-viable.

125. SMA-10 is a novel extracellular regulator of the Sma/Mab TGF-beta pathway in C. 

elegans 

Tina L. Gumienny, Cole M. Zimmerman, Andrew F. Roberts, Huang Wang, Lena Chin, Richard 

W. Padgett 

Waksman Institute, Rutgers University, 190 Frelinghuysen Road, Piscataway, New Jersey 

08854-8020 

Body size in C. elegans is regulated by a TGF-beta-like signaling pathway that controls both 

animal length (Sma) and male tail development (Mab). The Sma/Mab pathway is activated when 

its ligand, DBL-1, binds to a heteromeric complex of transmembrane receptor serine kinases 

(RSKs), DAF-4 and SMA-6, resulting in phosphorylation and nuclear accumulation of 

transcriptional regulators known as Smads, SMA-2, SMA-3, and SMA-4. While the ligand DBL-1 

is expressed in nerve cells, the focus of this pathway appears to act in the hypodermis, where the 

receptors and Smads are required. 

We isolated five alleles of sma-10 in a screen to identify novel members of this pathway. 

Mutant animals range in size from 60 to 75% the length of wild-type worms. Genetic analyses 

indicate that it functions between dbl-1 (ligand) and sma-6 (receptor), suggesting that SMA-10 

may function at the cell surface, upstream of SMA-6. 

To further elucidate its molecular function, we have cloned sma-10 using SNP mapping and 

germline transformation rescue. sma-10 encodes a protein with several leucine-rich repeats, 3 

IG-like domains, a single transmembrane domain, and an intracellular domain of only 19 amino 

acids. This structure suggests that SMA-10 and its homologs may function as extracellular 

cell-surface binding proteins for TGF-beta-related ligands or receptors. Predicted proteins with 

significant similarity to SMA-10 have been identified in animal genomes from fly to human, but 

none of these provides any additional insight into the possible function of SMA-10. The 

Drosophila gene rescues the phenotype of sma-10(lf). Notably, the gene for a closely related 

human protein has been mapped to a region that is deleted in several tumor lines, similar to other 

members of TGF signaling pathways that function as tumor suppressors. 

The rescuing translational fusion sma-10p::sma-10:gfp fluoresces brightly in the pharynx and 

weakly in the hypodermis. However, rescue of the body size defect is achieved with a 

hypodermal promoter. 

From these results, we propose that SMA-10 is a novel, conserved positive regulator of 

TGF-beta signaling.

126. LON-2 is a glypican heparan sulfate proteoglycan that regulates the Sma/Mab 

TGF-beta pathway in C. elegans 

Tina L. Gumienny, Huang Wang, Richard W. Padgett 

Waksman Institute, Rutgers University, 190 Frelinghuysen Road, Piscataway, New Jersey 

08854-8020 

The TGF-beta growth factor superfamily and its downstream core signaling components are 

found in all multicellular animal phyla studied, from nematodes to mammals, and are responsible 

for controlling several different developmental processes. They regulate pathways controlling cell 

cycle progression, apoptosis and immune functions, and suppress some types of cell growth. 

Because of such roles, they are frequently mutated in metastases. While the core components of 

this pathway have been identified and well-analyzed, the extracellular regulators of this pathway 

are not as well understood. 

Our lab focuses on the TGF-beta pathway that regulates body size and male tail ray 

development (the Sma/Mab pathway). Mutations in this pathway that prevent signal transduction 

result in small animals. lon-2(lf) mutant animals, however, are longer than the wild type. 

lon-2 acts genetically upstream of the TGF-beta superfamily member dbl-1. The protein 

contains the sequence motifs characteristic of glypican heparan sulfate proteoglycans, including 

an N-terminal signal sequence, 12 of the 14 conserved cysteines, a glycosaminoglycan 

attachment site, and a glycosyl phosphatidylinositol (GPI) linkage site. Unique to LON-2, though, 

is an RGD integrin-binding motif. It is closest in sequence homology to Drosophila dally (division 

abnormally delayed ) and mammalian Glypican-3, which have been shown to modulate TGF-beta 

superfamily member signaling. dally rescues the Lon phenotype of lon-2(lf) animals. The lon-2 

promoter driving GFP expresses in the intestine, as do the TGF-beta receptor genes sma-6 and 

daf-4 and the Smad gene sma-3 (though none is required in the intestine). However, a rescuing 

LON-2 translational fusion with GFP fluoresces in neurons in the head and ventral nerve cord, 

similar to dbl-1. 

Based on genetic, sequence, and functional analyses, we propose that LON-2 acts at the 

extracellular surface to negatively regulate DBL-1 signaling.

127. Reproductive isolation of C. briggsae haplotypes. 

Rachael M. Hampton, Scott E. Baird 

Department of Biological Sciences, Wright State University, Dayton OH 

Sequence comparisons of Cb-glp-1, Cb-tra-2, and Cb-COII have shown that C. briggsae 

strains tend to cluster into two distinct haplotypes with strains AF16 and VT847 comprising 

haplotype I and strains HK104, HK105, and PB800 grouping into a separate haplotype. We have 

confirmed this haplotype structure through sequence analyses at eleven additional nuclear loci for 

these strains. Evidence for reproductive isolation between haplotypes I and II has also been 

obtained from reciprocal crosses between strains AF16 and HK104. The intrinsic rate of increase, 

or r max, was used as a measure of fitness and the reproductive schedules of F1 and F2 

hermaphrodites were determined. Approximately one third of all F2 hybrids exhibit up to a 25% 

reduction in r max relative to the parental and F1 values. This decrease in fitness results from a 

delay in development. High frequencies of delayed F2s are generally obtained between AF16 

(haplotype I) and any strain in haplotype II. Loss of fitness in F2 hybrids is expected to result from 

dysgenic interactions among alleles in two or more genes, one of which is linked to Cb-egl-5. 

Recombinant inbred lines from AF16 and HK104 crosses selecting for rapid development have 

shown a significant skewing of the HK104 allele for Cb-egl-5 (p = 0.00016). The association of 

the AF16 allele of Cb-egl-5 with a loss of fitness has also been confirmed by directly genotyping 

23 delayed F2s. Of these, 14 were homozygous for AF16 and 9 were heterozygous. Additional 

genes involved in these interactions will be identified through bulk segregant analysis.

128. High Throughput TILLING, Ecotilling, Genotyping, and Sequencing Instrumentation 

Jeff Harford 1,2 

1LI-COR Biosciences, 4308 Progressive Avenue, Lincoln, NE 68504 

2email: jharford@licor.com 

Targeting Induced Local Lesions In Genomes, or TILLING R , is a novel high throughput 

technology for reverse genetics. TILLING uses chemical mutagenesis to yield a traditional allelic 

series of point mutations for virtually all genes. TILLING is of particular value for essential genes 

where sublethal alleles are required for phenotypic analysis. 

TILLING has become an established technique on many model organisms, including C. 

Elegans, Arabidopsis, Zebrafish, Maize, Rice, and others. 

A variation on TILLING is called Ecotilling, in which high throughput SNP discovery is 

performed by locating natural variations throughout the genome. 

LI-COR Biosciences is a manufacturer of DNA Analysis Instrumentation for high throughput 

TILLING and Ecotilling, as well as for DNA Sequencing, AFLP R , and Microsatellite analysis. 

LI-COR also manufactures instrumentation for infrared fluorescent protein imaging, offering 

superior quantification over standard chemiluminescence. 

Stop by our booth to pick up the latest literature and publications on our products. 

TILLING is a registered trademark of Anawah, Inc. AFLP is a registered trademark of 

KeyGene, N.V.

129. Analysis of synMuv Protein Complexes in vivo and Characterization of the Class B 

synMuv Gene lin-61 

Melissa M Harrison, Xiaowei Lu, Bob Horvitz 


Vulval induction in C. elegans is negatively regulated by at least three redundant functions 

provided by the synthetic Multivulva (synMuv) class A, B, and C genes. Loss-of-function 

mutations in members of any single class do not result in a Multivulva (Muv) phenotype, but 

animals with mutations in any two synMuv classes are Muv. The molecularly characterized 

synMuv class A genes encode novel proteins. Many of the class B and class C synMuv genes, 

including lin-35 Rb, dpl-1 DP, efl-1 E2F, lin-53 RbAp48, hda-1 HDAC, let-418 Mi-2, trr-1 TRRAP, 

mys-1 HAT, and epc-1 E(Pc), have homologs in other species that are involved in chromatin 

remodeling and transcriptional modulation. 

Some of the proteins that act in the synMuv B pathway have been shown to physically interact 

in vitro (1,2), in yeast two-hybrid assays (3), or in vivo based on co-immunoprecipitation 

experiments (4). These results along with homology to proteins in other species suggest that 

many synMuv proteins may function together in transcriptional regulatory complexes. We are 

using co-immunoprecipitation experiments to further explore in vivo physical interactions among 

the synMuv proteins. Such studies may allow us to better understand the functions of novel 

synMuv proteins, to analyze the effects of various synMuv mutations on physical interactions, and 

to identify potential sub-complexes among the synMuv proteins. We have focused initially on the 

gene products of the class B synMuv genes lin-37 and lin-61 as well as the class A synMuv gene 

lin-56,since null mutants in these genes are viable and can serve as negative controls for the 

immunoprecipitation experiments. LIN-37 is a small hydrophobic protein with no known homologs 

outside of nematodes. LIN-61 contains four MBT (malignant brain tumor) repeats, which are 

loosely defined sequences of approximately 100 amino acids found in a number of nuclear 

proteins including the Drosophila Polycomb group protein Sex Comb on Midleg. LIN-56 is a 

novel acidic protein with no canonical motifs. We have surveyed the interactions of these 

proteins with a number of class A and B synMuv proteins by immunoprecipitation followed by 

western blots and shown that a subset of class B proteins co-immunoprecipitate from embryo 

extract. We are developing reagents to perform immunoprecipitation studies of other synMuv 

proteins. We also hope to identify new proteins involved in vulval development on the basis of 

their interaction with the synMuv proteins by co-immunoprecipitations followed by mass 

spectrometry. 

We are also characterizing the class B synMuv gene lin-61 in detail as mutations in this gene 

cause a number of pleiotropic defects that differ from those of most other class B synMuv 

mutants. lin-61 loss-of-function alleles cause GFP transgene misexpression (see abstract by 

Schwartz, Wendell, and Horvitz), and Pothof et al. have shown that RNAi of lin-61 results in an 

increased mutation rate (5). We have made polyclonal antibodies that recognize LIN-61 and 

have shown that it is a ubiquitously expressed nuclear protein that is localized to condensed 

chromosomes in the germline. We have also shown that this localization remains unchanged in 

a large number of synMuv mutant backgrounds. 

(1) Ceol and Horvitz. Mol. Cell 7: 461-473, 2001. 

(2) Lu and Horvitz. Cell 95: 981-991, 1998. 

(3) Walhout et al. Science 287: 116-122, 2000. 

(4) Unihavaithaya et al. Cell 111: 991-1002, 2002. 

(5) Pothof et al. Genes Dev. 17:443-448, 2003.

130. Visualizing activity of C. elegans interneurons 

Gal Haspel, Anne C. Hart 

MGH Cancer Center, 149-7202 13th Street, Charlestown, MA 02129 

The location and anatomical connectivity of all the neurons in the adult C. elegans have been 

determined. This affords us the opportunity to address the neural circuitry underlying various 

behaviors. Specifically, we are interested in the rhythmic pattern of locomotion, its generation and 

its modulation by sensory input. Eight interneurons (4 pairs: AVA, AVB, AVD and PVC) have 

been associated, mostly by ablation studies, with locomotion and are suggested to comprise a 

central pattern generator (CPG) for locomotion. CPGs are neuronal networks that when activated 

can generate a rhythmic motor output without sensory or other input that carries specific timing 

information. CPGs underlie many rhythmic behaviors including breathing, digestion and 

locomotion. However, the generation of locomotive pattern in C. elegans might reside in a 

sensory feedback loop, in the motoneurons or even in the body wall muscles rather than in a 

central pattern generator. Recording the activity of the locomotory interneurons may directly 

answer this question. Simultaneously recording from the interneurons and their presynaptic 

sensory neurons (ASH) will allow us to address the synaptic efficacy of neuronal connections. 

To record activity from C. elegans neurons, we are expressing genetically encoded activity 

probes sensitive to calcium levels or the membrane potential. Cameleon is used to measure 

changes in calcium levels. It is composed of the calcium-binding domain of calmodulin attached 

to a pair of cyan and yellow fluorophores. In response to the binding of calcium, the voltage 

sensor alters the amount of fluorescence resonance energy transfer (FRET) between the 

fluorophores. Cameleons were previously used by Schafer et al to measure calcium levels in 

pharyngeal muscles and ASH neurons. Alternatively, we plan to use the Voltage Sensitive 

Fluorescent Protein 1 (VSFP1), another FRET-based probe, to measure changes in membrane 

potential. In VSFP1, a pair of cyan and yellow fluorophores is attached to the voltage-sensing 

domain of a potassium channel and FRET is affected by a change in the membrane potential. 

Optical recording of neuronal activity should allow us to directly address the function and 

modulation of the locomotion circuit, which is central to many C. elegans behaviors.

131. Multiple factors act in concert to initiate the cell death of the NSM sister cells 

Julia Hatzold, Barbara Conradt 

Dartmouth Medical School, Department of Genetics, 7400 Remsen Bldg., Hanover, NH 03755, 

USA 

During the development of a C. elegans hermaphrodite, 131 of the 1090 cells generated die 

due to programmed cell death, an important process conserved throughout the animal kingdom. 

Although a genetic pathway for programmed cell death has been established in C. elegans, not 

much is known about the signals that trigger cell death in cells destined to die. One particular 

cell-death event, the death of the NSM sister cell, occurs about 430 min after the first division of 

the zygote, just 20 min after its progenitor cell has undergone an asymmetric cell division. The 

sister of the NSM sister cell, the NSM, however, survives and differentiates into a serotonergic 

neuron located in the pharynx. We have previously shown that the cell-death activator egl-1 is 

expressed in the NSM sister cell, which is destined to die, but not in the surviving NSM, and that 

the NSM sister cell death is at least partially dependent on the activity of hlh-2 and hlh-3, which 

encode bHLH transcription factors. Our results suggest that a heterodimer composed of HLH-2 

and HLH 3 directly activates egl-1 expression by binding to a specific cis-regulatory region of the 

egl-1 locus. 

A weak hlh-2 loss-of function mutation, bx108, or a strong hlh-3 loss-of function mutation, 

bc248, only cause about 5% of the NSM sister cells to survive. In hlh-2(bx108); hlh-3(bc248) 

animals, however, 30% of the NSM sister cells survive, which suggests that hlh-2 and hlh-3 act 

together to kill the NSM sister cell. Nonetheless, these results indicate that additional factors 

might contribute to the NSM sister cell death. In order to identify these additional factors, we 

performed a forward genetic screen. In specific, we screened for mutations that enhance the 

NSM sister cell survival caused by hlh-2(bx108). We screened about 2,000 haploid genomes, and 

identified 14 mutations that significantly enhanced the hlh-2(bx108) phenotype. While four 

mutations block cell death in general, 10 mutations specifically cause the inappropriate survival of 

the NSM sister cells. Two of the 10 mutations are new loss-of-function mutations of ces-2, a gene 

previously shown to be required for the NSM sister cell death; the remaining eight mutations 

define at least five genes not previously implicated in this cell-death event. Most of the eight 

mutants exhibit a phenotype that is dependent on hlh-2(bx108). The cloning and characterization 

of the newly identified genes might elucidate new factors and mechanisms participating in the 

NSM sister cell death, such as direct or indirect regulators of egl-1 expression or factors involved 

in establishing asymmetry in the NSM progenitor cell.

132. An RNAi-based suppressor screen for components of the Aurora B kinase pathway 

Todd R. Heallen, Jill M. Schumacher 

Program in Genes and Development, Dept. of Molecular Genetics, UT-MD Anderson Cancer 

Center, Houston, TX 77030 

Proper execution of mitosis requires accurate segregation of newly replicated DNA into each of 

two newly formed cells. Genetic instability can be caused not only by defects in the segregation 

process itself, but also by failures in cytokinesis that can lead to the formation of aneuploid cells. 

Aneuploidy is seen in a variety of cancer types, and is associated with aberrant regulation of the 

cell cycle. Our studies focus on a highly conserved family of proteins, the Aurora kinases. AIR-2, 

the C. elegans homolog of Aurora B, is required for accurate chromosome segregation and 

cytokinesis during meiosis and mitosis (Schumacher et. al., 1998). Depletion of AIR-2 via 

RNA-mediated interference (RNAi) leads to embryonic lethality and characteristic unicellular 

polyploid embryos. Furthermore, a temperature-sensitive (ts) mutant strain, air-2(or207ts), 

phenocopies air-2(RNAi) embryos at the restrictive temperature (26°C) (Severson et. al., 2000). 

Our current goal is to identify components of the AIR-2 regulatory pathway that may mediate 

genome stability. We are utilizing a genome-wide RNAi-based screen to identify suppressors of 

air-2(or207ts) ts lethality by selecting for RNAi constructs that support the production of hatched, 

viable progeny at the restrictive temperature. Partial suppression is monitored by screening for 

multi-cellular embryos that bypass the air-2(or207ts) one-cell arrest. When suppression is 

observed, candidate genes are retested, and functional analyses then performed to determine the 

molecular relationship between these genes and air-2. To date, Chromosomes I-IV have been 

screened, and several candidate suppressors have been uncovered. These candidates are 

currently being analyzed at a semi-permissive temperature (20ºC) to ascertain the degree of 

air-2(or207ts) suppression. 

The Aurora kinases are likely to contribute to oncogenesis as human Aurora A and Aurora B 

are overexpressed in several cancer cell lines (Adams et. al., 2001). By identifying components of 

the AIR-2 regulatory pathway, we hope to contribute to the overall understanding of the cell cycle 

and learn how cells operate under normal conditions. This knowledge may then be used to 

generate novel directed therapies for many types of cancer.

133. Characterization of tissue-specific suppressors of cdc-25.1(gf). 

Michael Hebeisen, Roshni Basu, Richard Roy 

Department of Biology, McGill University, Montreal, Quebec, Canada 

We previously characterized a dominant, gain-of-function mutation in the positive cell cycle 

regulator cdc-25.1 phosphatase that gives rise to an ’’extra intestinal cell’’ phenotype that occurs 

during embryogenesis. This mutation causes an amino-acid substitution in a highly conserved 

putative F-box recruitment motif (DSGXXXS) that may target the CDC-25.1 phosphatase for 

SCF-mediated ubiquitination and subsequent proteasomal degradation. The perdurance of 

CDC-25.1(gf) detected in all embryonic blastomeres past the 40 cell stage is consistent with this 

notion, yet only the intestine is affected. The basis for this tissue-specificity remains unknown. 

To address this problem of tissue specificity, we performed a modifier screen to identify 

suppressors of the cdc-25.1(gf) intestinal phenotype. One intragenic and four extragenic 

suppressor mutations were isolated, each of which represents an individual locus, behaves 

recessively, and demonstrates a maternal effect. Among them, rr38 and rr40 showed the 

greatest penetrance, whereby in both cases about 80% of the worms displayed obvious 

suppression, of which 36% and 30% reverted to wild-type intestinal cell number, respectively. 

By performing lineage analysis with intestinal markers, we demonstrated that the suppression 

of the intestinal cell cycle defect associated with rr38 and rr40 is specific to the 

cdc-25.1(gf)-associated supernumerary embryonic division. Furthermore, neither of the 

suppressors showed a phenotype in the absence of cdc-25.1(gf), underscoring their specificity for 

this gfmutation. 

To see if a cdc-25.1(gf) extra-intestinal cell phenotype could be autonomously reproduced with 

a cdc-25.1 reporter driven specifically in the endoderm, we expressed a wild-type and 

gain-of-function gfp::cdc-25.1 translational fusion under the control of the end-3 GATA factor 

promoter. end-3 expression is temporally restricted to the early E lineage (from the 2E to the 8E 

stage) to specify the endodermal fate. While the GFP::CDC-25.1(wt) protein was not detectable 

after the 8E stage, the GFP::CDC-25.1(gf) protein perdured until the 32E stage phenocopying the 

cell cycle defect seen in cdc-25.1(gf) mutants. 

Interestingly, the cdc-25.1(gf) allele is also temperature sensitive, suggesting that the stability 

of the mutant protein may be affected by both temperature and the suppressor mutations. To test 

both destabilization effects, worms containing the end-3::gfp::cdc-25.1(gf) array will be put at 

25 o C or crossed with the suppressor mutations and the stability of the protein will be detected by 

means of GFP expression. 

Meiotic mapping indicated rr38 to be tightly linked to unc-29 on chromosome I, three-factor 

crosses are under way to refine its position. The mapping data for rr40 placed this suppressor to 

the left of unc-32 on chromosome III. A systematic analysis of the predicted genes in this region 

is currently in progress using the feeding RNAi library, in order to identify candidates that 

phenocopy rr38 and rr40 in a cdc-25.1(gf) background.

134. Form and function of glia-neuron interactions 

Maxwell G. Heiman, Shai Shaham 

Rockefeller University, Box 46, 1230 York Ave., New York NY 10021 

Glia are the primary source of brain tumors and are the most abundant cells in the brain, but 

their functions remain mysterious. To understand how glia interact with neurons and regulate 

neuronal behavior, we are studying the amphid sheath cell of Caenorhabditis elegans as a model 

glial cell. Like vertebrate glial cells, the amphid sheath cell is derived from a neuronal lineage, 

physically associates with neurons, and provides functions required for neuronal activity. 

The amphid, a sensory organ in the head of the worm, consists of 12 neurons and two glial-like 

cells, the sheath and the socket. The neurons project unbranched dendrites to an opening formed 

by the socket cell at the head of the worm. The sheath cell forms a process collateral with these 

dendrites, about 100 microns long in an adult animal, which terminates in a specialized structure 

that completely ensheathes all the dendritic endings. 

To identify genes that control the differentiation and morphogenesis of the glial cell and its 

association with neurons, we performed a genetic screen for mutants defective in glial 

development. We created a transgenic strain expressing fluorescent reporter genes in the amphid 

sheath cell and two amphid neurons. We performed a non-clonal visual screen and isolated a 

new class of mutants in which both the amphid sheath cell and the associated neuronal dendrites 

extend only a fraction of the wild-type length. Preliminary analysis suggests the glial cell may be 

required for dendrite extension. 

In a complementary approach, we are studying what signals the sheath cell supplies to the 

neurons. In wild-type animals, the sheath cell deposits an electron-dense matrix around the 

dendrites it ensheathes, while in che-12 mutants this matrix is severely lessened and neuronal 

functions are disrupted. To gain insight into the secretory pathway that connects the glial cell to 

neurons, we are fine mapping the che-12 mutation to identify the relevant gene. Additionally, we 

are attempting to isolate directly factors secreted by the amphid sheath cell.

135. Vulval and uterine development are not temporally coordinated in ku212 mutants 

Li Huang, Wendy Hanna-Rose 

406 Althouse Lab, Pennsylvania State University,University Park,16802 

We isolated ku212 in screens for Egl mutants with abnormal vulval morphology and named the 

corresponding gene cog-3 (connection of gonad defective) because no proper connection 

between the vulval and uterine lumens is formed in most mutant hermaphrodites, leading to the 

detected morphology defect at the apex of the vulva. Approximately 77% of ku212 mutants are 

Egl, 17% are Egl+ and 6% are sterile and, thus, cannot be evaluated for egg-laying. The gross 

morphology of the uterus is normal and vulva morphogenesis proceeds as expected until 

formation of the connection of the vulval lumen with the uterine lumen at a late stage of 

morphogenesis. However, the temporal control of vulval and uterine development is not 

coordinated in ku212 animals, with uterine development being delayed relative to that of the vulva 

in 92 % of animals. Thus, ku212 appears to have a heterochronic defect, and we speculate that 

loss of synchronism between vulval and uterine development contributes to the Egl phenotype. 

Our analysis is consistent with retarded uterine development as opposed to precocious vulval 

development. We are investigating ku212 to add to our understanding of the temporal control of 

gonad development. 

In addition to the Cog defect, ku212 animals also have a reduced brood size. Although the 

reduction in brood size for Egl animals is expected due to the shortened reproductive life of the 

mother, we are still investigating the cause of the low brood size (~90 progeny) in the Egl+ 

animals. We have determined that there is no embryonic lethality associated with the ku212 

mutation. Furthermore, cuticular structures and epicuticle formation is perturbed in ku212 

mutants. ku212 adults have the normal adult alae during early adulthood. However, older adults 

have alae that are L1-like in appearance. The animals are also lipophilic and have a "weak" 

cuticle that does not recover from puncture after injection. The weak, lipophilic cuticular defect is 

dominant whereas other phenotypes are recessive. We will report on our continued phenotypic 

analysis of ku212 as well as progress on molecular characterization.

136. COMPUTER MODELING, SIMULATION AND ANALYSIS OF C. elegans VULVAL 

INDUCTION 

Na’aman Kam 1,2 , Jasmin Fisher 1 , David Harel 1 , Amir Pnueli 1 , Michael J. Stern 3 , E. Jane 

Albert Hubbard 4 

1 Dept. of Computer Science & Applied Mathematics, The Weizmann Institute of Science, 

Rehovot, ISRAEL 

2 http://www.wisdom.weizmann.ac.il/~kam/CelegansModel/CelegansModel.htm 

3 Dept. of Genetics, Yale University, New Haven, CT, USA 

4 Dept. of Biology, New York University, New York, NY, USA 

We have been developing a new approach to modeling biological phenomena, focusing on the 

signaling events that influence vulval fate specification in C. elegans. Our motivation is the 

striking similarity between the methods and logic of developmental genetics with the languages 

and tools used in the design of complex computerized systems. Vulval precursor cell (VPC) fate 

specification is an excellent system to test our approach for two reasons: (1) the mechanisms 

underlying this process are sufficiently understood to allow meaningful models to be created; and 

(2) the multiple known inputs that determine fate output create a complexity that can adequately 

challenge the efficacy of the modeling approach. 

Developmental genetic data are difficult to make tractable for simulation and analysis by 

computers ("formalize"), since they are often in a condition-result form in which the precise 

mechanisms linking the conditions (eg. mutated genes) and the results (eg. phenotypes) are not 

fully established. We are using two complementary languages, statecharts and live sequence 

charts (LSCs), which describe system behaviors using time-constrained logical constructs. Here 

we present our model of VPC fate specification using the language of LSCs and the Play-Engine 

tool. The Play-Engine allows data and mechanistic models to be entered by the manipulation of a 

graphical user interface (GUI) that resembles the pictorial models that biologists draw (play in). 

The GUI also functions as a dynamic pictorial model that depicts computer-executed simulations 

(play out). Using these tools, we have succeeded in representing various types of data commonly 

obtained in VPC specification experiments, the variability of biological results, cell movement 

events, and the interdependence of biological behavior and anatomical structure. The model is 

easily extendable, either by including additional VPC specification data, by linking together GUIs 

that represent other developmental processes, or by creating links to models in the 

complementary language of statecharts. 

The construction and analysis of our model of VPC fate specification has enhanced our 

understanding of this biological process in several ways. At the simplest level, we have 

transformed biological data and mechanisms into a database of executable statements of system 

behavior. Beyond this, however, our modeling has revealed inconsistencies between the "rules" 

and the actual data in these established papers, and has highlighted interesting gaps in our 

understanding of this system that we have begun to investigate experimentally.

137. A Screen for Genes Synthetically Lethal with lin-35 Rb 

Mike Hurwitz 1 , Bob Horvitz 2 

1Department of Adult Oncology, DFCI, Boston, MA 02114, USA 

2HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA 

The class B synMuv gene lin-35encodes a C. elegans protein similar to Rb, a tumor suppressor 

inactivated in many human solid tumors. lin-35mutant animals provide a model for mammalian 

cells harboring mutant Rb genes. Since lin-35mutations are not lethal, we can screen for genes 

with functions required for viability in lin-35 mutants but not in wild-type animals to identify 

potential targets for cancer therapy. Pharmacologic inactivation of such target proteins could 

cause the specific deaths of Rb-deficient cells. 

We have screened the LGI RNAi library described by Fraser et al. (2000)1 for genes that are 

essential specifically in lin-35(n745) animals. n745 causes an early stop codon in lin-35 and is 

probably a null allele2. We have compared the phenotypes following RNAi of lin-35(n745) 

animals to the published RNAi phenotypes of the wild-type N2 strain1.Of the 2,445 dsRNAs we 

have screened, 2,128 did not cause abnormalities in either lin-35(n745) or N2 worms. 212 

dsRNAs caused the same abnormalities in lin-35(n745) and N2 worms. 105 dsRNAs resulted in 

abnormalities in lin-35(n745) worms but not in N2 worms. These 105 dsRNAs were tested in the 

RNAi-hypersensitive strain rrf-3(pk1426) to assess whether a more complete knockdown of gene 

function by the dsRNAs might generate more severe defects. 74 of them caused severe growth 

defects in rrf-3(pk1426) worms, suggesting that the effects seen in lin-35(n745) worms might be 

related to differential effects of RNAi in lin-35 worms. Of the remaining 31 genes, 20 have human 

counterparts, defined as having at least 25% identity over 75% of the length of the gene. These 

31 genes may function in Rb-containing or parallel pathways. We found no dsRNAs that were 

reported to have effects on the growth of N2 worms but did not have any effects on lin-35(n745) 

worms. 

Because lin-35(n745)animals are less healthy than N2 animals (decreased brood size and rare 

sterile animals)3, it is possible that some of the abnormal RNAi phenotypes seen in lin-35mutants 

but not in N2 animals are caused by the non-specific additive effects of two harmful mutations. 

For example, RNAi of some genes may affect one cell type and the lin-35mutation another so that 

together these two distinct defects result in severely affected animals. Since our goal is to identify 

pathways that are redundant within single cells, it is crucial to identify RNAs that cause 

cell-autonomous synthetic lethality. To find such genes, we are developing an assay to assess 

the effect of inactivation of lin-35 in a single tissue in combination with inactivation of any of the 

genes identified in our primary screen. 

1Fraseret al. Nature 408: 325-30, 2000. 

2Lu, X., and H. R. Horvitz. Cell 95: 981-91, 1998. 

3Fay et al. Genes & Develop 16: 503-17, 2002.

138. Genetic variation reveals differences among C. elegans isolates for ASH mediated 

behaviors 

Rhonda Hyde 1,2 , Anne C. Hart 1,3 

1MGH Cancer Center 149 13th Street, Charlestown, MA 

2Program in Neuroscience, Harvard Medical School, Boston, MA 

3Department of Pathology, Harvard Medical School, Boston, MA 

Genetic differences are one source of behavioral variation. Although some complex behaviors 

are quantitative in nature, several previous studies demonstrate that natural variation in C. 

elegans behavior is often monogenic. Single nucleotide polymorphisms (SNPs) in C. elegans 

isolates alter social feeding (1) and male mating behaviors (2) as well as germline sensitivity to 

RNAi (3). 

To address the genetic and molecular basis of behavior, we utilize the ASH circuit in C. 

elegans. The ASH neurons detect mechanical, osmotic and chemical stimuli; laser ablation 

studies indicate that they are primarily responsible for detecting light touch to the nose (4), high 

osmolarity (5) and certain volatile repellents (6). Activation of the ASH neurons by any of these 

sensory stimuli results in backward locomotion. The ASH neurons synapse onto a series of 

previously described interneurons which regulate spontaneous reversal rates (7) and are thought 

to be responsible for locomotion. We observed that animals from the CB4856 (Hawaiian) isolate 

are defective in their responses to these sensory stimuli and they have dramatically decreased 

spontaneous reversal rates as compared to N2 animals. These differences between strains are 

likely due to DNA polymorphisms in genes critical for neuronal function or development. 

We will describe our strategy to identify the genes that are responsible for the behavioral 

differences between the CB4856 and N2 isolates. Repeated outcrossing of the Hawaiian isolate 

to N2 and standard SNP analysis suggest that the nose touch behavioral difference is monogenic 

and maps to chromosome V. Tentatively, we have named the Hawaiian allele rt144. 

Recombination mapping has narrowed down the interval containing rt144 to five cosmids. 

We are interested in addressing the function of the gene corresponding to rt144 in the nervous 

system and determining whether the behavior traits seen in the CB4856 strain exist in other C. 

elegans isolates. This analysis will provide further insights in understanding genetic variation and 

its ramifications for behavior. 

1) de Bono and Bargmann Cell 1998; 2) Hodgkin and Doniach Genetics 1997; 3) Tijsterman et 

al Current Biology 2002; 4) Kaplan and Horvitz PNAS 1993; 5) Hart et al Nature 1995; 6) 

Bargmann et al Cold Spring Harb Symp Quant Biol 1990; 7) Zheng et al Neuron 1999.

139. Study of the Effects of Oxidative Damage Repair on Aging and Muscle HealthSpan 

Carolina Ibanez-Ventoso, Samuel Bassous, Peter J. Schmeisser, Suzhen Guo, Monica Driscoll 

Dept. of Mol. Biol. & Biochem., Rutgers University, Piscataway, NJ 

The rapidly growing elderly population inevitably confronts an increased risk for several 

debilitating diseases, possible cognitive decline, and virtually certain decline in muscle strength 

over time (a condition known as sarcopenia). We have found that muscle deterioration is a 

predominant feature of aging in the nematode Caenorhabditis elegans (Herndon et al., 2002). 

The cellular demise of aging nematode muscles occurs remarkably similarly to that of humans. 

Furthermore, both occur with mid-life onset. Analysis of age-related muscle decline in the simple 

nematode may thus provide novel insight into this common and debilitating condition of human 

aging. 

Oxidative damage of cellular macromolecules has been postulated to be a major contributing 

factor to aging. Reactive oxygen species (ROS) are generated in cells in the course of normal 

metabolism and interact with nucleic acids, proteins and lipids to impair their functions. 

Significantly, virtually all tested long-lived C. elegans mutants present enhanced resistance to 

oxidative stress. Moreover, mutants with high ROS loads can have short life spans. C. elegans 

mev-1(kn1) mutants have an increased ROS production, and thereby are hyper-sensitive to 

oxidative stress-inducing drugs, high oxygen and present short life spans. mev-1(kn1) is a point 

mutation in the gene cyt-1, which encodes a subunit of the succinate dehydrogenase cytochrome 

b of the mitochondrial electron transport complex II. We have observed that the mev-1(kn1) 

mutation accelerates cellular features of muscle aging, consistent with a role for oxidative stress 

in promoting tissue decline in vivo. 

An intrinsic antioxidant defense within cells is provided by peptide methionine sulfoxide 

reductases (Msr), which repair oxidized methionine in proteins by reducing methionine sulfoxide 

back to methionine. There are two classes of Msr enzymes, MsrA and MsrB, each specifically 

reduces one of the two possible oxidized enantiomers. There is evidence that increased function 

of MsrA by over-expression in Drosophila extends life span and confers higher resistance to 

oxidative stress, whereas loss-of-function of MsrA in mice shortens life span and renders the 

mutant animals more sensitive to oxidative stress. Interestingly, over-expression of MsrA in flies 

also delays the age-related onset of mobility and reproduction decline, and thus preserves youth 

until later stages in life. Together, these findings suggest that Msr proteins have a critical role in 

health span that may be conserved throughout evolution. We are studying the effects of Msr 

functions on C. elegans life span and muscle health span. Preliminary data have encouraged us 

to perform thorough life span and mobility analyses (routine in our lab) of several worm strains. 

We will present on our progress in the analysis of this problem.

140. Guidance and cell-matching of a migrating epidermal sheet during ventral enclosure 

by MAB-20 and PLX-2 

Richard Ikegami 1 , Kristin Simokat 2 , Louise Dixon 1 , Jeff Hardin 2 , Joseph Culotti 1 

1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, 

Ontario M5G 1X5, Canada 

2 Department of Zoology, University of Wisconsin 1117 W. Johnson Street, Madison WI 53706 

USA 

During epithelial morphogenesis, epithelial cells undergo dynamic changes in cell shape, 

cell-cell adhesions and cellular arrangement. Semaphorins, classically identified as repulsive 

axon guidance cues, play a major role in epithelial morphogenesis in C. elegans. Plexin-2/PLX-2 

was identified as one receptor for Semaphorin-2a/MAB-20 to function in a redundant manner with 

at least one other as yet unidentified receptor during male tail morphogenesis. We present 

phenotypic and genetic evidence consistent with the role of PLX-2 as one receptor for MAB-20 

signaling during ventral enclosure. 

Mutants of plx-2 including its null share similar but milder ventral enclosure defects compared 

to mutants of mab-20. At the cellular level, plx-2 mutants like mab-20 mutants show inappropriate 

regulation of adhesion between the P cells of the migrating epidermal sheet. In addition, the plx-2 

null is unable to enhance the embryonic lethality of the mab-20 null consistent with a genetic role 

in the same pathway yet suggests that mab-20 must also signal through a pathway parallel to 

plx-2. 

PLX-2 is expressed in a manner that could confer the cell-specific function of ubiquitously 

expressed MAB-20. PLX-2 is expressed in a discrete set of epidermal P cells (P3/4, P5/6, P9/10, 

P11/12) that migrate to meet their contralateral partners at the ventral midline. Concomitantly, 

PLX-2 is expressed in a subset of substrate neuroblasts of the ventral pocket. These neuroblasts 

undergo short-range migration and arrange into a band of a single cell width. This band bisects 

the ventral midline of the open pocket counterfacing the leading edge of the migrating PLX-2 

non-expressing P7/8 cell. In addition we show that VAB-1 Eph receptor may function to arrange 

the PLX-2 neuroblasts into a contiguous band just prior to the overlying migration of the 

epidermis. 

We present a model for the role of MAB-20/PLX-2 in guiding the migrating epidermal sheet to 

mediate contralateral cell-matching of opposing cells of the leading edge at the ventral midline 

during pocket closure.

141. Identification of Wnt Pathway Target Genes in C. elegans by Microarray Analysis 

Belinda M. Jackson, David M. Eisenmann 

1000 Hilltop Circle, University of Maryland Baltimore County, Baltimore, MD 21250 

During the well studied process of vulva development in C. elegans, six epithelial cells choose 

one of three cell fates based on activation of the RTK/Ras and Notch pathways in the vulva 

precursor cells (VPC). Our laboratory previously showed that a Wnt signaling pathway is also 

instrumental in vulva cell specification. We have shown that Axin (PRY-1), APC (APR-1), 

beta-catenin (BAR-1) and TCF (POP-1) act to maintain expression of the Hox gene LIN-39 in the 

VPCs. To identify other putative Wnt pathway targets important for vulva formation we will use 

Affymetrix microarray analysis. 

We previously showed that a heat shock inducible ΔNT BAR-1 protein leads to over-activation 

of Wnt signaling in the VPCs, causing extra vulva cell fates [1] . We will compare the gene 

expression profiles of worms over-expressing this activated BAR-1 product with that of wild-type 

worms and look for genes consistently up or down regulated. The caveat of this brute force 

approach will be a global affect on Wnt targets, including those not exclusive to the VPCs. In 

order to address this, we will also apply a strategy previously used to identify muscle cell specific 

gene expression in C. elegans [2] . Using a flag-tagged poly-A binding protein driven by a VPC 

specific promoter element, we will generate an mRNA pool derived predominantly from the vulva 

precursor cells, and analyze this by microarray analysis. 

Progress on both of these approaches will be reported. 

1. Gleason, J.E., H.C. Korswagen, and D.M. Eisenmann, Activation of Wnt signaling 

bypasses the requirement for RTK/Ras signaling during C. elegans vulval induction. Genes Dev, 

2002. 16(10): p. 1281-90. 

2. Roy, P.J., et al., Chromosomal clustering of muscle-expressed genes in Caenorhabditis 

elegans. Nature, 2002. 418(6901): p. 975-9.

142. Combinatorial control of lin-48 expression in the C. elegans excretory duct cell is 

mediated through Pax and bZip transcription factors 

Hongtao Jia 1 , Xiaodong Wang 2 , Helen M. Chamberlin 2 

1MCDB program, The Ohio State University, Columbus, OH 43210 

2Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210 

Transcription results from the combination of multiple factors that act together on a gene’s 

regulatory sequences. To investigate the regulatory logic that can result in cell- specific gene 

expression, we are studying the expression of the C. elegans lin-48 gene. lin-48 is expressed in 

several cells, including the hindgut, the excretory duct and cells of the head and phasmid. The 

regulation of this gene in each cell type is distinct. Our work has focused on regulation in the 

excretory duct cell. 

Previous experiments identified at least two lin-48 regulatory regions important for excretory 

duct cell expression. (Wang and Chamberlin, 2002). The Pax transcription factor EGL-38 was 

shown to act through one element, whereas the bZip transcription factor CES-2 acts through the 

other. Since bZip transcription factors can act as either homodimers or heterodimers, we sought 

to test whether other bZip factors might function with CES-2 to mediate lin-48 expression in the 

excretory duct cell. In a yeast two hybrid experiment, the C-terminal region of CES-2 which 

contains the leucine zipper dimerization domain and DNA binding domain was used as "bait" to 

screen C. elegans embryonic mRNA library, and four candidate partners were identified. 

(Metzstein, 1998) 

We used RNAi to knock down each of these four genes, and found that both K08F8.2 and 

ZC376.7 affect lin-48 expression and function in the excretory duct cell. Interestingly, K08F8.2 

has two bZip domains and whereas CES-2 and ZC376.7 each have one. Thus, our working 

hypothesis is that a complex of bZip transcription factors may contribute to the transactivation of 

lin-48 in the excretory duct cell. To test this idea, we have carried out in vitro DNA binding assays. 

We have found that both CES-2 homodimers and a heterodimer of CES-2/K08F2.2 can bind a 

bZip response element in the lin-48 sequences. The interaction is mediated between the 

C-terminal bZip domain of K08F2.2, and not the N-terminal domain. We are testing the possibility 

that ZC376.7 may interact with the N-terminal bZip domain of K08F8.02, either through another 

DNA element or as a co-transactivator. 

1. Metzstein, (1998) Ph.D thesis. Massachusetts Institute of Technology 

2. Wang, X. and Chamberlin, M. H. (2002) Genes and Development 16:2345-2349.

143. Structural and functional studies of the C.elegans Hsp90 ortholog DAF21 

Dayadevi Jirage, Harold Smith 

Center for Advanced Research in Biotechnology 9600 Gudelsky Way Rockville MD 20850 

The molecular chaperone Hsp90 is required for the function of multiple eukaryotic growth 

regulatory signaling proteins and in the cellular response to stress. The protein sequences of 

Hsp90s are highly conserved across species. Hsp90 protein is the target of the antitumor drug 

geldanamycin (GA) - a derivative of which is currently in clinical trials for cancer treatment. C. 

elegans possesses a single ortholog of Hsp90 called Daf21, whose protein sequence is 74% and 

76% identical to human Hsp90 alpha and beta respectively. Despite this conservation in protein 

sequence, DAF21 is the only Hsp90 examined to date that does not bind GA. The first goal of our 

research is to determine the 3-dimensional structure of DAF21 in order to identify the structural 

features that confer this resistance to GA. Our second goal is to test the function of human Hsp90 

in C. elegans daf21 mutants (RNAi and deletion) in order to develop a C. elegans-based system 

for drug assays and screening. 

For the structural studies, we have expressed His-tagged versions of the N-terminal domain 

(shown to be involved in GA-binding in other species) as well as the full-length DAF21 in E.coli, 

and purified the proteins to homogeneity using Ni-column affinity chromatography followed by 

ion-exchange chromatography. The structure of these proteins will be determined using X-ray 

crystallography and NMR-spectroscopy. 

For the functional studies, we first performed RNAi of daf21 using the feeding method. We 

observed a range of phenotypes - slow growth, embryonic lethality, vulval defects, dauer-like 

appearance and sterility. We made constructs comprising cDNAs of the human Hsp90 alpha and 

daf-21under the control of the daf-21 promoter. We have created transgenic worms expressing 

these plasmids using microparticle bombardment. We are currently analyzing these transgenic 

worms for complementation of the RNAi phenotypes.

144. GNA-2, Chitin and the Functionally-Redundant CEJ-1/B0280.5 are Required for the 

Synthesis of a Lipophobic Extraembryonic Matrix (EEM) and are Essential for 

Development and Polarity in the One-cell Embryo 

Wendy L. Johnston, Aldis Krizus, James W. Dennis 

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Ave, Toronto, ON, 

M5G 1X5, Canada 

Glucosamine 6-phosphate N-acetyltransferase (GNA-2) is essential for the biosynthesis of the 

amino sugar UDP-N-acetylglucosamine (UDP-GlcNAc), which is necessary to synthesise a wide 

variety of glycoconjugates, including N- and O-glycan-modified glycoproteins, proteoglycans, GPI 

linkers, O-GlcNAc-modified proteins and glycopolymers including chitin and hyaluronan. We have 

shown previously that a gna-2 allele deleted for the entire coding sequence (qa705) is Mel, with 

most embryos arrested as multinucleated single cells. Embryos are also defective in meiosis and 

polar body extrusion, and display reduced or mislocalised microtubules, polarity and osmotic 

defects (Pod), decreased eggshell WGA binding and decreased embryonic O-GlcNAc residues. 

In addition, gna-2(qa705) embryos lack the zone (extraembryonic matrix, EEM) between the 

embryo and the eggshell and are permeable to the lipophilic dye, FM 4-64. 

Chitin, a linear polymer of GlcNAc, is deposited in the eggshell very shortly after fertilisation. It 

was detected in N2 as a bright ring surrounding the embryo using a fluorescently labeled 

chitin-binding reagent (kindly donated by Y. Zhang, NEB). Conversely, in gna-2(qa705), chitin 

was undetectable. To test the hypothesis that chitin deficiency contributes to the gna-2(qa705) 

Mel phenotype, we performed RNAi with chi-1 and determined that eggshell chitin was missing 

and the embryonic lethal phenotype resembled gna-2(qa705). In the hermaphrodite germline, 

gna-2 RNA is bound and translationally repressed by GLD-1 1 . Other GLD-1 targets include the 

chitin-binding domain motif containing cej-1 and B0280.5, and cej-1/B0280.5 coRNAi resulted in 

an embryonic lethality that resembled that of gna-2 RNAi 1 . We performed cej-1/B0280.5 coRNAi 

and found that embryos were multinucleated, polarity-defective and inappropriately permeable to 

FM 4-64. These results suggest a model where gna-2 is made available for UDP-GlcNAc 

synthesis following relief of GLD-1 translational repression in the proximal germline. After 

fertilisation, chitin is synthesised from UDP-GlcNAc and extruded from the plasma membrane. 

CEJ-1 and B0280.5, synthesised following relief of GLD-1 RNA translational repression, are 

secreted and bind chitin, where, together with chitin, they support the production of a hydrophilic 

EEM. This EEM is required to complete meiosis and for further development, including 

polarisation. 

Using fluorescently labeled WGA, we previously reported the appearance of embryonic cortical 

patches in wildtype embryos that arise transiently at meiosis and disappear by the first cell 

division. These patches do not bind the chitin-binding reagent and they persist when a robust 

chitinous eggshell and an EEM have already formed. This suggests that they are not 

intermediates in the production of eggshell chitin, but rather, are non-chitin GlcNAc-containing 

structures that are dependent on GNA-2. Recently, Pellettieri et al. 2 demonstrated that the kinase 

MBK-2, which coordinates the degradation of a number of maternally supplied proteins (MEI-1, 

MEI-2, OMA-1, PIE-1), is present at meiosis in cortical patches. Using a GFP antibody in 

embryos carrying an MBK-2::GFP fusion, we determined that the WGA patches are coincident 

with MBK-2 patches during meiosis. We are currently testing the hypothesis that deficiency of 

GNA-2/chitin/CEJ-1/B0280.5 results in decreased MBK-2 activation, perhaps due to defects in 

the EEM. Decreased MBK-2 activation would then result in inappropriate perdurance of target 

maternal proteins, resulting in defects in development and polarisation. 

In summary, these results demonstrate that GNA-2 is necessary for eggshell chitin synthesis, 

and that chitin and the functionally redundant chitin-binding domain containing proteins, 

CEJ-1/B0280.5, are absolutely required for development and polarity at the one-cell stage. Our 

results also demonstrate that MBK-2 cortical patches colocalise with WGA and experiments are 

currently underway to investigate the development and composition of these patches. 

1 Lee, M.H. and Schedl, T. Genes Dev. 15, 2408-2420 (2001) 

2 Pellettieri, J., Reinke, V., Kim, S.K. and Seydoux, G. Dev. Cell 5, 451-462 (2003)

145. Biochemical and structure/function analysis of DGK-1, a neuronal diacylglycerol 

kinase and putative downstream effector of Gapha o signaling. 

Antony Jose, Michael Koelle 

MCDB, Yale University, New Haven CT 

Gα o, the most abundant G protein in the brain, mediates signaling by many neurotransmitter 

receptors, yet its signaling mechanism is not understood. GOA-1, the C. elegans ortholog of Gα o, 

is thought to inhibit neurotransmission by decreasing the levels of the second messenger 

diacylglycerol (DAG)(1). Diacylglycerol kinases are enzymes that inactivate DAG by 

phosphorylating it. The diacylglycerol kinase DGK-1 is a potential downstream component of 

GOA-1 signaling since dgk-1 mutants are phenotypically similar to goa-1(lf) mutants and dgk-1(lf) 

mutations are epistatic to goa-1(gf) transgenes. We have used biochemical approaches to test if 

GOA-1 signaling activates DGK-1 to reduce DAG levels and thereby inhibit neurotransmission. 

We also carried out a structure/function analysis of DGK-1. 

To test for a possible direct interaction between GOA-1 and DGK-1, the proteins were 

expressed recombinantly and purified. Purified DGK-1 phosphorylated diacylglycerol in vitro. 

However, the instability of purified DGK-1 precluded further biochemical analysis, and we thus 

have looked for effects of GOA-1 on native DGK-1 protein in C. elegans extracts. Fractionation of 

C. elegans extracts and western blotting indicated that DGK-1, which acts on the 

membrane-bound substrate diacylglycerol, is predominantly cytosolic. One hypothesis is that 

GOA-1 recruits the soluble DGK-1 to the membrane, and thus activates it. However, we were not 

able to detect any translocation of DGK-1 in mutant backgrounds that activate GOA-1 signaling, 

or after activating GOA-1 in wild-type extracts by treatment with GTP analogs. We were also 

unable to pull down DGK-1 from C. elegans extracts using purified recombinant GST::GOA-1 

fusion protein. Our results have thus failed to demonstrate any effect of GOA-1 on DGK-1 protein. 

It is possible that DGK-1 acts constitutively to shorten the half-life of DAG, and that GOA-1 acts 

by a mechanism independent and parallel to DGK-1. 

Diacylglycerol kinases have several conserved sequence motifs of unknown function. To 

understand the functional importance of these conserved sequence motifs in DGK-1, we 

identified the molecular lesions in 20 alleles of dgk-1 that have been generated by genetic 

screens [(2) and our lab]. We quantitated the behavioral defects caused by these mutations and 

also examined effect of these mutations on DGK-1 protein stability using western blotting. We 

identified missense mutations in conserved residues in the kinase domain and the cysteine-rich 

domains that dramatically reduce DGK-1 function but produce stable protein, suggesting that 

these domains are essential for the physiological function of DGK-1. We also identified an early 

stop codon mutation that disrupts all previously known dgk-1 isoforms yet retains significant 

function, and our preliminary analysis suggests that novel splice variants that skip the stop codon 

supply DGK-1 function in this mutant. Finally, we found a missense mutation that changes a 

serine to a valine in the kinase domain that resulted in a protein with altered mobility on 

SDS-PAGE. We are testing the possibility that phosphorylation of this serine residue affects the 

function of DGK-1 and alters its mobility on SDS-PAGE. 

(1). Nurrish S. et al., Neuron Vol. 24, 231-242. 

(2). Hajdu-Cronin Y. M. et al., Genes and Development Vol. 13, No. 14, 1780-1793.

146. Cytological screening of the germ lines of sterile mutants for meiotic defects 

Malek Jundi, Monique C. Zetka 

Department of Biology, McGill University, 1205 avenue Docteur Penfield, Montreal, QC H3A 1B1 

Extensive screening for mutations in essential genes has been carried out for defined 

chromosomal regions on LG I (Linkage Group; Rose et al. 1980: Howell et al. 1987; Howell and 

Rose 1990; McKim et al. 1992; Johnsen et al. 2000), LG II (Sigurdson et al. 1984), LG IV 

(Rogalski et al. 1982; Rogalski and Baillie 1985; Clark et al. 1988; Clark and Baillie 1992), LG V 

(Rosenbluth et al. 1988; Johnsen and Baillie 1991) and LG X (Meneely and Herman 1979, 1981). 

A subset of the alleles in this mutant library result in sterility and are likely to have identified genes 

essential for the development of the gonad and the germ line. Because the germ line of the 

nematode presents an orderly and predictable progression of nuclei through the various stages of 

proliferation and meiosis, the function of the wild-type gene can be inferred by examining the 

DAPI-stained nuclei of sterile animals for cytologically visible defects. We are screening a 

collection of 138 previously uncharacterized sterile mutants originating from these analyses for 

the presence of univalents or chromosome fragments at diakinesis, which is a general indicator of 

defects in upstream processes like meiotic cell-cycle progression, chromosome pairing, synapsis, 

recombination, and chromosome morphogenesis. To date, we have identified four sterile 

mutations that result in a high frequency of univalents at diakinesis and that have associated 

defects in conserved meiotic processes. 

Supported by a grant from the Canadian Institutes of Health Research (CIHR)

147. EGL-26 belongs to the NlpC/P60 superfamily of putative enzymes and is closely 

related to a mammalian acyl transferase 

Rasika Kalamegham, Wendy Hanna-Rose 

406 Althouse Lab , The Pennsylvanis State University , University Park PA 16802 

egl-26 was identified in a genetic screen to uncover mutants with vulval morphology defects. In 

egl-26 mutants the morphology of a single vulval toroid (vulF) is abnormal and a proper 

connection to the uterus is not made leading to the egg-laying defect. EGL-26 is a member of the 

NlpC/P60 superfamily of enzymes, which is characterized by a Histidine containing domain and a 

Cysteine containing domain (H-box and NC domain, respectively). EGL-26 along with other 

eukaryotic proteins belongs to a distinct subclass of NlpC/P60-related putative enzymes. The 

mammalian proteins lecithin: retinol acyltransferase or LRAT and H-ras revertant 107 or 

H-Rev107 are the most closely related to EGL-26. Both LRAT and H-Rev107 contain putative 

transmembrane domains in addition to the H-box and NC domains. Although EGL-26 contains no 

putative transmembrane domains, it is localized at the apical membrane of cells where it is 

expressed. Proper localization of LRAT within the retinal pigment epithelium is essential for its 

function. Significantly, an S-F substitution at amino acid 275 of EGL-26 found in the egl-26 (n481) 

allele causes mislocalization of an EGL-26::GFP fusion leading to general cytoplasmic expression 

as opposed to normal apical membrane localization. The corresponding Serine residue is 

conserved in both LRAT and H-Rev107. We are attempting to analyze the relationship between 

the mammalian proteins and EGL-26 by attempting a rescue of egl-26 mutants by expression of 

either LRAT or H-Rev107 or both. We plan to test the importance of membrane localization by 

restoring membrane localization to EGL-26n481 via addition of alternative membrane localization 

signals.

148. Genes controlling the developmental response to nutrients in L1 larvae. 

Gautam Kao 1,2 , Peter Naredi 2 , Simon Tuck 1 

1UCMP, Umea University, Umea S-90187, Sweden 

2Dept of Surgery, Umea university, Umea S-90187, Sweden 

Wild- type L1s hatched onto plates without food, arrest as L1s and only resume growth when 

food is provided. We are interested in finding out how this global control of development in 

response to nutrients is controlled at the L1 stage. More than 30 genes have been identified in 

global RNAi screens either as having a very slow growth phenotype or as arrested but viable L1s. 

We are re-evaluating these genes to see which are the genes with the most robust phenotypes 

and to discard genes which give a phenotype based on feeding defects. We are also examining 

whether the TGF-beta, insulin and the steroid hormone pathway have a role to play in this global 

response to food at the L1 stage as they do later in dauer development.

149. Characterization of F11A10.3, a RING domain protein that interacts with the 

transcription factor UNC-3 

Ozgur Karakuzu 1 , Brinda Prasad 2 , Scott Cameron 1 

1Departments of Pediatrics and Molecular Biology, The University of Texas Southwestern 

Medical Center at Dallas 

2New York University School of Medicine 

unc-3 encodes the C. elegans orthologue of a family of variant mammalian HLH transcription 

factors, the OLF/Ebf proteins, which determine aspects of terminal differentiation in neuronal and 

hematopoietic cells. Mutations in unc-3 result in a defect in VA and VB motor neuron 

differentiation such that VA and VB neurons can adopt a VC-like fate as assayed by expression 

of a P lin-11gfp reporter and the FMRF-amide neuropeptide, two characteristics of VC neurons. 

unc-3 is expressed in ASI chemosensory neurons and ventral nerve cord motor neurons. 

To examine the role of UNC-3 during neuronal differentiation, we used a yeast two-hybrid 

assay to identify proteins that interact with the C-terminal region of UNC-3, which contains the 

single helix domain of UNC-3. The helix domains of the neurogenic and myogenic bHLH proteins 

Mash1 and MyoD confer some of the specificity for these proteins in determining aspects of 

neurogenic or myogenic fate, suggesting that proteins that interact with these regions might be 

important for their function. We identified F11A10.3, a protein that contains a RING domain. We 

have begun to characterize the F11A10.3 gene and the interaction between UNC-3 and 

F11A10.3. 

Using the two hybrid assay we have mapped the interaction domain in F11A10.3 to the RING 

domain. Since many proteins that contain a RING domain function as E3 ligases, we are testing 

the hypothesis that F11A10.3 may modify UNC-3 through ubiquitylation or sumoylation. The 

F11A10.3 protein is encoded by the downstream gene in a two gene operon. We have obtained a 

deletion allele, n4275, which removes the start codon and the first 133 amino acids of the 

predicted protein. n4275mutants are homozygous viable, Egl and Mab. We are currently 

characterizing the nature of the n4275 allele and the phenotype of n4275 mutants.

150. Characterization of sel-6, a suppressor of lin-12 gain-of-function mutants 

Iskra Katic 1 , Iva Greenwald 2 

1 Department of Genetics and Development, Columbia University, New York, NY 10032 

2 Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, 

Columbia University, New York, NY 10032 

LIN-12/Notch proteins are receptors that mediate cell-cell interactions that specify cell fate. 

Isolation of extragenic suppressors and enhancers of various lin-12 alleles has been a powerful 

method for identifying core components of the signal transduction machinery and factors that 

influence LIN-12 activity. Suppressors of a lin-12 gain-of-function mutation previously defined the 

sel-6 gene (Tax et al., 1997). 

We positionally cloned sel-6 and found it to encode a protein containing a AAA domain 

(ATPases associated with diverse cellular activities) and is conserved in higher eukaryotes. We 

have found that sel-6 is necessary for proper embryonic and larval development, and that the 

suppressor alleles appear to be hypomorphs. We are performing genetic and molecular analyses 

to address the role of SEL-6 in the LIN-12/GLP-1 signaling pathways, in the AC/VU as well as 

other cell fate decisions during C. elegans development. We will report on our progress at the 

meeting.

151. How Does Ivermectin Induce Cell Death? 

Aamna Kaul, Joseph Dent 

Department of Biology, McGill University, 1205 Dr. Penfield Ave., Montreal, Quebec. H3A 2Z6 

Ivermectin is effective against a variety of parasitic nematodes, including Onchocerca volvulus 

(the roundworm causing river blindness). Ivermectin binds and hyperactivates 

invertebrate-specific glutamate-gated chloride channels (GluCls), causing paralysis of pharyngeal 

muscle and subsequent cessation of feeding and growth. 

Our work shows that even a brief exposure to ivermectin is sufficient to permanently paralyse 

C. elegans and prevent growth past the L1 stage. Additionally, ivermectin induces 

pharynx-specific cell death, which accumulates over the course of several days and shares 

morphological features of both apoptosis and necrosis. 

Screening of known GluCl mutants reveals that a mutant avr-15 (coding for a GluCl alpha 

subunit expressed in the pharynx) confers partial resistance to ivermectin. Screening of several 

well-known cell death mutants has revealed no obvious candidates for the cell death pathway 

involved. However, a mutant of eat-6 (coding for an Na/K ATPase) is more sensitive to ivermectin 

suggesting that perturbation of ion flux is a factor in this pathway. Cell corpses variably stain with 

Acridine Orange and Lysotracker indicating that ivermectin may cause dysfunction of acidic 

vesicles (possibly endosomes or lysosomes) following endocytosis of the hyperactivated 

receptor. 

The presence of AVR-15 appears sufficient for ivermectin to induce cell death; expression 

under neuron-specific promoters in the amphid and touch neurons targets these neurons for 

death. In these cases, however, the morphology of cell death appears apoptotic. The introduction 

of mutations in ced-3 and ced-4 (the executers of apoptosis) in these backgrounds will clarify the 

mechanism of neuronal cell death.

152. Expression, function and regulation of gon-2 

Ben Kemp, Rachel West, Diane Church, Samantha Schilling, Janet Lee, Robert Bruce III, 

Matthew Ambros, Eric Lambie 

Department of Biological Sciences, Dartmouth College, Hanover NH 03755 

gon-2 encodes a TRPM family cation channel that is required for the initiation of postembryonic 

divisions by the gonadal precursor cells. Our working hypothesis is that GON-2 protein is 

expressed in the somatic gonadal cells and mediates the uptake of cations such as calcium and 

magnesium that are required for cell cycle progression. To begin to test this hypothesis, we 

generated rabbit polyclonal antibodies against a peptide corresponding to part of the C-terminal 

cytoplasmic region of GON-2. Based on western blots with these antibodies, we find that GON-2 

is expressed in multiple tissues, including the adult gonad and intestine. Through mosaic 

analysis we found that gon-2activity is required within the MS lineage, but not the E lineage, in 

order for gonad development to proceed normally. This suggests that gon-2 function within the 

somatic gonad, but not the intestine, is likely to be required for gonad development. 

Hypomorphic mutations in gon-2 cause a maternal effect gonadless phenotype, but strong 

loss-of-function alleles cause zygotic sterility. These animals produce both sperm and oocytes, 

but very few fertilized eggs. Fertility cannot be rescued by mating with wild type males, 

suggesting that the oocytes may be refractory to fertilization or the ovulation program may be 

defective. We are examining the ovulation program of the zygotic sterile animals and testing the 

ability of an ipp-5 mutation that suppresses the ovulation defect of let-23(lf) to rescue sterility 

caused by gon-2(lf). 

We conducted a large scale reversion screen using gon-2(q388ts) in order to search for 

intragenic and extragenic suppressor mutations. We identified four loci that can mutate to 

suppress gon-2(q388), gem-1-4 (gon-2 extragenic modifier). Gain-of-function mutations in gem-1 

are capable of suppressing gon-2(0). Since gem-1encodes a multi-pass transmembrane protein, 

we suspect that GEM-1(gf) may bypass the requirement for GON-2 by providing an alternative 

pathway for cation uptake. We also identified six potential intragenic revertants of gon-2(q388). 

We are examining the sequence of gon-2 from these mutants in order to determine whether they 

suggest an interaction between the q388-containing region and another portion of the protein.

153. Centrosome Maturation and Duplication In C. elegans Require the Coiled-Coil Protein 

SPD-2 

Catherine A Kemp 1 , Kevin R. Kopish 1 , Peder Zipperlen 2 , Julie Ahringer 2 , Kevin F. O’Connell 1 

1 Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and 

Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0830 

2 Wellcome Trust/Cancer Research UK Institute, University of Cambridge, Tennis Court Road, 

CB2 1QR Cambridge, UK 

Centrosomes are major determinants of mitotic spindle structure but the mechanisms 

regulating their behavior remain poorly understood. The spd-2 gene of C. elegans is required for 

centrosome assembly or "maturation". Here we show that spd-2 encodes a coiled-coil protein that 

localizes within pericentriolar material (PCM) and in the immediate vicinity of centrioles. During 

maturation, SPD-2 gradually accumulates at the centrosome in a manner that is partially 

dependent on aurora-A kinase and cytoplasmic dynein. Interestingly, SPD-2 interacts genetically 

with dynein heavy chain and SPD-5, another coiled-coil protein required for centrosome 

maturation. SPD-2 and SPD-5 are co-dependent for localization to the PCM, but SPD-2 localizes 

to centrioles independently of SPD-5. Surprisingly, we also find that SPD-2 is required for 

centrosome duplication and genetically interacts with ZYG-1, a kinase required for duplication. 

Thus, we have identified SPD-2 as a factor critical for the two basic functions of the 

centrosome--microtubule organization and duplication.

154. nhr-67 and nhr-111, two NR2E nuclear receptors that may function in nervous system 

development 

Ryan Kennedy 1 , Kristy Reinert 1 , Genna Albert 1 , Chris Gissendanner 2 , Ann Sluder 3 , Bruce 

Wightman 1 

1Biology Department, Muhlenberg College, Allentown, PA 18104 

2New England Biolabs, Beverly, MA 01915 

3Cambria Biosciences, Woburn, MA 01801 

The nuclear hormone receptors (NHR’s) are a class of transcriptional regulators that typically 

contain a DNA binding domain (DBD) and a ligand binding domain (LBD). Nuclear hormone 

receptors can be classified based on amino acid sequence relationships in the DBD. Nuclear 

receptors in the NR2E class, such as tailless (tll) in Drosophila, Tlx in mice, PNR in vertebrates 

and fax-1 in C. elegans have been found to function in nervous system development. We have 

been investigating the function of the NR2E nuclear receptors nhr-67, the C. elegans tailless 

ortholog, and nhr-111, a novel nuclear receptor that is present in C. elegans but not C. briggsae. 

The DBD’s of fax-1 and nhr-67 are similar, although evidence from our laboratory suggests that 

they have different DNA-binding activities (see the abstract by DeMeo et al.) The DBD of nhr-111 

is somewhat diverged, but its LBD is similar to fax-1. Our goal is to determine the function and 

expression pattern of nhr-67 and nhr-111. Deletion of the nhr-111 gene does not produce an 

obvious phenotype, whereas deletion of nhr-67 results in L1 arrest. RNAi analysis of nhr-67 

reveals a postembryonic role in movement, larval growth, molting and vulval morphogenesis. 

GFP expression studies of nhr-67 and nhr-111 argue that both of these genes are expressed in 

the nervous system and may play a role in nervous system development: nhr-67::gfp reporters 

are expressed in a few pairs of head neurons, while nhr-111::gfp reporters are expressed in one 

or two pairs of head neurons and the somatic gonad. We are currently expressing recombinant 

NHR-67 protein in bacteria in order to produce polyclonal antibodies, which will allow us to 

determine the expression pattern of endogenous NHR-67 wild-type animals and various mutants 

by immunofluorescence studies.

155. The lin-4 homologue, mir-237, directs proper vulva and gonad development in C. 

elegans. 

Aurora Esquela Kerscher, Lei Bai, Frank J. Slack 

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 

06520 

The founding member of the microRNA family of gene regulators, lin-4, is a heterochronic gene 

essential for the L1/L2 transition in C. elegans. This gene shares strong sequence homology to 

the uncharacterized miRNA, mir-237. In order to determine the role of mir-237 during nematode 

development, the expression patterns and mutant phenotypes for mir-237 were obtained. Our 

results show that mir-237 RNA is first detected at the beginning of larval stage 3 (L3) and 

continues to be expressed in the adult by Northern blot analysis. In contrast, lin-4 is observed two 

stages earlier, at L1. Using mir::gfp fusion techniques, we found that mir-237 is normally 

expressed in the Z1 and Z4 derivatives of the somatic gonad at L1. By L3, mir-237 expression is 

noted in the vulval precursor cells (VPCs), the anchor cell and a subset of uterine cells, the distal 

tip cells of the gonad, neurons in the head and tail region, as well as in the seam cells. This data 

suggests that mir-237 plays multiple roles during vulva, gonad, neuron and skin development. 

Similar mir::gfp expression studies for lin-4, revealed that lin-4 is temporally expressed in the 

ventral cord neurons from L1 through the adult and in the seam cells from the L2 stage, which 

differs from the mir::gfp pattern observed for mir-237. Overexpression of mir-237 in nematodes 

resulted in a bursting vulva phenotype and a variety of gonad abnormalities, primarily effecting 

germ cell development. Potential targets for mir-237 have also been identified. These findings 

imply that mir-237 plays a distinct role from lin-4 despite their close sequence homology. We are 

currently studying the role of the mir-237 mammalian homologue, mir-125, in mice to determine if 

the biological function of this miRNA is evolutionarily conserved. Our Northern blot analysis 

revealed that mir-125 is expressed temporally during mouse development as well as in a wide 

range of adult tissues. In summary, our studies reveal that the lin-4 homologue, mir-237, is 

essential during nematode development to promote normal vulva and gonad morphogenesis.

156. Sex-specific centrosome inheritance requires cki-2 in C. elegans 

Dae Young Kim, Richard Roy 


Sexually reproducing organisms are faced with the challenge of reducing their chromosome 

copy number by half during meiosis to maintain correct ploidy in the zygote. Similarly, in those 

organisms that initiate embryogenesis following the contribution of the sperm centrosome at 

fertilisation, this is even further complicated: upon fertilisation the sperm centrioles split and 

subsequently duplicate to establish the poles of the bipolar spindle of the first zygotic division. 

Therefore, to avoid forming a multipolar spindle in the zygote, the maternal gametes must 

selectively eliminate their centrosomes to ensure that only a single centrosome (of paternal 

origin) is inherited by the zygote to generate a bipolar spindle. Little, however, is known about 

how this critical step is correctly maintained to avoid catastrophe at fertilisation. 

In C. elegans, two CIP/KIP family CDK inhibitors (CKIs) are encoded by the cki-1 and cki-2 

genes in tandem on chromosome II. While the role of cki-1 has been characterized in numerous 

developmental contexts in C. elegans, cki-2 has been difficult to characterize due to its 

refractoriness to RNAi. We used co-suppression to compromise cki-2 function in the germ line 

and in the early embryo cytoplasm. Affected embryos show multiple defects in meiotic and mitotic 

cell cycle progression. Most interestingly, we found that cki-2 is necessary for the appropriate 

elimination of the maternal centrosome during oogenesis, and in its absence, embryos form 

multipolar spindles that lead to severe aneuploidies following division. Our data indicate that cki-2 

is required to correctly establish the bipolar spindle in the C. elegans zygote, probably through 

blocking cyclin-dependent kinase activity in the oocyte, resulting in destabilisation of the maternal 

centrosome. CDK targets that may be involved in this process are currently being pursued, while 

further insight into how effective these maternal centrosomes may function in establishing 

embryonic polarity is under investigation.

157. Roles of PAR-3 and PKC-3 in establishment and maintenance of epithelial cell polarity 

in C. elegans 

Heon S. Kim 

433 Biotech Building, Cornell University, ITHACA, NY. 14850 

Cell polarity is required at many different stages of development, including early 

embryogenesis and organogenesis. Intercellular junctions are believed to be required to polarize 

epithelial cells. PAR-3/PKC-3/PAR-6 complex, which serves as one of the earlist polarity cues 

during the early embryogenesis in C. elegans, has been found to be localized at apical junctions 

of gut epithelium in C. elegans, Drosophila, and mammals. However, the function of 

PAR-3/PKC-3/PAR-6 in establishment and maintenance of epithelial cell polarity in C. elegans 

has not been studied, mainly because the loss of function of this protein complex perturbs early 

embryogenesis. I am trying to overcome this barrier in two different ways. Heat shock-induced 

par-3 RNAi, right after early embryogenesis will deplete par-3 mRNA and hopefully will result in 

par-3 knock-out phenotypes in late embryos or L1~L2 larvae. Alternatively, I am examining the 

effect of a mutation in pkc-3 on epitheial polarity. I have a pkc-3 mutant, which is newly generated 

by Vancouver Gene Knockout Lab. This mutant carries a 1.5kb deletion (ok544) that removes 

kinase domain. Homozygotes undergo normal embryogenesis due to maternal contribution, but 

they are arrested as L2 larvae. Currently I am trying to optimize the heat shock conditions for 

par-3 RNAi, and to identify the phenotypes of arrested pkc-3 mutants.

158. The Regulation of the CDC-6 Replication Licensing by CUL-4 

Jihyun Kim, Hui Feng, Edward T. Kipreos 

The University of Georgia, Athens, GA 30602 

In order for cells to maintain a stable genome, they must replicate their genomic DNA exactly 

once during each cell cycle. The tight fidelity of DNA replication is achieved through the temporal 

regulation of components that comprise the prereplicative complex, which assembles during G1 

phase at origins of replication. The replication licensing factors CDT1and CDC6 load onto the 

origins during G1 phase and are essential for the initiation of DNA replication. During S phase, 

these licensing factors are inactivated to ensure that DNA replication cannot re-initiate. In 

humans, C. elegans, and fission yeast, CDT1 is degraded during S phase. In contrast, while 

CDC6 is degraded in fission yeast, it is exported from the nucleus during S phase in humans. 

We have previously shown that CUL-4 is required for the degradation of CDT-1 during S phase 

(Zhong et al., 2002, Nature 423: 885). We have now found that the C. elegans homolog of CDC6 

is also required for DNA replication and is regulated by CUL-4. In cul-4(RNAi) larvae, CDC-6 fails 

to be exported from the nucleus during S phase. In humans, the trigger for CDC6 export during S 

phase is phosphorylation by the cyclin A/CDK2 kinase complex. By analogy, the failure of CDC-6 

nuclear export in cul-4(RNAi) larvae could either be due to a failure of phosphorylation of CDC-6 

or a failure of nuclear export after its phosphorylation. To determine if CDC-6 phosphorylation 

was affected by loss of CUL-4, we generated phospho-specific antibodies to three 

CDK-consensus sites in CDC-6. One of these antibodies detected S phase-specific 

phosphorylation of CDC-6. Using this antibody, we observed that phosphorylation of CDC-6 did 

not occur in cul-4(RNAi) larvae, suggesting that the failure of nuclear export was due to a failure 

of phosphorylation. 

We are currently focusing on the mechanism by which CDC-6 phosphorylation is regulated by 

CUL-4. We are also determining the functional relevance of CDC-6 phosphorylation and its 

nuclear export in preventing the re-initiation of DNA replication. We will present our latest findings 

at the meeting.

159. The O/E transcription factor UNC-3 specifies the identities of the ASI chemosensory 

neurons via cell-specific repression and activation mechanisms 

Kyuhyung Kim, Marc Colosimo, Piali Sengupta 

Dept. of Biology, Brandeis University, MS008, 415 South Street, Waltham, MA02454 

The C. elegans chemosensory system provides an excellent opportunity to investigate the 

development of individual chemosensory neuron subtypes and to understand the regulation of 

olfactory receptor gene expression at a single cell resolution. To identify genes that may have a 

role in the development or function of chemosensory neurons, transgenic animals carrying 

ceh-36::GFP arrays that were strongly expressed only in the AWC olfactory and ASE gustatory 

neurons, were used in genetic screens. ceh-36 encodes a member of the OTX/OTD family of 

homeodomain proteins and is required for the specification of the AWC and ASE neurons in C. 

elegans. More than 30 mutants showing altered expression of ceh-36::GFP were isolated by EMS 

mutagenesis. 

oy85 mutants exhibit ectopic expression of ceh-36::GFP in the ASI neurons. The ASI 

chemosensory neurons detect several water-soluble attractants and dauer pheromone and 

express the daf-7/TGFb ligand. oy85 was found to be allelic to the previously identified gene 

unc-3, which encodes a member of the family of O/E transcription factors. O/E family members 

have previously been implicated in olfactory gene regulation and olfactory neuron targeting in 

rodents. By examining the expression of other neuronal markers in unc-3 mutants, we found that 

the ASI neurons in unc-3 mutants fail to express a subset of ASI-specific genes including 

daf-7/TGFb gene and instead ectopically express genes specific to other chemosensory neurons. 

Ectopically expressed genes include markers of terminal differentiation such as olfactory 

receptors and neuropeptides. Interestingly, transcription factors known to specify the cell-fates of 

these chemosensory neurons were not ectopically expressed in the ASI neurons of unc-3 

mutants. Furthermore, we identified several putative UNC-3 binding sites in the promoters of 

ectopically expressed genes. Deletion of the UNC-3 binding sites in the promoter of the 

AWA-specific olfactory receptor gene odr-10 results in ectopic expression in the ASI neurons. 

These results strongly suggest that UNC-3 represses the expression of non-ASI-specific genes 

by directly binding their promoters. Therefore, in addition to cell-specific activation of specific cell 

fates, the identities of chemosensory neurons in C. elegans may also be specified via cell-specific 

repressive mechanisms. 

Interestingly, in well-fed unc-3 mutant adults which have bypassed the dauer stage, ectopic 

expression began at the L3 larval stage and is maintained thereafter. However, unc-3 mutant 

adults that have transiently passed through the dauer stage did not show ectopic expression. This 

result suggests that differential gene expression in the ASI neurons may serve as a cellular 

memory of the developmental history of the animal. Currently we are investigating the behavioral 

consequences for the altered gene expression profile and examining whether gene expression in 

other sensory neuron types are also differentially affected by developmental history.

160. Identification of CUL-4 complex components 

Youngjo Kim, Edward T. Kipreos 

University of Georgia, Athens, GA 30602 

CUL-4 is a key regulator of DNA replication in C. elegans. Inactivation of cul-4 by RNAi 

produces an L2 stage arrest with enlarged postembryonic somatic blast cells. The DNA content of 

these cells continues to increase in subsequent days post-hatch, with over 100C DNA content 

observed in seam cells. The increased ploidy results from DNA re-replication caused by origin 

refiring (Feng et al., 2003, Nature 423: 885). The re-replication is linked to a failure during S 

phase to degrade the DNA replication licensing factor CDT-1 and to export the licensing factor 

CDC-6 from the nucleus. 

Based on structural similarity with its cullin paralogs, CUL-4 is expected to form a ubiquitin 

ligase complex with multiple components. In both humans and yeast, CUL-4 orthologs interact 

with the DDB1 protein. We found that ddb-1 RNAi produced L2 larval arrest and re-replication 

phenotypes in C. elegans similar to that of cul-4 RNAi. In addition to increased DNA ploidy in 

seam cells, we have confirmed that CDT-1, which is normally degraded in S phase, is not 

degraded in ddb-1 RNAi larvae, similar to what is seen in cul-4 RNAi larvae. 

We are seeking additional CUL-4 complex components through genetic and biochemical 

approaches. In humans, many CUL4A-associated proteins that are proposed to function as 

substrate-binding components (SBCs) have WD repeats, e.g., CSA, DDB2, and hCOP1. We 

would like to determine if any C. elegans WD proteins interact with CUL-4. There are 153 WD 

repeat genes in the C. elegans genome. We initially screened for an RNAi re-replication 

phenotype, e.g., greatly enlarged seam cells with high DNA content. So far, of the 39 tested WD 

repeat genes, none have produced a re-replication phenotype upon RNAi. It is possible that 

distinct SBCs target CDT-1 and CDC-6 regulation and that inactivation of one SBC independently 

of the other will not induce re-replication. Therefore, we are also directly screening for defects of 

CDT-1 degradation and CDC-6 export during S-phase. 

In addition we are in the process of affinity purifying CUL-4 complexes to identify associated 

proteins. We have tagged CUL-4 with the epitopes MYC, FLAG and GFP and are using strains 

expressing these genes to isolate the CUL-4 complex with antibodies specific for the tag. 

Associated proteins will be identified by mass spectrometry. We have already identified the 

cullin-associated protein CAND1, which is known to bind the inactive, deneddylated form of 

cullins (Liu et al, 2002, Mol. Cell 10:1511).

161. Genetic pathways that affect C. elegans leaving, a mate searching behavior. 

Gunnar A. Kleemann, Ling yun Jia, Johnathan O .Lipton, Scott W. Emmons 

Department of Molecular Genetics, Albert Einstein College of Medicine,Bronx N.Y. 10461 

When placed alone on a spot of bacteria, a male C. elegans will leave the food source at a 

constant rate. We refer to this as leaving behavior. Since the presence of hermaphrodites 

inhibits male leaving behavior and hermaphrodites do not exhibit it, leaving behavior is thought of 

as a mate searching behavior. Three factors have been shown to reduce the male leaving rate: 

the presence of hermaphrodites, starvation, and the removal of the male germ line or gonad. 

Stimulus from each of these three inputs must be integrated within the worm and ultimately affect 

the execution of leaving behavior. Therefore, leaving mutants should have defects which affect at 

least one of five processes; (1-3) the inputs, (4) the integration of these signals and control of 

motivation or (5) the execution of the behavior. Currently, we are elaborating genetic pathways 

involved in leaving and assigning each gene to a step in the five-process scheme. We are 

characterizing four uncloned genes that affect leaving behavior: egl-35 (bx129), a putative 

component of the heterochronic pathway, unc-77 (bx122), las-1 (bx117) and bx121. Additionally, 

a number of cloned genes have been shown to affect leaving behavior. 

To describe the pathways affecting leaving behavior we are conducting epistasis analysis. A 

defect the insulin like growth factor (IGF) receptor, daf-2 (e1370) reduces the leaving rate. 

Animals without the serotonin biosynthetic enzyme tryptophan hydroxylase, tph-1 (mg280), are 

also slow leavers. Both tph-1and daf-2 defects are rescued by a null mutation in the forkhead 

transcription factor daf-16 (mgDf50), suggesting that they act upstream of daf-16. daf-16 (m26) is 

a fast leaver indicating that DAF-16 activity can inhibit leaving. Additionally, the removal of the 

monoamine vesicular transporter with cat-1 (e1111),suppresses the slow leaving phenotype of 

tph-1 suggesting that while serotonin increases leaving, another monoamine reduces leaving. 

Both egl-35, and unc-77 are also leaving defective but the daf-16 null does not rescue the defect, 

suggesting that these genes act downstream of daf-16. 

egl-4 (n477) and egl-35 leave in the presence of hermaphrodites, thus they appear to act in the 

hermaphrodite sensing portion of the pathway. egl-35 is particularly interesting since, 

hermaphrodites appear normal in their retention by food, but males leave slowly and ignore 

hermaphrodites (see Jia et. al. 2004 ECWM 781044). This suggests that egl-35 males may have 

defects in the motivation portion of the pathway. Males with null mutations in daf-16, or in both 

tph-1 and cat-1 stay with hermaphrodites suggesting that the neither daf-16transcription factor or 

monoamine signaling is required for retention by hermaphrodites. Starvation reduces leaving but 

this surprisingly does not require tph-1or daf-16activity because both the daf-16 null mutant, and 

the daf-16; tph-1 double null mutants show a reduced rate of leaving when starved.

162. A screen for suppressors of cyclin-D1 in C. elegans 

John Koreth 1 , Mike Boxem 1 , Huihong Xu 2 , Stuart H. Orkin 1 , Sander van den Heuvel 2 

1Dana Farber Cancer Institute, Boston, MA 02115 

2MGH Cancer Center, Charlestown, MA 02129 

Regulation of cell division is a critical aspect of development. In general, cells decide during the 

G1 phase whether to undergo a further round of division or to withdraw from the cell cycle. The 

cyclin D1/cyclin dependent kinase-4 (CYD-1/CDK-4) complex acts on downstream targets to 

enable progression through G1 and entry into S phase. Loss-of-function mutations in cyd-1 and 

cdk-4 abrogate post embryonic cell division and growth in C. elegans, which is rescued in part by 

inactivation of lin-35 Rb (Boxem, Dev. 2000). These results support not only that inactivation of 

lin-35 Rb is an important function of CYD-1/CDK-4, but also that cyclin D kinases must have 

additional functions. To identify additional targets of cyd-1/cdk-4, we have performed a forward 

genetic screen for mutations that suppress the sterility of lin-35(RNAi); cyd-1(he112) animals. In 

particular, we are interested in recessive mutations that bypass the requirement for cyd-1 

function. We have identified several candidate mutations in a substantial screen, including the 

recessive mutation he121, which restores complete fertility to lin-35; cyd-1 double mutant 

animals. Initial characterizations indicate that he121 defines a negative regulator of cell-cycle 

progression, which acts in parallel to lin-35 Rb. Similar functions have been attributed previously 

to CDK Inhibitors (CKIs) cki-1,2 of the Cip/Kip family. cki-1,2 Cip/Kip act in a pathway distinct 

from lin-35 Rb and likely downstream of cyd-1/cdk-4 (Boxem, Dev. 2000). However, SNP 

mapping has placed the gene affected by he121 to a region of Lg II distinct from cki-1,2 Cip/Kip, 

indicating that it represents a novel cell-cycle regulator. Further details on this screen and the 

he121 mutation will be presented.

163. Attempts to develop molecular genetic tools to study parasitic nematodes 

Kelly Kraus, Meera Sundaram 

University of Pennsylvania, Philadelphia PA 19104 

Parasitic nematodes have a tremendous negative impact on human and animal health. Modern 

molecular genetic tools for studying the biology of these parasites are currently lacking. We are 

collaborating with several parasitic helminthologists to try to develop transgenic, RNAi and/or 

morpholino-based approaches to study gene function in these parasites. So far our efforts have 

focused on the parasitic nematode Strongyloides stercoralis. These nematodes can survive for 

one generation outside of the host animal, and we can manipulate this free-living generation in 

ways similar to C. elegans. We are experimenting with DNA microinjections and bombardment 

with various GFP reporter transgenes, as well as testing morpholinos, and will report on our 

progress at the meeting.

164. Reiteration of a lineage branch generating right-sided amphid neurons in ref-1 bHLH 

mutants 

Anne Lanjuin, Julia K. Thompson, Piali Sengupta 

Biology Department, Brandeis University, Waltham, MA. 02454 

Genes that impart neuronal potential to developing cells are highly conserved across species. 

Proneural genes impart neuronal potential, whereas hairy-related neurogenic genes antagonize 

proneural gene activity in neighboring cells. The differential expression of these factors within 

neuronal lineages specify neuronal vs. non-neuronal cell-type identities. In C. elegans, the 

hairy-related gene lin-22 antagonizes lin-32 proneural gene expression in certain lineages and 

has been shown to repress the generation of postdeirids (V5 seam cell derived neuronal 

structures) from the V1-V4 seam cells postembryonically (Wrischnik and Kenyon, 1997). A gene 

more distantly related to hairy, ref-1, has also been shown to repress postdeirid lineage 

development from V6, in addition to regulating cell fusion events in the larval hypodermis (Alper 

and Kenyon, 2001). 

We have identified a novel role for ref-1 in the restriction of a branch of the embryonic lineage 

that generates multiple sensory neuron types. In ref-1 alleles, multiple lineally related sensory 

neurons, including the AWB, ADF, ASE, ASJ and ADL neurons, are generated ectopically. 

Tracing this defect back to the common precursor to this branch of the lineage suggests that 

REF-1 may be required in ABpraaap. The ectopic lineage generated in ref-1 alleles does not 

appear to form at the expense of other closely related lineages, suggesting that it may arise due 

to a lineage reiteration as opposed to transformation of a related lineage. 

Surprisingly, although the lineage that is reiterated in ref-1 mutants exhibits an identical pattern 

of cell divisions on both the left and the right side of the embryo, the ectopic lineage in ref-1 

mutants is generated only on the right. Interestingly, analysis of sensory neuron specific markers 

in ref-1 mutants reveals that the ectopic ASE neuron generated on the right side of the animal 

adopts characteristics of the left ASE neuron. The left identity in this neuron requires many of the 

genes previously shown to diversify the identities of the wild-type left and right ASE neurons, 

suggesting that left vs. right ASE identity might be established as early as the ~150 minute 

embryo, and may be maintained predominantly through lineage intrinsic mechanisms. 

Wrischnik, L. A. and Kenyon, C. J. (1997). Development 124, 2875-2888 

Alper, S. and Kenyon C. (2001). Development 128, 1793-1804

165. Identification and Characterization of Suppressors of him-3 

Ka-Lun Law, Monique Zetka 

Department of Biology, McGill University, 1205 Avenue Docteur Penfield, Montreal, QC, Canada. 

H3A 1B1 

Proper chromosome segregation at meiosis I depends on the initial alignment of homologous 

chromosomes, the stabilization of this alignment through synapsis and the formation of chiasmata 

between homologs. Previous studies have demonstrated that HIM-3, a structural component of 

the meiotic chromosome core, is required for these processes 1,2 . The him-3(vv6) mutation 

results in the substitution of a highly conserved amino acid of the HORMA domain 1 , believed to 

mediate protein-protein interactions. Despite this change, HIM-3 level in vv6 mutant germ 

linesappears to be normal and the protein is loaded to the chromosome core. Similar to other 

him-3 mutants, vv6 mutants still exhibit severe defects in homologue alignment, synapsis and 

chiasma formation, resulting in a high embryonic lethality and him phenotype as a consequence 

of chromosome missegregation. In addition, the nuclear spatial reorganization of early prophase 

nuclei in vv6 is disrupted as indicated by the extension of the transition zone that is populated by 

crescent shaped nuclei after DAPI staining. Our goal is to identify proteins that interact with 

HIM-3 in early meiotic processes by performing an EMS-based suppressor screen using the vv6 

allele. We screened for candidates that suppress the embryonic lethality characteristic of vv6. 

After screening 2382 genomes, we have isolated 4 dominant (vv38, vv39, vv41, vv50) 

suppressors and 1 semi-dominant (vv52) suppressor. In these suppressor strains, the number of 

progeny increased 3- to 6-fold, suggesting that the autosomal non-disjunction phenotype of vv6 

mutants is rescued. Furthermore, these suppressor strains exhibit various levels of X 

chromosome non-disjunction, suggesting that the suppressors may differentially affect the 

segregation of the sex chromosome. We are currently genetically mapping and characterizing 

these suppressor mutations. 

1. Couteau et. al. 2004. Current Biology 14, 585-592. 

2. Zetka et. al. 1999. Genes and Development 13, 2258-2270. 

This project is funded by NSERC and CIHR.

166. A him-8 mutation suppresses the PIE-1-induced synMuv defect 

Jungsoon Lee, Prashant Raghavan, Byung-Jae Park, Tae Ho Shin 

Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 

Recent evidence suggests that PIE-1, a nuclear protein enriched in the germline, binds to and 

inhibits a NuRD-like histone deacetylase complex, composed of at least three polypeptides, the 

zinc-finger protein MEP-1, the Mi-2 homologue LET-418 and the histone deacetylase HDA-1. 

Consistent with this model, ectopic expression of PIE-1 in the somatic cells causes penetrant 

multivulva defects in a lin-15A background, mimicking loss-of-function mutations in the mep-1 and 

let-418 genes. We propose that this repressive function of PIE-1 is essential for the maintenance 

of germline-specific chromatin organization during embryonic development. In order to 

understand the mechanism by which PIE-1 represses the MEP-1/LET-418/HDA-1 complex, we 

are carrying out a genetic screen for suppressors of the synthetic multivulva (synMuv) phenotype 

induced by the ectopic expression of PIE-1. During the course of this screen, we found that an 

existing allele of him-8, e1489, strongly suppresses the PIE-1-induced synMuv phenotype and 

that this suppression occurs without apparent changes in the level or the localization of the PIE-1 

protein. In contrast to him-8, mutations in him-3, him-4 or him-5do not significantly change the 

frequency of the multivulva animals caused by ectopic PIE-1 expression. Interestingly, 

him-8(e1489) fails to suppress the synMuv defect caused by mep-1(RNAi), raising the possibility 

that him-8 mediates the repressive function of PIE-1. Consistent with this possibility, our 

preliminary study indicates that approximately 80% of the pie-1/+; him-8/him-8animals produce 

sterile progeny (the proportion of sterile animals ranges from 5% to 100% of the total brood size) 

while nearly all progeny of the pie-1/+ and of the him-8/him-8 animals are fertile, suggesting that 

him-8, together with pie-1, contributes to proper development of the germ cells. 

him-8 mutants produce an increased number of male progeny owing to a high frequency of 

meiotic non-disjunction. Unlike most other him (high incidence of males) mutants, the him-8 

animals do not lay a large number of dead embryos, and the meiotic non-disjunction in the 

germline of the him-8 animals is apparently limited to the X chromosome. It is thus possible that 

the him-8gene product specifically participates in the organization of the X chromosome, which 

may bear particular significance for the maintenance and the expression of the germline potential. 

In this light, it is tempting to speculate that PIE-1 may also regulate chromatin structures of the X 

through the modulation of the MEP-1/LET-418/HDA-1 activity and perhaps in concert with the 

MES proteins.

167. Interaction between the SEK-1-PMK-1 p38 MAPK and DAF-2/DAF-16 insulin signaling 

pathways mediating pathogen resistance and longevity in C. elegans 

Dennis H. Kim 1 , Valerie Reinke 2 , Danielle A. Garsin 1 , Gary Ruvkun 1 , Siu Sylvia Lee 3 , 

Frederick M. Ausubel 1 

1 Department of Molecular Biology, Massachusetts General Hospital, and Department of 

Genetics, Harvard Medical School 

2 Department of Genetics, Yale University School of Medicine 

3 Department of Molecular Biology and Genetics, Cornell University 

An NSY-1-SEK-1-PMK-1 p38 MAPK pathway is required for C. elegans immunity to bacterial 

pathogens such as Pseudomonas aeruginosa andEnterococcus faecalis, whereas 

loss-of-function mutations in this signaling pathway do not compromise longevity under normal 

OP50 E. coliculturing conditions. We have also observed that long-lived daf-2 andglp (germline 

proliferation defective) mutants are resistant to bacterial pathogens. We have carried out 

epistasis analysis to evaluate the dependence of the pathogen resistance and longevity 

phenotypes of daf-2 and glp mutants on the PMK-1 p38 MAPK pathway. We show that the 

PMK-1 p38 MAPK pathway is required for both pathogen resistance and longevity phenotypes of 

the daf-2 and glpmutants. Further mechanistic studies, and implications for the relationship 

between immunity, stress resistance, and longevity, will be presented and discussed.

168. Global transcriptional changes caused by cognition enhancing compounds in C. 

elegans N2 

French A. Lewis, III, Brian A. Dougherty 

Department of Applied Genomics, Bristol-Myers Squibb Pharmaceutical Research Institute, 5 

Research Parkway, Wallingford, CT 06492 

Donepezil (Aricept) and mematine (Namenda) are current drugs used to treat the cognitive 

deficits caused by Alzheimer’s disease. These drugs act via different mechanisms of action, 

namely by the inhibition of cholinesterase (Aricept) or by blocking the effects of glutamate at the 

N-methyl-D-aspartate receptor (Namenda). However, treating N2 worms with either of these 

compounds produces the phenotype of paralysis. Like the cholinesterase aldicarb, Aricept 

rapidly induces paralysis, causing 100% paralysis by 10 minutes after treatment. The two 

compounds differ in that while the aldicarb paralysis remains for at least one hour, the Aricept 

induced paralysis starts at 100% and decreases to ~67% over the course of a one hour 

treatment. The N2s paralyzed by treatment with either of these cholinesterase inhibitors appear 

to be hyper contracted, which would be expected due to the over stimulation by acetyl choline. 

Mematine also causes paralysis by 10 minutes of exposure, however this compound only 

paralyzes approximately 58% of the worms treated. The phenotype of mematine paralysis is 

more of a hypotonic, flaccid worm. 

Piracetam, a non-Federal Drug Administration approved compound, has been implicated for 

enhancement of cognition. When we treated N2 worms with piracetam, we observed no 

paralysis phenotype. However, when we treated with the active metabolite of piracetam, 

2-pyrrolidinone, we observe approximately 17% paralysis, of a similar quality to mematine. 

We are currently generating and analyzing Affymetrix Gene Chip data from synchronized N2 

cultures treated with various cognition enhancing compounds.

169. Study of par-3 function in C. elegans 

Bingsi Li 

433 Biotech Building, Cornell University, Ithaca, NY14850 

Asymmetric divisions play fundamental roles in generating different cell types during 

development. The PAR proteins are required to establish and maintain cellular polarity in the C. 

elegans embryo. PAR-3, PAR-6 and PKC-3 interact to form a complex, which becomes restricted 

to the anterior cortex in response to a polarity cue from the sperm. Interestingly, in the anterior 

complex, PAR-3 is at the top of the hierarchy in terms of localization dependence. In order to 

study how PAR-3 is localized to the cortex asymmetrically, I am looking for putative PAR-3 

interactors in vivo by knocking down the homologues of newly reported human PAR-3 binding 

partners via RNAi in C. elegans. I will also use yeast-two-hybrid to identify possible PAR-3 

interactors in vitro. 

par-3 was regarded as a strict maternal-effect gene which is only essential in embryogenesis in 

C. elegans. However, Shinya Aono, a former postdoc in our lab found out that PAR-3 is also 

required for postembryonic development in C. elegans. Moreover, it might be expressed 

differently in terms of maternal form and zygotic form. I am trying to identify the isoforms of PAR-3 

by RACE-PCR.

170. sma-9, A Gene that Regulates Body Size Development in C. elegans 

Jun Liang, Ling Yu, Cathy Savage-Dunn 

Department of Biology, Queens College, Graduate Center, CUNY, Flushing, NY 11367 

Individual worm development follows the same pattern and steps, however the body size may 

be different leading to a small or a long animal. Changes of either the cell number or cell volume 

may contribute to these differences, which are regulated by different signaling cascades or living 

conditions. Among these is the DBL-1 pathway, a BMP/TGF-beta related signal transduction 

pathway. Loss of function of the pathway results in a small body size and an abnormal male tail. 

A new gene sma-9 regulates body size development in early larval stages via DBL-1 signaling. 

The Gene sma-9 encodes a zinc finger transcription factor. The SMA-9 N-terminus is rich in 

glutamine; the predicted C-terminus contains seven C2H2 zinc finger motifs. An alternative 

splicing of sma-9 was observed, which might give rise to different SMA-9 isoforms and contribute 

to different molecular functions in vivo. sma-9 transcriptional expression pattern overlaps with that 

of known pathway components, eg. sma-2, sma-3, sma-6 and daf-4, supporting its role in the 

DBL-1 pathway. It is widely expressed from L1 larval stage to adult, but not in the embryo. 

Further analysis of mutant phenotypes demonstrates that sma-9 functions in early but not late 

larval stages and acts downstream of the ligand dbl-1 in regulating body size. sma-9 is 

homologous to Drosophila Schnurri, which is required for Dpp/BMP pathway as a transcriptional 

cofactor of the Smad protein MAD. We propose that SMA-9 may act as a Smad cofactor 

mediating specific transcriptional responses to DBL-1 signaling for regulating body size 

development (Liang et al. Development 2003 130: 6453-64). .

171. Toward Identifying Targets of MAP Kinase During C. elegans germline Development 

Using Functional Proteomic Approaches 

Baiqing Lin, Valerie Reinke 

Department of Genetics, School of Medicine, Yale University, 333 Cedar Street, New Haven, CT 

06520 

MAP kinases are a widely conserved family of serine/threonine protein kinase that transduces 

extracellular signals to the nucleus. One route of the signal transduction is through 

phosphorylation of transcription factors. The phosphorylated transcription factors then alter gene 

expression patterns which result in the appropriate cellular responses. In the development of the 

C. elegans germline, MAP kinase signaling is required for progression through pachytene of 

meiosis I and subsequent differentiation into oocytes. We wish to identify the phosphorylation 

substrates of MAP kinase that mediate this effect. 

We speculated that transcriptional regulators expressed in the germline are the most likely 

candidates for transducing MAP kinase signals in oogenesis. Using genome-wide comparative 

analysis of DNA microarray experiments, we chose 178 genes encoding predicted regulatory 

factors that have germline-enriched expression. These include 164 candidates that have MAP 

kinase consensus phosphorylation sites, and 14 negative controls that do not. The candidates 

encode transcription factors, DNA-binding proteins, and chromatin modulatory proteins. We have 

initiated a project in which we will mass-produce those 178 candidate proteins along with lin-1 

and lin-31 as positive controls using a baculovirus expression system and purify proteins using a 

tandem-affinity purification procedure. We will print purified proteins on a solid platform, a protein 

chip. We plan to test them for in vitro phosphorylation using activated MAP kinase. These studies 

can be followed up by verification of their role as substrates of MAP kinase phosphorylation in 

vivo. This ongoing proteomic work will also be complemented by Dam ID analysis, which 

identifies DNA binding sites for chosen proteins. Additionally, the protein chip can be used to look 

at other post-translational modifications common to transcriptional regulators, such as acetylation 

and ubiquitination, as well as to analyze the ability of the attached proteins to bind consensus 

DNA sequences. Progress in protein expression and purification will be discussed in the 

conference.

172. CaM KII Regulates Neurotransmitter Release at the C. elegans Neuromuscular 

Junction 

Qiang Liu, Zhao-Wen Wang 

Department of Neuroscience, University of Connecticut Health Center, Farmington, CT 06030 

Ca 2+ /calmodulin-dependent kinase II (CaM KII) plays an important role in synaptic plasticity 

and development of the nervous system. Malfunction of CaM KII is associated with a variety of 

diseases including epilepsy. In the nervous system, CaM KII is expressed at both pre- and 

post-synaptic sites. Little is known about the potential role of CaM KII in neurotransmitter release. 

We have embarked on a project to study the function of CaM KII in neurotransmitter release 

using C. elegans as model system. C. elegans has a single CaM KII gene, unc-43. Both 

gain-of-function and loss-of-function mutants are available for analysis. To evaluate the function 

of CaM KII in neurotransmitter release, miniature and evoked postsynaptic currents (mPSCs and 

ePSCs) were recorded at the neuromuscular junction with the postsynaptic body-wall muscle cell 

clamped at -60 mV. We initially analyzed mPSCs and ePSCs in unc-43(n498), a gain-of-function 

mutant. Compared with the wild-type, the mutant showed a significant reduction in the amplitude 

of ePSCs (wild-type 1579 ± 129 pA, n = 8; n-498 721 ± 79 pA, n = 12) and in the frequency of 

mPSCs (wild-type 50.6 ± 3.4 Hz, n = 22; n-498 22.6 ± 3.4 Hz, n = 6). Postsynaptic receptor 

sensitivity appeared normal in the mutant since the amplitude of mPSCs did not change 

(wild-type 27.4 ± 1.6 pA, n = 22; n-498 28.4 ± 4.3 pA, n = 6). These results suggest that a normal 

function of CaM KII is to regulate neurotransmitter release. Experiments are under way to 

determine 1) whether unc-43 loss-of-function mutants show opposite changes of mPSCs and 

ePSCs compared with the gain-of-function mutant; 2) whether the apparent effect on 

neurotransmitter release is due to an action of presynaptic CaM KII, or a retrograde signal 

generated by postsynaptic CaM KII; and 3) what are the phosphorylation targets of CaM KII in 

regulating neurotransmitter release.

173. Control of aging and developmental arrest by TGF and insulin pathways during C. 

elegansdiapause 

Tao Liu, Manjing Pan, Garth Patterson 

Dept of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854 

When resources are scant, C. elegans larvae arrest as non-aging dauers under the control of 

insulin- and TGFß-related signaling pathways. However, relatively little is known about genes 

regulated by these pathways, and how they control complex downstream events. Recent 

experiments suggest that insulin- and TGFß-related pathways function in neurons to control the 

dauer decision (Wolkow et al., 2000 Science. 290:147; Inoue & Thomas 2000 Dev Biol 217:192; 

da Graca et al. 2003 Dev 131:435; Libina et al, 2003 Cell 115:489). We have previously 

presented our analysis of gene regulation at the time of the molt from L2d to the dauer L3. At this 

time, over 1200 genes are substantially regulated in a TGFß-pathway-dependent manner. Many 

insulin pathway and other known dauer regulatory genes are regulated by TGFß in a manner that 

suggests strong positive feedback. We have identified a large group of direct targets of the FOXO 

DAF-16 transcription factor, which are substantially different from the set of genes regulated by 

DAF-16 to control aging in adult animals. 

Our next approach is to use microarray analysis to compare wild-type, daf-2 (insulin receptor) 

and daf-7 (TGFß ligand) mutants from hatching through dauer. This analysis will allow us to 

distinguish between genes that function early in making the dauer decision from genes that 

function later in dauer morphogenesis or maintenance of the dauer fate. We are integrating 

functional analysis with expression analysis by using the RNAi feeding method to knock down 

expression of genes that show TGFß-dependent regulation. In the screen for dauer phenotypes, 

we have identified several genes whose RNAi increases dauer formation and life extensions (see 

abstract by Patterson et al.). We will present our detailed analysis of function of insulin ligands, 

putative non-kinase insulin receptors, and other genes regulated by the TGFß pathway.

174. End-to-end chromosome fusions in C elegans 

Mia R. Lowden, Bettina Meier, Shawn C. Ahmed 

Department of Biology 216 Fordham Hall University of North Carolina at Chapel Hill Chapel Hill, 

NC 27599-3280 USA 

Telomeres are DNA/protein complexes that protect the ends of eukaryotic linear chromosomes. 

Telomeres comprise a simple repetitive sequence, whose length is maintained by telomerase, an 

enzyme that is absent from most human somatic cells. We are working with C. elegans mutants 

that are defective for telomere replication, including several alleles of the trt-1 telomerase reverse 

transcriptase. Telomere attrition in such strains results in the formation of end-to-end 

chromosome fusions, which are stably transmitted, as C. elegans chromosomes are holocentric 

(i.e., the mitotic spindle attaches along the length of a chromosome). Most end-to-end fusions 

from trt-1 mutants are homozygous viable, allowing for physical analysis of the initial fusion event 

that occurs in the absence of telomere replication. In some cases, both fused ends are intact until 

the telomeric repeat tract begins. Typically, however, telomeric DNA from one of the fused ends 

has completely eroded and several kilobases of sub-telomeric DNA are missing. A hot-spot for 

end-to-end fusion may have been identified near the right end of the X chromosome. 

Non-homologous end-joining may be the pathway responsible for producing end-to-end fusions 

that occur as a consequence of telomerase defects. However, end-to-end fusions still occur in 

trt-1 mutants that lack DNA ligase IV, a protein that is essential for NHEJ. The breakpoints of 

such fusions are being examined to determine if their structure differs from those that occur in the 

presence of NHEJ.

175. An intracellular serpin, srp-6, is required for survival from hypoosmotic shock in 

Caenorhabditis elegans. 

Cliff J. Luke 1 , Stephen C. Pak 1 , Vasantha Kumar 1 , Sule Çataltepe 1 , Carmen M. Knebel 1 , 

Anthony Clark 1 , Deiter Brömme 2 , Gary A. Silverman 3 

1 Department of Pediatrics, Harvard Medical School and Division of Newborn Medicine, Children’s 

Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115 

2 Department of Human Genetics, Mount Sinai School of Medicine, Fifth Ave at 100th Street, New 

York, NY 10029 

3 Department of Pediatrics, Harvard Medical School and Division of Newborn Medicine, Children’s 

Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115 

Serpins are a superfamily of lysosomal cysteine and/or serine proteinase inhibitors. We 

identified one member of the C. elegansserpin family, srp-6, that inhibited the lysosomal cysteine 

proteinases. Srp-6 was expressed predominantly in the gland cell and the vulval muscles. The 

longevity and morphology of the srp-6 knockout animals were normal. However the serpin 

knockout animal had an adverse response to hypoosmotic stress. Compared to N2 worms, srp-6 

-/- animals show a marked decreased in survivability when placed in water (98% vs 10% viability, 

respectfully). A phenocopy induced by RNAi in adult worms showed that this defect was not due 

to a developmental abnormality. Using genetic and pharmacological approaches, we showed that 

hypoosmotic death in srp-6 -/- animals was associated with prolonged elevations in intracellular 

calcium and was independent of the classical apoptopic cell death pathway, but involves 

proteolysis by cysteine proteinases. We are now undertaking a genetic screen to determine other 

genes that may fall in this pathway. These data suggest that Srp-6 plays a role in protecting C. 

elegans from hypoosmotic shock.

176. Genetic Analysis of the Putative SUP-9/SUP-10/UNC-93 Two-Pore Domain K + Channel 

Complex 

Long Ma, Bob Horvitz 


sup-9, sup-10 and unc-93 encode components of a presumptive C. elegans two-pore domain 

K + channel complex. Rare gain-of-function mutations of each of these three genes cause 

abnormal body muscle contraction and are thought to activate the SUP-9 K + channel. The 

mutant animals are defective in egg laying, sluggish and exhibit the "rubberband" phenotype: 

when prodded on the head, the animals contract and relax along their entire bodies without 

moving backwards. The SUP-9 protein is similar to the mammalian Two-pore Acid Sensitive K + 

channels TASK-1 and TASK-3. sup-10 encodes a novel single transmembrane protein without 

apparent mammalian orthologs. unc-93 encodes a multiple transmembrane protein that defines a 

novel family of proteins conserved from C. elegans to mammals. Previous screens for recessive 

suppressors of the mutant phenotype of unc-93(e1500sd) animals identified only loss-of-function 

mutations of unc-93, sup-9 or sup-10. To seek essential genes that interact with sup-9, sup-10 

and/or unc-93, we screened ~10,000 EMS mutagenized F1 unc-93(e1500sd) animals clonally by 

picking animals with better locomotion and identified five partial suppressor strains. These five 

strains have better locomotion and a weaker rubberband phenotype, and are either homozygous 

sterile or carry mutations that cause sterility and that are closely linked to the suppressors. As an 

alternative approach to identify unc-93(e1500sd) suppressors essential for development and/or 

survival, we are currently screening using RNAi clones reported to cause sterility or lethality from 

the whole-genome RNAi library 1 . 

1 Kamath et al. (2003) Nature 421: 231-237.

177. Modeling and simulation of the behavior of proliferating C. elegans germ cells 

John Maciejowski 1 , Nadia Ugel 2 , Marco Isopi 2,3 , Bud Mishra 2,4 , E. Jane Albert Hubbard 1 

1Department of Biology, New York University 

2Courant Institute of Mathematical Sciences, New York University 

3Department of Mathematics, Università di Roma "la Sapienza" 

4Cold Spring Harbor Laboratory 

Most animals, including C. elegans, Drosophila, and mammals, maintain germline stem cells to 

provide a virtually endless supply of gametes. In mammals and Drosophila, individual germline 

stem cells are identified by their unique gene expression and their location within a somatic niche 

- a microenvironment crucial to the maintenance of germline stem cell fate. These stem cells 

divide asymmetrically to both self-renew and to contribute a cell to differentiation. In many cases, 

the differentiating daughter cell undergoes additional rounds of division (transit amplification 

divisions) prior to differentiation. 

The C. elegans germ line also requires a somatic niche, the distal tip cell (DTC), for 

maintenance of germline proliferation. However, molecular markers for individual stem cells are 

not yet available nor has it been determined whether transit amplification divisions - as distinct 

from stem cell divisions - do or do not occur. Relative to the number of cells in the distal mitotic 

zone of the adult, cell divisions are infrequently seen in fixed preparations, but they can be 

detected anywhere within the zone. Thus, for lack of more accurate means to define the distal 

mitotic zone, it is sometimes referred to as a germline "stem cell population". 

We would like to better define the behavior of cells in the mitotic zone and are taking both 

computational and laboratory-based approaches toward this end. The computational systems 

biology approach involves a model of the dynamics of cell division in the mitotic zone based on 

"snapshots" of the process. We have collected images from hundreds of fixed samples of adult 

gonads (in multiple focal planes per sample) that are stained for DNA and for cells in M phase. 

We are applying image-recognition software to extract quantitative information (cell counts and 

positions of actively dividing cells) from each focal plane. 

Besides using this information for statistical analyses, we use it as the starting point for building 

a simulation. The simulation models the behaviors of individual cells arranged on a grid or in a 

3-dimensional space as a function of their position (e.g., distance from the distal end or distance 

to other cells in the same gonad arm) and/or the surrounding environment. Our immediate aim is 

to address the issue of whether the mitotic zone is a homogeneous or heterogeneous population 

with respect to the probability of cell division based on distance from the distal end. Later studies 

will focus on earlier developmental stages and on mutants that display aberrant mitotic zones.

178. Nog mutants and early germline proliferation in C. elegans 

John Maciejowski, Giselle Cipriani, Ji-Inn Lee, James Ahn, Kerry Donny-Clark, E. Jane Albert 

Hubbard 

New York University Department of Biology 

In many animals, germ cell fate is specified very early in development, and primordial germ 

cells (PGCs) proliferate prior to germline differentiation. The molecular basis for early PGC 

proliferation is not well understood, and we are using C. elegans genetics to identify genes 

involved in this process. 

In C. elegans, as in other orgnanisms, germline proliferation is dependent on germline/soma 

interactions. After several divisions of the PGCs, germline proliferation is dependent on 

GLP-1-mediated interactions between the soma and germ line. glp-1 null mutants generate 4-8 

germ cells that all differentiate as sperm (Austin and Kimble, 1987). The molecular basis for the 

control of initial rounds of PGC proliferation is unknown, though early proliferation can be 

prevented by ablation of the two somatic cells that flank the PGCs in the L1 gonad primordium 

(Kimble and White, 1981). In addition, several mutants have been described in which PGCs 

undergo only a few rounds of division but do not enter meiosis, even in the absence of glp-1 (e.g., 

Beanan and Strome, 1992; Kadyk and Kimble, 1997), suggesting that the control of early 

proliferation and differentiation can be separated. 

To understand more about the molecular control of early germline proliferation, we have 

identified zygotic mutations that cause a Nog (no apparent germ line) phenotype. In our Nog 

mutants, the PGCs (Z2 and Z3) are generated and locate properly to the gonad primordium, but 

despite a morphologically normal somatic gonad, the germ cells neither divide nor differentiate. 

So far, we have cloned two nog mutants. Both (1) encode highly conserved critical factors for 

translation, and (2) are autosomal but have close paralogs on the X chromosome. Given that 

germline-intrinsic genes are underrepresented on the X chromosome (Reinke et al., 2000), this 

result suggests a soma/germline separation of cell-essential functions at the level of genome 

organization. Preliminary data support that the autosomal genes are germline-specific whereas 

their X-linked paralogs are expressed in the soma. That the X-linked paralog can perform all 

somatic functions in at least one of the mutants is further supported by lifespan extension in the 

mutant. Moreover, the requirement for robust translation in early germline proliferation is indicated 

by the severity of the mutant germline phenotype.

179. DNA replication and the proliferation versus meiotic development decision. 

Valarie Vought 1 , Min-Ho Lee 2 , Larissa Wirlo 1 , Katy Michalak 1 , Deborah Springer 1 , Ying Liu 1 , 

Valerie Reinke 3 , Tim Schedl 2 , Eleanor Maine 1 


2Department of Genetics, Washington University, St. Louis, MO 

3Department of Genetics, Yale University, New Haven, CT 

The late larval/adult C. elegans germline contains both proliferating and meiotic germ cells. 

Proliferating germ cells reside at the distal end of each gonad arm; as germ cells move 

proximally, they enter meiotic prophase and progress through gametogenesis. Germ cell 

proliferation is induced by GLP-1/Notch receptor signaling. GLP-1 signaling promotes proliferation 

by inhibiting the activities of the GLD-1 and GLD-2 pathways, which function redundantly to 

promote meiosis and/or inhibit proliferation. GLD-1 is likely to, at least in part, induce entry into 

meiotic prophase by inhibiting the translation of mRNAs that promote proliferation while GLD-2 is 

likely to, at least in part, promote entry into meiotic prophase by activating the translation of 

meiotic mRNAs. 

We previously identified several ego genes, which interact genetically with GLP-1 signaling 

[enhancers of weak glp-1 loss-of-function (lf)] and function to promote germline proliferation 

and/or inhibit meiotic development (Qiao et al. 1995; Smardon et al. 2000). ego genes encode a 

diverse set of proteins, including (1) LAG-1, a transcriptional regulator that is a core component of 

GLP-1/Notch signaling, (2) EGO-1, an RdRP that promotes several aspects of germline 

development (see abstract by Yu et al. ), and (3) ATX-2, a post-transcriptional regulator that 

promotes both proliferation and the oocyte fate (see abstract by She et al.). Here we identify the 

ego-5 gene as encoding the B subunit of the DNA polymerase (pol) alpha-primase complex, 

which is thought to function in assembly and nuclear transport of the complex but lacks catalytic 

activity. This finding has led us to investigate whether regulation of DNA replication proteins might 

be an aspect of the proliferation/meiosis decision. 

To investigate the specificity of the interaction between ego-5 and glp-1, we asked whether a 

weak glp-1 lf allele could be enhanced by knockdown of (1) other components of the DNA 

polymerase alpha-primase complex, (2) other DNA replication proteins, and (3) other proteins 

required for the mitotic cell cycle. In the absence of GLP-1 signaling, germ cells both prematurely 

cease proliferation AND prematurely enter meiosis; this phenotype is distinct from a simple 

mitotic defect, where germ cells cease proliferating, but do not enter meiosis. We find that weak 

glp-1 lf is also enhanced under conditions of partial RNAi-mediated knockdown of other (catalytic) 

subunits of the DNA pol alpha-primase complex and by depletion of some other DNA replication 

proteins. In contrast, depletion of various other cell cycle proteins (e.g. , cdk2) does not enhance 

weak glp-1 lf indicating that a general mitotic cell cycle progression defect does not induce 

meiotic entry. These results suggest that partial depletion of DNA replication factors may mimic a 

process that is a normal part of the switch to meiotic development. 

gld-1 promotes meiotic entry, and we have recently identified ~100 new putative GLD-1 mRNA 

targets based on a co-immunoprecipitation/amplification/microarray detection strategy. Among 

the targets are mRNAs that encode DNA replication proteins, including a component of the DNA 

pol alpha-primase complex. In other organisms, meiotic S phase is several-fold slower than 

mitotic S phase (see Forsburg, 2002). While the reasons for this difference are unclear, the 

reduced rate of DNA replication in meiotic S phase may be necessary for the association of 

factors with the chromosomes that are required for later meiotic events (e.g., recombination 

between homologs). Taken together, our data suggest a model where GLD-1 promotes entry into 

meiosis, in part, via translational repression of mRNAs that encode certain DNA replication 

proteins; reducing the level of certain replication proteins may decrease the rate of DNA 

replication, which in turn may be an important aspect of the switch from mitotic to meiotic S 

phase. 

Qiao, et al. (1995) Genetics 141, 551-569 

Smardon, et al. (2000) Current Biology 10, 169-178. 

Forsburg (2002) Mol Cell 9, 701-711.

180. The molecular analysis of ego-2. 

Ying Liu 1 , Deborah Swenton 1 , Dave Hansen 2 , Eleanor Maine 1 


2Department of Genetics, Washington University, St. Louis, MO 

In the C. elegans germ line, the GLP-1/Notch signaling pathway regulates the switch from 

proliferation to meiosis. GLP-1 is activated in the distal germ line by a signal from the somatic 

distal tip cell, and distal germ cells are thereby induced to proliferate. In the absence of GLP-1 

signaling, germ cells exit mitosis, enter meiosis, and differentiate. The ego screen was designed 

to identify components, regulators, and targets of the GLP-1-mediated signaling pathway in the 

germ line (Qiao et al., 1995). An ego-2 mutation, om33, was recovered as an enhancer of a weak 

glp-1 loss-of-function mutation in the germ line (Qiao et al. 1995). It also enhances a weak 

glp-1(lf) in the embryo (A. Smardon and E. Maine, unpublished data). 

ego-2(om33) was originally isolated on a chromosome with several other mutations; we have 

now recombined those mutations away, allowing us to examine the ego-2 phenotype in more 

detail. In a glp-1(+) background, ego-2(om33) has a temperature-sensitive Spe (spermatogenesis 

defective) phenotype. Preliminary data suggest that ego-2(om33) does not suppress the glp-1(gf) 

phenotype in the germ line, suggesting that ego-2 may act upstream of or in parallel with glp-1. 

We are now doing genetic analysis to elucidate the relationship between ego-2 and other genes 

that promote germ line proliferation (e.g., the GLP-1 signaling pathway and atx-2) and meiotic 

entry (e.g., gld-1 and gld-2). 

Previously, ego-2 was mapped between dpy-24 and unc-101 on the right arm of chromosome I 

(Qiao et al. 1995). To clone the gene, we have now mapped it relative to other cloned marker 

genes and single nucleotide polymorphisms (SNPs). We have localized ego-2 to an ~137 kb 

region between SNPs in M04D5 (11334980 bp) and ZK1025 (11471740 bp), which includes 21 

predicted genes. We are continuing the SNP mapping and using RNAi to deplete the gene 

products and test for enhancement of a weak glp-1(lf). 

Qiao et al. (1995) Genetics141, 551-569

181. Control of lipid accumulation by ciliated neurons in C. elegans 

Ho Yi Mak, Gary Ruvkun 

Department of Molecular Biology, Massachusetts General Hospital; Department of Genetics, 

Harvard Medical School, Boston MA 02114, USA. 

Energy balance and lipid homeostasis are controlled by the hypothalamus in mammals and 

there is emerging evidence that hypothalamic neurons are ciliated. Using a panel of mutants that 

have functional or structural defects in ciliated neurons, we report an evolutionarily conserved role 

for ciliated neurons in the control of lipid accumulation in C. elegans. 

The C. elegansorthologue of the obesity gene tubby,tub-1, is expressed in ciliated neurons. 

We previously found that tub-1 deletion mutant animals showed a mild increase in lipid 

accumulation that could be dramatically enhanced by loss of function mutations in kat-1, which 

encodes a 3-ketoacyl-coA thiolase involved in peroxisomal beta-oxidation. Based on the strong 

genetic interaction between tub-1 and kat-1, we reasoned that additional components of the tubby 

pathway might be identified in a kat-1 modifier screen. To this end, we screened 79200 haploid 

genomes after EMS mutagensis of kat-1(mg368)animals, for mutants that show synergistic 

increase in lipid accumulation in a kat-1(mg368) dependent manner. One isolate, mg409, is 

dye-filling defective indicating a failure in ciliated neuron differentiation. Accordingly, mg409 

mutant animals have a small body size and display excessive dwelling behaviour, two 

phenotypes that had been attributed to sensory deficits. We identified the gene mutated in 

mg409 by SNP mapping and found that it is orthologous to human BBS1, a gene mutated at high 

incidence in patients suffering from Bardet-Biedl syndrome, a complex genetic disease where 

obesity is one of the prominent clinical features. bbs-1 and tub-1function in the same genetic 

pathway, since bbs-1; tub-1animals do not accumulate more lipid than bbs-1 or tub-1single 

mutant animals. bbs-1 is expressed in ciliated neurons in C. elegans and a BBS-1::GFP fusion 

protein is localised to the transition zone, a structure similar to the basal body of cilia in other 

organisms. Besides bbs-1, a number of genes are known to be required for the differentiation 

and maintenance of ciliated neurons, such as che-2 and osm-5. Similar to bbs-1; kat-1mutant 

animals, kat-1; che-2 and kat-1;osm-5 animals showed a synergistic increase in lipid 

accumulation. 

Taken together, control of lipid accumulation depends on structural and functional integrity of 

ciliated neurons in C. elegans. We speculate that neuronal non-motile cilia may be a conserved 

structure that senses external and internal nutrient level.

182. n3263 is a mutant with persistent cell corpses that defines a candidate new 

engulfment gene 

Paolo M. Mangahas 1 , H. Robert Horvitz 2 , Zheng Zhou 1,3 

1 Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 

77030 

2 Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Ave, 

Cambridge, MA 02139 

3 Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of 

Medicine, One Baylor Plaza, Houston, TX 77030 

During the development of multi-cellular organisms, a large number of cells are eliminated by 

apoptosis or programmed cell death. These apoptotic cells must be efficiently cleared from the 

system in order to prevent them from undergoing secondary necrosis, leaking toxic substances 

and triggering autoimmune and inflammatory responses. 

Genetic studies in the nematode Caenorhabditis elegans have identified seven genes that 

function in the engulfment of dying cells: ced-1, -2, -5, -6, -7, -10, and -12. Double mutant 

analysis has organized these genes in two parallel and partially redundant pathways. In one 

pathway, CED-1, a transmembrane receptor expressed in engulfing cells recognizes cell corpses 

and clusters around them to mediate their recognition and subsequent engulfment. ced-7 

encodes an ABC-type transporter that may regulate the transport/distribution of signal molecules 

required for recognition by CED-1. ced-6 encodes a PTB-containing protein required in the 

engulfing cell and may function as an adaptor protein in signaling downstream of CED-1. In the 

second pathway, CED-2 (CrkII), CED-5 (DOCK180) and CED-12 (ELMO1) form a protein 

complex that regulates the activity of CED-10 (Rac GTPase). Activation of this signaling pathway 

results in Rac-mediated reorganization of the actin cytoskeleton in engulfing cells that results in 

phagocytosis of cell corpses. 

In order to identify new genes that function in the engulfment of apoptotic cells, we have 

performed genetic screens for mutants with persistent cell corpses. Here, we report the isolation 

and preliminary characterization of n3263, a recessive mutation that results in the persistence of 

a few cell corpses in embryos before hatching. This phenotype is subject to maternal-effect 

rescue by wild-type gene product. n3263 mutation might affect the efficiency of cell-corpse 

engulfment, or the timing or efficiency of the execution of programmed cell death. Mapping and 

complementation tests suggest that n3263 is neither one of the seven engulfment genes, nor is it 

an allele of ced-8, a gene that encodes a transmembrane protein that controls timing of cell 

death. We are currently in the process of cloning the gene and performing analyses of genetic 

interactions.

183. Localization of APH-1 Protein in Embryos 

David McGaughey, Valerie Hale, Caroline Goutte 

Department of Biology, Amherst College, Amherst MA 01002 

APH-1 is an essential component of the Notch signaling pathway. Loss of embryonic aph-1 

activity results in a fully penetrant embryonic lethal phenotype which resembles that caused by 

depletion of glp-1, presenilin (hop-1 and sel-12), or aph-2 activity. aph-1 is predicted to encode an 

evolutionarily conserved seven-pass transmembrane protein. Genetic and biochemical evidence 

suggest that APH-1 interacts with presenilins to facilitate the intramembranous cleavage of Notch 

receptor in response to ligand. The nature of this interaction and the specific role of APH-1 are 

not fully understood. We have developed APH-1-specific antisera to visualize native APH-1 

protein in cells that are known to be involved in Notch signaling events. We have begun our 

analysis in the embryo, where the localization of other signaling components, such as GLP-1 

receptor, APX-1 ligand, and APH-2, has been well characterized. We show here that APH-1 

protein can be detected in oocytes and all embryonic cells up to at least the 50-cell stage; in all 

cells APH-1 localizes to the outer cell membrane. This result supports the idea that APH-1 is a 

member of the cell-surface presenilin/APH-2 complex, and eliminates the possibility that APH-1 

might act only early in the secretory pathway to promote the assembly of such a complex, without 

itself remaining involved. Previous work had demonstrated that the APH-2 protein localizes to the 

outer cell membrane in a manner that is dependent on both APH-1 and presenilin proteins. Here 

we investigate the reciprocal relationship to determine whether APH-1 is dependent on APH-2 

and presenilins to translocate to the outer cell membrane.

184. EGL-32 Functions in Sperm to Regulate Egg-Laying through the TGF-beta Pathway in 

C.elegans 

Marie McGovern, Ling Yu, Cathy Savage-Dunn 

Queens College, CUNY, Flushing, NY 11367 

In C.elegans, egl-32 functions through the TGF-beta dauer pathway to effect egg laying. 

Provided conditions are favorable (plenty of food, and no overcrowding), the daf-7 signaling 

cascade represses daf-3 and daf-5 function, resulting in suppression of dauer formation in young 

animals and normal egg laying rates in adults. Mutations in the ligand, daf-7, the two receptors, 

daf-4 and daf-1, or the smads, daf-8 and daf-14, result in dauer constitutive and egg-laying 

defective phenotypes. These mutants are suppressed by mutations in daf-3 and daf-5. Although 

there are three pathways regulating the dauer decision (including the TGF-beta pathway) only the 

TGF-beta pathway is also involved in regulating egg-laying. We are interested in the role of this 

TGF-beta pathway in egg-laying. egl-32 is implicated in this TGF-beta pathway because its 

egg-laying defective phenotype is suppressed by daf-3 and daf-5. However, egl-32 is specific to 

the egg-laying branch of this pathway because egl-32 mutants do not show a dauer constitutive 

phenotype, only the egg-laying defective phenotype. Temperature shift assays on the 

temperature sensitive allele of egl-32(n155) reveal that the critical period for EGL-32 activity is at 

the L4 stage of development. 

The egl-32 mutant animals retain about twice as many eggs as wild-type animals. Normally 

eggs are laid by the comma stage of development. 41% of the eggs retained by egl-32 animals 

are at the comma stage or later. These eggs eventually hatch inside the mother resulting in a bag 

of worms as more larvae hatch internally and begin to eat their way out. We have begun 

experiments to identify the egl-32 gene product. In the course of this work, a linked interacting 

locus was identified whose transcript is enriched in sperm. In addition, the L4 stage, the critical 

stage for EGL-32 activity, is not the time when eggs are laid, but the only time when C.elegans 

hermaphrodites produce sperm. We were therefore interested in testing to see if egl-32 

functioned in sperm to effect egg-laying. When wild-type sperm was introduced, through mating 

with wild type males, into egl-32 hermaphrodites, the egl-32 hermaphrodites began to lay eggs at 

a normal rate. The number of animals bloated with eggs was reduced from 87% to 25%. This 

work is part of a growing literature on the influence of the germline on multiple aspects of adult 

development.

185. sns-10, the C. elegans ortholog of Aristaless/ARX, regulates sensory and motor 

neuron development 

Tal J. Melkman, Piali Sengupta 

Biology Department, Brandeis University, Waltham MA 02454 

Recent studies have shown that mutations in the Aristaless related gene, ARX, in humans are 

linked to mental retardation. While the cause for retardation is not clear, in mice, loss of ARX 

function causes reduced proliferation of neuronal precursors and abnormal migration and 

differentiation of GABAergic interneurons. The role of ARX in neuronal development and function 

is not fully understood. In Drosophila, mutations in Aristaless result in defects in patterning of a 

number of organs including the antennae. In particular, the most distal parts of the antennae, the 

aristae sensory organs, are lost. We have identified mutations in the C. elegans Aristaless/ARX 

ortholog, sns-10, and show it is also involved in neuronal development in C. elegans. 

sns-10 mutants show a variable loss of both AWA and ASG chemosensory neurons. Although 

AWA and ASG are sister cells, their loss in sns-10 mutants is not correlated suggesting that it is 

not due to a loss of their precursors. Therefore, SNS-10 affects AWA and ASG specification 

independently. In addition, the interaction of SNS-10 with the forkhead transcription factor 

UNC-130 andthe LIM homeodomain transcription factor LIN-11 appears to depend on the cell 

type. We have previously shown that UNC-130 acts in the AWA/ASG precursor cell to restrict the 

AWA potential to the daughter cell that will become AWA (Sarafi-Reinach and Sengupta, 2000). 

LIN-11 acts in AWA to initiate the expression of ODR-7, a nuclear hormone receptor transcription 

factor (Sarafi-Reinach et al., 2001) that is necessary for the specification of the AWA neurons 

(Sengupta et al., 1994). Mutations in lin-11also affect the differentiation of the ASG neurons. 

While in AWA development SNS-10 appears to function in parallel to UNC-130 and upstream of 

LIN-11, in ASG development, SNS-10 may act in parallel to both. Interestingly, in Drosophila, 

Aristaless was shown to act upstream of the LIN-11 ortholog dLim1. Moreover, ARX knockout 

mice lose expression of Lhx9, a LIN-11 related factor. Thus, understanding SNS-10 function in C. 

elegans may lead to insights into conserved cascades in other organisms. 

sns-10 mutants show various other defects. Of particular interest is the postembryonic 

formation of ectopic DD motoneurons. The fact that these motoneurons are GABAergic and that 

ARX knockout mice showed impaired GABAergic neuron development, suggests that sns-10 may 

have a common function in the specification of these cell types as well. We are currently 

determining the source of the ectopic DD motoneurons and the role sns-10 plays in this 

developmental pathway. Taken together, our understanding of SNS-10 function in various 

processes of fate specification in C. elegans may lead to insights into the function of its orthologs 

in other organisms and provide a better understanding of its role in neuronal development.

186. him genes and X chromosome meiosis 

Philip M. Meneely, Joshua Havassy, Kathryn Crozier 

Haverford College, Haverford PA 19041 

The him genes are proving to be a rich source of information about meiosis in C. elegans. Two 

genes, him-5 and him-8, affect the X chromosome much more strongly than they do the 

autosomes. However, they appear to do this by quite distinct methods. Recessive alleles of him-8 

resemble mutations of the X chromosome pairing center and have no effect on the autosomes. A 

collaboration between us and Abby Dernberg’s lab has shown that him-8 encodes a C2H2 zinc 

finger protein that binds specifically at the X chromosome pairing center. Binding of HIM-8 at this 

region is essential for X-chromosome pairing by an unknown mechanism. In contrast, him-5 

affects the X chromosome much more strongly than the autosomes, but autosomal effects are 

seen. We find that him-5 mutants are desynaptic, and the initial events of pairing and synapsis 

occur normally before the homologues come apart prematurely at diakinesis. him-5 encodes a 

small and extremely basic protein, with no significant similarity to any other protein but with 

overall similarity to many different chromosomal proteins. From antibody staining, HIM-5 is found 

exclusively in germline nuclei and may be loaded onto chromosomes extremely early in germline 

development in pre-meiotic nuclei. We have seen no evidence for specific localization of HIM-5 to 

a particular chromosome or a particular structure. This suggests that the effect of him-5 on 

meiosis is mediated through other properties of the X chromosome, not yet defined.

187. Characterization of an hlh-8 mutant 

Stephany G. Meyers, Ann K. Corsi 

Department of Biology Catholic University of America Washington D.C. 20064 

The hlh-8 gene encodes a basic helix-loop-helix (bHLH) transcription factor that is involved in 

mesoderm development. hlh-8 function is related to postembryonic M lineage patterning and 

enteric muscle development 1 . The M lineage cells differentiate into egg laying and body wall 

muscles as well as ceolomocytes. The hlh-8 gene is composed of 5 exons with a 2Kb intron after 

the first exon. We are currently characterizing a mutant, hlh-8(tm726), with a large deletion (646 

bp) in the large first intron of the gene. Two previous mutants have been characterized: 

hlh-8(n2170) 2 , a semidominant point mutation, and hlh-8 (nr2061) 1 , a presumptive null 

mutation. These mutations occur in the bHLH coding region and result in a phenotype that 

disrupts the proper development of the egg-laying and enteric muscles. Due to the improper 

muscle development, the previously characterized mutations result in constipated and egg laying 

deficient phenotypes. However, hlh-8(tm726) causes a constipated phenotype, but the animals 

are still able to lay eggs. Thus, we hypothesize that this phenotype may be due to a 

dosage-specific issue in the enteric muscles but not in the M lineage. 

We plan to introduce gfp constructs into tm726 animals to determine if the known downstream 

targets of HLH-8 are still activated. We will investigate the hlh-8 intron using a pes-10::gfp 

construct. pes-10::gfp contains the basic promoter machinery and can be activated in a variety of 

tissues by juxtaposition to a tissue-specific enhancer. Expression of a pes-10 construct made with 

intron 1, will yield insight into the enhancer elements of the intron region of hlh-8. Real Time PCR 

will also give a quantitative examination of the dosage of HLH-8 in the deletion mutation. Finally, 

Reverse transcription will be utilized to determine if the mutant mRNA differs from that of wild 

type hlh-8 mRNA. 

1 Corsi, A.K., Kostas, S.A., Fire, A. and Krause, M. (2000). Caenorhabditis elegans Twist plays 

an essential role in non-striated muscle development. Development. 127, 2041-2051. 

2 Corsi, A.K., Brodigan, T.M., Jorgensen, E.M., Krause, M. (2002). Characterization of a 

dominant negative C. elegans Twist mutant protein with implications for human Saethre-Chotzen 

syndrome. Development. 129, 2761-2772.

188. Functional Analysis of the MicroRNA Genes of C. elegans 

Eric A Miska 1 , Ezequiel Alvarez-Saavedra 1 , Allison L Abbott 2 , Andrew B Hellman 1 , Nelson C 

Lau 3 , David P Bartel 3 , Victor Ambros 2 , Bob Horvitz 1 

1 HHMI, Dept. Biology, MIT, Cambridge, MA 02139, USA 

2 Dept. Genetics, Dartmouth Medical School, Hanover, NH 03755, USA 

3 Whitehead Institute for Biomedical Research and Dept. Biology, MIT, Cambridge, MA 02142, 

USAWhitehead Institute for Biomedical Research and Dept. Biology, MIT, Cambridge, MA 

02142, USA 

The heterochronic genes lin-4 and let-7 encode small (21-22 nt) non-protein coding regulatory 

RNAs 1,2 . Strains carrying a mutation in either of these genes are heterochronic, displaying 

retarded development with some cell lineages having an altered temporal pattern of cell division 

and differentiation. lin-4 and let-7 normally inhibit translation of target genes that when mutated 

lead to a phenotype opposite that of lin-4 and let-7 mutants: precocious development and early 

expression of certain paths of cell division and differentiation. 

Recently, molecular and bioinformatic approaches have identified many genes encoding small 

RNAs in C. elegans, Drosophila and mammals 3-5 . All of these genes encode 21-25 nt RNAs 

derived from longer transcripts that contain partially double-stranded RNAs. These small RNAs, 

termed microRNAs (miRNAs, mirs), define a large, new class. 

To understand the biology of the C. elegans microRNA genes, we decided to combine the 

generation of loss-of-function mutants with GFP expression studies and target prediction using 

bioinformatics. To date we have generated deletion strains corresponding to 51 microRNAs. We 

will present the initial characterization of mutant phenotypes (for information about the mir-35 and 

the let-7 families of microRNAs, see poster by Alvarez-Saavedra et al.). One focus will be the 

issue of redundancy within families of microRNA genes. 

In a complementary approach we are generating knockout strains for the argonaute family of 

genes. The argonaute genes have been implicated both in RNAi and microRNA function 6,7 . We 

will present the initial characterization of the knockout phenotypes of two argonaute genes, prg-1 

and prg-2. We will present the defects of these mutants in germline development and the 

relationship of these genes to the microRNAs and to RNAi pathways. 

1. Lee, R. C., Feinbaum, R. L. & Ambros, V. The C. elegans heterochronic gene lin-4 encodes 

small RNAs with antisense complementarity to lin-14. Cell 75, 843-54 (1993). 

2. Reinhart, B. J. et al. The 21-nucleotide let-7 RNA regulates developmental timing in 

Caenorhabditis elegans. Nature 403, 901-6 (2000). 

3. Lagos-Quintana, M., Rauhut, R., Lendeckel, W. & Tuschl, T. Identification of novel genes 

coding for small expressed RNAs. Science 294, 853-8 (2001). 

4. Lau, N. C., Lim, L. P., Weinstein, E. G. & Bartel, D. P. An abundant class of tiny RNAs with 

probable regulatory roles in Caenorhabditis elegans. Science 294, 858-62 (2001). 

5. Mourelatos, Z. et al. miRNPs: a novel class of ribonucleoproteins containing numerous 

microRNAs. Genes Dev 16, 720-8 (2002). 

6. Grishok, A. et al. Genes and mechanisms related to RNA interference regulate expression of 

the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23-34 (2001). 

7. Tabara, H. et al. The rde-1 gene, RNA interference, and transposon silencing in C. elegans. 

Cell 99, 123-32 (1999).

189. Genetic and molecular analysis of the C2H2 zinc-finger gene ehn-3 

Kristin M. Morphy 1 , Judith Kimble 2,3 , Laura D. Mathies 1 

1Department of Genetics, North Carolina State University, Raleigh, NC 

2Department of Biochemistry, University of Wisconsin, Madison, WI 

3Howard Hughes Medical Institute, Madison, WI 

Development of the gonad begins with a simple four-celled primordium. The outer cells (Z1 and 

Z4) are somatic gonadal precursor cells (SGPs), which generate all somatic tissues of the gonad 

(e.g. uterus). The inner cells (Z2 and Z3) are primordial germ cells, which give rise to gametes. 

Past research has shown that the Hand bHLH gene (hnd-1) and the enhancer of hnd-1 (ehn-3) 

redundantly affect the position and presence of the SGPs (Mathies et al., 2003). In addition, 

ehn-3 acts redundantly with the sex-determining gene tra-1: whereas SGPs in tra-1 and ehn-3 

single mutants generate many descendants, those in tra-1; ehn-3 double mutants do not mature 

or divide. To identify additional genes acting in the tra-1 and hnd-1 pathways to control 

gonadogenesis, we are screening for genetic enhancers of ehn-3. From 1600 haploid genomes, 

we have identified six ehn-3 enhancers. Linkage mapping of these enhancers is in progress. 

The ehn-3 gene was cloned and found to encode two proteins, each containing two paired 

zinc-fingers, and no other recognizable motif. There are two existing alleles of ehn-3: q766 is a 

deletion removing the promoter and part of the first exon, q689 is an apparent promoter mutation. 

Curiously, the ehn-3(q766) deletion has a much weaker phenotype than ehn-3(q689). We are 

currently exploring the possibility that the ehn-3 gene contains additional upstream exons. We will 

report our progress at the meeting. 

Mathies et al. (2003) Development 130, 2881-2892.

190. Analysis of an UNC-13 protein expressed from an internal promoter 

Theresa Moser 1 , Bethany Stitt 2 , Kristin Servent 3 , Brooke Swalm 1 , Lydia Sanchez 1 , Monique 

Spencer 1 , Rebecca Kohn 1 

1Biology Department, Ursinus College, Collegeville, PA 

2Drexel University Medical School, Philadelphia, PA 

3Thomas Jefferson University, Philadelphia, PA 

Several protein products are expressed from unc-13. The predominant product localizes to 

synapses (Kohn, 2000) and is important for vesicle priming (Richmond, 2001). We are interested 

in understanding the roles of the other proteins expressed from the gene and are examining their 

expression patterns. Previous cosmid rescue experiments indicated that an internal promoter is 

used for expression of an UNC-13 protein product. We constructed a fusion of the putative 

promoter with the GFP gene and injected it into worms. We also developed antibodies to 

recognize the protein product expressed from the internal promoter. These antibodies bind 

UNC-13 proteins on Western transfers. GFP expression and immunohistochemical studies will 

determine the localization patterns of the less abundant form of UNC-13.

191. cwp-4, a novel male-specific C. elegans gene with a potential role in mating behavior 

William R. Mowrey 1 , Douglas S. Portman 2 


2 Center for Aging and Developmental Biology and Department of Biomedical Genetics, 

University of Rochester Medical Center, Rochester, NY 14642 

Male mating behavior in C. elegans comprises a series of steps whose execution is mediated 

by male-specific sensory structures. The RnB neurons of the male tail rays, as well as the 

hook-innervating HOB neuron and the CEM head sensory neurons, express the polycystin 

orthologs lov-1 and pkd-2, which are required both for the response of males to hermaphrodites 

and for the subsequent location of the hermaphrodite vulva. LOV-1 and PKD-2 are thought to 

form a channel complex in the ciliated endings of these neurons that transduces a calcium signal 

in response to sensory stimulation. In a microarray screen for new ray-expressed genes, we 

identified five novel genes that exhibit an anatomical expression pattern identical to that of lov-1 

and pkd-2, suggesting that the products of these genes could act in a common signaling pathway. 

We have dubbed these genes cwps (co-expressed with polycystins). The primary sequence of 

the products of the cwp genes indicates that they may have an extracellular site of action, 

consistent with being accessory to the function of the transmembrane polycystins. Like LOV-1, 

CWP-4 contains an S/T-rich mucin domain that is a potential target for glycosylation. To test the 

possibility that cwp-4 has a role in polycystin-mediated signal transduction, we are studying male 

mating behavior in animals homozygous for a large deletion in the cwp-4 locus. Preliminary 

behavioral analysis has indicated that cwp-4 males may be defective in the first step of male 

mating behavior, the response to contact with a hermaphrodite. Subsequent steps are currently 

being studied. Our findings are consistent with the possibility that CWP-4 has a role in sensing 

hermaphrodite contact. We are continuing to characterize cwp-4 mutant males to elucidate the 

role of this gene in mediating male-specific behaviors.

192. Identification of genes regulating chemosensory neuron-specific morphologies 

Saikat Mukhopadhyay, Anne Lanjuin, Piali Sengupta 

Department of Biology, Brandeis University, MS008, 415 South Street, Waltham, MA 02454, USA 

Amphid chemosensory neurons sense the external environment via their non-motile cilia, which 

are specialized for the particular neuron type. For example, AWB has characteristic forked cilia, 

while AWC has fan-like cilia. How are these structures specified and maintained? FKH-2, a 

forkhead domain transcription factor is expressed widely in the embryo (Molin et al., 2000) but 

expression is restricted to the AWB, ASK and ASI amphid neurons in larval stages and adults. In 

fkh-2 mutants, the ciliary structures of the AWB and AWC neurons but not other neuron types are 

affected. Dendritic development of AWB and AWC neurons is also affected in fkh-2 mutants, and 

this effect is manifested as stunting of the dendritic processes as early as the first larval stages. 

FKH-2 expression in the amphid neurons in early larval stages is regulated by DAF-19, an 

RFX-type transcription factor regulating general cilia development in all sensory neurons. 

However, the expression of fkh-2 is partly restored in daf-19 dauers and adults. It is possible that 

daf-19 could be regulating fkh-2 to determine cell-specific cilia morphology in AWB in addition to 

its general function in cilia formation. FKH-2 also acts in parallel with the OTX family 

homeodomain protein CEH-37 to specify the subtype identities of the AWB and ADF sensory 

neurons. Currently we are screening for additional mutants exhibiting altered cilia morphology 

specifically in the AWB neurons in order to characterize cell-specific factors necessary for their 

specialized cilia structure. We are also investigating the role of neuronal activity in the 

maintenance of ciliary structure. 

References: Molin L., Mounsey. A., Aslam, S., Bauer, P., Young, J., James, M., Sharma-Oates, 

A., Hope, I. (2000) Development 127, 4825-4835.

193. The Role of PDZ Domain Proteins in GLR-1 Localization 

Vidhya Munnamalai, Christopher Rongo 


Our goal is to understand how synaptic connections between neurons in the central nervous 

system are formed and modified during development, with particular emphasis on how glutamate 

receptors (GluR) are localized to synapses. We place particular emphasis on how glutamate 

receptors are localized to synapses because their trafficking plays an important role in synaptic 

plasticity, and in models of learning and memory. In C. elegans, GLR-1 and GLR-2 form 

glutamate-gated channels similar to GluRs found in the human brain. It is highly likely that the 

features that regulate GLR-1 and GLR-2 localization do so by physically interacting with GLR-2 

carboxy-terminal amino acids because these fifty-five amino acids are sufficient to direct 

localization when fused to a reporter protein. One family of molecules that could be interacting 

with the GLR-2 carboxy-terminus is the PDZ domain family, members of which are thought to 

bind to carboxy-termini of many cell surface molecules in both epithelia and neurons. I am 

interested in finding PDZ proteins that are required for glutamate receptor localization in the C. 

elegans nervous system. Using the Nematode Expression Pattern Database (NEXTDB), I have 

narrowed down a list of PDZ proteins to eight candidate proteins. So far, I have been able to 

show that a mutation in one of these PDZ proteins is required for proper GLR-1 localization. I am 

currently trying to understand the mechanism by which this protein facilitates GLR-1 localization.

194. Uncovering the role for sperm contributed SCU-1 in regulating meiotic exit and axis 

formation in the early Caenorhabditis elegans embryo 

MaryAnn Murrow, Anna Mazor, Rebecca Lyczak 

Department of Biology, Ursinus College, Collegeville, PA 19426 

During early development in the nematode Caenorhabditis elegans, cues from the sperm 

trigger completion of meiosis by the oocyte chromosomes and establishment of the 

anterior-posterior axis. Little is known about the control of these events and their possible 

interdependence. To study this, we are analyzing a mutant in C. elegans, called scu-1 (sperm cue 

abnormal) which has defects in both meiotic exit and anterior-posterior axis formation in the 

one-cell embryo. Interestingly, the scu-1 gene shows a partial paternal requirement, as reduced 

viability of embryos is observed from crosses involving either scu-1 mutant eggs with wild-type 

sperm or wild-type eggs with scu-1mutant sperm. 

P> P> 

In an attempt to better understand the maternal and paternal requirements for the scu-1 gene 

product in meiotic exit and polarity, we are performing mel and pel crosses in which either eggs or 

sperm are mutant for scu-1, and examining the resulting embryos via time-lapse imaging under 

DIC optics. As some embryos produced from these crosses survive to adulthood, it is not 

surprising that some of the embryos we’ve examined so far look completely wild-type during the 

first cell division. We are continuing these crosses in the hope of uncovering if any maternal or 

paternal specific defects will be observed. 

P> P> 

In addition to analysis of maternal and paternal scu-1 contributions in embryos, we are 

beginning to look at the meiotic defects of scu-1 mutants in more detail. Previously, meiotic 

defects have been observed in embryos produced by scu-1 mutant hermaphrodites. We are now 

in the process of determining if similar defects are observed during sperm production in scu-1 

mutant males. In addition, as chromosome segregation defects are occasionally observed during 

oocyte meiosis in scu-1 mutant embryos, we are testing to see if scu-1 mutant hermaphrodites 

produce an overabundance of males due to nondisjunction of the X chromosome. It is our hope 

that these studies will result in a clearer picture of the role for scu-1during meiosis and the role of 

maternal and paternal SCU-1 during early development.

195. Characterization of the identity and specificty of RGS protein targets in C. elegans 

Edith M. Myers, Michael R. Koelle 

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520 

Regulator of G protein signaling (RGS) proteins shorten the duration of signaling through 

heterotrimeric G proteins by stimulating the intrinsic GTPase activity of Gαproteins. In mammals, 

there are more than 25 RGS proteins and more than 20 Gα proteins. Each RGS protein contains 

an RGS domain sufficient to activate GTPase activity, and most contain other domains that may 

confer divergent functions to each of the RGS proteins. The role of the other domains remains 

unclear and, despite extensive in vitro and cell culture studies, it is still unclear whether full-length 

RGS proteins stimulate the GTPase activity of specific Gα proteins in vivo. In addition, little is 

known about proteins that may modify RGS protein activity, stability, or sub-cellular 

localization. We will purify and analyze RGS protein complexes from C. elegans. By 

characterizing the identity of RGS targets and the specificity of such interactions, our goal is to 

convert current genetic models for RGS function into concrete molecular mechanisms. 

We first plan to examine biochemically whether RGS proteins specifically bind their genetically 

defined Gα targets. Extensive genetic analysis of two antagonistic G protein signaling pathways 

in C. elegansdemonstrates that the RGS protein EAT-16 inhibits signaling through the Gα q 

EGL-30, while the RGS protein EGL-10 inhibits signaling through the Gα o GOA-1. We have 

expressed functional tagged EAT-16 and EGL-10 RGS proteins in C. elegans. After stabilizing 

the normally transient RGS . Gα interactions by treating C. elegans homogenates with 

GDP-AlF 4 - , we will affinity purify RGS . G protein complexes using the tandem affinity purification 

(TAP) tag. By Western blot analysis of the complexes, we will determine specifically whether 

EAT-16 and EGL-10 bind, and presumably activate, their genetically identified Gα targets, 

EGL-30 and GOA-1, respectively. 

Using this same experimental methodology, we also plan to test a model in which the RGS 

protein acts as a subunit in a new type of G protein heterotrimer. EAT-16 and EGL-10 are 

members of a family of RGS proteins that contain Gγ-like domains, through which they are 

constitutively bound to a Gβ 5 subunit. Such Gβ 5 . RGS dimers suggest that Gα . Gβ5 . RGS 

heterotrimers may form in addition to the analogous classical Gα . Gβ . Gγheterotrimers. The 

existence of Gα . Gβ 5 . RGS heterotrimers would provide an elegant mechanism for a switch 

between the antagonistic EGL-30 and GOA-1 pathways. In this proposed mechanism, the 

inactive heterotrimers would be released from the Gα subunit upon GTP binding. The released 

Gβ 5 . RGS dimers would activate the GTPase activity of the opposing Gα protein, thus turning off 

signaling through the antagonistic pathway. For instance, upon GTP binding, the Gβ 5 . EGL-10 

dimer would be released from GDP-bound EGL-30, and would activate the GTPase activity of 

activated GOA-1. We will affinity purify RGS complexes, and look for the EGL-30 . Gβ 5 . EGL-10 

or GOA-1 . Gβ 5 . EAT-16 heterotrimers with Western blot analysis. These assays will be 

performed in the absence of GDP-AlF 4 - , since our aim is to identify proteins interacting with the 

GDP-bound Gα subunit. 

Finally, we plan to identify other proteins in the purified RGS complexes and determine their 

role in G protein signaling. Components of such complexes will be identified by mass 

spectrometry, and their role in signaling will be determined after phenotypic analysis of animals 

carrying knockouts of the corresponding genes.

196. Evidence that lin-35 Rb functions in hyp7 to inhibit vulval fates 

Toshia R Myers, Iva Greenwald 

Columbia University 701 W 168th ST 720 HHSC NY NY 10032 

Class A and class B synthetic Multivulva (synMuv) genes function in genetically redundant 

pathways to inhibit vulval fate specification. Many synMuv genes encode proteins that have been 

implicated in transcriptional repression in other systems, such as the class B synMuv gene lin-35 

Rb (Lu and Horvitz, 1998). Knowing the cellular focus of synMuv gene activity is critical to 

understanding their roles during vulval development and the signaling events that lead to the 

invariant pattern of VPC fates. lin-35 Rb has been presumed to act in the vulval precursor cells 

(e.g. Ceol and Horvitz, 2001) but other SynMuv genes have been inferred to act in hyp7 (e.g. 

Herman and Hedgecock, 1990). Using a hyp7-specific promoter, we have obtained evidence that 

lin-35 Rb functions in hyp7 to inhibit vulval fates. We are currently performing mosaic analysis, as 

well as the complementary experiment with a VPC-specific promoter, and will report on our 

progress at the meeting

197. RNAi-mediated screen for meiotic genes in C. elegans 

Sandra Nagl, Allison Hurlburt, Mónica Colaiácovo 

Dept. of Genetics, Harvard Medical School, Boston, MA 02115 

Meiosis is the cell division program that allows diploid germ cells to generate haploid gametes. 

This process unfolds through a first (reductional) division and a second (equational) division. Our 

focus is on events occurring during prophase of meiosis I, during which homologous 

chromosomes pair, synapse and undergo meiotic recombination. A striking feature of this process 

is the formation of a proteinaceous structure known as the synaptonemal complex (SC) between 

paired and aligned homologous chromosomes. Crossover recombination is completed within the 

context of a fully formed SC, leading to the formation of chiasmata (physical connections between 

the homologs) that ensure proper alignment of homologs at the metaphase plate and subsequent 

proper segregation upon the first meiotic division. Errors in any of the processes mentioned 

above can lead to missegregation of chromosomes. 

We are interested in identifying new meiotic genes (in particular those involved in synapsis) 

and are continuing a functional genomics approach successfully used in Colaiácovo et al., 2002. 

We are taking advantage of genome-wide germline-enriched expression profiles presented in 

Reinke et al., 2003, based on microarrays encompassing 92% of the currently predicted genes in 

the C. elegans genome. The particular dataset we are using contains genes that are significantly 

enriched in the germline but are not differentially expressed in the male or female germlines. 

We have defined a subset of 192 genes that most closely match the behavior of known meiotic 

genes and are conducting an RNAi screen to identify those genes which elicit defects in meiotic 

prophase. Our screening strategy consists of: 1.) Scoring for increases in embryonic lethality 

associated with high frequencies of males (Him phenotype) on plates, produced by the F1 

generation of the injected animals; 2.) Monitoring for sterility among the F1 generation, as a result 

of an arrest during meiotic progression; 3.) Candidates that score positive for either one of the 

two criteria described above are subsequently subjected to cytological analysis allowing us to 

assess whether defects are specific to meiotic chromosomes and allowing us to assign them into 

distinct phenotypical categories. 

Our RNAi screen for meiotic genes is ongoing. A summary of the results will be presented

198. A germline-specific cell cycle inhibitor involved in dauer and adult lifespan 

Patrick Narbonne, Richard Roy 


Genetic analysis has demonstrated that the decision to execute dauer development is 

controlled by three parallel pathways. Little is known however concerning the downstream 

effectors common to these three pathways that mediate the important metabolic and 

morphological changes that take place during the formation of the dauer larva. One feature 

associated with this stage is the progressive establishment of cell cycle quiescence throughout 

the animal, including the cells of the germline. 

Surprisingly, we noticed that a lag-2::GFP transgene is strongly expressed in the DTCs 

throughout the dauer stage, suggesting that the germ cell nuclei presumably receive signals to 

proliferate and/or to block entry into meiosis, and should be free to divide mitotically in the dauer 

larva, yet they do not. We reasoned therefore that genes downstream of the dauer-inducing 

signals may be required to repress mitosis in the germline by affecting lag-2 or genes 

downstream. To identify these potential regulatory genes, we performed a pilot screen from which 

we have isolated and cloned one mutant (rr48) in which the dauer larvae show pronounced 

germline hyperplasia when induced by reduced TGF-beta or insulin signalling. Our genetic 

analysis suggests that rr48 acts in a dominant negative manner in the germline, and a 

time-course examination of germline proliferation revealed that rr48 is required to progressively 

block germline proliferation during dauer formation. Moreover, we observed that daf-2;rr48 dauer 

larvae die after 9-11 days in this stage, suggesting that rr48 is required to enhance survival of the 

dauer larva over extended periods. Consistent with this, daf-2;rr48 adults have a considerably 

reduced lifespan relative to daf-2 animals, indicating that rr48 is required for the full lifespan 

extension that is induced by low insulin-like signalling. The reduced dauer lifespan is not 

suppressed by germline ablation, indicating that germline overproliferation is not responsible for 

the observed rr48-mediated dauer lethality. 

To investigate what downstream genes rr48 may affect to repress the cell cycle specifically in 

the germline, we characterised its relationship with components of the Notch signalling pathway, 

which are known to be important in regulating cell divisions in this tissue. A hypomorphic mutation 

in lag-2 that significantly represses proliferation (without allowing germ cells to execute the 

meiotic programme) prior to dauer formation in daf-2 animals, partially suppressed the 

rr48-induced germline hyperplasia, arguing that a lag-2 signal is required for the mitotic 

proliferation observed in the rr48 mutant. We also found that rr48 is epistatic to glp-1 with respect 

to germline meiotic cell cycle progression. Germ cell nuclei are never seen beyond meiotic 

pachytene in daf-2 glp-1 dauers. However, they are often seen to have completed meiosis in 

daf-2 glp-1;rr48 dauers as they resemble sperm nuclei after DAPI staining. We believe that rr48 is 

acting in a daf-16 independent branch of the insulin-like pathway downstream of daf-2 to repress 

cell cycle progression in the germline. This may occur through altering the effect of Notch 

signalling at a step downstream of glp-1. How this crosstalk between the insulin-like pathway and 

germline cell cycle regulation may occur is currently under investigation and should be clarified 

when rr48 is characterised molecularly.

199. The C. elegans pumilio ortholog, puf-9, controls the timing of development 

Mona J. Nolde, Kristy Reinert, Nazli Saka, Frank J. Slack 

Dept of Molecular, Cellular and Developmental Biology, Yale University, PO Box 208103, New 

Haven, CT 06520 

Developmental timing is controlled by heterochronic genes, mutations in which cause changes 

in the relative timing of developmental events. Genetic studies in C. elegans have shown that the 

known heterochronic genes are ordered in a regulatory pathway. Late acting genes in this 

pathway include the small untranslated RNA, let-7; two of its target genes, lin-41 and hbl-1 (the C. 

elegans hunchback gene); and the transcription factor lin-29. LIN-29 is thought to be responsible 

for coordinating specific cellular fates at the larval to adult transition. Its expression appears to be 

posttranscriptionally repressed by lin-41 and hbl-1 until the L4 stage when LIN-29 protein is first 

observed. let-7 is thought to relieve lin-29 repression by binding to complementary regions in the 

3’UTRs of lin-41 and hbl-1, and act in an inhibitory manner to down regulate LIN-41 and HBL-1 

proteins. Translation of Drosophila hunchback is repressed in the embryo by binding of Nanos, 

Pumilio and Brat proteins at regulatory elements in the hunchback 3’UTR called Nanos Response 

Elements (NRE). Brat and LIN-41 belong to the same RBCC-NHL family of proteins, supporting 

the role of LIN-41 as a translational repressor. Members of this protein family are poorly 

understood and include many human disease genes including tumor suppressors and 

oncogenes. 

We have taken an RNAi approach to look for genetic interactions between lin-41 and the C. 

elegans homologs corresponding to Drosophila nanos and pumilio. We have evidence that puf-9, 

one of the C. elegans pumilio genes, acts downstream of lin-41 and coordinately with let-7 to 

negatively regulate hbl-1 and thus promote adult specific events. Preliminary data suggests that 

hbl-1 translation is regulated in a puf-9 dependent manner. In addition, the hbl-1 3’UTR contains 

multiple sequences related to NRE’s, the canonical Pumilio binding sites. We are testing the 

hypothesis that PUF-9 directly regulates hbl-1 and that lin-41 in turn regulates puf-9 activity.

200. High throughput genetic screen for suppressors of necrotic cell death 

Yury O. Nunez, Dewey Royal, MaryAnn Royal, Michael Lizzio Jr., Monica Driscoll 

Rutgers University, Department of Molecular Biology and Biochemistry, Nelson Biological 

Laboratories, 604 Allison Road, Piscataway, NJ 08854 

Necrotic cell death, often initiated by ion channel hyperactivation, plays a major role in the initial 

and prolonged death of neurons consequent to injury. Blocking or delaying such necrotic cell 

death would significantly limit this incapacitating neuronal damage. Our goal is to identify genes 

critical for the progression through ion channel-induced neuronal necrosis using a powerful 

experimental model -the nematode Caenorhabditis elegans. Like apoptotic cell death 

mechanisms, mechanisms of injury-induced necrosis appear conserved between nematodes and 

humans. Common features of invertebrate and vertebrate necrosis include induction by 

hyperactivated ion channels, essential rise in intracellular calcium levels and activation of calpain 

proteases. Much remains to be learned, however, about the molecular mechanisms operative. 

What we learn in C. elegans will likely provide novel insight into mammalian necrosis 

mechanisms. 

We are conducting a high-throughput saturation genetic screen to identify mutations that block 

necrotic cell death consequent to Na+ channel hyperactivation (the mutant MEC-4(d) channel) 

and to identify and genetically map mutations capable of reversing the death process. Our screen 

strategy is based on mec-4(d)-induced death of the touch receptor neurons. We begin with a 

strain that is Is5[pmec-4GFP]; mec-4(d) in which touch receptor neurons die. We then 

mutagenize and screen for rare animals in which touch neurons live, as evidence by restoration 

of touch neuron fluorescence. We have now exploited the automated screening capacity of the 

COPAS Biosort, based on sorting of fluorescent signals to screen large numbers of mutagenized 

genomes. We have screened 56,600 EMS-mutagenized genomes for mutations that suppress 

necrotic cell death (and plan to screen another 50,000 ENU-mutagenized genomes). This work 

so far identified 16 novel alleles that fail to complement known suppressors of mec-4(d)-induced 

necrosis, which define an absolute minimum of 3 new death suppressor genes (the number of 

distinct genes will very likely increase when genetic tests are completed). We will report on our 

mapping data and phenotypic characterization for such 3 new genes.

201. Activation of SKN-1 stress response by a chemoprotective antioxidant 

Riva P. Oliveira 1 , Jae Hyung An 1 , Rosana P. Baker 1 , Hideki Inoue 2 , Kunihiro Matsumoto 2 , T. 

Keith Blackwell 1 

1 Joslin Diabetes Center and Departament of Pathology, Harvard Medical School, 1 Joslin Place, 

Boston, MA 02215, USA 

2 Division of Biological Science, Nagoya University, Nagoya 464-8602, Japan 

In C elegans, a major oxidative stress defense is mediated by the transcription factor SKN-1, 

which is distantly related to and functions analogously to the vertebrate Nrf proteins. SKN-1 

accumulates in intestine nuclei and activates Phase II detoxification genes during oxidative 

stresses. The localization and activity of the SKN-1 protein are regulated under normal conditions 

and in response to stress by at least two different signaling mechanisms (p38 and GSK-3). In 

collaboration with our lab, the Matsumoto group has determined that arsenite activation of PMK-1 

MAPK (p38) is dependent upon its MAPKK SEK-1. Arsenite-induced accumulation of 

SKN-1::GFP in intestinal nuclei and activation of gcs-1, the rate-limiting enzyme for glutathione 

synthesis, are also sek-1-dependent. 

We have previously determined that intestinal expression of gcs-1 is upregulated in a SKN-1 

dependent manner in response to a wide range of different oxidative stress agents. These agents 

include heavy metals, hydrogen peroxide, and diamide. In addition to oxidative stress inducers, 

SKN-1 nuclear accumulation and upregulation of gcs-1 is also observed after treatment with 

sulforaphane, a non-toxic indirect protective antioxidant. In vertebrates, sulforaphane seems to 

disrupt the Keap1-Nrf2 complex either by reacting covalently with thiol groups present in the 

Keap1 protein. In C elegans there is no apparent direct ortholog of Keap1, suggesting that 

sulforaphane activates gcs-1 through SKN-1 in a Keap1-independent manner. We are currently 

investigating whether p38 is required for sulforaphane-induced nuclear localization of SKN-1 and 

concomitant expression of gcs-1. Preliminary experiments indicated that during sulforaphane 

treatment SKN-1 activation and expression of gcs-1 in the intestine is dependent upon sek-1. We 

are also investigating whether sulforaphane increase stress resistance and longevity in C 

elegans.

202. Some Dauer Formation, Social Feeding, and Chemotaxis Mutants are Abnormal in the 

Enhanced Slowing Response 

Daniel Omura, Bob Horvitz 


Well-fed animals encountering a bacterial lawn exhibit a dopamine-dependent basal slowing 

response mediated by the mechanosensory ADE, PDE, and CEP neurons. Acutely food-deprived 

animals exhibit a serotonin-dependent enhanced slowing response mediated by an undefined set 

of sensory neurons (Sawin, E. R., et al., Neuron 26, 619-31, 2000). The serotonin-gated 

chloride channel MOD-1 (modulation of locomotion defective) and the serotonin reuptake 

transporter MOD-5 are involved in propagating and attenuating the enhanced slowing response, 

respectively (Ranganathan, R., et al., J Neurosci 21, 5871-84, 2001; Ranganathan, R., et al., 

Nature408, 470-5, 2000). The mutation mod-6(n3076) causes animals to have a reduced 

enhanced slowing response and is likely an allele of che-3 (chemotaxis defective). Some but not 

all che-3 alleles cause defects in the enhanced slowing response. Our investigation of mutants 

bearing defects in sensory neurons in Mod behavior has led to the hypothesis that mutants 

broadly defective in sensory neuron function exhibit increased slowing, while mutants defective in 

a subset of chemosensory neurons are Mod and exhibit reduced enhanced slowing. Defects in a 

subset of chemosensory neurons that inhibit locomotion may specifically reduce the enhanced 

slowing response, while defects in a larger group of neurons that affect locomotion may result in 

greater slowing on bacteria. We are currently attempting to identify specific neurons and genetic 

pathways involved in the detection of bacteria when animals are food deprived. 

Acute food deprivation results in an internal state change that can be detected when animals 

are re-exposed to their food source, as outlined above. Mutants were previously identified that 

have a reduced enhanced slowing response after acute food deprivation. We have more recently 

screened for mutants that exhibit constitutive enhanced slowing. Such mutants would exhibit a 

serotonin-dependent slowing response in both the well-fed and food-deprived states, which could 

reflect a defect in the generation, storage, or signaling of a well-fed state. In this screen we 

sought mutations that conferred paralysis upon entering a bacterial lawn in the absence of acute 

food deprivation in a mod-5(n3314) background. Mutants that moved well in the absence of 

bacteria, grew at a roughly normal rate, and displayed a paralysis that was antagonized by the 

serotonin receptor antagonist methiothepin were saved for further analysis. Ten such mutants 

were isolated including a Mos1 transposon insertion allele of mrp-1 (multidrug resistance protein 

familiy). Studies by others have found that mrp-1 is synthetically dauer constitutive with unc-31 

(Yabe, T, et al., 2002 Japanese Worm Meeting abstract 5151). This finding led us to investigate 

the role of dauer genes in the enhanced slowing response. Thus far we have found dauer 

mutants that exhibit constitutive enhanced slowing as well as mutants that have reduced 

enhanced slowing. We are currently investigating the role of mrp-1 and dauer pathway genes in 

the enhanced slowing response.

203. Transcriptional regulation and stochasticity of eff-1 in fusing cell types. 

Eugene Opoku-Serebuoh, Victoria L. Scranton, William A. Mohler 

263 Farmington Ave, University of Connecticut Health Center, Dept. of Genetics and 

Developmental Biology, MC3301, Farmington, CT 06030 

In C. elegans, cell fusion requires the activity of the eff-1 gene. Nematodes that are mutant for 

this gene fail to fuse their hypodermal, vulval, and pharyngeal cells during development. We find 

that ectopic expression of eff-1 is lethal to embryos and larvae, suggesting that precise temporal 

regulation of eff-1 expression in these specific cell types is important for normal development. We 

have also found that full function of the eff-1 gene in rescuing mutant phenotypes requires ~7.5 of 

5’-upstream sequence. Thus we are interested in understanding the transcriptional control of eff-1 

expression. A transcriptional fusion of the eff-1 promoter to GFP (eff-1p::gfp) reveals a complex 

spatio-temporal expression pattern. Many cells that are fated to fuse express eff-1p::gfp in the 

moments leading up to cell fusion, suggesting that their fusion fate/competence may be limited 

specifically by expression of EFF-1 protein. Cell fusions occur during various developmental 

stages and in various tissues of the animal, and the eff-1 promoter appears to be differentially 

activated during the morphogenetic processes in which cell fusion is involved. We have created a 

series of transgenic lines that harbor progressively 5’-truncated versions of eff-1p::gfp. We have 

identified 0.5-kb regions in the promoter that are required for tissue-specific transcriptional activity 

of the transgene in the pharynx, vulva, and amphid sheath, and a separate region for repression 

of gut expression. Enhancer assay experiments have further been used to confirm the 

tissue-specific expression patterns conferred by some of these regions in the eff-1 promoter. As 

these upstream domains are likely to contain enhancing and silencing cis regulatory elements, we 

have now begun site-directed mutagenesis of recognizable transcription-factor binding motifs. In 

addition, we are currently performing yeast one-hybrid experiments to identify possible 

transcription factors that would regulate eff-1 promoter activity in a tissue-specific context. 

Our initial efforts indicate that PHA-4 may act directly upon eff-1 to drive pharyngeal 

expression. Interestingly, we observe a stochastic "all-or-none" effect on pharyngeal expression 

when two putative PHA-4 binding sites are mutated. The similar expression of eff-1p::gfp by each 

binucleate cell in the metacorpus of a given animal indicates the existence of a "noise-damping" 

signaling mechanism that tightly coordinates gene expression levels among these cells of quite 

distinct lineage.

204. Characterization of mutants defective in intestinal nuclear division. 


Department of Biology, McGill University, Montréal, Québec 

The appropriate coordination of developmental signals and the cell cycle machinery is required 

for the formation of a fertile adult organism. Our laboratory uses the intestinal lineage of C. 

elegans as a model system to understand this complex relationship due to the invariant 

developmentally-regulated cell cycle transitions that are characteristic of this lineage. We have 

performed screens using an intestinal-specific GFP marker (elt-2::GFP) in order to isolate 

mutants with an altered number of intestinal nuclei as a consequence of irregularities in the 

developmental control of cell/nuclear number. 

We have isolated a total of 5 mutants that affect the number of elt-2::GFP-expressing 

nuclei, all of which hatch with a wild-type number of instestinal cells, suggesting that these 

mutations do not affect embryonic mitosis. Rather, these mutations affect the cell cycle transition 

from mitosis to karyokinesis and finally to endoreplication that normally occurs at the first larval 

stage. The initial characterization of these mutants allowed us to classify them into two different 

groups based on their phenotype: more intestinal nuclei than wild-type (rr33 and rr45) and less 

intestinal nuclei (rr42, rr43 and rr44) than wild-type. Lineage analysis of rr33 suggests that the 

intestinal cells perform karyokinesis normally at the L1 stage but they fail to make the timely 

transition to endoreplication at the L1 moult. Consequently, they perform a second round of 

nuclear division early in the L2 stage to become tetranucleate. 

We performed epistasis analysis between the different mutants. We first tested the double 

mutant rr33; rr45 (two mutants with more intestinal nuclei) and found that they were synthetic 

lethal. rr42and rr43 shows non-allelic non-complementation suggesting that they are in the same 

pathway. We then crossed the mutants from the two groups. First, the double mutant rr33; rr42 

has an rr42 mutant phenotype, suggesting that rr42 is epistatic to rr33. However, the rr42 

mutation partially suppresses the rr45mutation and these double mutants have a wild-type 

number of intestinal nuclei. Finally, even though rr42 and rr43seem to be in the same pathway, 

rr43 does not suppress rr33. Therefore, we believe that rr33 is epistatic to rr43. 

In order to gain more knowledge of the pathway that controls these intestinal nuclear divisions, 

we mapped these mutants. rr33 mapped close to dpy-5 on LGI and rescue experiments are 

underway with cosmids covering this region. Meanwhile, rr45, which has a similar phenotype to 

rr33, mapped to an interval between bli-4and unc-24 on LGV. Injection of the cosmids covering 

the genetic position of rr45 is underway. rr42 (less intestinal nuclei due to a defect in 

karyokinesis) was mapped on LGV at around 2.5 MU. We found that a rescuing cosmid subclone 

containing only one gene, predicted to encode a rho-like GTPase, rescues the mutant 

phenotype. We performed RNAi with this gene and we were able to phenocopy the rr42mutant 

phenotype suggesting that rr42 encodes a rho-like gene. The rho-like gene family was shown to 

be involved in many different cellular processes, most of which involve microtubule dynamics. 

Elongated intestinal nuclei are observed in rr42 mutants suggesting that they may have a defect 

in destabilizing the central microtubule network during nuclear division. Mapping is also currently 

underway for the remaining mutants in this group (rr43 and rr44) to determine the molecular 

identity of the other members of this pathway required for post-embryonic intestinal nuclear 

division.

205. SRP-2 is an intracellular serpin that participates in postembryonic development 

Stephen C. Pak, Vasantha Kumar, Christopher Tsu, Cliff J. Luke, Yuko S. Askew, David J. 

Askew, David R. Mills, Anthony C. Clark, Gary Silverman 

Department of Pediatrics, Harvard Medical School, Children’s Hospital Boston, 300 Longwood 

Ave, Enders 950, Boston, MA 02115 

Serpins are high molecular weight serine proteinase inhibitors that inactivate target proteinases 

by a suicide substrate-like mechanism. Serpins can be categorized into two broad groups, 

intracellular and extracellular. Most extracellular serpins function in the circulation and regulate 

proteinases involved in blood coagulation, fibrinolysis, complement activation, inflammation, and 

extracellular matrix remodeling. In contrast, serpins belonging to the intracellular group have been 

implicated in regulating apoptosis, tumor progression, and metastasis. However, their functions in 

terms of an intact organism have not been well defined. Database analysis of the C. elegans 

genome reveals the presence of several intracellular serpins. To determine whether nematode 

serpins function as proteinase inhibitors, one family member, srp-2, was chosen for further 

characterization. Biochemical analysis of recombinant SRP-2 protein revealed SRP-2 to be a 

potent inhibitor of the lysosomal cysteine proteinases, cathepsins K, L, S, and V. Analysis of 

temporal and spatial expression indicated that SRP-2 was present during early embryonic 

development and highly expressed in the hypoderm of larval and adult worms. To determine 

whether SRP-2 plays a role in C. elegans development, null mutants and transgenic animals 

overexpressing SRP-2 were generated. Whereas null mutants showed no overt developmental 

phenotype, of the animals overexpressing SRP-2 ~30% displayed developmental abnormalities 

characterized by early (L1/L2) larval arrest/death, slow growth and molting defects at 25 ºC. 

However, at 27 ºC the phenotype was considerably more penetrant with ~95% of the animals 

being abnormal. Although the mode of SRP-2 action is currently unknown, we hypothesize that 

SRP-2 plays a role in regulating proteinase activity during development and that an imbalance in 

the serpin/proteinase equilibrium has deleterious consequences during C. elegans development.

206. The Identification of Factors Mediating the Downregulation of MEP-1 Function by 

PIE-1 

Byung-Jae Park, Prashant Raghavan, Seung-Il Kim, Jungsoon Lee, Keunhee Park, Tae Ho Shin 

Dept. of Mol and Cell Biol. Baylor College Of Medicine, Houston, TX, 77030 

Recent evidence suggests that a putative nucleosome remodeling complex containing MEP-1, 

LET-418 and HDA-1 (the MEP-1 complex) mediates the inactivation of germline potential in 

somatic cells of the C. elegans embryo. 1 In the germline, the MEP-1 complex appears to be 

inhibited by PIE-1, a germline-specific zinc finger protein, which directly interacts with MEP-1. We 

propose that this repression of MEP-1 function is a part of the mechanism that maintains the 

unique chromatin organization essential for the germline development. 

In order to identify factors that mediate the MEP-1 repression, we are conducting a genetic 

screen based on the phenotype that results from ectopic expression of PIE-1 in somatic cells. 

Animals expressing PIE-1 from the hsp16-41 (heat shock protein) promoter mimic the 

loss-of-function mep-1 mutant, causing the derepression of PGL-1 expression in numerous 

somatic cells and penetrant synMuvB (synthetic multivulva) defect resulting from the deregulation 

of the vulval differentiation potential. We are carrying out this screen in conjunction with four 

secondary screens, which examine the levels of PIE-1 expression, potential defects in the 

germline development, the expression of PGL-1 in somatic cells, and the epistatic relationship 

with known synMuvB genes. 

Using this strategy, 73 mutants have been recovered from approximately 20,000 haploid 

genomes analyzed. We broadly categorize these mutants into two classes. 

Class I: 17 mutants strongly suppress the PIE-1-induced Muv phenotype but do not suppress 

the Muv phenotype induced by RNAi directed to mep-1, let-418, lin-35(Rb), lin-36, lin-13 or 

lin-53(RbAp48). These are likely to be defective in the PIE-1 function itself (i.e. PIE-1 fails to 

inhibit the MEP-1/LET-418 complex) or in the negative regulation of the complex in somatic cells. 

Class II: 48 mutants suppress, partially or fully, the mep-1(RNAi)-induced Muv phenotype. 

Some of these mutants are expected to show enhanced activities of other synMuvB components. 

While others derepress the genes normally repressed by the MEP-1 complex. Presumably, these 

genes are in turn important for the non-P6.p cells to adopt the vulval fate. Of the 48 Class II 

suppressors, 13 also suppress the Muv phenotype caused by lin-35(Rb)(RNAi), 12 suppress the 

lin-36(RNAi)-induced Muv phenotype, and 4 suppress both the lin-35(RNAi)- and 

lin-36(RNAi)-induced Muv phenotype. 19 suppress neither lin-35(RNAi)- nor lin-36(RNAi)-induced 

Muv phenotype. These results suggest the presence of complex interactions among the synMuvB 

components, multiple critical targets regulated by the synMuvB pathway or both. 

We are primarily interested in the Class I mutants and some of the Class II mutants that 

specifically suppress the Muv phenotype induced by mep-1(RNAi). We will present the mapping, 

complementation, and genetic characterization of some of these suppressors. 

1. Unhavaithaya et al., (2002) Cell, 111, 991-1002.

207. A Screen for Mutants Resistant to Serotonin: a Search for Genes Involved in 

Neurotransmitter Signaling and Centrosome Movement 

Judy S. Pepper, Michael R. Koelle 

Department of Molecular Biophysics & Biochemistry, Yale University 

Serotonin is a neurotransmitter that influences human behaviors and psychiatric disorders, 

including depression. Several C. elegans behaviors are also mediated through serotonin 

signaling, including locomotion, egg-laying, and pharyngeal pumping. Serotonin signals in adult 

neurons through the Gα o protein, GOA-1, to control these behaviors (1). Gα o also regulates an 

essential early embryonic process, centrosome movement, during embryonic cell division (2). Our 

lab recently characterized maternal-effect lethal mutations in a gene, rgs-7, that regulates Gα o 

for its embryonic function (3). 

We hypothesize that there may be other Gα o signaling components used in both adult 

neurotransmitter signaling and embryonic cell divisions. We are carrying out a two-phase screen 

that will allow us to identify such signaling components. Our primary screen is to isolate mutants 

resistant to the paralytic effects of exogenous serotonin, which should identify adult animals 

defective for Gα o signaling. The screen is carried out in a "clonal" fashion, such that 

maternal-effect lethal mutations can be identified and recovered. Our secondary screen will 

identify mutations from the primary screen that also cause lethality due to defects in embryonic 

cell divisions. Previous screens would have missed Gα o signaling genes that function in both 

embryos and adult neurons because non-clonal screens require both viability and fertility. 

Our primary screen used the following approach: the F1 progeny of ethylmethane sulfonate 

mutagenized animals were cloned into liquid culture medium in 96-well microtiter dishes and 

allowed to grow for one generation, after which 30mM serotonin was added to each culture. 

30mM serotonin induces paralysis of wild-type animals within minutes, while mutants for the Gα o 

gene goa-1 or the serotonin-gated ion channel gene mod-1 continue to thrash under such 

treatment (4). We looked for new mutants that showed similar serotonin resistance. Wells 

containing ~25% resistant (F2 homozygous mutant) animals were identified, and we recovered 

the heterozygous mutant siblings found in the same wells to recover any maternal-effect lethal 

mutations 

We have completed a primary screen of 10,000 mutagenized haploid genomes and have 

recovered 13 isolates. Each mutation isolated will be outcrossed, genetically mapped, and 

analyzed in our secondary screen for defects in embryonic cell divisions. 

1. Segalat, L., Elkes, D.A. and Kaplan, J.M. (1995). Science 267: 1648-1651. 

2. Gotta M., and Ahringer, J. (2001). Nature Cell Biol. 3: 297-300. 

3. Hess, H.A. and Koelle, M.R., (2004) submitted 

4. Ranganathan, R, Canon S.C., and Horvitz, H.R. (2000). Nature 408: 470-475.

208. Investigating interacting partners of CeTwist 

Mary C. Philogene, Ann Corsi 

Department of Biology, Catholic University of America, Washington, DC. 

Twist is a transcription factor with a basic helix-loop-helix (bHLH) structure. Twist was originally 

identified in Drosophila, and has homologs in humans as well as in Caenorhadbitis elegans. Twist 

is predicted to dimerize with other bHLH proteins. A well-known partner of Twist is the 

ubiquitously expressed E protein. Twist forms heterodimers with E protein homologs in these 

organisms to activate downstream target genes necessary for mesoderm development. 

Significantly, Twist has been shown to function as homodimers in Drosophila (Castanon et al, 

2001). hlh-8, the gene that encodes CeTwist in C.elegans, is expressed in all cell derived from 

the M lineage. CeTwist is required during embryonic development for formation of enteric 

muscles, patterning of cells derived from the M mesoblast lineage, and formation of non-striated 

sex muscles during postembryonic development, (Corsi, 2000). The experiments proposed in this 

study are designed to test the hypothesis that CeTwist may also form homodimers with specific 

function in C.elegans. CeTwist homodimer formation in C.elegans has been observed in vitro 

using gel shift assays. In vivo reporter assays have also shown that overexpressed CeTwist can 

activate target genes in the absence of overexpressed E protein homolog, CeE/DA, in a wild type 

worm. We used RNAi feeding to remove expression of endogenous CeE/DA. Wild type worms 

containing a hs::hlh8 extrachromosomal array, which overexpresses CeTwist, were fed bacteria 

expressing hlh-2 dsRNA, the gene encoding CeE/DA. Preliminary evidence suggests that 

CeTwist can activate target genes such as arg-1::gfp and egl-15::gfp in worms fed with hlh-2 

dsRNA. To further investigate the function of these homodimers in vivo, we will use a tethered 

dimer construct. The bHLH monomers can be physically linked by a flexible glycine-serine 

polylinker, thereby facilitating dimer formation in vivo. Twist mutation in humans causes 

Saethre-Chotzen syndrome (SCS), which is one form of human craniosynostosis diseases. 

Investigating the many functions of Twist in C.elegans may shed some light into a better 

understanding of its function as well as further characterization of mutations associated with the 

various forms of human craniosynostosis diseases. 

Castanon I, Stetina S.V., Kass J., Baylies M.K. (2001). Dimerization partners determine the 

activity of the Twist bHLH protein during Drosophila mesoderm development, Development 128, 

3145-3159. Corsi, A.K., Kostas, S.A., Fire, A.,Krause, M. (2000). Caenorhabditis elegans Twist 

plays an essential role in non-striated muscle development, Development 127,2041-2051.

209. Genome wide RNAi screen for new synMuv genes 

Gino Poulin 1 , Yan Dong 1 , Andrew Fraser 1 , Neil Hopper 2 , Julie Ahringer 1 

1Wellcome Trust/CR UK Gurdon Institute, University of Cambridge, UK, Cambridge CB2 1QR 

2University of Southampton, Bassett Crescent East,Southampton, SO16 7PX 

Ras signalling promotes vulval development, and is antagonized by the functionally redundant 

class A and class B synthetic multivulval genes. Previous work showed that a Retinoblastoma 

protein and components of the NuRD histone deacetylase complex are encoded by some of the 

synthetic multivulval genes, indicating that negative transcriptional regulation of vulval 

development genes underlies the process of Ras antagonism. We carried out a genome-wide 

RNAi screen to identify systematically other components of this regulatory mechanism. After 

RNAi of 16,757 genes, we found 19 synMuv genes, 9 of which are new; 80% of previously known 

synMuv genes in the library were identified in the screen. Based on the sequence of the 9 newly 

identified genes, 7 appear to have a role in transcription regulation, and these have human 

homologs. For example, we identified a histone methytransferase and a homolog of lethal (3) 

malignant brain tumor, a tumor suppressor in Drosophila. We also found a requirement for 

components of a TRRAP/TIP60 histone acetyltransferase complex, suggesting that both 

acetlyation and deacetylation of histone tails may have roles in repression of vulval development. 

Ceol et al. (2004) have also recently identified this complex as part of a new synMuv C class. In 

addition, we show involvement of several components of the sumoylation pathway and find that 

the histone deacetylase HDA-1 is sumoylated. These data indicate that antagonism of Ras 

induced vulval development involves unexpectedly complex coordination among numerous 

chromatin regulators. As most of the genes identified in this screen are conserved in humans, we 

suggest that similar interactions are relevant for generating a repressed chromatin state to inhibit 

Ras signalling in mammalian cells.

210. A screen for axon branching and guidance mutants 

Brinda Prasad, Scott Clark 

Skirball Institute, NYU School of Medicine, New York, NY 

Although the axons of most neurons in C. elegans are relatively simple and unbranched, axons 

of some neurons have well defined, reproducible branched structures. The Zn finger 

homeodomain protein ZAG-1 acts as a transcriptional repressor and regulates the formation of 

stereotypic axon branches for several neuron types, including ALM and AVM. To identify 

additional genes needed for axon branching and other aspects of axonal development, we 

treated mec-4::gfp animals with EMS and picked roughly 2000 F1 animals. We screened their 

progeny for mutants with defects in the development of the touch receptor neurons ALM, PLM, 

AVM and PVM using a fluorescence dissecting stereomicroscope. Even though several screens 

to identify mutations affecting the development of these neurons have been performed over the 

years, most have not focused on specific aspects of axonal development. Thus, we believe that 

our F1 clonal, direct visual screening strategy will allow us to recover new genes. 

We isolated over 30 mutants with defects in axon branching and outgrowth, cell fate 

determination and migration of Q descendants. Based on our current analysis, many mutations 

are likely new alleles of previously identified genes, as would be expected. However, others 

appear to define new genes and are being characterized further.

211. Identification of Genetic Pathways Dependent on Protein N-glycosylation by 

GlcNAc-TV 

Justin M. Prien 1 , Justin M. Crocker 1 , Aldis Krizus 2 , James W. Dennis 2 , Charles E. Warren 1,3 

1Department of Biochemistry and Molecular Biology, University of New Hampshire 

2Mount Sinai Hospital Research Institute, University of Toronto, Canada 

3Genetics Program, University of New Hampshire 

UDP-N-acetylglucosamine:α-6-D-mannose β-1,6-N-acetylglucosaminyltransferase V 

(GlcNAc-TV) is a Golgi enzyme that catalyzes the addition of β-1,6-GlcNAc in the production of 

complex N-glycans. GlcNAc-TV is of particular interest because its activity is directly involved with 

cancer transformation and metastasis in mice and is a prognostic indicator of breast cancer 

survival in humans. Mice deficient in GlcNAc-TV via deletion in the Mgat5 locus experience 

dramatic suppression of metastasis. Analysis of the molecular mechanism of Mgat5 in mice is 

confounded by the pleiotropic nature of N-glycosylation. Perturbation of Mgat5 affects all 

β-6-GlcNAc substituted glycoproteins so that identification of specific molecules that control 

specific systems is difficult. The C. elegans genome contains an ortholog, gly-2, which can rescue 

CHO cells mutant at the Mgat5 locus. Strain XA766 gly-2(qa703)I qaEx743[C55B7.3 C55B7.10 

gly-2(+), rol-6(d)] was constructed and used in a pilot scale pre-complementation screen of 650 

haploid genomes to identify loci that are synthetic lethal in a gly-2 null background. Two alleles, 

not integrants, both of which are strongly dependent on the qaEx743 array were isolated. The 

allele nh4 is lethal in a gly-2 null background unless pre-complemented by the qaEx743 array. 

The second allele, nh3, exhibits a temperature-sensitive dauer constitutive phenotype that only 

exits dauer at restrictive temperature in the presence of the array. Both nh3 and nh4 are linked to 

gly-2 on LGI. One plausible explanation for the array dependence is that nh3 and nh4 are alleles 

of loci underneath the qaEx743 array. Formally then, nh3 or nh4 could interact to create a 

synthetic lethal phenotype with gly-2, rol-6 or be due to a single site mutation in C55B7.3 or 

C55B7.10. We are currently mapping the exact locations of nh3 and nh4. In the case that nh3 

and nh4 are gly-2 dependent, they should reconstruct with gly-20, the gene immediately 

upstream of gly-2 in the glycosylation pathway. We have separated nh3 from the gly-2 locus and 

are currently reconstructing it with gly-20.

212. Components of the dosage compensation machinery antagonize the MEP-1 complex 

function 

Prashant Raghavan, Jungsoon Lee, Byun-Jae Park, Seun-il Kim, Keunhee Park, Tae Ho Shin 

Department of Molecular and Cellular Biology,Baylor College of Medicine,Houston,Texas 

We previously proposed that the germline-specific PIE-1 protein inhibits the activity of the 

MEP-1 chromatin remodeling complex, which promotes somatic differentiation, and thus prevents 

the implementation of soma-specific, pro-differentiation chromatin structures, in place of the 

default germline-specific chromatin organization. In order to investigate this model further, we are 

examining the potential involvement of known and presumed chromatin regulators in the 

MEP-1-dependent developmental processes. 

MEP-1 and its binding partners LET-418 and HDA-1 are known to participate in vulva 

specification as a component of the genetically redundant process known as the synthetic 

multivulva B (synMuv B) pathway. Loss-of-function mutations in the mep-1, let-418 or hda-1 gene 

cause ectopic vulval specification in synMuv A mutant backgrounds. Similarly, the forced 

expression of PIE-1 in the somatic cells mimics the effect of synMuv B mutations. Using this 

phenotype as readout, we found that reducing the activity of the dosage compensation machinery 

increases the repression of the primary vulval fate in the vulva equivalent cells. For example, 

dpy-30(RNAi) reduces the occurrence of ectopic vulvae from ~85% to ~10% in animals 

ectopically expressing PIE-1. Similarly, RNAi directed against dpy-27, dpy-28 and sdc-2 result in 

significant suppression of the synMuv phenotype caused by ectopic PIE-1 expression and also by 

mep-1(RNAi), though for unknown reasons, the suppression in the latter case is much less 

dramatic. In addition, the dosage compensation machinery appears to be important for the 

derepression of the vulval fate when other synMuv B genes such as lin-35 and lin-36 are 

inactivated. 

Previous studies indicate that the MEP-1 complex antagonizes the function of MES proteins, a 

group of Polycomb group-related chromatin regulators, and that the effect of MES proteins is 

mediated, at least partly, by the repression of the X chromosome in the germline nuclei. One 

possible interpretation of these results is that activity levels of the X chromosome directly 

influence the developmental decision between the germline and somatic fates. The analogous 

antagonistic relationship between the MEP-1 complex and the X repression mechanism in the 

somatic cells raise an intriguing possibility that the major function of the MEP-1 complex may be 

to regulate the X chromosome, which in turn is consistent with the model that the X inactivity 

corresponds to the germline fate.

213. Behavioral quiescence during the L1 stage and its alteration in eat-7 mutants 

David M. Raizen 1,3 , Meera Sundaram 2,3 , Allan I. Pack 1,3 

1Center for Sleep and Respiratory Neurobiology 

2Department of Genetics 

3University of Pennsylvania Medical School 

Animals display periods of behavioral quiescence, when locomotion stops. In mammals, these 

periods of quiescence usually correspond to sleep, and are controlled by circadian and 

homeostatic processes. Behavioral quiescence in Drosophila is also controlled by these two 

processes and by some of the same neurochemicals that function in mammalian sleep(1,2), 

suggesting that the genetic control of behavioral quiescence is phylogenetically ancient. 

Locomotion of C. elegans has been observed to stop for prolonged periods during the 

lethargus periods, immediately prior to the molts(3). We have been measuring the quiescence 

associated with the L1 lethargus by videotaping single worms from the time of hatching. We 

digitize images at one-minute intervals and then track the path of the worm by sending the 

location of the developing gonad to a spreadsheet. 

We have found that in N2 worms grown at 22 deg, the longest consolidated quiescent period 

begins at 11.5+/-0.2, consistent with the reported time of onset of the L1 lethargus. When 

mechanically stimulated during its predicted consolidated quiescent period, the animal is able to 

move normally, indicating that this quiescent state is reversible. 

In eat-7, a mutant that was isolated based on small size, and decreased movement and eating 

when not stimulated(4), the duration of the L1 quiescent period is significantly prolonged while the 

time of onset of this quiescent period is unchanged. The fraction of quiescent periods in eat-7 

prior to the onset of its longest quiescent period is also slightly prolonged though this difference 

does not reach statistical significance. Hence, eat-7 mutants display increased behavioral 

quiescence during the L1 stage. In addition to this L1 quiescent phenotype, we have found that 

adult eat-7 mutants, when left unperturbed, make fewer tracks than N2 worms on a lawn of 

bacteria. When mechanically stimulated however, eat-7 mutants are capable of rapid and 

coordinated movement, indicating that the increased behavioral quiescence cannot be explained 

simply by an inability to move well. 

eat-7 is defined by a single dominant allele, ad450sd, which maps between ced-2 and lin-1(4), 

a genetic interval in which the gene egl-4 is found. Body size, life span, and roaming behaviors 

are increased in egl-4 mutants while these three phenotypes are decreased in eat-7 mutants, 

suggesting that ad450sd may be a hypermorphic egl-4 allele. This suggestion is supported by the 

finding that two egl-4 recessive alleles, n477 and n479, dominantly suppress the small body 

phenotype of eat-7(ad450sd)/+. We sequenced the EGL-4a cDNA in eat-7 mutants and have 

found a Glycine to Arginine mutation. This Glycine, located in the second cGMP binding domain, 

is conserved in all proteins containing a cGMP or cAMP binding domain. We will report the results 

of experiments testing the effect of expressing the EGL-4a cDNA with the G->R mutation in 

wild-type worms. 

(1)Hendricks et al, Neuron 2000. (2) Shaw et al, Science 2000. (3)Singh and Sulston, 

Nematologica 1978. (4)Avery, Genetics 1993.

214. Characterization of UNC-43/CaMKII transport and activity in neurons 

Paris Rapp, Toru Umemura, Christopher Rongo 


Calcium and calmodulin-dependent kinase type II (CaMKII) is an abundant brain protein 

involved in synaptic plasticity and thus learning and memory. CaMKII has a single homolog in C. 

elegans encoded by the unc-43 gene(1). UNC-43 activity is required for the anterograde 

trafficking of glutamate receptors(2). Moreover, UNC-43 itself is translocated from the neuron cell 

body to synapses in an activity-dependent manner. In the absence of calcium signaling, the 

autoregulatory domain of mammalian CaMKII associates with the catalytic domain, thus sterically 

hindering the binding of substrates. In the presence of calcium/calmodulin, the catalytic domain is 

released and the kinase is rendered active. We have expressed UNC-43 in S2 cells to examine 

its enzymatic activity and regulation. Like mammalian CaMKII, we find that UNC-43 can 

phosphorylate an autocamtide substrate in a calcium-dependent fashion. 

To better understand the mechanism by which UNC-43 is translocated from the neuron cell 

body to the synapse, we have performed a structure/function analysis of the protein. We have 

introduced several amino acid substitutions into the UNC-43 catalytic and autoregulatory 

domains, and tested the resulting mutant proteins for their ability to translocate in neurons. We 

have identified several key residues in the catalytic domain of UNC-43 that are required for its 

translocation upon calcium signaling. We have also expressed the mutant forms of the UNC-43 

kinase in S2 cells to examine the effects of the mutations on enzymatic activity. Our results have 

shown that translocation is irrespective of the relative level of calcium independence caused by 

the various mutants. Rather, we believe that key residues that interact with factors in the cell that 

facilitate UNC-43 translocation have been disrupted. We hypothesize that calcium signaling 

releases the catalytic domain from the autoregulatory domain, thereby allowing it to interact with 

the proteins involved in translocating the kinase out of the neuron cell body. 

1. D. J. Reiner, E. M. Newton, H. Tian, J. H. Thomas, Nature 402, 199-203 (1999). 

2. C. Rongo, J. K. Kaplan, Nature 402, 195-199 (1999).

215. Identification of factors required for germline silencing 

Tom Ratliff, Karissa McClinic, David Han, Bill Kelly 

Emory University, 1510 Clifton Road, Atlanta, GA 30322 

Transcriptional repression (silencing) is a major theme in the establishment and maintenance 

of the C. elegans germline. Loss of silencing in the embryonic germline can cause the 

transformation of germ lineage precursors to somatic cells while in the post-embryonic germline 

desilencing can adversely affect the differentiation of germ cells in the gonad. A loss of silencing 

in the adult germline can be assayed by expression of repetitive extrachromosomal arrays, which 

are normally strongly silenced in this tissue. To genetically identify factors involved in germline 

silencing, we have isolated mutations that allow germline GFP expression from a repetitive 

extrachromosomal array. To date, all sig (silencing in the germline-defective) mutants recovered 

are variably sterile, suggesting that the genes identified by these mutations are required for 

fertility and that the germline silencing mechanism is an essential requirement in C. elegans. In 

addition to isolating new sig mutations, we are focusing on the characterization and cloning of 

sig-2 and sig-7, each identified by a single mutation. The sig-2 mutant appears to have a 

germline-specific phenotype. In addition to the array desilencing defect, sig-2 animals are 

incompletely penetrant sterile. There is also a noticeable delay in the onset of oogenesis in the 

cohort of animals that eventually give rise to offspring. Mapping of sig-2 is in its very early 

stages. The sig-7 mutation causes a fully penetrant sterility and is notably the only sig mutation 

recovered thus far that has accompanying somatic defects. sig-7 animals are thin and have a 

protruding vulva, defects possibly related to our observation of increased transgene expression 

(let-858::gfp) in gut and vulval tissues. Using polymorphisms, we have physically mapped sig-7 to 

a small interval on LGI and are currently performing transformation rescue experiments.

216. Histone variant H2A.Z is essential for development in C.elegans 

Brianne J. Ray, William G. Kelly, Adam Raymond 

Emory University, Atlanta Ga 

There is growing interest in identifying and characterizing the roles of variant histone isoforms 

in organisms from yeast to mammals. While much is known about how modifications of canonical 

histones provide genome regulation through chromatin formation, the functions of variant 

histones are just beginning to come to light. Many histone variants have been identified and are 

usually conserved in makeup and function among various organisms. Some examples include the 

H2A.X and H2A.Z isoforms of the canonical histone H2A, and the H3.3 and CenpA variants of the 

canonical histone H3. 

Among the histone H2A variants, H2A.Z is highly conserved in both sequence and apparent 

essentiality. Although it only comprises ~10% of total H2A in most organisms, its depletion is a 

lethal event. The function of this variant is unclear, since it has been found to be enriched in both 

euchromatin and constitutive pericentric heterochromatin. Recent reports have indicated that its 

insertion into chromatin is highly regulated and involves ATP-dependent remodeling machinery. 

Regulated insertion of histone variants into chromatin could represent an alternative form of 

epigenetic regulation that could complement and/or influence histone tail modifications, and 

create a more stable chromatin architecture. We are interested in studying the role of H2A.Z in 

the development of C. elegans. We have identified a single homologue in worms with a high 

sequence identity to those of other organisms. RNAi depletion of the gene product results in a 

highly penetrant embryonic lethality with the rare escapers arresting as defective larvae. Peptide 

antibodies raised against a unique sequence of this protein yield an immunofluorescence pattern 

that is consistent with localization to transcriptionally competent chromatin. We are currently 

trying to understand the nature of the embryonic arrest, as well as studying the role of conserved 

components of the remodeling machinery that regulate its insertion in other organisms.

217. A Mos1 transposon mutagenesis screen for suppressors of the let-7 microRNA 

K. Reinert, F. J. Slack 

Department of Molecular, Cellular and Developmental Biology, Yale University, P.O. Box 208103 

New Haven, CT 06520 

The heterochronic pathway consists of a cascade of temporally regulated genes that specify 

the timing of developmental events. One of the members of this pathway, let-7, is a 21 nucleotide 

small temporally regulated RNA that is a member of the microRNA family and downregulates its 

target genes via a mechanism that involves binding to let-7 complementary sites (LCS) in their 3’ 

UTRs. To identify new targets of let-7, we performed a Mos1 mutagenesis screen to identify 

suppressors of the temperature sensitive lethal phenotype of let-7(n2853). We identified a 

transposon insertion in R08E3.4 that strongly suppresses this lethality. Subsequent analysis has 

shown that there is at least one LCS in the 3’ UTR of R08E3.4, highlighting the potential for a 

direct interaction with let-7. Experiments in which R08E3.4 has been knocked down by RNAi 

show no precocious seam cell fusion defects, though this knockdown is sufficient to suppress the 

lethal phenotype of let-7(n2853). R08E3.4 is a predicted transcription factor with homology to 

human oncogenes such as a zinc finger sarcoma gene, Wilms tumor 1, and B-cell lymphoma 6.

218. DKF-1 is A Diacylglycerol-regulated Kinase Involved in C.elegans Motility 

Min Ren, Hui Feng, Charles S. Rubin 

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461 

Structural and regulatory properties of protein kinase C isoforms (PKCs) have been extensively 

and thoroughly studied. In contrast, mechanisms by which PKCs generate unique, integrated 

physiological responses to DAG signals emanating from >100 types of receptors (in various 

differentiated cells) is poorly understood. A striking and unexpected relationship links protein 

kinase D (PKD) isoforms to PKC-mediated signaling pathways. One or more PKCs are evidently 

obligatory (direct or indirect) activators for ubiquitous PKDs. This raises the possibility that PKDs 

are involved in mediating many actions of DAG and DAG-activated PKCs. 

We have discovered and characterized a novel PKD family isoform from C. elegans. DKF-1 (for 

D Kinase Family-1) comprises 722 amino acids (Mr = 81,000), containing the characteristic 

features of the PKD family: two C1 domains, a PH domain and a predicted C terminal S/T protein 

kinase segment that is generated by the folding of ~260 amino acids. Cys-rich domains (C1a and 

C1b) at the N terminus of DKF-1 create Zn-finger domains that bind diacylglycerol (DAG) and 

phorbol esters (TPA). Ligation of TPA elicits translocation of cytoplasmic DKF-1 to plasma 

membrane, thereby promoting kinase activation via interaction with phosphatidylserine. 

Mutagenesis/expression experiments demonstrate that C1a in DKF-1 guides DKF-1 to plasma 

membrane, where activation occurs. In contrast to previously-studied PKDs, DKF-1 activation is 

PKC-independent. Chronic activation of DKF-1 results in proteasome mediated degradation. The 

PH domain does not bind phosphoinositides, but is absolutely essential for expression of 

phosphotransferase activity. Mutation of conserved residues in a central PH module generated 

inactive DKF-1, suggesting that this domain stabilizes the catalytically active conformation of 

DKF-1. Trans-phosphorylation of Thr588 in the activation loop of DKF-1 by an upstream kinase is 

a critical pre-requisite for the appearance of TPA-(DAG-) stimulated kinase activity. However, 

DKF-1 was activated by TPA or hormones when upstream PKCs were maintained in a basal 

state by potent PKC inhibitors. Thus, DKF-1 participates in a novel, DAG-controlled signaling 

pathway that is independent of PKCs. Activation of DKF-1 is tightly coupled with translocation to 

plasma membrane. A dkf-1 gene promoter/enhancer region was coupled to dkf-1 cDNA. A green 

fluorescent protein (GFP) reporter, DKF-1-GFP accumulates in neurons and gland cells in both 

the head and tail regions of post-embryonic nematodes. Both RNAi-mediated DKF-1 protein 

depletion and disruption dkf-1 gene disruption caused the same phenotype: loss of muscular 

control of lower body movement. DKF-1 protein expression was suppressed via RNA interference 

(RNAi) and knockout methodology. Normal sinusoidal movement of C. elegans on agar plates 

was abnormal in both DKF-1 RNAi and knockout animals. L4 and adult worms had severe 

immobility evident close to the tail region. This appears to be an unc phenotype. Introduction of a 

WT dkf-1 transgene into animals homozygous for the null allele rescued the movement defect. 

High-level overexpression of DKF-1-GFP also rescues uncoordinated movement, but elicits a 

sma phenotype (only 60% of N2 length). Thus, DKF-1 actions may impinge on the SMAD 

pathway. Many of the properties of DKF-1 are unprecedented for a member of the PKD family of 

isoforms.

219. Modulation of C. elegans Egg-laying Behavior by the Environment and Experience 

Niels Ringstad, Bob Horvitz 


The egg-laying behavior of C. elegans is modulated by the environment and experience. When 

a well-fed hermaphrodite is removed from a food source, the animal stops laying eggs. We have 

observed that upon return to food, the frequency of egg-laying events by food-deprived animals 

increases over that of animals left on food, and the magnitude of this increase is proportional to 

the duration of time spent away from food. 

We have scored egg-laying defective (Egl) mutants for defects in the modulation of egg-laying 

behavior. In particular, we have examined class C, D, and E Egl mutants (Trent et al., 1983), 

which have HSNs with apparently normal morphology, have functional sex muscles, and have a 

normal vulva, yet lay fewer eggs than wild-type animals. These mutants, like wild-type animals, 

lay eggs in response to exogenous serotonin and in response to the serotonin reuptake inhibitor 

imipramine, which is thought to potentiate the signaling from the serotonergic HSNs to the sex 

muscles. It therefore seems that these mutants release serotonin from the HSNs and possess an 

egg-laying neuromusculature able to respond to serotonin. One possibility is that mutations 

causing the egg-laying defects in serotonin- and imipramine-responsive Egl mutants act by 

inappropriately activating pathways that normally inhibit egg-laying behavior. 

We have found that egl-6(gf) and unc-31(lf) mutants, in addition to having reduced rates of 

egg-laying, fail to inhibit egg-laying in the absence of food. We have also found that egl-7(gf) 

mutants fail to upregulate egg-laying after a period of food deprivation. unc-31 has been cloned 

by others and encodes a CAPS-like protein implicated in the exocytosis of neuromodulators and 

neuropeptides stored in dense-core granules. We have cloned egl-6 and found that it encodes a 

putative neuropeptide receptor related to insect receptors for FMRFamide-containing 

peptides. These results suggest that the regulated secretion of neuropeptides is required both to 

stimulate egg laying in the presence of food and to inhibit egg laying in the absence of food. We 

have mapped egl-7(n575) to a small interval on LGIII and are pursuing its cloning and 

characterization.

220. Dissecting the role of CNK-1 in LIN-45 Raf activation 

Christian E. Rocheleau, Meera V. Sundaram 

Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 

The Ras GTPase plays a pivotal role relaying signals from cell surface receptors to the 

Raf/MEK/ERK kinase cascade. Activation of the Raf kinase by Ras is a complex process 

requiring membrane localization of Raf, Ras binding, and both phosphorylation and 

dephosphorylation of residues in the kinase and regulatory domains respectively. A number of 

proteins have been implicated in Raf activation, including KSR (a Raf/MEK/ERK scaffold), SUR-6 

(PP2A regulatory subunit), SUR-8 (Leu-rich repeat protein), and CNK (a multi-domain protein). In 

Drosophila, CNK has been shown to be an essential component of the Ras pathway and may 

regulate Raf activation via membrane localization. Defining the role of C. elegans cnk-1 in Ras 

signaling will provide insight into how CNK regulates Raf. 

We have found that C. elegans cnk-1 is a non-essential positive regulator of Ras signaling that 

acts genetically between ras and raf, a phenotype and epistasis also shared by ksr-1, sur-6, and 

sur-8. To define how cnk-1 contributes to Raf activation and determine how its contributions are 

different from these other positive regulators we are performing epistasis experiments with 

differentially activated lin-45 raf transgenes that can induce a multivulva phenotype. 

Consistent with a potential role for CNK-1 in protein localization we identified KIC-1, a putative 

RabGAP (Rab GTPase Activating Protein), in a yeast two hybrid screen for CNK-1 binding 

proteins. Rab GTPases regulate various step of vesicular trafficking. To determine the 

requirement for kic-1 in Ras signaling we have collaborated with Simon Tuck’s lab to isolate a 

deletion allele. We find that the kic-1(sv41) allele, like cnk-1, can enhance the ras-like larval lethal 

phenotype of ksr-1 suggesting that kic-1 promotes Ras signaling. However this enhancement of 

ksr-1 is weaker than what we see with cnk-1. We believe the sv41 deletion is likely not a null 

allele and is further complicated by the fact that it also deletes part of a neighboring gene. 

Therefore we are seeking to identify another deletion that specifically removes kic-1.

221. Progress Towards the Cloning of lin-38 and Identification of Novel Class A SynMuv 

Genes 

Adam Saffer, Ewa Davison, Bob Horvitz 

HHMI, Dept. Biology, Cambridge, MA 02139, USA 

Vulval induction in the C. elegans hermaphrodite requires an RTK/Ras signaling pathway and 

is antagonized by the products of the synthetic Multivulva (synMuv) genes. The synMuv genes 

are defined by at least three redundant classes: A, B, and C. Mutations in any one class are 

non-Muv, but mutations in any two of the classes together cause a Muv phenotype. Many of the 

cloned class B and C genes encode proteins predicted to act in chromatin remodeling and 

transcriptional repression. Four class A synMuv genes have been identified to date, three of 

which have been cloned and found to encode novel proteins. To further our understanding of the 

class A synMuv genes, we intend to clone lin-38 and identify new class A synMuv genes. 

The remaining uncloned class A synMuv gene, lin-38, had previously been mapped between 

rol-1 and unc-52 on chromosome II. Using SNP mapping we have placed lin-38 in a 30 kb 

region. We have generated an 18 kb PCR product that contains five genes and that rescues the 

Muv phenotype of a lin-38; lin-15B strain. Currently, we are building smaller rescuing constructs 

and determining the sequences of candidate genes. 

We are also seeking additional class A synMuv genes. All previously reported screens for 

class A genes were unable to recover mutations that also caused sterility, so we are doing a 

clonal screen to identify such mutations . We are screening using two different class B synMuv 

backgrounds, lin-15B(n744) and lin-52(n771). To date we have screened 19,500 haploid 

genomes clonally and isolated 28 independent mutations that cause a Muv phenotype, including 

three that either confer sterility or are closely linked to a sterile mutation. At least 22 of these 

mutations are in genes previously known to cause a Muv phenotype in a class B synMuv 

background. We are currently determining whether any of the other mutations define new class A 

synMuv genes.

222. C. elegans rme-3 encodes a clathrin heavy chain required for embryogenesis and 

neuro-muscular function 

Ken Sato 1,2 , Chih-Hsiung Chen 1 , Miyuki Sato 1 , Barth D. Grant 1 

1 Rutgers, The State University of New Jersey, Department of Molecular Biology and 

Biochemistry, 604 Allison Rd. Nelson Biological Laboratories, Room A307, Piscataway, NJ 

08854 USA 

2 Molecular Membrane Biology Lab., RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan 

To study the molecular mechanisms of receptor-mediated endocytosis in C. elegans, we 

developed a system to assess the efficiency of receptor-mediated endocytosis of yolk proteins by 

oocytes. We created a transgenic strain that expresses a GFP fusion gene with VIT-2, encoding 

the lipid binding yolk protein YP170. YP170-GFP is expressed in the intestinal cells and secreted 

into the pseudocoelom. In wild type worms, YP170-GFP is taken up by oocytes via 

receptor-mediated endocytosis. Using this assay, we isolated mutants that fell into 11 

complementation groups and named these genes rme for receptor-mediated endocytosis 

defective. One of these, the rme-3 (b1025) mutant, showed a temperature sensitive defect in yolk 

uptake and ts embryonic lethality. Cloning revealed that rme-3 (b1025) is a temperature sensitive 

allele of clathrin heavy chain (CHC), the first temperature sensitive CHC mutant in a higher 

eukaryote. Interestingly, rme-3 (b1025) also shows severe defects in neuro-muscular function. 

After incubation at 25 ¡ C for 24h, rme-3 (b1025) worms picked into liquid can thrash for a few 

minutes, but become almost completely paralyzed 3-5 min later, suggesting a progressive loss of 

synaptic function. rme-3 (b1025) also shows a moderate defect in pharyngeal pumping. A weaker 

CHC allele recovered in our screen, rme-3 (b1024), also shows similar but weaker defects. rme-3 

(b1025) is aldicarb-resistant and levamisole-sensitive, suggesting rme-3 functions 

pre-synaptically at neuro-muscular junctions. In the near future we plan on performing a detailed 

ultra-structural analysis of the synapses in the rme-3 mutants to determine the exact nature of 

these defects.

223. Characterization and mapping of sig-1, a new gene involved in germline-specific 

silencing of extrachromosomal transgenes 

Christine E. Schaner, William G. Kelly 

Emory University, Biology Department, Atlanta, GA. 

The post-embryonic germline has overlapping repressive mechanisms that are required to 

maintain germ cell function and fertility. One hallmark of these mechanisms is a germ cell-specific 

silencing that is observed with extrachromosomal transgenes. Most mutations that cause defects 

in transgene silencing also cause fertility defects, illustrating the essential nature of these 

processes for germ cell function. In a genetic screen for mutations that alleviate transgene 

silencing, several mutants, termed sig (silencers in germline) were isolated. One of these, sig-1, 

exhibits a non-maternal and progressive, temperature-sensitive sterility that accompanies the 

desilencing of repetitive transgenic arrays in the germline. sig-1 mutants also have decreased 

recombination rates, and exhibit both a high incidence of male (him) phenotype and heritable Tc1 

transposon mobilization in their germline (mutator phenotype), indicating sig-1 is likely to be 

involved in maintaining normal chromatin structure in germ cells. Interestingly, a growing subset 

of mutants (rde, mut, mes) that exhibit germline transgene desilencing, fertility defects, and/or 

germline transposon mobilization are also defective in RNA interference (RNAi). sig-1, differs 

from these mutants, as it is not RNAi defective, suggesting that sig-1 acts downstream from the 

common components, perhaps working at the DNA level. We have mapped sig-1 to a small 

region of chromosome II, and are currently working to identify the causative mutation to better 

understand the role of sig-1 in maintaining a functional germline.

224. The mutation bc202 blocks physiological as well as non-physiological germ cell death 

in the adult hermaphrodite gonad 

Claus Schertel, Barbara Conradt 

Dartmouth Medical School, Department of Genetics, 7400 Remsen Bldg., Hanover, NH 03755, 

USA 

In the gonad of adult C. elegans hermaphrodites about 50% of all germ cells are eliminated by 

programmed cell death (referred to as ’physiological germ cell death’). In contrast to the 

developmental death of somatic cells, physiological germ cell death is not determined by lineage 

and might be induced by cell-autonomous as well as cell non-autonomous factors. The genes 

ced-9, ced-4 and ced-3 (ced, cell death abnormal), which define the central machinery involved in 

developmental cell death, also act in the germ line: ced-3 and ced-4 loss of function (lf) mutations 

block physiological germ cell death: in contrast, ced-9 (lf) mutations lead to the massive death of 

germ cells. However, the factors that act upstream of the central cell death pathway to regulate its 

activation appear to differ in soma and germ line. For example, loss-of-function mutations in the 

cell-death activator gene egl-1 (egl, egg-laying defective), which acts upstream of ced-9, block 

developmental cell death but not physiological germ cell death. 

To identify genes that are required for physiological germ cell death, we performed a clonal F2 

screen in a ced-6 (n2095) background, in which the engulfment of apoptotic germ cells is partially 

blocked and in which persistent germ cells corpses consequently accumulate. To visualize and 

quantify the number of persistent germ cell corpses, the reporter Plim-7CED-1::GFP (lim, lim 

domain family) was used, which drives the expression of a CED-1::GFP fusion protein in the 

sheath cells, the cells that engulf apoptotic germ cells. We screened about 8,600 haploid 

genomes for mutations that cause a reduced number of persistent germ cell corpses, and 

isolated the mutation bc202. bc202 blocks most physiological germ cell death in the 

hermaphrodite gonad: 48 h after the L4 stage homozygous bc202; ced-6(n2095) animals have 

4.4+/-2.3 corpses compared to 46.9+/-3.3 corpses found in ced-6(n2095) animals. Furthermore, 

treatment with DNA damage inducing agents does not lead to an increase in the number of 

persistent germ cell corpses in bc202; ced-6(n2095) animals, suggesting that bc202 also blocks 

non-physiological germ cell death. Epistasis analysis suggests that bc202 acts downstream of or 

in parallel to ced-9 to kill germ cells. The gene defined by bc202 maps to a 0.5 m.u. interval on 

LG I. We are currently trying to clone the gene using transformation rescue and RNAi 

experiments.

225. Thrashing in liquid as a quantifiable measurement of aging 

Peter J. Schmeissner, Suzhen Guo, Shih-Hung Yu, Monica Driscoll 

Rutgers University, Department of Molecular Biology and Biochemistry, 604 Allison Road, 

Piscataway, New Jersey 08854 

The observation of declining motility with increasing age is well-documented in the literature 

(Croll et al. 1977, Hosono et al. 1980, Bolanowski et al. 1981, Johnson 1987, Herndon et al. 

2002). Declining motility has been shown to correlate not only with chronological age, but also 

more closely with anatomical/histological markers of aging decline, which can vary within an 

age-synchronized population. One of the major histological presentations of aging is the decline 

of muscle tissue. This decline is most easily observed by looking at the nuclear morphologies of 

the muscle tissues, when highlighted by GFP reporter proteins localized generally to the 

nucleoplasm. Both motility and histological biomarkers turn out to be better predictors of life 

expectancy than chronological age. Nematode motility on a plate can be subdivided into several 

different classes based upon movement after prodding, speed of movement, geometry of 

movement, forward/backward transitions, etc. However, scoring these class subdivisions is 

difficult and often holds some degree of observer subjectivity. Observations of age-related 

histological changes can be more precise, but this also is not without subjectivity. For example, 

variations always are present among the muscle nuclei from a single worm, thereby requiring 

observations of numerous muscle cell nuclei to achieve a general consensus on the state of 

decline of the muscle tissue for the entire organism. In order to achieve a more quantitative 

measure of aging that diminishes some of the subjectivity of the scoring, we have turned to 

studies of nematode motility in a liquid medium. Within seconds of being placed in liquid, 

nematodes uniformly initiate a thrashing motion characterized by both head and tail flexes 

towards the same direction with a mid-body bend (Miller et al., 1996, PNAS, 93(22): 12593-8). 

Counting the number of these thrashes relative to a starting position, within a given time frame, 

leads to very reproducible average numbers across age-synchronized populations of worms. 

Furthermore, that measure helps to delineate more easily the different motility classes of 

nematodes within a single population. Thrashing counts decline with chronological age and differ 

with motility class. We currently are using this readout of aging to track the influences of members 

of the insulin signaling pathway on aging.

226. The BarH Class Homeodomain Gene ceh-30 is Directly Regulated by tra-1 to Specify 

the Sexually Dimorphic Survival of the CEM Neurons 

Hillel Schwartz, Bob Horvitz 


While many genes involved in the execution of cell death have been identified, the mechanisms 

that control the commitment of specific cells to undergo programmed cell death are poorly 

understood. To identify genes that act in the specification of cell death, we performed a screen for 

hermaphrodites in which the male-specific CEM neurons, which die during normal hermaphrodite 

development but survive in males, fail to die. The reporter pkd-2::gfp (kindly provided by Maureen 

Barr and Paul Sternberg) is expressed in the CEMs of males and in the CEMs of hermaphrodites 

defective in programmed cell death. Using pkd-2::gfp as a marker for CEM survival, we screened 

60,000 mutagenized haploid genomes and recovered at least 154 independent mutations that 

cause survival of the CEMs, including at least 42 alleles of known cell-death genes and 64 

mutations that cause sexual transformation. 

Three mutations from this screen, n3713, n3714, and n3720, semidominantly cause CEM 

survival in hermaphrodites but cause no other obvious defects in programmed cell death or sex 

determination. By mapping and complementation studies, we found that these mutations affect a 

previously uncharacterized gene on LGX. The CEM survival caused by these mutations is not 

affected by loss of the fem genes, the most downstream genes required for masculinization, 

indicating that this gene may act downstream of sex determination. The n3714 CEM survival 

phenotype is not affected by a duplication, indicating n3714 causes increased wild-type function 

or altered function. 

In a screen for suppressors of n3714, we found one mutation, n4111, that is tightly linked to 

n3714 and dominantly suppresses the dominant CEM survival phenotype of n3714. In contrast to 

n3714 hermaphrodites, which inappropriately have surviving CEMs, n4111 n3714 males 

inappropriately lack CEMs. The CEMs of n4111 n3714 males are restored by mutations that 

prevent programmed cell death but not by a null mutation in tra-1, a gene required for 

feminization and the most downstream gene in the sex-determination pathway. Other deaths and 

sexually dimorphic characteristics are not affected by n4111 n3714. 

By performing transformation-rescue experiments and determining DNA sequences, we found 

that n4111 is a nonsense mutation in the BarH class homeodomain gene ceh-30. n3713, n3714, 

and n3720 are mutations in an evolutionarily conserved TRA-1 binding site in an intron of ceh-30. 

We propose that ceh-30 is specifically required for CEM survival in males and that in 

hermaphrodites ceh-30 is prevented from protecting the CEMs by direct transcriptional repression 

by TRA-1. It remains to be determined how ceh-30 protects the CEMs and to what extent this 

function of ceh-30 is shared by BarH class homeodomain genes in other organisms.

227. The "Green Pharynx" Phenotype of Transgene Misexpression Yields New Insight into 

the synMuv Genes 

Hillel Schwartz, Dawn Wendell, Bob Horvitz 


In the course of a screen to identify mutants defective in the control of the sex-specific deaths 

of the CEM neurons using the reporter pkd-2::gfp (see abstract by Schwartz and Horvitz), we 

found 29 independent isolates that had strong GFP expression in the pharynx, an organ that does 

not normally express this reporter. From this screen and further clonal and nonclonal screens, we 

have identified 68 mutants with the "green pharynx" phenotype. This transgene misexpression is 

not dependent on chromosomal integration, high transgene copy number, or choice of 

co-injection marker, and the phenotype can be seen with at least one other GFP reporter that 

contains a different promoter. The green pharynx phenotype is dependent on vector sequence in 

the reporter construct, consistent with previous reports of a cryptic pharyngeal promoter in the 

Fire vectors. In the wild type, pharyngeal expression from this cryptic promoter can be inhibited 

by the inclusion of an insert in the reporter construct; in the context of some but not all inserts, 

this inhibition depends on a mechanism absent in the green pharynx mutants. 

We found that mutations in certain synthetic Multivulva (synMuv) genes (see abstracts by 

Andersen and Horvitz and by Harrison, Lu, and Horvitz) could produce the green pharynx 

phenotype. The synMuv genes act to inhibit vulval development. Animals mutant in two classes of 

synMuv gene (of the three classes, A, B, and C), but not animals mutant in one or more members 

of the same class, display a Multivulva phenotype. Several class B and class C synMuv genes 

have been cloned and shown to encode genes with homologs implicated in transcriptional 

modification and chromatin remodeling. The synMuv genes able to cause pkd-2::gfp expression 

in the pharynx include one class A synMuv gene and three class B synMuv genes, a result that 

contrasts with the finding that genes in the A and B synMuv classes act separately and in parallel 

to prevent vulval cell fates. Mutations in 26 other synMuv genes have been tested and did not 

cause the green pharynx phenotype. 67 of the 68 mutations isolated based upon this phenotype 

appear to be alleles of three of these four synMuv genes; the fourth synMuv gene is maternally 

rescued for the green pharynx phenotype, and mutations in this gene were consequently not 

identified on the basis of this phenotype. 

n3599, the one green pharynx mutation that was not an allele of a synMuv gene, defined the 

gene pag-6 (pag, pattern of reporter gene expression abnormal). pag-6(n3599) mutants are not 

synMuv A, synMuv B, or synMuv C. Interestingly, pag-6(n3599) is synthetically lethal with a 

subset of class B synMuv mutations, including lin-35 Rb. This subset does not correspond to 

subsets associated with other phenotypes, including the subset of class B synMuv mutants 

defective in regulation of the cell cycle (Boxem and van den Heuvel, Curr Biol 12: 906-11, 2002; 

Fay, Keenan, and Han, Genes and Development 16: 503-517, 2002). It is possible that the 

synMuv genes that are synthetically lethal with pag-6(n3599) share a normal function distinct 

from both vulval development and cell cycle regulation but nonetheless involving transcriptional 

repression. pag-6(n3599) causes altered function of a gene encoding a novel protein with 

homology to other worm proteins. 

Further investigation into these transgene-misexpression and synthetic-lethal phenotypes may 

define new transcriptional silencing complexes that include novel proteins and proteins previously 

implicated in silencing and that act in new combinations.

228. Toward expression and biochemical characterization of EFF-1. 

Victoria L. Scranton, William A. Mohler 

University of Connecticut Health Center, Farmington, CT 06030 

Expression of integral transmembrane proteins has always presented a major challenge to 

biologists. We have explored several different systems for expressing and purifying the integral 

transmembrane protein EFF-1, with varying degrees of success. We have initially focused on 

bacterial expression, because of advantageous inducible expressions systems and a variety of 

sequence tags available for affinity purification. We have successfully expressed high levels of a 

truncated "solublilized" form of the protein (EFF-1-EC) using the pET bacterial expression 

system, and are using this pure product to raise specific antibodies to EFF-1. However, 

EFF-1-EC is confined to inclusion bodies (and likely not in a native conformation), and we have 

been unable to express the full length product - possibly due to known toxic effects of 

transmembrane proteins in bacterial expression systems. Another drawback is that proteins 

expressed in bacteria lack eukaryotic post-translational modifications. To overcome these issues 

and to incorporate eukaryotic post-translational modifications, we have also tried to express full 

length protein in the TnT Coupled Wheat Germ Extract System. The yields from this in vitro 

system will require further optimization to be useful.

229. atx-2 Promotes Germline Proliferation and the Female Fate 

Xingyu She 1 , Valarie E. Vought 1 , Dave Hansen 2 , Deborah Springer 1 , Eleanor M. Maine 1 

1Department of Biology, Syracuse University, Syracuse, NY 13244 

2Department of Genetics, Washington University School of Medicine, St. Louis, MO 

The interaction between GLP-1 signaling and the GLD-1 and GLD-2 pathways is important in 

regulating the switch from mitotic proliferation to meiosis in the germ line. The GLD-1 and GLD-2 

pathways function redundantly to promote meiosis and/or inhibit mitosis. In the distal germ line, 

GLP-1 signaling ultimately inhibits the activities of GLD-1 and GLD-2 pathways to promote 

proliferation. We found that atx-2(RNAi) causes a proliferation defect in wild type animals, 

enhances a weak allele of glp-1, and suppresses the gld-2 gld-1 meiotic entry defect. Thus, atx-2 

activity promotes germline proliferation and represses meiosis. We also find that atx-2(RNAi) 

causes a Mog (masculinization of the germ line) defect and suppresses fog-2, indicating that 

atx-2 promotes the spermatogenesis to oogenesis switch. It has previously been shown that atx-2 

is required for early embryogenesis (Gonczy et al. 2000; Kiehl. et al. 2000; Kamath et al. 2003). 

Our data suggest that ATX-2 is not a positive regulator of the GLP-1 pathway, neither is GLP-1 

the sole positive regulator of ATX-2. Our current hypothesis is that atx-2 functions in parallel with 

glp-1, and represses meiosis by working in opposition to the GLD-1 and GLD-2 pathways. In 

addition, it acts downsteam of fog-2 to promote the female germ cell fate. Based on analysis of 

mammalian ataxin-2, we hypothesize that ATX-2 regulates target genes at a post-transcriptional 

level to promote proliferation and the oocyte fate. 

Our experiments to date were done using atx-2(RNAi). Due to the limitations of RNAi, we are 

screening for atx-2 mutations. Using either UV or EMS as a mutagen, we are screening for 

suppressors of fog-2 that are linked to a marker near atx-2. We are also collaborating with the 

Conradt Lab (Dartmouth College) to recover a deletion allele. 

ATX-2 is a 959 amino acid protein that is related to mammalian ataxin-2. Ataxin-2-like proteins 

are found in mammals, insects and plants and contain two conserved regions, named the 

ATX2-N and ATX2-C domains (Satterfield et al. 2002). The ATX2-N domain is related to a portion 

of S. cerevisae PBP1 (Protein that Binds Poly(A)-binding Protein), while the ATX2-C domain is 

essentially a PAM2 (Poly(A)-Binding Protein, Cytoplasmic 1 Interacting Motif 2) sequence. 

Mammalian ataxin-2 has been reported to bind A2BP (Ataxin-2 Binding Protein), which is itself an 

RNA-binding protein (Pulst et al. 2000). We find that atx-2 interacts phenotypically with two C. 

elegans A2BP-related genes, fox-1 and spn-4. Therefore, we are now using a directed yeast 

two-hybrid approach to test whether FOX-1 and SPN-4 can physically interact with ATX-2. We 

hypothesize that these interactions may be important for regulating specific target mRNAs. 

Another likely interaction partner is PAB-1 (Poly(A)-Binding Protein), which we predict would bind 

to the ATX-2 PAM2 motif. pab-1(RNAi) produces a severe germline proliferation defect. Given 

these results, we are also testing whether PAB-1 binds to ATX-2 in our assay. 

Gonczy P et al. (2000) Nature 408, 331-336 

Kiehl TR et al. (2001) J MOL Neuroscience 15, 231-241. 

Kamath RS et al. (2003) Nature 421, 231-237. 

Satterfield TF et al. (2002) Genetics 162, 1687-1702. 

Pulst SM et al. (2000) Hum Mol Genet 9(9), 1303-1313.

230. The Unfolded Protein Response Regulates Glutamate Receptor Export from the ER 

Jaegal Shim, Toru Umemura, Erika Nothstein, Christopher Rongo 


AMPA-type glutamate receptors mediate the majority of excitatory signaling in the CNS. The 

functional properties of these receptors depend on their ability to assemble into a tetrameric 

channel, and on the subunit composition of such channels. Subunit assembly is thought to occur 

in the endoplasmic reticulum (ER), although we are just beginning to understand the underlying 

mechanism. We are studying the function and localization of GLR-1, a glutamate receptor subunit 

similar to mammalian AMPA-type subunits. From forward genetic screening, we have identified 

mutants with abnormal glutamate receptor localization using GLR-1::GFP transgenic worms. We 

have identified two unfolded protein response (UPR) genes, ire-1 and xbp-1, that are functionally 

required for GLR-1 export from the ER. We tested localization of various neuronal proteins 

including several glutamate receptors in these UPR mutant backgrounds. We find that neurons 

require signaling by the UPR to move GLR-1, GLR-2, and GLR-5 subunits out of the ER and 

through the secretory pathway. In contrast, other neuronal transmembrane proteins do not 

require UPR signaling for ER exit. Surprisingly, other ER stress signaling pathways, including 

pek-1, atf-6, and sel-1, are not required for GLR-1 ER export. Moreover, the requirement for the 

IRE-1/XBP-1 pathway is cell type and age dependent: impairment for receptor trafficking 

increases as animals age, and does not occur in all neurons. Expression of XBP-1, a component 

of the UPR pathway, is elevated in neurons during development. Our results suggest that UPR 

signaling is a critical step in neural differentiation that is needed for glutamate receptor assembly 

and secretion. Currently, we are trying to explore the mechanistic role of this pathway in GLR-1 

trafficking.

231. Microarrays analysis of two essential factors required for RNAi, RDE-1 and RDE-4 

Martin J. Simard 1 , Darryl Conte Jr 1 , Jennifer A. Keys 1,2 , Juerg Straubhaar 1 , Danila Ulyanov 1 , 

Craig C. Mello 1,2 

1Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, 

Massachusetts, USA, 01605 

2Howard Hughes Medical Institute 

Recently, RNA interference has emerged as a method of choice for specifically knocking out 

expression of studied genes in a myriad of biological fields. The RNA interference (RNAi) is 

produced by the destruction of cellular RNAs homologous to small RNAs species called small 

interfering RNA (siRNA). In Caenorhabditis elegans, the siRNAs are derived from a long dsRNA 

deliver into the animal either by injection or by feeding the nematode with bacteria expressing the 

dsRNA trigger. In previous genetic screens, we and others have identified several rde (RNA 

interference-deficient) mutants that are required for RNAi in C. elegans [Tabara et al, Cell 99: 

123-132 (1999), Ketting et al, Cell 99: 133-141 (1999)]. Among the isolated genes essential for 

RNA interference, the mutants alleles of rde-1 and rde-4 are required to produce RNAi initiated by 

exogenous dsRNA and are not required for transposon silencing like observed with mut-7. In 

order to uncover new functions for RDE-1 and RDE-4, we have conducted a microarrays analysis 

on a mutant allele of each of them. In the class of genes up- or down-regulated in both rde-1 and 

rde-4 mutant background, we are observing cluster of genes associated to innate immunity 

response in C. elegans. This observation may uncover a new function for RNAi pathway genes in 

nematode. We are currently testing the possible role of rde-1, rde-4 and other rde genes in worm 

immunity.

232. Regulation of gene expression by the Pax factor EGL-38 

Sama F. Sleiman 1 , Helen M. Chamberlin 2 

1MCDB Program, The Ohio State University, Columbus, OH., USA, 43210 

2Department of Molecular Genetics, The Ohio State University, Columbus, OH., USA, 43210 

Pax factors play an important role in organ development in all animals. To understand their 

specific role in organ development, it is important to characterize how they function with other 

transcription factors to regulate target genes. This study will identify factors that function with the 

C. elegans Pax factor EGL-38 as well as target genes. 

In C. elegans, EGL-38 is a Pax factor that plays a key role in the development of several 

organs, namely the egg-laying system, hindgut and the male tail. Pax factors have different 

targets in different cells because they are known to act in a combinatorial manner with other 

transcription factors. For example, lin-48 is a direct target of EGL-38 in the hindgut, but it is not 

expressed in other organs where EGL-38 is known to function such as the egg-laying system. 

Previously, two redundant cis regulatory elements important for lin-48 expression (lre1 and lre2) 

were shown to be sensitive to EGL-38 in vivo. However, EGL-38 only binds to lre2 in vitro. As lre1 

and lre2 are otherwise functionally similar, we hypothesize that EGL-38 acts through lre1 in 

combination with other factors. In this study, we show that lre1 activity in the hindgut requires a 

region of 33bps and that this newly defined area is sensitive to EGL-38 in vivo. Since Pax factors 

only bind to 20bps, this result is consistent with the idea that additional factors contribute to the 

activity of this element, To identify these factors, we conducted a genetic screen. This screen 

utilized animals carrying a lin-48::gfp with a mutant lre2 sequence. These animals express GFP 

normally since lre1 is intact. We sceened for mutans that lack hindgut expression to identify 

genes that act with egl-38 through the lre1 element. We have isolated two mutants that lack 

hindgut expression and are working to characterize the mutants and clone the genes. 

The different functions of egl-38 have been characterized using several non-null alleles. Each 

allele corresponds to a different amino acid substitution within the DNA binding domain of 

EGL-38, and preferentially disrupts different EGL-38 functions. These mutations affect the ability 

of EGL-38 to bind DNA in vitro (Zhang et al., in prep). To better understand the different roles of 

EGL-38, we are using an overexpression approach to identify its target genes. We tested the 

effect of ectopic expression of EGL-38 and its mutants in animals. Our results indicate that 

ectopic expression of EGL-38 in embryos induces lethality, whereas ectopic expression of its 

male tale specific alleles (sy287 and gu22) induce larval development delay and ectopic 

expression of its egg-laying defective allele (n578) does not have any effect. These defects could 

be due to misexpression of different EGL-38 targets in response to the different alleles. Using gfp 

reporter transgenes, we have shown that ectopic expression of EGL-38, induces ectopic 

expression of lin-48 (a known direct target) and lin-3 (a potential target). As the different EGL-38 

mutant forms exhibit different DNA binding affinities, we are planning to do microarray studies 

comparing RNA from strains expressing the different EGL-38 variants to identify potential EGL-38 

targets.

233. C.elegans recognizes protons as a nociceptive stimulus through the DEG/EnaC and 

TRP channel. 

Alfonso J. Apicella 1 , Robert D. Slone 2 , Monica Driscoll 2 , William R. Schafer 1 

1University of California, San Diego, La Jolla California 

2Molecular Bio. and Biochem., Rutgers University, Piscataway, NJ 

Acidic pH is known to activate both nociceptive neurons and acid taste chemosensory neurons 

in mammals. Previous experiments have shown that Caenorhabditis elegans also avoids an 

acidic environment (Sambongi et al. 2000). This avoidance behavior is dependent on multiple 

amphid chemosensory neurons, including the polymodal nociceptor ASH. The ASH sensory 

neurons are required to detect many water-soluble repellants that taste bitter to humans, as well 

as high osmolarity and touch to the nose. We have been interested in determining how pH 

sensation occurs in ASH, and how this sensory transduction process relates those involved in 

detecting other aversive stimuli. To address this question we have we used in vivo optical 

imaging with the calcium indicator cameleon (Kerr et al. Neuron 2000), together with behavioral 

experiments, to evaluate ASH sensory responses to acid pH and other stimuli. We observe 

robust calcium transients in ASH in response to stimulation with either acetic or hydrochloric acid; 

interestingly, after prolonged stimulation, we also observe a smaller "off" response following the 

return to neutral pH. 

In C.elegans members of the DEG/ENaC ion channel subunit superfamily (degenerin/epithelial 

Na+ channel) encode amiloride-sensitive sodium ion channels are implicated in 

mechanotransduction. The ASH neurons express three degenerin homologues: deg-1, C24G7.2, 

and ZK770.1 (Tavernarakis et al., IWM 662). By analyzing the effects of loss-of-function alleles of 

these genes, we determined that deg-1 and C24G7.2 function semi-redundantly in the detection 

of acidic pH; both single mutants exhibit abnormal acid-induced calcium influx in ASH, while the 

deg-1; C24G7.2 double mutant shows virtually no response to acid stimulation. Interestingly, 

deg-1 and C24G7.2 do not appear to affect ASH responses to chemical repellants such as 

copper; this contrasts with the ASH TRP channel OSM-9, which is required for all ASH sensory 

responses. Possible functional interactions between degenerin and TRP channels will be 

discussed.

234. Move or Die: Epidermal Migration in the Caenorhabditis elegans Embryo 

Esteban Chen*, Michael M. S. Huang*, Veronica Zappi, Martha C. Soto 

Department of Pathology and Laboratory Medicine, UMDNJ-Robert Wood Johnson Medical 

School, Pisctaway, NJ, 08854, USA 

One critical event in the development of Caenorhabditis elegans is the enclosure of the embryo 

by the epidermis. The ventral migration of epidermal cells leads to enclosure of the internal 

organs including the gut. Embryos defective in this process have a pronounced 

"gut-on-the-exterior," or Gex, phenotype. Polarized actin nucleation is thought to drive this ventral 

migration. Previous work has shown that loss of any of three genes, gex-1, gex-2, and gex-3, 

either by mutation or reduced expression via RNAi, leads to the Gex phenotype. We have found 

that GEX-1, GEX-2, and GEX-3 encode homologs of actin nucleation regulators: WAVE/SCAR, 

PIR121/SRA-1/P140, and KETTE/NAP1/HEM2, respectively. It has been proposed that the 

GEX-2 and GEX-3 mammalian homologs inhibit the GEX-1 homolog, WAVE/SCAR. However, 

our genetic data suggest the in vivo regulation in C. elegans is more complex. Our model is that 

GEX-2 and GEX-3 may affect GEX-1 by regulating localization or stability. To test this model we 

are using RNAi to make embryonic lysates depleted of GEX-1, GEX-2 or GEX-3 and studying if 

depletion of one GEX influences the others. We present preliminary evidence that GEX-2 serves 

to stabilize GEX-1. We are also testing by immunoprecipitation if GEX-1, GEX-2, and GEX-3 form 

a complex. We already know that GEX-2 and GEX-3 co-localize and co-immunoprecipitate, but 

we have yet to test if they interact with GEX-1. A second focus of this project is uncovering new 

players in the actin polymerization pathway by screening for additional mutants with the Gex 

phenotype. Gex mutants have zygotic Egl (egg-laying defective) and maternal effect lethal (Mel) 

phenotypes. Additional Egl Mel mutants could identify homologs of known players in the actin 

polymerization pathway, as well as novel components. For example, we presently know neither 

the signal nor the signaling receptor that regulates ventral migration. Our recent screens have 

isolated three new Gex mutants, pj1, pj2, and pj3. Preliminary mapping suggests that they do not 

map to the same location as any of our known mutants. 

* These authors contributed equally to the work.

235. Phenotypic characterization of egg-1, a molecule involved in fertilization 

Pavan Kadandale, Allison Stewart, Richard Klancer, Barth Grant, Andrew Singson 

Rutgers University, NJ 08854 

Standard forward genetic approaches have resulted in the isolation of a number of mutants of 

C. elegans that are defective in the process of fertilization. However, due to methodological 

limitations all of these mutants have been in sperm-specific molecules. Traditional mutagenesis 

screens have not yet identified any fertilization-defective mutants that affect molecules present in 

the oocyte. We have used a combination of bioinformatics and reverse genetics to obtain the first 

such mutant, which we have named egg-1. We have obtained a deletion allele (from the 

Japanese Knock-out Consortium) of egg-1, and this mutant has a temperature-sensitive fertility 

defect. At 16°C, egg-1 animals have brood sizes that are 91% of wild type. When shifted to 

25°C, however, these worms have only 7% of wild type brood sizes. egg-1 encodes a Type II 

transmembrane molecule that has multiple LDL-receptor repeats. We will present our ongoing 

analysis of the phenotypic analysis of egg-1 and its role in gamete interactions during fertilization.

236. How are cytoplasmic asymmetries achieved? In vivo studies of germ plasm 

localization dynamics during early embryogenesis 

Michael L. Stitzel 1 , Denis Wirtz 2 , Geraldine Seydoux 1 

1 Department of Molecular Biology & Genetics, Johns Hopkins University School of Medicine, 

Baltimore, MD 

2 Chemical & Biomolecular Engineering Department, Whiting School of Engineering, Johns 

Hopkins University, Baltimore, MD 

The CCCH finger proteins PIE-1, POS-1, and MEX-1 are maternally-encoded germ cell fate 

regulators that segregate with the germ lineage during early embryogenesis. These proteins, 

initially distributed throughout the zygote, become enriched at the posterior pole prior to mitosis 

and are maintained there through the first cell division. Genetic studies have indicated that 

asymmetric localization of cytoplasmic determinants depends on PAR-1 on the posterior cortex 

(Guo and Kemphues, 1995), and on MEX-5 and MEX-6, two homologous CCCH finger proteins 

that segregate opposite PIE-1, POS-1, and MEX-1 in the cytoplasm (Schubert et al., 2000). The 

molecular mechanisms that lead to cytoplasmic asymmetries in the zygote are not known. 

To begin to characterize these mechanisms, we are analyzing the in vivo localization dynamics 

of GFP fusions in wild type and mutant embryos using quantitative time-lapse microscopy and 

fluorescence recovery after photobleaching (FRAP). FRAP analysis of GFP:PIE-1 indicates that 

there is a larger immobile fraction of the fusion protein in the posterior compared to the anterior 

(35% vs. 0%) during mitosis, suggesting that PIE-1 may be tethered in the posterior region of the 

zygote. We are currently assessing the kinetics of recovery in par-1 and mex-5/-6 (RNAi) mutants 

to determine what roles they play in enrichment dynamics. 

References: 

1. Guo, S. and Kemphues, KJ. 1995. Cell. 81(4): 611-620. 

2. Schubert, C.M., et al., 2000. Mol. Cell. 5(4): 671-682.

237. Screen for Enhancers of ksr-2 Lethality 

Craig Stone, Meera Sundaram 

Dept. of Genetics, University of Pennsylvania Medical School 

Ras signaling is critical for normal development and growth, and is implicated in many forms of 

human cancer. The evolutionarily conserved KSR protein functions as a scaffold for several core 

Ras pathway components. Upon Ras activation, KSR localizes to the plasma membrane, and this 

movement is essential for Ras signaling. Two functionally redundant isoforms of KSR, KSR-1 and 

KSR-2, exist in C.elegans. They are important in Ras-dependent vulval and excretory duct 

development. Alterations in either of these processes lead to gross phenotypes. Previous genetic 

screens have used these phenotypes to identify mutations in Ras pathway components. 

Among known ras signaling pathway genes, ksr-1 is the only one which genetically interacts 

with ksr-2 to disrupt excretory duct function. Thus, any mutant identified by a genetic screen for 

enhancers of ksr-2 would likely be very specific to KSR function. I have thus far screened through 

600 F1 progeny of EMS mutagenized worms. I have picked 8 F2 progeny from each F1 animal to 

ksr-2 RNAi feeding plates, and looked for a defective excretory duct (rod-like) phenotype in the 

F3 generation. Thus far I have isolated two mutants, which are not alleles of ksr-1 based on 

complementation tests. Since there are no other obvious ksr-2 enhancer genes known besides, 

the chances of discovering a gene(s) with novel functions relative to this Ras pathway is high. 

One mutant segregates as an X-linked recessive mutation. On ksr-2 feeding plates it gives a 

greater than 50% rod-like lethality, which is comparable to ksr-1 (n2526) null animals. Almost all 

other larvae exhibit a growth arrest phenotype and do not become adults. The second mutant 

segregates as an autosomal dominant mutation. Homozygotes for the mutation are larval lethal. 

Further analysis will include genetic interaction analysis with ksr-1, and positionally mapping the 

candidates to single genes. In addition, about 3000 more F1 animals will be screened in an 

attempt to identify more enhancers of ksr-2, as well as additional alleles of the two current 

mutants.

238. EGL-26 controls vulF morphogenesis 

Hongliu Sun, Rita Sharma, Wendy Hanna-Rose 

Biochemistry and Molecular Biology, Penn State, University Park, PA 

Mutation of the egl-26 gene causes the vulF cell at the apex of the vulva to adopt an abnormal 

morphology, resulting in a blockage between the vulval and uterine lumens and an egg-laying 

defect. Because EGL-26::GFP expression is restricted to the apical edge of the vulE cell within 

the vulva while the egl-26 mutant has abnormal vulF morphology, it is hypothesized that EGL-26 

may function in a cell nonautonoumous manner. To test the cell nonautonomous model of 

EGL-26 function, we wish to confirm the expression pattern via other methods. Antibody and 

transgenic animals with tagged EGL-26 proteins are being generated to gather additional 

evidence about expression. 

We are also searching for interacting proteins via a yeast two-hybrid assay to shed light on 

EGL-26 function. Using full-length EGL-26 as bait, 7X10 5 clones were screened. Two candidates, 

M151.7 and R05D3.4, show sequence similarity to the rud3 and uso1 genes in S. cerevisiae, 

respectively. In yeast, rud3 was isolated as a multicopy suppressor of a mutation in uso1, which 

is required for assembly of the ER to Golgi SNARE complex in vivo. A third candidate K02D10.4 

encodes a putative t-SNARE of the plasma membrane, indicating that these three candidate 

EGL-26 interacting proteins (cEIPs) may have related functions. Thus, it is speculated that 

EGL-26 may interact with the secretion apparatus to facilitate production of a signal by vulE that 

subsequently affects vulF morphology. This would be consistent with the cell nonautonomous 

model of EGL-26. Alternatively, trafficking of EGL-26 to the apical membrane of vulE may be 

required for function and may be achieved by the SNARE-related apparatus. We are investigating 

these alternatives by RNAi of cEIPs to determine the function of the cEIPs individually and in 

combination. Furthermore, EGL-26 bait vectors incorporating the genetic lesions found in the 

ku211, ku228 and n481 mutants are being constructed to examine the specificity of interactions 

and interaction domains of EGL-26.

239. Development of Caenorhabditis elegans in CeHR Axenic Medium 

Maria Szilagyi, Hugh F. LaPenotiere, Eric D. Clegg 

US Army Center for Environmental Health Research, Ft. Detrick, MD 21702 

At the US Army Center for Environmental Health Research we have developed an axenic liquid 

culture medium; Caenorhabditis elegans Habitation and Reproduction (CeHR) medium. CeHR 

allows the growth, observation, exposure and harvest of large numbers of nematodes for 

toxicological experiments, and genomics and proteomics research. Also, the axenic liquid culture 

minimizes confounding from E. coli or liver homogenates. The developmental time from L1 to 

early gravid adult is 63 hours at 22 o C with approximately 100 offspring per worm. The lifespan is 

approximately 12 days. The pH range is 6.0-6.9 depending on the stages of worms present.

240. The Wnt genes egl-20 and cwn-1 are redundantly required for proper vulval cell fate 

specification. 

Elizabeth Szyleyko, Julie E. Gleason, David M. Eisenmann 

Dept. of Biological Sciences, University of Maryland Baltimore County, Baltimore MD 21250 

Our laboratory is interested in the mechanisms controlling cell fate specification by the Vulval 

Precursor Cells (VPCs) during vulval induction. Established work showed that RTK/Ras and 

Notch signaling pathways act during this process, and previous work from our laboratory and 

others has indicated that a Wnt signaling pathway is also required. The first evidence of this was 

the observation that mutations in bar-1, which encodes one of three C.elegans beta-catenin 

homologs, cause VPC fate specification defects that lead to Pvl and Egl phenotypes. One target 

of this pathway is the Hox gene lin-39, which is coordinately regulated at the transcriptional level 

by both Wnt and Ras pathways (see Wagmaister et al. abstract). Subsequently, we have shown 

that other canonical Wnt pathway components such as APC (APR-1), Axin (PRY-1) and TCF/LEF 

(POP-1), also function during VPC fate specification. However to date, we have not identified the 

Wnt signal used to activate this pathway, the source of this Wnt signal, or the Frizzled receptor 

used to transduce this signal in the VPCs. We predict that loss of the Wnt or frizzled gene activity 

would lead to a vulval phenotype resembling that of bar-1(ga80) or mig-14(ga62). 

There are five Wnt genes in the worm (lin-44, egl-20, mom-2, cwn-1 and cwn-2), and we took 

two approaches to determine which of these is acting on the VPCs. First, analysis of existing 

viable mutants showed that none of them had Pvl/Egl phenotypes like bar-1(ga80), however 

egl-20 mutants had a Fused fate phenotype at P3.p and P4.p like that observed in bar-1(ga80). 

This phenotype was not enhanced in a lin-44; egl-20 double mutant. To examine whether 

redundant Wnts might be used in VPC fate specification, we performed RNAi on cwn-1, cwn-2 or 

mom-2 in a lin-44; egl-20 background, and found that 21 - 36% of lin-44; egl-20; cwn-1(RNAi) 

animals had an Underinduced phenotype like bar-1(ga80), in which fewer than three VPCs 

adopted induced fates. Subsequently, we analyzed a cwn-1(ok546); egl-20(n585) double mutant 

strain (generous gift of Rik Korswagen) and found that 84% of these animals had Underinduced 

or Vulvaless phenotypes (versus 6% for cwn-1(ok546) and 3% for egl-20(n585)). These results 

suggest that the Wnt pathway in the VPCs is activated by two genetically redundant Wnt ligands, 

EGL-20 and CWN-1. 

Second, we created transcriptional GFP reporter constructs for all five Wnt genes to determine 

which, if any, were expressed in or around the VPCs at a time when they could influence fate 

specification. Analysis of the expression of these constructs showed that both cwn-1 and cwn-2 

are expressed in ventral cord neurons during the L2 and L3 stages, and could therefore be 

affecting the VPCs. Although egl-20 is expressed in the vulval region, we have not seen 

expression at a time early enough to corroborate our genetic evidence. This could be due to low 

level or transient expression, or to the lack of necessary genomic elements in our reporter. 

Data on the expression of the Wnt reporters, additional Wnt gene RNAi data, and similar 

experiments to identify the frizzled gene or genes (lin-17, mig-1, mom-5, or cfz-2) acting in the 

VPCs will be presented.

241. The G proteins GOA-1 and EGL-30 function antagonistically in the HSN neurons that 

regulate egg-laying behavior in C. elegans 

Jessica E. Tanis, James J. Moresco, Robert A. Lindquist, Michael R. Koelle 

Yale University, New Haven CT 

Neurotransmitters signal through heterotrimeric G proteins to alter neural activity. The C. 

elegans neural G proteins GOA-1 and EGL-30 (orthologs of mammalian Gαo and Gαq, 

respectively) have opposite effects on behavior, but the mechanism by which these G proteins 

modulate neural function is unclear. To analyze this mechanism, we are genetically manipulating 

G protein signaling in a single neuron in C. elegans and analyzing the effects on the function and 

synaptic structure of that neuron. 

Components of the GOA-1 and EGL-30 signaling pathways are expressed in the egg-laying 

system. EGL-30 loss-of-function mutants are egg laying defective, while GOA-1 loss-of-function 

mutants are hyperactive for egg laying. However, it is not known whether EGL-30 and GOA-1 

signal to antagonize each other directly in the same cell of the egg-laying system or indirectly by 

acting in different cells. Three cell types control egg laying: 1) the vulval muscles contract to lay 

eggs; 2) the HSN neurons synapse onto the vulval muscles and stimulate egg laying, and 3) the 

VC neurons synapse onto both the HSN neurons and the vulval muscles and inhibit egg laying. 

We developed promoters to drive transgene expression specifically in each of these three cell 

types. We have used these cell-specific promoters to determine in which cells of the egg-laying 

system the Gαo and Gαq pathways function. 

We found that GOA-1 and EGL-30 both function in the HSN neuron. Inactivation of GOA-1 in 

the HSNs of wild-type animals using the HSN-specific promoter to express the catalytic subunit of 

pertussis toxin resulted in hyperactive egg-laying behavior. Expression of a constitutively active 

form of GOA-1 in the HSN neurons of GOA-1 loss-of-function animals rescued the hyperactive 

phenotype. Additionally, HSN expression of EGL-10, a negative regulator of GOA-1, was 

sufficient to rescue the EGL-10 egg-laying defect. These results suggest that GOA-1 activity in 

the HSN is both necessary and sufficient to inhibit HSN function, and therefore to inhibit egg 

laying. Partial rescue of the egg-laying defective phenotype of egl-30 mutants was achieved by 

expressing EGL-30 in either the HSN neurons or the vulval muscles, suggesting a role for 

EGL-30 in both cell types. We will determine if co-expressing EGL-30 in both the HSNs and 

vulval muscles can achieve full rescue. We are further analyzing the sites of EGL-30 function by 

determining where EAT-16, the negative regulator of EGL-30, is required. We conclude that 

EGL-30 and GOA-1 function antagonistically in the HSN neurons to regulate egg laying. 

We are also analyzing the mechanism by which the G proteins affect neural function by 

determining how GOA-1 and EGL-30 signaling affects synapse morphology. Using the 

HSN-specific promoter to express fluorescently-tagged proteins, we can visualize the structure of 

the HSN neuron synapses and the localization of specific proteins required for synaptic vesicle 

release at those synapses.

242. SRY-box containing protein SOX-2 directly binds to the promoter of Hox gene egl-5 

and negatively regulates its expression 

Yingqi Teng, Scott W. Emmons 

Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park 

Avenue, Bronx, NY 10461 

Hox genes are important regulatory genes that specify regional identities in multi-cellular 

organisms. C. elegans Hox gene egl-5 is one of the posterior group genes, which is involved in 

multiple cell fate specification events. In the C. elegans male tail, egl-5 specifies the fate of the 

seam cell V6.ppp. In egl-5 mutant males, the fate of V6.ppp is transformed into that of its anterior 

counterpart V6.pap, generating two sensory rays instead of three. 

To understand how egl-5 is regulated in the V6 lineage, we have performed a promoter 

dissection study of egl-5, guided by the sequence comparison of C. elegans and C. briggsae 

egl-5 locus. We have located the V6 lineage regulatory activity to a 200bp region that is highly 

conserved between the two species, which we gave the name V6CRE (V6 cis-regulatory 

element). V6CRE can drive delta pes-10::gfp reporter in wild type egl-5 expression pattern in the 

V6 lineage. Further dissection of the V6CRE region suggests that V6CRE region bears functional 

redundancies and that important regulatory sites are spread over the whole V6CRE region. 

We performed a yeast one-hybrid assay in search of the transcription factors that 

directly bind to V6CRE. Out of 10 6 clones screened, we have recovered one clone that 

corresponds to the SRY box containing gene sox-2. Sox family genes share a conserved SRY 

box DNA binding domain but do not contain any known activation domain, therefore, they are 

considered structural components of DNA associated complexes. In addition, Sox genes are 

known regulators of neuronal development. We have identified three potential binding sites of 

SOX-2 in the V6CRE, and are currently assessing the importance of these sites. A translational 

sox-2::gfp reporter is expressed in the RnB neurons and structure cells of the developing ray 

sublineages. Expression of a hairpin dsRNA construct under the heatshock promoter causes a 

ray missing phenotype, suggesting that sox-2 is an important regulator in the male tail 

development. The relatively late expression of sox-2 compared to egl-5 suggests that sox-2 might 

serve to turn egl-5 off to allow proper differentiation of the ray group cells. Supporting this 

hypothesis, we have found that heatshock of sox-2 cDNA turns off egl-5::gfp reporter in the V6 

lineage. In conclusion, we have identified SOX-2 as a potential regulator of egl-5, which binds to 

the promoter of egl-5 and turns off its expression.

243. Identification of genes involved in the specification of the sexually dimorphic CEMs 

Tatiana Tomasi 1 , Stefanie Löser 2 , Phillip Grote 1 , Barbara Conradt 1 

1Dept. of Genetics, Dartmouth Medical School, 7400 Remsen Building, Hanover, NH, 03755, 

USA 

2Institute of Molecular Biotechnology (IMBA), Dr.Bohr-Gasse 3-5, A -1030 Wien, Austria 

Sexual dimorphism in the nervous system of C. elegans is a result of differential numbers of 

cell divisions, different cell fates, or programmed cell death (PCD). The cephalic companion 

neurons (CEMs), four sensory neurons thought to be involved in mating behaviour, and the 

hermaphrodite specific neurons (HSNs), two motoneurons necessary for egg laying, are sexually 

dimorphic as a result of PCD (Sulston and Horvitz, 1977). The HSNs and CEMs are born in 

embryos of both sexes but are subsequently eliminated by PCD in males (HSNs) or 

hermaphrodites (CEMs). The death of the CEMs and the survival of the HSNs in hermaphrodites 

are regulated by the most downstream factor of the sex-determination pathway, the zinc-finger 

transcription factor TRA-1 (tra, transformer), and by the key activator of the cell-death pathway, 

the BH3-only protein EGL-1(egl, egg-laying defective). Because TRA-1 functions to repress the 

egl-1 gene in the HSNs but to activate egl-1 in the CEMs, cell-specific factors must exist that act 

with TRA-1 to regulate PCD in these sexually dimorphic neurons. 

Gain-of-function (gf) mutations of the gene egl-41 cause the CEMs to inappropriately survive in 

hermaphrodites. In order to identify genes required for CEM survival, we performed a screen for 

mutations that cause the CEMs to be absent in egl-41(gf) hermaphrodites and identified three 

mutations, bc151, bc155 and bc159. All three mutations cause the CEMs to be absent in 

egl-41(gf) hermaphrodites and also in otherwise wild-type males. bc151, bc155 and bc159 affect 

the formation and/or specification of the CEMs rather than their sex specific death, since the 

absence of the CEMs can not be suppressed by a ced-3 (lf) mutation. 

bc151 represents an allele of unc-86, which codes for a POU homeodomain transcription factor 

previously shown to control neuronal specification in many neurons, including the HSNs and 

CEMs (Desai et al., 1988; Finney et al., 1988). bc155 causes 88% of the CEMs in egl-41(gf) 

hermaphrodites to be absent. We are in the process of mapping this mutation using SNP 

mapping. bc159 causes 100% of the CEMs in egl-41(gf) hermaphrodites to be absent. Moreover, 

bc159 animals are Egl and slightly Unc. The observation that the sisters of the dorsal CEMs, the 

dorsal URA neurons, are present in bc159 animals indicates that the last cell division, which gives 

rise to the dorsal URAs and the dorsal CEMs, takes place, but that the CEMs fail to differentiate. 

The reduced expression of the differentiation marker P glr-4glr-4::gfp in the URAs furthermore 

indicates that bc159 affects the fate of additional neurons. Epistasis analysis with cfi-1 (CEM fate 

inhibitor), a gene that represses the expression of CEM-specific genes in some neurons including 

the URAs (Shaham and Bargmann, 2002), revealed that the gene defined by bc159 acts 

downstream of or in parallel to cfi-1. The identification of this gene and further characterization of 

the bc159 phenotype will reveal new insights into the specification of the CEMs and other 

neurons. Using transformation rescue experiments, we have obtained cosmid rescue of the 

bc159 phenotype. 

Barr, M. M. and Sternberg, P. W. (1999). A polycystic kidney-disease gene homologue required 

for male mating behaviour in C. elegans. Nature 401, 386-9. 

Desai, C., Garriga, G., McIntire, S. L. and Horvitz, H. R. (1988). A genetic pathway for the 

development of the Caenorhabditis elegans HSN motor neurons. Nature 336, 638-646. 

Finney, M., Ruvkun, G. and Horvitz, H. R. (1988). The C. elegans cell lineage and 

differentiation gene unc-86 encodes a protein with a homeodomain and extended similarity to 

transcription factors. Cell 55, 757-69. 

Shaham, S. and Bargmann, C. I. (2002). Control of neuronal subtype identity by the C. elegans 

ARID protein CFI-1. Genes Dev 16, 972-83. 

Sulston, J. E. and Horvitz, H. R. (1977). Post-embryonic cell lineages of the nematode, 

Caenorhabditis elegans. Dev Biol 56, 110-56.

244. Genetic screen for factors functioning with EGL-38 Pax to regulate lin-48 expression 

in Caenorhabditis elegans 

Rong-Jeng Tseng, Helen M. Chamberlin 

Program in MCD Biology and Department of Molecular Genetics, The Ohio State University, 

Columbus, OH 43210 

EGL-38 is a Pax transcription factor important for development of the hindgut, the egg-laying 

system, and the male spicules. Genetic studies suggest EGL-38 has different target genes in the 

different tissues. To better understand how EGL-38 Pax regulates gene expression in a 

tissue-specific manner among many potential target genes, a hindgut specific target, lin-48, was 

utilized as a reporter. lin-48 encodes an Ovo-like zinc finger protein and is important for normal 

development of the hindgut. The lin-48::gfp expression in the hindgut is significantly reduced in 

egl-38 mutants. Likewise, mutations of EGL-38 binding elements in the lin-48 promoter disrupt 

hindgut expression. Since EGL-38 functions and is expressed in cells in addition to the hindgut, 

we predict it functions in combination with another factor(s) to regulate lin-48 specifically in 

hindgut cells. 

To identify factors functioning with EGL-38 to regulate the tissue-specific expression of lin-48, 

we performed an F2 genetic screen to isolate mutants with altered lin-48::gfp expression pattern. 

The isolated mutants can be grouped into two major classes: 1) reduced GFP expression in the 

hindgut; 2) ectopic or altered GFP expression pattern. The first class includes a new egl-38 allele 

and alleles in two additional complementary groups. We hypothesize the two new genes encode 

proteins important for EGL-38 expression or activity, or they may function with EGL-38 in the 

hindgut. In the second class, the mutated genes might include negative regulators of egl-38 or 

factors that affect lin-48 expression in other pathways. Currently, we are using SNP mapping and 

cosmid rescue to identify the two genes that with the reduced GFP expression mutant phenotype.

245. Transcriptional specification of neural subtype in the C. elegans male tail 

Carolyn Tyler 1 , Nicole Juskiw 2 , Douglas S. Portman 3 


2 GEBS Summer Scholar Program, University of Rochester Medical Center, Rochester, NY 

14642 

3 Center for Aging and Developmental Biology and Department of Biomedical Genetics, 

University of Rochester Medical Center, Rochester, NY 14642 

Rays are C. elegans male tail sensory organs thought to transduce chemo- and 

mechanosensory cues important for mating behavior. Each of the eighteen rays contains two 

neurons, RnA and RnB, and a structural cell, Rnst, that descend from a single ray precursor cell, 

Rn. The RnB neurons of all rays (except ray 6), along with the hook HOB and head CEM 

neurons, express lov-1 and pkd-2, genes that are required for the response of males to 

hermaphrodites and for the subsequent location of the hermaphrodite vulva 1 . In a 

microarray-based screen for new ray-expressed genes 2 , we identified five new genes, cwp-1 

through -5, that have a pattern of expression identical to that of lov-1 and pkd-2. The highly 

specialized and restricted expression pattern of these seven genes provides an opportunity to 

understand how the differential regulation of gene expression that defines neuronal subtype is 

implemented. We speculate that lov-1, pkd-2 and the five cwp genes share a common 

mechanism of transcriptional regulation. Using nested deletions and the pes-10 

enhancer-element assay, we sought to define minimal regulatory regions sufficient to activate 

transcription in the RnB neurons. With this approach, we have identified small regions from 

cwp-1, -2/3, -5 and pkd-2 that are sufficient to drive GFP expression in the RnBs as well as the 

CEMs and HOB. We have observed some variability in expression among these constructs which 

might result from pes-10 context effects or from the lack of motifs necessary for robust 

expression. We also found that a small region upstream of cwp-4 can drive expression in the IL2 

and URAD neurons as well as the RnB/HOB/CEM set. This suggests that this region may lack an 

element required for the function of the ARID protein CFI-1, which normally prevents expression 

of at least some of these genes in the IL2s and URADs 3 . We have not yet identified obvious 

sequence similarities in our minimal regions that could represent binding sites for a common RnB 

transcription factor. We are continuing to define the minimal requirements for RnB expression and 

will use bioinformatic and molecular strategies to identify the regulatory mechanisms that underlie 

the specific co-expression of this battery of genes. 

1 M. M. Barr and P. W. Sternberg (1999) Nature 401:386; M. M. Barr et al (2001) Curr Biol 

11:1341. 

2 D. S. Portman and S. W. Emmons (2004) Dev Biol, in press. 

3 S. Shaham and C. I. Bargmann (2002) Genes Dev 16:972.

246. Suppressors of pha-4/FoxA loss of function mutations define potential pha-4 

regulators 

Dustin L. Updike, Susan E. Mango 

University of Utah Salt Lake City, UT 84112 

Specification of pharyngeal cell fate depends on pha-4, which codes for a FoxA transcription 

factor homologue 1,2,3 . Fox proteins are characterized by conservation over 110 amino acids that 

encompass the DNA binding domain. Fox genes have been further subdivided into seventeen 

classes (A to Q) on the basis of additional sequence conservation. The FoxA subclass has been 

implicated in gut development in all animals examined, including vertebrates. Despite their 

importance, few co-factors or upstream regulatory components are known for FoxA proteins in 

any organism. 

To identify genes that encode regulators or co-factors of PHA-4, we have undertaken a screen 

for mutants that suppress a partial loss of pha-4 function. 27 suppressors from 15,000 haploid 

genomes were obtained using EMS as a mutagen and 29 more suppressors were found using 

the Mos1 transposon 4 . Surprisingly, none of our transposon-induced alleles carried a Mos1 

insertion. Secondary tests demonstrated that 17/27 EMS- and 26/29 Mos1-induced alleles were 

informational suppressors, leaving 13 suppressors that likely regulate the activity or expression of 

pha-4. Mapping data have revealed that the 13 suppressors represent at least 9 genes. 

We have begun to characterize two dominant pha-4 suppressors. One, px63, is a complex 

rearrangement in the second intron of pha-4. We propose that this mutation alters regulation of 

PHA-4 transcription. Another dominant suppressor, px34, contains a missense mutation in a 

predicted helicase. We are currently investigating the role of px34 for pha-4 activity and 

pharyngeal development. 

1. Mango et al., Development, 120:3019-3031 (1994). 2. Kalb et al., Development, 

125:2171-2180 (1998). 3. Horner et al., Genes Dev., 12:1947-1952 (1998). 4. Bessereau et al., 

Nature, 413:70-74 (2001).

247. Identification of genes involved in cell fate specification of the gonadal sheath cells in 


Laura G. Vallier 1,2 , Helaina Skop 1 , Lindsay Eisemann 1 

1Hofstra University, 114 Hofstra University, Gittleson Hall Rm. 103, Hempstead, NY 11549 

2email: biolgv@hofstra.edu 

The gonadal sheath is a tissue composed of five pairs of cells that covers the proximal half of 

each arm of the gonad. Its functions include rhythmic contractions crucial for ovulation and in 

meiotic progression of the germ cells as well as mitotic proliferation of the germline stem cell 

population. When one or more of these cells are absent, aberrations in these processes occur. To 

better understand the role this tissue performs, we are identifying the genes that are required for 

gonadal sheath cell development and identity combining an RNAi screen with a GFP reporter 

system that auto-fluoresces in the gonadal sheath cells (lim-7::GFP). To date, we have screened 

the majority of the genes on Chromosome I for those causing a loss of GFP fluorescence in the 

gonadal sheath and ensuing sterility after feeding with individual HT115 bacterial clones carrying 

the genes of Chromosome I in RNAi vectors. Currently, we have identified five genes that, when 

inactivated after exposure to RNAi, cause the gonads in lim-7::GFP worms to lose fluorescence, 

indicative that these animals did not make a gonadal sheath. These candidates were also 

examined for sterility, an expected result in an animal that does not have a gonadal sheath, and 

they were found to be sterile or have reduced numbers of progeny. Preliminary findings will be 

presented.

248. Genetic Screens for Suppressors of the ceh-30(n3714gf) Phenotype of Inappropriate 

Survival of the Male-Specific CEM Neurons in Hermaphrodites 

Johanna Varner, Hillel Schwartz, Bob Horvitz 


During wild-type hermaphrodite development, 131 somatic cells undergo programmed cell 

death. Many genes involved in the execution of cell death have been identified, but the 

mechanisms controlling the decision by specific cells to undergo programmed cell death are still 

poorly understood: only six genes that control the programmed deaths of ten cells have been 

described. The four CEM neurons are especially convenient for studies of the regulation of 

programmed cell death, because the CEMs die during hermaphrodite development but survive 

and differentiate in males, and the pkd-2::gfp cell-fate reporter (kindly provided by Maureen Barr 

and Paul Sternberg) can be used to rapidly score animals for the presence of CEMs. A genetic 

screen of 60,000 mutagenized haploid genomes recovered at least 154 independent mutations 

causing inappropriate survival of the CEMs in hermaphrodites. Three of these mutations, n3713, 

n3714, and n3720, were gain-of-function mutations in the gene ceh-30 (ceh, C. elegans 

homeobox), which is required for CEM survival in males. These three alleles disrupt an 

evolutionarily conserved binding site for the transcriptional repressor TRA-1, which is required in 

hermaphrodites to prevent masculinization, and these mutations appear to cause the 

inappropriate expression of ceh-30 in hermaphrodites and the consequent survival of the CEMs 

(see abstract by Schwartz and Horvitz). 

We performed several clonal screens, totaling nearly 7,000 mutagenized haploid genomes, for 

suppressors of ceh-30(n3714gf). From these screens, we recovered at least 92 independent 

mutations causing the absence of GFP-positive CEMs in pkd-2::gfp; ceh-30(n3714gf) 

hermaphrodites. These suppressors include at least one loss-of-function mutation in ceh-30, 

n4111, and at least one mutation, n4132, that prevents expression of the pkd-2::gfp reporter (see 

2003 International Worm Meeting abstract 461B by Wang and Barr). We are currently testing 

whether the disappearance of GFP-positive CEMs caused by our suppressors is affected by loss 

of function of ced-3 or ced-4 (ced, cell death abnormality), genes required for all programmed cell 

deaths, and observing the effects of our suppressor mutations upon male CEM development. We 

hope to identify mutations that cause the CEMs to die by programmed cell death despite the 

protection of the ceh-30(n3714gf) mutation and mutations in genes required specifically for the 

regulation of ceh-30 or that function downstream of or in parallel to ceh-30 to regulate the 

male-specific survival of the CEMs. We also expect to identify mutations in genes required to 

properly determine the fate of the CEM neurons.

249. Screens for suppressors of cul-2 and zyg-11 

Srividya Vasudevan, Edward T. Kipreos 

Dept. of Cellular Biology,University of Georgia, Athens, GA 30602 

Screens for suppressors of cul-2 and zyg-11 

P> P> 

PERSONNAME>GIVENNAME>SrividyaGIVENNAME> 

SN>PERSONNAME>VasudevanPERSONNAME>SN>PERSONNAME>,PERSONNAME>GIVENNAME>EdwardGIVENNAME> 

SN>KipreosSN>PERSONNAME> 

Dept. of Cellular Biology 

PLACE>PLACETYPE>UniversityPLACETYPE> of 

PLACENAME>GeorgiaPLACENAME>PLACE>,PLACE>CITY>AthensCITY>, 

STATE>GASTATE> POSTALCODE>30602POSTALCODE>PLACE> 

P> P> 

CUL-2 is a member of the cullin E3 ubiquitin-ligase family. In humans, CUL-2 is present in a 

protein complex with PERSONNAME>GIVENNAME>ElonginGIVENNAME> 

SN>BSN>PERSONNAME>, PERSONNAME>GIVENNAME>ElonginGIVENNAME> 

SN>CSN>PERSONNAME>, RBX1, and the tumor suppressor protein VHL, which targets the 

hypoxia inducible factor, HIF-1α for degradation (Lonergan et al. Mol. Cell. Biol., 1998). In 

C.elegans loss of the cul-2 gene causes a G1 phase germ cell arrest associated with an 

accumulation of the CDK inhibitor CKI-1. Inactivation of CUL-2 also causes a host of embryonic 

phenotypes such as failure to extrude the second polar body, cytoplasmic extensions, DNA 

bridges, multinuclei, and arrest at the 24-cell stage (Feng et al. Nat. Cell Biol., 1999). 

P> P> 

In fertilized cul-2 mutant zygotes, meiotic anaphase I occurs normally, but anaphase II is 

abolished or severely delayed. The meiotic delay is associated with the stabilization of cyclin B1, 

which is normally degraded during meiosis I and II in wild type. The extended meiosis II is also 

correlated with reversals in several aspects of AP polarity 

(PERSONNAME>GIVENNAME>JiGIVENNAME> SN>LiuSN>PERSONNAME>, 

GIVENNAME>S.V.GIVENNAME>, and E.T.K., Development, in press). 

P> P> 

Interestingly, inactivation of the leucine-rich repeat protein ZYG-11 produces a broad overlap 

with the cul-2 mutant phenotypes (Kemphues et al. Dev Biol, 1986). zyg-11 mutants do not have 

a germ cell arrest, but they do share meiotic and embryonic defects with cul-2 mutants, including 

stabilization of cyclin B1 and polarity reversals. Combining null alleles of cul-2 and zyg-11 does 

not enhance the meiotic delay observed for each mutant individually suggesting that the two 

genes function in the same pathway (PERSONNAME>GIVENNAME>JiGIVENNAME> 

SN>LiuSN>PERSONNAME>, GIVENNAME>S.V.GIVENNAME>, and E.T.K., Development, in 

press). We are interested in elucidating the molecular pathways by which CUL-2 and ZYG-11 

regulate meiosis and embryonic development. We are in the process of screening for CUL-2 and 

ZYG-11 genetic interactors using suppressor screens for cul-2 and zyg-11 mutants. The cul-2 

suppressor screen employs a cul-2(ek4) allele linked to unc-64 to clonally screen F2 progeny for 

embryos that have progressed beyond the 24-cell stage. The zyg-11 suppressor screen employs 

a ts allele of zyg-11 to obtain viable suppressors. So far we have screened 20,000 F2 genomes 

and obtained a single suppressor of zyg-11. We anticipate that these screens will provide 

information crucial to the understanding of how CUL-2 ubiquitin ligase activity regulates meiosis 

and embryonic development in C. elegans.

250. Half-molecule ATP-binding cassette transporter, CeHMT1, is required for 

PC-dependent heavy metal detoxification in Caenorhabditis elegans 

Olena K. Vatamaniuk 1 , Elizabeth A. Bucher 2 , Meera V. Sundaram 3 , Philip A. Rea 4 

1 Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA 

19104, USA; Department of Genetics, School of Medicine, University of Pennsylvania, 

Philadelphia, PA 19104, USA 

2 Department of Cell and Developmental Biology, School of Medicine, University of Pennsylvania, 

Philadelphia, PA 19104, USA 

3 Department of Genetics, School of Medicine, University of Pennsylvania, Philadelphia, PA 

19104, USA 

4 Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA 

19104, USA 

Phytochelatins (PCs), a family of small thiol-rich peptides play a central role in heavy metal, 

primarily Cd 2+ , detoxification in plants, some fungi such as Schizosaccharomyces pombe, and 

some invertebrates as exemplified by C. elegans. PCs are derived from the tripeptide glutathione 

by the action of a constitutively expressed dipeptidyl transpeptidase, phytochelatin synthase 

(PCS). PCs thiol-coordinate heavy metals and promote their sequestration into the 

vacuolysosomal compartment however, the transport processes associated with PC-dependent 

Cd 2+ detoxification remain to be defined precisely. Perhaps the best understood system is that of 

S. pombe. In this organism, a half-molecule vacuolar ATP-binding cassette (ABC) transporter, 

SpHMT1, participates in the detoxification of Cd 2+ by catalyzing the MgATP-energized transport 

of apo-PC and CdPC complexes into the vacuole (Ortiz et al 1992, 1995). 

Our recent discovery of the PC-dependent pathway for Cd 2+ detoxification in C. elegans has 

provided us with a unique opportunity to examine the role of HMT1-like proteins in organisms 

distinct from S. pombe. We identified one out of a total of 31 ORFs encoding half-molecule ABC 

transporters with an equivalent topology and bearing greater than 51% sequence similarity to 

SpHMT1. Designated ce-hmt-1, the 2.4 kb cDNA encodes a 90.7 kDa polypeptide which satisfies 

the requirements of a heavy metal tolerance factor involved in the PC-dependent heavy metal 

tolerance. When heterologously expressed in S. pombe, CeHMT1 localizes to the vacuolar 

membrane and alleviates the Cd 2+ -hypersensitivity of hmt 1- mutants. Crucially, CeHMT1 is 

required for Cd 2+ tolerance in the intact organism. The progeny of the wild type control worms 

develop into normal-sized, gravid adults even at high concentrations of Cd 2+ (50 and 100 µM) in 

the growth media, whereas the progeny of worms injected with dsce-hmt-1 RNA are acutely 

sensitive to Cd 2+ . They are developmentally arrested in the early L1-L2 stages and eventually 

die, never reaching adulthood even at the lowest Cd 2+ concentrations (5, 10 µM). Furthermore, 

the Cd 2+ -hypersensitivity of ce-hmt-1 RNAi worms is considerably greater than that of 

PCS-deficient worms (Vatamaniuk et al 2001). In addition, cellular morphological phenotypes of 

these two classes of RNAi mutant are readily distinguishable. Whereas the intestinal epithelial 

cells of ce-pcs-1 RNAi worms become necrotic upon exposure to Cd 2+ , the corresponding cells 

of ce-hmt-1 RNAi worms do not necrose per se but instead accumulate punctuate, apoptotic 

inclusions. 

These results and those from our previous investigations of the requirement for PCS for heavy 

metal tolerance in C. elegans demonstrate that PCS-dependent, HMT-1-mediated heavy metal 

detoxification pathways exist not only in S. pombe but also in some invertebrates, a possibility 

that had not even been speculated previously. Future studies of the tissue-specificity of ce-hmt-1 

RNA expression and the subcellular localization of its translation product will provide insights into 

which cell types, tissues, and subcellular compartments are responsible for the sequestration 

and/or elimination of Cd 2+ and other heavy metals not only in C. elegans but also in other 

animals that might deploy an equivalent mechanism for metal detoxification. 

This work was funded by NSF Grant No. MCB-0077838 awarded to P.A.R.

251. How are apoptotic cells recognized by their phagocytes? 

Victor Venegas, Zheng Zhou 

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of 

Medicine, One Baylor Plaza, Houston, TX 77030 

The nematode C. elegans hermaphrodite develops 1090 somatic cells of which 131 undergo 

programmed cell death, or apoptosis. Additionally more than 300 germ cells undergo 

programmed cell death during germ line development. All the dying cells are eliminated via 

phagocytosis by neighboring cells. Genetic screens have identified at least seven genes (ced-1, 

6, 7 and ced-2, 5, 10, 12) within two partially redundant pathways required for efficient engulfment 

of apoptotic cells. The focus of this study is the mechanism that leads engulfing cells to recognize 

apoptotic cells. 

CED-1 has been characterized molecularly and found to act as a phagocytic receptor that is 

required for the recognition and engulfment of cell corpses. The recognition of cell corpses by 

CED-1 is dependent on CED-7, a C. elegans homolog of mammalian ABC transporters. CED-7 is 

required on both engulfing and apoptotic cell. CED-6 is a candidate cytoplasmic adaptor 

proposed to play a role in downstream signaling pathway leading to the activation of the 

engulfment process. It is hypothesized that CED-7 promotes the presentation of a signal on the 

surface of cell corpses which is recognized by CED-1. The recognition of such a signal leads 

CED-1 to cluster around cell corpses. To further study apoptotic cell recognition we are 

conducting an EMS mutagenesis screen using a reporter construct P ced-1ced-1deltaC::GFP to 

distinguish the mutants of interest. CED-1deltaC::GFP clusters around cell corpses, but lacks 

components needed for CED-1 to promote cell-corpse engulfment. The screen aims at isolating 

mutants in which CED-1deltaC::GFP is not able to cluster around cell corpses. This screen will 

allow the identification of genes important for the generation, presentation, and recognition of the 

cell corpse signal or genes required for the generation of cell corpses. To date this screen has 

identified 5 possible mutants, two of which show no GFP circles and no cell corpses under 

Nomarski optics indicating that these two mutations block programmed cell death. We plan to 

continue this screen and to characterize the isolated mutants regarding their cell death 

abnormalities.

252. Modifiers of polyglutamine-mediated neurodegeneration 

Cindy Voisine, Adriana K. Jones, Anne C. Hart 

MGH Cancer Center, Charlestown, MA 02129 

At least eight hereditary neurodegenerative disorders, including Huntington’s disease, are 

caused by an expanded glutamine tract. We previously established a model system for studying 

polyglutamine (polyQ) neurotoxicity in C. elegans (PNAS 96, 179-184, 1999). The N-terminus of 

the mutant human huntingtin protein, which contains an expanded glutamine tract, is expressed 

in the well-characterized ASH neurons. Expression of the expanded huntingtin polyglutamine 

tract leads to age-dependent degeneration of the ASH neurons. To identify genes that modulate 

polyQ toxicity, we are performing an RNA interference (RNAi) screen in the sensitized rrf-3 

(pk1426) strain using a library provided by the Marc Vidal laboratory. We first validated our 

system by using a previously identified genetic enhancer of polyQ toxicity, pqe-1 (PNAS 99, 

17131-6, 2002). The vast majority of ASH neurons expressing expanded huntingtin fragments in 

pqe-1(rt13) are dead in young animals. In young rrf-3 mutant animals expressing huntingtin 

fragments, feeding dsRNA of pqe-1 causes 50% of ASH neurons to degenerate compared to 0% 

in the feeding control. In aged rrf-3 mutant animals expressing expanded huntingtin fragments, 

80% of ASH neurons are affected compared to 50% of ASH neurons in the control experiment. 

We are focusing on RNAi of those genes whose decrease in function results in sterility or reduced 

viability, since these candidates may have been missed in the original genetic screen for polyQ 

enhancers (PNAS 99, 17131-6, 2002). Candidate genes that enhance neurodegeneration to a 

similar level as feeding dsRNA of pqe-1 (80% ASH neurodegeneration) are being retested. 

Several interesting candidate genes have emerged. Reducing the expression of the molecular 

chaperone HSP40 (F39B2.10) enhances ASH neurodegeneration. Protein misfolding has been 

previously implicated in polyQ neurotoxicity in several systems, validating our experimental 

approach. RNAi of the B-chain of a sodium potassium transporting ATPase (C17E4.9) also 

enhances polyQ neurotoxicity. In a complementary approach, we designed an assay system to 

test the effect of pharmacological agents on polyQ neurotoxicity in C. elegans (collaboration with 

Hemant Varma, Stockwell lab, Whitehead Institute). Since our RNAi screen revealed C17E4.9 as 

an enhancer of polyQ toxicity, we are initially focusing the pharmacological tests on drugs that 

specifically inhibit the activity of the sodium potassium transporting ATPase. Identification and 

characterization of genes isolated from the RNAi screen will provide insight into pathogenic 

mechanisms underlying polyQ-induced neurodegeneration.

253. Disruption of germline pattern by forward and reverse genetics 

Roumen V. Voutev 1 , E. Jane Albert Hubbard 1,2 

1New York University, Biology Department, 100 Washington Square East, New York, NY 10003 

2e-mail: jane.hubbard@nyu.edu 

The germ line of C. elegans undergoes three spatially and temporally distinct phases before full 

maturation: 1) in the proliferative phase all germ cells divide mitotically; 2) in the second phase 

the proximally situated cells enter meiosis; 3) in the gametogenic phase a distal-to-proximal 

polarity is established with mitotic cells located distally and gametes located proximally in the 

mature gonad. Genetic disruptions in germline polarity can be used to learn more about the 

molecular basis of the establishment and maintenance of this polarity. 

The Pro phenotype (proximal proliferation) is characterized by ectopic mitotic proliferation in 

the proximal region of the gonad where gametogenesis normally occurs, resulting in sterility. 

Using RNAi against 91 candidate genes, we identified 28 genes that induce a Pro phenotype, 

albeit some at a low penetrance and accompanied by pleiotropic defects. In addition, two 

mutations that give a Pro phenotype, na27 and ar226, have been previously isolated in a forward 

genetic screen in our lab. They map to LG II and X, respectively. na27 complements pro-1(na48) 

and by three-factor mapping, lies in the region between unc-104 and unc-4. ar226 is on the right 

arm of the X chromosome. Both na27 and ar226 are temperature-sensitive alleles that show Pro 

phenotype only at 25ºC with 50% and 30% penetrance, respectively.

254. Regulation of Cell Death in the C. elegans Tail Spike Cell 

Carine Waase, Shai Shaham 

Rockefeller University, 1230 York Ave. Box 46, New York, N.Y. 10021 

Programmed cell death is a critical developmental and homeostatic process. While the cell 

death execution machinery has been well characterized in C. elegans, the upstream factors that 

activate this machinery are, for the most part, unknown. We are interested in exploring how a cell 

destined to die activates its cell death machinery at the appropriate time in development, and are 

using the C. elegans tail spike cell to probe this question. 

113 cells in C. elegans die embryonically. The vast majority of these cells die as 

undifferentiated cells within minutes of being born. In contrast, the tail spike cell persists for over 

five hours after birth before dying, and during this time exhibits an elaborate morphological 

program. We hypothesize that upstream cellular events may play a critical role in tail spike cell 

death, and wish to better understand how this cell dies. 

Using a ced-3p::gfp marker we have developed that is expressed in the tail spike cell, we have 

determined that tail spike cell death is strongly dependent on functional ced-3 andced-4, partially 

dependent on functional egl-1, and only weakly affected by a ced-9(n1950) mutation. We have 

performed time-lapse studies on N2 embryos expressing our marker, and intriguingly, observe 

that gfp expression begins minutes before the tail spike cell exhibits the corpse morphology 

stereotypical of a dying cell. We therefore speculate that transcriptional upregulation of ced-3 

may be critical for initiation of tail spike cell death. Regulation of caspases has been 

characterized at the post-translational level, but their transcriptional regulation has remained 

generally unaddressed. Studies assessing the functional relevance of ced-3upregulation in the 

tail spike cell are presently underway. The minimal ced-3 promoter driving expression of our 

marker is highly conserved between C. elegans and C. briggsae. In an attempt to define the 

factors involved in ced-3 transcriptional upregulation, we have performed promoter deletion 

studies on this minimal conserved region. We have identified three15 bp regions necessary for 

reporter expression, and are presently testing whether these sites are required for 

ced-3upregulation. We have taken a bioinformatics approach to identify putative transcription 

factors, and are currently testing these candidates. We also plan to screen for factors required for 

ced-3 transcriptional upregulation. We hope to identify the transcription factors and upstream 

signaling factors implicated in this novel cell death paradigm.

255. A suppressor screen for genes involved in UNC-6 mediated guidance 

Gauri Kulkarni, Chaunte Cannon, William G. Wadsworth 

Department of Pathology, Robert Wood Johnson Medical School, Piscataway, NJ 08854 

UNC-6 is an extracellular matrix guidance cue that directs circumferential cell and axon 

migrations. We have developed a suppressor screen to identify new players in unc-6 mediated 

guidance. Using the unc-6 (rh46) allele, which introduces an alanine to proline change in domain 

VI, we have identified a few potential suppressors. The ur280 suppressor partially reverts the 

paralyzed phenotype of unc-6(rh46) animals and partially restores circumferential axon guidance. 

While ur280 suppresses unc-6(rh46) phenotypes, it will not suppress unc-6(ev400), a null allele. 

ur280 has a slight dumpy-like phenotype that is enhanced by unc-6(rh46), further suggesting 

interactions. The mutation has been mapped to a small region of chromosome V where genes 

involved in axon guidance have not been described.

256. Regulation of RNA Polymerase II in C. elegans embryos and germline 

Amy K. Walker, T. Keith Blackwell 

Joslin Diabetes Center, One Joslin Place, Boston, MA 02115 

In C. elegans embryos, mRNA transcription initiates at the 3-4 cell stage in the somatic cells. In 

the germline precursor, the transcriptional repressor PIE-1 prevents gene expression, possibly by 

interfering with phosphorylation of the C-terminal domain (CTD) of RNA Polymerase II. The Pol II 

CTD is composed of a heptapeptide repeat (YSPTSPS), which is differentially phosphorylated 

during the transcription cycle; unphosphorylated Pol II is recruited to a promoter and as the 

transcription pre-initiation complex forms, CDK-7/CyclinH phosphorylates the CTD serine 5. 

During the transition to mRNA elongation, CTD phosphorylation shifts to serine 2 due to the 

activity of CDK-9/CyclinT. While the CTD may be modified by a variety of factors, the 

mechanisms regulating the transcription cycle have not been well described in vivo. 

During early embryogenesis the somatic and germline precursors have distinct patterns of 

Pser5 and Pser2 levels and localization. Pser2 is detected in somatic cells after zygotic 

transcription begins, but is absent in the transcriptionally silent germline precursor. Pser5 levels 

parallel Pser2 in the somatic nuclei. The germline precursors have little or no nucleoplasmic 

Pser5, however they do contain two distinct foci, similar to those in the transcriptionally silent 

germline cells of Drosophila embryos (Seydoux and Dunn (1997) Devt 124:2191). We have 

examined regulation of Pol II during early embryogenesis and studied the Pser5 foci in more 

detail. Appearance of these foci depends on parts of the transcription pre-initiation complex, such 

as TFIIB and the mediator complex component RGR-1. However, the PSer5 foci appear in all 

somatic cells when transcription is inhibited through interference with TFIID components, which 

function at the last step in transcription initiation. Thus, these structures may represent stalled 

transcriptional loci or storage/recycling areas that accumulate factors such as Pol II from aborted 

transcription events. 

To better understand the Pser5 foci, we have examined other nuclear structures. Consistent 

with a role in stalled or aborted transcription, the Pser5 foci did not co-localize with antibodies to 

nuclear speckles, nor did they co-localize with antibodies specific to nucleoli. Cajal bodies have 

been described as sites for recycling components of the transcription machinery, however these 

structures are not well described in C. elegans. To approach this, we have used RNAi to inhibit 

expression of smn-1, which is found in Cajal bodies or related structures known as Gems. While 

overall transcription levels are diminished in the smn-1(RNAi) embryos, the Pser5 foci are not 

affected. Thus, the Pser5 foci may represent a discrete functional compartment of a structure 

such as a Cajal body, or a distinct type of nuclear structure. 

We have also examined the regulation of CTD phosphorylation in embryonic and germline 

nuclei. Mitotic and pachytene regions of the germline have high levels of Pser5 and Pser2, 

reflecting the robust transcription in these nuclei. However, the most proximal oocytes, which 

have arrested in diakinesis prior, have no detectable Pser5 or Pser2. Suprisingly, we have found 

that disruption of the ubiquitination pathway through interference with ubiquitin activating enzyme, 

uba-1, or a ubiquitin conjugating enzyme, ubc-2(let-70), causes Pser5, but not Pser2, to appear in 

the proximal oocytes and in 1-2 cell embryos. This increase in Pser5 in uba-1(RNAi) oocytes and 

embryos may represent not inappropriate transcription because Pser2 levels are not increased 

and because the early embryonic reporter PES-10::GFP appears at the appropriate time. We 

have also investigated to role of several ubiquitin conjugating factors that modify Pol II in yeast. 

Interference with expression of these genes in C. elegans did not affect Pser5 levels, suggesting 

that the ubiquitination pathway may not be directly modifying Pol II. In addition, levels of 

unphosphorylated Pol II are normal in the uba-1 or ubc-2/let-70(RNAi) oocytes and embryos. 

These results suggest that ubiquitination is important for regulating early steps of the transcription 

cycle and that loss of ubiquitination causes persistent or inappropriate serine 5 phosphorylation.

257. Identification and characterization of the downstream target genes of CeTwist and its 

partner CeE/DA 

Peng Wang, Ann Corsi 

Department of Biology, Catholic University of America, Washington, DC. 20064 

CeTwist, encoded by the hlh-8 gene, is a member of the basic helix-loop-helix (bHLH) family of 

transcription factors and prefers to function as a heterodimer with CeE/DA, another bHLH protein 

encoded by the hlh-2 gene (1). Identification of new downstream target genes of CeTwist will 

shed more light on the function of CeTwist in C. elegans mesoderm development. In addition, 

several known CeTwist target genes have human homologues. Mutations in these homologues 

are believed to cause several syndromes of human craniosynostotic disease. We predict the new 

CeTwist target genes which are found in our work might identify genetic defects of other related 

syndromes. To find new target genes, we built the strains: experimental group containing pRF4 

pHS::hlh-8 pHS::hlh-2 and control group containing pRF4 only. All of the constructs were 

integrated onto chromosomes through gamma irradiation. The strategy is to overexpress both 

CeTwist and CeE/DA by heat shock, and then detect gene expression changes compared to 

controls using Affymetrix oligonucleotide microarrays of C. elegans genome. We used RT-PCR to 

investigate the kinetics of the known target gene arg-1 under heat shock treatment, and found 

that a 20 min heat shock treatment at 33 Celcius degree followed by a 40 min recovery at 20 

Celcius degree is the optimal experimental condition for observing the known target mRNA 

upregulation but not the appearance of protein. The expectation is that unknown targets will be 

expressed under these conditions as well. Microarrays were done with isolated mRNA from the 

experimental and control groups that were either subjected to heat shock or kept at 20 Celcius 

degree in three independent experiments. By analyzing the microarrays data with Microarray 

Suite 5.0 (Affymetrix) and Genespring, we identified genes that overlapped between the different 

methods, and further confirmed the gene expression changes with SYBR Green real-time PCR. 

Now we have several candidate target genes and are using gfp construct to study their 

expression pattern and RNAi to investigate their functions. 

1. Harfe, B. D., Gomes, A. V., Kenyon, C., Liu, J., Krause, M. and Fire, A. (1998). Analysis of a 

Caenorhabditis elegans Twist homolog identifies conserved and divergent aspects of 

mesodermal patterning. Genes Dev 12, 2623-2635.

258. Identification of regulatory sequences necessary for egl-1 function in the ventral 

nerve cord 

Erin Webster, Tamara Strauss, Kelly Liu, Scott Cameron 

Departments of Pediatrics and Molecular Biology, The University of Texas Southwestern Medical 

Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390 

egl1 is required for programmed cell death of somatic cells in C. elegans, but little is known 

about what controls the function of this gene. Only two direct regulators have been identified. 

The first is tra1, a sex specific regulator of egl1 in the HSNs. A human homolog of tra1is Gli, 

which is amplified in glioblastoma. The second is ces1,which directly regulates egl1in the NSM 

sister cells of the pharynx. ces1 is in turn regulated by ces2. ces1 andces2 are homologous to 

the human genes slugand Hepatic Leukemia Factor (hlf)respectively, both of which have been 

implicated in leukemogenesis. The homology of these regulatory genes to those implicated in 

cancers indicates that an understanding of the control mechanisms of egl1 could contribute to our 

knowledge of the development of cancer. 

We are applying two strategies to identify regions of egl1 that are important for its function 

during the development of the ventral nerve cord. The first approach is to delete various portions 

of the egl1 regulatory sequences and assay the ability of these constructs to rescue egl1 function 

in the ventral nerve cord. Using this assay we can identify regions necessary for the function of 

egl1. 

In a complementary approach, genomic sequences of egl1 from C. elegansand C. briggsae 

were compared. This alignment revealed several regions of homology in the egl1promoter. We 

are placing these regions into minimal promoter vectors containing gfp, injecting them into worms, 

and assaying gfp expression in the ventral nerve cord. In this way we hope to identify elements 

sufficient to drive egl1 expression in the ventral nerve cord.

259. Identification and Characterization of MAPK Signaling Targets in the C. elegans 

Germline 

Stefanie West, Valerie Reinke 

Yale University, Department of Genetics, New Haven, CT 06520 

The Ras/MAPK pathway is required for pachytene exit during meiosis in the germline of C. 

elegans. We have used a temperature-sensitive MAPK mutant allele, mpk-1(ga111), which 

produces a phenotype only in the germline, resulting in pachytene arrest of germ cells at 25 o C. 

Shifting mpk-1(ga111) adults back to the permissive temperature restores MAPK signaling, as 

evidenced by resumption of meiotic progression and production of functional oocytes by 15 hours 

at 15 o C. 

By comparing gene expression of mpk-1(ga111) and control animals through a timecourse 

microarray analysis, we can examine the global genome response to loss and restoration of 

MAPK signaling in the germline. We have compared mRNA extracts from control and mutant 

adults raised at 25 o C and then at 3,6,9,12, and 15 hours after shifting the mpk-1(ga111) mutants 

to 15 o C. We also collected mRNA from control animals after 15 hours at 15 o C. We required that 

candidate genes must be enriched in the control as compared to the mpk-1 mutant (1.5-fold, 

p

260. The nuclear receptor gene fax-1 and homeobox gene unc-42 coordinate interneuron 

identity by regulating the expression of glutamate receptor subunits and other 


Bruce Wightman, Sheila Clever 

Biology Department, Muhlenberg College, Allentown, PA 18104 

The fax-1 gene encodes a conserved nuclear receptor that is the ortholog of the human PNR 

gene, and functions in the specification of neuron identities. Mutations in fax-1 result in 

uncoordinated locomotion. FAX-1 protein accumulates in the nuclei of eighteen neurons and 

transiently in two non-neuronal cell types. The neurons that express FAX-1 include the AVA, 

AVB, and AVE interneuron pairs that coordinate body movements. The identities of AVA and AVE 

interneurons are defective in fax-1 mutants; neither neuron expresses the NMDA receptor 

subunits nmr-1 and nmr-2. Other ionotropic glutamate receptors, such as glr-1, glr-2 and glr-4 are 

expressed normally in the AVA and AVE neurons. The unc-42 homeobox gene also regulates 

AVA and AVE identity, however unc-42 mutants display the complementary phenotype; NMDA 

receptor subunit expression is normal, but some non-NMDA glutamate receptors are not 

expressed. These observations support a combinatorial role for fax-1 and unc-42 in specifying 

AVA and AVE identity. However, in four types of neurons, fax-1 is regulated by unc-42, and both 

transcriptional regulators function in the regulation of the opt-3 gene in the AVE neurons and the 

flp-1 and ncs-1 genes in the AVK neurons. Therefore, while fax-1 and unc-42 act in 

complementary parallel pathways in some cells, they also function in overlapping or linear 

pathways in other cellular contexts, suggesting that combinatorial relationships among 

transcriptional regulators are complex and cannot be generalized from one neuron type to 

another. 

P> P> 

In addition to the AVA, AVB, and AVE "command" interneurons, FAX-1 protein also 

accumulates in the M4 pharyngeal motorneuron, two pairs of neurons in the head that we 

tentatively identify as the SIBD and SIBV pairs, the AVK and RIC interneuron pairs, and the DVA 

neuron. We also observe strong, but transient expression of FAX-1 in the distal tip cells from L2 

through L4 and in two bilateral pairs of vulval cells (VulE or VulF) during L4. While this expression 

correlates with the time during which these non-neuronal cell types are migrating or undergoing 

movements associate with morphogenesis, we have not observed gonadal or vulval defects in 

fax-1 mutants. Thus fax-1 function in these cell types, if any, appears to be redundant with 

another factor, possibly another nuclear receptor (which could have overlapping DNA-binding 

activity). 

P> P> 

fax-1 is required for normal axon pathfinding by the AVK interneurons (hence the name 

fasciculation of axons defective), as is unc-42. The unc-42 homeobox gene is also required for 

normal pathfinding by some or all of the "command" interneurons. The expression of glr::gfp 

transgenes in the AVA, AVB, and AVE interneurons of fax-1 mutants allowed us to evaluate the 

requirement for fax-1 in axon pathfinding by these neuron types. In contrast to our results for 

AVK, axon anatomy appeared grossly normal for the AVA, AVB, and AVE interneurons of fax-1 

mutants. Therefore, while unc-42 functions in regulating axon pathfinding by AVA, AVB, and/or 

AVE interneurons, fax-1 does not. This observation underscores the overlapping contributions to 

transcriptional regulation of neuron identity by fax-1 and unc-42.

261. Characterization of loci that control or depend upon N-glycosylation in C. elegans. 

William C. Wiswall Jr 1 , Kristin M.D. Shaw 1 , Weston B. Struwe 1 , Charles E. Warren 1,2 

1Department of Biochemistry & Molecular Biology 

2Genetics Program, University of New Hampshire 

N-glycosylation is an essential process in which complex carbohydrate structures, termed 

N-glycans, are covalently attached to specific proteins. Glycosylation occurs in three phases; 

synthesis of the lipid linked oligosaccharide (LLO) in the endoplasmic reticulum, the transfer of 

the oligosaccharide to the nascent polypeptide, followed by extensive processing in the Golgi 

apparatus. Defects in the biosynthetic pathway potentially alter all glycoprotein structures and 

may cause multisystemic syndromes, for example, the group of hereditary diseases known as 

congenital disorders of glycosylation (CDG). 

The clinical manifestation of CDG is pleiotropic, confounding the identification of specific 

defective glycoprotein interactions. Utilizing a chemical genetic approach in C. elegans, we are 

modeling CDG type Ij. In practice, we use a tunicamycin dose (an antibiotic that inhibits 

biosynthesis of the LLO at the initial step) that induces a sub-clinical case of CDG type Ij in wild 

type worms and seek mutants that are hypersensitive; dying at doses where non-mutants are 

essentially unaffected. 

By screening for tunicamycin hypersensitive (Tmh) animals, we would expect to isolate alleles 

of four classes: mutations affecting tunicamycin pharmacokinetics, non-epistatic loci in the 

glycosylation pathway, intracellular defects that interact with glycosylation (e.g. UPR), and loci in 

a glycoprotein dependent pathway. 

To test this hypothesis we examined tunicamycin sensitivity through RNA interference on 

known genes in LLO biosynthesis, endoplasmic reticulum quality control and other aspects of 

glycoprotein maturation. As predicted, all the loci tested confirmed Tmh. We therefore are 

conducting a genome-wide RNAi screen for tunicamycin hypersensitive genes. 

We are also pursuing a complementary forward screen. We observed that N2 larval arrested 

animals on high doses of tunicamycin can be rescued by picking to drug free plates. Therefore, 

200,000 EMS mutagenized haploid genomes were screened for Tmh by growing synchronized L1 

animals on NGM plates containing 2µg/ml of the antibiotic. From this, four strong Lva candidates 

were selected for further characterization. One of these, tmh(nh1) maps to LG IV where no Tmh 

loci are currently known to reside.

262. sid-5 is required for robust environmental RNAi 

Amanda J. Wright, Craig P. Hunter 

Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, 

Cambridge, MA 02138 

In a screen for C. elegans systemic RNAi deficient mutants (Winston et al., 2001), two alleles of 

sid-5 were identified. The mutants were isolated as defective in silencing GFP expressed in body 

wall muscles when fed bacteria expressing GFP dsRNA. sid-5 worms injected into either the 

gonad or intestine with mex-3 or unc-22 dsRNA produce affected progeny, indicating that sid-5 is 

not strictly required for general or systemic RNAi. Therefore, we explored the requirement for 

sid-5 in environmental RNAi, where the dsRNA is introduced via feeding or soaking. sid-5 worms 

fed bacteria expressing dsRNA that corresponds to endogenous targets (unc-22, unc-54, or 

pal-1) are less affected than wild-type controls but the longer the worms are exposed to the food, 

the stronger the silencing becomes. In contrast, sid-5worms soaked in dsRNA (unc-22 or pal-1) 

display a strong RNAi effect which, relative to wild-type controls, diminishes over time. These 

results indicate that sid-5 is needed for robust environmental RNAi. 

The sid-5 gene was mapped to a small interval on linkage group X and rescued by 

injection of a genomic fragment that encompassed two genes, one of which was mutated in both 

sid-5 alleles. sid-5 is predicted to encode a 67 amino acid protein with a single transmembrane 

domain and no signal sequence. Preliminary analysis of a full-length SID-5::GFP fusion indicates 

that SID-5 is expressed in the excretory cell, spermatheca, and rectum. Current experiments are 

focused on refining the RNAi phenotype and SID-5 expression pattern more fully. 

Winston, W. M., C. M., Molodowitch, C. P. Hunter. 2002. Systemic RNAi in C. elegans 

requires the putative transmembrane domain protein SID-1. Science. 295:2456-59.

263. LIN-10 inhibits the synaptic delivery of GLR-1 

Tricia Wright, Henry Schaefer, Keri Martinowich, Howard Chang, Douglas Beach, Christopher 

Rongo 


A fundamental question in neurobiology is how synaptic connections between neurons in the 

central nervous system are formed and regulated. In particular, we are interested in how 

glutamate receptors are localized to synapses. Glutamate receptors in C. elegans are conserved 

in the nematode, where they transduce signals between mechanosensory neurons and their 

interneuron targets(1, 2). Previous studies have shown that LIN-10, a PDZ-domain protein, is 

required for the proper synaptic localization of the GLR-1 glutamate receptor in C. elegans(3, 4). 

LIN-10 does not directly bind to GLR-1, so presumably there are additional proteins involved in 

the localization process that have yet to be identified. LIN-10 is also required for LET-23 

basolateral localization in epithelial cells(4). 

Mutants that lack LIN-10 accumulate high levels of GLR-1 throughout their neurites. We have 

examined whether the lin-10 phenotype is due to impaired turnover of GLR-1 in lin-10 mutants. 

Ubiquitination and subsequent endocytosis regulate the synaptic abundance of GLR-1(5). We 

find that GLR-1 is sensitive to ubiquitin-mediated turnover in lin-10 mutants, unlike what has been 

found for endocytosis mutants like unc-11. To determine whether the lin-10 phenotype is due to 

facilitated delivery of GLR-1 in lin-10 mutants, we have used FRAP to monitor the recovery of 

photobleached GLR-1::GFP at synapses. We find that GLR-1 recovers from FRAP more rapidly 

in lin-10 mutants compared to wild-type worms, suggesting that the normal role of LIN-10 is to 

inhibit GLR-1 delivery. To have a better understanding of the role of LIN-10 in GLR-1 localization, 

we are also performing a structure/function analysis of the different domains of the protein. We 

have generated amino acid substitutions in the carboxy-terminal region of LIN-10 that impair the 

function of LIN-10 for GLR-1 localization in neurons, but do not impair the vulval induction 

function of LIN-10 in epithelia. We have also identified a domain in the amino-terminus of LIN-10 

that is responsible for its colocalization with GLR-1. Our future goal is to identify the relevant 

proteins that interact with LIN-10 to facilitate GLR-1 localization. 

1. A. C. Hart, S. Sims, J. M. Kaplan, Nature, 82-84 (1995). 

2. P. J. Brockie, D. M. Madsen, Y. Zheng, J. Mellem, A. V. Maricq, J Neurosci 21, 1510-22. 

(2001). 

3. C. Rongo, C. W. Whitfield, A. Rodal, S. K. Kim, J. M. Kaplan, Cell 94, 751-9 (1998). 

4. C. W. Whitfield, C. Benard, T. Barnes, S. Hekimi, S. K. Kim, Mol Biol Cell 10, 2087-100 

(1999). 

5. M. Burbea, L. Dreier, J. S. Dittman, M. E. Grunwald, J. M. Kaplan, Neuron 35, 107-20 (Jul 3, 

2002).

264. MSP signals microtubule reorganization in C. elegans oocytes prior to fertilization 

Jana E. Harris, Ikuko Yamamoto, David Greenstein 

Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN USA 

The microtubule cytoskeleton of most animal oocytes differs from that of somatic cells in that 

the centrioles are lost during oogenesis. In most cases, the meiotic spindle develops without 

centrosomes serving as microtubule-organizing centers. In addition, microtubules play critical 

roles in controlling cell shape, protein trafficking, RNA localization, and cell polarity. Despite these 

essential functions, it is not well understood how extracellular signals regulate microtubule 

organization and function. It is essential to uncover the signaling mechanisms regulating 

microtubules in oocytes because of the striking age-related increase in non-disjunction during 

meiosis I in human females leading to congenital birth defects or miscarriage. In order to address 

this issue, we have been analyzing the behavior of microtubules during oocyte meiotic maturation 

in C.elegans. 

In C. elegans, oocyte meiotic maturation is triggered by the major sperm proteins (MSPs) 

released from sperm. MSPs elicit several cellular responses in oocytes including MAPK 

activation, M-phase entry, nuclear envelope breakdown, and meiotic spindle assembly. MSP 

signaling transpires through two parallel pathways defined by VAB-1, an MSP/Eph receptor, and 

CEH-18, a POU-homeoprotein required for gonadal sheath cell differentiation and function. In the 

absence of MSP, both pathways negatively regulate meiotic maturation, whereas in its presence, 

the inhibition is relieved allowing ovulation and fertilization. To examine MSP’s role in facilitating 

meiotic spindle assembly, we investigated the microtubule cytoskeleton in oocytes. We observed 

that the microtubule arrangement in oocytes differs in the presence and absence of sperm. In the 

presence of sperm, microtubules disperse evenly in a net-like fashion throughout the cytoplasm. 

By contrast, in the absence of sperm, microtubules are enriched 1.5-fold at the proximal-distal 

cortical edges of the oocyte. We also examined microtubules in oocytes of hermaphrodites over 

time, and observed that as sperm and MSP are depleted, cortical microtubule enrichment 

progressively increased from proximal to distal oocytes, correlating with the MSP distribution. To 

test whether MSP is sufficient to signal microtubule reorganization in oocytes, we injected female 

animals with purified MSP and analyzed microtubule distribution by confocal microscopy. 

Whereas, oocytes of buffer-injected females exhibited cortically enriched microtubules, oocytes of 

MSP-injected females did not. Based on these results, we propose that MSP signaling affects 

microtubule localization and/or dynamics in oocytes prior to fertilization. For future studies, our 

goal will be to trace the signal transduction pathway from the cell surface to the microtubule.

265. The conserved DEAD-box helicase CGH-1 negatively regulates MAP kinase activation 

in C. elegans oocytes 

Ikuko Yamamoto, David I. Greenstein 

Dept. of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 

Oocyte meiotic cell cycle progression must be precisely coordinated with ovulation and 

fertilization in order to form a diploid zygote. In C. elegans, fully-grown oocytes arrest at 

diakinesis of meiotic prophase I, and this arrest is released by an extracellular signal provided by 

the sperm, the major sperm protein (MSP). MSP signaling activates the conserved MAP kinase 

(MAPK) pathway in oocytes and promotes diverse meiotic responses, including M-phase entry, 

cortical cytoskeletal rearrangement, and gonadal sheath cell contraction. 

To clarify the mechanisms of MSP signal transduction and to identify downstream components 

in meiotic maturation regulatory pathways, we took a genetic approach. We isolated a dominant 

mutant std-1(tn691d,ts) (stuck in diakinesis) that affects MSP signaling responses, interferes with 

normal oocyte meiotic maturation processes, and disrupts meiotic chromosome segregation. 

Positional cloning of the gene revealed that std-1 corresponds to cgh-1 (1), which encodes a 

member of a highly conserved small subfamily of DEAD-box RNA helicases associated with 

germline development and meiotic progression. 

According to phenotypic and molecular analyses, cgh-1(tn691d,ts) possesses 

dominant-negative character. Analysis of a protein null mutant, cgh-1(ok492), indicates cgh-1 is a 

negative regulator of MAPK activation in oocytes. MSP signaling activates MAPK in the most 

proximal one to three oocytes in the wild type. In contrast, in the cgh-1 mutants, MAPK activation 

is observed in not only proximal but also distal oocytes. In females, in which the MSP signal is 

absent, MAPK activation is not observed; however, in feminized cgh-1 mutants 

[cgh-1(tn691d,ts);fog-2(q71) and cgh-1(ok492);fog-3(q443)] signal independent MAPK activation 

is detected. Thus, cgh-1 is required for: (i) the establishment of the response threshold in the 

presence of the MSP signal; and (ii) the inhibition of MAPK activation in the absence of sperm. 

OMA-1 and OMA-2 are two zinc finger proteins redundantly required for meiotic maturation (2). 

Without oma-1/oma-2 function, MAPK activation in proximal oocytes is not observed. In either 

cgh-1(RNAi);oma-1(te33);oma-2(te51) or oma-1(RNAi);oma-2(RNAi);cgh-1(ok492), MAPK 

activation is also not observed. Therefore, cgh-1 functions upstream or in parallel to oma-1 and 

oma-2 for MAPK activation. In addition to germline phenotypes, cgh-1 mutants exhibit elevated 

gonadal sheath cell contractions in both the presence and absence of sperm. Elevated sheath 

cell contractions are also observed in cgh-1(RNAi);rrf-1(pk1417) hermaphrodites and females, 

indicating that cgh-1 functions in the germ line to modulate sheath cell response to MSP. 

CGH-1 protein is specifically expressed in the germ line and localizes to the cytoplasm in 

proximal oocytes. In the wild type, CGH-1 is localized to a subcortical band that encircles the 

oocyte. In contrast, in the absence of sperm, CGH-1 localizes subcortically especially to large 

distinctive cytoplasmic foci. Similarly, in the dominant-negative mutant, cgh-1(tn691d,ts), CGH-1 

localizes to large subcortical cytoplasmic foci with and without sperm. Thus, CGH-1 could be in 

different kind of complexes or associated with different factors in the presence and absence of 

the MSP signal, and the dominant-negative mutation may alter such associations. 

(1) Navarro et al. Development 128: 3221, 2001. (2) Detwiler et al. Dev. Cell 1: 87, 2001.

266. Functional genomic characterization of germline stem cells in C. elegans 

Zhang Yang, Michelle Banfill, Valerie Reinke 

Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520 

We have used a functional genomics approach to investigate the properties of germline stem 

cells in C. elegans. A gain-of-function mutation in the GLP-1(Notch) receptor causes these cells 

to maintain their stem cell characteristics independent of the niche environment, resulting in a 

gonad filled with stem cells at the expense of meiotic and differentiating germ cells (Berry et al., 

1997). We used whole-genome DNA microarrays to compare the gene expression profiles of 

glp-1(gf) and wild type animals, and identified 207 genes with increased expression in animals 

bearing excess stem cells. Of these, 166 genes show expression in the wild type germline based 

on additional expression analysis. Investigation of in situ hybridization patterns in wild type 

animals show that of 85 examined, 76 are expressed at the distal end of the gonad, where the 

stem cell population resides. The stem cell-enriched transcripts primarily encode proteins 

predicted to function in chromatin and transcriptional regulation, RNA binding and regulation, and 

proteolysis. The genes include several with previously established roles in germline stem cells, 

including puf-8, fbf-1/fbf-2, and gpr-1. Fully 57% of these C. elegans stem cell-enriched factors 

have significant similarity (>30% over >50% of length) to mammalian proteins. 

To investigate the functions of the corresponding gene products, we performed a systematic 

RNA-mediated interference (RNAi) screen in which we soaked third-stage (L3) larvae in 

gene-specific dsRNA and examined the treated animals for highly-penetrant sterility as adults. 

For the 47 genes whose depletion produced sterile phenotypes, we fixed affected animals and 

stained with DAPI to visualize germ cell nuclei using fluorescent microscopy. Depletion of 

essentially all the genes showed a similar array of germline defects, primarily under-proliferation, 

which was sometimes accompanied by aberrant gamete differentiation and/or abnormal nuclear 

morphology. 

We have chosen two genes for in-depth characterization based on potential roles for the 

corresponding mammalian orthologs in stem cell regulation. Mammalian nucleostemin is a 

nucleolar GTPase whose expression affects the proliferation of neural stem cells and cancer 

cells. A deletion mutant of C. elegans nucleostemin, nst-1, results in a larval arrest. Homozygous 

nst-1 mutants, whose somatic, but not germline, expression of nst-1 has been rescued using an 

extrachromosomal transgene have only a very few immature germ cells. The second gene, 

byn-1, is an ortholog of mammalian bystin. Bystin is expressed in haematopoetic, embryonic, and 

neural stem cells in mammals, but its function is unknown. A deletion mutant of byn-1 also 

exhibits a larval developmental arrest similar to that of nst-1. Thus, initial RNAi as well as deletion 

mutant analysis suggest that both nst-1 and byn-1 play roles in regulating cellular proliferation in 

both germline and somatic tissues.

267. The Function of rde-1 homologs in C. elegans 

Erbay Yigit 1 , Martin Simard 1 , Ka Ming Pang 1 , Ngan K. Vo 1 , Shih-Chang Tsai 1 , Shohei Mitani 2 , 

Craig C. Mello 3 

1 Department of Molecular Medicine, UMASS Medical School, Worcester, MA 01605 

2 Department of Physiology, Tokyo Women’s Medical University School of Medicine 8-1, 

Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan 

3 Howard Hughes Medical Institute 

In a number of organisms, the introduction of double-stranded RNA into cells causes the 

post-transcriptional silencing of the corresponding gene. This experimental phenomenon is called 

RNA interference (RNAi). We are particularly interested in the molecular mechanism of this 

phenomenon. In order to study RNAi we screened for rde (RNAi deficient) strains. Among the 

genes identified to date, rde-1 and rde-4 have been cloned and analyzed further. RDE-1 is a 

novel protein with PAZ and PIWI domains found in numerous other proteins implicated in gene 

silencing and development. RDE-4 has two copies of a double stranded RNA binding motif. 

Genetic analysis by (Grishok et al., 2000) suggest that rde-1 and rde-4 are required at an 

upstream step in the RNAi pathway. Biochemical studies indicate that RDE-4 associates with at 

least three other proteins in vivo, RDE-1, DCR-1 and DRH-1 (Tabara et al., 2002). DCR-1 is a 

conserved RNase III related protein implicated in RNAi in several organisms. DRH-1 is a 

conserved DExH box helicase that appears to be required for RNAi in both the soma and the 

germline in C. elegans. RDE-1 appears to be necessary for RDE-4 to interact with the long-trigger 

dsRNA in vivo. However, in other organisms RDE-1 homologs appear to interact with siRNA 

products that function downstream in mRNA destruction. We therefore decided to ask if other 

RDE-1 homologs in C. elegans are required for RNAi. 

To date, we have identified a total 26 homologues of rde-1 in the C. elegans genome. In order 

to analyze the potential functions of these genes, we have injected dsRNA targeting all 26 genes. 

We have found three subclasses within the rde-1 gene family (including rde-1) that appear to be 

required for RNAi. Two other subclasses are required for embryonic development, and another 

one is required for proper germline function. We have screened a PCR deletion library to obtain 

deletion allele of each gene in these subclasses. 

We are in the process of identifying alleles of the remaining genes. We plan to determine what 

proteins and RNA species interact with these proteins to mediate their specific functions. These 

studies should shed further light on how members of this interesting family of RDE-1-related 

proteins function in RNAi and in essential developmental pathways.

268. Alteration of Pax protein DNA binding properties affects tissue-specific activities of 

the C. elegans gene egl-38 

Guojuan Zhang 1 , Rong-Jeng Tseng 2 , Helen M. Chamberlin 1 

1Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210 

2MCDB program, The Ohio State University, Columbus, OH 43210 

Pax transcription factors play an important role in organ development in all animals. Most Pax 

proteins function in the development of more than one organ or cell type. However, it is not well 

characterized how they can mediate different effects in different cell types. 

To better understand target gene selection by Pax proteins, we have characterized the function 

of the C. elegans Pax gene egl-38. egl-38 plays an important role in the development of several 

C. elegans organs, including the egg-laying system, the excretory system, the male spicules, and 

the hindgut. Genetic analysis showed that four non-null alleles of egl-38 ( n578, sy287, sy294, 

and gu22) disrupt different functions in different tissues, suggesting that EGL-38 regulates 

different target genes in different tissues. Each of these mutant alleles corresponds to an amino 

acid change in the DNA-binding domain of the EGL-38. In a previous study, we characterized 

lin-48, a direct, tissue-specific target for EGL-38 in hindgut cells, and identified one regulatory 

element in the lin-48 promoter that binds EGL-38 (Johnson et al, 2001). We have used this 

EGL-38 binding sequence (termed lre2) to understand the different properties of the egl-38 tissue 

preferential alleles. 

Using EMSA analysis, we have demonstrated that n578 and gu22 proteins retained the 

DNA-binding affinity to lre2, while sy287 protein showed much lower affinity and sy294 showed 

no detectable binding. These in vitro results are consistent with in vivo analysis of egl-38 alleles, 

which showed that among these mutants, sy294 disrupted hindgut development and lin-48::gfp 

transcription to the greatest extent, whereas n578 had little effect. These in vitro data also 

indicate that the mutations affect egl-38 activity in the hindgut by altering EGL-38 DNA-binding 

properties. 

Using the in vitro random DNA oligomer selection method, we identified the DNA sequences 

bound by the EGL-38 wild type (wt) and mutant proteins. The mutant sy294 or n578 protein was 

found to select a single sequence each, while, like wt, sy287 and gu22 proteins selected multiple 

sequences. The consensus sequence selected by wt protein is similar to that selected by Pax2 

protein (Xu et al, 1995). All of the mutant proteins select sequences that differ from those 

selected by wt in the nucleotides contacted by ß-hairpin and linker region of the Pax DNA binding 

domain. To test the in vivo activity associated with these selected sequences, we used modified 

lin-48::gfp reporter genes. We replaced lre2 with the consensus sequence selected by wt protein 

or the sequence selected by sy294 or n578 protein. In animals bearing the modified transgenes, 

lin-48::gfp was expressed in the hindgut cells but at levels much lower than transgenes bearing 

the endogenous lre2. This result indicates that DNA binding affinity is just one feature that 

determines the sequence of a Pax-response element. These results provide additional 

information to better understand the interaction between Pax family proteins and DNA. 

1. Johnson et al. (2001). Development 128, 2857-2865. 

2. Xu et al. (1995). Cell 8, 639-650.

269. The regulation of egl-5 expression in C. elegans by sop-2 

Hongjie Zhang, Yingqi Teng, Scott Emmons 

Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park 

Avenue, Bronx, NY 10461 

Transcriptional repressors of the polycomb group (PcG), together with the counteracting 

trithorax group (trxG) proteins, have been shown to regulate hox gene expression in flies and 

mammals. PcG proteins constitute two complexes, polycomb repressive complex 1 (PRC1) and 

the ESC-E(Z) complex. Sop-2, a PcG like gene in C. elegans, encodes a protein with a SAM 

domain, a self-associating protein domain also found in PRC1 components polyhomeotic (PH) 

and sex combs on midleg (SCM). The function of sop-2 is found to be analogous to that of PRC1. 

A sop-2 loss of function mutation results in the ectopic expression of several hox genes. For 

example, in a sop-2 mutant, C. elegans hox gene egl-5 (Abd-B homolog) is expressed not only in 

several posterior tissues, but also ectopically in head neurons and anterior seam cells. SOP-2 is 

identified to be a RNA binding protein, having interaction with a zinc-finger RNA binding protein 

MEP-1 (Zhang et al., Mol. Cell, in press). Based on the above evidences, we hypothesize that 

sop-2 might regulate egl-5 expression post-transcriptionally via a regulatory element in the egl-5 

3 ’ UTR. 

In order to asses whether regulation can occur via 3 ’ UTR, we compared the expression 

patterns of two reporters using the V6 lineage cis-regulatory element (V6CRE), an enhancer of 

egl-5, to drive delta pes-10::gfp expression with unc-54 3 ’ UTR and elg-5 3 ’ UTR, respectively. We 

found that whereas in the V6 lineage their expression is the same, the expression patterns of 

V6CRE-GFP-unc-54 3 ’ UTR and V6CRE-GFP-egl-5 3 ’ UTR are different in head neurons and gut 

cells. V6CRE-GFP-unc-54 3 ’ UTR is expressed in many head neurons and gut cells, while 

V6CRE-GFP-egl-5 3 ’ UTR is not. These differences indicate that regulation can occur through the 

3 ’ UTR. Moreover, in a sop-2 mutant, V6CRE-GFP-egl-5 3 ’ UTR is expressed ectopically in 

anterior seam cells, and more head neurons than in wildtype, but far less than 

V6CRE-GFP-unc-54 3 ’ UTR. This result suggests that sop-2 and other factors are recruited to 

regulatory elements on the egl-5 3 ’ UTR to regulate expression. 

In our effort to find the regulatory elements in the egl-5 3 ’ UTR, we identified a 5bp region 

required for repression in head neurons. This region has the same sequence as the point mutant 

element (PME) in fem-3 3 ’ UTR. The PME site is a binding site for FBF protein, which controls the 

switch from spermatogenesis to oogenesis in hermaphrodite worms by the repression of fem-3 

mRNA activity. With deletion of the PME site in the egl-5 3 ’ UTR, V6CRE-GFP-egl-5 3 ’ UTR is 

expressed in many more head neurons. However, it is likely that there are no specific sequences 

in the egl-5 3 ’ UTR responsible for repression in gut cells. Deletion of 1.5kb, 1.8kb and 2.5kb of 

downstream sequences in the egl-5 3 ’ UTR and even insertion of a 120bp sequence of the egl-5 

3 ’ UTR before the unc-54 3 ’ UTR, had no observed effect on the expression pattern of gut cells. 

The binding sites for sop-2, other factors regulating expression as well as the regulation 

mechanism are still under investigation.

270. Characterizing the Cell Biology of mec-4(d)-induced Necrotic Cell Death in C. elegans 

Wenying Zhang, Monica Driscoll 

Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ. 

Neurons may die as a normal physiological process during development or as a pathological 

process in disease. There are two major morphological categories of cell death: apoptosis and 

necrosis. Apoptosis is characterized by cell shrinkage, chromatin condensation and DNA 

degradation. Necrosis is an injury-associated death involving cellular swelling, distortion of 

organelles and loss of membrane integrity. The mechanism of apoptosis is fairly well-understood, 

whereas the mechanism of necrosis remains sketchy. 

In C. elegans mutations substituting large sidechain amino acids for a highly conserved small 

residue near the pore in ion channel subunit MEC-4 cause channel hyperactivation and excess 

ion influx, which eventually leads to necrotic-like cell death in touch neurons. Previous work has 

shown that necrotic cell death induced by MEC-4(d) is the result of elevated cytosolic Ca 2+ 

leading to the activation of calpains (calcium-activated proteases), which in turn may initiate a 

proteolytic cascade by activating cathepsins (Xu et al., Neuron. 2001 957-71. Syntichaki et al., 

Nature. 2002 939-44.). 

As part of our effort to better understand neuronal necrosis in response to ion 

channel-mediated insults, we have been characterizing the cell biology of necrosis using markers 

for specific organelles. 

The lysosome is considered as a "suicide bag" that contains more than 80 hydrolytic enzymes, 

including cathepsins. Disruption of lysosomes, leading to release of lysosomal hydrolases, is 

extremely cytotoxic. It has been proposed that lysosomal dysfunction plays a role in 

neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease and Huntington’s 

disease, and that calpains may provoke lysosomal protease release (Yamashima et al., 

Hippocampus. 2003 791-800). I used a touch neuron-specific GFP fusion gene that localizes to 

the lysosomal membrane to visualize the lysosome morphology in live worms. I observed that the 

number of small lysosome-like structures per pair of PLM touch neurons decreases significantly 

in mec-4(d) strains, whereas the individual size of marked compartment increases in mec-4(d) 

strains. It appears that the lysosomal compartment is markedly expanded in dying touch neurons. 

Changes in the lysosomal compartment also can be observed in cultured mec-4(d) neurons. Also, 

more lysosome-like structures traveling along the PLM axons were observed in mec-4(d) 

background, indicating lysosome may play a role during axon degeneration. 

Mitochondrial swelling is often associated with necrosis. I have also expressed a mitochondrial 

reporter in touch neurons and compared wild type with mec-4(d). I will report on our quantitative 

analysis.

271. The Roles of Chitin and Chitin Synthases in C. elegans and Parasitic Nematodes 

Yinhua Zhang, Jeremy Foster, Laura Nelson, Dong Ma, Clotilde Carlow 

New England Biolabs, 32 Tozer Rd, Beverly, MA 01915 

Chitin is a homopolymer of beta-1,4 linked N-acetyl-D-glucosamine synthesized by membrane 

bound chitin synthases. Chitin is known to provide structural support and an impermeable barrier 

in cell walls of fungi and exoskeletons of many invertebrates. In nematode, it is only known 

conclusively that chitin is present in the eggshell. It is unclear if chitin also plays a role in other 

cells during development in nematodes. 

We are studying chitin and chitin synthases in C. elegans as a model to address their functions 

in nematodes. We probed the function of chitin synthases by knocking down individual chitin 

synthase transcripts with RNAi and by GFP transcription reporter analysis. We also examined the 

localization of chitin in different tissues of wild type worms using a novel chitin-specific detection 

method. There are two predicted chitin synthases (F48A11.1 and T25G3.2) in the C. elegans 

genome. Like the chitin synthases found in fungi and other organisms, the nematode enzymes 

contain about 15-transmembrane domains and show some similarity to each other. Promoter 

reporter analysis showed that F48A11.1 was strongly expressed in the pharyngeal muscle cells. 

Knockdown of F48A11.1 by RNAi led to a developmental arrest at the L1 stage and the arrested 

worms appeared to be starved. Interestingly, chitin was detected in the lumen wall of discrete 

regions of the pharynx using our chitin probe, suggesting that F48A11.1 is responsible for the 

deposition of chitin in the pharynx and that chitin may be required for proper development of the 

pharynx or necessary for feeding. Knockdown of T25G3.2 by RNAi led to sterilized 

hermaphrodites that laid dead embryos and fractured eggs. These embryos were highly 

permeable to molecules in the environment compared with normal embryos, indicating a loss in 

eggshell integrity and increased permeability. The results suggest that chitin provides both 

mechanical support and a chemical barrier for developing embryos. This is consistent with that 

chitin is known to be a major component of the nematode eggshell, and that chitin was detected 

in the eggshell using our in situ chitin staining procedure. By studying cDNAs from parasitic 

nematodes we found that they appear to have corresponding orthologs of the two chitin 

synthases found in C. elegans. We conclude that chitin is essential in nematode biology and 

many nematodes likely possess 2 forms of chitin synthase with independent roles in the pharynx 

and eggshell.

272. Identification and characterization of a mutation which causes arg-1 to be expressed 

ectopically in C. elegans 

Jie Zhao, Ann Corsi 

the Catholic University of America Washington,D.C. 

In C.elegans,arg-1 is one of the downstream target genes of the hlh-8 gene product, CeTwist. It 

encodes a member of the DSL family of transmembrane signaling ligands and is closely related 

to apx-1 and lag-2 (Mello et al. 1994). Previous studies showed that ARG-1 is involved in cell fate 

decision during early embryogenesis and possibly plays a role in mesoderm development as well. 

In wild type worms, ARG-1 is expressed in the two intestinal muscles, anal sphincter, anal 

depressor, vm1 vulval muscles and the head mesodermal cell. However, the regulatory 

mechanism and related factors involved in the expression of this gene are largely unknown. In 

our lab, we obtained a strain which shows ectopic expression of arg-1::gfp during a screen of 

EMS induced mutations. Our initial attempt was to screen for CeTwist suppressor using a hlh-8 

mutant containing arg-1::gfp where expression of the latter is abolished due to a mutation on the 

hlh-8 gene. We recovered a single mutant that expresses arg-1::gfp in two ectopic cells in the 

head (which are possibly neurons) but not in any of its normal places. Moreover, worms with the 

same mutation in an otherwise wild type background show both normal and ectopic expression of 

arg-1::gfp. No other detectable phenotypes are found in the mutants. Preliminary data suggests 

that the mutation is recessive and is independent of hlh-8 or its protein product. My project is to 

first identify the mutation using SNP and traditional three-point mapping strategies. Currently, it 

has been located to the middle of chromosome III and fine mapping is in progress. Once the 

mutated gene is identified, I will study its expression pattern using GFP constructs and its 

potential functions using RNAi. We believe that identification and characterization of this mutation 

will help us to understand how this gene is regulated. 

[Reference] Mello, C.C., Draper, B.W. and Priess, J.R. (1994). The maternal genes apx-1 and 

glp-1 and establishment of dorsal ¨Cventral polarity in the early C.elegans embryo. Cell 77, 

95-106.

273. Neuropeptide modulation of C. elegans male mating behavior 

Tiewen Liu 1 , Maureen M. Barr 1,2 

1Laboratory of Genetics, University of Wisconsin, Madison, WI 53706 

2School of Pharmacy, University of Wisconsin, Madison, WI 53705 

The C. elegans male mating behavior comprises the steps of response, backing, turning, 

location of vulva, spicule insertion and sperm transfer. Cell ablation experiments have identified 

sensory neurons responsible for each step (1), making this behavior an attractive model for 

studying synaptic transmission in a defined neural circuit. Neuropeptides represent a large and 

diverse set of non-classical neural transmitters. They can modulate synaptic transmission by 

regulating presynaptic neurotransmitter release and/or postsynaptic neurotransmitter receptor 

responsiveness. The C. elegans genome is predicted to encode 23 FMRFamide-like 

neuropeptide (flp) genes (2). To determine the function of these flp genes in male mating 

behavior, we characterized their expression patterns in adult C. elegans males with GFP 

reporters. Several flp genes are expressed in male-specific neurons, suggesting a role in the 

male nervous system. We also characterized the male mating behavior of a number of flp 

mutants. Four of them, flp-8, flp-10, flp-12 and flp-20, exhibit an abnormal "stutter" turning 

phenotype. Currently, we are trying to determine the cellular basis of this turning defect. 

We are also interested in the biosynthesis and secretion of neuropeptides in C. elegans. To this 

end, we have found that C. elegans proprotein convertase egl-3 (3) has overlapping expression 

pattern with some of the flp genes and egl-3 mutant males also have a stutter turning phenotype. 

We also found that ida-1, the C. elegans homolog of mammalian dense core vesicle IA-2 (4), is 

expressed in a subset of FMRFamide-like neuropeptide expressing neurons, and ida-1 mutant 

males exhibit stutter turning phenotype. We are currently investigating the mechanisms by which 

EGL-3 and IDA-1 regulate male turning behavior. 

We are grateful to Dr. Chris Li for the flp::GFP strains and flp mutant strains. 1) Liu and 

Sternberg 1995, Neuron 14, 79-89. 2) Li et al 1999, Ann. NY Acad. Sci. 897, 239-252. 3) Thacker 

and Rose 2000, Bioessays 22, 545-553. 4) Zahn et al 2001, J. Comp. Neurol. 429, 127-143.


Diana David-Rus 1 , Peter J. Schmeissner 1 , Beate Hartmann 2 , Christophe Grundschober 2 , Uri 

Einav 3 , Eytan Domany 3 , Patrick Nef 2 , Garth Patterson 1 , Monica Driscoll 1 

1Rutgers University, Piscataway, New Jersey 

2Hoffmann-LaRoche, Basel, Switzerland 

3Weizmann Institute of Science, Rehovot, Israel 

In an effort to better understand the biology of aging with an emphasis on mid-life changes that 

influence healthspan, we have undertaken a DNA microarray analysis of global gene expression 

profiles over time using Affymetrix gene chip arrays. Our experiment includes time points 

including the reproductive and post-reproductive periods, with a series of consecutive mid-life 

time points being covered. Studies in our lab and others have suggested that critical events 

during the mid-life of the nematode can influence the aging of this organism. For our analyses, we 

used supervised and unsupervised methods, including a clustering method developed in the 

Domany lab. Interestingly, we find a sharp change in the transcriptional levels of numerous genes 

at about 10 days post egg-lay. This abrupt change in gene expression on day 10 is consistent 

with a window of time identified previously by our lab as a time when mutations in the age-1 gene 

are able to delay the deterioration of muscle tissue (Herndon et al., 2002). The abrupt change 

also correlates with a transition point at which autofluorescent biomarkers accumulate (see 

abstract by Gerstbrein et al., this volume). Since two similar microarray experiments already have 

been performed (Lund et al., 2001, Curr Biol. 12(18): 1566-73; Murphy et al., 2003, Nature, 

424(6946):277-83), we attempted a detailed cross-comparison between data from all three 

experiments, using non-parametric techniques. We found approximately 100 genes showing 

changes in gene expression over adulthood common to all three studies. These expression 

changes might be relevant to rapid end-stage deterioration in old nematodes.

275. Isolation of Mutations that Cause Mini-Chromosome Loss 

Christine Barbishs, Sandi-Jo Galati, Stephanie Keller, Stacey Eggert, Mary Howe 

Department of Biology and Program in Cell Biology and Bichemistry, Bucknell University, 

Lewisburg, PA 17837 

Faithful mitotic chromosome separation requires that sister chromatids establish bipolar 

attachment to a bilateral spindle. Proper attachment depends on kinetochore, spindle, and 

centrosome function. We are using a genetic approach to investigate these structures and identify 

proteins that mediate the establishment of proper sister chromatid attachment to the mitotic 

spindle. Specifically we are screening for mutations that cause an increase in the somatic loss of 

a sur-5:GFP extrachromosomal array,kuEx77. This array serves as a nonessential 

mini-chromosome, the mitotic transmission of which is more sensitive to genetic perturbation than 

intact chromosomes. 

We have screened approximately 3,000 haploid genomes and identified 17 putative mutant 

populations with high rates of mosaic sur-5 GFP expression. This screen is highly sensitive; we 

included and recovered positive controls, unmarked him-10(e1511ts) populations, in our screens. 

This result suggests that we will recover most mutations that have a phenotype similar to that of 

the kinetochore mutant him-10 (e1511ts). We have developed cytological and genetic assays to 

eliminate false positives from our pool of putative mutants. 

Thus far three mutant alleles have been identified as strong candidates for 

mini-chromosome-loss mutations. Alleles qa5300 and qa5301 are homozygous viable alleles that 

cause recessive defects in germline and somatic transmission of the sur-5;GFP array. The 

qa5302 allele causes a recessive lethal phenotype and dominant mini-chromosome loss. Current 

efforts focus on the characterization and mapping of these three alleles.

276. Abstract for Leica Microsystems Inc. 

Lon Nelson 

Leica Microsystems Inc., 2345 Waukegan Road, Bannockburn, IL 60015 

Leica Microsystems offers a new tool for better imaging of C.elegans and other model 

organisms: the MZ16 FA. This automated fluorescence stereomicroscope provides greater 

resolution than ever before possible from macroscopic to the sub-cellular level. Patented 

TripleBeam TM technology yields the highest contrast, and for experiments in time lapse or 

extended depth of focus, faster answers are obtained through Intelligent Automation of the MZ16 

FA’s motorized features. Leica will also exhibit the DFC300 FX digital camera that offers 

extended range sensitivity with active Peltier cooling and long exposure capability for the most 

challenging fluorescence imaging applications. 

Leica Microsystems is a leading global designer and producer of innovative high-tech precision 

optics systems for the analysis of microstructures. For more information on Leica 

stereomicroscope solutions and other high-performance imaging products for your laboratory, 

visit http://www.leica-microsystems.com.

Author Index 

Abbott, Allison L 62, 188 

Abe, Namiko 58 

Abraham, Mary C 59 

Ahmed, Shawn C 56, 174 

Ahn, James 178 

Ahringer, Julie 153, 209 

Albert, Genna 154 

Alkema, Mark 60 

Allen, Eleanor 61 

Altun, Zeynep F 32 

Alvarez-Saavedra, Ezequiel 62, 188 

Ambros, Matthew 152 

Ambros, Victor 62, 188 

An, Jae Hyung 41, 201 

Andersen, Erik 63-64 

Antonio, Celia 65 

Apicella, Alfonso J 233 

Armstrong, Kristin R 66 

Askew, David J 205 

Askew, Yuko S 205 

Ausubel, Frederick M 167 

Bacaj, Taulant 67 

Bai, Lei 155 

Baird, Scott E 127 

Baird, Scott Everet 68 

Baker, Rosana P 201 

Baker, Rosanna 41 

Bakoulis, Anastasia 61 

Baldi, Chris 69 

Balklava, Zita 70 

Banerjee, Diya 71 

Banfill, Michelle 266 

Bany, Amy I 72 

Barbishs, Christine 275 

Barr, Maureen M 273 

Bartel, David P 62, 188 

Bassous, Samuel 139 

Basu, Roshni 133 

Bates, Emily A 73 

Baugh, L Ryan 94 

Beach, Douglas 263 

Bei, Yanxia 41 

Benard, Claire 74 

Bennett, Daniel C 75 

Berdichevsky, Ala 76 

Bernstein, David S 2 

Bernstein, Yelena 14 

Bianchi, Laura 27, 77 

Blackwell, T Keith 41, 78, 201, 256 

Boag, Peter R 78

Boxem, Mike 79, 162 

Boyd, Windy A 80 

Branda, Catherine S 45 

Brodigan, Thomas 112 

Brömme, Deiter 175 

Brown, Adam 81-82 

Bruce, Robert III 152 

Bucher, Elizabeth A 250 

Byrd, Dana 96 

Byrne, Alexandra 83 

Cameron, Scott 149, 258 

Campo, Jacob J del 102 

Cannon, Chaunte 255 

Carlow, Clotilde 271 

Carter, Katherine O 84 

Cassata, Giuseppe 38 

Çataltepe, Sule 175 

Ceron, Julian 55, 85 

Chamberlin, Helen M 35, 66, 142, 232, 244, 268 

Chandra, Abha 85 

Chang, Howard 86, 263 

Chao, Michael Y 26, 87 

Chase, Daniel 88 

Chatterjee, Indrani 5 

Checchi, Paula M 89 

Chen, Carlos Chih-Hsiung 90 

Chen, Chih-Hsiung 222 

Chen, Esteban 234 

Chen, Nansheng 31 

Chen, Pei-Lung 91-92 

Chi, Woo 9 

Chin, Lena 125 

Cho, Soochin 33, 69 

Choi, Eun-Young 15 

Chow, David K 93 

Chuang, Chin-Hua 50 

Church, Diane 152 

Cipriani, Giselle 178 

Claggett, Julia M 94 

Clark, Anthony C 175, 205 

Clark, Damon 82 

Clark, Scott 61, 63, 210 

Clegg, Eric D 239 

Clever, Sheila 103, 260 

Colaiácovo, Mónica 105, 197 

Colosimo, Marc 81, 159 

Conradt, Barbara 124, 131, 224, 243 

Conte, Darryl Jr 13, 231 

Corsi, Ann K 187, 208, 257, 272 

Cram, Erin J 95 

Crittenden, Sarah L 2, 96

Croce, Assunta 38 

Crocker, Justin M 211 

Crozier, Kathryn 186 

Cuenca, Adrian 97 

Cui, Yuxia 98 

Culotti, Joseph 140 

da Graca, Li Sun 18 

D’Agostino, Ingrid 91-92 

Dahlen, Alex 114 

David-Rus, Diana 99, 274 

Davison, Ewa 221 

Day, Tovah A 57, 100 

de Souza, Natalie 101 

DeMeo, Stephen 103 

Dennis, James W 144, 211 

Dent, Joseph 151 

Deplancke, B 104 

DiFiore, Pier P 38 

Dionne, Heather M 87 

Dixon, Louise 140 

Dixon, Scott J 44, 83 

Domany, Eytan 99, 274 

Dong, Yan 209 

Donny-Clark, Kerry 178 

Dougherty, Brian A 168 

Driscoll, Monica 25, 27, 77, 99, 117-118, 139, 200, 225, 233, 270, 274 

Dupuy, D 104 

Eggert, Stacey 275 

Einav, Uri 99, 274 

Eisemann, Lindsay 247 

Eisenmann, David M 37, 141, 240 

Eizinger, Andreas 105 

Ellis, Ronald E 33, 69 

Emmons, Scott W 29, 106, 119, 161, 242, 269 

Engebrecht, JoAnne 105 

Fang, Chunhui 106 

Fares, Hanna 101 

Feng, Hui 107, 158, 218 

Fernandes, Raynah 44 

Fidalgo, Manuel A 108 

Finger, Fern P 53, 111 

Firestein, Bonnie L 120 

Fisher, Jasmin 136 

Fitch, David H A 34 

Fitzgerald, Michael C 109 

Foehr, Marisa L 110 

Fonarev, Paul Andre 52 

Foster, Jeremy 271 

Fraser, Andrew 209 

Freedman, Jonathan H 80, 98 

Fu, Iwen 111

Fukushige, Tetsunari 112 

Fukuto, Hana S 87 

Gabel, Chris 82, 113-114 

Galati, Sandi-Jo 275 

Galvin, Brendan 51 

Gami, Minaxi S 115 

Garsin, Danielle A 167 

Gavin, Nicholas P 34 

Geldziler, Brian 116 

Gerstbrein, Beate 27, 117-118 

Gerstein, Mark 123 

Ghosh, Rajarshi 106, 119 

Gissendanner, Chris R 4, 154 

Gleason, Julie E 37, 240 

Glodowski, Doreen R 120 

Golden, Andy 121 

Goodman, S Jay 45 

Goodyer, William 54 

Goutte, Caroline 183 

Grabowski, Melissa M 122 

Grant, Barth D 52, 70, 90, 222, 235 

Greenstein, David I 8, 264-265 

Greenwald, Iva 101, 150, 196 

Grosshans, Helge 123 

Grote, Phillip 124, 243 

Grundschober, Christophe 99, 274 

Guarente, Leonard 76 

Gumienny, Tina L 125-126 

Guo, Suzhen 139, 225 

Hajnal, Alex 38 

Hale, Valerie 183 

Hall, David H 32 

Hampton, Rachael M 127 

Han, David 215 

Han, Min 102 

Hanna-Rose, Wendy 48, 135, 147, 238 

Hanselman, Keaton 115 

Hansen, Dave 180, 229 

Hardin, Jeff 140 

Harel, David 136 

Harford, Jeff 128 

Harris, Jana E 264 

Harrison, Melissa M 129 

Hart, Anne C 26, 73, 87, 130, 138, 252 

Hartmann, Beate 99, 274 

Hartwieg, Erika 51 

Haspel, Gal 130 

Hatzold, Julia 131 

Havassy, Joshua 186 

Heallen, Todd R 132 

Hebeisen, Michael 133

Heiman, Maxwell G 134 

Hellman, Andrew B 188 

Hess, Heather A 10 

Higginbotham, Megan 22 

Hill, David 49 

Hisamoto, Naoki 41 

Hobert, Oliver 65, 74 

Hoener, Marius 4 

Hoier, Erika Fröhli 38 

Hopper, Neil 209 

Horvitz, Bob 22, 51, 58, 60, 62-64, 76, 129, 137, 176, 188, 202, 219, 221, 

226-227, 248 

Horvitz, H Robert 50, 182 

Howe, Mary 275 

Hu, Patrick J 16 

Huang, Li 135 

Huang, Michael M S 234 

Huang, Peng 45 

Hubbard, E Jane Albert 3, 136, 177-178, 253 

Huber, Claudia 124 

Hunt, Dave 61 

Hunter, Craig P 94, 109, 262 

Hurd, Daryl D 30 

Hurlburt, Allison 105, 197 

Hurwitz, Mike 137 

Hyde, Rhonda 138 

Ibanez-Ventoso, Carolina 139 

Ikegami, Richard 140 

Inoue, Hideki 41, 201 

Isaacson, Ariel B 12 

Ishidate, Takao 11 

Isopi, Marco 177 

Issacson, Ariel B 102 

Jackson, Belinda M 141 

Jerome, Jay 8 

Jia, Hongtao 142 

Jia, Ling yun 161 

Jia, Lingyun 29 

Jiang, Yuan 46 

Jirage, Dayadevi 143 

Johnson, Ted 123 

Johnston, Wendy L 144 

Jones, Adriana K 252 

Jose, Antony 145 

Julie Y Koh 77 

Jundi, Malek 146 

Juskiw, Nicole 30, 245 

Kadandale, Pavan 235 

Kalamegham, Rasika 147 

Kam, Na’aman 136 

Kao, Albert 113

Kao, Gautam 148 

Karakuzu, Ozgur 149 

Katic, Iskra 150 

Kaul, Aamna 151 

Keller, Stephanie 275 

Kelly, Bill 1, 215 

Kelly, William G 89, 216, 223 

Kemp, Ben 152 

Kemp, Catherine A 153 

Kemper, Keven 42 

Kennedy, Ryan 154 

Kerscher, Aurora Esquela 155 

Keys, Jennifer A 231 

Killian, Darrell J 3 

Kim, Dae Young 156 

Kim, Dennis H 167 

Kim, Heon S 157 

Kim, Jihyun 158 

Kim, Kyuhyung 159 

Kim, Saechin 51 

Kim, Seung-Il 206 

Kim, Seun-il 212 

Kim, Youngjo 160 

Kimble, Judith 2, 96, 189 

Kiontke, Karin C 34 

Kipreos, Edward T 158, 160, 249 

Klancer, Richard 235 

Kleemann, Gunnar A 161 

Knebel, Carmen M 175 

Koelle, Michael R 10, 72, 88, 145, 195, 207, 241 

Kohn, Rebecca 190 

Kollias, Nikiforos 118 

Komatsu, Hidetoshi 87 

Kopish, Kevin R 153 

Koreth, John 162 

Kornfeld, Kerry 37 

Kosinski, Mary 8 

Kraus, Kelly 163 

Krause, Michael 112 

Krizus, Aldis 144, 211 

Kroft, Tim L 7 

Kulkarni, Gauri 255 

Kumar, Vasantha 175, 205 

Kwok, Alvin 71 

Lambie, Eric 90, 152 

Lamont, Liana B 2 

Land, Marianne 40 

Lanjuin, Anne 81, 164, 192 

LaPenotiere, Hugh F 239 

Larkins-Ford, Jonah 26 

Lau, Nelson C 62, 188

Law, Ka-Lun 165 

Lee, Janet 152 

Lee, Ji-Inn 178 

Lee, Jungsoon 166, 206, 212 

Lee, Kwi Yeon 30 

Lee, Min-Ho 179 

Lee, Siu Sylvia 167 

Lee, Wei-Hsiang 27, 77 

Leng, Xiaohong 50 

Leonhard, Kim 96 

Lewis, French A, III 168 

L’Hernault, Steven W 7 

Li, Bingsi 169 

Li, Siming 49 

Liang, Jun 170 

Lin, Baiqing 171 

Lin, Shin-Yi 71, 84 

Lin, Steven 82 

Lindquist, Robert A 241 

Lints, Robyn 32 

Lipton, Johnathan O, . 161 

Liu, Jun 110 

Liu, Jun Kelly 46 

Liu, Kelly 258 

Liu, Qiang 23, 172 

Liu, Tao 18, 173 

Liu, Tiewen 273 

Liu, Ying 179-180 

Lizzio, Michael Jr. 200 

Lo, Te-Wen 45 

Lo, Vienna 117 

Lombel, Rebecca 103 

Löser, Stefanie 243 

Lowden, Mia R 174 

Lu, Xiaowei 63, 129 

Luke, Cliff J 175, 205 

Lyczak, Rebecca 194 

Ma, Dong 271 

Ma, Long 176 

Maciejowski, John 177-178 

Maina, Claude V 4 

Maine, Eleanor M 1, 179-180, 229 

Mak, Ho Yi 181 

Mangahas, Paolo M 182 

Mango, Susan E 246 

Mano, Itzhak 25 

Martinowich, Keri 263 

Mathies, Laura D 47, 189 

Matsumoto, Kunihiro 41, 201 

Mazor, Anna 194 

McBride, Sandra J 80, 98

McClinic, Karissa 215 

McDermott, Joan 112 

McDonald, Kent 8 

McGaughey, David 183 

McGovern, Marie 184 

Meier, Bettina 56, 174 

Melkman, Tal J 185 

Mello, Craig C 11, 13, 41, 231, 267 

Meneely, Philip M 186 

Mense, Sarah 56 

Meyers, Stephany G 187 

Michalak, Katy 179 

Miley, Ginger R 37 

Miller, David M, III 77 

Miller, Leilani M 37 

Mills, David R 205 

Milstein, Stuart 49 

Mishra, Bud 177 

Miska, Eric A 62, 188 

Mitani, Shohei 267 

Moffat, Jason 83 

Mohler, William A 12, 102, 203, 228 

Moore, Landon L 57, 100 

Moresco, James J 241 

Morphy, Kristin M 189 

Morris, Corey A 37 

Moser, Theresa 190 

Moss, Eric G 42 

Mowrey, William R 30, 191 

Mukherjee, Gargi 27 

Mukhopadhyay, Saikat 81, 192 

Munnamalai, Vidhya 193 

Munoz, Manuel J 108 

Murrow, MaryAnn 194 

Myers, Edith M 195 

Myers, Toshia R 196 

Nagl, Sandra 197 

Nakamura, Kuniaki 11 

Narbonne, Patrick 198 

Naredi, Peter 148 

Nef, Patrick 99, 274 

Nelms, Brian L 48 

Nelson, Laura 271 

Nelson, Lon 276 

Nguyen, Tri Q 4 

Nolan, Katie 20 

Nolde, Mona J 199 

Nonet, Michael 23 

Nothstein, Erika 230 

Nunez, Yury O 200 

O’Connell, Kevin F 153

Odera, Sampeter 50 

Oh, Seung Wook 19 

Oliveira, Riva P 41, 201 

Omura, Daniel 202 

Opoku-Serebuoh, Eugene 203 

Orkin, Stuart H 162 

Ouellet, Jimmy 17, 204 

Pack, Allan I 213 

Padgett, Richard W 125-126 

Pak, Stephen C 175, 205 

Pan, Manjing 18, 173 

Pang, Ka Ming 267 

Park, Byung-Jae 166, 206 

Park, Byun-Jae 212 

Park, Keunhee 206, 212 

Patterson, Garth I 18, 99, 173, 274 

Pavlichin, Dmitri 113 

Peach, Bethan 36 

Pepper, Judy S 88, 207 

Perens, Elliot A 43 

Perides, Ares 82 

Perreault, Audrey 36 

Philogene, Mary C 208 

Piano, Fabio 34 

Pnueli, Amir 136 

Portman, Douglas S 30, 191, 245 

Poulin, Gino 209 

Prasad, Brinda 149, 210 

Prien, Justin M 211 

Quinn, Christopher C 21 

Raghavan, Prashant 166, 206, 212 

Raizen, David M 213 

Ramaswamy, 

15 

Gopalakrishna 

Rapp, Paris 214 

Ratliff, Tom 1, 215 

Ray, Brianne J 216 

Raymond, Adam 216 

Raynes, Yevgeniy 34 

Rea, Philip A 250 

Reddy, Kirthi 105 

Reinert, K 217 

Reinert, Kristy 84, 154, 199 

Reinke, Valerie 9, 55, 167, 171, 179, 259, 266 

Ren, Min 107, 218 

Reuter, Jason W 111 

Rice, Julie R 80 

Richmond, Alissa 6 

Ringstad, Niels 58, 60, 219 

Roberts, Andrew F 125 

Rocheleau, Christian E 14, 220

Roehrig, Casey 34 

Rongo, Chris 86 

Rongo, Christopher 24, 120, 193, 214, 230, 263 

Rosen, David 57 

Roy, Peter J 44, 83 

Roy, Richard 17, 133, 156, 198, 204 

Royal, Dewey 27, 200 

Royal, MaryAnn 200 

Royal, Maryanne 27 

Rual, Jean-Francois 85 

Rubin, Charles S 40, 107, 218 

Ruvkun, Gary 16, 167, 181 

Ryder, Elizabeth F 21 

Saffer, Adam 221 

Saito, R Mako 36 

Saka, Nazli 199 

Salkoff, Lawrence 23 

Samuel, Aravi 82, 113-114 

Sanchez, Lydia 190 

Sasson, Isaac E 45, 75 

Sato, Ken 52, 222 

Sato, Miyuki 52, 222 

Satterlee, John S 36 

Savage-Dunn, Cathy 39, 170, 184 

Schaefer, Henry 24, 263 

Schafer, William R 233 

Schaner, Christine E 89, 223 

Schedl, Tim 179 

Schertel, Claus 224 

Schilling, Samantha 152 

Schmeissner, Peter J 99, 139, 225, 274 

Schumacher, Jill M 132 

Schvarzstein, Mara 47 

Schwartz, Hillel 226-227, 248 

Schwarzbauer, Jean E 95 

Schweinsberg, Peter 90 

Scranton, Victoria L 203, 228 

Sengupta, Piali 20, 81-82, 159, 164, 185, 192 

Servent, Kristin 190 

Seydoux, Geraldine 91-92, 97, 236 

Shaham, Shai 43, 59, 67, 134, 254 

Shakes, Diane C 6 

Shan, Ge 28 

Sharma, Rita 238 

Shaw, Kristin MD 261 

She, Xingyu 229 

Shi, Yang 55, 73 

Shim, Jaegal 230 

Shin, Tae Ho 166, 206, 212 

Shirayama, Masaki 11 

Silverman, Gary A 175, 205

Simard, Martin J 231, 267 

Simokat, Kristin 140 

Singson, Andrew W 5, 235 

Singson, Andy 116 

Skop, Helaina 247 

Slack, F J 217 

Slack, Frank J 15, 71, 84, 123, 155, 199 

Sleiman, Sama F 232 

Slone, Dan 77 

Slone, Robert D 233 

Sluder, Ann E 4, 154 

Smith, Eric 103 

Smith, Harold 143 

Snowflack, Danielle 103 

Soto, Martha C 234 

Spence, Andrew 47 

Spencer, Monique 190 

Spice, Christine M 68 

Springer, Deborah 179, 229 

Stamatas, Georgios 118 

Stankiewicz, Matt 57 

Stein, Lincoln D 31 

Stephney, Gloria 32 

Stern, Michael J 45, 75, 136 

Stetak, Attila 38 

Stewart, Allison 235 

Stitt, Bethany 190 

Stitzel, Michael L 236 

Stone, Craig 237 

Stovall, Elizabeth 21 

Straubhaar, Juerg 231 

Straud, Sarah 25 

Strauss, Tamara 258 

Struwe, Weston B 261 

Sun, Hongliu 238 

Sundaram, Meera V 14, 163, 213, 220, 237, 250 

Svrzikapa, Nenad 19, 122 

Swalm, Brooke 190 

Swenton, Deborah 180 

Szilagyi, Maria 239 

Szyleyko, Elizabeth 240 

Tanis, Jessica E 241 

Teng, Yingqi 242, 269 

Thompson, Julia K 164 

Tissenbaum, Heidi A 19, 122 

Tomasi, Tatiana 243 

Tsai, Shih-Chang 267 

Tseng, Rong-Jeng 244, 268 

Tsu, Christopher 205 

Tuck, Simon 148 

Tucker, Morgan 102

Tyler, Carolyn 30, 245 

Ugel, Nadia 177 

Ulyanov, Danila 231 

Umemura, Toru 214, 230 

Unhavathaya, Yingdee 13 

Updike, Dustin L 246 

Vallier, Laura G 101, 247 

van den Heuvel, Sander 36, 85, 162 

van der Linden, Alexander 

20 

M 

Varner, Johanna 248 

Vasudevan, Srividya 249 

Vatamaniuk, Olena K 250 

Venegas, Victor 251 

Victor, Martin 73 

Vidal, M 104 

Vidal, Marc 49, 79, 85 

Vidalain, Pierre-Olivier 49 

Villeneuve, Anne 105 

Vo, Ngan K 267 

Voisine, Cindy 73 

Voisine, Cindy 252 

Volaski, Maurice 32 

Vought, Valarie E 1, 179, 229 

Voutev, Roumen V 253 

Vranas, Kelly 41 

Waase, Carine 254 

Wadsworth, William G 21, 255 

Wagmaister, Javier A 37 

Wagner, Aaron 103 

Walhout, AJ Marian 104 

Walker, Amy K 256 

Walthall, Bill 28 

Wang, Huang 125-126 

Wang, Jianjun 39 

Wang, Peng 257 

Wang, Xiaodong 35, 142 

Wang, Zhao-Wen 23, 172 

Wani, Khursheed 85 

Warren, Charles E 211, 261 

Wasilenko, Jamie 1 

Webster, Erin 258 

Wendell, Dawn 227 

Weng, Huawei 32 

West, Rachel 152 

West, Stefanie 259 

Whetstine, Johnathan R 55 

Wickens, Marvin 2 

Wightman, Bruce 103, 154, 260 

Wirlo, Larissa 179 

Wirtz, Denis 236

Wiswall, William C, Jr 261 

Wolkow, Catherine A 93, 115 

WormBase Consortium 31 

Wright, Amanda J 262 

Wright, Tricia 263 

Wu, Hai 30 

Xu, Huihong 162 

Xu, Jinling 16 

Xu, Ming 110 

Xue, Jian 27 

Yamamoto, Ikuko 264-265 

Yang, Zhang 266 

Yigit, Erbay 267 

Yu, Ling 170, 184 

Yu, Shih-Hung 225 

Yu, Xiang 1 

Yu, Xiaomeng 50 

Zappi, Veronica 234 

Zetka, Monique C 54, 146, 165 

Zhang, Guojuan 268 

Zhang, Hongjie 269 

Zhang, Wenying 270 

Zhang, Yinhua 271 

Zhao, Jie 272 

Zhao, Yan 56 

Zhou, Zheng 50, 182, 251 

Zimmerman, Cole M 125 

Zipperlen, Peder 153

Program of the 2004 East Coast Worm Meeting - Caenorhabditis ...

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