West Coast Worm Meeting Abstracts - Caenorhabditis elegans ...

West Coast Worm Meeting 2000
West Coast Worm Meeting Abstracts
1. Meiotic Pairing and Synapsis [p 11]
Amy MacQueen, Anne M. Villeneuve
2. Conditional mitotic spindle mutants in C. elegans [p 12]
Danielle R. Hamill, Bruce Bowerman
3. Mitotic Chromosome Segregation by a Conserved Protein Complex [p 13]
K. Hagstrom, R. Chan, D. Pasqualone, B.J. Meyer
4. HIM-10 a Probable Kinetochore Protein Involved in Mitotic and Meiotic Chromosome Segregation [p
14] M. Howe, D G. Albertson, B. J. Meyer
5. A C. elegans chromokinesin required for chromosome segregation [p 15]
Jim Powers, Bill Saxton, Susan Strome
6. Nuclear Envelope Dynamics in Caenorhabditis elegans [p 16]
Kenneth Lee, Yosef Gruenbaum, Katherine L. Wilson
7. The formin protein CYK-1 acts in parallel to an aurora-like kinase/MKLP-1 pathway to execute
cytokinesis in early Caenorhabditis elegans embryos [p 17]
Aaron F. Severson, Danielle R. Hamill, Bruce Bowerman
8. The L type Cyclin SAG-4 is required for heat-shock induced protein expression [p 18]
Wen J. Chen, Yvonne M. Hajdu-Cronin, Paul W. Sternberg
9. cdl-1 encodes a stem- loop binding protein (SLBP) homolog and may be essential for core histone
expression. [p 19]
Yuki Kodama, Asako Sugimoto, Joel Rothman, Masayuki Yamamoto
10. UNC-23 is a member of the BAG family of chaperone regulators [p 20]
Poupak Rahmani, Donald Moerman
11. Maternal UNC-45 protein co-localizes with NMY-2, a non-muscle myosin at the cleavage furrow of
early embryos [p 21]
Wanyuan Ao, Dave Pilgrim
12. Polyunsaturated fatty acids requirements for proper functioning of the nervous system [p 22]
Jenny Watts, John Browse
13. Testing functions of phagocytosis receptor homologs in cell corpse elimination and gonadal outgrowth
[p 23]
Sambath Chung, Monica Driscoll
14. Regulation of Cell Fusion in C. elegans [p 24]
Scott Alper, Cynthia Kenyon
15. Ethanol sensitivity genes in Caenorhabditis elegans [p 25]
MinGi Hong, JaeYoung Kwon, InYoung Lee, MinSung Choi, Junho Lee
16. State-dependent learning in C. elegans. [p 26]
Jill C. Bettinger, Steven L. McIntire
17. The ut236 mutant in C. elegans has defects in the interaction of two sensory signals and an
associative learning. [p 27]
Takeshi Ishihara, Yuichi Iino, Isao Katsura
18. Mutation in the LIM homeobox gene lim-6 disrupts asymmetric function of the ASE chemosensory
neurons [p 28]
J.T. Pierce-Shimomura, M.R. Gaston, B.J. Pearson, S.R. Lockery
19. Information Coding in the C. elegans Olfactory System [p 29]
PD Wes, A Sagasti, G Jansen, RHA Plasterk, CI Bargmann
20. Roles of osm-9/capsaicin receptor family members in sensory behaviors [p 30]
D. Tobin, D. Madsen, G. Moulder, R. Barstead, A.V. Maricq, M. deBono, C. Bargmann
21. Execution and regulation of male C. elegans spicule muscle contractions during mating [p 31]
L. René García, Paul W. Sternberg
22. Genetic Analysis of Nicotine Adaptation in C. elegans. [p 32]
Jinah Kim, Laura E. Waggoner, Kari A. Dickinson, Daniel S. Poole, William R. Schafer
23. Neural control of locomotion in C. elegans [p 33]
Saleem Mukhtar, Jane Mendel, Jehoshua (Shuki) Bruck, Paul W. Sternberg
24. Analysis of glutamatergic neurotransmission by knockout of glutamate transporter genes. [p 34]
Itzhak Mano, Monica Driscoll
25. Electrophysiological analysis of C. elegans ionotropic glutamate receptors [p 35]
Jerry E. Mellem, Penelope J. Brockie, David M. Madsen, Andres V. Maricq
26. Electrophysiological analysis of unc-18 mutants. [p 36]
J. E. Richmond, R. Weimer, W. S. Davis, E. M. Jorgensen
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West Coast Worm Meeting 2000
27. Electrophysiological analysis of serotonin modulation of body wall neuromuscular physiology. [p 37]
Jon Madison, Joshua Kaplan
28. Serotonin signaling in the pharynx [p 38]
Timothy Niacaris, Leon Avery
29. A Muscarinic Contribution to the Regulation of Feeding [p 39]
Kate Steger, Leon Avery
30. WormBase: From ACeDB to a more complete and usable database [p 40]
Paul W. Sternberg, Erich Schwarz, Norma Foltz, WormBase Consortium
31. The C. elegans ORFeome project [p 41]
Jerome Reboul, Philippe Vaglio, Cindy Jackson, Troy Moore, Jean Thierry-Mieg, Danielle
Thierry-Mieg, Jim Hartley, Gary Temple, Mike Brasch, Nia Tzellas, Marc Vidal
32. Analysis of splicing and regulatory elements using the Intronerator [p 42]
W. James Kent, Alan M. Zahler
33. A global profile of germ line gene expression using microarrays reveals germ line-specific regulation
of the X chromosome in males and hermaphrodites [p 43]
Valerie Reinke, Harold E. Smith, Jeremy Nance, Abby F. Dernburg, Anne M. Villeneuve, Samuel
Ward, Stuart K. Kim
34. The promise and peril of genomics: sperm development as model system [p 44]
Harold Smith, Marci Millhouse, Sam Ward
35. Functional Analysis of Chromosome I [p 45]
Andrew Fraser, Ravi Kamath, Peder Zipperlen, Maruxa Martinez-Campos, Julie Ahringer
36. Optimizing the mutagenic properties of the mos1 transposon in C. elegans [p 46]
Daniel C. Williams, Jean-Louis Bessereau, Erik M. Jorgensen
37. SNAP-25, a protein implicated genetically in C. elegans anesthetic mechanisms, binds the general
anesthetic isoflurane [p 47]
Jason Berilgen, Mike Crowder
38. Happy worms: further characterization of fluoxetine (prozac) resistant mutants [p 48]
Robert K.M. Choy, James H. Thomas
39. unc-43 Ca2+/Calmodulin-dependent kinase II (CaMKII) mutant worms have convulsions in response
to the seizure-inducing drug PTZ [p 49]
Elizabeth M. Newton, James H. Thomas
40. Neurotoxin sensitivity of dopaminergic neurons in C. elegans: role of the dopamine transporter and
cell death pathways [p 50]
R. Nass, J. Duerr, , J. Rand, D. M. Miller, R. D. Blakely
41. Gene expression in transgenic C. elegans animals expressing the human beta amyloid peptide. [p 51]
Chris Link, Carolyn Johnson, Amy Fluet, Kyle K. Duke, Stuart K. Kim
42. A nematode model for mitochondrial diseases [p 52]
William Y. Tsang, Bernard D. Lemire
43. Bacillus toxin (Bt) susceptibility and resistance in C. elegans [p 53]
Lisa Marroquin, Dino Elyassnia, Joel Griffitts, Johanna O’Dell, Jerald Feitelson, Raffi Aroian
44. New dauer genes and pathways [p 54]
Michael Ailion, James H. Thomas
45. Temporal regulation of aging in the nematode C. elegans [p 55]
Andrew Dillin, Cynthia Kenyon
46. A longitudinal analysis of adult neurons in C. elegans [p 56]
Mark I. Snow, Pamela L. Larsen
47. Germ-line cells that regulate aging in C. elegans [p 57]
Nuno Arantes-Oliveira, Javier Apfeld, Cynthia Kenyon
48. A screen for genes that control programmed cell death in the germ line [p 58]
S Milstein, A Gartner, M Hengartner
49. C. elegans p53: requirement for radiation-induced programmed cell death, stress resistance, and
normal adult lifespan following diapause. [p 59]
W. Brent Derry, Aaron Putzke, Joel H. Rothman
50. Identification of cell-specific regulators of programmed cell death in C. elegans. [p 60]
Shai Shaham, Cori Bargmann
51. Biochemical, structural, and genetic analyses of the activation of programmed cell death [p 61]
Jay Parrish, Betsy Metters, Lin Chen, Ding Xue
52. Analysis of RNA associated with P granules in germ cells of C. elegans adults [p 62]
Jennifer A. Schisa, Jason N. Pitt, James R. Priess
53. The splicing Sm proteins colocalize with P granules in germ cells and participate in P granule
localization in the early embryo [p 63]
Scott A. Barbee, Alex L. Lublin, Thomas C. Evans
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West Coast Worm Meeting 2000
54. pod-2 defines a new class of mutants required for antero-posterior asymmetry in the early
Caenorhabditis elegans embryo [p 64]
Akiko Tagawa, Raffi V. Aroian
55. ooc-5 encodes a putative ATPase required for the reestablishment of asymmetric PAR protein
localization in two-cell embryos [p 65]
Stephen E. Basham, Lesilee S. Rose
56. RIC-8 (Synembryn): A novel regulator of G Protein signaling [p 66]
Kenneth G. Miller, Melanie D. Emerson, John R. McManus, James B. Rand
57. MED-1 AND -2 act at the convergence point of SKN-1 and POS-1 to specify MS and E identity [p 67]
Morris F. Maduro, Regina Broitman-Maduro, Joel H. Rothman
58. Mass spectrometric identification of PLP-1 and its role in mesendoderm specification [p 68]
E. Witze, E. Field, D. Hunt, J.H. Rothman
59. The C. elegans NeuroD homolog cnd-1 functions in multiple aspects of motor neuron fate
specification [p 69]
Steven Hallam, Emily Singer, David Waring, Yishi Jin
60. Left-right asymmetry in C. elegans intestinal organogenesis involves a LIN-12/Notch signaling
pathway [p 70]
Greg J. Hermann, Ben Leung, James R. Priess
61. The pho-1 Gene and Three Kinds of Gut Polarity [p 71]
Tetsunari Fukushige, James D. McGhee
62. A role for dishevelled in asymmetric cell division. [p 72]
Nancy Hawkins, Gregory Ellis, Bruce Bowerman, Gian Garriga
63. rho-1, a target of the exchange factor unc-73, is required for cell migrations during C. elegans
development [p 73]
Andrew G. Spencer, Christian J. Malone, Satoshi Orita, Min Han
64. PTP-1, a LAR-like receptor protein tyrosine phosphatase, may act in parallel with C. elegans Eph
signaling to direct morphogenesis [p 74]
Robert J. Harrington, Michael Gutch, Michael Hengartner, Nicholas Tonks, Andrew Chisholm
65. GEX-2 and GEX-3 define a conserved protein complex required for tissue morphogenesis and cell
migrations in C. elegans [p 75]
Martha Soto, Katsuhisa Kasuya, Hiroshi Qadota, Kozo Kaibuchi, Craig C. Mello
66. Pharyngeal extension: the short and the long of it [p 76]
MF Portereiko, SE Mango
67. A VAB-8/UNC-51/UNC-14 complex mediates axon outgrowth [p 77]
Tina Lai, Gian Garriga
68. Cytoskeletal Signalling in Response to the UNC-6 Axonal Attractant [p 78]
Zemer Gitai, Erik Lundquist, Marc Tessier-Lavigne, Cori Bargmann
69. Identifying genes involved in axonal branching in C. elegans [p 79]
Joe C. Hao, Marc Tessier-Lavigne, Cornelia I. Bargmann
70. UNC-119 suppresses supernumerary branching in C. elegans [p 80]
Karla Knobel, Warren Davis, Michael Bastiani, Erik Jorgensen
71. UNC-119 and axon outgrowth: Toward a mechanism [p 81]
Wayne Materi, Dave Pilgrim
72. Three distinct functions of beta-spectrin (UNC-70) [p 82]
Marc Hammarlund, Warren S. Davis, Erik M. Jorgensen
73. RPM-1, a conserved novel protein, regulates presynaptic terminal formation [p 83]
Xun Huang, Mei Zhen, Bruce Bamber, Yishi Jin
74. A C. elegans Inositol 5- Phosphatase Homologue Involved In Inositol 1,4,5-triphosphate Signaling and
Ovulation. [p 84]
Yen Kim Bui, Paul W. Sternberg
75. Mechanisms regulating the timing and specificity of anchor cell attachment to the vulval epithelium [p
85] David R. Sherwood, Paul W. Sternberg
76. Mutations in cyclin E reveal coordination between cell-cycle control and vulval development. [p 86]
David S. Fay, Min Han
77. Novel cell-cell interactions during vulva development in Pristionchus pacificus [p 87]
Benno Jungblut, Ralf J Sommer
78. Cellular and genetic analysis of Gq mediated signaling pathways in C. elegans [p 88]
C. A. Bastiani, S. Gharib, P.W. Sternberg, M.I. Simon
79. Calcium/calmodulin-dependent protein Kinase II regulates C. elegans locomotion in concert with a
G-protein signaling network [p 89]
Merrilee Robatzek, James H. Thomas
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West Coast Worm Meeting 2000
80. A novel lateral signaling pathway determines asymmetric olfactory neuron fates [p 90]
Alvaro Sagasti, Cori Bargmann
81. The search for dosage compensation complex binding sites on X chromosomes [p 91]
Raymond C. Chan, Tammy F. Wu, Barbara J. Meyer
82. Recognition and Assembly of SDC Protein Complexes onto Specific DNA Target Sites [p 92]
Diana Chu, Heather Dawes, Jason Lieb, Annie Kuo, Barbara J. Meyer
83. The TBP-like Factor CeTLF is Required to Activate RNA Polymerase II Transcription in C. elegans
Embryos [p 93]
Linda S. Kaltenbach, Susan E. Mango
84. The intracellular domain of the feminising receptor TRA-2A interacts directly with the transcription
factor TRA-1A [p 94]
David H. Lum, P. Kuwabara, D. Zarkower, A.M. Spence
85. chw-1 encodes a novel protein that interacts with pha-4 [p 95]
Michael Horner, Linda Kaltenbach, Susan Mango
86. The UNC-4 homeoprotein and its transcriptional co-repressor UNC-37/Groucho regulate
neurotransmitter vesicles in cholinergic motor neurons [p 96]
Kim Lickteig, Janet Duerr, Dennis Frisby, David Hall, Jim Rand, David Miller
87. The components of sensory cilia in C. elegans [p 97]
Peter Swoboda, Kerry Bubb, James H. Thomas
88. In vivo imaging of HSN outgrowth [p 98]
Carolyn E. Adler, Cornelia I. Bargmann
89. Temporal and spatial requirement of sensory cilia in the regulation of worm lifespan [p 99]
Joy Alcedo, Javier Apfeld, Bella Albinder, Jennifer Dorman, Honor Hsin, Bernadine Tsung, Cynthia
Kenyon
90. THE TTX-3 LIM HOMEOBOX GENE IS A CENTRAL REGULATOR OF INTERNEURON CELL FATE
[p 100]
Z. Altun-Gultekin, O. Hobert
91. The heterochronic gene pathway: Regulatory interactions and regulatory outputs. [p 101]
Victor Ambros, Marta Hristova, Rosalind Lee, Eric Moss
92. Genetic and phenotypic characterization of evl-14 and evl-20, genes involved in C. elegans vulva
development [p 102]
Igor Antoshechkin, Min Han
93. Caenorhabditis elegans T05H10.5, a homologue of yeast ubiquitin fusion degradation protein
(UDF-2), is expressed throughout the nervous system and in the gut [p 103]
Wanyuan Ao, Dave Pilgrim
94. Genetic analysis of neuroendocrine controls of fat metabolism in C. elegans [p 104]
Kaveh Ashrafi, Gary Ruvkun
95. zig genes and the PVT guidepost neuron [p 105]
Oscar Aurelio, Oliver Hobert
96. Analysis of GABA receptor plasticity in C. elegans [p 106]
Bruce A. Bamber, Janet E. Richmond, Pierrette K. Danieu
97. Isolation of suppressors of a dominant synapse defective mutant, syd-5(ju89) [p 107]
Renee Baran, Yishi Jin
98. Cargo recognition by synaptic vesicle kinesin [p 108]
Ewa Bednarek, Erik M. Jorgensen
99. The exp-1 locus may encode a subunit of an excitatory GABA receptor [p 109]
Asim A. Beg, Erik M. Jorgensen
100. The life span gene clk-2 is essential for embryonic development [p 110]
Claire Bènard, Brent McCright, Yue Zhang, Stephanie Felkai, Siegfried Hekimi
101. Characterization of Caenorhabditis elegans gamma-tubulin in dividing cells and differentiated tissues
[p 111]
Yves Bobinnec, Makoto Fukuda, Eisuke Nishida
102. Does CEH-20, an Exd/Pbx homolog in C. elegans, play a role in worm embryogenesis? [p 111]
Q.F. Boese, W.B. Wood
103. A-domain-containing protein family in C. elegans. [p 112]
Michael Brannan, Joaquin Muriel, Kathryn Taylor, Gordon Lithgow, Danny Tuckwell
104. Distribution and Regulation of Glutamate Receptors in the Locomotory Control Circuit of C. elegans.
[p 113]
Penelope J. Brockie, David M. Madsen, Yi Zheng, Jerry E. Mellem, Andres V. Maricq
105. Mutations That Affect Synaptic Localization Of Glr-1 [p 114]
Michelle Burbea, Joshua M. Kaplan
106. Regulation of C. elegans dauer formation by an RNA quality control pathway component [p 115]
J Burgess, JC Labbe, S Hekimi
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West Coast Worm Meeting 2000
107. Synaptic vesicle localization is misregulated in unc-16 mutants [p 116]
DT Byrd, Y Jin
108. The egl-21 gene encodes a carboxypeptidase E, which is required for pro-neuropeptide processing
[p 117]
Tija Carey, Joshua M. Kaplan
109. How are anterior cell migrations guided by mig-13? [p 118]
QueeLim Ch’ng, Cynthia Kenyon
110. New Screens for Negative Regulators of let-23 [p 119]
Monica Chan, Marie Tiongsen, Romel C. Castro, Vanessa Lee, Gregg Jongeward
111. C. elegans MRE-11 is required for meiotic recombination and DNA repair but not for the meiotic G2
DNA damage checkpoint [p 120]
Gregory Chin, Anne Villeneuve
112. Suppressor Analysis of Eph/Ephrin Defective Signaling in C. elegans [p 121]
Ian Chin-Sang, Julie McCleery, Andrew Chisholm
113. RNAi Screen for Components of the C. elegans Meiotic Machinery [p 122]
Mónica Colaiácovo, Gillian Stanfield, Kirthi Reddy, Anne Villeneuve
114. Exploring the role of PINCH/UNC-97 in muscle development and focal adhesion assembly in
Caenorhabditis elegans and mammalian tissue culture cell lines [p 123]
Shaun Cordes, May Dang-Lawson, Poupak Rahmani, Linda Matsuuchi, Donald G. Moerman
115. The SAD-1 kinase regulates presynaptic vesicle clustering in C. elegans [p 124]
Justin Gage Crump, Mei Zhen, Kang Shen, Yishi Jin, Cornelia I. Bargmann
116. Mutants with altered sensitivity to the effects of ethanol on locomotion [p 125]
Andrew G. Davies, Tod R. Thiele, Catharine Eastman, Steven L. McIntire
117. A screen for DD/DV axonal morphology defects [p 126]
M. Wayne Davis, Erik M. Jorgensen
118. spn-2 AND spn-3 FUNCTION TO ORIENT THE SPINDLE DURING EARLY CLEAVAGES [p 127]
Leah R. DeBella, Lesilee S. Rose
119. Molecules acting in parallel with UNC-34 to control cell migration [p 128]
Megan Dell, N Chugh, N Hawkins, E Kong, J Hardin, G Garriga
120. Insights into the role of C. elegans protein UNC-119 in axonogenesis [p 129]
Chantal Denholm, Wayne Materi, Daniel Gietz, David Pilgrim
121. The defecation gene aex-1 may regulate a retrograde signaling pathway at neuromusclular
junctions. [p 130]
Motomichi Doi, Kouichi Iwasaki
122. Cosuppression in the Germline: Silencing is Golden [p 131]
Abby F. Dernburg, Mónica P. Colaiácovo, Jonathan Zalevsky, Anne M. Villeneuve
123. sur-9 a Suppressor of Activated let-60(n1046) in the C.elegans Vulva. [p 132]
Dennis Eastburn, Min Han
124. Knockouts In C. elegans: Madness and Methodology [p 133]
Mark Edgley, Erin Gilchrist, Greg Mullen, Bin Shen, Margaret Kotarska, Don Moerman, Steven
Jones, Anil Dsouza, Gary Moulder, Malini Viswanathan, Martin Lansdale, Robert Barstead
125. Using DNA microarrays to identify targets of homeobox genes in C. elegans [p 134]
Andreas Eizinger, Tibor Vellai, Fritz Müller, Stuart K. Kim
126. ded Genes Disrupt Cell Division Timing and Patterning in C. elegans Embryos [p 135]
Sandra Encalada, Paula Martin, Jennifer Phillips, Rebecca Lyzcak, Danielle Hamill, Kathryn Swan,
Bruce Bowerman
127. Voltage-dependent currents in homologous chemosensory neurons with different functions in C.
elegans [p 136]
S Faumont, S.R. Lockery
128. VAV is required for pharyngeal muscle contraction in C. elegans [p 137]
R.T. Fazzio, J.E. Mellem, M.C. Beckerle, A.V. Maricq
129. Regulation of C. elegans Body Size by Sensory Cues [p 138]
Manabi Fujiwara, Hoan Phan, Steven L. McIntire
130. Regulation of intracellular dynamics of MAPKAPK2 in living C.elegans [p 139]
Makoto Fukuda, Yves Bobinnec, Eisuke Nishida
131. Role of cki-1 in terminal embryonic differentiation and cell-cycle arrest [p 140]
Masamitsu Fukuyama, W. Brent Derry, Joel H. Rothman
132. sax-1 and sax-2 act in parallel with unc-34 to Maintain Neuron Polarity. [p 141]
Maria E. Gallegos, Jennifer A. Zallen, Cori Bargmann
133. Identifying pharyngeal targets of PHA-4 using DNA microarrays [p 142]
Jeb Gaudet, Michael Horner, Stuart Kim, Susan E. Mango
134. An overview of predicted cytochrome P450 genes in C. elegans [p 143]
Erin Gilchrist
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West Coast Worm Meeting 2000
135. Clues toward understanding EGF/ Wnt signal integration in the specification of P12 fate: analysis of
the egl-5 promoter [p 144]
Lisa Girard, Henrique B. Ferreira, Scott Emmons, Paul Sternberg
136. spn-4: a gene required for mitotic spindle orientation in the 2-cell stage C. elegans embryo [p 145]
José E. Gomes, Kathryn A. Swan, Christopher A. Shelton, Bruce Bowerman
137. Ca2+-signalling via the neuron-specific Ca2+ sensor NCS-1 is essential for thermotaxis, a form of
associative learning and memory in C. elegans [p 146]
Marie Gomez, Edouard De Castro, Ernesto Guarin, Patrick Nef
138. Characterizing the Neural Circuitry of Chemotaxis to Volatile Odorants [p 147]
Jesse Gray, Maria Gallegos, Tim Yu, Cori Bargmann
139. Studies on the Nematicidal Bacillus thuringiensis Toxins [p 148]
Joel S. Griffitts, Raffi V. Aroian
140. Synaptic localization of the glutamate-gated chloride channel GBR-2 [p 149]
Maria E. Grunwald, Joshua M. Kaplan
141. Regulation and function of lin-11 in C. elegans vulval development [p 150]
Bhagwati P Gupta, Paul W. Sternberg
142. sur-7, a gene that suppresses activated ras [p 151]
Eric Hague, Min Han
143. Characterization and Suppression of eat-16; sag-1/dgk-1 lethality [p 152]
Yvonne M. Hajdu-Cronin, Wen J. Chen, Paul W. Sternberg
144. Improved Tissue Preservation Using Metal Mirror Freezing or High Pressure Freezing for TEM [p
153]
David H. Hall, Frank Macaluso, Gloria Stepheney, Marie-Christine Paupard
145. Role of the Caenorhabditis elegans homologs of cdk5 and p35 in migration and axon outgrowth [p
154]
Thomas Harbaugh, Gian Garriga
146. Regulation of egg-laying by sensory cues [p 155]
Laura Anne Hardaker, William R. Schafer
147. Characterization of the C. elegans Serotonin-Synthetic Aromatic Amino Acid Decarboxylase Gene
bas-1 [p 156]
Emily Hare, Curtis M. Loer
148. XOL-1 Files [p 157]
Christian A. Hassig, Barbara J. Meyer
149. Y41G9a.1, the C. elegans Homologue of Tg737, is Expressed in Ciliated Neurons [p 158]
Courtney J. Haycraft, Patrick D. Taulman, Stephen M. Krum, Bradley K. Yoder
150. let-381 is a forkhead gene [p 159]
Marika Hellqvist-Greberg, Ann M Rose, David L Baillie
151. Genetic analysis of dynamic search behavior in C. elegans [p 160]
T.T. Hills, F. Adler, A. V. Maricq
152. Multiple roles for the Ras-MAPK signal transduction pathway in chemotaxis to odorants? [p 161]
Takaaki Hirotsu, Satoshi Saeki, Yuichi Iino
153. mab-26 encodes the C. elegans ephrin EFN-4 [p 162]
Thomas Holcomb, Sean E. George, Ian Chin-Sang, Mei Ding, Andrew Chisholm
154. syd-8, a new player in axon guidance. [p 163]
Xun Huang, Mei Zhen, Yishi Jin
155. Analysis of gcy-31, a putative soluble guanylyl cyclase gene in Caenorhabditis elegans [p 164]
Martin L Hudson, David S. Karow, Michael A. Marletta, David B. Morton
156. Using C. elegans to Determine the Mechanism of Action of Pharmaceuticals and Pesticides [p 165]
Tak Hung, Ben Burley, Emery Dora, Dan Elkes, Steve Gendreau, Denise Jacobus, Rachel Kindt,
Mark Lackner, Lisa Moore, Scott Ogg, Dianne Parry, Roxanna Peng, Ellyn Pham, Jenny Kopczynski
157. In vivo characterization of the effects of the unc-64(md130) mutation on anesthetic sensitivity. [p
166]
Hunt S.J., Mike Crowder
158. Regulation of the C. elegans epidermal growth factor homolog LIN-3 [p 167]
Byung Joon Hwang, Paul W. Sternberg
159. Characterization of the regulatory elements required for neuron-specific expression of SNAP-25 in
the nematode [p 168]
Soon Baek Hwang, Junho Lee
160. Analysis of 2° vulval lineage execution [p 169]
Takao Inoue, Paul W. Sternberg
161. Developing a C. briggsae genetic map [p 170]
B. Johnsen, S. Gharib, A. Mah, K. Brown, D. Baillie, P. Sternberg
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West Coast Worm Meeting 2000
162. Coenzyme Q and aging in the nematode Caenorhabditis elegans. [p 171]
Tanya Jonassen, Pamela L. Larsen, Catherine F. Clarke
163. osm-9 signaling: who’s involved? [p 172]
Amanda H. Kahn, David Tobin, Cornelia I. Bargmann
164. Looking for synergy with PHA-4 on the myo-2 promoter [p 173]
John Kalb, Pete Okkema, Jim McGhee
165. Initial Characterization of Soluble Guanylate Cyclases in C. elegans [p 174]
David Karow, Jennifer Chang, Scott Nicholls, Ronald Ellis, Martin Hudson, David Morton, Michael
Marletta
166. Multiple regulatory elements activate end-1 expression in the E lineage [p 175]
Jodie J. Kasmir, Morris Maduro, Joel H. Rothman
167. Mutations that perturb the effect of octopamine/serotonin on pharyngeal activity. [p 176]
John Keane, Leon Avery
168. Pheromone Regulation of Neuroendocrine Outputs in C. elegans [p 177]
Scott Kennedy, Gabriel Hayes, Gary Ruvkun
169. Calcium Imaging in Excitable Cells of C. elegans. [p 178]
Rex Kerr, Varda Lev-Ram, Roger Y. Tsien, William R. Schafer
170. A genetic analysis of the effects of ethanol on egg laying [p 179]
Hongkyun Kim, M. Christina Yu, James Kim, Steven L. McIntire
171. Genes affecting the activity of nicotinic receptors involved in egg-laying behavior [p 180]
Jinah Kim, Daniel S. Poole, Laura E. Waggoner, Alexandra Treschow, William R. Schafer
172. Sensory axon guidance defects in C. elegans [p 181]
Susan Kirch, Gage Crump, Cori Bargmann
173. Isolation of a third lin-4 allele from a lin-3A overexpression line [p 182]
Martha Kirouac, Paul Sternberg
174. elt-5 and elt-6 are essential for development of seam cells, the vulva, and the male tail. [p 183]
Kyunghee Koh, Joel H. Rothman
175. A genetic screen for genes involved in gut development and differentiation in Caenorhabditis
elegans [p 184]
Jay D. Kormish, James D. McGhee
176. An E1-like activating enzyme is involved in cell division processes in the early C. elegans embryo. [p
185]
Thimo K. Kurz, Danielle R. Hamill, Bruce Bowerman
177. Olfactory Adaptation [p 186]
Noelle L’Etoile, Cori Bargmann
178. You can’t get there from here: a gene required for pharyngeal extension. [p 187]
SK Lange, JR Saam, SE Mango
179. Signaling by the VAB-1 Eph receptor intracellular domain [p 188]
Kristoffer Larsen,, Sean George, Andrew Chisholm
180. mdf-1 suppressors that may play a role in the metaphase to anaphase checkpoint [p 189]
Elaine Law, Risa Kitagawa, Ann M. Rose
181. Characterization of a C. elegans Defecation Mutant [p 190]
Anne Lehtela, Garry Wong
182. Organogenesis of the C. elegans Intestine [p 191]
Benjamin Leung, Greg J. Hermann, James R. Priess
183. Expression and regulation of daf-16::gfp constructs [p 192]
Kui Lin, Cynthia Kenyon
184. Identification of novel unc-64 (syntaxin) alleles [p 193]
Christine Liu, C. Michael Crowder
185. Mutations that cause neurite sprouting of the DVB motor neuron [p 194]
Loria, P., Boulin, T., Conte, S., Hobert, O.
186. A b -tubulin gene, tbb-2, functions as an activator of mei-1 and mei-2 in female meiotic spindle
formation in Caenorhabditis elegans. [p 195]
Chenggang Lu, Martin Srayko, Paul E. Mains
187. Global profile of gene expression during aging [p 196]
James Lund, Pamela Larsen, Pat Tedesco, Thomas Johnson, Stuart Kim
188. Conditional mutations affecting mitotic spindle positioning and polarity in the C. elegans embryo [p
197]
Rebecca Lyczak, Bruce Bowerman
189. Role of PDZ domain proteins in establishing gut epithelial polarity [p 198]
Kathleen E. Mach, Stuart K. Kim
190. Genetic analysis of NMDA receptor expression in C. elegans [p 199]
David M. Madsen, Chingju Lin, Penelope J. Brockie, Andres V. Maricq
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West Coast Worm Meeting 2000
191. Large Scale Reverse Genetic Approach Using RNAi [p 200]
Sarah Mahoney, Alex Phan, Mark Maxwell, Candace Swimmer, Jonathan Heller, Brett Milash, Kate
McKusick, Monique Nicoll
192. Sequence Confirmation of 182 snps between C. elegans N2 and CB4856 Strains and Plans for
Generation of 1000 New snps. [p 201]
Penny Mapa, Kathryn Swan, Mike Ellis
193. Building a dictionary for C. elegans promoter sequences [p 202]
Steven McCarroll, Hao Li, Cori Bargmann
194. High Pressure Freezing Methods for C. elegans Embryo Ultrastructure and EM Immunolabeling [p
203] Kent L. McDonald, Thomas Mueller-Reichert, Akiko Tagawa, Chad A. Rappleye, Raffi Aroian
195. Molecular Identification of Transcriptional Targets of the DAF-16 Winged Helix Transcription Factor
[p 204]
Joshua J. McElwee, James H. Thomas
196. Functional conservation of C. elegans UNC-30 and mouse Pitx2 in GABAergic neuron specification
[p 205]
Jason McEwen, Yishi Jin
197. Genes involved in nicotinic neurotransmission in the pharynx [p 206]
Jim McKay, David Raizen, Leon Avery
198. Genetic analysis of the functions of a GSK-3ß homolog called sgg-1 and a ß-TRCP/slimb homolog
during C. elegans embryogenesis [p 207]
Marc Meneghini, Greg Ellis, Ann Schlesinger, Bruce Bowerman
199. The Effect of Nonimmobilizers on C. elegans [p 208]
Laura B. Metz, Mike Crowder
200. Isolation and characterization of mutations that enhance let-23(sa62gf) during vulval development [p
209] Nadeem Moghal, Paul W. Sternberg
201. The trampoline assay: A new method for measuring the step response of the chemotaxis
mechanism in C. elegans. [p 210]
Moravec, M.L, Cervantes, J., Lockery, S.R.
202. Identification of genes regulating body length in the DBL-1 pathway by differential hybridization of
arrayed cDNAs [p 211]
Kiyokazu Morita, Makoto Mochii, Yukiko Sugihara, Satoru Yoshida, Yo Suzuki, William B. Wood, Yuji
Kohara, Naoto Ueno
203. Mutations in the ephrin mab-26/efn-4 cause defects in closure of the gastrulation cleft and in
epidermal enclosure [p 212]
Sarah L. Moseley, Andrew Chisholm
204. Cellular and developmental events required to generate functional muscle in C. elegans. [p 213]
K. Norman, S. Cordes, G. Mullen, P. Rahmani, T. Rogalski, D. Moerman
205. Is the DAG kinase DGK-1 an effector of Go alpha (GOA-1)? [p 214]
Stephen Nurrish, Michael Dybbs, Joshua Kaplan
206. Transforming Nematodes into Insects: Understanding Bt-resistance [p 215]
Johanna O’Dell, Raffi Aroian
207. The cytoskeletal protein zk370.3 may contribute to oocyte development and fertilization [p 216]
Alex Parker, Ann M. Rose
208. Oxidant Stress Responses in C. elegans [p 217]
Farhang Payvar, Andrew DeMatteo, Tom Hazinski
209. Pharyngeal pumping defects in unc-103 mutants [p 218]
Christina I. Petersen, David J. Reiner, Elizabeth M. Newton., James H. Thomas, Jeffrey R. Balser
210. A requirement for C. elegans Rho-binding kinase in early cleavage [p 219]
Alisa J. Piekny, Paul E. Mains
211. Function of the receptor tyrosine kinase CAM-1/KIN-8 in coordinated movement [p 220]
S. Poulson, D. Madsen, A.V. Maricq
212. The Autosomal Sex Signal in C. elegans? [p 221]
Jennifer R. Powell, Barbara J. Meyer
213. Got the blues? Try another genetic screen! [p 222]
Chad Rappleye, Rebecca Lyczak, Bruce Bowerman, Raffi Aroian
214. Identification of Components of the Meiotic Machinery in C. elegans [p 223]
Kirthi Reddy, Monica Colaiacovo, Gillian Stanfield, Anne Villeneuve
215. Novel and Atypical Receptor Tyrosine Kinases in Morphogenesis. [p 224]
David J. Reiner, Lewis Leng, Barbara J. Meyer
216. Differential effects of heat shock and cold shock following massed and distributed long-term
habituation training in C. elegans [p 225]
Jacqueline Rose, Kenneth Eng, Catharine Rankin
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West Coast Worm Meeting 2000
217. A new en masse training procedure to study long-term habituation in C. elegans [p 226]
Jacqueline Rose, Catharine Rankin
218. Global patterns of expression patterns in muscle using mRNA-Tagging [p 227]
Peter J. Roy, Stuart Kim
219. Cooperation between unc-26/synaptojanin and the dynamin-related protein DRP-1 during
mitochondrial division [p 228]
Dan Rube, Todd Harris, Erik Jorgensen, Alexander van der Bliek
220. Calcium dynamics of fertilization in C. elegans [p 229]
Aravinthan D.T. Samuel, Venkatesh N. Murthy, Michael O. Hengartner
221. Mutants in Thermosensory Neuron Specification and Function [p 230]
John S. Satterlee, Piali Sengupta
222. Vesicular GABA transport in C. elegans requires two proteins UNC-47 and UNC-46 [p 231]
Kim Schuske, Erik M. Jorgensen
223. Utilizing two approaches, genetic and genomic, to identify the vesicular glutamate transporter [p 232]
Kim Schuske, Dan Williams, Erik M. Jorgensen
224. Actin-dependent processes in the early C. elegans embryo require the profilin gene pfn-1, the FH
gene cyk-1, and bel-1 [p 233]
Aaron F. Severson, Rebecca Lyczak, David L. Baillie, Bruce Bowerman
225. LIN-12 post-transcriptional downregulation during VPC specification [p 234]
DD Shaye, I Greenwald
226. Distint and redundant functions of mu1 medium chains of AP-1 clathrin-associated protein complex
in the nematode Caenorhabditis elegans [p 235]
Jaegal Shim, Junho Lee
227. Molecular Mechanisms of Daf-12 Action: Identificationof Response Elements and Functional
Analysis of the Protein [p 236]
Yuriy Shostak, Adam Antebi, Marc R. van Gilst, Kieth R. Yamamoto
228. Serotonin-resistant egg-laying mutants and a receptor knockout in progress [p 237]
Stanley Shyn, William Schafer
229. A novel genetic screen for synaptic transmission genes acting in the diacyglycerol pathway [p 238]
Derek S. Sieburth, Wendy Cham, Josh M. Kaplan
230. Evidence of a Mate-finding Cue in the Free-Living Soil Nematode C. elegans [p 239]
Jasper M. Simon, Paul W. Sternberg
231. Understanding C27H5.1: From sequence to sense [p 240]
Jessica Smith, David Pilgrim
232. Genetic screens for novel components involved in blastomere asymmetry in the early C. elegans
embryo [p 241]
Martha Soto, Craig C. Mello
233. Pax be with you - patterning the pharynx [p 242]
Jeff Stevenson, Andrew Chisholm, Susan E. Mango
234. The evolution and expression of FEM-2 [p 243]
Paul Stothard, Dave Hansen, Tamara Checkland, Dave Pilgrim
235. Forming a gut: the view from an elt and two odds [p 244]
Keith Strohmaier, Morris Maduro, Joel Rothman
236. C. elegans homologue of protein phosphatase 4 is required in spermatogenesis [p 245]
Eisuke Sumiyoshi, Asako Sugimoto, Masayuki Yamamoto
237. Transcriptional regulation of the tryptophan hydroxylase gene tph-1 [p 246]
Ji Ying Sze
238. Searching for new genes involved in dosage compensation [p 247]
Chun Tsai, Barbara J. Meyer
239. Characterizing the role of let-99 in spindle orientation [p 248]
Meng-Fu Tsou, Adam Hayashi, Lesilee S. Rose
240. Characterization and cloning of the muscle activation gene unc-58 [p 249]
Monika Tzoneva, James H. Thomas
241. The structure/function relationship of clk-1 in the nematode Caenorhabditis elegans [p 250]
Antonio Ubach, Siegfried Hekimi
242. Characterization of transcriptional regulation by a class of monomeric nuclear receptors found in C.
elegans [p 251]
Marc R. Van Gilst, Keith R. Yamamoto
243. UNC-4 targets ACR-5 and DEL-1: Are they determinants of synaptic choice? [p 252]
Stephen E. Von Stetina, David M. Miller, III
244. Nicotine adaptation: a process involving PKC-dependant regulation of nAChR protein levels. [p 253]
Laura Waggoner, Kari Dickonson, Daniel Poole, Bill Schafer
9

245. Analysis of GLR-7, GLR-5, Ionotropic Glutamate Receptor Subunits [p 254]
Craig S. Walker, David M. Madsen, Penelope J. Brockie, Andres V. Maricq
246. Microarray analysis of gene expression patterns in dauer larvae [p 255]
John Wang, Stuart K. Kim
247. Characterization of CAN cell and excretory canal defects in mig-10(ct41) mutants [p 256]
Nicole Washington, Jim Manser
248. ric-7 encodes a novel presynaptic protein required for neurotransmission [p 257]
Robby M. Weimer, Erik M. Jorgensen
249. The requirement of synaptic vesicle loading for synaptic vesicle exocytosis [p 258]
Robby M. Weimer, Janet E. Richmond, Erik M. Jorgensen
250. Unraveling the biological role of DMWD, a gene close to the unstable CTG-repeat in the myotonic
dystrophy locus. [p 259]
J. Westerlaken, B. Wieringa, P.E. Mains
251. Calcium imaging of the defecation rhythm in C. elegans [p 260]
Jeanna M. Wheeler, James H. Thomas
252. Establishing the left/right asymmetry of Q neuroblast polarisation and migration in C. elegans [p 261]
Lisa Williams, Lee Honigberg, Cynthia Kenyon
253. A screen for cell migration and axon outgrowth mutants [p 262]
Jim Withee, Gian Garriga
254. Mapping and Characterization of had-1, an HSN Axon Guidance Gene [p 263]
Lianna Wong, Jim Rader, Gian Garriga
255. Recognition of X-chromosome-enriched DNA elements by dosage compensation proteins [p 264]
Tammy F. Wu, Jason D. Lieb, Barbara J. Meyer
256. Rac-like GTPases and cell migration [p 265]
Yi-Chun Wu, Li-Chun Cheng, Nei-Yin Weng, Ting-Wen Cheng
257. Two new genes regulating neuroblast migration in C. elegans [p 266]
Lucie Yang, Mary Sym, Queelim Ch’ng, Cynthia Kenyon
258. Identification and characterization of telomere binding proteins in the nematode C. elegans [p 267]
Su Young Yi, Seunghyun Kim, Junho Lee
259. Molecular Analysis of the Dosage Compensation Gene dpy-21 [p 268]
Stephanie Yonker, Edith Cookson, Barbara J. Meyer
260. A search for lethal synaptic function mutants using a sensitized background [p 269]
Karen Yook, Erik Jorgensen
261. Identification of downstream target genes in daf-2 pathway [p 270]
Hui Yu, Pamela L. Larsen
262. Fate specification in male P(9-11).p equivalence group [p 271]
Hui Yu, Paul W. Sternberg
263. Loss of a dynamin related protein MGM-1 causes excessive mitochondrial fragmentation [p 272]
Mauro Zappaterra, Alexander van der Bliek
264. Isolation and phenotypic analysis of syd-7 [p 273]
Mei Zhen, Nikki Alvarez, Yishi Jin
265. A screen to identify genes that regulate the activity of the C. elegans glutamate receptor GLR-1. [p
274] Yi Zheng, Heng Xie, Pene J. Brockie, Andres V. Maricq
266. A resource for C. elegans microarrays [p 275]
Stuart K. Kim, Min Jiang, Kyle Duke
Leon Avery (Leon@eatworms.swmed.edu)
Last modified: Mon Jul 24 15:24:42 2000
West Coast Worm Meeting 2000
10

West Coast Worm Meeting 2000
MEIOTIC PAIRING AND SYNAPSIS
Amy MacQueen, Anne M. Villeneuve
Department of Developmental Biology, Stanford University, Stanford CA 94305
Successful meiotic chromosome segregation relies upon a prior association between homologous
chromosomes. We want to understand how homologs establish and maintain functional associations
throughout meiotic prophase. Using cytological tools, we screened through a collection of meiotic
chromosome segregation mutants to identify mutations that specifically disrupt homologous pairing and
synapsis.
We have identified mutations in three complementation groups that cause defects in homologous
synapsis. hal-1 (homolog alignment) mutants contain chromosomes that are asynapsed in the pachytene
region of the germline, where partner chromosomes are normally aligned in parallel tracks with one
another. Fluorescence in situ hybridization (FISH) studies indicate that homologous pairing is abolished in
hal-1 germlines. Further, chromatin in hal-1 early meiotic nuclei does not undergo the distinct
morphological transition that normally accompanies the onset of pairing, suggesting that hal-1 disrupts an
early step required for the initiation of pairing.
Chromatin in nuclei entering meiosis in sys-1 or sys-2 (synapsis) mutants do exhibit typical transition
morphology, and homologs initially pair. However, as nuclei progress to later stages of meiotic prophase,
paired associations are lost. Failure to stabilize homolog pairing suggests a role for the sys-1 and sys-2
genes in chromosome synapsis. Further, the sys-1 gene encodes a protein consisting mainly of a
predicted coiled-coil domain, suggesting that it is likely a structural component of the synaptonemal
complex. Interestingly, a timecourse analysis of sys-1 meiotic nuclei using FISH probes near to either end
of chromosome I revealed that the "pairing center" end consistently achieves a higher degree of
association during meiotic prophase compared with the opposite end of the chromosome. This differential
behavior of opposite ends of a chromosome strengthens prior notions that synapsis along a chromosome
initiates at the "pairing center"end, and suggests a role for the sys-1 gene in synapsis.
Our analysis demonstrates that homolog pairing and synapsis can be subdivided into discrete steps: hal-1
gene function is required for the early establishment of homologous associations, while sys-1 and other
genes of its class are required for the maintenance of these pairing interactions.
11

West Coast Worm Meeting 2000
CONDITIONAL MITOTIC SPINDLE MUTANTS IN C. ELEGANS
Danielle R. Hamill, Bruce Bowerman
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403
The large size of the transparent early embryos and the powerful genetics of C. elegans, make it
attractive for studies of cell division. Therefore, we have isolated and observed early embryonic cell
divisions in approximately 600 temperature-sensitive, embryonic-lethal mutants in an ongoing screen in
the lab. About 35 mutants appear defective in microtubule- and/or microfilament-dependent processes,
including pronuclear migration, centrosome function, mitotic spindle assembly or orientation, and
cytokinesis. We isolated ts-alleles of several genes known to affect these processes, including an
aurora-like kinase (air-2) and an MKLP1-like kinesin (zen-4) [see abstract by A.F. Severson], as well as
several previously unidentified genes [see abstracts by J.E. Gomes and R. Lyczak].
Here we describe two temperature-sensitive mutants, spd-4 and spd-5, that are required for mitotic
spindle assembly and function (spd=spindle-defective). spd-4 mutant embryos assemble bipolar mitotic
spindles, but they are shorter than wild type and do not elongate. spd-4 mutant embryos also have
defects in DNA segregation and cytokinesis. Pronuclear migration is defective in spd-5 mutant embryos, a
mitotic spindle does not form, and the first cell division fails. Intriguingly, spd-4 and spd-5 show genetic
interactions that suggest they function together in a complex to regulate mitotic spindle assembly in the
early C. elegans embryo.
From the map position and phenotype of spd-4, as well as genetic complementation analysis (in
collaboration with D. Schmidt, S. Strome, and W. Saxton, Indiana University) we believe that spd-4 might
encode a dynein heavy chain gene, although this still needs to be confirmed. Injection of dsRNA
corresponding to the Genefinder locus F56A3.4 phenocopies the spd-5 mutant phenotype. We are
sequencing this locus to determine the molecular nature of the lesion. F56A3.4 encodes a coiled-coil
protein with no significant similarity to other proteins apart from this motif. Therefore, if spd-4 is dynein
heavy chain, spd-5 likely represents a novel dynein regulator.
12

MITOTIC CHROMOSOME SEGREGATION BY A CONSERVED
PROTEIN COMPLEX
K. Hagstrom, R. Chan, D. Pasqualone, B.J. Meyer
HHMI & MCB, UC Berkeley, Berkeley, CA 94720
West Coast Worm Meeting 2000
From bacteria to man, the highly conserved SMC (structural maintenance of chromosomes) protein family
is required for chromosome segregation and cell division. In C. elegans SMC proteins also direct X
chromosome dosage compensation. We are studying the composition and function of SMC protein
complexes and how individual SMC proteins participate in more than one chromosomal process. For
example, how does the SMC protein MIX-1, essential for both mitosis and dosage compensation, achieve
its dual function within a single cell? MIX-1 requires the SMC protein DPY-27 for its role in dosage
compensation and X localization, but DPY-27 plays no role in mitosis. Thus, it seemed likely that MIX-1
would have a different SMC partner for mitosis. Searching the C. elegans genome revealed another SMC
homolog, SLP-2 (SMC-like protein-2.)
The RNAi phenotype and protein localization of SLP-2 suggested its involvement in mitosis. Like MIX-1,
SLP-2 RNAi produces dead embryos with defects such as chromatin bridges and abnormally large nuclei.
Time-lapse microscopy shows a failure in chromosome segregation, and fluorescent in situ hybridization
reveals nuclei with abnormally high DNA content. Thus, loss of SLP-2 prevents chromosome segregation,
but not DNA replication and cell cycle progression. SLP-2 co-localizes with MIX-1 on mitotic
chromosomes in embryos and in the germline. SLP-2 and MIX-1 surround chromosomes as they
condense, then appear on the poleward face of chromosomes aligned at metaphase, where they remain
until they disappear at telophase.
The idea that MIX-1 partners with SLP-2 for mitosis, but with DPY-27 for dosage compensation, is further
supported by immunoprecipitation (IP) results. Co-IP from embryonic extracts is observed between SLP-2
and MIX-1, but not between SLP-2 and DPY-27. Moreover, IPs show that SLP-2 and MIX-1 are part of a
large protein complex. The identities of these subunits are being explored by mass spectrometry. One
subunit (see R. Chan, et al.) shares homology with both a conserved component of the mitotic complex in
other organisms, and a component of the C. elegans dosage compensation complex. It will be interesting
to learn if the mitotic and dosage compensation complexes share additional components, and to what
extent the biochemical activities of these complexes are related.
13

West Coast Worm Meeting 2000
HIM-10 A PROBABLE KINETOCHORE PROTEIN INVOLVED IN
MITOTIC AND MEIOTIC CHROMOSOME SEGREGATION
M. Howe 1 , D G. Albertson 2 , B. J. Meyer 1
1HHMI & Dept. of Mol. Cell Bio. UCB, Berkeley, CA 94720
2CRI, UCSF, San Francisco, CA 94143
The mitotic and meiotic chromosomes of C. elegans are atypical. These mitotic chromosomes are
holocentric, that is, the kinetochore (the structure mediating chromosome attachment to the spindle)
extends along the length of the chromosome. The ultrastructure of these long kinetochores is similar to
those of monocentic chromosomes common to other animals. C. elegans meiotic chromosomes have no
discernable kinetochores at the ultrastructural level. Investigation of C. elegans chromosomes may
identify conserved features of kinetochores essential to chromosome segregation in mitosis and meiosis.
To understand C. elegans kinetochores we have characterized him-10, a gene implicated in mitotic
kinetochore function by an allele that increases free duplication loss. Cloning him-10 revealed that the
gene encodes a novel protein. Protein localization and loss-of-function phenotypes are consistent with
HIM-10 playing a direct role in kinetochore function in mitosis and meiosis.
HIM-10 appears to associate with the kinetochore face of mitotic chromosomes from prophase through
anaphase. HIM-10 staining partially overlaps with a conserved centromeric histone variant HPC-3, with
HIM-10 localizing to the kinetochore region, distal to HPC-3.
him-10 RNAi treatment caused the progeny from injected animals to die as embryos. Fluorescent in situ
hybridization (FISH) to these dead embryos revealed severe aneuploidy suggesting that lethality is due to
aberrant embryonic mitosis. Tubulin staining of dsRNAi embryos showed displaced metaphase
chromosomes, unipolar chromosome attachments, and irregular nuclei. FISH and tubulin staining suggest
that loss of him-10 function causes segregation defects consistent with kinetochore failure.
him-10 is also required during meiosis. The hypomorphic mutation, him-10 (e1511ts), causes a sterility
that can be rescued by mating with wild-type males, suggesting a role for the protein in sperm meiosis.
FISH to dead embryos from the ts sterile adults revealed monosomic and trisomic embryos, suggesting
that the mutation causes chromosome loss in meiosis, not embryonic mitosis. HIM-10 encases
spermatocyte and oocyte chromosomes and duplications, suggesting that a kinetochore-like structure
functions in C. elegans meiosis.
14

West Coast Worm Meeting 2000
A C. ELEGANS CHROMOKINESIN REQUIRED FOR
CHROMOSOME SEGREGATION
Jim Powers, Bill Saxton, Susan Strome
Biology Department, Indiana University, Bloomington, IN 47401
A search of the C. elegans genome, using the motor domain of kinesin heavy chain (unc-116), which is a
microtubule motor, identified 18 distinct kinesins. Two of these, CeChromoK-A and B, are most closely
related to the vertebrate chromokinesins. Vertebrate chromokinesins bind chromosomes and can bind
microtubules in an ATP-sensitive manner. Therefore, they may function as mitotic motors. However,
strong evidence that chromokinesins actually move along microtubules has not been reported. The
CeChromoKs lack the putative DNA-binding domain that is found in vertebrate chromokinesins.
We have tested the functions of CeChromoKs in oogenesis and early embryogenesis by RNA
interference (RNAi). We detected no phenotypes after RNAi of CeChromoK-A. However, RNAi of
CeChromoK-B causes marked defects in mitosis. Anti-tubulin and anti-histone staining and observation of
GFP-tagged histone in embryos suggest that spindle formation is normal, but that chromosomes
congress poorly to form a loose metaphase plate. During anaphase, chromosomes do not disjoin
accurately, stretching along the pole-to-pole axis to form numerous anaphase bridges. Some
chromosomes fragment, giving rise to multiple micronuclei during telophase.
Antibodies to CeChromoK-B show bright staining of mitotic nuclei in the distal germline and of oocyte
nuclei in the oviduct. After fertilization, CeChromoK-B becomes concentrated between paired meiotic
chromosomes, and is left near the spindle equator as anaphase proceeds. A similar pattern is seen
during mitosis. CeChromoK-B becomes associated with chromosomes during late prophase, remains on
the chromosomes during metaphase and early anaphase, and later becomes concentrated at the spindle
equator, appearing to associate with the overlapping microtubules of the developing telophase bridge.
This concentration persists in the midbody after cytokinesis.
Our results suggest that despite the lack of a recognizable DNA-binding domain, CeChromoK-B
associates with mitotic chromatin and is important for chromosome-microtubule interactions that ensure
an ordered metaphase plate and accurate anaphase separation of chromosomes. We suspect that
CeChromoK-B modulates the interactions of microtubules with chromosome arms to resolve bipolar
chromatid attachment or chromosome catenation.
15

NUCLEAR ENVELOPE DYNAMICS IN CAENORHABDITIS
ELEGANS
Kenneth Lee, Yosef Gruenbaum, Katherine L. Wilson
*No Address*
West Coast Worm Meeting 2000
Emerin, MAN1 and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share
a 43-residue N-terminal motif, termed the LEM-domain. We found three putative LEM-domain genes in
Caenorhabditis elegans, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes
Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-Emerin and Ce-MAN1 migrate on SDS-PAGE as 17
and 52 kDa proteins, respectively. Based on their biochemical extraction properties and
immunolocalization, both Ce-emerin and Ce-MAN1 are integral membrane proteins localized at the
nuclear envelope. We used antibodies against Ce-MAN1, Ce-emerin, nucleoporins, and Ce-lamin to
determine the timing of nuclear envelope breakdown during mitosis in C. elegans. The C. elegans nuclear
envelope disassembles very late, compared to vertebrates and Drosophila. The nuclear membranes
remained intact everywhere except near spindle poles during metaphase and early anaphase, fully
disassembling only during mid-late anaphase. Disassembly of pore complexes, and to a lesser extent the
lamina, depended on embryo age: pore complexes were absent during metaphase in >30-cell embryos,
but exist until anaphase in 2-24 cell embryos. Intranuclear mRNA splicing factors disassembled after
prophase. The timing of nuclear disassembly in C. elegans is novel, and may reflect its evolutionary
position between unicellular and more complex eukaryotes.
16

THE FORMIN PROTEIN CYK-1 ACTS IN PARALLEL TO AN
AURORA-LIKE KINASE/MKLP-1 PATHWAY TO EXECUTE
CYTOKINESIS IN EARLY CAENORHABDITIS ELEGANS
EMBRYOS
Aaron F. Severson, Danielle R. Hamill, Bruce Bowerman
University of Oregon, Eugene, Oregon 97403 USA
West Coast Worm Meeting 2000
The C. elegans Formin Homology protein CYK-1 localizes to cleavage furrows in dividing embryonic cells
and, based on an analysis of partial loss-of-function mutations, is required late in cytokinesis (Swan et al.
1998). Analysis of a more severe allele indicates that CYK-1 also is required early in cytokinesis for
contractile ring assembly or function. In addition to CYK-1, embryonic cytokinesis in C. elegans requires
the Aurora-like kinase AIR-2 and the mitotic kinesin-like protein ZEN-4. Genetic interactions involving
these loci suggest that an AIR-2/ZEN-4 mitotic spindle pathway functions in parallel to a contractile ring
pathway that includes CYK-1. We have identified temperature-sensitive alleles of both air-2 and zen-4. A
temporal analysis of their function suggests that AIR-2 acts in metaphase or early anaphase, to localize
ZEN-4 to the spindle interzone, while ZEN-4 acts in cytokinesis, during late anaphase or telophase.
Intriguingly, ZEN-4 may also be required well after the apparent completion of cytokinesis, to maintain the
separation of daughter cells. We are currently using the yeast two-hybrid system to determine if AIR-2
and ZEN-4 interact directly. Additionally, we are collaborating with Dr. Jill Schumacher (University of
Texas) to determine if AIR-2 and ZEN-4 associate in stable complexes that can be immunprecipitated.
Our analysis provides genetic evidence that separable, parallel pathways coordinate microfilament and
microtubule functions during cytokinesis in an animal embryo.
17

West Coast Worm Meeting 2000
THE L TYPE CYCLIN SAG-4 IS REQUIRED FOR HEAT-SHOCK
INDUCED PROTEIN EXPRESSION
Wen J. Chen, Yvonne M. Hajdu-Cronin, Paul W. Sternberg
HHMI and Dept. of Biology, Caltech, Pasadena, CA91125, USA
In a screen for suppressors of activated GOA-1 under the control of a heat shock promoter, we identified
four genetic loci that affect heat-shock induction of GOA-1. sag-4 and sag-8 are wild type in appearance,
while sag-3 and sag-5 are egg-laying defective. Western analysis indicated that sag-4 or sag-8 mutations
suppress activated Goa by decreasing heat-shock induced protein expression. Although endogenous
GOA-1 expression is not affected, heat-shock induction of GOA-1 decreased in the suppressor strains.
We cloned sag-4 locus, which encodes a cyclin most similar to cyclin L. The latter is a novel type of cyclin
with unknown function, but also similar to cyclin T, K or C, which was identified as a subunit of TFIIH, part
of RNA polymerase II complex and functions in basal transcription. Only transgenes with hsp16-2
promoter can be affected by sag-4. These results suggest that sag-4 must suppress heat-shock GOA-1
phenotypes by preventing heat- shock mediated transcription in C. elegans. We propose that cyclin L is
the type of cyclin acting in TFIIH during heat-shock induced mRNA transcription, which carries function
similar to cyclin T, K or C during basal transcription. sag-3, sag-5 and sag-8 might also be involved in
similar processes.
18

West Coast Worm Meeting 2000
CDL-1 ENCODES A STEM- LOOP BINDING PROTEIN (SLBP)
HOMOLOG AND MAY BE ESSENTIAL FOR CORE HISTONE
EXPRESSION.
Yuki Kodama 1 , Asako Sugimoto 1 , Joel Rothman 2 , Masayuki
Yamamoto 1
1Dept. of Biophys. & Biochem., Grad. School of Science, Univ. of Tokyo, Tokyo 113, JAPAN
2Neuroscience Research Institute, Univ. of California-Santa Barbara, CA93106
cdl-1 (cell death lethal) mutants show several embryonic defects: 1) delay in appearance of cell corpses
and accumulation of cell corpses in late embryogenesis, 2) defects in elongation, 3) failure in attachment
of the pharynx to the buccal cavity. To understand the cdl-1 function, we cloned the cdl-1 gene. By
transformation assay, we found that a cosmid T19E10 could rescue the cdl-1 phenotypes. RNAi for ORFs
on T19E10 revealed that some of R06F6.1(RNAi) F1s showed cdl-1-like phenotype, although most of
them arrested at the early embryonic stage. We sequenced the corresponding region from cdl-1 mutants
and identified mutations in two alleles, thus concluding that R06F6.1 is the cdl-1 gene.
cdl-1 encodes a member of the stem-loop binding protein(SLBP) family, which binds to the 3’-stem-loop
of core histone mRNAs. It has been described that metazoan core histone mRNAs have a stem-loop
structure instead of a poly-A sequence, and SLBPs have been implicated in post-transcriptional regulation
of core histone mRNAs. In C. elegans, 58 of core histone genes contain a conserved stem-loop sequence
in their 3’-UTR sequence. We confirmed the interaction between CDL-1 protein and the stem-loop
structure by yeast three-hybrid system. This result suggests that CDL-1 may also function in the
post-transcriptional regulation of core histones.
To examine the early embryonic phenotypes in cdl-1(RNAi) embryos, we observed them by DAPI staining
and Nomarski optics. In these embryos, chromosomes were less condensed during mitosis, cytokinesis
occurred before completion of the nuclear division, and nuclear fragments existed in some blastmeres.
These observations suggest that the chromatin structure, especially its condensation, might be defective
in cdl-1(RNAi) embryos. We then performed RNAi with core histone genes, the probable targets of
CDL-1. Most RNAi embryos showed early arrest phenotype similar to cdl-1(RNAi) embryos, which
supports the hypothesis that CDL-1 regulates the expression of core histones.
We are currently trying to examine whether the original cdl-1 phenotypes are also caused by the defect of
core histone expression.
19

West Coast Worm Meeting 2000
UNC-23 IS A MEMBER OF THE BAG FAMILY OF
CHAPERONE REGULATORS
Poupak Rahmani, Donald Moerman
Department of Zoology. University of British Columbia, 6270 University Blvd. Vancouver, BC Canada.
V6T 1Z4
Mutations in the unc-23 gene result in detachment of the anterior body wall musculature and a bent-head
phenotype (Waterston et al, 1980). This phenotype is not observed when animals are grown in liquid
culture (Bullerjahn and Riddle, pers. comm.) Neither muscle cell positioning nor myofilament assembly is
affected in liquid grown unc-23 animals; muscle cell attachment, however, is affected since a small
amount of stress applied results in detachment of the muscle cells from the hypodermis.The result of
immunological staining of unc-23 animals with antibodies to basement membrane and hypodermal
components suggest that the primary defect in unc-23 animals is located within the hypodermis. We have
recently cloned unc-23, and found it to be a protein most similar to a chaperone regulator known as
BAG-2 (BCL2-associated athanogene-2). In humans, the BAG family of chaperone regulators contains a
conserved 45 amino acid region near their C termini (the BAG domain) that binds Hsp70/Hsc70 and
control their chaperone activity (Takayama et al. 1999). Human BAG-2 and UNC-23 share 40% amino
acid identity and 62% similarity over the BAG domain and its upstream region. We are currently
attempting to obtain a full-length unc-23::GFP transgenic line to study the spatial and temporal expression
pattern of this gene
20

MATERNAL UNC-45 PROTEIN CO-LOCALIZES WITH NMY-2,
A NON-MUSCLE MYOSIN AT THE CLEAVAGE FURROW OF
EARLY EMBRYOS
Wanyuan Ao, Dave Pilgrim
Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada.
unc-45 is an essential gene for normal body wall muscle thick filament development and mutants show a
partial maternal effect. The unc-45 protein product (UNC-45) contains tetratricopeptide (TPR) repeats and
similarity to fungal proteins, but its biochemical function is still unknown. We have previously shown that
UNC-45 is a component of muscle thick filaments and co-localizes with myosin heavy chain B but not
myosin heavy chain A in the body wall muscles of adult worms [1] . Previous genetic evidence also
suggests that UNC-45 may interact with myosin heavy chain isoforms in the muscle cells [2] . We show
here that UNC-45 is also contributed maternally to the embryos and present in all cells of the early
embryo. Zygotic UNC-45 is only detected in the developing muscle cells of the embryo.
Moreover, our yeast two-hybrid screens show that UNC-45 interacts specifically with NMY-2, a
non-muscle myosin. These two proteins are also co-localized at the cleavage furrow of the early embryos.
The localization of UNC-45 at the cleavage furrow is dependent on the presence of NMY-2. NMY-2 has
been previously shown to be required for embryonic polarity and cytokinesis [3,4] . Our results suggest that
the maternal UNC-45 may have a function in the early embryo which is independent of muscle function.
References
West Coast Worm Meeting 2000
1. Ao, W., and Pilgrim, D. (2000). J. Cell Biol. 148, 375-384.
2. Venolia, L., and R.H. Waterston. (1990). Genetics. 126, 345-354.
3. Guo, S., and Kemphues, K. J. (1996). Nature, 382, 455-458.
4. Shelton, C. A., Carter, J. C., Ellis, G. C., and Bowerman, B. (1999). J. Cell Biol. 146, 439-451.
21

POLYUNSATURATED FATTY ACIDS REQUIREMENTS FOR
PROPER FUNCTIONING OF THE NERVOUS SYSTEM
Jenny Watts, John Browse
West Coast Worm Meeting 2000
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340
Polyunsaturated phospholipids are critical for the function of excitable membranes. Membrane-mediated
information transfers are intimately related to the biochemical events that occur within the neuronal
plasma membrane. Polyunsaturated fatty acid components of phospholipids are necessary to create a
fluid environment as well as to provide precursors of second messenger signaling molecules. C. elegans
can synthesize a wide range of polyunsaturated fatty acids using only saturated and monounsaturated
fatty acids from E. coli as precursors. In order to study the role of polyunsaturated fatty acids in the
nervous system, we designed a unique biochemical screen which enabled us to isolate a number of
mutant lines exhibiting a range of altered fatty acid compositions. We discovered that many of the
mutations are in known desaturase genes that encode enzymes responsible for inserting double bonds
into a fatty acid chain. The phenotypes of these strains range from no apparent defects in strains lacking
specific classes of 20-carbon polyunsaturated fatty acids to severe locomotion defects and impaired
defecation in strains with more extreme alterations in fatty acid composition. These more extreme strains
also grow slowly and display temperature sensitive embryonic lethality. Providing the worms with dietary
polyunsaturated fatty acids rescues these defects. We are currently performing assays of neurological
function on the whole range of mutants, both unsupplemented and supplemented with various fatty acids.
Comparison of the worm fatty acid composition with the severity of neurological defects will allow us to
determine the polyunsaturated fatty acid requirements for proper movement and behavior.
22

West Coast Worm Meeting 2000
TESTING FUNCTIONS OF PHAGOCYTOSIS RECEPTOR
HOMOLOGS IN CELL CORPSE ELIMINATION AND GONADAL
OUTGROWTH
Sambath Chung 1 , Monica Driscoll 2
1UMDNJ-Graduate School of Biomedical Sciences, Piscataway, NJ 08855
2Rutgers University
Cell death can occur as a normal event in development or as a consequence of cell injury. Effective
elimination of cell corpses is essential for maintaining tissue homeostasis, recycling cellular metabolites,
and removing potentially harmful residual cellular contents. Both C. elegans programmed cell death
corpses and necrotic-like corpses (such as those generated by mec-4(d), deg-3(d) and other stimuli) are
removed via the action of seven engulfment ced genes--ced-1, ced-2, ced-5, ced-6, ced-7, ced-10, and
ced-12. We have been interested in identifying genes that might specifically be involved in the
recognition/elimination of necrotic cell corpses. Because the receptors that initially mediate recognition of
the necrotic cells might be different from those recognizing the morphologically distinct programmed cell
death corpses, we considered the hypothesis that a subset of nematode genes related to phagocytosis
receptor genes in other organisms might be required for recognition of necrotic cell corpses.
Mammalian CD36 and Drosophila Croquemort are related scavenger receptors that function in cell corpse
removal. We searched the C. elegans genomic database and identified six homologs of the
CD36/Croquemort family. We generated a deletion mutation affecting the gene most closely related to
CD36/Croquemort. This allele harbors a deletion of approximately 1kb, starting about 200 bp upstream of
the receptor open reading frame. We named this locus scr-1, for scavenger receptor-like. The scr-1
deletion mutant does not exhibit necrotic or programmed cell death corpse persistence, nor does this
mutation enhance corpse persistence when present in combination with any of the seven engulfment ced
mutations. Interestingly, however, a significant percentage of scr-1 mutants arrest at the L1 larval stage
and appear to have programmed cell death corpses throughout their bodies. scr-1 mutants do exhibit
distinctive defects in distal tip cell migration, similar to that observed in ced-2, ced-5, ced-10, and ced-12
engulfment mutants. This observation suggests that SCR-1 might function as a receptor important in
gonadal outgrowth in the process involving CED-2, -5, -10, -12.
We have also tested for effects of the other five scr homologs using RNAi.
23

REGULATION OF CELL FUSION IN C. ELEGANS
Scott Alper, Cynthia Kenyon
University of California, San Francisco
West Coast Worm Meeting 2000
Cell fusion is a common process in C. elegans. The C. elegans hypodermis consists of several
multinucleate syncytia that are generated by the fusion of cells throughout development. The largest
syncytium is hyp7, which spans most of the length of the worm and which contains more than 130 nuclei.
We are carrying out two screens to identify mutations affecting cell fusion. First, we screened for
mutations that prevent fusion of the Pn.p cells with hyp7 on the ventral surface of the worm. Mutations
identified in this screen affect the decision of these cells to fuse. Second, in order to identify mutations in
genes that carry out the cell fusion process, we are screening for mutations that affect fusion of the seam
cells that line the lateral surface of the worm.
We identified several mutations that affect the pattern of Pn.p cell fusion and have been characterizing
two mutations that affect Pn.p cell fusion by altering Hox protein activity. The fusion decision of the 12
Pn.p cells is controlled by two Hox genes, lin-39 and mab-5. lin-39 is expressed in the mid-body [P(3-8).p]
and in hermaphrodites prevents fusion of these cells. mab-5 is expressed more posteriorly [in P(7-11).p]
in both sexes, but is not active in hermaphrodite Pn.ps. In ref-1(mu220) (REgulator of Fusion)
hermaphrodites, P9.p and P10.p fail to fuse with hyp7. This is due, in part, to inappropriate activation of
MAB-5 in ref-1 hermaphrodites. ref-1 encodes a gene with two basic-helix-loop-helix DNA binding
domains of the hairy/E(spl) family.
In males, lin-39 and mab-5 each individually prevent Pn.p cell fusion in P(3-6).p and P(9-11).p,
respectively. However, in P7.p and P8.p, where both Hox genes are expressed in the same cell, they
somehow neutralize one another’s activities, so that P7.p and P8.p fuse with hyp7. In ref-2(mu218)
males, P7.p and P8.p fail to fuse with hyp7, perhaps because LIN-39 and MAB-5 fail to cancel each
others activities. ref-2 has been mapped to a 40 kb region on the center of the X chromosome and
transformation rescue experiments are in progress.
Descendants of the seam cells fuse with hyp7 at all larval stages and ultimately the seam cells fuse with
each other during L4. We are currently using a membrane localized gfp fusion expressed in the seam
cells to identify mutants in which seam cell fusions fail to occur.
24

ETHANOL SENSITIVITY GENES IN CAENORHABDITIS
ELEGANS
MinGi Hong, JaeYoung Kwon, InYoung Lee, MinSung Choi, Junho Lee
Department of Biology, Yonsei University
West Coast Worm Meeting 2000
The mechanisms and sites of action of volatile anesthetics and ethanol are not fully understood. In the
hope of understanding the mechanisms of ethanol, we first identified genes that control sensitivity to
ethanol and anesthetics in the invertebrate system Caenorhabditis elegans. We identified 10 mutations
that confer ethanol resistance either by EMS mutagenesis or transposon insertion mutagenesis. The
genes are being cloned by positional cloning and analyses on the mutations are under way. In the next
experiments, we used the cDNA microarray to identify genes that are either up-regulated or
down-regulated by exposure of the animals to 7 % ethanol for 6 hours. Several gene families including
heat-shock protein family, glutamate receptor family, and gene families with unknown function, were
up-regulated by ethanol. Also, there are gene families down-regulated. We are now examining these
candidate ethanol-affected genes by northen analysis and GFP reporter analysis. To establish an
experimental system by which one can study Fetal Alcohol Syndrome using the nematode, we examined
the effect of ethanol on embryogenesis. After incubating adult hermaphrodites in 7% EtOH for 12 hours,
we observed egg-laying defects and abnormal embryogenesis. Based on this preliminary data, we will
investigate and characterize the ethanol sensitivity genes involved in embryogenesis. In summary, we
identified ethanol resistance genes by EMS or tensposon mutagenesis; we identified genes whose
transcription levels are altered by ethanol in the microarray analysis; and we established an experimental
model system to study Fetal Alcohol Syndrome.
25

STATE-DEPENDENT LEARNING IN C. ELEGANS.
Jill C. Bettinger, Steven L. McIntire
Gallo Center and Program in Biological Sciences, Department of Neurology, UCSF
The execution of learned behaviors may be triggered by contextual information, consisting of
environmental cues or the internal state of the organism. State-dependent learning refers to the ability of
an organism to more effectively execute learned behaviors if the organism experiences internal contextual
influences similar to those experienced when the learning occurred. Such contexts can be
pharmacologically manipulated by treating with cholinergic compounds, opiates, cocaine and
amphetamines, and with neurodepressants such as ethanol and barbituates 1 . Worms become
intoxicated by ethanol in a manner similar to that of most other organisms tested (see abstracts by A.
Davies and H. Kim, this meeting). We have sought to study the mechanisms of ethanol-induced
state-dependent learning in worms.
Olfactory adaptation in C. elegans is a decrease in the chemotaxis response to an odorant as a result of
prior exposure to the odorant 2 . We demonstrate a form of state-dependent learning in worms by pairing
olfactory adaptation and ethanol administration. Ethanol does not interfere with olfactory adaptation,
however, worms exposed to an odorant while being treated with ethanol will only show subsequent
adaptation to the odorant if ethanol is again administered during chemotaxis testing. If the odorant is
presented without ethanol during testing, the animals behave as naïve animals and therefore fail to alter
their behavior based on their previous experience or prior exposure to the odorant.
Further, we demonstrate that the state-dependent effects of ethanol require normal dopaminergic
function. The dopamine-defective cat-1 4 and cat-2 5 mutants are able to adapt to volatile odorants,
however, they do not show state-dependency when they are adapted to volatile odorants while
intoxicated by ethanol. These results suggest that C. elegans is capable of a form of learning and indicate
a conserved role of dopamine in the modulation of behavioral responses to ethanol.
1 Izquierdo. in Neurobiology of Learning and Memory (eds Lynch, McGaugh, and Wienberger) 333
(Guilford, New York, 1984).
2 Shulz, Sosnik, Ego, Haidarliu and Ahissar (2000) Nature 403, 549, and references therein.
3 Colbert and Bargmann (1995) Neuron 14, 803.
4 Duerr et al. (1999) J. Neuroscience 19, 72.
West Coast Worm Meeting 2000
5 Lints and Emmons (1999) Development 126, 5819.
26

West Coast Worm Meeting 2000
THE UT236 MUTANT IN C. ELEGANS HAS DEFECTS IN THE
INTERACTION OF TWO SENSORY SIGNALS AND AN
ASSOCIATIVE LEARNING.
Takeshi Ishihara 1 , Yuichi Iino 2 , Isao Katsura 1
1Struct.Biol.Cent., Natl.Inst.Genet., Dept of Genet. Grad.Univ.Adv.Stud.
2Mol. Genet. Res. Lab., Univ. of Tokyo
Among many environmental stimuli, C. elegans seems to respond to only a few stimuli simultaneously. To
elucidate how the neural circuit processes many sensory signals to respond properly, we have designed a
new assay system to analyze interaction between two responses: chemotaxis to odorants and avoidance
from Cu 2+ ion. In this assay, the behavior depends on the concentration of both stimuli, which are sensed
by different neurons. Our observation suggests that these two responses repress each other. We can
argue that this mutual interaction takes place in a defined part of the neural circuit, which consists of
about 10 pairs of neurons.
One mutant, ut236, has defects in this interaction. It prefers to avoid Cu 2+ ion rather than to go to
odorants, while its dose responses to the odorants and Cu 2+ ion are indistinguishable from those of the
wild type. Positional cloning revealed that the ut236 gene encodes a novel secretory protein (C36B7.7)
with an LDL receptor ligand binding motif. Expression studies with a functional GFP fusion gene and by
immunostaining show that it is expressed in a few sensory neurons and AIY neurons. By using cell type
specific promoter, we found that the expression of C36B7.7 in AIY or ASE is sufficient for the rescue of
this phenotype. Also, by using a heatshock promoter construct, it is shown that the expression of
C36B7.7 in embryonic stage is not sufficient for the rescue but the expression in the late larval or adult
stage is. These results suggest that C36B7.7 is necessary for the neuronal function of these neurons, but
is not for the development of the nervous system.
The ut236 mutant seems to have another defect in an associative learning by paired presentation of NaCl
and starvation. This phenotype can be also rescued by the C36B7.7 transgene. Therefore, we postulate
that this novel secretory protein is involved in the modification of sensory signals in the neural circuit to
regulate their preference in chemical stimuli.
27

MUTATION IN THE LIM HOMEOBOX GENE LIM-6 DISRUPTS
ASYMMETRIC FUNCTION OF THE ASE CHEMOSENSORY
NEURONS
J.T. Pierce-Shimomura, M.R. Gaston, B.J. Pearson, S.R. Lockery
Institute of Neurosci, Univ of Oregon, Eugene, OR 97405
C. elegans detects chemicals with 11 pairs of bilaterally symmetric sensory neurons. All of the right-left
members of these pairs share similarity in lineage, morphology, and synaptic connectivity. However, one
of these pairs (ASE) expresses genes asymmetrically 1 . ASEL expresses two putative chemoreceptor
guanylyl cyclases gcy-6&7 and the homeobox gene lim-6, while ASER expresses a different guanylyl
cyclase gcy-5. The ASE neurons are important for chemotaxis to soluble attractants including salts 3 . We
tested whether the asymmetry in expression pattern correlated with an asymmetry in function by ablating
ASER, ASEL, or both neurons in individual worms and tracked each animal during chemotaxis in
gradients of ammonium chloride, sodium acetate, potassium acetate, and ammonium acetate. To
measure chance performance, we tracked worms (n=74) in the absence of a gradient, but analyzed them
as if they were in a gradient. Most ASER- (n=29) and ASE- (n=27) worms failed to reach the peak of the
NH 4Cl gradient, while most ASEL- worms (n=22) reached the peak similar to sham worms
(anaesthetized and recovered but not ablated; n=35). Likewise, most ASER- (n=17) and ASE- (n=15)
worms failed to reach the peak of the K-acetate gradient, while most ASEL- worms (n=20) reached the
peak similar to sham worms. Surprisingly, most ASEL- (n=26) and ASE- (n=12) worms failed to reach the
peak of the Na-acetate gradient, while most ASER- worms (n=25) reached the peak similar to sham
worms. Worms did not perform better than chance in the ammonium-acetate gradient (n=37). Thus,
ASER controls chemotaxis to Cl - and K + , while ASEL controls chemotaxis to Na + . In lim-6 deletion
mutants gcy-5 is ectopically expressed in ASEL, while gcy-6&7 expression is correctly restricted to
ASEL 2 . To examine if LIM-6 specifies asymmetry in ASE function, we tested whether ASE neurons retain
their asymmetric function in lim-6 mutants. In contrast to wildtype worms, many ASER- lim-6 worms
(n=28) were able to reach the peak of the ammonium chloride gradient. Thus, a homeobox gene is
involved in breaking symmetry in neuronal function.
1. Yu et al (1997) PNAS 95:3384-7
2. Hobert et al (1999) Dev 126:1547-62
3. Bargmann & Horvitz (1991) Neuron 7:729-42
West Coast Worm Meeting 2000
28

West Coast Worm Meeting 2000
INFORMATION CODING IN THE C. ELEGANS OLFACTORY
SYSTEM
PD Wes, A Sagasti, G Jansen, RHA Plasterk, CI Bargmann
HHMI and Department of Anatomy. University of California, San Francisco, CA 94143-0452
In order to better understand the molecular and cellular basis of behavior, we have begun to genetically
scrutinize odorant discrimination in C. elegans. C. elegans senses a broad array of attractive odors with
just two pairs of sensory neurons, AWA and AWC. Each member of the pair are thought to be largely
identical, except that the G protein-coupled receptor, STR-2, is stochastically expressed in either the left
or right AWC neuron. A number of odorants activate each neuron. Isoamyl alcohol (iaa),
2,3-pentanedione (pd), butanone (bu) and benzaldehyde (bz) all activate AWC. Each odorant utilizes the
same cGMP signaling cascade, yet animals retain the ability to distinguish amongst some of these
odorants. For instance, when placed in a uniform field of bu, animals will no longer chemotax toward a
point source of bu (defined as saturation), yet will chemotax toward bz. A screen was conducted to
identify mutants that failed to chemotax toward a point source of bz in a field of bu (defined as
cross-saturation). One mutant, ky542, displayed total cross-saturation, as well as defects in pd
chemotaxis and olfactory adaptation. ky542 was cloned and found to be an allele of nsy-1, a "neuronal
symmetry" mutant in which STR-2 is expressed in both AWC neurons. Other nsy mutants, including
nsy-2, nsy-3 and egl-2(gf), also showed cross-saturation phenotypes. One model proposes that odorant
discrimination is achieved by expressing the bu receptor in the STR-2 "ON" cell, the pd receptor in the
STR-2 "OFF", and the bz receptor in both cells. A variety of forward and reverse genetic studies and
ablation experiments support this model. Nevertheless, neuronal asymmetry cannot fully account for
odorant discrimination since the 2 AWC neurons can distinguish at least 4 classes of odorants. For
instance, iaa is distinguished from bu, bz and pd. Therefore, informational processing must also occur
intracellularly. Since at least 6 Ga proteins are expressed in AWC, we reasoned that this protein family
may provide the requisite degree of diversity for signaling specificity. Indeed, gpa-5 is required for
discrimination between iaa and bu in AWC, suggesting that different odorants may employ specific
modulatory signaling pathways within single cells.
29

ROLES OF OSM-9/CAPSAICIN RECEPTOR FAMILY
MEMBERS IN SENSORY BEHAVIORS
D. Tobin 1 , D. Madsen 2 , G. Moulder 3 , R. Barstead 3 , A.V. Maricq 2 , M.
deBono 4 , C. Bargmann 1
1University of California San Francisco, HHMI
2University of Utah
3Oklahoma Medical Research Foundation
4MRC, Cambridge
West Coast Worm Meeting 2000
The osm-9 gene is required for a wide range of sensory modalities in C. elegans including
chemosensation, mechanosensation, osmosensation, and certain forms of olfactory adaptation. osm-9
encodes a putative ion channel with striking homology to the vertebrate capsaicin receptor, a cation
channel expressed in pain-sensing neurons and gated by the active component of chili peppers. In the
amphid neuron AWA, OSM-9 localizes to sensory cilia, suggesting a direct role in sensory transduction.
The vertebrate capsaicin receptor VR1 responds to heat, capsaicin, and low pH. Expression of
mamamlian VR1 under an AWA-specific promoter partially rescued osm-9’s AWA defects. Thus, the
homology between the two proteins may have functional relevance. However, OSM-9 is not sensitive to
capsaicin. We found that expression of mammalian VR1 in C. elegans nociceptive neurons created a
robust capsaicin-avoidance behavior. Heterologous expression of VR1 should be a useful tool for specific,
drug-inducible neuronal activation.
Four relatives of osm-9 are each expressed in subsets of osm-9-expressing cells. We call these genes
ocr (osm-9/capsaicin receptor-related)genes and have generated mutations in two of them. ocr-1 is
expressed primarily in AWA while ocr-2 is expressed in AWA, ASH, ADL, and ADF. Based on their
expression patterns and similarity to osm-9 we hypothesized that the OCR channels might coassemble
with OSM-9 to form heteromultimeric complexes. Mutations in ocr-2 recapitulate some of osm-9’s defects:
there are dramatic defects in AWA-mediated chemotaxis and ASH-mediated avoidance behaviors. We
propose that different combinations of subunits may account for the distinct functions of osm-9 in different
sensory neurons.
Interestingly, ocr-2 has a role in C. elegans social behavior. npr-1 encodes a putative neuropeptide
receptor that regulates the choice between solitary and social foraging behavior. osm-9 and ocr-2 mutants
suppress the clumping behavior of npr-1 mutants. We have performed single-cell rescue experiments to
identify the neurons in which ocr-2 expression is required for social behavior. Identification of these
neurons should help define the sensory signals and neuronal circuitry that mediate clumping.
30

EXECUTION AND REGULATION OF MALE C. ELEGANS
SPICULE MUSCLE CONTRACTIONS DURING MATING
L. René García, Paul W. Sternberg
West Coast Worm Meeting 2000
HHMI & California Institute of Technology, Division of Biology, Pasadena, CA 91125 U.S.A.
As the C. elegans male attempts to penetrate the hermaphrodite vulva with his spicules, the protractor
muscles that are attached to these copulatory structures contract and relax rapidly such that the spicules
prod the vulva slit at a frequency of 7.2 ± 1.3 Hz. Phasic protractor muscle contractions persist until the
vulva slit is partially penetrated. Once the vulva is breached, the protractor muscles contract tonically and
the spicules extend completely into the hermaphrodite. The spicule muscles stay contracted for 75 ± 20
seconds, sufficient time for sperm to transfer into the hermaphrodite. The male protractor muscles are
innervated by the SPC motor neurons (2), and removal of these cells results in male impotency (1). The
SPC neurons are essential for tonic, but not phasic spicule muscle contractions; the spicules of
SPC-ablated males can not fully extend into the hermaphrodite, but can prod the vulva at a frequency of
5.1 ± 1.1 Hz.
The ACh agonists levamisole, nicotine, arecoline, and oxotremorine can stimulate the protractor muscles
to contract; and the SPC motor neurons support the expression of the unc-17-encoded VAChT. Therefore
ACh most likely regulates the contractile state of the spicule muscles. The ACh agonists differentially
require egl-19 and unc-68-encoded calcium channels to mediate the behavioral output; thus suggesting
that calcium mobilization from these channels might have non-overlapping roles during copulation.
During mating the spicules of egl-19 males can prod the vulva at a frequency of 5.0 ± 1.5 Hz, but they can
not breach the vulva lips. In contrast, the spicules of unc-68 males can insert into the hermaphrodite, but
prior to penetration, the spicules prod the vulva at a frequency of 0.48 ± 1.4 Hz. Therefore, the UNC-68
channel is utilized in phasic muscle contractions, and the EGL-19 channel is required for tonic muscle
contractions.
1. Liu, K.S., and Sternberg, P.W. (1995).Neuron 14, 79-89.
2. Sulston, J.E., Albertson, D.G., and Thomson, J.N. (1980). Dev. Biol. 78, 542-576.
31

West Coast Worm Meeting 2000
GENETIC ANALYSIS OF NICOTINE ADAPTATION IN C.
ELEGANS.
Jinah Kim 1 , Laura E. Waggoner 2 , Kari A. Dickinson 2 , Daniel S.
Poole 2 , William R. Schafer 3
1Department of Neuroscience, University of California, San Diego, La Jolla, CA.
2Department of Biology, University of California, San Diego, La Jolla, CA.
3Departments of Biology and Neuroscience, University of California, San Diego, La Jolla, CA.
In many organisms, prolonged exposure to nicotine causes long-lasting changes in the abundance and
functional activity of nicotinic receptors, processes thought to underlie nicotine addiction in humans. At
present, the molecular basis for nicotine adaptation is poorly understood. We have begun using a genetic
approach to identify molecules required for nicotine adaptation in the egg-laying circuitry of C. elegans.
Acute exposure to nicotine or the nicotinic agonist levamisole have dramatic effects on behavior, including
stimulation of egg-laying. These acute effects of nicotine on egg-laying require the activity of a nicotinic
receptor containing subunits unc-29, unc-38, and lev-1, as mutations in these genes confer resistance to
nicotine and levamisole. This receptor appears to function in the vulval muscle, since expression of an
unc-29 transgene under a vulval muscle-specific promoter in unc-29 mutant animals restored egg-laying
sensitivity to levamisole. Upon long-term nicotine treatment, wildtype worms undergo adaptation, and
acquire a long-lasting loss of egg-laying sensitivity. This effect is at least partially mediated at the level of
receptor abundance, as the expression of an UNC-29:GFP chimera was drastically reduced upon
overnight treatment with nicotine.
In order to identify molecules required for the long-term effects of nicotine, we examined known mutants
and screened for new mutants with defects in adaptation. From the known mutants, tpa-1, a PKC
homologue, was shown to be necessary for the control of UNC-29 receptor abundance, since tpa-1
mutants remained sensitive to levamisole and retained high UNC-29 receptor levels in the vulval muscles
even after long-term nicotine treatment. We also identified a new gene, nic-1, in a screen for adaptation
defective mutants. These mutants displayed hypersensitivity to nicotine and a defect in adaptation, as
well as an unusual locomotive phenotype and a deficiency in male mating ability, all of which are
consistent with a defect in cholinergic function in the neuromusculature. Currently, we are in the process
of mapping nic-1 in order to clone it, and hope to discover the identity of the molecule encoded by this
gene.
32

NEURAL CONTROL OF LOCOMOTION IN C. ELEGANS
Saleem Mukhtar 1 , Jane Mendel 2 , Jehoshua (Shuki) Bruck 1 , Paul W.
Sternberg 2
1Mail Code 136-93, Caltech, Pasadena. CA 91125.
2Biology 156-29, Caltech, Pasadena. CA 91125.
West Coast Worm Meeting 2000
Our long-term goal is to understand the functioning of the neural circuit responsible for movement in C.
elegans. We hope to accomplish this by perturbing the nervous system and observing what effect this has
on the dynamics of the worm.
We have developed an automated worm tracking system that enables us to record moving worms. We
have also developed an automated worm recognition system that enables us to extract quantitative data
describing the worm’s movement from these videos. Using these tools we have been able to uncover
important constraints on the dynamics of wildtype worms that are imposed by the underlying neural
circuitry. Based on these constraints we have formulated a set of physically relevant metrics that can be
used to quantitatively compare the dynamics of two sets of worms.
By applying these tools to the full spectrum of perturbations - genetic, pharmacological and cell ablations,
we hope to elucidate the functioning of the ventral cord circuit for locomotion.
33

ANALYSIS OF GLUTAMATERGIC NEUROTRANSMISSION BY
KNOCKOUT OF GLUTAMATE TRANSPORTER GENES.
Itzhak Mano, Monica Driscoll
West Coast Worm Meeting 2000
Department of Molecular Biology & Biochemistry, Rutgers University, Piscataway, NJ.
The amino acid L-Glutamate (Glu) is a neurotransmitter that mediates most of the excitatory
neurotransmission in the human brain and is thus central to development, basic physiology and higher
brain functions. Exaggerated activation of glutamatergic transmission leads to neuronal cell death (in a
process termed excitotoxicity) and is believed to be a cause or a contributing factor to many acute and
chronic neurological disorders, including stroke and ALS. In order to ensure accurate response to rapid
Glu signals and to avoid buildup of toxic levels of this transmitter, synaptic Glu is rigorously pumped out of
the synapse by specialized Glu Transporters (GluTs). The molecular components of Glu synapses, and in
particular GluTs, are highly conserved from nematodes to humans, suggesting that a detailed description
of the molecules and the processes involved in normal and pathological Glu neurotransmission in C.
elegans might be used to gain insight to these processes in higher animals.
Since a key feature common to the initial steps of many neurodegenerative disorders is a decline in GluT
efficacy, we decided to create synaptic Glu buildup by systematic knockout of C. elegans GluT genes. We
have so far knocked out 3 of the 6 GluT genes in the worm, and we are continuously screening additional
deletion libraries for more GluT mutants. Analysis of the phenotypes of the current GluT deletion mutants
by themselves and in combination with other Glu-related mutants is underway. Initial observations
indicate that GluTs are key regulators of pharyngeal pumping and of the responses to a range of
chemical, osmotic and mechanical stimuli. Together with other studies of Glu neurotransmission in C.
elegans, these observations serve as initial steps for a detailed molecular and cellular description of key
processes of synaptic function and behavior. Furthermore, the phenotypes of these GluT knockout
mutants can serve as the bases for genetic screens aimed at the identification of additional components
of the pathways involved in normal and pathological Glu neurotransmission.
34

West Coast Worm Meeting 2000
ELECTROPHYSIOLOGICAL ANALYSIS OF C. ELEGANS
IONOTROPIC GLUTAMATE RECEPTORS
Jerry E. Mellem, Penelope J. Brockie, David M. Madsen, Andres V.
Maricq
Dept. of Biology University of Utah 257 S. 1400 E. Salt Lake City UT 84112
A fundamental problem in neurobiology is to understand how neuronal circuits function to control
behavior. A simple neuronal circuit that controls movement has been identified in the nematode
Caenorhabditis elegans. In this circuit, the command interneurons AVA, AVB, AVD, AVE, and PVC are
essential for coordinated movement and escape from tactile stimuli.
Our lab has shown that six putative ionotropic glutamate receptor subunits are expressed in the command
interneurons and that perturbation of the subunits nmr-1 and glr-1 lead to altered locomotion (see abstract
by Brockie et. al.). To better understand how ionotropic glutamate receptors modulate the activity of the
locomotory control circuit, we are undertaking an electrophysiological analysis of the receptors expressed
in the command interneuron AVA.
We have modified the slit worm dissection to isolate neurons of the locomotory control circuit. Using
patch-clamp techniques, we have recorded glutamate-dependent currents from the neuron AVA. The
current can be elicited by glutamate, kainate, and NMDA and can be partially blocked by selective
pharmacological antagonists. The current rapidly desensitizes to glutamate and recovery from
desensitization is slow. The current evoked by NMDA is voltage dependent and exhibits an outward
rectification.
We have also determined the electrophysiological defects underlying the behavioral phenotypes observed
in the glr-1 and nmr-1 deletion mutants. Both mutants display a diminshed response to glutamate. The
nmr-1 mutant, in addition, has an aberrant response to NMDA.
We will describe the dissection used to isolate the command interneuron circuitry, our findings on how
glutamate receptors function in wild type and mutant animals, and our initial insights into how glutamate
receptors modulate the activity of the locomotory control circuit.
35

ELECTROPHYSIOLOGICAL ANALYSIS OF UNC-18
MUTANTS.
J. E. Richmond, R. Weimer, W. S. Davis, E. M. Jorgensen
University of Utah, Salt Lake City, Utah 84112
West Coast Worm Meeting 2000
UNC-18 is a C. elegans neuron-specific protein belonging to a conserved protein family implicated in
vesicle fusion. UNC-18 interacts with the SNARE protein syntaxin, which is believed to mediate fusion of
synaptic vesicles with the plasma membrane. Mutations in unc-18 result in severe locomotory defects and
acetylcholine accumulation, implying a critical role for UNC-18 in synaptic transmission. Three mutants
were examined, unc-18(e81 and e234) which have premature stop codons and unc-18(b403) which
disrupts binding to syntaxin. The mutant phenotype was not caused by developmental abnormalities since
GFP-expression patterns revealed motor neurons had normal morphology and exhibited wild-type
distributions of synapses. We have used electrophysiological techniques to analyze synaptic function in
these unc-18 mutants. Evoked release at the C. elegans neuromuscular junction was reduced to 5% of
wild-type amplitudes (from 1800pA to 100pA) in all three mutants. A similar reduction in endogenous
synaptic event frequency (from 44Hz to 5 Hz) was observed. The mutant synaptic event amplitudes were
unaffected demonstrating that the reduction in the evoked response was not due to a defect in vesicle
neurotransmitter filling or in post-synaptic receptor sensitivity. Preliminary EM data showed an
accumulation of vesicles at the neuromuscular synapses of unc-18 mutants consistent with a defect in
exocytosis as opposed to defects in vesicle biogenesis or retrieval. In the absence of external Ca2+, the
rates of spontaneous fusion events were also reduced, suggesting that either the docking or fusion
competence of unc-18 mutants is defective. Given these data, how might UNC-18 function in exocytosis?
UNC-18 forms a heterodimer with syntaxin, which is mutually exclusive of SNARE complex formation.
Several studies suggest that the interaction of UNC-18 and syntaxin is an essential step in regulated
release, and that this interaction may be required to promote a conformational change in syntaxin which
facilitates SNARE complex formation. To test this hypothesis, we have engineered a double mutation in
syntaxin, which, in vitro eliminates UNC-18 binding, and induces the syntaxin open configuration. We are
currently examining the effects of this mutation in wild-type, unc-64 (syntaxin) and unc-18 mutants.
36

ELECTROPHYSIOLOGICAL ANALYSIS OF SEROTONIN
MODULATION OF BODY WALL NEUROMUSCULAR
PHYSIOLOGY.
Jon Madison, Joshua Kaplan
361 LSA, Univ. of Cal., Berkeley, CA 94720
The ability of neurons to alter their synaptic function underlies our ability to control behavior. We are
interested in understanding how neurotransmitters like serotonin (5-HT) cause changes in neuron and
synapse function to effect behavioral changes. In C. elegans, 5-HT modulates the rate of locomotion.
Recent analysis has shown that 5-HT acts presynaptically on motor neurons through a G-protein Gao
subunit, GOA-1, to reduce acetylcholine (ACh) release at body wall neuromuscular junctions (NMJs) [1].
Using a recently developed dissection technique and whole cell voltage clamp recordings [2], we have
recorded the effects of 5-HT on adult body wall NMJ physiology.
We have recorded from body wall muscle both miniature excitatory post-synaptic currents (mEPSCs) and
muscle responses to ACh application in the presence of 5-HT. We see two effects of 5-HT: a reduction in
EPSC frequency and a reduction in ACh-activated current amplitude. The amplitude of ACh-activated
current is reduced by 40% in the presence of 5-HT. Previous physiological analysis by Richmond and
Jorgensen has shown there to be two classes of ACh receptors at the body wall NMJ; one ACh receptor
class is sensitive to the cholinergic agonist levamisole and one receptor is insensitive [2]. Recordings of
levamisole-activated current show that 5-HT reduces this current up to 90%. These data suggest that the
levamisole activated ACh receptors may be specifically modulated by 5-HT. To resolve the contribution of
post-synaptic modulation by 5-HT to presynaptic changes in mEPSC frequency and to further understand
the mechanism of the post-synaptic response, we have recorded from mutants that might eliminate the
post-synaptic ACh receptor modulation. Since 5-HT’s downstream effects are mediated by G-protein
coupled pathways, we have tested a number of candidate signal transduction mutants expressed in
muscle for defects in ACh receptor modulation. We have tested the G-protein Ga subunit mutants, gpa-14
and gpa-7, and an adenylate cyclase, acy-1, and all are normal for ACh receptor modulation by 5-HT.
Future physiologic and genetic experiments will help further our understanding of 5-HT’s modulation of
synaptic function and behavior.
1. Nurrish et al., Neuron 24: 231-242 1999.
West Coast Worm Meeting 2000
2. Richmond and Jorgensen, Nat Neurosci 2: 791-797 1999.
37

SEROTONIN SIGNALING IN THE PHARYNX
Timothy Niacaris 1,2 , Leon Avery 1,3
West Coast Worm Meeting 2000
1University of Texas Southwestern Medical Center, Department of Molecular Biology, Dallas, TX
75390-9148
2tim@eatworms.swmed.edu
3leon@eatworms.swmed.edu
Serotonin increases the rate of pharyngeal pumping. This effect occurs by at least two different
mechanisms. Serotonin can indirectly stimulate pharyngeal muscle by increasing the activity of the
pharyngeal motorneuron MC. Serotonin can also act independent of the pharyngeal nervous system to
increase pharyngeal pumping rate, presumably by acting directly on pharyngeal muscle. Serotonin has
two additional MC-independent effects on the pharynx. It decreases the duration of pharyngeal
contractions and enhances the effect of the pharyngeal motorneuron M3. To identify the receptor that
mediates these effects, we have searched the genomic sequence for candidate serotonin receptors and
determined their expression patterns. One strong candidate for mediating the effects of serotonin on
pharyngeal muscle is ser-1. ser-1 is expressed predominantly in pharyngeal muscle and has recently
been determined to be responsive to serotonin in a heterologous expression system 1 . We have isolated
a deletion that eliminates the 3’ end and UTR of ser-1. ser-1(ad1675) truncates the receptor and
eliminates a large portion of the C-terminal intracellular tail, a region important for several aspects of
receptor desensitization. ser-1(ad1675) mutants have an exaggerated response to exogenous serotonin,
consistent with an inability to desensitize in the presence of serotonin. We are currently characterizing
worms defective in other components of the G-protein signaling cascade to identify proteins responsible
for mediating the effects of serotonin on pharyngeal muscle.
1 Hamdan et al. (1999) J. Neurochemistry 72(4):1372-83
38

A MUSCARINIC CONTRIBUTION TO THE REGULATION OF
FEEDING
Kate Steger 1,2 , Leon Avery 3
West Coast Worm Meeting 2000
1Department of Biology, McGill University. 1205 Dr. Penfield Ave. Montreal, QC, Canada
2kate@eatworms.swmed.edu
3Department of Molecular Biology, UT Southwestern Medical Center. 6000 Harry Hines Blvd. Dallas, TX
Acetylcholine (Ach) is essential for worms’ survival and can stimulate feeding through nicotinic receptors
on the pharyngeal muscle. However, we suspect that Ach affects the pharynx through muscarinic
receptors as well. Worms that lack Ach (e.g. cha-1 mutants) have a more severe feeding defect than
worms that lack pharyngeal nicotinic transmission (eat-2; eat-18). Atropine, a muscarinic antagonist,
causes worms to appear starved, although it increases their pumping rate.
Three ORFs in the worm genome closely resemble vertebrate muscarinic receptors: C15B12.5 (acm-1),
F47D12.2 (acm-2) and C53A5.12 (acm-3). We have examined the expression of these three genes: all
three are expressed in pharyngeal tissue (muscle, neurons or both) and in the extra-pharyngeal nerve
ring.
We have obtained mutant alleles of two putative muscarinic receptors: acm-1, and acm-2 (a gift from
Stefan Eimer and Rolf Baumeister). The two mutations have very different effects on worm physiology.
acm-2 worms are hypersensitive to aldicarb (an inhibitor of cholinesterase) and to nicotine. They pump
more rapidly than wild type worms in the absence of drug, and decrease their pumping rate in the
presence of atropine. We suspect that acm-2 mutants are defective in down-regulating nicotinic
transmission. We hypothesize that acm-2, which is expressed in neurons both inside and outside the
pharynx, acts as a negative regulator at nicotinic synapses.
acm-1 mutants, in contrast, are resistant to aldicarb, and are not hypersensitive to nicotine. In an unc-17
background of reduced cholinergic transmission, acm-1 worms are hypersensitive to atropine. We
suspect that acm-1, probably in combination with acm-3, affects pumping efficiency or the adaptation of
feeding behavior to particular circumstances. We are currently designing assays and appropriate genetic
backgrounds to examine these possibilities
39

West Coast Worm Meeting 2000
WORMBASE: FROM ACEDB TO A MORE COMPLETE AND
USABLE DATABASE
Paul W. Sternberg, Erich Schwarz, Norma Foltz, WormBase
Consortium
Division of Biology 156-29, Caltech, Pasadena, CA 91125
We briefly describe the current status and plans for WormBase, initially an extension of the existing
ACeDB database with a new user interface. The WormBase consortium includes the team that developed
ACeDB (Richard Durbin and colleagues at the Sanger Centre; Jean Thierry-Mieg and colleagues at
Montpellier); Lincoln Stein and colleagues at Cold Spring Harbor, who developed the current web
interface for WormBase; and John Spieth and colleagues at the Genome Sequencing Center at
Washington University, who along with the Sanger Centre team, continue to annotate the genomic
sequence. The Caltech group will curate new data including cell function in development, behavior and
physiology, gene expression at a cellular level, and gene interactions. Data will be extracted from the
literature, as well as by community submission. We look forward to providing the C. elegans and broader
research community easy access to vast quantities of high quality data on C. elegans. Also, we welcome
your suggestions and criticism at any time. WormBase can be accessed at www.wormbase.org.
40

West Coast Worm Meeting 2000
THE C. ELEGANS ORFEOME PROJECT
Jerome Reboul 1 , Philippe Vaglio 1 , Cindy Jackson 2 , Troy Moore 2 ,
Jean Thierry-Mieg 3 , Danielle Thierry-Mieg 3 , Jim Hartley 4 , Gary
Temple 4 , Mike Brasch 4 , Nia Tzellas 1 , Marc Vidal 1
1Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, MA, USA
2Research Genetics, Huntsville, AL, USA
3IGM-CNRS, Montpellier, France
4Life Technologies Inc., Rockville, MD, USA
In addition to gene-based functional genomics approaches such as large-scale gene knock-outs and
microarray or chip analysis, it is also important to develop protein-based approaches, e.g. protein
interaction mapping, protein localization mapping, and biochemical and structural genomics. Most of
protein-based approaches rely upon the availability of near complete set of open reading frames
("ORFeomes") cloned into various expression vectors (i.e., for each ORF: the sequence between the start
and the stop codons, in the absence of 5’ and 3’ untranslated sequences and introns).
To clone the C. elegans ORFeome into various expression vectors, we use a Recombination Cloning
technique (RC) referred to as Gateway TM (Walhout et al., 2000, Science, 287, 166-122). RC allows both
the initial cloning of ORFs and their subsequent transfer into different expression vectors by site-specific
recombination in vitro. In addition, RC is amenable to automation in 96-well (or 384-) plate settings, which
is crucial for large-scale ORFeome cloning. So far we have cloned 2,000 C. elegans ORFs. At the current
throughput (~400 ORFs/week), ~70% of the C. elegans ORFeome should be cloned by the end of the
year. We will present: i) the details of the method used, ii) illustrations of our current throughput, iii) a
description of the cloning quality, iv) how this resource will be made available to the community, and v)
how the ORFeome project will help the protein interaction mapping project (see abstract by Walhout et
al).
41

ANALYSIS OF SPLICING AND REGULATORY ELEMENTS
USING THE INTRONERATOR
W. James Kent, Alan M. Zahler
University of California, Santa Cruz
West Coast Worm Meeting 2000
The Intronerator (http://www.cse.ucsc.edu/~kent/intronerator/) is a set of web-based tools for exploring
RNA splicing, gene structure, and cross-species alignments in C. elegans. It features a display of
mRNA/DNA and cross-species DNA/DNA alignments. It also includes a flexible tool for extracting
sequences, a database of introns, and a catalog of alternatively spliced genes. This catalog of 845
alternatively spliced genes identified through comparisons of ESTs with genomic sequence is currently
the largest database of alternatively spliced genes available for any organism. The use of the Intronerator
will be demonstrated and we will explain in brief the algorithms behind the program, and the use of the
program to explore regulatory regions. We will present observations made during the alignment of 8
million bases of C briggsae genomic DNA with the C. elegans genome, including characteristics of introns
present in one species but not the other, the ability to confirm the identification of genes for which no EST
data is available, and the degree of rearrangement that has occurred in the ~50 million years since the
two species diverged.
42

West Coast Worm Meeting 2000
A GLOBAL PROFILE OF GERM LINE GENE EXPRESSION
USING MICROARRAYS REVEALS GERM LINE-SPECIFIC
REGULATION OF THE X CHROMOSOME IN MALES AND
HERMAPHRODITES
Valerie Reinke 1 , Harold E. Smith 2 , Jeremy Nance 2 , Abby F.
Dernburg 1 , Anne M. Villeneuve 1 , Samuel Ward 2 , Stuart K. Kim 1
1Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305
2Department of Molecular and Cellular Biology, University of Arizona, Tuscon, AZ 85721
We have produced DNA microarrays containing genomic PCR products corresponding to 11,917 C.
elegans genes. To identify genes expressed in the germ line, we compared wild-type gene expression
levels to that of glp-4 mutants, in which the germ line precursor cells do not proliferate. We also compared
a mutant strain making only sperm, fem-3(gf), to a mutant strain producing only oocytes, fem-1(lf), to
identify both sperm-enriched and oocyte-enriched genes. Using a statistical criterion for significance,
these experiments together define 1416 germ line-expressed genes that fall into three categories: 650
sperm-enriched, 258 oocyte-enriched, and 508 germ line-intrinsic. We have determined the temporal
expression pattern of each of these genes during development. These germ line genes comprise a
molecular definition of germ line components, and also provide the framework to identify individual genes
involved in specific germ line functions. The sperm-enriched group contains an unusually large number of
protein kinases and phosphatases. The oocyte-enriched group includes potentially new components of
embryonic signaling pathways. The germ line-intrinsic group, defined as genes expressed similarly in
germ lines making only sperm or only oocytes, contains a family of piwi-related genes that may be
important for stem cell proliferation. Surprisingly, we found evidence for germ line-specific regulation of
the X chromosome. Sperm-enriched and germ line-intrinsic genes are nearly absent from the X
chromosome, and X-linked oocyte-enriched genes are expressed at about three fold lower levels than
autosomal genes. Further, a marker for active gene expression (acetylated histone H4) is detectable on
autosomes but staining is strongly reduced on the X chromosomes in germ line nuclei.
43

THE PROMISE AND PERIL OF GENOMICS: SPERM
DEVELOPMENT AS MODEL SYSTEM
Harold Smith, Marci Millhouse, Sam Ward
University of Arizona, Tucson, AZ 85721
West Coast Worm Meeting 2000
Microarray screening allows the investigator to profile expression patterns on a genome-wide scale.
However, even a successful screen can generate a daunting amount of data. In our effort to identify
genes involved in sperm development, we compared expression levels in fem-3(gf) mutants, which make
only sperm, to fem-1(lf) mutants, which make only oocytes. We identified 650 sperm-enriched genes (as
well as 258 oocyte-enriched genes) out of 11,917 genes screened (see Reinke et al.). Now, how do we
use this information? We have focused our efforts in two areas: 1) obtaining deletion mutations in
potentially interesting genes; and 2) identifying sperm promoter elements and their relevant transcription
factors.
Prior work with the calmodulin inhibitor trifluoperazine (TFP) had implicated Ca++ function in both sperm
activation and motility; therefore, we generated a deletion allele of the sperm-enriched Ca++ channel
gene K01A11.4 (since named spe-39). The spe-39 mutant exhibits reduced fertility and an aberrant
sperm morphology that mimics TFP treatment. We can now examine the role of other spe mutants in
Ca++ sensing, sperm activation, and motility.
We are using an in silico approach to identify potential sperm promoter elements, and molecular and
genetic analyses to confirm functional significance. The algorithm (written by John Anderson, NCBI)
determines which sequences are over-represented in the 5’ upstream sequences of sperm genes
compared to non-sperm genes. One of these sperm-enriched sequences contains two potential binding
sites for the GATA transcription factor elt-1. Prior work has shown that elt-1 is required to specify
hypodermal cell fates in the developing embryo; however, our microarray data also identified elt-1 as a
sperm-enriched gene. Yeast one-hybrid screening demonstrates that elt-1 binds to and activates
transcription from these putative promoter elements. In collaboration with Barbara Page, we are currently
investigating the role of elt-1 in sperm development.
44

West Coast Worm Meeting 2000
FUNCTIONAL ANALYSIS OF CHROMOSOME I
Andrew Fraser, Ravi Kamath, Peder Zipperlen, Maruxa
Martinez-Campos, Julie Ahringer
Wellcome-CRC Institute, Tennis Court Road, Cambridge, UK
RNAi is a powerful and specific means of inhibiting gene function. However, the most widely used
technique for RNAi, the injection of dsRNA into adult animals, is too labour-intensive to allow efficient
genome-wide screening for gene function by RNAi. An alternative approach for RNAi is to feed bacteria
expressing dsRNA to worms. We have determined conditions for which RNAi by bacterial feeding is as
potent as RNAi by injection. We have constructed a library of dsRNA-expressing bacteria that can be
used to target 90% of genes on chromosome I by RNAi. This reagent can be used for an unlimited
number of low-cost screens for gene function. We have used this library to screen for all genes on
chromosome I that give a clear phenotype when targeted by RNAi. We will present our results and
discuss their implications for genome-wide functional analysis of C. elegans genes.
45

West Coast Worm Meeting 2000
OPTIMIZING THE MUTAGENIC PROPERTIES OF THE MOS1
TRANSPOSON IN C. ELEGANS
Daniel C. Williams, Jean-Louis Bessereau, Erik M. Jorgensen
Department of Biology, University of Utah, Salt Lake City, UT, 84112
Forward genetic analysis in C. elegans provides an unbiased method to determine the function of
individual genes. However, identifying the physical location and molecular nature of a mutation that
results in a specific phenotype is quite laborious. One way to avoid genetic mapping is to use
transposable elements as mutagens, which provide a tag at the mutation site. The C. elegans genome
contains multiple copies of endogenous transposons, which hinder efforts to isolate the insertion of
interest. Recently, we demonstrated that Mos1 (a Tc1/mariner family member from D. mauritiana) can
hop in C. elegans. Mos1 insertions in the germ-line result in a unique tag at the precise location of gene
disruption. Unfortunately, these results also demonstrate that Mos1 is an inadequate mutagen for two
reasons: (1) the frequency of transposition is too low for genetic screens and (2) Mos1 insertions may be
phenotypically silent.
We are taking a number of approaches to make Mos1 a more effective mutagen. In order to increase
transposition frequency, we determined whether the local DNA structure around Mos1 substrate
molecules affects hopping. We determined that transposition of Mos1 can occur if substrate molecules
are located in repetitive or complex arrays. Current experiments address whether Mos1 copy number
within an array correlates with transposition frequency
Exonic Mos1 insertions may be phenotypically silent because they are spliced out during mRNA
processing in the same manner as Tc1 elements (Rushforth et al. 1996). We generated recombinant
Mos1 elements that contain a polyadenylation signal in the middle of the transposon that should prevent
splicing of the insertion and result in a truncated transcript. These experiments also allow characterization
of the cis factors present within Mos1 that are necessary for transposition. During these experiments we
isolated an allele of dpy-17. Using inverse PCR the location of this insertion was determined to be 176
base pairs upstream of the putative DPY-17 open reading frame F54D8.1. Determination of the physical
location of this mutation was extremely rapid, suggesting that Mos1 will be a valuable tool for forward
genetic analysis.
Rushforth, A. M. and P. Anderson (1996). Mol Cell Biol 16(1): 422-9.
46

SNAP-25, A PROTEIN IMPLICATED GENETICALLY IN C.
ELEGANS ANESTHETIC MECHANISMS, BINDS THE
GENERAL ANESTHETIC ISOFLURANE
Jason Berilgen 1 , Mike Crowder 1,2
West Coast Worm Meeting 2000
1Department of Anesthesiology, Washington University School of Medicine
2Department Molecular Biology/Pharmacology, Washington University School of Medicine
Mutations in the neuronal syntaxin gene unc-64 profoundly alter the volatile anesthetic (VA) sensitivity of
C. elegans. Two hypomorphic unc-64 alleles confer hypersensitivity to the VAs isoflurane and halothane,
but a third unc-64 hypomorph, md130, which produces a truncated syntaxin product, is VA resistant. The
difference between the isoflurane EC 50s of the hypersensitive and resistant alleles is over 30-fold; this is
by far the biggest allelic variation in VA sensitivity thusfar seen in any animal. Given that these allelic
differences cannot be explained by indirect effects on synaptic transmission and can be rescued and
knocked-in, we hypothesized that syntaxin or a syntaxin-binding protein binds VAs and that the truncated
md130 product somehow interferes with that binding. We have looked for isoflurane binding to syntaxin
and to two syntaxin-binding proteins, VAMP and SNAP-25, which form a ternary complex with syntaxin to
mediate synaptic vesicle fusion. Recombinant expression proteins (C. elegans syntaxin and VAMP -
coded for by snb-1, and rat SNAP-25 - plasmids all kindly provided by M. Nonet) without their
transmembrane domains or lipid modifications were used. We used 19 F-NMR to measure binding of
isoflurane to the purified proteins. The transverse relaxation time (T2) of a nucleus is known to decrease
when its Brownian motion decreases; thus, binding of small molecules (eg. isoflurane) to larger ones (eg.,
a protein) shortens T2s. We found that the T2 of the CF 3-moiety of isoflurane was significantly shorter in
solutions containing SNAP-25 than in buffer alone and was protein and isoflurane concentration
dependent, changing over the in vivo relevant isoflurane concentration range. Recombinant syntaxin had
no effect on the T2. We are currently gathering data for VAMP and the ternary complex. Thus, our data
show that SNAP-25 but not syntaxin binds isoflurane at relevant concentrations; SNAP-25 is the first
neuronal protein shown to bind VAs. Given our genetic data in C. elegans and electrophysiologic data in
vertebrates showing that neurotransmitter release is reduced by VAs, we think that SNAP-25 may be a
VA target that mediates some aspects of general anesthesia.
47

West Coast Worm Meeting 2000
HAPPY WORMS: FURTHER CHARACTERIZATION OF
FLUOXETINE (PROZAC) RESISTANT MUTANTS
Robert K.M. Choy 1,2 , James H. Thomas 2,3
1Program in Molecular and Cellular Biology, Box 357360, University of Washington, Seattle, WA
98195-7360
2rchoy@genetics.washington.edu
3Department of Genetics, Box 357360, University of Washington, Seattle, WA 98195-7360
Although over 20 million people have taken fluoxetine for a wide range of mental disorders, its molecular
mechanism of action remains unproven. Fluoxetine is a member of the selective serotonin reuptake
inhibitor (SSRI) class of antidepressants, which inhibit the presynaptic serotonin reuptake transporter.
However, it is still unclear whether this inhibition is responsible for their antidepressant action.
Furthermore, the targets responsible for the various side-effects of SSRIs are poorly characterized.
We previously reported that in C. elegans, SSRIs induce contraction of nose and body-wall muscles by
acting on a non-serotonergic target. We also reported the isolation and characterization of several
mutants that are nose resistant to fluoxetine (Nrf). These mutants are cross-resistant to other SSRIs, but
are fully sensitive to several other drugs that also induce nose muscle contraction. Therefore, these Nrf
mutations may identify novel genes relevant to antidepressant action.
Mutations in three of the Nrf genes (nrf-5, nrf-6 and ndg-4) have a common secondary phenotype of
producing pale eggs (Peg). These mutants are defective in yolk transport and accumulate yolk in the
pseudocoelomic space. nrf-6 and ndg-4 both encode homologous transmembrane proteins and define a
novel gene family of several dozen members in C. elegans and Drosophila. By a combination of mosaic
analysis and tissue specific promoters, we have found that nrf-6 function is required in the intestine for
both fluoxetine-induced nose contraction and yolk transport. We have also cloned nrf-5 and found that it
is homologous to the mammalian BPI/CETP family of secreted lipid binding proteins. One possibility is
that these genes are involved in a novel aspect of antidepressant transport. Alternatively, they may
mediate some aspect of lipid metabolism that is required for antidepressant-induced nose muscle
contraction.
48

West Coast Worm Meeting 2000
UNC-43 CA2+/CALMODULIN-DEPENDENT KINASE II
(CAMKII) MUTANT WORMS HAVE CONVULSIONS IN
RESPONSE TO THE SEIZURE-INDUCING DRUG PTZ
Elizabeth M. Newton, James H. Thomas
Dept. of Genetics, University of Washington, Seattle WA 98195
Epilepsy is one of the most common neurological disorders, effecting an estimated 50 million people
worldwide. While most seizures are treatable with present anti-convulsant drugs, approximately 20-30%
of patients remain refractory to drug therapy. Recent work in mouse models has begun to identify genes
involved in seizure susceptibility, but much about the mechanism of seizure susceptibility and the
mechanism of anti-epileptic drugs remains to be elucidated.
Null mutations in C. elegans unc-43 CaM Kinase II cause worms to appear nervous and jerky with
occasional spontaneous muscle contractions that resemble convulsions. To test if these contractions
could be related to seizure-like convulsions, we put unc-43 null worms on plates containing
pentylenetetrazole (PTZ), a seizure-inducing drug commonly used in rodent epilepsy models. The
phenotype observed is dramatic: the worms begin to have fast, strong simultaneous contractions of the
anterior and posterior body muscles in an uncontrolled fashion, completely disrupting normal locomotion.
(A videotape of this phenotype will be shown.) Wild-type worms appear mildly hyperactive in response to
PTZ, as expected for a CNS stimulant, and become slightly jerky and less coordinated over time but
never have convulsions, even at doses 10 fold higher than used to induce convulsions in unc-43 mutants.
Other drugs that induce muscle contraction but are not convulsive in mouse models, such as aldicarb and
levamisole, do not induce convulsions in unc-43 mutants but instead induce the expected
hypercontraction/paralysis phenotype. a-CaM Kinase II knock-out mice are epileptic, supporting the idea
that worm convulsions may be related to seizures in rodent models. The unc-43 convulsion phenotype
has a neuronal basis as it is rescued by a transgene expressing an unc-43 cDNA from the aex-3
promoter, which drives expression in all neurons but not muscle.
We tested a number of different mutant strains that might be expected to have hyper-excited neuronal
activity, such as loss of function mutations in potassium channels and mutations in the goa-1 pathway,
but none had convulsions in response to PTZ. We are currently screening for more seizure-susceptible
mutants to determine if this phenotype is specific to unc-43 CaM kinase II. We have also begun screening
for suppressors of the unc-43 PTZ-induced convulsion phenotype, and we have isolated several
independent mutants, indicating that it is likely that there are many genes involved in this response.
Studying suppressors of convulsions may identify new theraputic targets for the future.
49

West Coast Worm Meeting 2000
NEUROTOXIN SENSITIVITY OF DOPAMINERGIC NEURONS
IN C. ELEGANS: ROLE OF THE DOPAMINE TRANSPORTER
AND CELL DEATH PATHWAYS
R. Nass 1,2 , J. Duerr 3 , , J. Rand 3 , D. M. Miller 2,4 , R. D. Blakely 1,2
1Dept of Pharmacol, Vanderbilt U. Med. School, Nashville, TN
2Ctr. for Molecular Neuroscience, Vanderbilt U. Med. School, Nashville, TN
3Program in Molecular and Cell Biology, OMRF, Oklahoma City, OK
4Dept of Cell Biology, Vanderbilt U. Med. School, Nashville, TN
The dopamine transporter (DAT) constitutes the primary mechanism for the inactivation of dopamine (DA)
neurotransmission in the brain. DATs are targets for many psychoactive drugs and the cellular gateway
for the accumulation of the neurotoxin 6-hydroxydopamine (6-OHDA) which evokes neuronal death and
Parkinson-like syndrome in animal models. We have previously cloned and tagged the C. elegans DAT
(CeDAT), and have shown that it is functionally similar to mammalian DATs and expressed exclusively in
the DA neurons (Jayanthi et al. 1998, Nass et al. 1999). We have also developed WT and DAT knockout
transgenic lines which target a CeDAT promotor-GFP fusion to all 8 DA neurons in the hermaphrodite.
Intense GFP expression is seen throughout the axons and dendrites in the live animals. We now show
that a brief exposure to 6-OHDA results in the blebbing of DA neuronal processes, swelling of DA
neuronal cell bodies, and the loss of CeDAT-GFP expression, while co-exposure with CeDAT substrates
or antagonists significantly reduces the 6-OHDA induced response. We also show that this response is
dependent on the expression of CeDAT, since the CeDAT knockout line is insensitive to the effects of the
neurotoxin. Furthermore, the effect appears to be dependent on a necrotic cell death pathway, since the 6
OHDA induced response occurs in a caspase-deficient ced-3 knockout background. These studies as
well as our progress on toxin-based genetic screens for regulators of CeDAT function, localization, and
the toxin mediated cell death will be presented. Supported by MH19732-07 (RN), GM38679 (JR),
NS26115 (DMM), MH58921 (RDB)
50

West Coast Worm Meeting 2000
GENE EXPRESSION IN TRANSGENIC C. ELEGANS ANIMALS
EXPRESSING THE HUMAN BETA AMYLOID PEPTIDE.
Chris Link 1 , Carolyn Johnson 1 , Amy Fluet 1 , Kyle K. Duke 2 , Stuart K.
Kim 2
1Institute for Behavioral Genetics, University of Colorado, Boulder, CO 80309
2Department of Developmental Biology, Stanford Medical School, Stanford, CA, 94305-5427
We have used Andy Fire’s smg-1 dependent expression vectors to engineer worms with inducible
muscle-specific expression of the human beta amyloid peptide. These animals appear wild type at the
permissive temperature (16 degrees C), but become paralyzed approximately 24 hrs after upshift to the
non-permissive temperature (23 degrees C). As expected, this upshift is accompanied by a large increase
in the synthesis of the beta amyloid peptide. We have examined gene expression profiles in these
animals using DNA microarrays containing probes for all predicted C. elegans genes. For our initial
experiments, we compared staged L4 populations from uninduced animals and animals upshifted 22 hr
(harvested before onset of paralysis) or 29 hr (all animals arrested), prepared independently in triplicate.
Approximately 150 genes show an average expression increase > 2X at both induced time points, while
approximately 100 genes show an average expression decrease >2X at both timepoints. We have not yet
independently confirmed these putative gene expression changes, with the exception of HSP-16-2, which
is clearly also upregulated at the protein level, as demonstrated by immunoblots. Perhaps the most
obvious pattern of expression changes is observed in the down-regulated class, where there appears to
be a coordinated decrease in expression of a number of genes involved in energy production and
mitochondrial function (e.g., enolase, GPDH, citrate synthase, succinate dehydrogenase, etc.) (These
changes precede any obvious changes in motility.) These results are consistent with reports of abnormal
glucose metabolism in the brains of Alzheimer patients. to send it in.
51

West Coast Worm Meeting 2000
A NEMATODE MODEL FOR MITOCHONDRIAL DISEASES
William Y. Tsang, Bernard D. Lemire
Department of Biochemistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
The mitochondrial respiratory chain (MRC) is composed of 5 protein complexes capable of generating
ATP for most tissues. Its biogenesis requires the coordinate expression of genes from both the nuclear
and the mitochondrial genomes. Defective MRCs are often associated with myopathies, neuromuscular
and heart diseases. We are developing Caenorhabditis elegans as our model system to investigate the
biochemical, genetic, and phenotypic consequences of mutations affecting the MRC.
We have identified and cloned 3 MRC mutations. The first two mutations are nuclear deletions in the
nuo-1 (C09H10.3) and in the atp-2 (C34E10.6) genes encoding the active site subunits of Complex I and
V, respectively. Both mutations are homozygous lethal and arrest at the L3 stage. atp2 homozygotes
have a normal life span and cannot enter dauer. Furthermore, development of these animals to L3
requires maternal contribution of atp-2 mRNA.
The third mutation is a mitochondrial DNA (mtDNA) deletion that removes 4 MRC and 7 tRNA genes.
These animals are heteroplasmic and aphenotypic despite the different proportions of mutant mtDNA
(~20-80%). However, exposure of N2 gravid adults to ethidium bromide, an inhibitor of mtDNA replication,
results in L3 arrest progeny with characteristics similar to the nuclear mutants. The arrested animals
exhibit a progressive decrease in the steady-state level of mtDNA with age.
We speculate that the L3 arrest phenotype is due the failure of a common energy-requiring step in
development. In support of this hypothesis, we have determined that the passage from L3 to L4 is
associated with a 3-fold increase in the mtDNA copy numbers, as well as a significant increase in the
levels of ATP-2. In addition, we believe that mitochondrial energy metabolism is linked to the regulation of
dauer formation.
52

BACILLUS TOXIN (BT) SUSCEPTIBILITY AND RESISTANCE
IN C. ELEGANS
Lisa Marroquin 1 , Dino Elyassnia 1 , Joel Griffitts 1 , Johanna O’Dell 1 ,
Jerald Feitelson 2 , Raffi Aroian 1
1U.C. San Diego
2Akkadix Corporation
West Coast Worm Meeting 2000
The protein toxins produced by Bacillus thuringiensis (Bt) are the most widely used natural insecticides in
agriculture and have been expressed in transgenic corn, potato, and cotton to provide organic crop
protection against insect pests. Despite successful and extensive use of these toxins, little is known about
toxicity and resistance pathways in target insects since these organisms are not ideal for molecular
genetic studies. To address this limitation and to investigate the potential use of these toxins to control
parasitic nematodes, we are studying Bt toxin action and resistance in Caenorhabditis elegans. We
demonstrate for the first time that a single Bt toxin can target a nematode. When fed Bt toxin, C. elegans
hermaphrodites undergo extensive damage to the gut, a decrease in fertility, and death, consistent with
toxin effects in insects. We have screened for and isolated ten recessive mutants that resist the toxin’s
effects on the intestine, on fertility, and on viability. These mutants define five genes, indicating that more
components are required for Bt toxicity than previously known. We find that a second, unrelated
nematicidal Bt toxin may utilize a different toxicity pathway. Our data indicate that C. elegans can be
used to undertake detailed molecular genetic analysis of Bt toxin pathways and that Bt toxins hold
promise as nematicides. More recently, we have achieved rescue of one of these mutants with a
subclone containing a single predicted open reading frame. Once confirmed by sequencing of mutant
alleles, this would result in the first definitive identification of a gene required for Bt toxin action in any
organism.
53

West Coast Worm Meeting 2000
NEW DAUER GENES AND PATHWAYS
Michael Ailion 1 , James H. Thomas 2
1Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195
2Department of Genetics, University of Washington, Seattle, WA 98195
We have isolated and characterized mutants that are strongly Daf-c at 27° but that are not Daf-c at 25°,
a phenotype we call Hid (high temperature-induced dauer formation). Previously identified mutants with
this phenotype include unc-64, unc-31, unc-3, egl-4, daf-3 and the dyf mutants. We screened for new Hid
mutants at 27° and isolated 100 mutants, including twenty-three alleles of known Daf-c genes (many
weak alleles), sixteen alleles of dyf or other Daf-d genes and five alleles of unc-31 or unc-3. We also
isolated alleles of a number of new dauer genes. These include alleles of the genes pdk-1, akt-1, aex-6,
kin-8 and hid-1.
Many of the Hid mutants are fully suppressed by mutations in daf-16, suggesting that these genes act in
the daf-2/age-1 (insulin-receptor /PI3 kinase) pathway. These include unc-64 and unc-31, which encode
neurosecretory proteins that may be involved in insulin secretion, and pdk-1and akt-1 which encode
PI3-dependent protein kinases that act downstream of age-1. The aex-6 and hid-1 genes may also act in
this pathway. In addition to their Hid phenotype, the aex-6 and hid-1 mutants also have defects in
movement and defecation, suggesting that they may function in a common process to regulate multiple
behaviors. We cloned hid-1 and found that it encodes a novel protein with many transmembrane
domains. The HID-1 protein is strongly conserved in Drosophila and humans, with each organism
appearing to carry only one gene of this type.
Reexamining the epistatic interactions of strong Daf-c genes at 27° leads to several new inferences.
Mutations in the TGF-b pathway Daf-c genes and daf-2 are completely suppressed by daf-5 and daf-16,
respectively, at 25° but only partially suppressed at 27°. This suggests that there are additional branches
of the TGF-b and insulin pathways that are not detected at 25°. Furthermore, epistasis results based on
pheromone response at 25° show qualitative differences from epistasis results at 27°, indicating that
gene interactions inferred from epistasis experiments performed under one set of conditions may not be
the same as those under a different set of environmental conditions.
54

TEMPORAL REGULATION OF AGING IN THE NEMATODE C.
ELEGANS
Andrew Dillin, Cynthia Kenyon
West Coast Worm Meeting 2000
Department of Biochemistry and Biophysics, University of California at San Francisco
It has long been assumed that the aging process is stochastic, beginning early in life and eventually
gaining enough activity to cause the eventual decline and death of an organism. In contrast, aging can be
viewed as a developmental process, much like early embryonic development, involving signals, receptive
cues and timely action of these processes during discrete periods of an animal’s life cycle. I have tested
these opposing theories by determining when several genes required for lifespan extension actually
function. Aging in C. elegans is regulated by a subset of genes that form a signal transduction pathway.
daf-2 is an insulin-like receptor that acts upstream of a PI3-kinase, age-1. Both genes normally function to
limit life span by down regulating the activity of daf-16, a Forkhead-like transcription factor. daf-16
normally functions to increase life span. I will present data indicating that the aging function of the genes
age-1, daf-2 and daf-16 is exerted during a specific period of the worm’s life cycle. These results suggest
that aging, at least in C. elegans, is regulated during specific periods in life. In addition, I have also started
work on a novel screen to identify other genes required for life span regulation. Once these genes are
identified I will test when they function.
55

A LONGITUDINAL ANALYSIS OF ADULT NEURONS IN C.
ELEGANS
Mark I. Snow, Pamela L. Larsen
West Coast Worm Meeting 2000
Molecular Biology Program and Division of Biogerontology, University of Southern California, Los
Angeles, CA 90089
An intriguing question that remains to be answered is what happens to the cells of the nervous system in
an individual as it ages? Previous studies following the neuron number in rodents and humans have used
a cross sectional design and the correlative conclusions drawn from these experiments are controversial
with regard to whether or not there is a decline in neuron number with advancing age. C. elegans show a
progressive decline in neuronally regulated behaviors as they age, so we chose to follow specific neurons
longitudinally in worms by using integrated transgenic GFP arrays.
The analysis of GFP expression for the 26 GABA and the 8 dopaminergic neurons in hermaphrodites was
performed at early adulthood and again just before death. We found that 84% of the old animals with a
wild-type background do not lose GFP expression in their GABA neurons. For the 16% of animals that
express GFP in less than 26 GABA neurons just before dying, the loss of expression appears to be
cell-specific. In animals expressing GFP in the dopaminergic neurons, 94% do not lose GFP expression
with age. The small number that did display changes were in specific neurons.
Two daf-2 alleles, m41 and e1370, were introduced into the strains expressing GFP in either the GABA or
dopaminergic neurons. The GFP expression patterns of the double mutant strains were not altered and
the life spans of the animals were that of the daf-2 alleles. No change in the number of neurons
expressing GFP was observed in any of the animals with the daf-2(m41) or daf-2(e1370) alleles. The
daf-2 mutation appears to prevent loss of GFP expression in older animals.
The experiments described here are the first to follow neurons in a longitudinal manner in individuals as
they age. Assuming that the endogenous gene and the transgene are similarly regulated, absence of
GFP expression may indicate functional loss because the unc-25 and cat-2 genes encode enzymes
necessary for neurotransmitter biosynthesis. From this approach, we conclude that loss of GFP
expression is not widespread and the daf-2 mutations are neural protective.
56

West Coast Worm Meeting 2000
GERM-LINE CELLS THAT REGULATE AGING IN C. ELEGANS
Nuno Arantes-Oliveira, Javier Apfeld, Cynthia Kenyon
Department of Biochemistry and Biophysics, University of California San Francisco
The lifespan of C. elegans is regulated by a hormonal pathway that integrates signals from the nervous
and reproductive systems of the animal. Ablation of the germ-line precursor cells produces a lifespan
extension of approximately 60%. This extension is dependent on the presence of the somatic tissues of
the gonad and thus is not likely to be caused by sterility. We have used a variety of genetic approaches,
RNAi and laser ablations to ask which cells in the germ-line (sperm, oocytes, mitotic precursors) regulate
lifespan. Our findings support the interpretation that mitotically-dividing germ-line cells produce the signals
that regulate lifespan. These signals may act by down-regulating daf-16.
57

West Coast Worm Meeting 2000
A SCREEN FOR GENES THAT CONTROL PROGRAMMED
CELL DEATH IN THE GERM LINE
S Milstein 1,2,3 , A Gartner 4 , M Hengartner 5
1Program in Genetics, SUNY Stony Brook
2Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 USA.
3milstein@cshl.org
4Max Planck Institut for Biochemie, D-82152 Martinsried, Germany
5Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 USA
Although we know a great deal about the apoptotic program in C. elegans, we know very little about how
the program is regulated. Since this cell fate is responsible for the demise of about 10% of the somatic
cells, and as many as half of the germ cell, we are interested in defining the genes that may be involved
in the decision of a cell to die.
In order to find such genes, we developed a screen using the vital dye acridine orange, which specifically
stains apoptotic cells in the germ line. Using this screen we have looked at approximately 40,000
genomes and have identified 21 mutants that have increased levels of germ cell death. The ten mutants
that have thus far been mapped represent eight complementation groups, only one of which has multiple
alleles. Of these I am currently cloning two by single nucleotide polymorphism mapping, gla-1(op212) and
gla-3(op234).
In order to determine if these mutants are specifically defective in the regulation apoptosis, or if they are
pathologically compromised, and thus have damage that causes the cells to undergo apoptosis, we made
double mutations with ced-3 and rad-5. Most of the mutations examined are suppressed in the ced-3
background, suggesting that the damage is non pathological (we would have expected necrosis or some
other gross morphological consequence if this were the case). In the rad-5 background, a mutation that
we have shown to render worms insensitive to DNA damage, these mutations are not suppressed. This
again suggests specificity since mutations that somehow result in DNA damage would be suppressed.
58

West Coast Worm Meeting 2000
C. ELEGANS P53: REQUIREMENT FOR
RADIATION-INDUCED PROGRAMMED CELL DEATH,
STRESS RESISTANCE, AND NORMAL ADULT LIFESPAN
FOLLOWING DIAPAUSE.
W. Brent Derry, Aaron Putzke, Joel H. Rothman
Department of Molecular, Cellular & Developmental Biology, University of California, Santa Barbara, CA
93106 USA
We have identified the apparent C. elegans homologue of the p53 tumor suppressor gene, cep-1, and
have begun to characterize it genetically. CEP-1 appears to be the only p53 family member present in the
completely sequenced C. elegans genome and includes all of the signature domains of p53, including 4
of the 5 conserved arginine residues frequently mutated in human cancer. A cep-1::gfp reporter shows
ubiquitous expression throughout embryonic development, which becomes restricted to a subset of
pharyngeal muscle and neuronal cells postembryonically. Ectopic expression of cep-1 or of human p53
fails to induce cell cycle arrest but potently promotes the rapid ced-3-independent necrotic death of all
somatic cells, demonstrating that regulation of cep-1 expression in a critical range is essential for survival.
We isolated a TMP-induced cep-1 mutation that removes the conserved DNA binding domains. Though
cep-1(-) homozygotes show a low level of embryonic lethality, they are generally viable. However, they
are highly resistant to radiation-induced apoptosis of germ cells. Cep-1 deletion mutants also show
enhanced hypoxia-induced lethality, indicating that C. elegans p53 is essential for normal physiological
stress response. Furthermore, cep-1 mutants display both reduced viability and reduced lifespan after
release from starvation-induced L1 diapause. These results provide the first evidence linking p53 to
longevity of an animal in response to physiological stress.
59

IDENTIFICATION OF CELL-SPECIFIC REGULATORS OF
PROGRAMMED CELL DEATH IN C. ELEGANS.
Shai Shaham, Cori Bargmann
Dept. of Anatomy, UCSF
West Coast Worm Meeting 2000
Programmed cell death (PCD) is a common metazoan cell fate. In C. elegans 12% of somatic cells and
half of germ cells born undergo PCD. Excess or insufficient PCD can lead to a variety of human diseases.
In C. elegans PCD is regulated by egl-1 (a BH3 domain protein), ced-9 (a bcl-2 family member), ced-4
(similar to human Apaf-1), and ced-3 (a caspase protease). Although much is known about the machinery
that executes PCD, only in a few instances have the signal transduction pathways leading to activation of
this machinery been elucidated.
Several lines of evidence suggest that ced-9, ced-4, and ced-3 are expressed in most if not all cells. We
are interested in defining molecular events that trigger PCD activation in specific cells. As a first step, we
identified a novel gene (cip-1) whose protein product interacts with the CED-9 protein. CIP-1 (CED-9
interacting protein) binds to CED-9 in a two-hybrid assay. Precipitation experiments using GST-CED-9
and 35 S-labelled CIP-1 also indicate that these proteins may interact. Reminiscent of C. elegans EGL-1,
CIP-1 may contain a BH3 domain (bcl-2 homology domain 3) mediating interaction with CED-9.
To understand the function of CIP-1 we expressed the protein in worms using heat-shock promoters.
Animals expressing HS-CIP-1 are dead. This inviability is rescued by ced-3, ced-4, or ced-9(gf)
mutations. Thus, CIP-1 may act upstream of the global PCD machinery to regulate PCD activation.
Preliminary data suggest that cip-1::GFP is expressed in a small number of cells, suggesting that this
gene may well be a cell-specific PCD activator.
In addition to characterizing cip-1, we are screening for genes controlling PCD of the male-specific CEM
neurons, which normally die in hermaphrodites. Using a pkd-2::GFP transgene specifically expressed in
the CEMs, we have isolated 25 independent mutants that abnormally express pkd-2::GFP in
hermaphrodites. Of these, five are alleles of the PCD activators ced-3 and ced-4, and one is an allele of
the tra-2 sex-determination gene. Seventeen mutations promote CEM survival and do not result in any
other gross abnormalities. These mutations may affect either CEM-specific PCD regulators or CEM cell
fate genes. Two allelic mutations define a novel gene required to suppress CEM-like development of
other head neurons.
60

West Coast Worm Meeting 2000
BIOCHEMICAL, STRUCTURAL, AND GENETIC ANALYSES
OF THE ACTIVATION OF PROGRAMMED CELL DEATH
Jay Parrish 1 , Betsy Metters 1 , Lin Chen 2 , Ding Xue 1
1Dept. of MCD Biology, University of Colorado, Boulder, Colorado 80309
2Dept. of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309
In the nematode C. elegans, three genes egl-1, ced-3, and ced-4, are required for activation of
programmed cell death whereas one gene, ced-9, prevents programmed cell death. Molecular
characterization of these genes has shown that egl-1 encodes a BH3-only (Bcl-2 homology domain 3) cell
death activator, ced-3 encodes an aspartate-specific cysteine protease (caspase), ced-4 encodes an
Apaf-1-like (apoptotic protease activating factor-1) protein, and ced-9 encodes a Bcl-2 homologue.
Genetic studies have placed these genes in a negative regulatory pathway, with egl-1 antagonizing the
activity of ced-9, which inhibits the activity o f ced-4 to activate CED-3 and eventually cell death. In
combination with in vitro biochemical studies, these genetic findings suggest that a protein interaction
cascade involving EGL-1, CED-9, CED-4, and CED-3 regulates the activation of programmed cell death
in C. elegans. We have initiated biochemical, structural, and genetic analyses to study the mechanisms
that activate the death protease CED-3, including the protein interactions involved in this activation
process. Specifically, we are attempting to reconstitute the events necessary for activation of CED-3 in
vitro using purified recombinant proteins. Furthermore, we are using a structural modeling approach to
analyze these protein interactions and guide our efforts to demonstrate the importance of these protein
interactions in vivo. So far, we have been able to recapitulate the early events of cell death activation in
vitro, including the EGL-1 mediated release of CED-4 from CED-4/CED-9 complexes and are in the
process of purifying additional factors that are required for the activation of CED-3. Structural modeling of
the EGL-1/CED-9 complex and corresponding biochemical analyses have revealed important molecular
interactions between EGL-1 and CED-9 and the nature of an unusual gain-of-function mutation in ced-9,
facilitating successful design of its second-site suppressor mutations. Further studies of this kind should
help address the central question of how cell death and the cell death protease CED-3 are activated in
the appropriate cells and at the right time.
61

ANALYSIS OF RNA ASSOCIATED WITH P GRANULES IN
GERM CELLS OF C. ELEGANS ADULTS
Jennifer A. Schisa 1 , Jason N. Pitt 2 , James R. Priess 2
1Fred Hutchinson Cancer Research Center
2Howard Hughes Medical Institute, Seattle, WA 98109
The germ plasm of many species contains germline-specific cytoplasmic granules. These granules (called
P granules in C. elegans) have been hypothesized to have some function in germline development; and
several of the proteins that are required for germline specification in the embryo (PIE-1, POS-1, MEX-1)
transiently associate with P granules during the first few cell divisions. Because P granules are associated
with germ cell nuclei during most of development, we have begun a characterization of the
nuclear-associated P granules in adult gonads. These studies have shown that P granules in the gonad
are tightly associated with clusters of nuclear pores 1 .
Several protein components of P granules have been identified and found to have potential RNA-binding
motifs. We find that P granules in the gonad contain RNA using a probe for SL1, a transpliced leader
found on many C. elegans RNAs. We have also examined sterile animals lacking glh-1 and glh-2; germ
cells in these gonads do not appear to assemble P granules, as ascertained by immunostaining with
several P granule antibodies 2 . Animals lacking glh-1 and glh-2 show hybridization of SL1 on perinuclear
foci. We examined the germ nuclei in these animals by electron microscopy and found perinuclear foci of
abnormal, electron dense material that appeared to be associated with nuclear pores. Thus, it appears
that the putative RNA-binding proteins GLH-1 and GLH-2 are not essential for RNA to localize outside the
nuclear envelope.
We examined various classes of maternally-expressed RNAs by fluorescence in situ hybridization to
determine which specific mRNAs are in P granules. While ribosomal RNAs and some abundant
housekeeping mRNAs do not appear to be enriched in P granules above the general level in the
cytoplasm, several maternal mRNAs that are translated only in the early embryo appear to accumulate on
germ cell P granules.
1 Pitt et al., 2000. Dev. Biol. 219: 315-333.
2 Gruidl et al., 1996. PNAS 93, 13837-13842.
West Coast Worm Meeting 2000
62

West Coast Worm Meeting 2000
THE SPLICING SM PROTEINS COLOCALIZE WITH P
GRANULES IN GERM CELLS AND PARTICIPATE IN P
GRANULE LOCALIZATION IN THE EARLY EMBRYO
Scott A. Barbee, Alex L. Lublin, Thomas C. Evans
Department of Cellular and Structural Biology, University of Colorado Health Science Center, Denver,
Colorado, 80262
Early embryonic polarity in C. elegans is initiated by asymmetric cell division which leads to the
localization of maternally contributed proteins and mRNAs. In turn, these factors control the temporal and
spatial expression of gene products that determine the developmental fate of cells. Conventional genetic
screening has identified more than 20 genes involved in this process. However, these screens may not
uncover pleiotropic genes that have a necessary function at other stages. With this in mind, we have used
RNA interference (RNAi) to screen a cDNA library for new genes involved in generating asymmetries in
the early embryo.
One gene identified in the RNAi screen is a homolog to human SNRPE (SmE). SmE is one of several Sm
proteins which form a complex required for mRNA splicing and import of snRNAs (U1, U2, U4, and U5)
into the nucleus. RNAi of SmE and other Sm proteins, which together form a subcomplex, resulted in
disruption of P granule segregation in the embryo. P granules are cytoplasmic ribonucleoprotein particles
that segregate to the posterior germline precursor cells. RNAi of several subunits of the Sm complex
caused mislocalization of P granules at various stages. Other aspects of early polarity may also be
disrupted. However, RNAi of other distinct Sm proteins had no effect. RNAi of the core splicing factors
U170K and U2AF65, or of RNA polymerase II (ama-1), also had no effect on P granule localization.
These data suggest that the P granule phenotype is not the result of a general splicing defect. Therefore,
Sm proteins may have a role in generating early embryonic polarity that is independent of snRNA import
and splicing. Interestingly, using a monoclonal antibody against mammalian Sm proteins, we found
colocalization of the Sm’s with PGL-1 in P granules at all stages of development. Sm proteins, therefore,
may be P granule components that participate in P-granule localization and/or other polarity functions.
63

POD-2 DEFINES A NEW CLASS OF MUTANTS REQUIRED
FOR ANTERO-POSTERIOR ASYMMETRY IN THE EARLY
CAENORHABDITIS ELEGANS EMBRYO
Akiko Tagawa, Raffi V. Aroian
West Coast Worm Meeting 2000
University of California, San Diego. La Jolla, CA 92093-0349
The generation of asymmetry in the one-cell C. elegans embryo is indispensable for a proper segregation
of developmental determinants and thus development of the multicellular organism. We have isolated a
mutation in a previously unidentified gene, pod-2 (for polarity and osmotic defect), through a screen for
cold sensitive embryonic lethals. Many pod-2 mutant embryos resemble par mutant embryos and show a
loss of physical and developmental asymmetry at the one and two-cell stage. More strikingly, pod-2
resembles another mutant discovered in our lab, pod-1, in that mutants embryos are also osmotically
sensitive. Double mutant analysis indicates these two genes work in the same pathway thus defining
pod-1 and pod-2 as a new class of polarity genes and a new pathway important for early embryonic
development. In addition to polarity and osmotic defects, pod-2 mutation also causes abnormal
localization of germline components such that in 25% of mutant embryos, germline components are
located to a single cell where EMS normally would be. By GFP analysis, we show that this defect can be
traced back to abnormal localization of germline components in the one-cell embryo. Our data suggests
that in the mutant, germline components remain bundled but do not move to the proper position in the
one-cell embryo. Thus pod-2 appears to be required for proper positioning of developmental components
and perhaps other asymmetries. Cloning of pod-2 is underway, and we have recently achieved rescue of
the mutant with a single cosmid.
64

West Coast Worm Meeting 2000
OOC-5 ENCODES A PUTATIVE ATPASE REQUIRED FOR THE
REESTABLISHMENT OF ASYMMETRIC PAR PROTEIN
LOCALIZATION IN TWO-CELL EMBRYOS
Stephen E. Basham, Lesilee S. Rose
Section of Molecular and Cellular Biology, University of California, Davis, CA 95616 USA
C. elegans embryos display two distinct patterns of spindle orientation. Divisions in the AB lineage occur
in an orthogonal pattern while some division in the P lineage repeatedly occur on the same axis, due to a
rotation of the nuclear-centrosome complex. Genetic and molecular analyses suggests that PAR-3 protein
plays an important role in preventing nuclear rotation in the AB cell, while PAR-2 protein functions to allow
nuclear rotation in P1 by restricting PAR-3 localization within this cell.
We have screened maternal-effect lethal mutants for alterations in cleavage pattern. Hermaphrodites
homozygous for mutations in the ooc-5 or ooc-3 gene produce reduced sized embryos that fail to undergo
P1 nuclear rotation. Our phenotypic characterization of ooc-5 and ooc-3 mutants indicates that polarity is
perturbed in the P1 cell of 2-cell mutant embryos. In particular, we have found that in most ooc-5 and
ooc-3 mutant embryos, PAR-3 localization appears normal at the 1-cell stage but is no longer restricted to
the anterior of the P1 cell in 2-cell embryos. PAR-2 is also normal in 1-cell mutant embryos, but is absent
from the periphery of the P1 cell in most 2-cell ooc mutant embryos. Finally, P granules are localized
normally in 1-cell mutant embryos, but often fail to localize normally to the posterior of the P1 cell in 2-cell
mutant embryos. Thus, ooc-5 and ooc-3 may function (directly or indirectly) in polarizing the P1 cell. One
model for OOC-5 and OOC-3 is that they function in P1 to maintain PAR-2 at the posterior of P1, thus
restricting PAR-3 localization in this cell. We have cloned ooc-5 and it is predicted to encode a 350 amino
acid protein containing an amino-terminal signal sequence, a transmembrane spanning region and an
ATP binding domain. A BLAST search indicates that ooc-5 is related to two other predicted genes in the
C. elegans genome and is a member of a family of predicted ATPases that have been identified in a
variety of species. A mutation in one of the human orthologs of this gene results in a neuromuscular
disease, but the biological basis for this disease is not well understood. We are currently generating GFP
reporter constructs and anti-OOC-5 antibodies to examine OOC-5 expression and localization.
65

West Coast Worm Meeting 2000
RIC-8 (SYNEMBRYN): A NOVEL REGULATOR OF G PROTEIN
SIGNALING
Kenneth G. Miller, Melanie D. Emerson, John R. McManus, James B.
Rand
Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK
73104
Recent studies describe a network of signaling proteins centered around GOA-1 (G oa) and EGL-30
(G qa) that regulates neurotransmitter secretion in C. elegans by controlling the production and
consumption of diacylglycerol (DAG). We sought other components of the G oa-G qa signaling network by
screening for aldicarb resistant mutants with phenotypes similar to egl-30 (G qa) mutants. In so doing we
identified ric-8, which encodes a novel protein named RIC-8 (synembryn). Through cDNA analysis we
show that RIC-8 is conserved in vertebrates. Through immunostaining we show that RIC-8 is
concentrated in the cytoplasm of neurons. Exogenous application of phorbol esters or loss of DGK-1
(diacylglycerol kinase) rescues ric-8 mutant phenotypes. A genetic analysis suggests that RIC-8 functions
upstream of EGL-30 (G qa), or in a parallel intersecting pathway. Both ric-8 and goa-1 reduction of
function mutants also exhibit partial embryonic lethality. Furthermore, the embryonic lethality of ric-8
mutants is enhanced to 95-100% by a 50% reduction in maternal goa-1 gene dosage. In a separate study
we investigated the roles of RIC-8 and GOA-1 in early embryos (pre 8-cell stage) and found that goa-1
and ric-8 mutant embryos exhibit defects in the movements of centrosomes. Comparing the roles of
RIC-8 and GOA-1 in the nervous system and the embryo reveals potentially informative differences
between the two pathways. First, EGL-30 (G qa) does not appear to play a role in the embryonic pathway.
Second, in the embryonic pathway, reduction of function mutations in goa-1 and ric-8 lead to similar
phenotypes that are enhanced in goa-1; ric-8 double mutants, whereas in the adult neuronal pathway the
same goa-1 and ric-8 mutants have opposite phenotypes and suppress each other. Based on these
findings, we propose that RIC-8 is required, directly or indirectly, for proper activation of G proteins in the
early embryo and in the nervous system.
66

West Coast Worm Meeting 2000
MED-1 AND -2 ACT AT THE CONVERGENCE POINT OF
SKN-1 AND POS-1 TO SPECIFY MS AND E IDENTITY
Morris F. Maduro, Regina Broitman-Maduro, Joel H. Rothman
Department of MCD Biology, UC Santa Barbara, Santa Barbara, CA 93106
EMS produces daughters with different fates: E produces the endoderm while MS makes mesoderm,
including the posterior pharynx. Depletion of the zygotically expressed MED-1 and -2 GATA transcription
factors by RNAi results in a penetrant MS ’ C transformation and an impenetrant E ’ C transformation,
similar to that seen in maternal skn-1 mutants. However, unlike skn-1 mutants, ABa still makes anterior
pharynx in med-1,2(RNAi) embryos, indicating that the med genes are not required for the
GLP-1-mediated signal that induces ABa pharynx. pos-1 mutants also misspecify MS, yet retain
GLP-1-dependent pharynx (Tabara et al. 1999). These observations suggest that POS-1 is also required
for an activity downstream of SKN-1.
The med genes appear to act at the convergence of SKN-1 and POS-1 function in the EMS lineage.
While expression of a med-1::GFP::MED-1 reporter is detected in the early E and MS lineages, it is
undetectable in both skn-1(RNAi) and pos-1(RNAi) embryos. Ectopic med expression is detected in
embryos expressing SKN-1 ectopically. In addition, expression requires a cluster of upstream conserved
SKN-1 binding sites, which bind bacterially expressed SKN-1 in vitro. We propose that POS-1, which is
primarily cytoplasmic, modulates an activity required for transcriptional activation of the meds by SKN-1.
end-1 and end-3 specify E fate, and are likely downstream targets of med-1,2 in the E lineage. Indeed,
GFP-tagged MED-1 binds in vivo to an extrachromosomal array containing the end-3 promoter. This
interaction also occurs in MS, where end-1 and end-3 are known to be repressed by POP-1. Thus, the
mechanism by which POP-1 represses E fate in MS does not preclude binding of MED-1 to end-3.
Our data indicate that SKN-1 directly activates the med genes through a POS-1-requiring mechanism. As
the meds are required for both E and MS specification, their activity appears to be modulated by POP-1,
the target of the Wnt/MAPK pathways in the E blastomere, perhaps at a step after their binding to target
promoters.
67

West Coast Worm Meeting 2000
MASS SPECTROMETRIC IDENTIFICATION OF PLP-1 AND ITS
ROLE IN MESENDODERM SPECIFICATION
E. Witze 1 , E. Field 2 , D. Hunt 2 , J.H. Rothman 1
1Department of MCD Biology, University of California, Santa Barbara, CA
2Department of Chemistry, University of Virginia, Charlottesville, VA
end-1 encodes a GATA type transcription factor that is sufficient to specify the endoderm progenitor, E.
end-1 is activated in the E lineage by the combined action of SKN-1 and the Wnt/MAP kinase-activated
form of POP-1 (see abstract by Kasmir et al.). It is repressed in MS, the sister of E, by POP-1 in the
absence of the Wnt/MAPK signal.
We are taking a biochemical approach to characterize additional factors that activate end-1 in the E
lineage and repress it in MS. We developed procedures for isolating factors from early developing C.
elegans embryos that bind key regulatory elements in end-1. Mass spectrometry of affinity-purified
proteins identified several end-1 binding factors, one of which, encoded by the plp-1 gene, is an apparent
homologue of a mammalian transcription factor called pur alpha, whose in vivo function is unknown.
We performed RNAi to examine the role of PLP-1 in mesendoderm development. Less than 10% of
plp-1(RNAi) embryos arrest before hatching. A fraction of these contain extra intestinal cells and show a
concomitant loss of MS-derived (posterior) pharynx. This phenotype appears to result from an MS to E
transformation, consistent with our finding that end-1 is sometimes expressed in MS and its descendants
in plp-1(RNAi) embryos. Thus, plp-1 appears to function in part to repress end-1 expression in the MS
lineage. As such, it may act as a corepressor with POP-1.
Paradoxically, we also observe an impenetrant (

West Coast Worm Meeting 2000
THE C. ELEGANS NEUROD HOMOLOG CND-1 FUNCTIONS
IN MULTIPLE ASPECTS OF MOTOR NEURON FATE
SPECIFICATION
Steven Hallam 1 , Emily Singer 1 , David Waring 2 , Yishi Jin 1
1Department of Biology, Sinsheimer Laboratories, University of California Santa Cruz, California 95064
2Fred Hutchinson Cancer Research Center, Seattle, Washington 98109
The basic Helix-Loop-Helix transcription factor NeuroD has been implicated in neuronal fate
determination, differentiation and survival. Here we report the expression and phenotypic analysis of
cnd-1, a C. elegans NeuroD homolog*. cnd-1 expression was first detected in neuroblasts of the AB
lineage in 14 cell embryos and maintained in many neuronal descendents of the AB lineage during
embryogenesis, diminishing in most terminally differentiated neurons prior to hatching. Specifically, cnd-1
reporter genes were expressed in the precursors of embryonic ventral cord motor neurons including
ABplpp, ABprpp and their progeny. A loss of function mutant, cnd-1(ju29), exhibited multiple defects in
the ventral cord motor neurons. First, the number of motor neurons was reduced, apparently caused by
the premature withdrawal of the precursors from mitotic cycles. Second, the strict correlation between the
fate of a motor neuron with respect to its lineage and position in the ventral cord was disrupted, as
manifested by the variable spatial and temporal expression patterns of motor neuron fate specific
markers. Third, motor neurons also exhibited defects in terminal differentiation characteristics including
axonal morphology and synaptic connectivity. Finally, the expression patterns of three neuronal
type-specific transcription factors, unc-3, unc-4 and unc-30, were altered. Our data suggest that cnd-1
may specify the identity of ventral cord motor neurons both by maintaining the mitotic competence of their
precursors and by modulating the expression of neuronal type-specific determination factors. cnd-1
appears to have combined the functions of several vertebrate neurogenic bHLH proteins, and may
represent an ancestral form of this protein family.
* Singer E et al., Worm Breeder’s Gazette 16(1): 52 (October 1, 1999)
69

West Coast Worm Meeting 2000
LEFT-RIGHT ASYMMETRY IN C. ELEGANS INTESTINAL
ORGANOGENESIS INVOLVES A LIN-12/NOTCH SIGNALING
PATHWAY
Greg J. Hermann, Ben Leung, James R. Priess
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 and Howard Hughes Medical
Institute
The C. elegans intestine is a simple tube consisting of a monolayer of polarized epithelial cells. During
embryogenesis, cells in the anterior of the intestinal primordium undergo reproducible movements that
create an invariant, asymmetrical "twist" in the intestine. We have analyzed the development of twist to
determine how left-right and anterior-posterior asymmetries are generated within the intestinal
primordium. The twist requires the LIN-12/Notch-like signaling pathway of C. elegans. All cells within the
intestinal primordium initially express LIN-12, a receptor related to Notch. However, only cells in the left
half of the intestinal primordium contact external, non-intestinal cells that express LAG-2, a ligand related
to Delta. LIN-12 and LAG-2-mediated interactions result in the left primordial cells expressing lower levels
of LIN-12 than the right primordial cells. We propose that this asymmetrical pattern of LIN-12 expression
is the basis for asymmetry in later cell-cell interactions within the intestinal primordium that lead directly to
intestinal twist. Like the interactions that initially establish LIN-12 asymmetry, the later interactions are
mediated by LIN-12. However, the later interactions involve a different Delta-related ligand, called APX-1.
We show that the anterior-posterior asymmetry in intestinal twist involves the kinase LIT-1, which is part
of a signaling pathway in early embryogenesis that generates anterior-posterior differences between
sister cells.
70

West Coast Worm Meeting 2000
THE PHO-1 GENE AND THREE KINDS OF GUT POLARITY
Tetsunari Fukushige, James D. McGhee
Department of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Dr. NW, Calgary,
Alberta, CANADA T2N 4N1
Many years ago (Beh et al., 1991), we described an acid phosphatase activity (pho-1) that was expressed
along the intestinal brush border in every gut cell except int-1 and int-2, beginning at the three fold stage
of embryogenesis. Thus, compared to early gut differentiation markers such as the ges-1 esterase, pho-1
should allow us to investigate three different polarities within the gut: anterior-posterior, apical-basal, and
temporal. The pho-1 gene was cloned and its expression pattern is now being analyzed. First of all,
fusions of the pho-1 promoter to GFP reporters reflect the endogenous expression pattern and do not
express in int-1 and int-2; in other words, the anterior-posterior patterning is at the level of the pho-1
promoter. We have previously shown that A/P patterned expression of a modified ges-1 reporter depends
on zygotic pop-1 activity (Schroeder and McGhee, 1998). This patterning within the E-lineage is
gut-autonomous and occurs after the Wnt-dependent P2-EMS contact of the four cell stage. We are now
investigating whether pho-1 responds to the same A/P patterning cues but in an opposite manner, i.e. we
would expect that high level of pop-1 within the gut cell nuclei would repress pho-1 expression, whereas
high level of pop-1 in gut nuclei activates the modified ges-1 reporter. An experiment to test this model is
to inject lit-1 doublestranded RNA into a pop-1(zu189) mother. The gut is still formed (zu189 results in low
maternal pop-1 and hence a double E cell) but loss of lit-1 activity should prevent downregulation of
zygotic pop-1 within the gut. Our prediction is that pho-1 should be repressed throughout the gut because
of high pop-1 and (in preliminary experiments) this is indeed what we see. The availability of a
temperature sensitive allele of lit-1 should allow us to investigate the gut patterning events in much finer
detail. To investigate apical-basal asymmetry, we are attempting to find a GFP construct that will reflect
the endogenous pho-1 distribution, in order to allow for genetic screens of polarity defects. At the
moment, all we know is that mutations in lin-2, lin-7 and lin-10 (shown by Stuart Kim’s lab to be involved
in epithelial polarity) have no apparent effect on the normal distribution of pho-1 activity. Finally, we are
investigating why pho-1 is expressed 2-3 E-cell divisions later than ges-1. There are GATA sites in the
promoter, that might be a target for elt-2. However, ectopic expression of either elt-2 or end-1 does not
activate ectopic expression of either endogenous pho-1 gene or pho-1 reporter gene, suggesting other
factors or events may be required. We are analyzing the pho-1 promoter to identify the site of action of
these timing factors or timing events.
71

West Coast Worm Meeting 2000
A ROLE FOR DISHEVELLED IN ASYMMETRIC CELL
DIVISION.
Nancy Hawkins 1 , Gregory Ellis 2 , Bruce Bowerman 2 , Gian Garriga 1
1Dept. of Mol. and Cell Biology, University of California, Berkeley, CA 94720
2Institute of Mol. Biology, University of Oregon, Eugene, OR 97403
Both intrinsic and extrinsic mechanisms can polarize asymmetrically dividing cells. Asymmetrically
distributed intracellular molecules are known to segregate fate to daughter cells during mitosis. Wnt
signaling also polarizes asymmetrically dividing cells by an unknown mechanism. We propose that in C.
elegans, Wnt signaling may segregate intracellular molecules that distribute developmental potential.
In the lineage that generates the HSN and PHB neurons, an HSN/PHB neuroblast divides asymmetrically
to generate an anterior daughter cell that dies and a posterior daughter cell, the HSN/PHB precursor. This
precursor then divides to produce the HSN and PHB neurons. In ham-1 mutants, the HSN/PHB
neuroblast frequently divides symmetrically producing two HSN/PHB precursors. ham-1 encodes a novel
protein that is expressed in a subset of cells during embryogenesis, and in mitotic cells, is often restricted
to one side in a crescent shaped pattern. In the HSN/PHB neuroblast, HAM-1 is asymmetrically localized
and is inherited by the HSN/PHB precursor.
To determine if Wnt signaling is also involved in asymmetric division of the HSN/PHB neuroblast, we
focused on the three C. elegans dishevelled (dsh) homologs. Upon Wnt stimulation, Dsh becomes
hyperphosphorylated and recruited to the cell membrane. Several lines of evidence indicate that dsh-2
(C27A2.6) is necessary for asymmetric division of the HSN/PHB neuroblast. First, both RNAi with dsh-2,
as well as a deletion allele, result in a Ham-1 phenotype in the HSN/PHB lineage. Second,
overexpression of dsh-2 produces weak defects in asymmetric cell division. Finally, DSH-2 appears to be
primarily membrane associated and is expressed from approximately the 4-6 cell stage until the 1 1/2 fold
stage. In cells expressing HAM-1, the two proteins co-localize and when HAM-1 is asymmetric, DSH-2
localization also appears to be asymmetric. We are performing co-immunoprecipitation and in vitro
binding experiments to determine if HAM-1 and DSH-2 physically interact.
72

West Coast Worm Meeting 2000
RHO-1, A TARGET OF THE EXCHANGE FACTOR UNC-73, IS
REQUIRED FOR CELL MIGRATIONS DURING C. ELEGANS
DEVELOPMENT
Andrew G. Spencer, Christian J. Malone, Satoshi Orita, Min Han
Howard Hughes Medical Institute, Department of Molecular, Cellular, and Developmental Biology,
University of Colorado, Boulder, CO, 80309-0347
Members of the rho family of small GTPases have been implicated and characterized in a variety of
cytoskeletal regulatory events. However, the spatial and temporal regulation of the activities of such
proteins in vivo is not well understood and represents an important aspect of the cell movements and
morphogenesis required during animal development. By expressing dominant-negative and
dominant-active versions of the C. elegans rhoA homologue rho-1 from a tissue specific promoter
(col-10), we establish a role for rho-1 during P cell migration to the ventral cord during the first larval
stage. Expression of rho-1 (dn) causes a strong P cell migration defect. This effect appears to be
specifically the result of rho-1 inhibition since expression of the C3 exoenzyme causes a similar P cell
phenotype. Genetic and biochemical analyses of unc-73, a GDP/GTP exchange factor, indicate that
unc-73 acts as an exchange factor for rho-1 during P cell migration in vivo. Another rho family GTPase,
mig-2, is not required for P cell migrations but may contribute to a common pathway with rho-1 in some
manner. This is based on observations that (a) mig-2 null alleles exacerbate a weak P cell migration
defect in unc-73 mutants (Zipkin, et al., 1997), which is partially rescued by rho-1 (gf) (Zipkin, Kindt, and
Keynon, 1997 1 ), and (b) mig-2 gain-of-function alleles cause a P cell migration defect, presumably by
interfering with members of the rho-1 pathway. In order to identify molecules acting downstream of rho-1
during P cell migration, we have studied multiple alleles of let-502. Putative null alleles of let-502 and
let-502 RNAi result in a weak P cell migration defect in worms surviving to L2. A temperature-sensitive
hypomorphic allele causes a similar P cell phenotype. We propose that unc-73 and rho-1 act together,
partially through let-502, to direct P cell migration in C. elegans.
1 Zipkin, Ilan D., Rachel M. Kindt, and Cynthia J. Kenyon Cell 1997 90: 883-894.
73

West Coast Worm Meeting 2000
PTP-1, A LAR-LIKE RECEPTOR PROTEIN TYROSINE
PHOSPHATASE, MAY ACT IN PARALLEL WITH C. ELEGANS
EPH SIGNALING TO DIRECT MORPHOGENESIS
Robert J. Harrington 1 , Michael Gutch 2 , Michael Hengartner 2 ,
Nicholas Tonks 2 , Andrew Chisholm 1
1Dept. of Biology, University of California, Santa Cruz CA 95064
2Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724
Mutations in the C. elegans Eph receptor tyrosine kinase, VAB-1, and the ephrins, VAB-2 and MAB-26,
cause defects in neural and epidermal morphogenesis. These defects are incompletely penetrant,
suggesting that other pathways may function in parallel. To identify components of such parallel pathways
we investigated whether vab-1 mutations displayed synthetic lethality with other morphogenetic mutants.
A mutation in a C. elegans receptor tyrosine phosphatase, ptp-1(op147), causes mild morphogenetic
defects similar to those of weak vab-1 alleles. We have found that ptp-1(op147) synergizes with vab-1
mutations, in particular, kinase domain mutations. ptp-1(op147) also synergizes to some degree with
mutations in vab-2 and mab-26, suggesting that PTP-1 may act in parallel with Eph signaling to regulate
morphogenesis. We are testing the specificity of this interaction with other morphogenetic mutants that
are not known Eph signaling components.
PTP-1 is most similar to LAR-like receptor protein tyrosine phosphatases. We have found that the ptp-1
locus encodes two isoforms, PTP-1A and PTP-1B. PTP-1A contains 3 Ig-like domains, 8 fibronectin type
III repeats, and two tandem cytoplasmic phosphatase domains. PTP-1B contains only 5 FNIII repeats and
the two phosphatase domains. Both isoforms are expressed in neurons.
We have analyzed the phenotype of ptp-1 single mutants and vab-1 ptp-1 double mutants at the 4D
microscopy level. We have found that ptp-1 mutants have gastrulation and ventral closure defects similar
to Eph signaling mutants. vab-1 ptp-1 double mutants show the same defects, only at a higher
occurrence. Our data suggests that PTP-1 and Eph signaling may function in parallel in neurons to direct
morphogenesis.
74

West Coast Worm Meeting 2000
GEX-2 AND GEX-3 DEFINE A CONSERVED PROTEIN
COMPLEX REQUIRED FOR TISSUE MORPHOGENESIS AND
CELL MIGRATIONS IN C. ELEGANS
Martha Soto 1 , Katsuhisa Kasuya 2 , Hiroshi Qadota 2 , Kozo Kaibuchi 2 ,
Craig C. Mello 1
1University of Massachusetts Medical Center, Worcester, MA 01605, USA
2Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma,
Nara, 630-0101, Japan
Morphogenesis during C. elegans embryogenesis requires coordinated cell movements. We report the
identification of two genes, gex-2 and gex-3, necessary for tissue morphogenesis and cell migrations
during C. elegans embryogenesis and for cell migrations in adult worms. Inactivation of gex-2 or gex-3
with the RNAi technique or a loss of function mutation in gex-3 caused a maternal effect embryonic
lethality with failure of dorsal intercalation and of ventral migrations of the epidermis. The gex-3 mutant
larva showed zygotic phenotypes including an egg laying defect (Egl) and defects in distal tip cell
migration.
gex-2 and gex-3 encode proteins with no known motifs that are conserved from nematodes to humans,
suggesting conserved essential functions for GEX-2 and GEX-3. The mammalian homolog of GEX-2,
Sra-1 was identified as a specific interactor of GTP-bound Rac. GEX-3 has a human homolog;
HEM2/NAP1/NCKAP1 is implicated in Rac signaling and is reported to bind the SH2-SH3 adaptor protein
NCK/DOCK. The GEX-3 fly homolog, HEM2/KETTE affects axonal cell migrations, actin distribution and
interacts genetically with NCK/DOCK.
Immunostaining with anti-GEX-2 and GEX-3 antibodies showed that GEX-2 and GEX-3 colocalize to
cell-cell contact sites of all embryonic cells. GEX-2 interacts with GEX-3 in the two hybrid system, and
GEX-2 and GEX-3 proteins can immunoprecipitate each other, suggesting GEX-2 and GEX-3 form a
complex. Our findings suggest that a novel protein complex including GEX-2 and GEX-3 localizes to cell
boundaries to regulate cell migrations and cell shape changes required for embryonic tissue
morphogenesis and for adult cell migrations. This complex may reorganize the actin cytoskeleton to
control cell shape changes necessary for dorsal intercalation, migration of the hypodermal cells in the
embryo and migration of the distal tip cells post-embryonically.
75

PHARYNGEAL EXTENSION: THE SHORT AND THE LONG OF
IT
MF Portereiko, SE Mango
West Coast Worm Meeting 2000
Huntsman Cancer Institute Center for Children and Department of Oncological, Sciences, University of
Utah, Salt Lake City, UT 84112
Pharyngeal morphogenesis initiates during mid-embryogenesis, when 78 of the 80 pharyngeal cells have
been born, and the embryo has begun to elongate. At this time, the pharyngeal precursors form a
compact ball that is attached to the nascent midgut and surrounded by a basement membrane that
encloses the entire primordium except for a gap at the anterior. Over the next 80 minutes, the pharyngeal
precursors alter their morphology and position to form a linear tube that is linked to the buccal cavity
anteriorly and the midgut posteriorly. We call this process pharyngeal extension. We have used
time-lapse videomicroscopy and GFP reporter constructs to follow the behavior of the pharyngeal
precursors during pharyngeal extension. Our studies demonstrate that pharyngeal extension can be
loosely divided into three stages i) reorganization of cellular polarity within pharyngeal cells (specifically
the pharyngeal epithelial precursors), ii) formation of an epithelium that mechanically couples the
pharyngeal cells to arcade cells in the nascent buccal cavity, and iii) an apparent contraction that shifts
the buccal cavity posteriorly and the pharynx anteriorly. We are considering a ’purse string’ model to
explain the cellular behaviors we see during contraction. Our findings suggest that pharyngeal extension
is the result of ’pulling’ by anterior pharyngeal cells rather than ’pushing’ by posterior pharyngeal cells. To
test this idea, we eliminated the posterior pharynx via genetic and physical means and demonstrated that
pharyngeal extension still occurs normally. Our model also predicts that tension between cells of the
pharynx and buccal cavity is required for the movement we observe during contraction. We tested this
idea by destroying the arcade cells. As expected, this manipulation blocked the anterior-directed
movement of the pharyngeal cells and reduced the posterior-directed movement of the buccal cavity. Our
current goal is to identify molecules required for pharyngeal extension using forward and reverse genetic
approaches.
76

A VAB-8/UNC-51/UNC-14 COMPLEX MEDIATES AXON
OUTGROWTH
Tina Lai, Gian Garriga
Molecular and Cell Biology, University of California, Berkeley, CA 94720
In C. elegans most posteriorly directed cell and growth cone migrations require vab-8, a gene that
encodes at least two protein products known as VAB-8L and VAB-8S. VAB-8L is a 1066 amino acid
protein that contains an N-terminal kinesin-like motor domain and functions in vab-8-dependent growth
cone migrations. VAB-8S is colinear with the C-terminal half of VAB-8L, and lacks the kinesin-like motor
domain. VAB-8S is necessary for certain vab-8-dependent cell migrations.
To identify VAB-8-interacting proteins, we conducted a yeast two-hybrid screen using full length VAB-8L
as bait. One protein identified was UNC-51, a serine/threonine kinase required for proper axon outgrowth
(Ogura et al., 1994). In yeast and in vitro, a region between the kinesin and shared domains of VAB-8L
interacts with the C-terminal half of UNC-51, which lacks the kinase domain.
Ogura et al. (1997) have shown that the C-terminal half of UNC-51 also interacts with UNC-14. Using
yeast two-hybrid, we found that a portion of VAB-8L interacts with UNC-14, but full length VAB-8L does
not. These observations suggest that UNC-14 interaction sites are masked in the full length VAB-8
protein.
Several observations suggest that VAB-8, UNC-14 and UNC-51 also interact in C. elegans. First, vab-8,
unc-14 and unc-51 mutants display axon outgrowth defects. Second, all three genes are expressed in
neurons that require vab-8 function. Finally, misexpression of the UNC-51-binding domain of VAB-8L
under control of the ceh-23 promoter results in highly penetrant CAN cell and growth cone migration
defects, presumably by interfering with UNC-51 binding with wildtype VAB-8L. We are in the process of
determining whether this misexpression phenotype could be suppressed by simultaneous misexpression
of unc-51.
Our results and those of Ogura et al. (1997) argue that VAB-8, UNC-14 and UNC-51 form a complex that
functions in the outgrowth of certain axons. We have observed that overexpression of vab-8 can suppress
axon outgrowth defects of an unc-51 mutant, suggesting a positive regulatory relationship between the
two genes. Two possibilities we are testing are whether VAB-8L is an UNC-51 target and whether VAB-8L
can regulate UNC-51 kinase activity.
Ogura et al. (1994) Genes & Dev. 8: 2389-2400.
Ogura et al. (1997) Genes & Dev. 11: 1801-1811.
West Coast Worm Meeting 2000
77

CYTOSKELETAL SIGNALLING IN RESPONSE TO THE UNC-6
AXONAL ATTRACTANT
Zemer Gitai 1 , Erik Lundquist 2 , Marc Tessier-Lavigne 1 , Cori
Bargmann 1
1HHMI, UCSF, San Francisco, CA 94143-0452
2Univeristy of Kansas, Lawrence, KS 66045
West Coast Worm Meeting 2000
The netrin UNC-6 is involved in attraction and repulsion of circumferentially migrating cells and axons in
C. elegans. Attraction to netrin/UNC-6 is mediated by the UNC-40 receptor, but the signalling events that
occur upon UNC-40 activation remain unknown.
In wild type animals UNC-6 is expressed ventrally, and several axons are directed ventrally via the
UNC-40 receptor. This ventral migration is disrupted in unc-6 and unc-40 mutants. To probe the
mechanism of UNC-40 activation by UNC-6, we deleted the extracellular domain of an unc-40 transgene
and added a myristylation signal to the cytoplasmic domain, generating what we refer to as
MYR::UNC-40.
When expressed under the control of the mec-7 promoter, MYR::UNC-40 produced a variety of novel
phenotypes in all mec-7-expressing touch cells. These phenotypes included exuberant axon outgrowth
and branching, defective axon guidance, and enlarged and deformed cell bodies. The effects are
independent of the endogenous UNC-6 ligand and UNC-40 receptor. These results lead us to believe that
the MYR::UNC-40 represents a ligand-independent, constitutively active form of UNC-40.
The generation of a gain-of-function UNC-40 molecule allowed us to search for downstream components
of the UNC-40 signalling pathway by looking for mutations that suppressed the MYR::UNC-40-induced
phenotype. One such mutation was in unc-115, a gene encoding a molecule with three LIM domains and
an actin-binding villin headpiece. Both unc-40 and unc-115 mutants alone had defects in the ventral
migration of the AVM axon. In the unc-40; unc-115 double mutant, the AVM axon guidance defect was
not enhanced, again suggesting that UNC-115 might function in the UNC-40 pathway. The agreement of
the loss-of-function double mutant phenotype with the MYR::UNC-40 suppression further supports the
idea that MYR::UNC-40 represents a gain-of-function of UNC-40.
Biochemical and cell biological experiments are currently underway to determine whether UNC-40 and
UNC-115 physically associate. Structure/function experiments are also being conducted to determine the
portion of the UNC-40 cytoplasmic domain responsible for signalling to UNC-115. Finally, we are
continuing to search for new components of the UNC-40 signalling pathway by looking for additional
suppressors of MYR::UNC-40.
78

West Coast Worm Meeting 2000
IDENTIFYING GENES INVOLVED IN AXONAL BRANCHING IN
C. ELEGANS
Joe C. Hao, Marc Tessier-Lavigne, Cornelia I. Bargmann
Department of Anatomy and HHMI, UCSF, San Francisco, CA 94143-0452, USA
In both the developing and mature nervous system, neurons can innervate multiple targets by sprouting
secondary axon collaterals, or branches, from a primary axon shaft. Although positive and negative
regulators of primary axonal growth cone guidance have been identified, little is known about the
molecular mechanisms mediating axonal branching. In order to elucidate genes that may control axonal
branching, we have performed a screen to identify mutants defective in branch formation by using a
reporter expressed in the chemosensory neuron ADL. Unlike other amphid neurons, whose axons enter
the nerve ring via the amphid commissures, the axon of ADL projects into the nerve ring laterally, where it
then branches into both a dorsal and a ventral process.
As a first step, we have examined the effects of known axon guidance genes on ADL axonal morphology.
Interestingly, the unc-6/unc-40 pathway may mediate ADL ventral branch formation. In unc-6 or unc-40
mutants, the majority of ADL axons lack the ventral axonal process. sax-3 mutants also exhibit defects in
branch formation, ranging from the loss of both branches to the absence of either process.
From our screen, we have isolated 36 mutants defective in ADL axonal branching that represent at least
17 complementation groups. These fall into three classes: i. mutants defective in ventral branching; ii.
mutants defective in dorsal branching; and iii. mutants with multiple branching defects. We have named
these the branching of axon defective, or bad mutants. In addition to known axon outgrowth and guidance
mutants, we have also isolated several novel mutants defective in axon guidance and/or branching.
We are currently attempting to clone and further characterize bad-1, a mutant with identical ADL
branching phenotypes as those observed in unc-6 and unc-40 mutants. These three mutants appear to
act in the same genetic pathway and also affect AVM axon guidance and PLM axon branching. However,
bad-1 animals are not uncoordinated and display normal motor neuron axon guidance, suggesting that
unc-5-dependent repulsion is preserved in these mutants. These results suggest a role for bad-1 in a
subset of unc-6/unc-40-mediated processes. We are also mapping and characterizing several other bad
mutants.
79

West Coast Worm Meeting 2000
UNC-119 SUPPRESSES SUPERNUMERARY BRANCHING IN
C. ELEGANS
Karla Knobel, Warren Davis, Michael Bastiani, Erik Jorgensen
Biology Dept., University of Utah, Salt Lake City, UT 84112
We previously characterized the behavior of migrating GABA motorneuron growth cones in C. elegans
larvae using time-lapse confocal microscopy. VD growth cones exhibit specific behaviors that result from
the interaction between growth cones and different cellular substrates encountered during migration . The
most dramatic behavior exhibited by migrating VD motorneuron growth cones in vivo is their collapse at,
and subsequent extension beyond the dorsal body wall muscle. To identify molecules required for specific
growth cone behaviors such as collapse we characterized the motorneuron axon outgrowth phenotype of
existing uncoordinated mutants. One mutant, unc-119(ed3), possessed abnormally branched axons, most
of which were located at the body wall muscle. This suggested that unc-119(ed3) growth cones extend
supernumerary branches between the muscle and epidermis. Analysis of the UNC-119 expression
pattern indicated that UNC-119 is expressed in neurons. Expression of UNC-119 under heterologous
promoters demonstrated that it functions cell-autonomously to suppress axon branching. However,
time-lapse analysis of motor neuron development and axon outgrowth indicated that UNC-119 does not
function during growth cone migration. Comparison of axon outgrowth patterns 1 hour and 48 hours after
the completion of growth cone migration indicated that axon branching occurs after axon outgrowth is
completed in unc-119(ed3) mutants. Time-lapse analysis of established axons demonstrated that
secondary growth cones sprout from motorneuron axons and cell bodies. These secondary growth cones
extend extra branches to the dorsal nerve cord. Concurrently axonal extensions along the dorsal nerve
cord are retracted. As a result, unc-119(ed3) adults have many commissural branches as well as large
gaps in the dorsal nerve cord. Synapse development occurs after outgrowth is completed. To determine if
synaptogenesis is affected we characterized synapses in unc-119(ed3) mutants. Synapses form and are
morphologically normal as determined by electron microscopy. However, synapses are localized to
inappropriate locations of the axon, including branches and dendritic regions. These data suggest that
UNC-119 is a member of a new class of cell intrinsic factors that preserve the architecture of the nervous
system and regulate synapse localization.
80

UNC-119 AND AXON OUTGROWTH: TOWARD A
MECHANISM
Wayne Materi, Dave Pilgrim
West Coast Worm Meeting 2000
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9
UNC-119 is crucial for the correct development of the nematode nervous system. Worms mutant for the
neuronally-expressed unc-119 gene show locomotory and sensory abnormalities that are explained by
structural defects resulting from aberrant nervous system development.
Direct examination of nervous system structure using GFP reporters shows that unc-119 mutants have a
variety of neurite outgrowth defects. The axons of chemosensory amphids exhibit highly penetrant ventral
elongation defects within the nerve ring while the axons of anterior mechanosensory neurons have similar
elongation defects in the dorsal direction. These defects suggest a disruption of normal synaptic patterns
that is sufficient to explain both the inability of unc-119 mutants to form dauer larvae and their inability to
respond to touch. The ventral nerve cord is moderately to severely defasciculated in 50% of mutant
worms, supporting a general role for UNC-119 in axon guidance.
Defects in the anteroposterior position of dorsolateral turns suggests an aberrant response to choice
points. However, there are no circumferential pathfinding defects in motor neurons, similar to those seen
in unc-6 , unc-5 or unc-40 mutants. Indeed, although some cell bodies are misplaced in unc-119 mutants,
their axons are often correctly targeted, implying that turning at choice points is somewhat independent of
other pathfinding mechanisms.
Expression and rescue experiments with functional UNC-119::GFP constructs suggest that it acts cell
autonomously. Although sub-cellular localization of the rat homologue, RRG4, has been demonstrated
within photoreceptor ribbon synapses, antibody studies in the worm are still in progress. Yeast two-hybrid
experiments suggest that UNC-119 is involved in a novel neurite development signaling pathway that
mediates interaction between basement membrane collagen’s and an uncharacterized zinc-finger protein.
We have used PCR-based reverse genetics to isolate strains bearing deletions in the latter of these and
have observed a lack of exploratory behavior but otherwise normal locomotion and response to touch
stimuli.
81

THREE DISTINCT FUNCTIONS OF BETA-SPECTRIN (UNC-70)
Marc Hammarlund, Warren S. Davis, Erik M. Jorgensen
University of Utah, SLC UT 84112
We cloned unc-70 and found that it encodes the C. elegans homolog of beta-spectrin, an essential
component of the membrane skeleton.1 The membrane skeleton is a protein mesh that binds to the
plasma membrane and that interacts with a large number of membrane and cytosolic proteins. It has
been proposed that the membrane skeleton functions to support the plasma membrane or to generate
cell polarity. However, we found that null mutations in unc-70 do not result in general membrane or
polarity defects. unc-70 null animals have a severe paralyzed, dumpy phenotype. By analyzing this
phenotype we have identified at least three specific processes that are perturbed by loss of beta-spectrin.
First, we found defects in axonal morphology, suggesting that the membrane skeleton plays an essential
role in neuronal development. We suspect that loss of beta-spectrin may specifically affect growth cone
motility and are conducting time-lapse studies of unc-70 growth cones to test this hypothesis. Second,
we found that loss of beta-spectrin causes defects in the myofilament attachment structures of body wall
muscle (dense bodies and M lines). We are using a panel of antibodies against components of these
structures to understand how the membrane skeleton functions in muscle. Finally, the membrane
skeleton appears to function in synaptic transmission. We found that rare dominant alleles of unc-70 do
not exhibit the null phenotypes described above; rather, they have abnormally high levels of
neurotransmission. Preliminary evidence suggests that synaptic vesicles are mislocalized within the
nerve terminal of these mutants. We are analyzing the synaptic ultrastructure of these mutants, and
using electrophysiology to relate structural defects to possible functional roles for beta-spectrin in nerve
terminals.
1. Hammarlund, M. et al., J. Cell Biol., 149(4), 2000.
West Coast Worm Meeting 2000
82

RPM-1, A CONSERVED NOVEL PROTEIN, REGULATES
PRESYNAPTIC TERMINAL FORMATION
Xun Huang 1 , Mei Zhen 1 , Bruce Bamber 2 , Yishi Jin 1
1Department of Biology, University of California, Santa Cruz, CA 95064
2Department of Biology, University of Utah, Salt Lake City, UT 84112
Presynaptic terminals contain highly specialized subcellular structures to facilitate neurotransmitter
release. We used the synaptic vesicle tagged GFP marker under the unc-25 promoter to visualize the
presynaptic varicosities of the GABAergic DD and VD motor neurons 1 . In wild-type animals GFP
expression is observed as distinct fluorescent puncta, corresponding to individual neuromuscular
junctions along the dorsal and ventral nerve cords. rpm-1 mutations were isolated in a screen for
abnormal morphology of the GFP puncta.
In rpm-1 mutants the total number of these GFP puncta is reduced to various extents depending on the
strength of the mutation, and regions along the dorsal and ventral nerve cords often contained no GFP
puncta. Moreover, the remaining GFP puncta were variable in size and shape. The overall axonal
morphology of the DD and VD neurons appeared normal. At the ultrastructural level, we observed two
defects at GABAergic NMJs in the rpm-1 mutants: 1) "over-developed" synapses that contained more
than two individual electron-dense presynaptic active zones within the same varicosity, 2)
"under-developed" synapses that had few synaptic vesicles and instead were filled with electron-dense
debris-like material. In cholinergic NMJs, we observed the presynaptic terminals with longer active zones.
rpm-1 encodes a large protein similar to Drosophila Highwire (Hiw) and mammalian Pam. All three
proteins have a putative GEF domain and a highly conserved C-terminal with several zinc finger domains.
RPM-1 is localized to the presynaptic region independently of synaptic vesicles. Mosaic analysis indicated
that RPM-1 functions cell-autonomously. Using a temperature sensitive allele of rpm-1, we found that
RPM-1 is required around the time of the formation of presynaptic terminals. Our results suggest that
RPM-1 may regulate the distribution of presynaptic terminals, or function to prevent the formation of
excessive presynaptic structures.
1. Zhen, M and Jin, Y. Nature 1999
West Coast Worm Meeting 2000
83

A C. ELEGANS INOSITOL 5- PHOSPHATASE HOMOLOGUE
INVOLVED IN INOSITOL 1,4,5-TRIPHOSPHATE SIGNALING
AND OVULATION.
Yen Kim Bui, Paul W. Sternberg
West Coast Worm Meeting 2000
Howard Hughes Medical Institute and Division of Biology, California Institute of Technology, Pasadena,
CA, 91125 USA.
Understanding the basis of positive and negative regulation of the inositol signaling pathway may
increase our understanding of the control of diverse biological processes dependent on inositol
1,4,5-triphosphate (IP3). J. McCarter in T. Schedl’s lab demonstrated that mutations in lin-3/EGF or let-23
/Receptor Tyrosine Kinase (RTK) result in a sterile Emo phenotype whereby the oocytes fail to be
ovulated and become trapped in the gonad arm and undergo multiple rounds of DNA synthesis. T.
Clandinin in our lab, showed that either a loss of function mutation in thelfe-2 /IP-3 kinase or gain of
function (gf) in the lfe-1/IP3 Receptor can bypass the requirement for RTK signaling indicating that
ovulation in C. elegans is dependent on IP3 signaling. Inositol 5-phosphatase plays a role in
dephosphorylating IP3 to inositol 1,4-phosphate. However the contribution of this step in terminating IP3
signaling in vivo remains unclear. The C. elegans genome predicts a putative ortholog of the human Type
I inositol 5-phosphatase (CO9B8.1).
Using a PCR based strategy, we screened for a deletion (D) mutant of the type I C. elegans inositol
5-phosphatase. In contrast to the lfe-2 null or lfe-1 (gf), the 5-Ptase D mutant shows a novel ovulation
phenotype whereby two oocytes enter the spermetheca during a single ovulation cylce; thus suggesting a
crucial role for the 5-Ptase in regulating IP3 mediated ovulation. In contrast to the lfe-2 overexpression
phenotype whereby the oocyte remains stuck in the dilated spermetheca, overexpression of the 5-Ptase
has no noticeable affect. We are characterizing the mechanism by which it regulates ovulation by genetic
analysis. Our genetic data support the hypothesis that increased IP3 signaling causes more ovulation,
and that either too much signaling or too little signaling prevents ovulation. Like the lfe-2 null mutant, the
5-Ptase D mutant suppresses the sterile defect of lin-3(n1058). This suggests that lin-3 sterile mutants fail
to generate sufficient IP3 to execute ovulation. Furthermore, the 5-Ptase D mutant synthetically interacts
with others mutants that increase IP3 signaling to produce a sterile Emo phenotype.
84

West Coast Worm Meeting 2000
MECHANISMS REGULATING THE TIMING AND SPECIFICITY
OF ANCHOR CELL ATTACHMENT TO THE VULVAL
EPITHELIUM
David R. Sherwood, Paul W. Sternberg
Division of Biology and HHMI, California Institute of Technology, Pasadena CA
We are interested in understanding the basic cellular and molecular mechanisms that guide
morphogenetic processes during development. Towards this goal we are examining the initial steps
involved in the connection of the uterus and vulva during C.elegans development. The initial contact
between the developing uterus and the vulva is established by the anchor cell (AC), which crosses the
basement membranes separating both tissues and specifically attaches to the progeny of the P6.p cell
during the mid- to late L3 stage. Following attachment, the AC invades between the inner descendants of
the P6.p. cell and becomes positioned at the apex of the developing vulva. Using mutants lacking vulval
induction, we have found that the descendants of the underlying induced vulval precursor cell, P6.p, send
a signal to trigger the attachment behavior in the AC. We have also discovered that the competence of
the AC to respond to this signal is regulated: AC attachment is either absent or delayed in the
heterochronic mutant lin-28, which causes precocious vulval development. To understand the molecular
mechanisms that regulate AC attachment, we have examined many known mutants, as well as initiated a
genetic screen to search for new mutants with defective attachment behavior. Through these studies we
have found that the evl-5 mutant, originally isolated in a screen for sterile p-vul’s (Seydoux et al., (1993)
Dev. Biol. 157(2): 423-36), has a defect in AC attachment to the vulval epithelium. In evl-5 animals vulval
induction and AC positioning over the P6.p cell is normal; however, AC attachment either does not occur
or is severely delayed. Visualization of AC behaviour in this mutant revealed the extension of cellular
processes from the AC toward the vulva, indicating that the AC is still attracted to the developing vulva.
However, in many cases the AC processes appeared to flatten or broaden out at the basement
membrane of the developing gonad, suggesting that the AC may not be able to cross this basement
membrane. We are currently molecularly cloning evl-5.
85

MUTATIONS IN CYCLIN E REVEAL COORDINATION
BETWEEN CELL-CYCLE CONTROL AND VULVAL
DEVELOPMENT.
David S. Fay, Min Han
West Coast Worm Meeting 2000
Howard Hughes Medical Institute and Department of Molecular, Cellular, and Developmental Biology,
University of Colorado, Boulder CO 80309-0347
In screens for mutations affecting vulval development, we have identified strong loss-of-function
mutations in the C. elegans cyclin E gene, cye-1. Mutations in cye-1 lead to the under-proliferation of
many postembryonic blast lineages as well as defects in fertility and gut-cell endoreduplication. In
addition, cye-1 is required maternally, but not zygotically for embryonic development. Our analysis of
vulval development in cye-1 mutants suggests that a timing mechanism may control the onset of vulval
cell terminal differentiation: once induced, these cells appear to differentiate after a set amount of time,
rather than a specific number of division cycles. cye-1 mutants also show an increase in the percentage
of vulval precursor cells (VPCs) that adopt vulval cell fates, indicating that cell-cycle length can play a role
in the proper patterning of vulval cells. By analyzing cul-1 mutants, we further demonstrate that vulval cell
terminal differentiation can be uncoupled from associated changes in vulval cell division planes.
86

West Coast Worm Meeting 2000
NOVEL CELL-CELL INTERACTIONS DURING VULVA
DEVELOPMENT IN PRISTIONCHUS PACIFICUS
Benno Jungblut 1,2 , Ralf J Sommer 1,3
1Max-Planck Institut für Entwicklungsbiologie, Abt. Evolutionsbiologie, Spemannstrasse 35, 72076
Tübingen, Germany
2email: benno.jungblut@tuebingen.mpg.de
3email: ralf.sommer@tuebingen.mpg.de
Comparative analysis of vulva development revealed several differences between the two nematode
species Caenorhabditis elegans and Pristionchus pacificus including changes in cell fates and inductive
processes. For example, seven of 12 ventral epidermal cells in P. pacificus die of apoptosis, whereas
homologous cells in C. elegans fuse with the hypodermal syncytium. In addition vulva induction by the
anchor cell is a one-step process in C. elegans, but requires an interaction of both anchor cell and the
gonad with the vulval precursor cells in P. pacificus. We have identified novel cell-cell interactions while
carrying out combinatiorial cell ablation studies of the vulval precursor cells. In particular the ventral
epidermal cell P8.p and the mesoblast M contribute to vulval pattern formation. In contrast to the
homologous cell in C. elegans, P8.p in P. pacificus is incompetent to respond to inductive signaling from
the gonad. Nonetheless it can be induced to adopt a vulval fate by lateral signaling from a neighboring
vulval precursor cell. Immunofluorescence studies showed that P8.p fuses with the hypodermis a few
hours before the birth of the anchor cell. Using cell ablation studies we show that P8.p provides an
inhibitory signal that limits the developmental competence of P(5, 7).p. This lateral inhibition also requires
the mesoblast M. In Ppa-mab-5 mutants, M is misspecified and provides inductive signaling to the vulval
precursor cells, including P8.p. Therefore P8.p differentiates gonad independently in these mutants.
Taken together, vulva development in P. pacificus displays novel cell-cell interactions involving the
mesoblast M and P8.p. In particular, P8. represents a new ventral epidermal cell fate, which is
characterized by novel interactions and a specific response to gonadal signaling.
87

West Coast Worm Meeting 2000
CELLULAR AND GENETIC ANALYSIS OF G Q MEDIATED
SIGNALING PATHWAYS IN C. ELEGANS
C. A. Bastiani, S. Gharib, P.W. Sternberg, M.I. Simon
Division of Biology, California Institue of Technology, Pasadena, CA 91125
egl-30 encodes the C. elegans homologue of the mammalian heterotrimeric G protein alpha subunit, Gq.
Reduction-of-function and loss-of-function mutations in egl-30 affect diverse behaviors in C. elegans.
These include: viability, egg-laying, pharyngeal pumping, movement, and spicule protraction. In a screen
for suppressors of the reduction-of-function mutation, egl-30(md186), two intragenic suppressor mutations
were recovered. One of the mutations lies in a region in close proximity to residues involved in guanine
ring binding. The other mutation is in a region that might affect receptor interaction, and is not expected to
make contacts with the region that contains the original egl-30(md186) mutation. These mutants
phenotypically resemble worms that overexpress egl-30, and have been used as a tool to characterize
genes that function in a pathway with egl-30. From these studies, it is clear that EGL-30 regulates egg
laying via downstream signaling pathways distinct from downstream pathways so far defined that control
movement (1), viability (2), and spicule protraction (3). A rescuing gfp::egl-30 transgene confirms that
egl-30 is coexpressed in some cells with egl-8 and lfe-1/itr-1/dec-4, two genes that encode signaling
molecules defined as downstream of Gq in mammalian systems, and that appear to be downstream of
egl-30 with respect to movement and viability, respectively (1,2). However, egl-30 is also uniquely
localized and expressed in some cells. To determine the basis for the differences in genetic interactions
with respect to different behaviors, we are develooping a system to characterize cell-type-specific
molecular interactions with EGL-30 in vivo .
1. Lackner MR, Nurrish SJ, Kaplan JM; Neuron 24: 335-346 1999
2. Hajdu-Cronin YM and Sternberg PW, personal communication
3. Garcia LR and Sternberg PW, personal communication
88

West Coast Worm Meeting 2000
CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE II
REGULATES C. ELEGANS LOCOMOTION IN CONCERT WITH
A G-PROTEIN SIGNALING NETWORK
Merrilee Robatzek, James H. Thomas
Department of Genetics, University of Washington, Seattle, WA 98195 USA
Calcium/calmodulin-dependent protein kinase II (CaMKII) is an important regulator of synaptic strength
based on studies in mollusks, Drosophila, and mouse. Although the phosphorylation of many gene
products has been attributed to CaMKII from in vitro assays, the mechanism of CaMKII activity in vivo has
not been fully defined. We are using a genetic approach to study the in vivo function of the C. elegans
CaMKII homolog unc-43. UNC-43 is 83% identical in the kinase domain to mammalian CaMKII, indicating
that the targets of this kinase are probably conserved.
unc-43 regulates several behaviors, including locomotion. A kinase-activated allele of unc-43, n498,
causes severe lethargy, as well as body-wall muscle hypercontraction, reduced egg laying, and reduced
defecation. Several of these effects are genetically separable, suggesting that unc-43 regulates
defecation, body-wall muscle tone, and locomotion rate through different effectors. To understand how
unc-43 controls locomotion rate in C. elegans, we performed a genetic suppressor screen with
unc-43(n498) to identify genes that act with unc-43 to control locomotion rate. From a screen of 28,000
EMS-mutagenized haploid genomes, we recovered 19 recessive extragenic suppressors.
Complementation tests showed that we had recovered multiple alleles of the genes goa-1, dgk-1, and
eat-16, all involved in the goa-1/egl-30 heterotrimeric G-protein network , , , and alleles of a fourth gene,
eat-11, that probably affects this same pathway. In addition to suppressing the unc-43(n498) lethargy,
mutations in these genes also suppress the unc-43(n498) egg-laying defect. Quantitative analysis of the
suppression indicates that UNC-43 may control locomotion rate and egg-laying activity by regulating this
G-protein signaling network.
89

A NOVEL LATERAL SIGNALING PATHWAY DETERMINES
ASYMMETRIC OLFACTORY NEURON FATES
Alvaro Sagasti, Cori Bargmann
UCSF and HHMI
West Coast Worm Meeting 2000
The AWC olfactory neurons in C. elegans are a bilaterally symmetric pair of cells required for sensing
certain attractive volatile odorants. Although bilaterally homologous neurons are usually thought to be
identical, the candidate seven transmembrane olfactory receptor str-2 is expressed asymmetrically in the
AWC neurons. About half of a population of worms expresses str-2 in the left homolog of the AWC pair,
and half expresses it in the right homolog. The AWC cells are able to coordinate their fates by
communicating with each other, probably through their axons which contact each other in the nerve ring.
To study the signaling mechanisms by which this communication is accomplished we conducted a screen
for mutant worms that express a str-2::GFP reporter in both AWC neurons. The screen yielded mutations
in three novel neuronal symmetry genes (nsy-1, nsy-2, and nsy-3) and three known genes that affect
calcium signaling: alleles of the voltage-gated calcium channel subunits unc-2 and unc-36 and an allele of
the calcium/calmodulin-dependent protein kinase II homolog unc-43. This suggests that calcium signals
are an important component of the AWC asymmetry signaling pathway. Genes in a cGMP signaling
pathway necessary for olfaction have the phenotype opposite of the calcium signaling genes. They are
required for any expression of str-2 in AWC, perhaps in an activity-dependent mechanism used for
maintenance of receptor expression. Epistasis analysis is consistent with nsy-1 and nsy-2 acting after
calcium signaling but before cGMP signaling. In contrast, the dominant mutant nsy-3 seems to act after
axon outgrowth but before calcium signaling. We recently cloned the nsy-1 gene and found that it
encodes a protein kinase. We are currently performing experiments to determine where nsy-1 is
expressed, whether it is acting in the AWC cells, and how it is activated by CaMKII. We are also setting
up a system that will permit us to identify easily AWC left/right mosaics, allowing us to determine which
genes in this pathway act permissively and which act instructively
90

West Coast Worm Meeting 2000
THE SEARCH FOR DOSAGE COMPENSATION COMPLEX
BINDING SITES ON X CHROMOSOMES
Raymond C. Chan, Tammy F. Wu, Barbara J. Meyer
HHMI and Dept. of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3204
Dosage compensation (DC) equalizes the level of X-linked gene products between XX hermaphrodites
and XO males. A protein complex (DCC), with remarkable similarity to the Xenopus mitotic 13S
condensin complex, is directed by the sex-determination and dosage compensation (SDC) proteins to the
hermaphrodite X chromosomes, where it represses gene expression. A mechanistic link between dosage
compensation and mitotic chromosome condensation is further indicated by the dual roles of many DCC
subunits in mitosis or meiosis. This link raises an intriguing question of how the DCC specifically
recognizes X chromosomes to effect global repression of gene expression. To study X recognition, we
developed a chromatin immunoprecipitation (ChIP) protocol to isolate DNA bound by the DCC. We
initially devised an affinity purification method to precipitate DC proteins using peptide antibodies against
the DCC. The IP not only confirmed the association of DCC subunits, it also provided a means to isolate
large quantities of DC and mitotic complexes for identifying novel co-purifying factors (see Hagstrom et
al.). When combined with in vivo formaldehyde crosslinking, we can also recover DNA in the precipitate,
thus enabling us to identify candidate X recognition sequences.
To validate this ChIP assay, we tested the known interaction between the SDC proteins and the her-1
promoter. Aside from DC, the SDC proteins bind and repress the promoter of her-1 to establish the
female fate in young XX embryos. In the sdc-3(y52Tra) mutant strain, SDC proteins fail to bind the her-1
promoter leading to the masculinization of XX animals. With our current protocol, we detected her-1
promoter sequences by PCR in both SDC-2 and SDC-3 ChIP. Moreover, ChIP performed on extracts
from sdc-3(Tra) embryos revealed drastically reduced levels of her-1 DNA consistent with the loss of
her-1 binding in these mutants.
We are now focused on identifying X sequences bound by the SDC and DCC proteins. We have
examined 180 kb around myo-2, a known dosage compensated gene on X, and we found an
approximately 4-kb region enriched in the SDC and DCC ChIP. Experiments are in progress to further
define this region and to confirm SDC and DCC protein binding in vivo and by additional in vitro methods.
91

West Coast Worm Meeting 2000
RECOGNITION AND ASSEMBLY OF SDC PROTEIN
COMPLEXES ONTO SPECIFIC DNA TARGET SITES
Diana Chu 1 , Heather Dawes 1 , Jason Lieb 2 , Annie Kuo 1 , Barbara J.
Meyer 1
1HHMI and Department of Molecular and Cell Biology
2HHMI and Department of Biochemistry, Stanford University Medical Center, Stanford, CA 94305
SDC (Sex and Dosage Compensation) proteins trigger hermaphrodite development by activating dosage
compensation and repressing the male sex determination gene her-1. We have biochemically defined an
SDC protein complex including SDC-1, SDC-2, and SDC-3. This complex localizes to the her-1 promoter,
where it represses transcription at least 20-fold. It also associates along the entire length of the
hermaphrodite X chromosomes, where it recruits other dosage compensation proteins (DC) such as
DPY-26, DPY-27, DPY-28 and MIX-1 to repress X-linked gene expression 2-fold. We are interested in
how the SDC complex can recognize and act differentially at these separate sites.
Despite the fact that DC proteins are not required for transcriptional repression of her-1, we have found
that the SDC proteins recruit these DC proteins to the her-1 promoter. We showed this co-localization in
transgenic animals that express a GFP-LacI fusion protein from extrachromosomal arrays that also
contain both lac operator repeats (lacO) and the her-1 promoter. Analysis of SDC and DC protein
localization to the her-1 promoter arrays in SDC and DC mutant backgrounds has revealed sequential
requirements for recognition and assembly of the SDC and DC proteins onto target sequences.
To define how SDC proteins recognize specific target sites, we mapped regions within the her-1 promoter
that are important for SDC protein recognition using the assay above. To test the functional significance
of the three regions we identified, we mutated them in the context of a full length her-1 rescuing construct.
Stable transgenic XX strains transformed with a wild-type her-1 show little masculinization. In contrast,
stable transgenic XX strains transformed with mutated her-1 show varying degrees of transformation to
the male fate, including induction of male tail structures and decreased fertility. We interpret this
masculinization as an indication of her-1 derepression. Interestingly, sequences in the regions important
to support SDC-2 localization are not homologous to sequences on the X chromosome. Thus, recognition
of the X chromosome and the her-1 promoter appears to be specified by distinct mechanisms.
92

West Coast Worm Meeting 2000
THE TBP-LIKE FACTOR CETLF IS REQUIRED TO ACTIVATE
RNA POLYMERASE II TRANSCRIPTION IN C. ELEGANS
EMBRYOS
Linda S. Kaltenbach, Susan E. Mango
Huntsman Cancer Institute and Department of Oncological Sciences, University of Utah, Salt Lake City,
UT, 84112
Modulation of transcription is crucial to establish differences between cells during development. While the
importance of activators and repressors for regulating transcription is well known, recent data suggest
coregulators and general transcription factors may provide an additional source of control. TATA-binding
protein (TBP) is a component of the basal transcription machinery that is ubiquitously expressed in
metazoans and essential for all transcription in yeast. Recently, two variants of TBP have been
discovered: 1) TBP-Related Factor, known only in Drosophila, which may act as a cell-type specific TBP
and 2) TBP-Like Factor (TLF), found in most, if not all, metazoans. Given the central role of TBP, what is
the function of the TBP paralogs?
On the basis of biochemical studies, four models have been proposed for the role of TLF in RNA
polymerase II (pol II) transcription: i) TLF and TBP could function redundantly, ii) TLF could antagonize
TBP, iii) TLF could be a tissue-specific TBP or iv) TLF and TBP could have unique activities. To
distinguish between these models, we examined the function of C. elegans TLF (CeTLF) in vivo.
We have found that CeTLF has an essential role to activate pol II transcription in early embryos. Loss of
CeTLF activity by RNAi results in embryonic arrest at the 70-200 cell stage with no evidence of
differentiation. This phenotype and global expression of CeTLF demonstrate that CeTLF is not a
tissue-specific TBP. Embryos lacking CeTLF fail to gastrulate, a phenotype that resembles loss of ama-1,
which encodes the large subunit of pol II. These embryos also express a marker of elongating pol II at a
reduced level, suggesting that CeTLF is required for some but not all pol II transcription. To test this idea,
we have examined whether CeTLF is necessary to express individual genes normally transcribed in the
gastrula-stage embryo and find that CeTLF is required to activate two of four surveyed genes. Preliminary
experiments indicate that CeTLF physically associates with at least one of these genes in vivo. These
results suggest that CeTLF performs a unique function to activate pol II transcription at some promoters,
and that this activity is distinct from that of CeTBP.
93

West Coast Worm Meeting 2000
THE INTRACELLULAR DOMAIN OF THE FEMINISING
RECEPTOR TRA-2A INTERACTS DIRECTLY WITH THE
TRANSCRIPTION FACTOR TRA-1A
David H. Lum 1 , P. Kuwabara 2 , D. Zarkower 3 , A.M. Spence 1
1Dept. of Molecular and Medical Genetics, University of Toronto, ON, M5S 1A8, CANADA.
2MRC Laboratory of Molecular Biology, Hills Road, Cambridge , CB2 2QH.
3Institute of Human Genetics, Minneapolis, MN 55455, U.S.A.
In XX animals the tra-2 gene negatively regulates the activity of the three fem genes. The absence of fem
activity in XX animals frees tra-1, the terminal regulator of somatic sex determination, to promote female
development. The tra-2 gene encodes two proteins TRA-2A and TRA-2B. The larger of the two proteins,
TRA-2A, is a predicted transmembrane protein with a large C-terminal intracellular domain. TRA-2B is
expressed in the hermaphrodite germline and is predicted to be a soluble protein consisting of just the
C-terminal domain of TRA-2A.
The intracellular domain of TRA-2A negatively regulates the FEMs through a direct interaction with
FEM-3. Overexpression of the C-terminal domain of TRA-2A (TRA-2Ac) is sufficient for FEM negative
regulation and the transformation of XO animals into females. To investigate the mechanism of TRA-2Ac
feminizing activity we overexpressed various TRA-2Ac fragments. Surprisingly, a C-terminal fragment of
TRA-2Ac (TRA-2AcD 5), incapable of interacting with FEM-3 in the yeast 2-hybrid system, maintained
weak feminizing activity. Both yeast 2-hybrid and biochemical data demonstrate that TRA-2AcD 5
physically interacts with TRA-1A. tra-2(mx) mutations affect tra-2 activity in both the soma and germline.
Three mx mutations all disrupted the TRA-2Ac/TRA-1A interaction and reduce or eliminate the feminising
activity of TRA-2AcD 5 in overexpression assays.
Our surprising results suggest that in the soma TRA-2A plays a role in regulating TRA-1A through a direct
interaction. TRA-1A has been demonstrated to directly regulate transcription of several genes and is
predicted to be nuclear localized. We observe strong nuclear localization of both GFP::TRA-1A and
Myc::TRA-1A in transgenic animals. Consistent with a nuclear role for TRA-2A regulation of TRA-1A, we
observe nuclear localization of a highly active GFP::TRA-2Ac fusion protein. Our data raise the intriguing
possibility that in the soma TRA-2A is cleaved to allow the intracellular domain to enter the nucleus and
interact with TRA-1A.
94

West Coast Worm Meeting 2000
CHW-1 ENCODES A NOVEL PROTEIN THAT INTERACTS
WITH PHA-4
Michael Horner, Linda Kaltenbach, Susan Mango
Huntsman Cancer Institute and Department of Oncological Sciences, University of Utah, Salt Lake City,
Utah 84112
The winged-helix transcription factor PHA-4 specifies organ identity during pharynx and rectal
development. Embryos lacking pha-4 activity produce no pharyngeal or rectal cells while embryos
expressing ectopic pha-4 generate extra pharyngeal and rectal cells at the expense of other cell types.
PHA-4 is also expressed in the gonad, but its function in this organ is unknown.
Homologs of pha-4 are found in most if not all metazoans, where they also regulate gut development.
Proteins belonging to this family are predicted to be transcription factors, with a well-characterized DNA
binding domain. However, little is known about how these proteins regulate transcription to produce such
profound effects on development. To begin to address this issue, we have initiated an analysis of PHA-4
protein and PHA-4 cofactors. Our characterization of five pha-4 alleles demonstrates that the amino
terminus is critical for PHA-4 function. Surprisingly, the carboxyl terminus, which is conserved with
vertebrate HNF-3 proteins and Ce-DISTALLESS, is dispensable.
Since transcription factors often function by recruiting other proteins to target genes, we used a yeast
2-hybrid screen to identify proteins that bind the PHA-4 amino terminus. We isolated six potential
cofactors from a screen of >1x106 clones and have chosen one of these proteins for further study (the
chw genes for Components that Heterodimerize with a Winged helix protein). We have confirmed that
CHW-1 can bind PHA-4 in vitro, suggesting these proteins contact each other directly.
PHA-4 and CHW-1 may function together to regulate expression of a subset of PHA-4 target genes.
CHW-1 is a novel, glutamine-rich protein that is expressed in the intestine, rectum and pm6 pharyngeal
muscles, as is PHA-4. In addition, CHW-1::GFP is expressed in body wall muscles where PHA-4 is not
found. We have used RNAi to show that loss of chw-1 activity affects cells that normally express PHA-4.
In many chw-1(RNAi) animals, the pharyngeal grinder is deformed, larvae appear starved, and gonad
migration is defective. Interestingly, these phenotypes are strongly enhanced when PHA-4 activity is
compromised. We suggest that PHA-4 may bind distinct CHW factors to regulate different target genes in
the pharynx, rectum or gonad.
95

West Coast Worm Meeting 2000
THE UNC-4 HOMEOPROTEIN AND ITS TRANSCRIPTIONAL
CO-REPRESSOR UNC-37/GROUCHO REGULATE
NEUROTRANSMITTER VESICLES IN CHOLINERGIC MOTOR
NEURONS
Kim Lickteig 1 , Janet Duerr 2 , Dennis Frisby 3 , David Hall 4 , Jim Rand 2 ,
David Miller 1
1Vanderbilt University, Nashville, TN
2Oklahoma Medical Research Foundation, Oklahoma City, OK
3Univ. of Central Oklahoma, Edmond, OK
4Albert Einstein College of Medicine, Bronx, NY
The UNC-4 homeoprotein is expressed in VA motor neurons to prevent the adoption of synaptic inputs
normally reserved for their lineal sisters, the VB neurons. Work from this laboratory has shown that
UNC-4 functions as a negative regulator of VB-specific genes and that this activity depends on physical
interaction with the ubiquitously expressed UNC-37/Groucho co-repressor protein. UNC-4 is also
expressed in other motor neurons (DA, SAB, VC) but these cells (e.g. DAs) are not miswired in unc-4
mutants. Here we show that unc-4 and unc-37 mutations result in decreased levels of synaptic vesicle
(SV) proteins in "unc-4-expressing motor neurons" and that this deficit impairs the function of these cells.
Antibody staining reveals that five vesicular proteins (UNC-17, ChAT, synaptotagmin, synaptobrevin,
RAB-3) are substantially reduced in unc-4 and unc-37 mutants whereas other non-vesicular presynaptic
proteins (syntaxin, UNC-18, UNC-11) are not affected. This finding is consistent with ultrastructural
analysis of VA motor neurons in unc-4(e120) which show a 40% reduction in SVs at presynaptic
densities. Because the UNC-4/UNC-37 complex has been shown to mediate trancriptional repression, we
believe that these effects must be executed through an intermediate gene. Furthermore, our results
indicate that this intermediate gene ("Gene-X") functions as a negative regulator of SV biogenesis or
stability. Experiments with a temperature sensitive unc-4 mutant indicate that the adult level of SV
proteins strictly depends on unc-4 function during a critical early period of motor neuron differentiation.
unc-4 activity during this sensitive larval stage (L2 to mid L3) is also required for the creation of proper
synaptic inputs to the VA neurons. The temporal correlation of these events offers the intriguing possibility
that a common regulatory mechanism involving unc-4 may account for the specificity of synaptic input as
well as the strength of synaptic output for the VA motor neurons.
96

West Coast Worm Meeting 2000
THE COMPONENTS OF SENSORY CILIA IN C. ELEGANS
Peter Swoboda, Kerry Bubb, James H. Thomas
Department of Genetics, University of Washington, Seattle, WA 98195
Cilia are an important cellular differentiation of sensory neurons for receiving information from the
environment. 60 of the 302 C. elegans neurons have ciliated endings. Mutations in a large number of
genes have been identified that affect the structure of these sensory cilia. Of these, daf-19 mutations are
unique in completely lacking all sensory cilia. Aside from the absence of sensory cilia, the neurons seem
to be morphologically normal. We previously reported that daf-19 encodes an RFX-type transcription
factor that is expressed in ciliated sensory neurons.We also showed that DAF-19 regulates via its target
site in promoter regions, the x-box, a number of effector genes involved in sensory cilium formation.
These effector genes are expressed in essentially all ciliated sensory neurons and, when mutated, cause
morphological defects in sensory cilia (e.g. che-2, osm-1, osm-6). Our results strongly suggest that
DAF-19 regulates the differentiation of sensory cilia by activating the transcription of a battery of genes
whose products form the sensory cilium.
Cilia and flagella are structurally very similar subcellular organelles. Work done on the unicellular,
flagellated green alga Chlamydomonas lead to an estimate of around 300 different protein components
required for structure and function of flagella. We expect a similar number for cilium structure and
function. We aim to initiate the identification of all ciliary components by analyzing genes that have an
appropriately spaced x-box promoter element: so called xbx genes, hypothesized to be regulated by
daf-19. In a first search through the C. elegans genome sequence, using an algorithm designed to find
promoter elements, we uncovered more than 200 candidate xbx genes, several of which had C. briggsae
homologues that also had an appropriately positioned x-box promoter element. Initial expression analyses
of a subgroup of these xbx genes showed that, indeed, some of them were specifically expressed in
ciliated sensory neurons in a daf-19 dependent manner. In another approach, using oligo-nucleotide
arrays representing nearly all genes of C. elegans (1), we compared the gene expression profile of wild
type and daf-19 mutants. A small group of genes was reproducibly down-regulated in a daf-19
background, among them known cilium-structure genes and several xbx genes. In combination with
previous data about sensory cilia, these approaches have the potential to reveal all the genes required for
sensory cilium function and structure.
(1) in collaboration with Allan Jones (Rosetta Inpharmatics, Kirkland, WA, U.S.A.)
97

West Coast Worm Meeting 2000
IN VIVO IMAGING OF HSN OUTGROWTH
Carolyn E. Adler, Cornelia I. Bargmann
Department of Anatomy, UCSF, San Francisco, CA 94143
Axons in developing organisms must migrate and extend long distances in order to synapse onto their
proper targets. Little is known about how extracellular guidance cues elicit the changes in cytoskeletal
dynamics that drive axon outgrowth. In order to understand the factors that control the development of the
HSN motorneuron, we are undertaking an observational approach. We are utilizing two-photon
microscopy to analyze the trajectory of HSN as it grows ventrally in late L2/early L3 stage worms. Using
worms expressing GFP under the unc-86 promoter, we are able to visualize HSN during the course of its
outgrowth.
Preliminary imaging indicates that HSN initially extends filopodia in all directions from its cell body, but
that only those filopodia facing the ventral side persist. Eventually these filopodia develop into a large
lamellopod that extends ventrally and stalls at the ventral muscle barrier. A few processes protrude
through the body wall muscle attachment sites, as seen in VD and DD motorneuron growth through the
dorsal muscle attachment sites.1 Although multiple processes extend toward the ventral nerve cord, only
one is stabilized and develops into the axon.
We plan to characterize HSN outgrowth over the course of its development. With this information, we can
analyze the HSN axon in mutant worms known to have defects in ventral growth. In particular, we will
examine worms containing mutations in the extracellular guidance cues to which HSN responds as well
as in cytoplasmic proteins thought to participate in the regulation of cytoskeletal dynamics. In addition, we
plan to label the actin and microtubule cytoskeletons and visualize their dynamics during HSN outgrowth.
This approach will allow us to elucidate the roles of particular proteins in the process of HSN outgrowth.
1. Knobel KM, Jorgensen EM, Bastiani MJ. Development. 1999 Oct;126(20):4489-98.
98

West Coast Worm Meeting 2000
TEMPORAL AND SPATIAL REQUIREMENT OF SENSORY
CILIA IN THE REGULATION OF WORM LIFESPAN
Joy Alcedo, Javier Apfeld, Bella Albinder, Jennifer Dorman, Honor
Hsin, Bernadine Tsung, Cynthia Kenyon
Department of Biochemistry and Biophysics, UCSF, San Francisco, CA 94143
C. elegans is an excellent organism in which to study the regulation of lifespan. Genetic analyses have
already shown that an insulin/IGF-like hormonal control system, the DAF-2 pathway, regulates lifespan in
the worm. However, many components of the pathway, whose existence has been inferred from previous
studies (Apfeld and Kenyon, (1999). Nature 402, 804-809; and Hsin and Kenyon, (1999) Nature 399,
362-366) have not been identified. To search for more genes that would help to elucidate mechanisms
that control lifespan in the worm, our group has done an EMS mutagenesis screen for long-lived worms
and found 29 independent long-lived mutants.
At least one of the mutants, mu377(IV), maps to and fails to complement the gene daf-10, which is
required for the normal development of the worm’s sensory cilia. Apfeld and Kenyon (1999) have recently
shown that worms defective in sensory cilia, including daf-10 mutants, live longer than normal worms,
which suggests that the lifespan of C. elegans might be regulated by the perception of a signal(s) from
the environment. mu377(IV) has a temperature-sensitive defect in its sensory cilia, which is linked to its
temperature-sensitive lifespan phenotype. At the restrictive temperature, its lifespan is longer than
wild-type and it exhibits a defect in its sensory cilia; whereas at the permissive temperature, its lifespan is
the same as wild-type and it exhibits normal sensory cilia. Since the defect in the sensory cilia of
mu377(IV) is reversible, we determined the temporal requirement for the gene product of mu377(IV) in
regulating the lifespan of the worm. To gain insight into the spatial requirement for sensory perception in
controlling the worm’s lifespan, we are also in the process of determining which sensory neurons are
necessary to regulate its lifespan by expressing wild-type sensory genes in subsets of mutant sensory
neurons.
99

THE TTX-3 LIM HOMEOBOX GENE IS A CENTRAL
REGULATOR OF INTERNEURON CELL FATE
Z. Altun-Gultekin, O. Hobert
Columbia University, College of Physicians & Surgeons, Center for Neurobiology and Behavior, New
York, NY 10032
The LIM homeobox (Lhx) gene ttx-3 is required for correct thermotactic behavior (1,2). In ttx-3 mutants,
the AIY interneuron, a component of the thermoregulatory neural circuit, is structurally and functionally
defective. ttx-3::gfp reporter gene fusions are expressed in AIY. We have set out to test what cellular role
the ttx-3 gene plays and present here our analysis of the execution of the AIY cell fate in ttx-3 mutants.
Previously we have shown that in ttx-3 mutants maintenance of postembryonic ttx-3 expression is lost in
AIY due to autoregulation. Using GFP reporter gene constructs, we have now found that expression of
other AIY cell fate markers including a 7-TM receptor, sra-11, a homeobox gene, ceh-23, and a secreted
protein, C36B7.7 (kindly provided by T. Ishihara) is also downregulated in AIY in ttx-3 mutants. A GFP
fusion to the octopamine/serotonin receptor ser-2 (kindly provided by T. Niacaris), which we identified as
being expressed in AIY as well, is also downregulated. Additionally, AIY loses its cholinergic phenotype in
ttx-3 mutants as detected by VAchT antibody staining (antibodies kindly provided by J.Duerr). These
results suggest that AIY fails to differentiate correctly in ttx-3 mutants. So far it is unclear whether ttx-3
directly controls the expression of the genes described above or functions through intermediary
transcription factors. We are addressing this question by delineating and comparing the ttx-3 -dependent
regulatory elements in the distinct AIY cell fate markers.
Previously it was shown that mutations in the Lhx gene lim-4 lead to a switch of the fate of the AWB
sensory neuron to AWC (3). Although many if not all aspects of the correct fate of AIY are lost in ttx-3
mutants, our preliminary tests for a cell fate switch of AIY into any of its lineal, structural or functional
homologs suggest that AIY has not taken over the identity of AIM, AIZ, ASE or AWC. In contrast to AWB
in lim-4 and AIY in ttx-3 mutants, the DVB motor neuron maintains its correct fate in animals mutant for
the Lhx gene lim-6 (O. H., unpubl.). Hence, in Lhx mutants the identity of a neuron may be maintained,
lost or switched.
References:
(1) Hobert et al., 1997, Neuron 19, 345
(2) Mori and Oshima, 1995, Nature 376, 344
(3) Sagasti et al., 1999, Genes Dev 13, 1794
West Coast Worm Meeting 2000
100

West Coast Worm Meeting 2000
THE HETEROCHRONIC GENE PATHWAY: REGULATORY
INTERACTIONS AND REGULATORY OUTPUTS.
Victor Ambros 1 , Marta Hristova 1 , Rosalind Lee 1 , Eric Moss 2
1Department of Biology, Dartmouth College, Hanover, NH 03755
2Fox Chase Cancer Center, Philadelphia PA
The heterochronic gene pathway controls the timing of larval developmental events. Central to the action
of this pathway is the developmental down-regulation of lin-14 and lin-28, whereby high levels specify L1
events, and lower levels specify L2 and L3 events. The decrease in lin-14 and lin-28 results from
translational repression by the small RNA product of lin-4. lin-4 RNA is complementary to sequences in
the 3’ UTR of its target mRNAs, and recent experiments suggest that lin-4 acts by gating access to the
3’UTR by other positive and negative inputs. These other inputs include a mutual positive feedback
between lin-14 and lin-28 and a daf-12-dependent negative regulatory input.
The outputs of the heterochronic gene pathway are the expression of cell-type specific developmental
programs. To seek downstream targets of lin-14 for L2 events, we employed a microarray of 12,000
ORFs prepared by the Kim lab at Stanford University. Probes were prepared from mRNAs of
synchronized mid-L1 populations of N2 and lin-14(n179ts) animals. Among the genes that displayed
lin-14-dependent expression by microarray analysis, we chose four genes for follow-up by Northern and
GFP-fusions. By Northern analysis, three of these four genes show precocious mRNA expression in
lin-14(lf) animals, consistent with their temporal regulation by lin-14 in the wild type. The expression of
GFP promoter fusions suggests that at least one of the genes, ins-33, appears to be developmentally
regulated by lin-14 at the transcriptional level.
For L3 events, the chief effectors downstream of lin-14 and lin-28 appear to be daf-12 and lin-46. lin-46
encodes a member of a conserved family of proteins involved in protein-protein interactions, suggesting a
complex of regulators controling L3 targets. In the vulva precursor cells, L3 events controled by the
heterochronic genes are progress through the G1/S phase of the cell cycle and the acqusition of
competence to express vulval cell fates. VPC G1/S is regulated by the transcriptional activation of a cyclin
kinase inhibitor gene, cki-1. We are identifying regions of the cki-1 promoter which mediate the
VPC-specific control of cki-1 transcription by the heterochronic gene pathway.
101

GENETIC AND PHENOTYPIC CHARACTERIZATION OF
EVL-14 AND EVL-20, GENES INVOLVED IN C. ELEGANS
VULVA DEVELOPMENT
Igor Antoshechkin, Min Han
West Coast Worm Meeting 2000
Department of MCD Biology, University of Colorado, Boulder, CO 80309
In an attempt to identify new genes that participate in C. elegans vulva development, we carried out a
screen for sterile mutants with protruding vulva. The mutants were then examined using Nomarski optics
for abnormal vulva morphology at the "Christmas tree" stage. Several of isolated mutations were alleles of
evl genes previously identified by Geraldine Seydoux . Here we report genetic and phenotypic
characterization of two such genes, evl-14 and evl-20. Both mutations are completely recessive and result
in a 100 % sterile phenotype. Their vulva structures are variably abnormal and are missing anywhere
from 0 to 10 cells suggesting either under induction or cell cycle/division defect. In order to distinguish
between these possibilities we are making doubles with members of Ras signaling pathway and GFP
reporters for vulval cell fates. We are also performing detailed lineage analysis of the mutants. evl-20 also
displays gonad migration defect not seen in evl-14. evl-14 maps to linkage group III just left of pal-1.
evl-20 is located on chromosome II next to rol-6. We have injected cosmids from these regions and
identified cosmid pools that rescue both evl-14 and evl-20. We are in the process of injecting single
cosmids and making subclones to identify single open reading frames that will rescue the mutants.
102

CAENORHABDITIS ELEGANS T05H10.5, A HOMOLOGUE OF
YEAST UBIQUITIN FUSION DEGRADATION PROTEIN
(UDF-2), IS EXPRESSED THROUGHOUT THE NERVOUS
SYSTEM AND IN THE GUT
Wanyuan Ao, Dave Pilgrim
West Coast Worm Meeting 2000
Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9, Canada
C. elegans UNC-45 is a component of muscle thick filaments and co-localizes with myosin heavy chain B
in body wall muscles. The N-terminal of UNC-45 contains three tetratricopeptide repeats (TPR) and its
C-terminal shows limited similarity to fungal proteins. In our yeast two-hybrid screens using unc-45 cDNA
as a bait, we detected that UNC-45 interacts with T05H10.5 and the TRP domain of UNC-45 is sufficient
for that interaction. The predicted T05H10.5 gene encodes a 113.2 KD protein that is a homologue of
yeast ubiquitin fusion degradation protein (UDF-2). In further characterization of T05H10.5, We found that
the GFP reporter driven by T05H10.5 promoter is expressed throughout the nervous system and also
strongly in the gut of the adult worm. The UDF-2-like proteins are highly conserved from yeast to human
and its putative human homologue is expressed in brain tissues. Our results indicates that T05H10.5 is a
nerve cell and gut-specific gene and may interact with TRP-containing proteins (like UNC-45).
103

GENETIC ANALYSIS OF NEUROENDOCRINE CONTROLS OF
FAT METABOLISM IN C. ELEGANS
Kaveh Ashrafi, Gary Ruvkun
West Coast Worm Meeting 2000
Dept. of Molecular Biology, Massachusetts General Hospital and Dept. of Genetics, Harvard Medical
School, Boston, MA 02114
Body fat regulation is a complex process that affects behavior, physiology, and metabolism of an
organism in order to balance energy intake with energy expenditure. C. elegans is an attractive choice for
the systematic identification of components of fat homeostasis and cell biology. As in mammals, an insulin
signaling pathway in C. elegans couples nutritional status to the tempo and mode of metabolism, while
neuronal outputs, e.g. serotonin, translate food sensation to various motor and endocrine outputs.
We have used the phenoxazine dye Nile Red to visualize fat droplets in the intestinal and hypodermal
cells of living C. elegans. The Nile Red staining pattern closely matches the pattern observed with the
traditional methods of fat staining in C. elegans (e.g Sudan Black B). Moreover, the known and predicted
patterns of fat accumulation arising from mutations in insulin-like (daf-2 / daf-16) and TGF-b like (daf-7 /
daf-3) signaling pathways are well recapitulated by Nile Red staining. Thus, Nile Red staining of living
animals provides a means for genetic screens based on fat metabolism and homeostasis. Two such
screens are presented:
(i) To identify genes involved in the biogenesis and cell biology of fat storage, we screened
EMS-mutagenized N2 animals for mutant worms with abnormalities in lipid droplet size or number. In a
screen of 15,000 mutagenized F2, we identified four mutants that display abnormally large droplet sizes.
(ii) The C. elegans insulin signaling pathway determines whether larvae grow to a fast metabolizing adult
stage or enter a slow metabolizing dauer stage. Reduced signaling through the DAF-2 insulin-like
receptor promotes a shift in metabolism to fat storage and dauer arrest. The forkhead transcription factor
DAF-16, is the key target of DAF-2 signaling pathway and is required for the shift in fat metabolism and
dauer arrest. Mutant adult daf-2 animals have a diffuse pattern of Nile Red staining. In contrast, Nile Red
appears in very discrete droplet spots in daf-16 animals. To identify downstream metabolic targets of
DAF-16, we have begun screening EMS-mutagenized daf-16 worms to find mutants that recapitulate the
daf-2 pattern of staining.
104

ZIG GENES AND THE PVT GUIDEPOST NEURON
Oscar Aurelio, Oliver Hobert
Columbia University,New York, NY 10032
West Coast Worm Meeting 2000
We have identified a novel family of putative cell adhesion and/or signaling molecules which are defined
by the presence of a signal sequence and 2 Ig domains; none of these molecules contain a predicted
transmembrane sequence, but several contain consensus sites for GPI anchorage. C.elegans contains 9
members of this family, termed the zig gene family. We hypothesize that these genes may play a role in
neural patterning. As part of receptor/ligand complexes, they may be involved in cell recognition or,
alternatively, they may serve as secreted and diffusible signaling molecules.
GFP fusion to the promoter region of these 9 genes reveals that 6 of these genes are expressed almost
exclusively in very specific subdomains of the nervous system, while 2 of them are exclusively expressed
in body wall muscles. One zig gene is expressed ubiquitously in the nervous system and in body wall
muscles.
The most intriguing aspect of zig gene expression in the nervous system relates to the PVT neuron: 5 of
the 9 zig genes are expressed in PVT. Aside from the panneuronally expressed zig-1 reporter gene,
zig-2, zig-4 and zig-9 are expressed in few other cells than PVT, while zig-5 is expressed in about one
dozen additional head neurons. PVT acts during embryonic patterning as a guidepost cell for pioneer
neurons of the ventral nerve cord (Durbin, 1987) and is a source of unc-6 expression (Wadsworth et al.,
1996). Given the embryonic role of PVT, we were surprised to find that at least two of the five
PVT-expressed zig genes, zig-2 and zig-4, are not expressed embryonically; instead their expression is
turned on right after hatching and presists throughout adulthood. Additionally, we found that maintenance,
but not initiation of zig-2 and zig-4 expression requires the lim-6 LIM homeobox gene, which is expressed
in PVT.
The postembryonic expression of zig-2 and zig-4 suggests that PVT subserves a postembryonic role in
axonal patterning. What could this role be? In hermaphrodites relatively few neurons extend their axon
into the ventral nerve cord postembryonically; however, in males, many neurons are born
postembryonically in the tail and presumably require cues to extend their axon along the ventral nerve
cord. We will test whether PVT plays a role in this process by driving expression of cytotoxic agents under
control of the postembryonic zig-2 promoter.
105

West Coast Worm Meeting 2000
ANALYSIS OF GABA RECEPTOR PLASTICITY IN C.
ELEGANS
Bruce A. Bamber 1 , Janet E. Richmond 2 , Pierrette K. Danieu 1
1 Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, UT, 84112
2 Department of Biology, University of Utah, Salt Lake City, UT, 84112
Neuronal activity depends on the balance of excitatory and inhibitory neurotransmission. One way for a
neuron to regulate its activity level is to alter its sensitivity to inhibitory neurotransmitters. Most inhibitory
neurotransmission in the mammalian brain is mediated by g-aminobutyric acid (GABA), which acts mainly
through GABA A receptors (ligand-gated chloride channels). Neurons regulate their GABA sensitivity by
regulating the density and GABA-responsiveness of synaptic GABA A receptors. This receptor plasticity is
important for regulating neuronal excitability in both normal and diseased nervous systems. The
mechanisms of GABA A receptor plasticity are not well understood, in part because it is difficult to apply
genetic techniques to study mammalian synapse function. The C. elegans neuromuscular junction is a
powerful model system to study GABA receptor plasticity. A GABA A receptor homolog, consisting of
UNC-49B and UNC-49C subunits, functions in C. elegans muscle cells. This receptor exhibits
mammalian-like plasticity: The GABA receptor agonist muscimol acutely paralyzes C. elegans, but
chronic exposure results in adaptation (i.e. the appearance of muscimol resistance, and a GABA
receptor-defective phenotype). Patch-clamp analysis of muscle cells in adapted animals revealed an 85%
decrease in GABA-evoked currents compared to untreated animals, indicating that GABA receptor
downregulation underlies adaptation. Muscimol exposure also caused GFP-tagged UNC-49 receptors to
form clusters in extra-synaptic muscle membranes, which implicates the clathrin-mediated endocytosis
pathway in downregulation. We are pursuing two strategies to study GABA receptor plasticity in C.
elegans. First, we are identifying UNC-49 sequences required for synaptic localization and
downregulation. Our results have demonstrated that the UNC-49B subunit is necessary and sufficient for
both processes. Second, we are performing a yeast two-hybrid screen to identify GABA
receptor-associated molecules in C. elegans. We have identified several interesting candidates, and we
are using RNA interference to determine which of these proteins are important for GABA receptor
regulation.
106

ISOLATION OF SUPPRESSORS OF A DOMINANT SYNAPSE
DEFECTIVE MUTANT, SYD-5(JU89)
Renee Baran, Yishi Jin
Department of Biology, Sinsheimer Labs, University of California, Santa Cruz, CA 95064
syd-5 (ju89)was identified in a screen for mutations that alter the differentiation of GABAergic synapses in
C. elegans using the presynaptic vesicle marker, Punc-25::SNB-1-GFP 1,2 . In adult wild-type worms,
Punc-25-SNB-1-GFP is expressed as puncta of uniform size and spacing along the dorsal and ventral
nerve cords, corresponding to neuromuscular junctions made by the GABAergic DD and VD neurons with
dorsal and ventral body wall muscles, respectively. syd-5(ju89) mutant worms are uncoordinated and
exhibit abnormally large and small SNB-1-GFP puncta dispersed in an irregular pattern along the nerve
cords. This phenotype closely resembles the SNB-1 GFP phenotype of loss-of-function syd-3/rpm-1
mutants, which have overdeveloped synapses with multiple active zones 3 . syd-5 maps to chromosome I
between egl-33 and lin-11. syd-5( ju89) behaves genetically as a weak gain-of-function mutation: Df/+
worms are wild-type, but heterozygous ju89/+ worms exhibit a locomotion and SNB-1 GFP phenotype
that is intermediate between wild-type and homozygous ju89 worms. A suppressor screen was performed
to identify loss-of-function syd-5 alleles and genes that may function in the same pathway. syd-5(ju89)
worms were mutagenized with EMS, and 30,000 F1 progeny were screened for suppression of the
syd-5(ju89) uncoordinated phenotype. Twenty-four mutants were isolated that suppress both the syd-5
locomotion phenotype and the synaptic vesicle marker defect. Four suppressors are linked to ju89 and
may represent loss-of-function syd-5 alleles. Preliminary characterization of the syd-5 suppressors will be
presented, along with progress in the cloning and molecular characterization of syd-5.
1 Zhen & Jin. 1999. Nature
2 Nonet, M. 1999. Neurosci. Methods
3 Zhen et al. 2000. Neuron
West Coast Worm Meeting 2000
107

CARGO RECOGNITION BY SYNAPTIC VESICLE KINESIN
Ewa Bednarek, Erik M. Jorgensen
West Coast Worm Meeting 2000
Department of Biology, University of Utah, Salt Lake City, UT 84112, USA
Synaptic vesicles are transported to the synapse by kinesin, an ATP-dependent motor protein that moves
along axonal microtubules. In the cell body the kinesin must specifically bind vesicles destined for the
nerve terminal. We would like to identify the molecular basis for vesicle cargo recognition by UNC-104, a
Caenorhabditis elegans homolog of the motor protein kinesin. One theory suggests that UNC-104
recognizes specific protein molecules associated with the vesicle membranes. Alternatively, we
hypothesize that UNC-104 recognizes synaptic vesicles by binding specific lipids. This is supported by the
fact that UNC-104 has a pleckstrin homology (PH) domain, which is a lipid binding moiety. If PH
domain-lipid binding determines cargo recognition, the following predictions can be made: the PH domain
should bind lipids specific to synaptic membranes, and the PH domain should be required for cargo
recognition. To test these predictions we are first expressing the PH domain in bacteria and determining
whether it binds phosphoinositides. Second, we are testing whether mutations in the PH domain disrupt
synaptic vesicle trafficking.
108

THE EXP-1 LOCUS MAY ENCODE A SUBUNIT OF AN
EXCITATORY GABA RECEPTOR
Asim A. Beg, Erik M. Jorgensen
West Coast Worm Meeting 2000
Dept. of Biology, University of Utah 257 South 1400 East Salt Lake City, UT 84112
Gamma-aminobutyric-acid (GABA) is a major neurotransmitter of inhibitory synapses. In the worm, as in
vertebrates, GABA mediates inhibitory neurotransmission (VD/DD to body muscles). However, in the
enteric muscles, excitatory neurotransmission seems to be mediated by GABA. The GABAergic neurons
AVL and DVB directly synapse on the enteric muscles and are the only neurons required for excitation of
the enteric muscles. We have cloned a potential excitatory GABA receptor subunit, exp-1. Of the six
genes required for GABA neurotransmission, only exp-1 specifically eliminated the excitatory function of
GABA while leaving all other functions intact. We hypothesize that EXP-1 is a subunit of a novel
excitatory GABA receptor expressed in the enteric muscles.
We cloned the exp-1 gene by microinjection rescue. Genetic map data showed that exp-1 is located on
Chromosome II between lin-4 and lin-23. One fosmid in this region, H35N03, contains a gene related to
GABA receptors. Transgenes bearing a subclone of this single open reading frame (ORF) completely
rescued exp-1(sa6) mutants (gift of J. Thomas). In addition, we identified point mutations in this ORF in
three alleles, which confirmed that exp-1 encodes this GABA receptor related protein. We isolated exp-1
cDNAs which show that EXP-1 has a divergent transmembrane 2 (TM2) from all known GABA receptor
subunits. TM2 has been shown to confer ion selectivity to the ligand-gated ion channel superfamily.
Consistent with our hypothesis that EXP-1 is an excitatory GABA receptor subunit, TM2 has numerous
negatively charged residues, suggesting this subunit might form a receptor that passes cations rather
than the anion chloride.
The majority of physiological GABA receptors are heteromultimers. By BLAST search we have identified
a second putative subunit that is very similar to EXP-1. Specifically, TM2 of the predicted subunit is
nearly identical to EXP-1, suggesting this might be the physiological partner of EXP-1. We are currently
performing RT-PCR to isolate cDNAs for this subunit. We will coinject Xenopus laevis oocytes with
cRNAs from both subunits and determine GABA gating and ion permeability of the channel using
electrophysiological analysis.
109

West Coast Worm Meeting 2000
THE LIFE SPAN GENE CLK-2 IS ESSENTIAL FOR
EMBRYONIC DEVELOPMENT
Claire Bènard, Brent McCright, Yue Zhang, Stephanie Felkai, Siegfried
Hekimi
Department of Biology, McGill University, 1205 Dr Penfield Ave., Montreal, Quebec, Canada
Mutations in the C. elegans maternal-effect genes clk-1, clk-2, clk-3, and gro-1 are highly pleiotropic
resulting in an average slowing of development, adult rhythmic behaviors, reproduction, as well as in an
extended life span. Here we will present the genetic and molecular characterization of the gene clk-2.
This gene is defined by one recessive mutation, qm37, which was isolated in a screen for viable
maternal-effect mutations (Hekimi et al., 1995). At 20C, the duration of embryonic and post-embryonic
development of clk-2(qm37) mutants is lengthened and adult behaviors such as defecation, pharyngeal
pumping, and egg laying are slower. In addition, they live long. In contrast, at 25C, clk-2(qm37) mutants
die as embryos. Moreover, at 20C, clk-2(qm37) mutants can be fully rescued both zygotically and
maternally, while at 25C there is a strict-maternal effect. We believe that clk-2(qm37) is a
temperature-sensitive mutation that behaves as a hypomorph at 20C and as a null at 25C. As we have
found that clk-2 is not required for the development of the germline per se at 25C, we have explored the
origin of the embryonic lethality at 25C by carrying out temperature shift experiments. We have shown
that clk-2 is required before the two-cell stage of embryogenesis. The temporal and spatial expression
pattern of clk-2 deduced from northern and western analyses, and from the use of clk-2::gfp reporter
fusions, is consistent with a maternal role of clk-2 early in development and throughout the life of the
worm.
110

DOES CEH-20, AN EXD/PBX HOMOLOG IN C. ELEGANS,
PLAY A ROLE IN WORM EMBRYOGENESIS?
Q.F. Boese, W.B. Wood
Dept of MCD Biology, University of Colorado, Boulder, CO 80309
Several studies have shown that the TALE (three-amino-acid-loop-extension) homeodomain (HD)
proteins, Exd and Hth in Drosophila and their mammalian homologs, Pbx1-3 and Meis, complex with Hox
proteins to enhance binding specificity for target promoter sites. Studies have further shown that Hth/Meis
interacts with Exd/Pbx to promote nuclear localization facilitating their roles as co-factors in the nucleus.
Recent work in our lab revealed that loss-of-function (lf) mutations in the C. elegans Hth/Meis homolog,
unc-62, result in pleiotropic defects including defects in embryonic development. To investigate whether
the Exd/Pbx homolog, CEH-20 might also be involved in embryonic patterning, we have begun
characterizing the defects of two strong ceh-20(lf) alleles (gift from M. Stern) with respect to their potential
interactions with unc-62 and other embryonically required genes (Hox or non-Hox). Both ceh-20 alleles
result in low-penetrance larval lethality and no apparent embryonic defects suggesting that ceh-20 is not
normally required for embryogenesis. Injection of double-stranded ceh-20 RNA into wild-type
hermaphrodites [ceh-20 (RNAi)] results in an Unc Egl phenotype but no embryonic defects.
To test for an interaction with the embryonically required posterior Hox paralog gene nob-1 (1), ceh-20
(RNAi) was performed in the background of a weak allele, nob-1(ct230). No enhancement of the mutant
phenotype was observed. Whether this reflects compensatory functions of two other Exd/Pbx homologs,
ceh-40 and F22A3.x, in the absence of ceh-20, is under investigation.
To test for interactions with unc-62, ceh-20 RNAi was performed in the background of the weak allele
unc-62 (e644). The low penetrance e644 lethal phenotype was enhanced over the uninjected control,
consistent with an interaction between these genes during embryogenesis. To test for interactions with
other non-Hox HD proteins important for embryogenesis, ceh-20 RNAi was performed in the background
of a weak pal-1 allele (e2091). The e2091 mutant phenotype was also enhanced suggesting a previously
undescribed interaction between Exd/Pbx and Caudal/CDX homologs. Further tests for ceh-20
interactions with other embryonically required genes are in progress.
(1) Van Auken, K. et al., 2000, PNAS 91: 4499-503
West Coast Worm Meeting 2000
111

West Coast Worm Meeting 2000
A-DOMAIN-CONTAINING PROTEIN FAMILY IN C. ELEGANS.
Michael Brannan, Joaquin Muriel, Kathryn Taylor, Gordon Lithgow,
Danny Tuckwell
School of Biological Sciences, University of Manchester, Stopford Building, Manchester M13 9PT, UK.
The extracellular matrix (ECM) plays an important structural and functional role in metazoans and its
major components include collagen, laminin, and proteoglycan. The cuticle and basement membrane are
the major forms of ECM in C. elegans: the cuticle is an exoskeleton important for shape and motility, and
it is also a barrier between the worm and its environment. The basement membrane provides structural
support and tissue separation. Very little is known about the molecules that organise the ECM or the cell
receptors for ECM molecules in C. elegans.
A-domains are 200-amino acids modules identified in a range of mammalian ECM proteins. We have
previously studied the A-domains of the integrin a subunits. Integrins a 1b 1 and a 2b 1 are the two major
receptors in the human body for collagens. We have showed that it is the A-domains found within the a
subunits that are responsible for collagen binding, and also for the interaction of these integrins with other
ECM proteins such as laminin. Studies of a number of other A-domain-containing proteins indicate a
collagen or ECM-binding function.
Using bioinformatics approaches we have identified 8 novel A-domain-containing proteins in the C.
elegans genome. For some of the novel proteins, regions flanking the A-domains were found to resemble
mucin core protein-like sequence and cuticulin domains. Since mucins and cuticulins are ECM
components, this suggests that a number of the novel proteins are likely to be ECM molecules. Genetical
and biochemical characterisation of these molecules will give insights into the function of these proteins in
ECM organisation and cell-ECM interactions.
112

West Coast Worm Meeting 2000
DISTRIBUTION AND REGULATION OF GLUTAMATE
RECEPTORS IN THE LOCOMOTORY CONTROL CIRCUIT OF
C. ELEGANS.
Penelope J. Brockie, David M. Madsen, Yi Zheng, Jerry E. Mellem,
Andres V. Maricq
Department of Biology, University of Utah, Salt Lake City, UT 84112
Ionotropic glutamate receptors are ligand gated ion channels that mediate rapid excitatory synaptic
communication in most nervous systems. Ten putative ionotropic glutamate receptor subunits (glr-1 -
glr-8, nmr-1 and nmr-2) have been identified in C. elegans. Analysis of the predicted amino acid
sequence suggests that GLR-1 - GLR-8 are members of the non-NMDA class of glutamate receptors,
where NMR-1 and NMR-2 are most similar to members of the NMDA class. Using GFP fusions we have
shown that the subunits are differentially expressed in the nervous system with some found in only a
single neuron.
The NMDA subunits and four of the non-NMDA subunits (GLR-1 - GLR-4) are expressed in at least one
of the five pairs of command interneurons that regulate worm locomotion (AVA, AVB, AVD, AVE and
PVC). This provides insight into the possible combinations of receptor subunits that form ion channels in
vivo, and suggests that a diversity of both NMDA and non-NMDA type glutamate receptors function in the
locomotory control circuit. What regulates expression of the glutamate receptors in this circuit? It has
recently been shown that the homeodomain protein UNC-42 regulates glr-1 expression in a number of
neurons, including AVA, AVD and AVE (1). We show that unc-42 is also required for glr-3 and glr-4
expression in these neurons, but not for the expression of glr-2, nmr-1 or nmr-2. This indicates that
expression of glutamate receptors in the command interneurons is differentially regulated.
Like glr-1 mutants, unc-42 mutants are nose touch defective and show additional mechanosensory and
locomotory defects. Using the nmr-1::gfp fusion, we show that unc-42 mutants also have dramatic defects
in axon outgrowth in the command interneurons. In wild-type worms, these neurons send their axons
along the ventral cord. In unc-42 mutants, axons are dorsally, laterally and anteriorally misplaced. Using
the nmr-1 promoter to express the unc-42 cDNA does not rescue the axon defects. This suggests that
expressing UNC-42 in the command interneurons alone is not sufficient to direct proper axon outgrowth in
these cells.
1. Baran, et al., Development 126, 2241-2251 (1999)
113

West Coast Worm Meeting 2000
MUTATIONS THAT AFFECT SYNAPTIC LOCALIZATION OF
GLR-1
Michelle Burbea, Joshua M. Kaplan
Department of Molecular and Cellular Biology, 361 Life Sciences Addition, UC Berkeley, Berkeley, CA
94720-3200
The orderly flow of information in the nervous system requires that proteins are specifically targeted to
pre- and post-synaptic specializations. We are interested in identifying the molecules that target and
maintain proteins to synapses. We have used the GLR-1 AMPA type glutamate receptor as a marker for
post-synaptic localization. A wild type animal expressing a GLR-1::GFP fusion exhibits punctate spots
along the ventral nerve chord representing clustering of the glutamate receptor at specific synaptic
terminii. To identify genes involved in synaptogenesis, we are screening mutagenized GLR-1::GFP
worms for defects in GLR-1 localization.
We predict classes of mutants to represent trafficking, targeting, localization and stabilization molecules
necessary to initiate and maintain synaptic architecture. Approximately 7000 genomes have been clonally
screened so that sterile or inviable mutants may be recovered as heterozygously. Nine complementation
groups have been isolated which define loci important for the proper localization of GLR-1 in the nervous
system. Several of the loci are in genes whose identity is already known. These include mutations in
unc-11, an AP180 homologue, and unc-101, a medium chain subunit of the AP-1 clathrin adaptin
complex. The implication of these mutants on receptor trafficking and maintanance at the synapse will be
discussed.
114

REGULATION OF C. ELEGANS DAUER FORMATION BY AN
RNA QUALITY CONTROL PATHWAY COMPONENT
J Burgess 1 , JC Labbe 2 , S Hekimi 1
West Coast Worm Meeting 2000
1Department of Biology, Mc Gill University, 1205 Dr. Penfield Ave, Montreal, Quebec, Canada H3A1B1
2Department of Biology, University of North Carolina at Chapel Hill, 3280 Coker Hall, Chapel Hill, North
Carolina. 27599-3280, USA
Under conditions that do not favor developmental growth and reproduction, worms can enter an alternate
third-larval stage termed the dauer stage. Dauer larvae are developmentally arrested and are adapted for
long term survival and dispersal. In order to make this decision, animals monitor a variety of
chemosensory cues including the concentration of a constitutively secreted pheromone, as well as food
and temperature. It is currently unknown whether internal states, such as the amount of molecular
damage accumulated, is also taken into account when making the decision to enter the dauer stage. The
gene rop-1 encodes the worm homologue of the human Ro ribonucleoprotein 60-kDa constituent, which
has previously been shown to participate in the quality control of 5S ribosomal RNA. Here, we present
biochemical evidence that rop-1 is cleaved at the L2/L3 molt concommittant with entry into L3 but is not
cleaved when animals enter the dauer stage. In addition, rop-1 is not cleaved in daf-2 mutants suggesting
cleavage of rop-1 may require functional insulin-like signaling. Furthermore, we provide evidence that
rop-1 genetically interacts with the insulin receptor-like kinase daf-2 in an allele-specific manner as well as
with the TGF-b transforming growth factor daf-7 in a temperature dependent manner. Finally, we show
that the enhancement of dauer formation by rop-1 in daf-2(e1370) is daf-16 dependent. However, the
processing of rop-1 appears to be daf-16 independent. We suggest that an RNA quality control
checkpoint may exist for dauer formation.
115

SYNAPTIC VESICLE LOCALIZATION IS MISREGULATED IN
UNC-16 MUTANTS
DT Byrd, Y Jin
University of California, Santa Cruz, Dept. Biol., Sinsheimer Labs, CA 95064
The DD motoneurons provide a model in which to study synaptic development and remodeling. The DDs
are born embryonically, innervate ventral body wall muscles in L1 animals, and remodel to innervate
dorsal body wall muscles in L2 through adult animals. We visualize DD synapses and the process of DD
synaptic remodeling in vivo with a synaptic vesicle marker expressed by the DDs and other GABAergic
neurons1. In wild type L1s, the synaptic vesicle marker is strictly localized to the ventral processes of the
DDs until just before the molt to L22. After the L1 molt, the synaptic vesicle marker is localized along the
dorsal processes of the DDs, indicating that the DDs have remodeled.
We isolated a partial loss-of-function mutation in unc-16 in a screen for dorsal localization of the synaptic
vesicle marker in the DDs of early L1 animals. unc-16 mutant L1 animals exhibit full ventral localization of
the synaptic vesicle marker as well as varying amounts of dorsal localization in the DDs. Seven alleles of
unc-16 form an allelic series in which n730, e109, ju79, and s1072 are partial loss-of-function alleles of
increasing severity, respectively, and s453, s529, and s222 are genetic null alleles. unc-16 encodes a
pan-neurally expressed novel protein with interesting motifs that may interact with a number of signaling
pathways. We are currently determining the molecular lesions in the unc-16 mutants.
We hypothesize that the vesicle localization defect may be caused by disruptions in synaptic vesicle
cargo selection, transport, or docking. Supporting this hypothesis, we found that weak unc-16 mutations
partially suppress partial loss-of-function mutations in unc-104, a gene encoding a kinesin. This suggests
that in the DDs, unc-16 may play a role in regulating synaptic vesicle transport.
1. Nonet ML. 1999. J Neurosci Methods 89(1):33-40.
2. Hallam and Jin. 1998. Nature 395:78-82.
West Coast Worm Meeting 2000
116

THE EGL-21 GENE ENCODES A CARBOXYPEPTIDASE E,
WHICH IS REQUIRED FOR PRO-NEUROPEPTIDE
PROCESSING
Tija Carey, Joshua M. Kaplan
West Coast Worm Meeting 2000
UC Berkeley, MCB Dept , 361 LSA, Berkeley CA 94720
We are interested in understanding the mechanisms for neurotransmitter and neuropeptide modulation of
C. elegans behavior, including locomotion, defecation, and egg laying. We previously reported that the
egl-3 gene encodes a prohormone convertase involved in proteolytic processing of pro-neuropeptides
(Kass and Kaplan IWM99). We will present evidence that the egl-21 gene encodes a carboxypeptidase
E(CPE), which mediates the next enzymatic step in peptide processing, removal of C-terminal basic
residues.
Mutations in egl-21 were first isolated in a screen for egg laying defective mutants, but also showed
defective defecation and uncoordinated locomotion (Trent, Genetics 104: 619-647 1983). The various
defects in egl-21 mutants show differing drug sensitivity: the egg laying defect is resistant to serotonin and
imipramine, while the locomotion phenotype is sensitive to both.
We found that egl-21 mutations map very close to mec-3. The genome sequence of this region indicated
that a gene very similar to vertebrate CPE mapped in this region. We found that a cosmid containing this
CPE rescued the egl-21 defecation defects. Next, we showed that two egl-21 alleles correspond to
mutations in the CPE gene. The n576 allele alters an invariant G in a splice donor sequence whereas the
n476 allele corresponds to a 122 bp in-frame deletion. Since egl-21 encodes the only apparent CPE in
the worm genome, we would expect that the phenotype of egl-21 mutants corresponds to a neuropeptide
null phenotype. We are currently studying the role of egl-21 CPE in modulation of several behaviors.
117

HOW ARE ANTERIOR CELL MIGRATIONS GUIDED BY
MIG-13?
QueeLim Ch’ng, Cynthia Kenyon
Department of Biochemistry, UC San Francisco, CA 94143-0448
mig-13 was previously identified as a guidance factor specifically required for the anterior migrations of
the QR descendants. mig-13 acts by promoting cell migrations in the anterior direction (Sym et. al., 1999).
We are taking several approaches to understand further how the anterior migrations of the QR
descendants are guided by mig-13. mig-13 is predicted to encode a novel transmembrane protein
containing a CUB domain and LDL-receptor repeat in the extracellular region as well as a proline-rich
domain in the intracellular region (Sym et. al., 1999). Our structure/function studies suggest that the
extracellular domain of MIG-13 alone can confer partial function in guiding the QR descendants to the
anterior.
A rescuing mig-13::GFP fusion was previously found to be expressed in the anterior and mid-body ventral
cord motor neurons, which cross the migratory track of the QR descendants. Consistent with this
expression pattern, mosaic analysis revealed that mig-13 acts non-autonomously to direct the migrations
of the QR lineage (Sym et. al., 1999). To determine where mig-13 expression is sufficient to guide the
migrating cells, we are expressing mig-13 in broad sets of tissues, as well as in specific subsets of cells
that express mig-13::GFP. We also intend to refine previous mosaic analysis to pinpoint the cells in which
mig-13 acts.
Reference
West Coast Worm Meeting 2000
Sym M, Robinson N and Kenyon C. Cell, 1999 Jul 9, 98(1):25-36.
118

West Coast Worm Meeting 2000
NEW SCREENS FOR NEGATIVE REGULATORS OF LET-23
Monica Chan, Marie Tiongsen, Romel C. Castro, Vanessa Lee, Gregg
Jongeward
University of the Pacific, Department of Biological Sciences, 3601 Pacific Avenue, Stockton, Cal 95211
We are trying to identify additional genes that act to negatively regulate the Epidermal Growth Factor
Receptor (EGF-receptor) during vulval induction in Caenorabditis elegans. The gene let-23 encodes the
C. elegans homolog of the EGF-receptor. Mutations in let-23 generally result in a number of defects,
including larval arrest, sterility, and defects in the vulva and the male tail. At 20 o C, animals homozygous
for the allele let-23(n1045) display excess vulval differentiation. A similar phenotype is observed in
animals homozygous for lin-2(n768). lin-2 activity is required to properly localize EGF-receptor on the
vulval precursor cell. For as yet unknown reasons, animals homozygous for both let-23(n1045)and
lin-2(n768) display a highly penetrant vulval defect unlike that seen in either homozygote. These doubly
mutant animals fail to form any vulval tissue. We have begun to screen for new alleles that suppress the
egg-laying defect associated with the inability to form vulval tissue in animals of this genotype. Thus far,
we have recovered four new alleles. At least one of these alleles appears to be a new lin-1 mutation, as
animals homozygous for the suppressor display excessive vulval differentiation even if there are no other
mutations present. A second (independent) suppressor causes a similar phenotype. The other two alleles
restore approximately normal vulval differentiation. Animals of the genotype let-23(n1045); lin-2(n768);
new suppressor are generally able to lay eggs but lack prominent pseudovulvae. We are currently
characterizing these new alleles to determine if they are mutations in previously identified genes.
119

C. ELEGANS MRE-11 IS REQUIRED FOR MEIOTIC
RECOMBINATION AND DNA REPAIR BUT NOT FOR THE
MEIOTIC G2 DNA DAMAGE CHECKPOINT
Gregory Chin, Anne Villeneuve
West Coast Worm Meeting 2000
Department of Developmental Biology, Stanford University School of Medicine Stanford, CA 94305
We have isolated and analyzed mutants defective in the nematode ortholog of yeast MRE11, a
multifunctional protein with roles in diverse cellular processes required to maintain genome integrity,
including meiotic recombination, DNA repair, and telomere length maintenance. While MRE11 is highly
conserved among several species and has been linked to genetic disease in humans, exploration of its in
vivo roles in metazoan systems has been hampered by the fact that vertebrate cells that lack MRE11 are
inviable. We have found that worms homozygous for an mre-11 null mutation are viable, allowing us to
demonstrate an in vivo requirement for MRE-11 in meiotic recombination and DNA repair. In mre-11
mutants, crossovers are not detected and chromosomes lack chiasmata at diakinesis but appear
otherwise intact. Irradiation of mre-11 mutants not only fails to induce chiasmata but also eliminates
progeny survivorship and leads to cytologically visible chromosomal abnormalities including
fragmentation. These results indicate a defect in the ability of mre-11 mutant germ cells to repair
radiation-induced damage. While they are repair-deficient, we show that mre-11 mutant germ cells retain
function of the meiotic G2 DNA damage checkpoint.
Although mre-11 homozyogtes derived from heterozygous parents are fully viable and produce a normal
number of embryos, there is a marked drop both in the number and in the survivorship of embryos
produced by succeeding generations. As a result, the strain cannot be propagated as a homozygous
stock. This progressive loss of fecundity and viability sets mre-11 mutants apart from many other meiotic
recombination-defective mutants, and indicates that MRE-11 performs an additional essential function in
maintaining reproductive capacity in the species.
120

West Coast Worm Meeting 2000
SUPPRESSOR ANALYSIS OF EPH/EPHRIN DEFECTIVE
SIGNALING IN C. ELEGANS
Ian Chin-Sang, Julie McCleery, Andrew Chisholm
Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064, USA
Mutations in the genes vab-1 and vab-2 cause similar defects in neural and epidermal morphogenesis.
The phenotypes range from embryonic lethality, due to epidermal enclosure defects, and a ventral
notched head in larvae and adults. vab-1 encodes an Eph receptor tyrosine kinase that is expressed in
the developing nervous system, and is required in neurons for proper epidermal morphogenesis (George
et al., Cell 92:633). vab-2/efn-1 encodes a GPI anchored ephrin that specifically interacts with VAB-1 in
neurons to regulate neuronal and epidermal morphogenesis (Chin-Sang et al., Cell 99:781). What are the
molecular consequences of the VAB-1/VAB-2 interaction? We have taken a genetic approach to identify
genes that might act in the VAB-1/2 Eph/Ephrin signaling pathway or in parallel pathways. The incomplete
penetrance and variability of phenotypes displayed by vab-1 and vab-2 mutants, made it difficult in the
past to carry out genetic modifier screens. Therefore we sought to create strains that would make vab-1
and vab-2 mutants more suitable for genetic modifier screens. A mutation in a LAR like receptor tyrosine
phosphatase, ptp-1, results in a synthetic lethality with both vab-1and vab-2, suggesting that PTP-1 may
function redundantly with Eph signaling (see abstract by Harrington et al.). The ptp-1(op147);vab-2(ju1)
double mutant is temperature sensitive- viable at 15 0 C and 20 0 C, however, at 25 0 C almost completely
inviable. We screened for suppressors of the ptp-1(op147);vab-2(ju1) lethality at 25 0 C and isolated at
least 19 suppressors from a screen of over 67,000 F1 animals. The suppressors are of two classes: those
that reduce the lethality and those that suppress both the lethality and the notched head phenotype of
vab-2. Five suppressors of the second class map to LGIV and are dominant suppressors of vab-2.
Intriguingly, these suppressors display a low penetrance notched head phenotype (about 2-5% of the
animals), however, unlike vab-1 and vab-2 the notch is always on the dorsal side. We are currently
determining the specificity of the suppression effect. We will present results from the characterization of
these suppressors.
121

West Coast Worm Meeting 2000
RNAI SCREEN FOR COMPONENTS OF THE C. ELEGANS
MEIOTIC MACHINERY
Mónica Colaiácovo, Gillian Stanfield, Kirthi Reddy, Anne Villeneuve
Dept. of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305
Meiosis is the specialized cell division process by which diploid organisms generate haploid gametes. In
preparation for the meiosis I division, homologous chromosomes condense, recognize each other,
synapse, and undergo recombination. During this process a proteinaceous structure known as the
synaptonemal complex (SC) forms between homologs maintaining them in a side-by-side alignment.
Crossing over occurs in the context of the SC, leading to formation of chiasmata that connect the
homologs and allow them to orient toward opposite poles of the meiosis I spindle.
We are using a functional genomics strategy to identify genes required for these key meiotic prophase
events, taking advantage of a genome-wide survey of germline gene expression conducted by V. Reinke
and collaborators in the lab of S. Kim. We defined a subset of 206 germline-enriched genes whose
expression profiles most closely match those of known meiosis genes, and designed a screen to identify
those genes for which RNAi elicits defects in meiotic prophase. Defects in pairing, synapsis or
recombination that lead to chromosome non-disjunction are initially identified by the production of inviable
embryos and an increased frequency of male progeny. Defects that lead to arrest in meiotic progression
are initially identified by a sterile phenotype. Candidates fitting either of these profiles are then subjected
to cytological analysis to investigate possible meiotic defects. The efficacy of our screening procedure has
been validated using genes encoding known meiotic recombination and synapsis components as positive
controls.
Our initial screening has already identified several distinct phenotypic classes with defects in
chromosome behavior and/or morphology. These define genes with putative roles in meiotic chromosome
synapsis (see accompanying poster by Reddy et al.), as well as genes that may have both mitotic and
meiotic roles. A summary of the results of this ongoing screen will be presented.
122

West Coast Worm Meeting 2000
EXPLORING THE ROLE OF PINCH/UNC-97 IN MUSCLE
DEVELOPMENT AND FOCAL ADHESION ASSEMBLY IN
CAENORHABDITIS ELEGANS AND MAMMALIAN TISSUE
CULTURE CELL LINES
Shaun Cordes 1 , May Dang-Lawson 1 , Poupak Rahmani 1 , Linda
Matsuuchi 1 , Donald G. Moerman 1,2
1Department of Zoology, U.B.C., Vancouver, B.C., Canada
2Biotechnology Laboratory, U.B.C., Vancouver, B.C., Canada
PINCH/UNC-97 is a phylogenetically conserved adaptor protein consisting entirely of 5 LIM domains
which has been implicated in the assembly of dense bodies, focal adhesion (FA)-like structures in worm
muscle. An examination of the subcellular localization of UNC-97 in body wall muscles using a full length
UNC-97::GFP reporter construct reveals that the protein co-localizes with integrin-containing attachment
structures and that it is also in the nucleus. Studies of partial and complete loss of function mutations in
unc-97 demonstrate that the protein is necessary for the integrity of FA-like attachment structures found in
nematode body wall muscles (JCB 144: 45-57). We are using a combination of genetics in C. elegans
and expression studies in mammalian tissue culture cells to further investigate the role of PINCH in
muscle development and FA assembly. Moreover, we are investigating the functional importance of
nuclear-localized UNC-97 in the regulation of these processes.
To identify the regions of PINCH responsible for its localization, we have designed altered UNC-97::GFP
reporter constructs which delete one or multiple UNC-97 LIM domains. Preliminary results in worms
suggest that LIM2 and/or LIM3 are involved in nuclear localization. When a full length UNC-97::GFP
reporter construct is transiently transfected into COS fibroblasts, the pattern of expression in the
cytoplasm is similar to that of the FA associated protein alpha-actinin. Preliminary results in COS cells
suggest that UNC-97 requires LIM1 for cytoplasmic localization to punctate cytoplasmic structures. Our
results suggest that the interaction between PINCH and its LIM1 binding partners (such as the
integrin-linked kinase; Mol. Cell Biol. 19: 2425-34), is important for the recruitment of UNC-97 to specific
locations in the cytosol. Future experiments will explore the localization of our reporter constructs in the
mouse myoblast cell line C2C12.
123

West Coast Worm Meeting 2000
THE SAD-1 KINASE REGULATES PRESYNAPTIC VESICLE
CLUSTERING IN C. ELEGANS
Justin Gage Crump 1 , Mei Zhen 2 , Kang Shen 1 , Yishi Jin 2 , Cornelia I.
Bargmann 1
1Howard Hughes Medical Institute and Department of Anatomy, University of California, San Francisco,
CA 94143-0452 USA
2Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz 95064, USA
Synaptic development is a multistep process that includes the presynaptic clustering of vesicles and
active zone proteins, the postsynaptic clustering of neurotransmitter receptors and regulatory proteins,
and the termination of axon outgrowth as neurons recognize their targets. We report here the
identification of a novel serine/threonine kinase, SAD-1, that regulates several aspects of presynaptic
differentiation in C. elegans. In sad-1 mutant animals presynaptic vesicle clusters in sensory neurons and
motor neurons are disorganized and more diffuse. Sensory axons fail to terminate in sad-1 mutants,
whereas overexpression of SAD-1 causes axons to terminate prematurely. SAD-1 is related to PAR-1, a
kinase that regulates cell polarity during asymmetric cell division. SAD-1 is expressed in the nervous
system at the time of synaptogenesis and localizes to synapse-rich regions of the axons, where it can
function cell-autonomously in presynaptic neurons. Strikingly, overexpression of SAD-1 can induce the
formation of well-organized, evenly spaced vesicle clusters in dendrites, which are normally devoid of
synaptic vesicles. These results reveal a potent function for sad-1 in specifying synaptic regions in
polarized neurons. We have also started to study the molecular events that coordinate the presynaptic
and postsynaptic specialization.
124

West Coast Worm Meeting 2000
MUTANTS WITH ALTERED SENSITIVITY TO THE EFFECTS
OF ETHANOL ON LOCOMOTION
Andrew G. Davies, Tod R. Thiele, Catharine Eastman, Steven L.
McIntire
Gallo Center and Program in Biological Sciences, Dept. of Neurology, UCSF
Ethanol is reported to have multiple targets in the nervous system, a factor that has complicated efforts to
understand the actions of ethanol that bring about behavioral effects in mammalian systems. We are
using C. elegans to confirm suspected targets of ethanol and identify new targets and pathways that can
be affected by ethanol to produce behavioral changes. We have concentrated on the effects of ethanol on
locomotion. With increasing doses of ethanol in a plate assay, wild-type animals display a decrease in
activity, velocity and amplitude of the body waveform. Non-saturating screens for mutants with altered
sensitivity to ethanol for locomotion have produced eight mutants with increased resistance to ethanol
and four mutants with increased sensitivity to ethanol. We have characterized the locomotion, egg laying
and pharyngeal pumping behaviors of these mutants in the presence and absence of ethanol and
examined nervous system function by pharmacological analysis.
Mapping and cloning of the genes mutated in these strains is underway using positioned Tc1
polymorphisms or single nucleotide polymorphisms that result in alterations to restriction enzyme sites
(thanks to Stephen Wicks for unpublished data). We have identified one of the resistance genes as the
calcium-activated potassium channel gene slo-1. Potassium channels of the slo class are thought to
regulate the excitability of neurons. There is in vitro evidence for a direct activating interaction of ethanol
on channels of this class derived from mammalian systems (reviewed by Dopico et al. 1999, Neurochem.
Int. 35:103-106) raising the possibility that we may have identified a conserved direct target of ethanol in
the nervous system of the worm.
125

West Coast Worm Meeting 2000
A SCREEN FOR DD/DV AXONAL MORPHOLOGY DEFECTS
M. Wayne Davis, Erik M. Jorgensen
Department of Biology, University of Utah, 257 South 1400 East Salt Lake City, UT 84112-0840
Growth cones undergo a series of morphological changes as they migrate from the neuronal cell body to
their target. VD motor neuron growth cones slow down and change their shape when they encounter the
lateral nerve cord and body wall muscle (Knobel et al. Development 1999). In order to pass through the
narrow spaces between the body wall muscles and the hypodermis, the growth cone stops and projects
filopodia through these spaces. When the forward tip of one filopodium reaches the other side of the
muscle the growth cone behind the muscle collapses and re-forms at the tip of this projection. The axon
then branches at the dorsal nerve cord and synapses onto the dorsal muscles.
Using an integrated unc-47::GFP construct that specifically labels the DD and VD axons, I screened for
mutations that disrupt DD/VD axon morphology. In this screen, I identified two main classes of mutations.
The mutations in the first class cause wandering of the axons; mutations in the second class cause
premature termination and branching, either at the lateral nerve cord or at the dorsal muscle boundary.
Most of the wandering axon mutations cause an Unc phenotype, and many of these are likely to
represent alleles of previously known genes. However, the premature branching mutants have no other
obvious phenotype. These mutations may identify new genes necessary for migrating growth cones to
deal with obstacles or changes in substrate.
I have done this screen in the CB4856 Hawaiian background. This should allow the use of
single-nucleotide polymorphisms between this strain and N2 to facilitate mapping of the mutations with
subtle phenotypes.
126

SPN-2 AND SPN-3 FUNCTION TO ORIENT THE SPINDLE
DURING EARLY CLEAVAGES
Leah R. DeBella, Lesilee S. Rose
West Coast Worm Meeting 2000
Section of Molecular and Cellular Biology, University of California, Davis CA 95616 USA
Orientation of cell division is critical for partitioning cytoplasmic factors and setting up cellular interactions
necessary for proper development. In C. elegans, the first cleavage bisects the long axis of the embryo,
generating the AB and P1 cells. Subsequent cleavages in the AB lineage are perpendicular to the
previous axis of division. Cleavages in the P1 lineage occur repeatedly on the same axis, due to a nuclear
rotation event. Previous studies have shown actin, microtubules, dynein, and components of the dynactin
complex to be required for proper spindle orientation in some cells. However, the precise mechanism by
which spindles align is not understood.
We are studying two genes, spn-2 and spn-3, which are required for proper spindle orientation in
embryos. spn-2 embryos show defects in spindle orientation at the 2 and 4-cell stage in both AB and P1
lineages, while spn-2/sDf130 worms show defects in the generation of fertilized embryos. Together these
phenotypes suggest a continuing role for spn-2 in spindle orientation during embryogenesis, as well as a
role prior to embryonic division. Some spn-3 embryos fail to rotate at the 2-cell stage while others have
ectopic rotation at the 4-cell stage. However, spn-3/sDf127 embryos are defective in positioning the first
mitotic spindle. This data suggests spn-3 plays a role in spindle orientation during the first three divisions
after fertilization.
An examination of polarity markers in homozygous spn mutants indicates these markers are localized
normally prior to abnormal spindle alignment. In addition, cell divisions are asymmetric and cell cycle
timing is normal, suggesting that overall polarity is not affected in these embryos.
Our working hypothesis is that the spn genes play a role specifically in spindle orientation by regulating
interactions between the microtubules and actin cytoskeleton. As a first step towards testing our model of
spn gene function, we are isolating these genes using a positional cloning approach. spn-2 has been
placed within 220 kb and spn-3 within 11 kb. Examination of the spn genes, combined with the analysis of
other genes in our lab, will provide a basis for understanding the mechanism behind spindle orientation in
all blastomeres of the early embryo.
127

MOLECULES ACTING IN PARALLEL WITH UNC-34 TO
CONTROL CELL MIGRATION
Megan Dell 1 , N Chugh 1 , N Hawkins 1 , E Kong 1 , J Hardin 2 , G Garriga 1
1MCB, UC Berkeley
2Dept Zoology, U Wisconsin-Madison
West Coast Worm Meeting 2000
Guidance cues and their receptors direct cell and growth cone migrations by regulating cytoskeletal
dynamics. Several signaling molecules that function downstream of guidance receptors to regulate actin
have been described. We have found that three signaling molecules, UNC-34, the C. elegans homolog of
Enabled, WAS-1, a homolog of the Wiskott-Aldrich Syndrome Protein (WASP), and DAB-1, a homolog of
Disabled, act in parallel to regulate cell migrations in C. elegans.
Drosophila Enabled (Ena) and its mouse homolog Mena function in axon outgrowth and fasciculation. The
phenotypes of unc-34 mutants show that this gene functions not only in these processes but also in cell
migrations. unc-34 null mutants are viable and display only partially penetrant defects in cell and growth
cone migrations, suggesting the existance of parallel pathways in C. elegans that can compensate for the
absence of UNC-34. By conducting RNAi experiments in an unc-34 background, we have found that
WAS-1 and DAB-1 act in parallel to UNC-34.
Because both WASP and Ena have an EVH-1 domain at their N-termini and regulate actin assembly, we
injected was-1 RNA into wild-type and unc-34 mutant animals. While was-1(RNAi) animals appeared
normal, was-1(RNAi); unc-34(null) animals died as embryos due to ventral enclosure defects. Injection of
was-1 RNA into a temperature-sensitive allele of unc-34 resulted in temperature-dependent lethality.
Injection into worms raised at 25° , the restrictive temperature, resulted in embryonic lethality, while
injected worms raised at 20° showed synthetic cell migration defects and partial lethality. These results
show that UNC-34 and WAS-1 act in parallel pathways to control ventral enclosure and cell migration. A
screen for mutations that result in synthetic lethal and migration defects in the unc-34(ts) background has
yielded several potential mutants.
Disabled is a negative regulator of Ena in flies. dab-1(RNAi) animals show defects in embryonic neuronal
migrations, and injection of dab-1 RNA into unc-34(null) mutants results in enhanced migration defects.
This result is different from the genetic evidence in Drosophila in which Disabled mutations suppress the
Ena phenotype and suggests that unc-34 and dab-1 act in parallel pathways to regulate neuronal
migrations in C. elegans.
128

West Coast Worm Meeting 2000
INSIGHTS INTO THE ROLE OF C. ELEGANS PROTEIN
UNC-119 IN AXONOGENESIS
Chantal Denholm 1 , Wayne Materi 1 , Daniel Gietz 2 , David Pilgrim 1
1Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada, T6G 2E9
2Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba,
Canada, R3E 0W3
UNC-119 has widespread effects on the development and maintenance of the C. elegans nervous
system. Unc-119 mutants have several movement, sensory, and behavioral abnormalities due to
structural and functional defects in the nervous system. Homologues have also been identified in humans,
rat, Drosophila, and zebrafish, and their functional conservation suggests that this protein plays a
fundamental role in neurogenesis.
Human protein HRG4 has 57% identity with UNC-119 and is a novel photoreceptor enriched protein
which begins to be expressed at the time of photoreceptor differentiation in the ribbon synapse. It is
thought to be involved in visual neural signal transmission, however it is also expressed at lower levels in
other tisues including fetal brain.
Although the phenotype of unc-119 mutants has been well described, the biological role of UNC-119 and
HRG4 remains to be determined because their sequences give no clues to their function(s). To gain
insights about how this protein exerts its effects in vivo, yeast-two hybrid screens have been conducted
using UNC-119 and its functional homologue HRG4 to determine with what proteins they interact. Both
screens have yielded good candidate interactions which are consistent with these proteins’ involvement in
neurite outgrowth, and these results, along with possible models of UNC-119/HRG4 function will be
discussed.
129

THE DEFECATION GENE AEX-1 MAY REGULATE A
RETROGRADE SIGNALING PATHWAY AT
NEUROMUSCLULAR JUNCTIONS.
Motomichi Doi, Kouichi Iwasaki
National Institute of Bioscience, Japan
Communication between pre- and postsynaptic cells is crucial for synaptic transmission regulation. In one
regulatory mechanism, retrograde signaling, signals from postsynaptic cells control synaptic connectivity
and transmission of presynaptic cells. Although there is evidence that retrograde signaling occurs at
neuromuscular junctions in C. elegans 1) , its mechanism is unknown. Here we report that the defecation
gene aex-1 may be a regulatory component of C. elegans retrograde signaling.
aex-1 mutants show phenotypes similar to those in aex-3 mutants, which were consistent with
presynaptic defects. These phenotypes include defecation defects, reduced male mating, and resistance
to the acetylcholinesterase inhibitor aldicarb. Based on these observations we initially hypothesized that
aex-1 encodes a component which functions at presynaptic terminals.
To determine the molecular function of AEX-1, we cloned and characterized aex-1. aex-1 encodes a
novel protein of 1027 amino acids whose C-terminus is similar to the second C2 domain of the mouse
protein Munc-13-4 and the human protein BAP-3. Using GFP fusion constructs we unexpectedly found
that aex-1 was expressed primarily in body wall muscles. Furthermore, aex-1 expression driven by the
muscle-specific unc-54 promoter conferred hypersensitivity to aldicarb but did not change sensitivity to
the acetylcholine agonist levamisole. This suggests that AEX-1 affects presynaptic activities at
neuromuscular junctions.
To investigate whether AEX-1 regulates retrograde signaling, we generated egl-30(gf);aex-1 double
mutants and tested whether or not hyperexcitation of ventral cord motor neurons suppresses the
aldicarb-resistance phenotype of aex-1 mutants. egl-30(gf) expression in ventral cord motor neurons
confers hypersentivity to aldicarb 2) . Our prediction was that if aex-1 functions downstream of ventral cord
motor neurons then the egl-30(gf) mutation would not suppress the aex-1 phenotype. However, the
egl-30(gf) mutation could suppress the aldicarb-resistance phenotype of aex-1 mutants, suggesting that
aex-1 acts upstream of the egl-30(gf) mutation. This observation is consistent with the hypothesis that
AEX-1 regulates retrograde signaling at neuromuscular junctions.
1) Zhao, H. and Nonet, M. L. Development 127 1253-1266 (2000)
2) Lackner, M.R. et al., Neuron 24 335-346 (1999)
@
West Coast Worm Meeting 2000
130

West Coast Worm Meeting 2000
COSUPPRESSION IN THE GERMLINE: SILENCING IS
GOLDEN
Abby F. Dernburg 1 , Mónica P. Colaiácovo 1 , Jonathan Zalevsky 2 ,
Anne M. Villeneuve 1
1 Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA
94305-5329
2 Current address: Department of Biochemistry and Biophysics, University of California, San Francisco,
San Francisco, CA 94143-0448
When genes required in the nematode germline are introduced as trangenes, they show notoriously poor
expression. Paradoxically, the presence of such an array in a wild-type worm can have a bizarre
outcome: it can produce specific phenocopy of loss-of-function mutations in the homologous gene. Similar
"cosuppression" or "quelling" phenomena, in which transgenes induce functional silencing of endogenous
genes, have been reported in diverse organisms, from plants to flies to fungi. We have demonstrated the
generality of this phenomenon and characterized its rules. We show that functional repression is not a
consequence of persistent physical association between transgenes and endogenous genes, or of
mutations in affected genes. Constructing transgenes with different regulatory and coding information, we
obtained evidence that cosuppression is likely to require transcription from the array to generate an RNA
mediator that dictates its target specificity. Because this potential RNA involvement is reminiscent of
RNAi, we asked whether mutations that abrogate RNAi have any effect on cosuppression. This analysis
demonstrated that the genetic requirements for these two silencing phenomena do overlap but show key
differences. Specifically, we find that both rde-2 and mut-7 are essential for cosuppression, but that the
function of rde-1 is dispensable.
Transgene-mediated cosuppression provides a valuable alternative to RNAi as a technique for probing
gene function. Targeting interesting candidate genes, we have elicited diverse germline phenotypes,
including defective chromosome synapsis, failures in spindle assembly, or altered meiotic progression.
Because the effects of cosuppression appear to be largely restricted to the germline, this approach can
reveal germline-specific functions for essential genes for which mutation or RNAi would result in lethality.
131

SUR-9 A SUPPRESSOR OF ACTIVATED LET-60(N1046) IN
THE C.ELEGANS VULVA.
Dennis Eastburn, Min Han
West Coast Worm Meeting 2000
HHMI, Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder
The adult C. elegans vulva is induced by an EGF like signal which activates the Ras/MAPK pathway.
Constitutively active alleles of ras result in a multivulva (Muv) phenotype where numerous pseudovulvae
are formed from vulval precursor cells (VPCs) that are normally not induced in a wild-type background. By
initiating suppressor screens of activated let-60 ras, many previously unknown components of this
pathway have been identified. One gene, sur-9, has been isolated from such a screen and is currently
being cloned and characterized. sur-9(ku258) has been mapped to chromosome III and is currently being
fine mapped. The ku258 allele can suppress the Muv phenotype of let-60(n1046) from 80% to
approximately 1%. In addition, ku258 is dominant with respect to its suppression. Cloning and elucidation
of sur-9’s molecular identity could contribute to our knowledge of a highly conserved and important
biological pathway.
132

West Coast Worm Meeting 2000
KNOCKOUTS IN C. ELEGANS: MADNESS AND
METHODOLOGY
Mark Edgley 1 , Erin Gilchrist 1 , Greg Mullen 1 , Bin Shen 1 , Margaret
Kotarska 1 , Don Moerman 1,2 , Steven Jones 3 , Anil Dsouza 4 , Gary
Moulder 4 , Malini Viswanathan 4 , Martin Lansdale 4 , Robert Barstead 4
1Biotechnology Laboratory,U.B.C., Vancoouver, B.C., Canada
2Department of Zoology, U.B.C., Vancouver, B.C., Canada
3Genome Sequence Centre, Vancouver, B.C., Canada.
4Department of Molecular and Cell Biology, OMRF, Oklahoma City, Oklahoma
As the first multicellular metazoan to have its genome sequenced the nematode C. elegans offers an
unprecedented opportunity to investigate larger scale developmental problems including tissue formation
and organogenesis. The C. elegans genome contains approximately 19,000 ORF’s, of which only a
fraction have been characterized through genetic mutational analysis. Functional analysis of the complete
genome may be feasible using reverse genetic methodology. Random chemical mutagenesis with
trimethylpsoralen and UV irradiation, coupled to PCR amplification using primers to a specific gene, can
allow for targeted gene disruption. Potential deletions are detected using agarose gel electrophoresis.
The approach is sensitive enough to detect deletions in single animals in complex populations. Through a
process of "sib-selection" one can derive a clone of animals bearing a specific deletion. The mutations are
then sequenced and mutations conferring lethality are balanced using (where possible) a GFP-balancer
chromosome.
Our laboratories are part of the C. elegans Gene Knockout Consortium, an international group of labs that
do gene knockouts by request from the worm community [see web site:
http://www.cigenomics.bc.ca/elegans]. In the past year the consortium has received 662 requests for
targeted gene disruptions. From this request list consortium labs have eliminated the function of
approximately 80 genes using the chemical mutagenesis approach [as of January, 2000]. All mutants and
data provided by consortium laboratories are in the public domain and freely available to all researchers.
133

West Coast Worm Meeting 2000
USING DNA MICROARRAYS TO IDENTIFY TARGETS OF
HOMEOBOX GENES IN C. ELEGANS
Andreas Eizinger 1 , Tibor Vellai 2 , Fritz Müller 2 , Stuart K. Kim 1
1Stanford University Medical Center, Developmental Biology, 279 Campus Drive B369, Stanford, CA
94305, USA
2University of Fribourg, Institute of Zoology, Perolles, Fribourg CH-1700, Switzerland
Homeobox genes are necessary to specify anterioposterior identity in metazoa. Despite their importance,
targets of homeobox genes are poorly understood. Using our DNA microarrays, which contain nearly
every gene of C. elegans, we wish to identify a comprehensive list of homeobox target genes in C.
elegans. Currently we are focusing on the four major HOX genes ceh-13, lin-39, mab-5 and egl-5.
Preliminary data showed that heat shock expression of homeobox genes in embryos cause strong
lethality and malformation of the hatched larvae. This shows that embryonic cells are responsive to
excessive or ectopic HOX gene expression. We will use our DNA microarrays to identify the earliest gene
expression changes following induction of HOX gene expression by heat shock. By comparing the gene
expression profiles from heat shock treatment of HOX transgenic embryos to control strains, we will be
able to separate the heat shock and HOX responses. We are also interested in the specificity of HOX
genes. Elucidation of the targets of each of the four major HOX genes will show which targets are
common between all HOX genes and which are specific to individual HOX genes.
134

West Coast Worm Meeting 2000
DED GENES DISRUPT CELL DIVISION TIMING AND
PATTERNING IN C. ELEGANS EMBRYOS
Sandra Encalada, Paula Martin, Jennifer Phillips, Rebecca Lyzcak,
Danielle Hamill, Kathryn Swan, Bruce Bowerman
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403
In early C. elegans embryos, asymmetric cell divisions produce descendants with characteristic cell cycle
times. To understand how these differences in cell cycle timing arise, and to investigate their role in
pattern formation, we are characterizing ded (delayed division) mutants, in which embryonic cell divisions
are delayed and cell fate patterning is abnormal.
In genetic screens for both non-conditional and temperature-sensitive embryonic lethal mutations, we
identified 25 ded alleles. Twelve mutations define nine genes (ded-1 through ded-9), based on genetic
mapping and complementation tests. A common phenotype of these mutants is a prolonged three cell
stage due to a delay in the division of P1. The terminal phenotype of some ded mutants resembles skn-1
mutants: they produce differentiated cells but lack pharynx and gut and instead make extra hypodermis.
The skn-1 gene encodes a transcription factor that specifies mesoderm and endoderm fate in the EMS
blastomere in a wild-type 4-cell embryo. The protein PIE-1 normally blocks SKN-1 function in the
germiline precursor P2. Analysis of a ded-1;pie-1::GFP strain shows that PIE-1 accumulates to
abnormally high levels in EMS and C in ded mutant embryos. These observations suggest that PIE-1 is
blocking SKN-1 function in both P2 and EMS. Consistent with this conclusion, ded-1;pie-1 double mutants
resemble pie-1 mutants, producing extra phrynx and intestine. Finally, the delayed cell divisions of ded-1
mutants also result in a loss of asymmetry in the size of the daughters of P1, and in the mislocalization of
P-granules.
We have cloned ded-1, and it encodes the B subunit of the DNA polymerase-primase complex. Based on
RNA interference experiments with other DNA replication genes, the other loci we have identified likely
define a set of DNA replication genes. Based on these results, we conclude that (1) interference with DNA
replication delays the onset of mitosis, suggesting that a checkpoint monitors the completion of DNA
replication, and (2) proper control of cell division timing is important for multiple aspects of asymmetric cell
division in early C. elegans embryos.
135

VOLTAGE-DEPENDENT CURRENTS IN HOMOLOGOUS
CHEMOSENSORY NEURONS WITH DIFFERENT FUNCTIONS
IN C. ELEGANS
S Faumont, S.R. Lockery
West Coast Worm Meeting 2000
Institute of Neurosci, Univ of Oregon, Eugene, OR 97405
In C. elegans, chemotaxis to the attractant NaCl is largely controlled by a pair of left-right homologous
chemosensory neurons ASER and ASEL. Although the two neurons are bilaterally symetric in their
morphology and pattern of connectivity , recent work argues for functional asymetry between these
neurons in chemotaxis (Pierce-Shinomura et al., abstr. WCWM). Laser ablation of ASER greatly reduces
chemotaxis to Cl- but not Na+; conversely, ablation of ASEL greatly reduces chemotaxis to Na+ but not
Cl-. To determine if this functional asymetry reflects a difference in the ionic currents expressed in the two
neurons, we made whole-cell voltage clamp recordings fron ASEL (n=20) for comparison with previous
recording from ASER (Goodman et al., Neuron 20: 763-72, 1998).
We found that ASEL is similar to ASER in three main respects. First, ASEL exhibits an outward current
activated by depolarisation (-30 to 100 mV) and an inward current activated by hyperpolarisation below
-70 mV. Little or no current is activated in the region between -70 and -30 mV. Second, the outward
current comprises inactivating and sustained components which are probably carried by K+ ions, because
both components are eliminated by substitution of N-methyl-glutamine (NMG) for K+ in the recording
pipette. Third, outward current inactivation is voltage-dependent, because prepulses more positive than
-70mV decreases peak amplitude of the outward current. These results suggest that the functional
asymetry reflect differences in the response to chemical stimuli rather than differences in voltage
dependent ionic current. Support: NIMH 51383 and NSF IBN-9458102.
136

West Coast Worm Meeting 2000
VAV IS REQUIRED FOR PHARYNGEAL MUSCLE
CONTRACTION IN C. ELEGANS
R.T. Fazzio, J.E. Mellem, M.C. Beckerle, A.V. Maricq
Dept. of Biology and Huntsman Cancer Institute, Univ. of Utah, SLC, UT 84112
In vertebrates, VAV, VAV-2 and VAV-3 are guanine nucleotide exchange factors for Rho-family GTPases.
Nucleotide exchange is a key mechanism by which Rho GTPases are regulated during cytoskeletal
remodeling. Disruption of this regulation by specific mutations in VAV results in the constituative activation
of Rho-family members and oncogenesis. To better understand the role of VAV in cytoskeletal control, we
have undertaken a genetic and molecular analysis of VAV in C. elegans.
We have cloned the C. elegans homolog of vav, vav-1, from first-strand cDNA. vav-1 encodes a 975
amino acid protein that shares 34% overall identity with human VAV and contains the predicted exchange
factor domain and other important functional motifs. We have previously reported vav-1 expression in
pharyngeal tissue, body wall muscle, vulval tissue and somatic gonad. In the pharynx, VAV-1 is found in
muscle, in some neuronal cells and in the g1 gland cells. Subcellular localization of VAV-1 will be studied
using a mouse monoclonal antibody generated to this protein.
We have engineered a deletion mutation in vav-1 that removes 85% of the mature protein product. This
disruption yields a pharyngeal contraction defect that ultimately results in early larval lethality. Mutant
animals display asynchronous pharyngeal muscle contraction or, in some cases, complete pharyngeal
paralysis. Electrophysiological analysis of mutant pharyngeal muscle confirms the importance of VAV-1
for synchronous contraction. We can rescue this defect by the transgenic expression of a genomic DNA
fragment that contains the vav-1 open reading frame.
We are currently pursuing experiments designed to provide a better understanding of the vav-1 null
phenotype: Through tissue-specific expression of vav-1 in the null background, and possibly mosaic
analysis, we aim to identify the pharyngeal cells (and other cells) that require VAV-1. In addition, we aim
to complete a mutational analysis of the vav-1 gene to determine the domains/residues of VAV-1 that are
important for function. The generation of multiple alleles in vav-1 will provide the reagents necessary for a
genetic screen designed to identify proteins that function in the VAV signal transduction pathway.
137

West Coast Worm Meeting 2000
REGULATION OF C. ELEGANS BODY SIZE BY SENSORY
CUES
Manabi Fujiwara, Hoan Phan, Steven L. McIntire
Gallo Center and Program in Biological Sciences, Dept. of Neurology, UCSF, San Francisco, CA94608
In C. elegans, body size may be regulated by the nervous system. Lewis and Hodgkin initially reported
that cilium-defective mutants such as che-2 and che-3 are smaller than wild-type animals. A smaller body
size is also observed in other cilium-defective mutants and in the tax-4 mutant (tax-4 encodes a
cGMP-gated channel that is necessary for chemosensation). Furthermore, DBL-1/CET-1 TGF-b, which is
involved in body size regulation, is expressed in neurons. These observations suggest that if an animal
cannot sense an environmental cue such as food, body size may be reduced through altered neural
activity. Such regulation may be useful if a smaller body is economical. Recently Apfeld and Kenyon
reported that sensory cues also regulate aging of C. elegans.
In order to analyze the putative neural regulation of body size, we isolated suppressor mutants of the
che-2 small body size phenotype (chb). 15,000 haploid genomes were screened, yielding 28 candidates.
These suppressor mutants do not suppress the dye-filling defect of che-2, but suppress the small body
size of che-2. Some of these suppressors show the same body size with or without the che-2 mutation in
the background. Such mutants may result from defects downstream of sensory signals. Nine strains of
this category are being mapped using the snipSNP method (Wicks and Plasterk). One of these, chb-1,
maps to the left arm of chromosome IV and may be allelic with odr-9/egl-4. odr-9/egl-4 maps to the same
region. chb-1 has the chemotaxis defect to diacetyl that is found in odr-9/egl-4. chb-1 and odr-9/egl-4 both
have a slightly larger body than wild type. We have mapped chb-1 to a reigion that is less than 100kb and
are attempting to rescue with cosmids covering this region.
138

West Coast Worm Meeting 2000
REGULATION OF INTRACELLULAR DYNAMICS OF
MAPKAPK2 IN LIVING C.ELEGANS
Makoto Fukuda, Yves Bobinnec, Eisuke Nishida
Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University,
Sakyo-ku, Kyoto 606-8502, Japan
MAPKAPK2, a target of p38 MAPK, is shown to be exported from the nucleus to the cytoplasm upon its
activation in mammalian cells. This nuclear export is believed to be dependent on its nuclear export signal
(NES). Here, we have examined intracellular dynamics of C. elegans MAPKAPK2 (C44C8.6) during
whole processes of development. To visualize subcellular localization of MAPKAPK2 in living C. elegans,
we constructed a fusion between GFP and MAPKAPK2 under the control of its own promoter. This
construct was expressed in late embryo, all larval stages and adults. In adults, MAPKAPK2-GFP was
expressed mainly in neuron, neuroblast and vulva. MAPKAPK2-GFP was present mainly in the nucleus of
those cells. However, in response to extracellular stresses that induce activation of P38 MAPK and
MAPKAPK2, MAPKAPK2-GFP was completely exported from the nucleus to the cytoplasm. An
NES-disrupted form of MAPKAPK2 was not exported from the nucleus under any conditions. In addition,
RNAi with C. elegans CRM1, which is known as an NES receptor, resulted in the inhibition of nuclear
export of MAPKAPK2. These results define the requirement of the NES of MAPKAPK2 for its nuclear
export. Moreover, we found that MAPKAPK2 was exclusively localized in the cytoplasm in some neuronal
cells under normal condition, suggesting that MAPKAPK2 is activated in these cells. It is possible that
nuclear export (= cytoplasmic localization) of MAPKAPK2 is tightly associated with activation of p38
MAPK and MAPKAPK2. We are currently examining when and where the p38 MAPK/MAPKAPK2
pathway is functioning in living C. elegans by this method.
139

West Coast Worm Meeting 2000
ROLE OF CKI-1 IN TERMINAL EMBRYONIC
DIFFERENTIATION AND CELL-CYCLE ARREST
Masamitsu Fukuyama, W. Brent Derry, Joel H. Rothman
Department of MCD Biology, UC Santa Barbara, Santa Barbara, CA 93106
The CIP/KIP family of Cyclin-dependent Kinase Inhibitors (CKIs) function to arrest the cell cycle at the
appropriate time during metazoan development. In C. elegans, two apparent CIP/KIP CKIs are encoded
by the adjacent cki-1 and cki-2 genes (1). We previously reported that deficiencies that remove both cki
genes cause extra embryonic cell divisions in multiple tissues and accumulation of excess cell corpses,
and that a cosmid containing these genes can rescue these deficiency phenotypes (2). We recently found
that the cki-1 transgene alone can rescue the hyperplastic phenotype of homozygous deficiency embryos,
while the cki-2 transgene shows only very weak rescuing activity, suggesting that cki-1 is essential for
embryonic cell cycle arrest. In support of this hypothesis, cki-1(RNAi) but not cki-2(RNAi) leads to
embryonic hyperplasia in most tissues and the appearance of many cell corpses. Interestingly, embryonic
germ cells arrest the cell cycle in a CKI-1-independent manner.
The CIP/KIP CKIs in vertebrates have been implicated in terminal differentiation as well as exit from the
cell cycle. We previously demonstrated that cki-1(RNAi) causes loss of a differentiation marker in the
adult somatic gonad (3). We recently found that cki-1(RNAi) can eliminate expression of two late
differentiation markers during embryogenesis, one in seam cells and one in the gut, suggesting that cki-1
is required to promote terminal stages of differentiation. We are currently attempting to determine (1)
which stage in differentiation is blocked by loss of cki-1 activity, (2) whether terminal differentiation in the
other lineages requires cki-1 activity, and (3) whether CKI-1 promotes differentiation through a
cyclin-dependent kinase activity.
1. Hong, Y et al. (1998). Development 125, 3585-3597. Feng, H et al. (1999). Nature Cell. Biol. 1,
486-492.
2. Gendreau, SB and Rothman, JH. (1997). 11th International Worm Meeting.
3. Fukuyama, M et al (1998). 1998 West Coast Worm Meeting.
140

West Coast Worm Meeting 2000
SAX-1 AND SAX-2 ACT IN PARALLEL WITH UNC-34 TO
MAINTAIN NEURON POLARITY.
Maria E. Gallegos 1 , Jennifer A. Zallen 2 , Cori Bargmann 1
1HHMI, Dept. of Anatomy, UCSF, San Francisco, CA 94143
2Dept of Molecular Biology, Princeton University, Princeton, New Jersey, 08544
During development each neuron in C. elegans extends a precise number of processes from its cell body.
This polarity is established normally in sax-1 and sax-2 mutants, but later in development ectopic neurites
often emerge from the soma of select neurons. In addition, sax-1 and sax-2 mutants have neurons with
enlarged, irregularly shaped cell bodies (Zallen et al. 1999). Whereas similar phenotypes have been seen
in a variety of mutants known to disrupt neuron function, neurons in sax-1 and sax-2 mutants function
normally suggesting that sax-1 and sax-2 may act more directly with the actin cytoskeleton to maintain
neuron polarity.
To ask if the late-emerging neurites in sax-1 and sax-2 mutants result from a deregulated axon guidance
pathway, I made double mutants of sax-1 and sax-2 with various axon guidance mutants including
unc-34, a homolog of Enabled (M. Dell and G. Garriga, pers. comm.). unc-34 lf mutants have axon
guidance and early axon termination defects suggesting that unc-34 functions to direct axon guidance
and promote axon outgrowth during development. Surprisingly, unc-34;sax-1 and unc-34;sax-2 double
mutants are enhanced for the ectopic neurite outgrowth defect. Furthermore, these ectopic neurites
emerge after initial polarity has been established. These results suggest that sax-1 and sax-2 act in
parallel with unc-34 to inhibit ectopic neurite outgrowth. Since the early axon termination defects of
unc-34 suggest that it functions to promote axon outgrowth one interesting possibility is that unc-34 is
bifunctional. These results also demonstrate that unc-34 plays a role during the establishment and
maintenance of neuron polarity.
sax-1 encodes a S/T kinase related to the Ndr protein kinase in humans (62% id.) and flies (60 % id.)
(Zallen and Bargmann submitted). While the function of the Ndr kinases is unknown, other closely related
kinases have been shown to play a role in cell shape and polarity in nonneuronal cells. Since double
mutant analysis places sax-1 and sax-2 in the same genetic pathway, I am in the process of cloning sax-2
in order to identify other components of this kinase pathway. Progress in cloning and additional
phenotypic characterization will be presented.
141

West Coast Worm Meeting 2000
IDENTIFYING PHARYNGEAL TARGETS OF PHA-4 USING
DNA MICROARRAYS
Jeb Gaudet 1 , Michael Horner 1 , Stuart Kim 2 , Susan E. Mango 1
1Huntsman Cancer Institute and Department of Oncological Sciences, University of Utah, Salt Lake City,
UT 84112
2Department of Developmental Biology, Stanford University Medical School, Stanford, CA 94305-5427
Development of the C. elegans pharynx requires the activity of the gene pha-4. Animals that lack pha-4
activity fail to make a pharynx, while ectopic expression of pha-4 leads to the ectopic production of
pharyngeal markers at the expense of other cell types. These data indicate that pha-4 specifies
pharyngeal organ identity.
The PHA-4 product is a member of the family of winged helix transcription factor and is expressed in all
pharyngeal cells. Several genes are known to be specifically expressed in the pharynx, and at least two of
these (ceh-22 and myo-2) require PHA-4 for their expression. Therefore, pha-4 probably functions by
activating pharynx-specific gene expression.
For pha-4 to direct development of the entire pharynx, it must act either directly or indirectly on all
pharyngeal genes. One possibility is that pha-4 directly activates a small subset of pharyngeal genes,
which then activate other downstream targets. This model proposes a regulatory hierarchy with PHA-4
directly activating those genes immediately downstream of it. Another hypothesis is that pha-4 directly
regulates all pharyngeal genes, whether they act early or late in pharyngeal development. A third
possibility is that pha-4 directly activates a subset of genes at multiple levels in the hierarchy and
indirectly activates others.
To understand how pha-4 directs organogenesis we have identified potential targets of PHA-4 using
microarray experiments. Probes were made from RNA from skn-1 embryos, which produce no pharyngeal
cells, and from par-1 embryos, which produce excess pharyngeal cells.
The results of these experiments have identified ~500 genes whose transcripts are more abundant in
par-1 versus skn-1 embryos. Among these genes are known targets of PHA-4 (such as myo-2) as well as
other genes that are involved in pharyngeal development, including pha-4 itself and ceh-22. We are
currently analyzing the other positives to determine whether they are expressed in the pharynx, and
whether they are direct or indirect targets of PHA-4.
142

AN OVERVIEW OF PREDICTED CYTOCHROME P450 GENES
IN C. ELEGANS
Erin Gilchrist
West Coast Worm Meeting 2000
C. elegans Reverse Genetics Core Facility, Biotechnology Laboratory, U.B.C., Vancouver, B.C., Canada
P450 heme-thiolate genes (more commonly known as cytochromes P450) are found in all eukaryotes and
most bacteria and Archaea. They encode a family of heme proteins that are responsible for metabolizing
a wide range of endogenous and exogenous compounds. These enzymes have been implicated in
carcinogenesis, and in the bioactivation or detoxification of many drugs and other xenobiotics.
The Wormpep database has identified 75 predicted proteins that encode P450 enzymes in C. elegans.
These can be sorted into 16 different classes based on their sequence similarity to each other and to
P450s in other systems. The genes in different classes are likely to be involved in different metabolic
pathways, therefore, I have compared the sequences of the C. elegans P450 genes with the known
substrate-binding domains of P450s in other systems. This indicates which processes the different
classes of C. elegans proteins may be involved in. Future biochemical and genetic analysis of the C.
elegans loci will be used to resolve which amino acids in the substrate-binding domain are essential for
the binding of that substrate.
Several classes of P450 proteins in the worm are believed to be specific to C. elegans, or at least to
nematodes, while others have orthologues in vertebrate or other invertebrate systems. Some of the
worm’s P450 genes are more than 90% identical to each other, and several of them are clustered,
suggesting that they evolved by gene duplication of some ancestral heme-thiolate locus. In addition, the
polycistronic nature of some of these clusters indicates that their expression is likely to be coordinated
and that their function may be redundant. I am endeavoring to generate a knockout of one of these
clusters of P450-encoding genes: the C. elegans-specific CYP13 cluster on cosmid T10B9. I am also
comparing the ORFs and flanking DNA of the C. elegans P450 sequences with sequences from C.
briggsae to predict sequences that are important for regulating the expression of the genes in this family.
143

West Coast Worm Meeting 2000
CLUES TOWARD UNDERSTANDING EGF/ WNT SIGNAL
INTEGRATION IN THE SPECIFICATION OF P12 FATE:
ANALYSIS OF THE EGL-5 PROMOTER
Lisa Girard 1 , Henrique B. Ferreira 2 , Scott Emmons 2 , Paul Sternberg 1
1Howard Hughes Medical Institute and California Institute of Technology Pasadena, CA 91125
2Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York 10461
The C. elegans Hox gene egl-5 is most similar, based on sequence analysis, to Abdominal-B. Consistent
with its assignment into this paralog group, egl-5 is expressed in the posterior region of the worm.
Immuno-staining results have shown that in the hermaphrodite, egl-5 is expressed in the hermaphrodite
specific neuron, body wall muscle, posterior lateral microtubule neuron, PVC interneuron, M, V6, the
rectal epithelial cells K, F, B, U and the P12 neuroectoblast cell.
The two most posterior P cells are P11 and P12. The anterior products of their first division are both
neuroblasts. P11.p fuses with the epidermal syncytium and P12.p divides again during late L1. The
anterior division products, the epidermal cells P12.pa and P11.p are distinguishable by their distinct
nuclear morphologies and positions.
Earlier ablation experiments have shown that before interdigitating at the ventral midline during early L1,
either of the two most posterior P cells can adopt the P12 fate. Previous genetic analysis indicates that
P12 fate specification requires the synergistic action of the EGF and the Wnt signaling pathways.
Reduction - or loss of function mutations in components of the EGF or the Wnt pathway result in partially
penetrant P12 to P11 or P11 to P12 transformations. Double mutants of EGF and Wnt pathway
components significantly enhance the frequency of transformation. P12 is not specified in an egl-5(lf)
mutant and overexpression of egl-5 can rescue the loss of P12 specification phenotype of let-23 mutants.
In order to understand how information from the EGF and Wnt pathway are integrated at a cis-regulatory
level, we have undertaken an analysis of the egl-5 promoter to determine elements required for egl-5
expression in P12. Do the two pathways converge on the egl-5 promoter or upstream of it? We have
identified an approximately 1.3 kb. fragment of egl-5 promoter sufficient to drive expression of a
heterologous promoter in the cells K, F, B, U, body wall muscle and P12. We are further dissecting this
region in an attempt to identify P12 specific elements. Additionally, we hope to utilize this P12 enhancer
as a tool to identify additional players involved in egl-5 activation in P12.
144

West Coast Worm Meeting 2000
SPN-4: A GENE REQUIRED FOR MITOTIC SPINDLE
ORIENTATION IN THE 2-CELL STAGE C. ELEGANS EMBRYO
José E. Gomes, Kathryn A. Swan, Christopher A. Shelton, Bruce
Bowerman
Institute of Molecular Biology, University of Oregon, 1390 Franklin Blvd, Eugene OR 97403
Mitotic spindle orientation is crucial during development for proper segregation of cytoplasmic factors
upon asymmetric cell division. Nevertheless the mechanism underlying the specification of spindle
orientation is not well understood. In the C. elegans embryo the P1 blastomere divides asymmetrically
with the mitotic spindle oriented along the anterior-posterior (a-p) axis. The centrosomes, after duplication
and migration, are initially positioned transversely to a-p axis and rotate subsequently, setting up the
spindle longitudinally. The remnant site, a structure derived from the completion of cytokinesis, has been
proposed to be necessary for this centrosome-nucleus complex rotation, by capturing the astral
microtubules, in a process involving the actin cytoskeleton. In addition mutations in the par genes exhibit
defects on spindle orientation in the 2-cell stage suggesting that these genes also play a role in P1
spindle orientation. In genetic screens for non-conditional and for temperature sensitive embryonic lethal
mutations we found three mutant alleles (or25, or80 and or191ts) of a gene, spn-4, exhibiting defects on
mitotic spindle orientation in the P1 blastomere. In spn-4 mutant embryos the P1 spindle fails to set up
along the a-p axis due to a deficiency in centrosome-nucleus complex rotation. In contrast to par mutants,
other aspects of polarity in the 1-cell stage are not affected. Like in wild type embryos the spindle in the
first division is oriented along the a-p axis, AB and P1 blastomeres are unequal in size and divide
asynchronously. Thus spn-4 may be more specifically required for the process of mitotic spindle
alignment in P1. spn-4 maps to linkage group V, position +0.1 map units. We are using a combination of
RNA mediated interference and cosmid rescue to determine its molecular identity. Using the or191ts
allele we have identified set of 8 cosmids that is able to rescue the mutant phenotype.
145

West Coast Worm Meeting 2000
CA 2+ -SIGNALLING VIA THE NEURON-SPECIFIC CA 2+
SENSOR NCS-1 IS ESSENTIAL FOR THERMOTAXIS, A FORM
OF ASSOCIATIVE LEARNING AND MEMORY IN C. ELEGANS
Marie Gomez 1 , Edouard De Castro 2 , Ernesto Guarin 1 , Patrick Nef 1
1Department of Central Nervous System, F. Hoffmann-La Roche, Basel, Switzerland
2Department of Institute for Behavioral Genetics, University of Colorado at Boulder, USA
A form of associative memory in Caenorhabditis elegans is the pairing of the temperature of growth with
food. After conditioning, worms seek food at the temperature of growth in a very precise circular fashion
on a radial gradient of temperature. This phenotype is called the isothermal behavior. Here we show that
NCS-1, a neuron-specific calcium sensor highly conserved throughout evolution, is essential for proper
isothermal behavior. ncs-1 knockout animals are unable to perform isothermal track behavior, although
their loco motor and thermal avoidance behaviors are normal. The knockout phenotype is rescued by
re-introducing wild-type NCS-1, but is not rescued by a loss-of-function form of NCS-1 unable to bind
calcium. Calcium signaling via NCS-1 is therefore essential for proper thermotaxis, a pavlovian form of
associative learning and memory in C. elegans.
146

CHARACTERIZING THE NEURAL CIRCUITRY OF
CHEMOTAXIS TO VOLATILE ODORANTS
Jesse Gray, Maria Gallegos, Tim Yu, Cori Bargmann
UCSF and HHMI, San Francisco, CA 94143
The C. elegans chemotaxis circuit offers the possibility of applying genetics to understand a functionally
complex neural circuit. Further characterization of this circuit, as well as an in vivo physiological assay,
would help relate new results to knowledge of circuitry in other systems. Anatomically, the chemotaxis
circuit can be defined as having three main layers: sensory neurons, interneurons, and motorneurons.
The sensory input to the chemotaxis circuit is relatively well-understood. As a first step in characterizing
the remainder of the circuit, we are using laser ablations to identify functionally important interneurons,
motorneurons, and muscles.
We are also developing new behavioral assays to more specifically control chemotaxis circuit input and
more directly assay circuit output. By flowing odorants over an airtight plate, we can create a temporal
gradient of volatile odorant. It has recently been shown that ASE-mediated (water-soluble) chemotaxis is
accomplished by modulating the frequency of pirouettes (i.e., omega bends and reversals) (3). Using a
video camera to record worm movement during odorant application and subsequently counting omega
bends and reversals, we have begun to characterize the kinetics of the pirouette response to volatile
odorants.
Finally, we will apply imaging techniques to assay physiology. We have constructed a myo3::cameleon
(1) that expresses in body wall muscles. We will use myo3::cameleon worms to identify muscles
important for omega bends and will follow up by ablating the corresponding muscles and motorneurons.
We hope to extend this analysis to neurons in order to characterize circuit activity during olfactory
stimulation and adaptation. We are also exploring other imaging technologies.
1. Miyawaki et al. (1997) Nature. 388, 882-887.
West Coast Worm Meeting 2000
2. Pierce-Shimomura et al. (1999) J Neurosci. 19(21), 9557-69.
147

STUDIES ON THE NEMATICIDAL BACILLUS THURINGIENSIS
TOXINS
Joel S. Griffitts, Raffi V. Aroian
UC San Diego, Department of Biology
West Coast Worm Meeting 2000
The insecticidal crystal (Cry) proteins produced by the soil bacterium Bacillus thuringiensis have been
used for decades to control herbivorous pests. The ability to produce these toxins in transgenic crop
plants has given rise to more extensive application in recent years. Little has been accomplished in
elucidating the mode of action of Cry toxins, and even less in determining how pests generate resistance
to them. One reason for this is that agricultural pests are generally not amenable to molecular genetic
analysis. That C. elegans is susceptible to a subset of the toxins in this family makes it a promising
system in which to approach these questions genetically. We present findings based on two toxins, Cry5B
and Cry21. These toxins are similar in their amino acid sequence (42% identical) and in their destructive
effects on the worm gut. Previously, our laboratory has characterized the susceptibility of C. elegans to
Cry5B, and isolated mutations in five complementation groups that confer resistance (see abstract by
Marroquin et al). Here, we discuss the cloning of one of these resistance genes, bre-5. Mutations in the
bre-5 gene result in strong resistance to Cry5B toxin. We have mapped the bre-5 gene to a small interval
on chromosome IV. Based on homology, we have identified a candidate gene in this region. We are
currently working to determine whether this gene, or some other nearby gene, is the bre-5 gene. We have
started to characterize the toxicity of Cry21. It appears to be a more potent nematicide than Cry5B,
inducing remarkable destruction of the gut. Additionally, we have not found animals in large mutagenized
populations which escape the toxicity of Cry21, even over a series of dilutions. The results outlined incite
fundamental questions about what molecules in the worm mediate the toxicity response and the
generation of resistance, and about what element or elements in the two toxins under study account for
the aforementioned differences in spite of overall high sequence similarity. The experimental approach we
have developed will undoubtedly shed light on these questions, and perhaps on issues of normal gut
development and maintenance in C. elegans.
148

West Coast Worm Meeting 2000
SYNAPTIC LOCALIZATION OF THE GLUTAMATE-GATED
CHLORIDE CHANNEL GBR-2
Maria E. Grunwald, Joshua M. Kaplan
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
In C. elegans and other invertebrates, glutamate receptors are likely to play a dual role at excitatory, as
well as at inhibitory synapses. Recently, an AMPA-type glutamate receptor (GLR-1) has been shown to
be localized to excitatory synapses (Rongo et al., 1998), and glutamate-gated chloride channels (GluCls)
have been implicated in the formation of inhibitory synapses (Lee et al., 1999). We are interested whether
these different types of glutamate receptors are in fact localized to functionally diverse synapses and if so,
how this differential targeting is achieved.
We are comparing the synaptic targeting of GLR-1 receptors and the glutamate-gated chloride channel
GBR-2, encoded by the avr-14 gene (Dent and Avery, WCWM 1998). Using a GFP-tagged version of
GBR-2, we found that these receptors are expressed in interneurons and localized to distinct synapses
along the ventral cord. In addition, GBR-2 is expressed in motorneurons and in the sensory neurons ALM
and PVD. Thus, compared to GLR-1, which is mainly expressed in interneurons, GBR-2 is more broadly
expressed. We have also compared the subcellular localization of GBR-2 to that of GLR-1. Interestingly,
GBR-2 and GLR-1 receptors are targeted to different synapses. GBR-2 does not co-localize with GLR-1
and only a minor fraction of GBR-2 was found at touch neuron-interneuron synapses, where GLR-1 is
found. However, a significant fraction of GBR-2 was identified at interneuron-interneuron synapses in the
ventral cord. We speculate that these GBR-2-containing synapses correspond to the reciprocal synapses
between forward and backward command interneurons, and that these putatively inhibitory synapses
coordinate transitions between forward and reverse locomotion. We are presently investigating this
hypothesis. Furthermore, we are interested in what causes the differential targeting of GBR-2 molecularly.
Recently, we were able to show that the PDZ protein LIN-10 is responsible for GLR-1 localization (Rongo
et al., 1998). GBR-2 is likely to use alternative pathways for its localization. We are presently searching
for proteins that are responsible for its clustering using EMS mutagenesis, yeast-two-hybrid screening
and co-localization assays with candidate proteins.
149

West Coast Worm Meeting 2000
REGULATION AND FUNCTION OF LIN-11 IN C. ELEGANS
VULVAL DEVELOPMENT
Bhagwati P Gupta, Paul W. Sternberg
HHMI and Division of Biology, California Institute of Technology, Pasadena, California, USA
The C. elegans hermaphrodite vulva has been a well-established model system to address the
mechanisms of cell fate specification. The highly invariant lineage of the vulval precursor cells (VPCs)
provides an opportunity to study the molecular interactions of genes that control cell types so precisely.
We are interested in understanding the mechanism of the nuclear factors that function to specify the
terminal fate of the vulval cells. Towards this we have been analyzing the role of lin-11 that encodes a
LIM Homeobox family of transcription factor. Mutations in lin-11 gene cause egg-laying defective (Egl)
phenotype. Cell lineage analysis and reporter gene expression experiments suggest its possible function
in the specification of a subset of 2 o vulval cells. In addition, the gene is also required for the normal
development of uterine p precursors that ultimately form vulva-uterus connection, and a subset of
neurons. In order to understand the function and regulation of lin-11, we have characterized its regulatory
region and identified distinct cis-elements for the vulva and uterine - specific function. We have also
re-examined its expression pattern using multiple reporter constructs and the gene function in egg-laying
behavior. Our results show that lin-11 is likely to function in many more cells and at multiple times than
previously reported. In an effort to further understand its function, we have searched the C. elegans
genome database to identify a putative binding partner of LIN-11 - LBP-1 (LIM Binding Protein-1) - and
currently investigating its function in vulval morphogenesis. We plan to carry out genetic screens using
different genetic backgrounds to identify the upstream and downstream genes of the lin-11 pathway.
These experiments are likely to provide a better understanding of the function of LIM Homeobox family
members that act to specify diverse cell types in multiple organisms.
150

SUR-7, A GENE THAT SUPPRESSES ACTIVATED RAS
Eric Hague 1 , Min Han 2
West Coast Worm Meeting 2000
1Department of MCD Biology, University of Colorado at Boulder
2HHMI, Department of MCD Biology, University of Colorado at Boulder
Suppressors of let-60 (n1046) ras have proven to be important in the regulation of the ras signal
transduction pathway. The sur (suppressor of activated ras) genes include sur-1/mpk-1, sur-2
(downstream of mpk-1), sur-3/ksr-1, sur-6 (a PP2A regulatory subunit) and sur-8 (a leucine rich repeat
containing protein). ksr-1, sur-6 and sur-8 appear to act positively between ras and raf.
I am attempting to clone sur-7using single nucleotide polymorphisms (SNP’s). sur-7 has no phenotype
alone, but suppresses n1046 better than 99%. sur-7 maps between sdc-1 and sup-10 on LGX.
151

CHARACTERIZATION AND SUPPRESSION OF EAT-16;
SAG-1/DGK-1 LETHALITY
Yvonne M. Hajdu-Cronin, Wen J. Chen, Paul W. Sternberg
HHMI and Division of Biology, Cal Tech, Pasadena CA 91125
goa-1 (Ga o) modulates many behaviors in C. elegans, including locomotion, egg-laying, and feeding.
Reducing function in goa-1 causes hyperactive locomotion and constitutive egg laying (1, 2); in contrast,
overexpressing the constitutively activated Q205L mutation causes lethargy and cessation of egg laying
(1). Previously, we screened for suppressors of the paralysis of syIs17, an integrated transgene
containing goa-1(Q205L) under control of a heat shock promoter. Hyperactive mutants were isolated in
two genes, eat-16 and sag-1/dgk-1. We found that in addition to suppressing the phenotype of activated
Ga o, eat-16 and sag-1/dgk-1 mutations also partially suppressed the phenotype of several reduction of
function mutations in egl-30 ( C. elegans Ga q). eat-16 encodes a regulator of G protein signaling, which
we believe functions as a GAP for EGL-30, and sag-1/dgk-1 encodes a diacyl glycerol kinase (3) which
likely functions to reduce the levels of diacyl glycerol (DAG), a second messenger produced upon
stimulation of PLCb by activated EGL-30.
eat-16(sy438) and sag-1/dgk-1(sy428) double mutants arrest during larval development. This lethal
phenotype is highly penetrant (>99%). We hypothesized that the lethality of eat-16; sag-1/dgk-1 is caused
by excessive levels of second messengers produced downstream of Ga q, such as DAG. In support of
this hypothesis, the triple mutant egl-30(md186) eat-16(sy438); sag-1/dgk-1(sy428) is viable to adulthood
and fertile. To further understand the cause of death and identify more components in the pathway, we
are seeking other suppressors of eat-16; sag/dgk-1 lethality besides egl-30, both by testing mutations in
genes that have already been described, and by screening for new suppressors. To date we have
screened about 7000 genomes and have backcrossed 7 suppressor mutants. We are beginning to
characterize the lethal phenotype by Nomarski optics and plan to determine the site of action.
References:
1. Mendel et al., 1995. Science 267: 1652-1655.
2. Segalat et al., 1995. Science 267: 1648-1651.
3. Nurrish et al., 1999. Neuron 24: 231-242
West Coast Worm Meeting 2000
152

IMPROVED TISSUE PRESERVATION USING METAL MIRROR
FREEZING OR HIGH PRESSURE FREEZING FOR TEM
David H. Hall 1 , Frank Macaluso 2 , Gloria Stepheney 1 , Marie-Christine
Paupard 1
1Center for C. elegans Anatomy, Albert Einstein College of Medicine, Bronx NY 10461
2Analytical Imaging Facility, Albert Einstein College of Medicine, Bronx NY 10461
Several recent reports (see below) have demonstrated that C. elegans tissues can be very well preserved
for electron microscopy by high pressure freezing (HPF) followed by freeze substitution, perhaps
substantially better than by standard chemical immersion fixation. HPF shows the potential to capture a
more "life-like" view of the worm’s ultrastructure. We have been testing both HPF and a related technique,
rapid freezing on a metal mirror (MMF) followed by freeze substitution. Both methods obtain similar high
quality fixation, although there are some freezing artifacts using the metal mirror device that are
eliminated in HPF. For MMF, live animals are concentrated on a small piece of filter paper and plunged
against a metal mirror at liquid nitrogen temperature. While freezing damage often occurs about 5-15
microns into the worms, some animals are very well frozen throughout. The frozen samples are held at
low temperature and freeze substituted into 1% osmium tetroxide in acetone, then embedded into plastic
resin and cured for thin sectioning. For HPF, we have tried two methods to concentrate live animals into
small metal planchette, either holding the animals within fine strands of dialysis tubing (C. Lavin, pers.
comm.), or mixing them into a slurry of yeast paste to form a space-filling solid support (McDonald, 1999).
Examination of fast-frozen specimens by TEM reveals excellent views of membrane events and
organelles. For instance, we see many omega figures on coelomocytes which are indicative of active
endocytosis, events which are not commonly captured by chemical fixation. Synaptic active zones and
vesicles are well preserved, as are their relationships to microtubules. A network of microtubules can also
been seen extending to the periphery of hypodermis. Basal laminae look strikingly different, much looser
and more mesh-like when compared to chemical fixation. Sample images are shown on our website
[www.aecom.yu.edu/wormem/new.html].
These two preparation methods, HPF and MMF, also hold great promise for high resolution immuno-EM.
By reducing the osmium content and adding a dilute aldheyde fixation to the freeze substitution medium,
we can obtain better resolution than is currently possible by our microwave technique. We have
successfully localized epitopes in thin sections from HPF samples. MMF equipment is available here at
Einstein campus. We are conducting HPF trials with the help of Stan Erlandson and Ya Chen at the
University of Minnesota. As our skills improve, we will be happy to offer such services to the C. elegans
community.
For further information on HPF, we recommend the following sources:
Colleen Lavin’s website at www.geology.wisc.edu/~uwmr/caoting.html
Martin Muller’s website at www.em.bio.ethz.ch/
West Coast Worm Meeting 2000
Kent McDonald, Methods in Molecular Biology, vol 117, pp. 77-97 (Human Press) 1999.
In the U.S., there are HPF machines open to the outside users in Madison, Berkeley, Minneapolis and
Albany.
153

ROLE OF THE CAENORHABDITIS ELEGANS HOMOLOGS OF
CDK5 AND P35 IN MIGRATION AND AXON OUTGROWTH
Thomas Harbaugh, Gian Garriga
Department of Molecular and Cell Biology, 401 Barker Hall, University of California, Berkeley, CA 94720
While cdk5 was originally identified in vertebrates based on its homology to the cell cycle regulated cdc2,
further study has demonstrated a function not in the cell cycle but instead in post-mitotic neurons. Mouse
knockouts of cdk5 and its activator, p35, result in defects in the pattern of neuronal migration in the cortex
(1,2). Studies in cultured rat neurons demonstrated that the cdk5/p35 kinase activity can promote neurite
outgrowth (3).
The C. elegans homolog of cdk5 is 74% identical to mouse, and the p35 homolog contains a 159 AA
region that is 54% identical. Translational GFP reporter constructs for cdk5 and p35 are expressed in the
cytoplasm of most neurons beginning around the two-fold stage of embryogenesis and continuing into
adulthood.
Simultaneous overexpression of both cdk5 and p35 produces a strongly uncoordinated phenotype, with
defects in fasciculation and axon pathfinding. Along with the expression pattern, these phenotypes
suggest a role for cdk5 and p35 in neuronal development.
The mig-18(k140) mutation was identified based on defects in distal tip cell migration (4) and may
represent a Ce-cdk5 allele. Extrachromosomal arrays containing the Ce-cdk5 genomic region can
partially rescue the distal tip cell migration defect of mig-18(k140). Furthermore, sequencing of the
Ce-cdk5 genomic region revealed a point mutation predicted to change a conserved histidine into a
tyrosine. To confirm that k140 is a Ce-cdk5 allele, we plan to isolate additional mig-18 mutants and
determine whether they contain Ce-cdk5 molecular lesions. Also, we will screen a deletion library for
alleles of Ce-cdk5 and its activator Ce-p35.
(1) Gilmore EC, Ohshima T, Goffinet AM, Kulkarni AB, Herrup K, J Neurosci, 18:6370-7 (1998)
(2) Chae T, Kwon YT, Bronson R, Dikkes P, Li E, Tsai LH, Neuron, 18:29-42 (1997)
(3) Nikolic M, Dudek H, Kwon YT, Ramos YF, Tsai LH, Genes Dev, 10:816-25 (1996)
(4) Nishiwaki K, Genetics, 152:985-97 (1999)
West Coast Worm Meeting 2000
154

West Coast Worm Meeting 2000
REGULATION OF EGG-LAYING BY SENSORY CUES
Laura Anne Hardaker, William R. Schafer
Dept. of Biology, University of California at San Diego, La Jolla, CA 92093-0349
Egg-laying in C. elegans is regulated by sensory cues. For instance, it has been found that food
deprivation increases the length of the inactive phase of egg-laying. However, the exact mechanisms by
which this occurs are not known. We are investigating this phenomenon by focussing on two pathways,
the flp-1 pathway and the type II TGF-beta dauer pathway.
The gene flp-1 encodes a precursor of FMRFamide related neuropeptides, and it was shown by Chris Li
to be expressed in a specific subset of head neurons. She found that flp-1 knockouts affect a variety of
behaviors, including egg-laying. When we tested flp-1 worms, we found that they had a lengthened
inactive phase (similar to the food-deprived worms). In addition, their egg-laying rate did not change
depending on the presence or absence of food. Ablation experiments have shown that the effects of flp-1
encoded peptides on egg-laying are independent of the presence of the HSN. Thus, we believe that the
effects of FLP-1 may be mediated, at least in part, by a hormonal mechanism.
Jim Thomas’ lab showed that the type II TGF-beta daf (dauer formation) mutants also displayed defects in
egg-laying. As the Ruvkun and Riddle labs found that the TGF-beta pathway functions in sensory
neurons, it seems to be a reasonable candidate to be involved in the regulation of egg-laying by sensory
cues. Interestingly, we found that the egg-laying patterns of the Daf-c mutants (daf-1, daf-4, daf-7, and
daf-8) are similar to that of flp-1: all have a lengthened inactive period of egg-laying. In addition, the
egg-laying of daf-4 and daf-8 (but not daf-1 or daf-7) is unable to be modulated by the presence or
absence of food. Furthermore, we found that the Daf-d mutation in daf-3 does not cause a defect in food
modulation, and in fact has the ability to suppress the food insensitivity of daf-4.
These results suggest that there may be two hormonal pathways involved with relaying sensory cues to
the egg-laying circuitry, one involving flp-1 and the other involving the Daf-c genes. We are currently
investigating connections between the flp-1 and daf pathways by constructing double mutants, and the
results will be presented.
155

CHARACTERIZATION OF THE C. ELEGANS
SEROTONIN-SYNTHETIC AROMATIC AMINO ACID
DECARBOXYLASE GENE BAS-1
Emily Hare, Curtis M. Loer
West Coast Worm Meeting 2000
Dept. of Biology, Univ. of San Diego, 5998 Alcala Park, San Diego, CA 92110
We are attempting to identify mechanisms by which neurons choose a neurotransmitter during
development. We are currently characterizing genes used by the serotonergic neurons in C. elegans to
learn how they are regulated. Worms with mutations in the bas-1 gene (biogenic amine synthesis
abnormal) are serotonin- and dopamine-deficient. Wildtype serotonin can be restored by the application of
exogenous serotonin, but not its immediate precursor 5-HTP; this phenotype is consistent with the loss of
serotonin-synthetic aromatic amino acid decarboxylase (AAADC) activity. We have previously shown that
wildtype serotonin immunofluroescence can be rescued in bas-1 mutants by injection of a 15.1 kb
genomic clone (C05D2XN) containing two adjacent AAADC homologous sequences. The genomic
structure of the region suggests the two AAADC sequences (called C05D2.4 and C05D2.3) will be
transcribed together as an operon (e.g., the two coding regions are less than 400 bp apart). The pair of
sequences must be from an ancient gene duplication since the predicted amino acid sequences are only
66% identical. Interestingly, the second sequence (C05D2.3) is missing a highly conserved segment of
protein, suggesting it may not be able to function as an AAADC. The existence of a partial cDNA for
C05D2.3, however, does demonstrate the gene is expressed. We are currently attempting to determine
which of the two predicted sequences is necessary for serotonin synthesis and how the AAADC gene is
expressed and regulated. Two bas-1 mutant alleles sequenced to date contain point mutations in
C05D2.4 coding sequence resulting in premature stop codons. Furthermore, a construct in which
C05D2.4 is mutated does not rescue bas-1 mutants whereas a construct mutated in C05D2.3 does
rescue bas-1 mutants. Our preliminary results strongly suggest that C05D2.4 constitutes the bas-1 gene;
we are still attempting to determine what role C05D2.3 might play. This work is supported by the Fletcher
Jones Foundation and an NIH (AREA) Grant.
156

XOL-1 FILES
West Coast Worm Meeting 2000
Christian A. Hassig, Barbara J. Meyer
HHMI and Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
Sexual differentiation in C. elegans is mediated by the developmental switch gene xol-1. XOL-1 represses
one or more of the hermaphrodite-specific sdc genes or products, thereby directing the male modes of
sex-determination and dosage compensation. XOL-1 has no known homologs and the primary structure
offers no clues towards understanding its molecular function. Experiments are under way to determine the
precise molecular mechanism(s) of this biological alien.
The primary sex-determination signal (X:A ratio) is transduced by a set of signal elements, including sex-1
and fox-1, that determine the level of XOL-1 protein. This X chromosome counting mechanism results in
10-fold higher XOL-1 levels in XO versus XX embryos. Genetic studies suggest that both sex-1 and fox-1
function as xol-1 repressors even in XO embryos. Consistent with these results, our antibodies detect
XOL-1 in XO embryos carrying reduced copies of sex-1 (and 2 extra copies of xol-1), but not in wildtype
XO embryos. Staining occurs in nearly all nuclei of embryos between the 100-500 cell stage. Similar
experiments are being performed with fox-1 mutants. These results will establish a direct correlation
between X-signal element dose and XOL-1 levels.
Experiments are consistent with a role for XOL-1 as a repressor of sdc-2. Several features distinguish
SDC-2 from other SDC proteins and make it a likely target for XOL-1 regulation: 1) SDC-2 directs the
assembly of the dosage compensation complex on the hermaphrodite X chromosome, 2) SDC-2 is
undetectable in wildtype XO embryos, 3) overexpression of SDC-2 causes male-specific lethality. These
results suggest that XOL-1 could interfere with the expression of sdc-2 and thereby inhibit the formation of
the dosage compensation complex in XO embryos.
Our experiments using transgenic strains further support a role for XOL-1 in repressing sdc-2.
Hermaphrodite lethality caused by overexpression of XOL-1 is partially suppressed by SDC-2 expressed
from a heterologous promoter. This suppression is not seen with overexpression of SDC-3 and suggests
that XOL-1 might regulate sdc-2 at the transcriptional level. Transgenic lines will be used to determine if
XOL-1 associates with the sdc-2 locus in vivo. Results from these experiments will direct future inquiries
into the XOL-1 Files.
157

West Coast Worm Meeting 2000
Y41G9A.1, THE C. ELEGANS HOMOLOGUE OF TG737, IS
EXPRESSED IN CILIATED NEURONS
Courtney J. Haycraft 1 , Patrick D. Taulman 1 , Stephen M. Krum 1 ,
Bradley K. Yoder 2
1MCLM 639, 1530 3rd Ave. South, Birmingham, AL 35294-0005
2MCLM 656, 1530 3rd Ave. South, Birmingham, AL 35294-0005
The C. elegans gene Y41G9a.1 encodes the homologue of the mouse gene Tg737 which was identified
through a BLAST search of the C. elegans genome database. Y41G9a.1 (CeTg737) was isolated from a
mixed stage C. elegans cDNA library. CeTg737 encodes a predicted 823 amino acid protein with 45%
identity to the mouse protein (Polaris). As determined for Polaris, CeTg737 contains two blocks of
tetratricopeptide motifs (TPR) thought to be involved in protein-protein interactions. Northern blot analysis
reveals a 2.6 kb transcript with the highest level of expression during embryogenesis and early larval
stages. Transcriptional Y41G9a.1::GFP fusions using 400 bp upstream of the predicted ATG show
expression in ciliated neurons including the amphids and phasmids. Analysis of the promoter region
identified a potential X-box sequence approximately 100 bp upstream of the ATG. X-boxes are targets for
the DAF-19 transcription factor and have been identified in the promoters of several genes involved in
sensory cilia formation or maintenance. The role of DAF-19 in regulation of CeTg737 is currently being
explored. Consistent with GFP expression analysis, monoclonal antibodies raised against recombinant
CeTg737 detect a protein in the dendrites and cilia of sensory neurons and the sensory rays of the male
tail. Overall the results in the worm are in agreement with our analysis in the mouse where Polaris
localizes to the base of cilia and its disruption results in a loss of cilia.
158

West Coast Worm Meeting 2000
LET-381 IS A FORKHEAD GENE
Marika Hellqvist-Greberg 1 , Ann M Rose 2 , David L Baillie 1
1IMBB, Simon Fraser University, Burnaby, B.C, V5A 1S6, Canada
2Dep.of Medical Genetics, UBC, 6174 University Blvd., Vancouver, B.C V6T 1Z3 Canada
Forkhead genes encode transcription factors characterized by a highly conserved region of 100 amino
acids necessary for DNA binding. Members of the forkhead gene family have been identified in eukaryotic
organisms ranging from yeast to mammals. They have proved to be an important gene family involved in
development and tumorigenesis.
We are studying this gene family in C.elegans. We have identified at least 15 members of the forkhead
gene family in the finished sequence of C.elegans. 5 of them have known mutants, daf-16, pha-4, pes-1,
unc-130 and lin-31.
RNAi was used for three of the forkhead genes not yet described, T14G12.4, F26B1.7 and K03C7.2.
F26B1.7 had a lethal phenotype that resembled the phenotype of let-381 identified in Ann Roses lab.
Therefore we sequenced one of the alleles of let-381, h107, and found a point mutation in the splice site
of exon 3 in the F26B1.7 gene. We are now analyzing the phenotype and the expression pattern in order
to understand the function and identify target genes for this forkhead protein.
159

GENETIC ANALYSIS OF DYNAMIC SEARCH BEHAVIOR IN C.
ELEGANS
T.T. Hills, F. Adler, A. V. Maricq
West Coast Worm Meeting 2000
Dept. of Biology, University of Utah, Salt Lake City, UT 84112
We are interested in identifying genes that control dynamic behavior modification underlying exploratory
search in C. elegans. Immediately following removal from food, worms engage in an area-restricted
search (ARS), characterized by an initial high frequency of sharp turns that then decays over time. This
causes the search path to shift over time from local and overlapping to globally directed with infrequent
overlap. ARS is found in a wide variety of animals and is theoretically supported as an efficient search
strategy for finding resources that are distributed in patches. Using motion-sensitive computer analysis,
we observed worms immediately after removal from food and quantified the temporal dynamics of their
search paths including such features as velocity, radial displacement, and turning angle distribution.
These features change continuously with time, but stabilize by approximately 40 minutes. This search
behavior is experience dependent, changing significantly in a predictable way across worms reared in
different resource distributions. Worms grown in liquid also display ARS, suggesting a genetic
predisposition for the behavior.
To identify genes controlling this dynamic behavior modification, we quantified ARS in worms with
mutations in candidate genes. Mutations in the glutamate receptor glr-1 and the amino acid
decarboxylase bas-1 cause significant changes in the dynamic aspects of ARS, suggesting that the
dynamics of ARS are modified by mutations in a single gene. To further identify genes controlling this
behavior, we are developing a genetic screen to isolate mutants that lack the local search following
removal from food. We are isolating worms that disperse to a point significantly farther than wild type
following removal from food. bas-1 mutants, which lack the local search, are being used to calibrate the
distance and time that provides the most efficient separation between wild type and potential mutants. In
conclusion, the area-restricted search phenotype has been quantified in C. elegans, shows predictable
interactions with the environment, and appears to be under the control of specific genes which we are
currently in the process of identifying.
160

West Coast Worm Meeting 2000
MULTIPLE ROLES FOR THE RAS-MAPK SIGNAL
TRANSDUCTION PATHWAY IN CHEMOTAXIS TO
ODORANTS?
Takaaki Hirotsu 1 , Satoshi Saeki 2 , Yuichi Iino 1
1Molecular Genetics Research Laboratory, The University of Tokyo, Tokyo 113-0033, JAPAN
2Present address: Tokyo Research Laboratories, Kyowahakkokogyo Co. Ltd., Machida 194-8533,
JAPAN
We have previously reported that mutants affected in the Ras-MAPK pathway show defects in chemotaxis
to volatile odorants. Experiments in which let-60 ras was expressed using a heat shock promoter and a
cell-specific promoter indicated that the normal activity of LET-60 Ras is required in mature olfactory
neurons.
To determine how the Ras-MAPK pathway is activated in olfactory neurons, we observed accumulation of
activated MAPK by immunofluorescence. The activation of MAPK in the AWC neurons was detected after
10 seconds of application of isoamylalcohol (which is sensed by AWC) at 10 -4 dilution. This activation of
MAPK was dependent on the function of the G protein alpha subunit ODR-3, cyclic-nucleotide gated
channel TAX-2/TAX-4 and the voltage-activated calcium channel subunit UNC-2. These results suggest
that odorant-induced neuronal activity is essential for the activation of the Ras-MAPK pathway.
When animals were exposed to higher concentration (10 -2 dilution) of isoamylalcohol for 10 seconds and
stained for activated MAPK, the AWB neurons, as well as AWC, were stained. Interestingly, the activation
of MAPK in AWC was shut off by longer exposure (for 5min) to 10 -2 isoamylalcohol, while the activation in
AWB was sustained. On the other hands, the application of 10 -4 isoamylalcohol induced activation of
MAPK in AWC, but not in AWB.
AWB neurons are known to mediate avoidance of volatile odorants. Our immunofluorescence analysis
indicated the possibility that high concentration of isoamylalcohol might induce avoidance behavior.
Indeed, worms show avoidance behavior when large amount of undiluted isoamylalcohol is presented.
The let-60(gf), lin-45(lf) and mek-2(0) mutants exhibited defects in this behavior, suggesting that the
pathway is also important for avoidance of odorants.
When wild-type worms are pre-exposed to isoamylalcohol at 10 -4 dilution for 5min, they show avoidance
of isoamylalcohol at the normally attractive concentration. The let-60(lf) mutants showed defects in this
behavior. The Ras-MAPK pathway may also play roles in this behavioral plasticity.
161

West Coast Worm Meeting 2000
MAB-26 ENCODES THE C. ELEGANS EPHRIN EFN-4
Thomas Holcomb, Sean E. George, Ian Chin-Sang, Mei Ding, Andrew
Chisholm
Department of Biology, University of California, Santa Cruz, California 95064
In C. elegans, Eph signaling functions in embryonic neuroblast movements after gastrulation, and in
epidermal enclosure. The C. elegans genome encodes one Eph receptor, VAB-1, and four ephrins:
VAB-2/EFN-1, EFN-2, EFN-3, and EFN-4. mab-26 mutants have defects in male tail morphogenesis
(Chow and Emmons 1994). The predicted ephrin gene efn-4 (aka F56A11.3), was located in the same
region as mab-26. A genomic clone containing F56A11.3 rescued Mab-26 phenotypes in transgenic
arrays. These data combined with identification of mutant alleles demonstrate that mab-26 and efn-4 are
the same gene.
The EFN-4 protein encodes a divergent ephrin. EFN-4 overall is most similar to murine Ephrin-B2 (27%
identity and 45% similarity). Like other worm ephrins, EFN-4 is predicted to be GPI-anchored. Unlike all
other ephrins EFN-4 contains a 24 amino acid insert in the putative receptor-binding domain.
The mutation bx80 is a deletion of second exon of efn-4, and thus is likely a null mutation. Three other
alleles from the Cambridge collection cause alterations in efn-4: e36 causes an early nonsense mutation,
and the weak alleles e660 and e1746 (provided by Jonathan Hodgkin) cause missense alterations in the
receptor binding domain.
The phenotypes of efn-4 mutants indicate that like other eph signaling proteins it is required for embryonic
morphogenesis. Genetic interactions with the other ephrins and vab-1 are inconsistent with EFN-4 acting
only in the VAB-1 pathway (see abstract by Moseley and Chisholm). These observations have led us to
examine the the EFN-4/VAB-1 binding interaction using cell culture experiments. Previously the EFN-1
ephrin was shown to bind VAB-1::AP fusion proteins with high affinity in mammalian cell culture assays
(Chin-Sang et al 1999). We are using parallel approaches to test EFN-4/VAB-1 interactions.
An EFN-4::GFP reporter gene is expressed in a subset of neurons. This expression pattern suggests that
EFN-4/VAB-1 signaling in the embryo occurs between neurons and is thus required non-autonomously for
epidermal development. We will test this idea using genetic mosaic analysis and tissue-specific rescue
experiments.
162

SYD-8, A NEW PLAYER IN AXON GUIDANCE.
Xun Huang, Mei Zhen, Yishi Jin
Department of Biology, University of California, Santa Cruz, CA95064
Growth cones sense and respond to various extracellular cues to be guided to their targets. Many such
cues and their receptors have been identified in recent years, including netrins, slits, and ephrins.
However, the signal transduction pathways of these cues are not well understood. We describe here a
gene that may function in the downstream signaling of axon guidance.
syd-8 (ju39) was isolated in a genetic screen for abnormal synapse mutants 1 . syd-8 (ju39) animals have
wild-type mating, moving and egg-laying behaviors. In syd-8 (ju39) animals, the synaptic GFP puncta are
discontinuous in the dorsal cord. Analysis of axonal morphology in syd-8 mutant revealed several axonal
defects in DD and VD neurons: 1) commissures terminate prematurely before reaching the dorsal cord, 2)
commissures branch or wander to the lateral region, 3) axons defasciculate in both dorsal and ventral
cords. Similar defects are also observed in the DA and DB axons, but to lesser extents. We also observed
defects in pathfinding by the HSNs. In wild type animals, HSNs send out processes along the ventral
nerve cord to the nerve ring . Normally, HSNL extends along the left small bundle of ventral cord. HSNR
extends along the large right bundle of ventral cord. In syd-8(ju39) animals, HSNs have two types of
defects: 1) HSN, mostly the HSNL, wander in the lateral region, and 2) HSNs fasciculate on the ventral
cord. In 20% of syd-8 (ju39) animals, the HSNs are in the large right bundle of ventral cord. The touch
neurons appear to be normal as judged by the Pmec-7-GFP marker.
ju39 is a recessive, partial loss-of-function mutation. We have mapped syd-8 on Chromosome V between
lin-25 and him-5. arDf1, itDf1 and yDf8 complement syd-8; yDf12 and nDf42 fail to complement it. Cloning
of syd-8 is underway and we will report further molecular analysis of syd-8 at the meeting.
1. Zhen, M and Jin, Y. Nature 1999
West Coast Worm Meeting 2000
163

West Coast Worm Meeting 2000
ANALYSIS OF GCY-31, A PUTATIVE SOLUBLE GUANYLYL
CYCLASE GENE IN CAENORHABDITIS ELEGANS
Martin L Hudson 1,2 , David S. Karow 1,2 , Michael A. Marletta 1,2 , David
B. Morton 1,2
1 Department of Biological Structure and Function, Oregon Health Sciences University, 611 SW Campus
Drive, Portland, OR. 97201
2 Howard Hughes Medical Institute and Department of Cell & Molecular Biology, University of Michigan,
Ann Arbor, MI 48109
Soluble guanylyl cyclases (sGCs) are involved in many cellular and physiological roles including neuronal
signaling, axon guidance and maintenance of vascular tone. Many of these functions are carried out
through activation of sGCs by the gaseous signaling molecule, nitric oxide (NO). Recently, a non-NO
activated sGC (MsGCb-3) has been isolated from the developing larval nervous system of the tobacco
hawk moth, Manduca sexta. By BLAST analysis, its closest homologue in C. elegans is the putative sGC,
gcy-31. Previous work by Yu et al. failed to demonstrate any expression data for this gene using
GFP-reporter constructs. In our hands, gcy-31::GFP constructs show expression in a bilaterally
symmetrical pair of cells in the C. elegans head. These have processes that lead anteriorly to the tip of
the pharynx, as well as distinctive circular projections posteriorly into the nerve ring. From this
morphology, we have tentatively identified these as either OLL or IL1 neurons.
cDNAs corresponding to gcy-31 have been isolated and show multiple splice variants. However,
heterologous expression of the cDNAs in COS-7 cells has so far failed to show any cyclase activity. Apart
from MsGCb3, all sGCs previously isolated have been obligate heterodimers. These are composed of an
a and b subunit, hence an absent subunit partner may explain the lack of activity in this assay. To answer
this, we are cloning and mapping the expression patterns of the remaining sGCs in C. elegans. Inverted
repeat constructs under the control of heat-shock promoters are also being made to generate in-situ
dsRNA corresponding to gcy-31, allowing us to generate and analyze a null mutation in this gene.
164

West Coast Worm Meeting 2000
USING C. ELEGANS TO DETERMINE THE MECHANISM OF
ACTION OF PHARMACEUTICALS AND PESTICIDES
Tak Hung, Ben Burley, Emery Dora, Dan Elkes, Steve Gendreau,
Denise Jacobus, Rachel Kindt, Mark Lackner, Lisa Moore, Scott Ogg,
Dianne Parry, Roxanna Peng, Ellyn Pham, Jenny Kopczynski
Exelixis, Inc., 170 Harbor Way, P.O. Box 511, South San Francisco, CA 94083-0511 (www.exelixis.com)
The primary goal of the mechanism of action program at Exelixis is to identify the cognate targets (binding
partners) of compounds with unknown mechanisms of action. We start the process by identifying
mutations in genes whose activity modulates the response of a model organism -- C. elegans, Drosophila,
yeast, algae or plants -- to the drug, insecticide, fungicide or herbicide. We then clone these genes and
their homologs in human or pest species, and use biochemical approaches to distinguish the cognate
targets from the other genes in the response pathways. Once a cognate target has been identified, new
compounds with increased activity, less toxicity and better delivery properties can be developed.
Secondary goals of the program include identifying other genes in the response pathways that may also
be drug or pesticide targets, and identifying genes that mediate undesirable side-effects of drugs.
We have corporate partnerships with Bayer, Pharmacia and Bristol-Myers Squibb to identify the pathways
and cognate targets of pharmaceuticals and agrochemicals. These partnerships typically start with a
feasibility study to establish that a particular model system can be used to determine the mechanism of
action for a particular compound. Roughly half of tested compounds cause genetically tractable
phenotypes in C. elegans. Based on the observed phenotypes, mutations and RNAi of candidate genes
in the response pathway are tested for resistance or hypersensitivity to the compound. Also, forward and
reverse genetic screens are carried out to identify target pathways and candidate cognate targets.
In addition to our corporate partnerships, we have an internally funded program to identify the cognate
targets of pharmaceuticals. A significant number of drugs, with annual sales over $100 million, act by
unknown mechanisms. Many more compounds- with unknown mechanisms of action- have demonstrated
promising activities in clinical or pre-clinical studies. The development of second generation drugs with
improved efficacy, fewer side effects, and lower production costs is often hindered by a lack of
understanding of how these compounds act. One advantage of starting with compounds rather than
mutations for target discovery is that we know in advance that at least the cognate targets will be
druggable and therapeutic. We will present our analysis of C. elegans phenotypes caused by
oncology-related compounds.
165

IN VIVO CHARACTERIZATION OF THE EFFECTS OF THE
UNC-64(MD130) MUTATION ON ANESTHETIC SENSITIVITY.
Hunt S.J., Mike Crowder
West Coast Worm Meeting 2000
Department of Anesthesiology, Washington University School of Medicine
We have previously shown that mutations in the neuronal syntaxin gene unc-64 profoundly alter the
volatile anesthetic (VA) sensitivity of C. elegans. Two hypomorphic unc-64 alleles confer hypersensitivity
to the VAs isoflurane and halothane but a third unc-64 hypomorph is VA resistant. The difference
between the isoflurane EC 50s (the concentration where the effect is half maximal) of the hypersensitive
and resistant alleles is over 30-fold. The high-level resistance produced by the unc-64(md130) mutation
indicates that VAs act specifically through a single major mechanism to disrupt coordinated locomotion in
C. elegans. The md130 mutation is a single base change at the splice-donor site of the sixth intron,
causing some aberrant splicing. By RT-PCR, md130 mutants produce truncated RNAs, in addition to
wild-type unc-64 mRNAs. The predicted truncated forms of syntaxin are believed nonfunctional as they
lack the transmembrane and part of the H3 domains proven vital for syntaxin’s ability to function in
neurotransmitter release. The behavioral phenotype of md130 is recessive and similar to other weak
reduction-of-function unc-64 alleles. However, the VA-resistance phenotype of md130 is semidominant
and behaves as a gain-of-function mutation, consistent with the truncated forms acting dominantly to
block VA action. The current studies attempt to address in vivo the molecular mechanism(s) by which the
md130 mutation confers this resistance. A plasmid was constructed containing the full-length genomic
unc-64(md130) sequence and used to transform wild-type and unc-64(null) animals. These animals are
significantly VA resistant. Next we addressed if a truncated product alone could produce the VA
phenotype. We constructed a plasmid to produce solely truncated and not wild-type unc-64 product by
introducing mutations at both the donor and acceptor splice consensus sequence of the sixth intron. This
plasmid was unable to rescue null syntaxin mutants, consistent with no wild-type product being produced;
however it successfully knocked-in VA resistance in both the wild-type and null/wild-type backgrounds.
Additional plasmids for expressing various syntaxin fragments are currently being constructed to define
the specific region(s) of syntaxin that are regulating VA action.
166

West Coast Worm Meeting 2000
REGULATION OF THE C. ELEGANS EPIDERMAL GROWTH
FACTOR HOMOLOG LIN-3
Byung Joon Hwang, Paul W. Sternberg
Division of Biology, California Institute of Technology, Pasadena, California 91125
The C. elegans lin-3 gene, a homolog of the epidermal growth factors (EGFs), is required for multiple
aspects of C. elegans development. LIN-3 is required for vulval induction, ovulation, and cell fate
specification of the P12 neuroblast of hermaphrodites. It is also required for cell fate specification of B cell
lineage in males, and for viability of males and hermaphrodites.
LIN-3 has an extracellular domain with one EGF motif, a transmembrane domain, and a cytoplasmic
domain that is longer than that of most other growth factors containing an EGF motif. Little is known about
the mechanisms that regulate LIN-3.
We are taking two approaches to understand the mechanisms regulating lin-3 expression. First, deletion
analysis of lin-3 regulatory region has been performed to identify cis-acting elements for its tissue-specific
expression. Second, a cis-acting element of lin-3 which is mutated in e1417 allele of lin-3 has been
identified by sequencing the lin-3 regulatory regions. The wild type sequence of e1417 element inserted
into an enhancer assay vector supports expression of lin-3::gfp in anchor cell, but the e1417 mutation
abolishes enhancer activity in this assay. Currently, we are looking for proteins that bind to this
AC-specific element of lin-3.
167

CHARACTERIZATION OF THE REGULATORY ELEMENTS
REQUIRED FOR NEURON-SPECIFIC EXPRESSION OF
SNAP-25 IN THE NEMATODE
Soon Baek Hwang, Junho Lee
West Coast Worm Meeting 2000
Department of Biology, Yonsei University, Seoul, Korea 120-749
SNAP-25 is a presynaptic protein exclusively expressed in the nervous system of the nematode. We
wanted to characterize the regulatory elements that are required for this tissue-specific expression. We
determined the whole sequence of the C. elegans SNAP-25 gene including the 5’ upstream region and
the relatively large first intron. In order to obtain a C. briggsae SNAP-25 homolog, we screened a genomic
library and obtained a fosmid clone named G01P23 that contained the full genomic sequence of the C.
briggsae homolog. We obtained the entire sequence of the fosmid from the genome center. As
transformation of G01P23 into the C. elegans SNAP-25 mutants complements the mutant phenotypes,
specific transcription factors of C. elegans may be able to bind the cis-acting elements in the C. briggsae
SNAP-25 gene, and the cis-acting elements may be conserved in these two species. By both examining
serially-deleted 5’ upstream regions and the first intron of SNAP-25 and comparing the sequences
conserved both in the C. elegans and C. briggsae SNAP-25 genes, we were able to identify the
regulatory elements required for neuron-specific expression of SNAP-25. We defined two sequence
motifs in the promoter region and two cis-acting regulatory motifs in the first intron. The -1096-1058 region
of 5’ upstream was required in motor neurons of the body region and the -207-190 region was required in
amphid and phasmid neurons. A 1.3Kb patch of the first intron may act in pharyngeal neurons and
another 1.6Kb patch may act in mechanosensory neurons. We expect that each motif confers binding site
for transcription activator(s) in different subsets of neuron cells, which may differ in cell lineage and
function.
168

ANALYSIS OF 2° VULVAL LINEAGE EXECUTION
Takao Inoue, Paul W. Sternberg
West Coast Worm Meeting 2000
California Institute of Technology, Division of Biology MC 156-29, Pasadena, CA 91125
During the development of the hermaphrodite vulva, P6.p adopts the 1° cell fate and P5.p and P7.p adopt
the 2° cell fate. Cells that adopt the 2° cell fate divide twice longitudinally, and then in a characteristic
LLTN (P5.p) or NTLL (P7.p) pattern. Five distinct cell types are generated from this lineage; vulA (from
outer L), vulB1 and vulB2 (inner L) vulC (T) and vulD (N). Our goal is to understand the mechanism of 2°
lineage execution; specifically how these cell types are specified through a network of genetic interations.
To facilitate the identification of different vulval cell types, we are assembling a panel of chromosomally
integrated gfp reporter constructs which express in subsets of vulval cells. These markers will be
examined under various conditions (e.g. mutant background and/or laser ablation of key cells) to
determine how they are regulated. Furthermore, to identify genes involved, screens for mutations which
alter the expression pattern will be carried out. Previously, egl-29 mutations were shown to affect the
expression of egl-17::gfp in the 2° lineage (M. Wang and P.W.S). Since this gene may be involved in 2°
lineage patterning, we are cloning it.
169

DEVELOPING A C. BRIGGSAE GENETIC MAP
B. Johnsen 1 , S. Gharib 2 , A. Mah 1 , K. Brown 2 , D. Baillie 1 , P.
Sternberg 2
1Simon Fraser University
2Caltech
West Coast Worm Meeting 2000
We are developing a genetic map of Caenorhabditis briggsae in order to facilitate comparative studies
with Caenorhabditis elegans. The two nematode species are morphologically similar but are
approximately 25 - 40 million years apart, which is about one-half the 60 - 90 million year evolutionary
distance between mice and humans. Insights gained by the studies of the two nematode species should
help in the studies of the two mammals. The 100 million base pair C. elegans genome is effectively
completely sequenced revealing approximately 19,000 genes. Over 9.4 million base pairs (about 12%) of
the C. briggsae genome has been sequenced and roughly another 4 million bases pair should be
sequenced over the next year (M. Marra pers. comm.). The two nematodes show 30-50% divergence of
unselected nucleotide sequence. Blocks of unaltered sequence are, presumably, under selective
pressure to remain unchanged. The comparisons of the genetic maps of the two species should reveal
how selective pressure constrains evolution of the linkage groups. For example, in C. elegans’
chromosomes there are regions of low and high rates of recombination. Highly conserved genes might
more likely be in regions of low recombination whereas rapidly evolving genes more likely reside in
regions of high recombination. If this were true we would expect a similar gene distribution in C. briggsae.
Although the two species are similar there are some differences. We have noted that C. briggsae matures
quicker and lays its first eggs at about age three days, about one-half day sooner than C. elegans. C.
briggsae also lives longer then C. elegans (averaging 29.7 days versus 18.6 days @ 20° for our lab’s
strains). We have over 100 C. briggsae visible mutations. So far we have identified 15 cby, 7mip, 1 rot
and 1 sml genes (the C. briggsae versions of dpy, unc, rol and sma respectively). Five of the cby genes,
three of the mips and the rot are on the X-chromosome while the other 10 cbys, 4 mips and sml map to
autosomes. We hypothesize that mip-1 is C. briggsae unc-22 and cby-4 is dpy-1. We have also shown
that mip-1, mip-3, mip-6, cby-7 and cby-8 are linked.
170

West Coast Worm Meeting 2000
COENZYME Q AND AGING IN THE NEMATODE
CAENORHABDITIS ELEGANS.
Tanya Jonassen 1 , Pamela L. Larsen 2 , Catherine F. Clarke 1
1UCLA Department of Chemistry and Biochemistry, Los Angeles, CA 90095.
2The USC Andrus Gerontology Center, Los Angeles, CA 90089.
The nematode Caenorhabditis elegans has been used as a model for the genetic studies of aging.
Mutations in the clk-1 gene of C. elegans result in slowed development and rhythmic behaviors, and an
extended life span. The C. elegans clk-1 gene is a homologue of COQ7, a gene in Saccharomyces
cerevisiae required for the biosynthesis of ubiquinone (coenzyme Q). Coenzyme Q is a redox active lipid
that functions in the electron transport chains of mitochondria and plasma membranes, and plays an
important role as an antioxidant. The nematode, rat and human homologues of clk-1/COQ7 all function to
restore coenzyme Q biosynthesis in the yeast coq7 null mutant. Given the functional conservation of
yeast rat, human and C. elegans CLK-1/Coq7 polypeptides, it is crucial to test whether changes in the
level of coenzyme Q may be responsible for the slowed development, behavior and rate of aging in the
nematode model. We have examined whether mutations in the clk-1 gene effect the level of coenzyme Q
in C. elegans. We have found conditions where the level of coenzyme Q affects developmental timing,
behavior and lifespan. The studies to be presented show that the clk-1 mutations do impact the level of
coenzyme Q in the nematode system. This system provides a model that is ideal for evaluating the
relationship between coenzyme Q and aging. These studies also indicate that C. elegans provides a
metazoan model uniquely suited to address questions regarding Q uptake, metabolism and redistribution.
171

West Coast Worm Meeting 2000
OSM-9 SIGNALING: WHO’S INVOLVED?
Amanda H. Kahn, David Tobin, Cornelia I. Bargmann
UCSF, 513 Parnassus Ave, San Francisco, CA 94143-0452
osm-9 encodes a predicted cation channel that is involved in multiple C. elegans sensory modalities. Loss
of osm-9 function eliminates chemotaxis to the AWA-sensed odorant diacetyl, compromises adaptation to
a subset of AWC-sensed odorants, and diminishes avoidance of ASH-sensed noxious stimuli. osm-9
mutants also exhibit reduced AWA expression of a Green Fluorescent Protein (GFP) transgene. OSM-9 is
similar to sensory channels in other species, including the Drosophila TRP/TRPL phototransduction
channels and a novel fly homolog. OSM-9 bears closest homology to the mammalian nociceptive
receptors VR1/VRL-1. Although signaling through the TRP/TRPL channels has been well-studied,
relatively little is understood about the mechanisms of osm-9 signaling. Thus, we are using forward and
candidate genetic approaches to elucidate osm-9 signaling pathways.
We employed the osm-9 AWA GFP phenotype in a visual screen for molecules that interact genetically
with osm-9. The screen utilized an allele of osm-9 containing a charge substitution in a conserved residue
of an ankyrin protein-protein interaction motif. In anticipation of identifying gain-of-function mutations, we
screened for dominant suppressors of this allele -- that is, F1 worms with restored expression of the AWA
GFP transgene. At present, we are mapping and characterizing three highly penetrant dominant
suppressors of osm-9. Interestingly, one mutant (ky440) is suppressed for both the AWA GFP and ASH
avoidance phenotypes of osm-9. We will present recent progress in characterizing and mapping these
mutants.
We have also taken a candidate approach to studying osm-9 signaling, using existing C. elegans
signaling mutants. The osm-9 AWA GFP phenotype can be suppressed by overexpression of the G_
protein odr-3 and by a gain-of-function allele of the calcium-calmodulin dependent kinase unc-43. Our
provisional model is that ODR-3 is an upstream activator of OSM-9, which allows Ca2+ entry to regulate
UNC-43 and gene expression. We are presently characterizing the effects of these suppressors on osm-9
mediated behaviors.
These studies will intertwine novel and well-studied transduction components in sensory signaling
pathways, with the eventual goal of understanding how C. elegans employs similar -- or different -elements
in individual sensory neurons.
172

West Coast Worm Meeting 2000
LOOKING FOR SYNERGY WITH PHA-4 ON THE MYO-2
PROMOTER
John Kalb 1 , Pete Okkema 2 , Jim McGhee 1
1Department of Biochemistry & Molecular Biology, University of Calgary, Calgary, Alberta, CANADA
2Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois
PHA-4 is a fork head transcription factor that is essential for pharyngeal development. Forced expression
of PHA-4 outside of the pharynx can activate pharynx-specific genes. For example, it has been previously
shown that PHA-4 activates myo-2, pharyngeal myosin, through the C-subelement in the myo-2 promoter.
Ectopic PHA-4 expression leads to strong ectopic expression of a C-subelement regulated reporter gene.
However, it only weakly activates endogenous myo-2; with ectopic expression of PHA-4 throughout the
embryo, ectopic expression of myo-2 is only detected in body wall muscles, showing factors in addition to
PHA-4 are necessary for myo-2 expression. Thus, we suggest that PHA-4 works with co-factors to
activate gene transcription. One likely candidate to be a co-factor with PHA-4 is PEB-1. PEB-1 is a novel
DNA binding protein expressed in the pharynx and was identified by its ability to bind to the C-subelement
in the myo-2 promoter (Thatcher, Fernandez, Beaster-Jones, Haun and Okkema). The binding sites for
PHA-4 and PEB-1 overlap in the C-subelement. To test if PHA-4 and PEB-1 act synergistically via the
C-subelement to activate gene expression, we have been assembling the transcriptional regulatory
network in yeast. We find that both PHA-4 and PEB-1 are (rather weak) transcriptional activators on their
own. However, when both PHA-4 and PEB-1 are expressed together, we find no synergism, either
positive or negative. To continue our investigation into the biochemical basis of PHA-4 action, we have
produced all three forms of PHA-4 in baculovirus to define preferred binding sites.
173

West Coast Worm Meeting 2000
INITIAL CHARACTERIZATION OF SOLUBLE GUANYLATE
CYCLASES IN C. ELEGANS
David Karow 1 , Jennifer Chang 2 , Scott Nicholls 2 , Ronald Ellis 3 ,
Martin Hudson 4 , David Morton 4 , Michael Marletta 5
1Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI 48109
2Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI 48109
3Department of Biology, University of Michigan, Ann Arbor, MI 48109
4Department of Biological Structure and Function, Oregon Health Sciences University, Portland, OR
97201
5Cellular and Molecular Biology, Howard Hughes Medical Institute, University of Michigan, Ann Arbor, MI
48109
Soluble guanylate cyclases (sGCs) catalyze the conversion of GTP to cGMP. In organisms other than C.
elegans, biochemically characterized sGCs are heterodimers composed of a1 and b1 subunits. The b1
subunit is responsible for binding heme, which is ligated to a histidine residue (His 105). Nitric oxide (NO)
binds to the heme and stimulates the enzyme 400-fold, increasing cGMP levels.
In contrast to the previously characterized heterodimers described above, analysis of the completed C.
elegans genome revealed seven putative sGC b subunits but no a subunits. Each of the putative b
subunits contains the residues necessary for catalysis, binding GTP and binding heme (His 105).
However, no open reading frame for nitric oxide synthase was found. This finding raises the possibility
that these putative b subunits might function as novel NO-insensitive isoforms.
Our preliminary data are consistent with this hypothesis. We have identified NO-insensitive sGC activity in
C. elegans lysates. For further analyses, we have begun by focusing on one subunit -- T04D3.4.
Preliminary characterization of the over-produced putative heme domain from T04D3.4 suggests that it
does bind heme. In addition, promoter::GFP fusion studies suggest that this subunit is expressed in
specific ciliated sensory neurons and possibly some mechanosensory neurons.
These cyclases are conserved in Drosophila. They are also more homologous to the relatively
uncharacterized mammalian b2 subunit than to the b1 subunit. If our preliminary evidence proves correct,
these sGCs would define the first class of heme-binding cyclases that are insensitive to NO.
174

West Coast Worm Meeting 2000
MULTIPLE REGULATORY ELEMENTS ACTIVATE END-1
EXPRESSION IN THE E LINEAGE
Jodie J. Kasmir, Morris Maduro, Joel H. Rothman
University of California-Santa Barbara, Santa Barbara, CA 93106
end-1 encodes a GATA factor that is sufficient to specify the identity of the sole endoderm precursor, the
E blastomere. By dissecting the end-1 regulatory region, we have identified elements that control the
levels and specificity of end-1 expression.
end-1 is activated by at least one maternal factor, SKN-1. Heat-shock-driven expression of SKN-1 results
in widespread end-1 expression. In addition, end-1 appears to be activated by zygotically expressed
GATA factors: ectopic expression of either END-3, the genetically redundant partner of END-1, or MED-1,
another zygotic GATA factor (see abstract by Maduro and Rothman), also result in widespread end-1
expression. We are testing whether conserved SKN-1 and GATA consensus binding sites are directly
responsive to SKN-1, END-3 and MED-1 action.
While embryos lacking SKN-1 do not express detectable MED-1, end-1 is still expressed, albeit at low
levels, and gut is still made 30% of the time. This observation suggests the existence of yet another
activator of end-1. Previously, we reported that POP-1, a Lef-1 like protein that represses end-1
expression in MS, appears to activate end-1 and gut differentiation in the E lineage in response to the
MAPK/Wnt signal that induces endoderm. However, we have been unable to identify a single region in
the end-1 promoter that is necessary for this POP-1-dependent activation of end-1. Indeed, our genetic
and biochemical data indicate that multiple sites may be necessary for activation of end-1 in the E lineage
by Wnt/MAPK-activated POP-1.
Our further analysis has uncovered several important regulatory elements that are necessary to establish
normal levels of end-1 expression. Some of these elements contain no consensus sites for known
regulators, further indicating the existence of multiple positive inputs.
175

MUTATIONS THAT PERTURB THE EFFECT OF
OCTOPAMINE/SEROTONIN ON PHARYNGEAL ACTIVITY.
John Keane, Leon Avery
University of Texas Southwestern Medical Center, Department of Molecular Biology, Dallas, TX
75390-9148, USA
We have identified some mutants which increase the rate of pharyngeal pumping in C. elegans, most
notably during the dauer larval stage. Pharynxes of these mutants (unc-9, unc-7 and goa-1) also respond
abnormally to the application of exogenous octopamine. The neuromodulators serotonin and octopamine
have previously been reported to act antagonistically (1). It is possible that these mutants are either
hypersensitive to serotonin (normally stimulates pumping) or resistant to octopamine (inhibits pumping) or
both. To address this we are examining the effect on pharyngeal activity of mutations which are reported
to reduce octopamine (osm-3, che-3) and serotonin (tph-1) levels.
Efforts to identify where unc-7, unc-9 and goa-1 mediate the observed effect have involved combining
these mutants with eat-2, a mutant that knocks out excitatory synaptic communication between motor
neuron MC and pharyngeal muscle. The double mutants reveal that MC is necessary for the observed
increase in pumping rates. As yet, we have not determined whether this means that the motor neuron is
more active in these mutants or whether the muscle has become more receptive to neuronal stimulation.
As the increase in dauer pumping rate in these mutants is small compared to wild type, it is unlikely that
octopamine has a major role in inhibiting pharyngeal activity during this developmental stage.
(1) Horvitz R. et al. Science 216: 1012-1014 (1982)
West Coast Worm Meeting 2000
176

West Coast Worm Meeting 2000
PHEROMONE REGULATION OF NEUROENDOCRINE
OUTPUTS IN C. ELEGANS
Scott Kennedy, Gabriel Hayes, Gary Ruvkun
Dept. of Molecular Biology, Massachusetts General Hospital and Dept. of Genetics, Harvard Medical
School, Boston MA 02114
A major regulator of the decision to enter the dauer stage is the environmental concentration of a
constitutively secreted dauer promoting pheromone. One mechanism by which dauer pheromone controls
dauer formation is via the regulation of the expression levels of the TGF-b like ligand DAF-7. To
determine the mechanism(s) by which pheromone regulates the expression of DAF-7 we undertook a
genetic screen to identify genes that when mutated would lead to both a loss of DAF-7 expression and a
Daf-c (dauer constitutive) phenotype. In order to monitor DAF-7 expression we have utilized a daf-7p::gfp
reporter construct, which is expressed predominantly in the ASI amphid neuron. From an initial screen of
20,000 haploid genomes we identified one mutation, mg295. Mapping data indicates that mg295 maps to
LGV, very near the daf-11 locus. In addition, mg295 fails to complement daf-11 (m47). Consequently we
conclude that mg295 is very likely to be a mutation in daf-11. Daf-11 encodes a guanylyl cyclase , an
enzyme that converts GTP to the intracellular second messenger cGMP. This suggests the possibility that
pheromone signaling in C. elegans involves the second messenger cGMP, a molecule previously
identified as essential for mammalian photoreceptor signal transduction.
To further our understanding of the mechanism(s) by which pheromone and daf-11 control daf-7
expression we have begun two additional genetic screens. The first screen seeks to identify mutants that
lead to both a loss of daf-7p::GFP expression and a Daf-c phenotype under more stringent environmental
conditions (27É). We anticipate that this screen will identify additional genes involved in the regulation of
dauer formation and daf-7 expression. Progress on this screen will be reported. We have also begun a
genetic screen to identify mutations that both suppress the daf-11 Daf-c phenotype and lead to a
reversion in daf-7p::GFP expression. From an initial screen of 250,000 haploid genomes we have
identified two mutations; tentatively named PollyAnna-1 (mg296), and PollyAnna-2 (mg297), that satisfy
these criteria. Progress of this screen and on the characterization and mapping of mg296 and mg297 will
be reported.
177

West Coast Worm Meeting 2000
CALCIUM IMAGING IN EXCITABLE CELLS OF C. ELEGANS.
Rex Kerr 1 , Varda Lev-Ram 2 , Roger Y. Tsien 2 , William R. Schafer 1
1Dept. of Biology, UCSD, La Jolla, CA 92093-0349
2Dept. of Pharmacology, UCSD, La Jolla, CA 92093-0617
It is desirable in behavioral studies to record the activity of neurons and muscles, but this has traditionally
been difficult in C. elegans. We are using a transfectable calcium indicator, cameleon [Miyawaki et al.,
Nature 388:882], to overcome this barrier.
To develop the technique, we focused on the pharyngeal muscle. The pharynx has been previously
characterized behaviorally and electrically, primarily by the Avery lab, and is large enough to simplify
imaging. In addition, calcium influx in muscles causes contraction, which provides an independent
measure of activity. Using the myo-2 promoter, we expressed cameleon in the pharynx and recorded
distinctive calcium transients coupled to muscle contraction in intact animals. The duration of these
transients varied in mutants of egl-19, a voltage-gated calcium channel, consistent with previous electrical
recordings [Lee et al., EMBO 16:1066]. In mutants of unc-36, a channel-associated a2 subunit, we found
that transients were substantially increased in magnitude, suggesting an inhibitory role for the wild-type
UNC-36 protein. This was surprising as coexpression studies of vertebrate homologues in Xenopus
oocytes had found that the a2 had many relatively subtle effects, including increasing expression levels of
the pore-forming subunit. We are currently extending our study of muscular calcium transients to
investigate the effects of neurotransmitters and mutations on the vulval muscles and their role in
egg-laying behavior.
In addition, we expressed cameleon using the pan-neuronal promoter unc-119. We provided direct
electrical stimulation through an extracellular electrode inserted through the cuticle and positioned near
the nerve ring. Transients were observed coupled to the stimulation, indicating that calcium influx through
voltage-gated calcium channels can be observed in neurons using this method. This raises the possibility
of constructing a functional map of neuronal connectivity by electrically stimulating single neurons and
recording the activity of their synaptic partners using cameleon.
178

West Coast Worm Meeting 2000
A GENETIC ANALYSIS OF THE EFFECTS OF ETHANOL ON
EGG LAYING
Hongkyun Kim, M. Christina Yu, James Kim, Steven L. McIntire
Gallo Center and Program in Biological Sciences, Department of Neurology, UCSF, CA 94608
Ethanol causes dose-dependent suppression of egg laying in C. elegans. To elucidate the neuronal
targets of ethanol, we screened for mutants showing ethanol-resistant or -inducible egg-laying behavior in
a plate assay. F2 progeny of mutagenized animals were exposed to a dose of ethanol that strongly
suppresses egg laying of wild type animals. Eggs were collected and allowed to hatch in the absence of
ethanol. Once the F3 animals had matured to adulthood, the process was repeated for 2 cycles. By using
this enrichment strategy, we isolated four different classes of mutants. Class I mutants are hyperactive
and exhibit hyperforaging behavior and are aldicarb-hypersensitive. At least 4 mutants of class I belong to
the same complementation group and are alleles of slo-1. Class II mutants show high amplitude
sinusoidal tracks and slightly exaggerated head movement in the direction of the forward movement. This
class exhibits variable aldicarb responses and normal foraging behavior. This class includes at least three
complementation groups. The finding of the same behavioral defects in the different mutants of this class
suggests that the same, ethanol-sensitive pathway may be affected by each of the mutations. Class III
mutants have egg-laying defects in the absence of ethanol. Class IV mutants do not have any obvious
phenotype. Egg laying of class I and II mutants is induced at relatively low ethanol concentrations that
moderately suppress egg laying of N2. More eggs are laid in the presence than in the absence of ethanol
in these mutants. Further mapping and characterization of these mutants may lead to the identification of
the molecular targets of ethanol in C. elegans.
179

West Coast Worm Meeting 2000
GENES AFFECTING THE ACTIVITY OF NICOTINIC
RECEPTORS INVOLVED IN EGG-LAYING BEHAVIOR
Jinah Kim 1 , Daniel S. Poole 2 , Laura E. Waggoner 2 , Alexandra
Treschow 2 , William R. Schafer 2
1Group in Neuroscience, University of California at San Diego, La Jolla, CA 92093-0349
2Dept. of Biology, University of California at San Diego, La Jolla, CA 92093-0349
Egg-laying behavior in C. elegans is regulated by multiple neurotransmitters, including acetylcholine and
serotonin. Agonists of nicotinic acetylcholine receptors such as nicotine and levamisole stimulate
egg-laying; however, the genetic and molecular basis for cholinergic neurotransmission in the egg-laying
circuitry is not well understood. We have examined the egg-laying phenotypes of eight known levamisole
resistance genes which may mediate or regulate nicotinic receptor activity in the egg-laying
neuromusculature. Five "strong" levamisole resistance genes, including unc-63, unc-74, and the nicotinic
receptor subunit genes unc-29, unc-38, and lev-1, were essential for the stimulation of egg-laying by
levamisole. In the absence of drug these mutants retained the characteristic biphasic pattern of
egg-laying and caused only subtle shifts in the timing of egg-laying behavior. Worms mutant for two or
three of these nicotinic receptor genes also had a generally wild-type pattern of egg-laying. These genes
appear to encode components of a levamisole-sensitive nicotinic receptor that promotes egg-laying but is
not necessary for egg-laying muscle contraction. unc-29 and unc-38 mutants were also hypersensitive to
the stimulation of egg-laying by serotonin, suggesting that nicotinic receptors might negatively regulate
serotonin response pathways in the egg-laying muscles or neurons. Three "weak" levamisole resistance
genes, lev-8, lev-9, and lev-10, had effects on egg-laying that were distinct from those of the levamisole
receptor genes. Phenotypic analysis indicated that lev-8, lev-9, and lev-10 may encode regulatory
molecules that control the functional activity of nicotinic receptors, since their loss of function attenuates
nicotinic receptor function without seeming to eliminate the receptors themselves.
180

West Coast Worm Meeting 2000
SENSORY AXON GUIDANCE DEFECTS IN C. ELEGANS
Susan Kirch, Gage Crump, Cori Bargmann
HHMI and Department of Anatomy; UCSF; San Francisco, CA 94131
Worms depend on taste, touch and smell to sense and explore their environment. Appropriate responses
to information received through these different modalities depends on precise connectivity between
sensory neurons and downstream effector neurons in the nerve ring. Little is known about how axons
migrate through this nerve ring neuropil during development to find their partners.
How is positional information interpreted by an extending axon so that desired synaptic partners are
found? We focused on the guidance of the ASI chemosensory neurons to their appropriate positions in
the nerve ring. A mutant screen led to the isolation of 16 candidate mutant strains that appeared to have
ASI axon guidance and termination phenotypes. These mutations define at least five new genes which
we have named sax-10-14 (sensory axon guidance). We have isolated five alleles of sax-10. The
canonical allele, ky297, has axon defects in several axons in the nerve ring including the chemosensory
neurons AWB, ASI and ASH and the interneuron PVQ, as well as an altered expression pattern of the
AWC- specific gene str-2. ky297 is normal for the VD, DD and HSN motoneurons, the chemosensory
neuron ADL and the phasmids. sax-12 suffers from defects in ASI and ADL, but is normal for AWC
structure. sax-13 displays abnormal axon structures when we examine ASI, ADL, AWB, ASH and PVQ,
but appears wild-type for AWC. Mutants display either one or a combination of phenotypes (including:
premature termination, branching, thickening, wandering and inappropriate pathfinding) with a penetrance
that ranges from 13% to 100%. Dye filling of sensory neurons with the fluorescent dye DiI, and
examination of neurons with other cell-specific GFP constructs in these mutants reveals that the overall
morphology of the nerve ring is normal in many sax-10, sax-12, and sax-13 animals, and that the
mutations disrupting ASI are probably in genes specifically required by ASI or a subset of neurons.
sax-14, however, has more severe dye filling phenotypes that are being characterized further. To facilitate
genetic analysis and cloning of these new sax genes, we are mapping the genes and currently trying to
rescue the sax-10 phenotype by cosmid injections and germline transformation.
181

ISOLATION OF A THIRD LIN-4 ALLELE FROM A LIN-3A
OVEREXPRESSION LINE
Martha Kirouac, Paul Sternberg
West Coast Worm Meeting 2000
HHMI and Division of Biology, 156-29, Caltech, Pasadena, CA 91125
In order to investigate the role of LIN-3 in cell fate specification, we conducted a F2 screen using a
reduction of function lin-3(n378) strain which contains an integrated transgene that carries a mutation that
presumably eliminates lin-3B splicing and should, therefore, only produce lin-3A. The line carrying the
integrated transgene is fertile and 100% multivulva. Animals that lack lin-3A are sterile (Jing Liu & PWS,
unpubl.). To potentiate rescuing any lines that are sterile because they lack lin-3A, lfe-1 was incorporated
into the strain background. lfe-1(sy290) is a gain of function allele that partially rescues the sterility
caused by let-23 loss of function animals. The strain is fertile and 100% multivulva. From these screens,
we looked for animals which were non-Muv and isolated two mutations.
One of these mutations still displayed some induction, but displayed neither invagination nor
morphogenesis. Since these animals were 100% vulvaless, we artifically created a hole connecting the
outside to the uterus using a typical injection scope and needle. After obtaining cross progeny and
outcrossing this mutant, it displayed characteristic heterochronic defects; extra molts, late divisions, and
no adult cuticle. Having preliminarily mapped this mutation to linkage group II, we tested the hypothesis
that it was a lin-4 allele by performing single worm PCR on the homozygous animals using primers to
lin-4. Indeed, there was a mutation in the lin-4 gene, C516T. Though there are only two other alleles of
lin-4 ,this is the second time that this mutation has been isolated (the other allele was isolated in Victor
Ambros lab and is known as ma161). Lin-4 is a heterochronic retarded mutant which is critical to the
controlled coordination of cell division in postembryonic divisions. What is most interesting about lin-4 is
that it encodes a 22 nt. RNA form to block translation of other proteins such as lin-14 and lin-28. The fact
that this allele has been isolated twice is indicative of the importance of this site in the function of the lin-4
RNA. It has been hypothesized (Ha, I. and colleagues) that this C forms a bulge which is recognized by
another, as of yet unknown, factor. When this C is replaced with a T, this bulge does not form, and does
not inhibit the translation of lin-14 and lin-28.
182

ELT-5 AND ELT-6 ARE ESSENTIAL FOR DEVELOPMENT OF
SEAM CELLS, THE VULVA, AND THE MALE TAIL.
Kyunghee Koh, Joel H. Rothman
West Coast Worm Meeting 2000
Neuroscience Research Institute, University of California, Santa Barbara, CA 93106
Patterning of the embryonic epidermis into the major cell types (syncytial, P, and seam cells), and
regulation of their post-embryonic development, involve a complex series of regulatory events. We
previously described the role of elt-5 and -6, adjacent genes encoding GATA transcription factors, in
embryonic seam cell development (WCWM, 1998). We since found that they perform a variety of
essential functions in embryonic and post-embryonic ectodermal development.
elt-5 and -6 show complex and dynamic expression patterns. GFP reporters express 1) in many anterior
and ventral cells during embryogenesis, 2) in seam cells, 3) in a subset of sheath and socket cells, 4) at
low levels in the P cells and higher levels in the descendants of the vulval precursor cells, and 5) in the
developing male tail. Expression patterns of elt-5:gfp and elt-6:gfp overlap, but are not identical. ELT-5
and -6 antibodies corroborate some of these observations.
High levels of elt-5 dsRNA result in penetrant late embryonic or L1 lethality, in which the affected animals
are lumpy, dumpy, uncoordinated, and show fusion of seam cells with epidermal syncytia. Anterior seam
cells fuse more frequently than those in the posterior, suggesting that an A/P patterning system in the
seam or surrounding syncytia regulates their propensity for fusion. Many, but not all seam-specific
markers are eliminated in the affected animals, and elt-3, normally expressed in non-seam epidermis, is
occasionally expressed in unfused seam cells.
At lower doses of dsRNA, surviving vulvaless adults are observed. Examination of the vulval precursor
cells shows that some inappropriately fuse with hyp7. In addition, migration of the gonad is aberrant, and
rays in the male tail are often missing.
Ectopic expression of elt-5 or -6 during mid-embryogenesis results in an excess of seam cells, while
ectopic expression in L1s and L2s often results in adult males with missing rays and hermaphrodites that
herniate through a malformed vulva.
Collectively, these findings suggest that ELT-5 and -6 may collaborate with A/P patterning systems and
inductive processes to regulate development of the embryonic ectoderm and post-embryonic genitalia.
183

A GENETIC SCREEN FOR GENES INVOLVED IN GUT
DEVELOPMENT AND DIFFERENTIATION IN
CAENORHABDITIS ELEGANS
Jay D. Kormish, James D. McGhee
West Coast Worm Meeting 2000
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1
Canada
Our lab is currently interested in studying genes, in particular transcription factors, that direct the
development and differentiation of these cells into a functioning organ. I have developed a genetic screen
to isolate zygotically expressed genes that when mutated result in a gut obstructed "gob" defect. When
mutagenized worms are fed a mixture of bacteria and polystyrene fluorescent microspheres, mutant
worms with an obstructed gut can be identified under the dissecting microscope because of the highly
fluorescent beads that collect in the terminal bulb of the pharynx and in the anterior intestine. The
indication that screening for such a phenotype could isolate genes specifically involved in gut
development came from our observation that mutations in a gut specific GATA transcription factor gene
elt-2 resulted in the gob phenotype. I am currently in the middle of a saturation screen; five thousand
genomes have already been screened and the remaining five thousand are expected to be completed in
the near future. During the characterization of my initial round of candidates, I have found three strains
that appear to give gut specific defects. The strain with the highest degree of penetrance, 26-3(8), has
been chosen for mapping and cloning of the gene. 26-3(8) has been mapped to the X chromosome and is
being tested for complementation with elt-2. Electron microscopy of lumen cross sections and gut
morphology markers are being used to describe the lumen morphology of the mutants.
184

West Coast Worm Meeting 2000
AN E1-LIKE ACTIVATING ENZYME IS INVOLVED IN CELL
DIVISION PROCESSES IN THE EARLY C. ELEGANS
EMBRYO.
Thimo K. Kurz, Danielle R. Hamill, Bruce Bowerman
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403
In a screen for temperature-sensitive embryonic lethal mutations, we isolated a mutant called or198ts that
has several defects in early C. elegans embryos. In or198ts mutant embryos, the first mitotic spindle often
is delayed in aligning along the anterior-posterior axis, and shortly after the spindle begins to elongate, it
is no longer clearly visible by DIC videomicroscopy. In about half of or198ts mutant embryos, the first
attempt at cytokinesis fails. In embryos in which cytokinesis succeeds, we observe penetrant defects in
nuclear positioning and in mitotic spindle orientation at the two cell stage. In addition, in interphase partial
ectopic cleavage furrows may form; there is extra pinching and blebbing at the cell membranes, and the
cells are misshapen.
or198ts is on LGIII at approximately --0.8 m.u., and corresponds to the Genefinder locus F11H8.1. We
have identified a single base lesion in the mutant that converts a valine to alanine at amino acid position
269. F11H8.1 encodes an E1-like activating enzyme with around 30% amino acid identity to its yeast and
human homologs. E1-like activating enzymes are involved in conjugating ubiquitin-related proteins (e.g.
SUMO-1/Smt3p) to a small subset of other cellular proteins in a manner similar, although not identical to
ubiquitination. In contrast to ubiquitin, which targets proteins for degradation, post-translational
modification by ubiquitin-like proteins may regulate the activity and intracellular localization of the proteins
they modify. In both yeast and mammals, Smt3p/SUMO modifies multiple proteins. Perhaps the
pleiotropic defects observed in or198ts embryos reflect multiple targets of this post-translational
modification. To our knowledge, this is the first example of a ubiquitin-like modification being important for
cell division processes in multicellular eukaryotes.
185

OLFACTORY ADAPTATION
Noelle L’Etoile, Cori Bargmann
West Coast Worm Meeting 2000
HHMI/Dept. Anatomy, UCSF, San Francisco, CA 94143
To extract information from a complex environment, an organism continuously adjusts its sensitivity to
different stimuli. One strategy for integrating olfactory information over time is to down-regulate odorant
perception at the level of the sensory neuron. Another strategy requires a circuit in which interneurons
provide feedback inhibition. I have been focusing my efforts on examining adaptation to odorants sensed
by the olfactory neuron AWC, a process that may take place within the sensory neuron itself.
The pair of AWC neurons allows the animal to respond to at least five different odorants. The signal
transduction pathway within the AWCs includes putative seven transmembrane G-protein coupled
odorant receptors, at least two heterotrimeric G-proteins, at least two guanylyl cyclases and a
cGMP-gated cation channel that may open in response to cyclase stimulation. Intriguingly, adaptation to
each odorant is independent of the others sensed by this pair. I have identified a mutant, pkg-1, that fails
to adapt to all AWC-sensed odorants tested. I mapped the mutation and was able to obtain rescue of the
phenotype with an extrachromosomal array of a cosmid containing one of the two C. elegans genes
encoding cGMP-dependent protein kinases (PKGs) present in the genome.
I am attempting to identify the molecular lesion by sequencing the locus that encodes the PKG in our
existing allele. I am also generating additional alleles using a non-complementation screen.
What is the target of PKG-1 phosphorylation in olfactory adaptation? I am taking a candidate approach to
this question by mutating potential PKG phosphorylated residues within members of the signal
transduction pathway. Animals that express only the mutated form of the protein should be adaptation
defective if that protein’s function is regulated by phosphorylation. I hope to examine the phosphorylation
state of these candidates in extracts made from both N2 and pkg-1 worms. An important conundrum
remains: how does the worm utilize a shared adaptation component to specifically down regulate its
response to one of five odorants sensed by the same pair of neurons? This will be addressed by
identifying PKG-1’s targets, binding partners and examining their localization within the pair of AWC
neurons during adaptation.
186

YOU CAN’T GET THERE FROM HERE: A GENE REQUIRED
FOR PHARYNGEAL EXTENSION.
SK Lange, JR Saam, SE Mango
West Coast Worm Meeting 2000
Huntsman Cancer Institute Center for Children and Department of Oncological Sciences, University of
Utah, Salt Lake City, UT 84112
During pharyngeal extension, a ball of pharyngeal precursors is converted into a linear tube connected to
the buccal cavity anteriorly and the midgut posteriorly. Time-lapse videomicroscopy has shown that this
process depends on the reorientation of the pharyngeal epithelial cells and their adhesion to neighboring
arcade cells in the buccal cavity (see abstract by Portereiko and Mango). Intriguingly, mutants that disrupt
cell-cell or cell-substratum adhesion do not affect pharyngeal extension.
In a survey of homozygous deficiencies, we discovered one locus, mnDf90, with defective pharyngeal
extension. Nevertheless, homozygous mutant embryos form a pharyngeal primordium, differentiate
normally and produce the correct number of PHA-4+ cells. This phenotype suggests that cell fate
specification is unaffected and that the defect is specific for morphogenesis. We have identified three
EMS-generated alleles that phenocopy the pharyngeal extension phenotype and map near mnDf90. The
map position and phenotype resemble cdl-1 (Cell Death Lethal), discovered by the Yamamoto lab, and so
we have provisionally called our locus cdl-1.
Homozygous cdl-1 embryos arrest at the 3-fold or L1 stage with an unattached pharynx. This phenotype
suggests that the locus is not required for at least two other morphogenetic processes: ventral closure
and embryonic elongation. Surprisingly, two of our alleles also accumulate cell corpses. This phenotype is
likely to reflect a defect in cell corpse engulfment, a process thought to depend on cytoskeletal
reorganization to extend filopodia around the corpse. Since mnDf90 homozygotes do not show the cell
corpse defect, one possibility is that cdl-1 is not required for engulfment of corpses, but that our cdl-1
alleles produce altered proteins that interfere with normal engulfment. We suggest that cdl-1 is involved in
regulation or function of the cytoskeleton.
Our current goal is to determine what aspect of pharyngeal extension is disrupted by cdl-1 and whether
other morphogenetic processes (e.g. migration) are also compromised. In addition, we are using standard
approaches to clone cdl-1 and initiate a molecular analysis of pharyngeal extension.
187

West Coast Worm Meeting 2000
SIGNALING BY THE VAB-1 EPH RECEPTOR
INTRACELLULAR DOMAIN
Kristoffer Larsen,, Sean George, Andrew Chisholm
Department of Biology, University of California, Santa Cruz, CA 95064
The VAB-1 Eph receptor tyrosine kinase functions in embryonic morphogenesis and axon guidance. The
VAB-1 intracellular domain (ICD), like those of other Eph receptors, includes a conserved juxtamembrane
motif containing phosphotyrosines, a tyrosine kinase domain, and a C-terminal region with weak similarity
to SAM domains. Genetic analysis suggests that VAB-1 has both kinase-dependent and
kinase-independent functions. We are using two-hybrid and genetic screens to identify components of the
VAB-1 kinase-dependent pathway.
The full-length VAB-1 ICD could not be used as bait in two-hybrid screens because it caused reporter
gene activation in the absence of prey. After extensive subcloning we identified fragments of the VAB-1
ICD that did not cause self-activation. One of these contains the complete kinase domain and C-terminal
tail, and has been the basis for a pilot screen from which we identified several positive clones. We will
present our analysis of these potential interactors.
Several proteins have been previously identified as binding to activated Eph receptor ICDs. These include
SH2-domain containing adaptor proteins (Nck, etc), and other proteins whose mode of interaction is less
clear. We have begun testing C. elegans homologs to ask if these interactions have been conserved.
Finally, we are using genetics to ask whether the VAB-1 kinase-dependent pathway shares components
with other RTK signaling pathways characterized in C. elegans. We have found that activating mutations
in LET-60 Ras do not significantly suppress vab-1 loss-of-function phenotypes, suggesting that VAB-1
does not signal via Ras activation. We will present results of additional double mutant experiments.
188

West Coast Worm Meeting 2000
MDF-1 SUPPRESSORS THAT MAY PLAY A ROLE IN THE
METAPHASE TO ANAPHASE CHECKPOINT
Elaine Law, Risa Kitagawa, Ann M. Rose
Department of Medical Genetics, University of British Columbia, Vancouver V6T 1Z3
The spindle assembly checkpoint is an evolutionarily conserved mechanism that ensures accurate
chromosome segregation. This mechanism inhibits cell-cycle progression in response to a signal
generated by mitotic spindle damage or by kinetochores that have not attached to microtubules prior to
sister-chromatid separation. Such arrest allows cells to complete essential events for the maintenance of
genetic fidelity. The spindle checkpoint mechanism has been studied in yeast and mammalian tissue
culture systems, but little is known about it in multi-cellular organisms. We have identified mdf-1 and
mdf-2 (mitotic arrest defective) as homologues of yeast spindle checkpoint genes MAD1 and MAD2 in
Caenorhabditis elegans. Both genes are essential for the long-term survival and fertility of C. elegans.
Loss of function of either gene leads to the accumulation of a variety of defects, which ultimately results in
genetic lethality. To better understand how the checkpoint functions in C. elegans, and to identify
additional components of the checkpoint, we screened for suppressors capable of rescuing the mdf-1
(gk2) loss of function phenotype. Sixteen suppressors were recovered, which increased the number of
fertile progeny and the survival of the genetic strain. The suppressors are being genetically mapped. One
dominant suppressor was recovered, h1983. We have positioned this mutation and are currently
examining the molecular basis for the suppression. One hypothesis, which we are now testing, is that the
suppressor gene product is a downstream target of MDF-1 and acts compensatory to the spindle
checkpoint defect to reduce chromosome missegregation.
189

CHARACTERIZATION OF A C. ELEGANS DEFECATION
MUTANT
Anne Lehtela, Garry Wong
West Coast Worm Meeting 2000
A.I. Virtanen Institute, Kuopio University, PL 1627, Kuopio 70211, FINLAND
Defecation in C. elegans is a tightly regulated and complex behavior. The defecation behavior is
generated from neuronal signals and is transmitted into movement of multiple body muscles in a highly
coordinated fashion. Defecation occurs in 3 steps: contraction of posterior body muscles (pBoc);
contraction of anterior body muscles (aBoc); expulsion of intestinal contents (exp). The defecation cycle
occurs at regular 50-60 second intervals when wildtype Bristol N2 worms are feeding.
A C. elegans defecation mutant was identified in a mutagenesis screen. Six-thousand genomes were
mutagenized with 50 mM ethyl methanesulfonate for 4 h. F2’s were selected for resistance to 0.7 mM
aldicarb, an acetylcholine esterase inhibitor. A single mutant, which appeared to be constipated, was
cloned and then outcrossed to N2.
The defecation mutant, in comparison to N2 per 30 min, had fewer pBOC (15.3 ± 2 vs. 32.8 ± 1), fewer
exp (6.5 ± 1 vs. 31.8 ± 0), and fewer successful complete defecation motor program events (32 ± 9% vs.
97 ± 2%), respectively. When pBOC was followed by an exp event, the latency appeared longer in the
mutant (8-10 sec) compared to N2 (2-3 sec). On the few occasions when the complete defecation motor
program was intact in mutants, the interval was longer (80-90 sec) than N2 (55-60 sec). Genetic analysis
has tentatively identified the location of the mutation on chromosome III.
These results, describing a derangement of the defecation motor program, combined with identification of
the gene mutation responsible for this phenotype, should provide insights into the control of complex
neuronal and neuromuscular controlled behaviors.
190

West Coast Worm Meeting 2000
ORGANOGENESIS OF THE C. ELEGANS INTESTINE
Benjamin Leung 1 , Greg J. Hermann 2 , James R. Priess 3
1MCB Program, University of Washington and Fred Hutchinson Cancer Research Center, Seattle, WA
98109.
2Fred Hutchinson Cancer Research Center, Seattle, WA 98109
3MCB Program, University of Washington and Fred Hutchinson Cancer Research Center, HHMI, Seattle,
WA 98109.
The C. elegans intestine is a bilaterally symmetric tube of 20 polarized epithelial cells derived from a
single early blastomere called E. Cell polarity in the intestine begins with reorganization of the microtubule
cytoskeleton, followed by migration of the intestinal nuclei apically, and other organelles basally. Small
apical membrane separations coalesce into a continuous compartment to form the lumen of the intestine.
The early primordium consists of two tiers of polarized cells; specific cell intercalations rearrange the
primordium into a single elongated tier.
An E blastomere cultured in isolation produces a cyst of polarized cells, but the aggregate lacks the
bilateral symmetry seen in the normal intestine. Thus the ability to polarize appears to be an intrinsic
property of intestinal cells, but bilateral symmetry requires interactions with cells that normally surround
the intestine. Cell killing experiments suggest these interactions must occur at an early stage in intestinal
morphogenesis. We are currently using the technique of RNAi to determine whether genes implicated in
epithelial polarity in other systems are required for proper development of the C. elegans intestine.
191

EXPRESSION AND REGULATION OF DAF-16::GFP
CONSTRUCTS
Kui Lin, Cynthia Kenyon
West Coast Worm Meeting 2000
Department of Biochemistry & Biophysics, University of California at San Francisco, San Francisco, CA
94143-0448
Wild-type C.elegans ages rapidly, undergoing development, senescence, and death in less than 3 weeks.
In contrast, mutants with reduced activity of daf-2, a homolog of the insulin and insulin-like growth factor
(IGF-1) receptors (Kimura et al., 1997), age more slowly and live more than twice as long (Kenyon et al.,
1993). Wild-type DAF-2 activity also promotes growth to adulthood and prevents dauer formation, since
severe loss of daf-2 function causes the animals to become dauers in the presence of food. Both the
lifespan extension and dauer-constitutive phenotypes caused by daf-2 mutations are dependent on the
activity of daf-16, which encodes an HNF-3/forkhead family member (Ogg et al., 1997; Lin et al., 1997).
We have created GFP-tagged daf-16 constructs and used them to study the expression and regulation of
daf-16. We will report our progress on this study.
192

IDENTIFICATION OF NOVEL UNC-64 (SYNTAXIN) ALLELES
Christine Liu, C. Michael Crowder
West Coast Worm Meeting 2000
Department of Anesthesiology, Washington University in St. Louis, St. Louis, MO 63110-1093
unc-64 encodes a homolog of vertebrate syntaxin 1A. Syntaxin is expressed ubiquitously in the nervous
system of the nematode. unc-64 contains a high degree of homology with human and Drosophila
syntaxin, suggesting that this molecule is conserved across species and performs similar functions.
Syntaxin is involved in membrane fusion of synaptic vesicles and controls neurotransmitter release, in
part through its H3 helical domain, which interacts with SNAP-25 and synaptobrevin. Very few viable
syntaxin alleles have been identified in any animal, including C. elegans. In C. elegans, the null allele,
js115, confers larval lethality: the nematode completes embryogenesis, but dies as L1 larva. Currently,
only four viable syntaxin alleles have been identified in C. elegans: e246, md1259, js21, md130. js21 and
e246 contain different C to T missense mutations in exon 7; md130 and md1259 contain G to A splice
site mutations at the splice donors of intron 6 and intron 3, respectively. All these previously identified
mutations affect the H3 domain of syntaxin or cause reduced expression. While all viable syntaxin alleles
appear to reduce neurotransmitter release, large allelic differences are seen in their sensitivities to volatile
general anesthetics: md130 is resistant to volatile anesthetics while js21 and md1259 are hypersensitive.
In addition to the H3 domain, syntaxin also contains N-terminal H ABC domains and a hinge region that are
also thought to regulate membrane fusion events. However, no mutations have been identified in these
regions. To better define these portions of syntaxin that regulate transmitter release and anesthetic
sensitivity, a non-complementation screen using EMS as the mutagen was conducted using the unc-64
(e246) allele. Three potentially new unc-64 alleles have been isolated out of approximately 4800 F1
genomes screened. These animals exhibit phenotypes consistent with syntaxin hypomorphic alleles,
including being uncoordinated and failure on re-testing to complement e246 allele. Other experiments to
characterize these putative new viable syntaxin alleles are currently in progress.
193

West Coast Worm Meeting 2000
MUTATIONS THAT CAUSE NEURITE SPROUTING OF THE
DVB MOTOR NEURON
Loria, P., Boulin, T., Conte, S., Hobert, O.
Columbia University, College of Physicians & Surgeons, Center for Neurobiology and Behavior, New
York, NY 10032
Neuroanatomical studies in vertebrates long ago revealed that certain forms of insult such as axotomy or
activity blockade result in the outgrowth of additional neurites. These observations suggest that
postmitotic neurons contain an intrinsic capacity to assess their integrity and to modulate their anatomy
accordingly. In C.elegans, several genetic lesions have been described that lead to the outgrowth of
additional neurites ("neurite sprouting")(1,2). We have found that a null mutation in the lim-6 LIM
homeobox gene causes neurite sprouting in the DVB motor neuron (3). Using this observation as a
starting point, we have set out to understand the molecular components of neurite sprouting and to further
dissect the role of lim-6 in this process. First, we used a candidate gene approach to test mutations in
proteins involved in different aspects of neuron and muscle function. Second, we performed an unbiased
genetic screen for mutants that phenocopy the lim-6 sprouting defect in DVB.
Since activity blockade at the neuromuscular junction in vertebrates and flies causes neurite sprouting,
we tested whether loss of the neurotransmitter GABA causes sprouting of the GABAergic DVB
motorneuron. We indeed find that mutations in unc-25 cause neurite sprouting of DVB. These defects can
be enhanced by mutations in unc-31, which presumably abolishes peptinergic neurotransmission.
Furthermore, blocking the activity of the enteric muscle targets of DVB with an egl-2(gf) mutation, which
activates a K-channel (4), leads to neurite sprouting of DVB, as does the complete removal of the enteric
muscles in hlh-8/twist mutants. We also find that a gain-of-function mutation in CamKII/unc-43, a gene
whose role in neurite outgrowth has been described extensively in vertebrates, causes neurite sprouting
in DVB.
Using a clonal, fluorescence microscope-based screen of 3300 haploid genomes, we identified nine
mutants, ot1 through ot9, that affect neurite sprouting of DVB. These mutants fall into at least 4
complementation groups and are not allelic to any of the genes described above. The mutants do not
cause sprouting in other defined sensory or interneurons tested. We also find that the as yet uncloned
unc-122 gene causes specific neurite sprouting of DVB. We are in the process of mapping and cloning
these genes.
194

West Coast Worm Meeting 2000
A B -TUBULIN GENE, TBB-2, FUNCTIONS AS AN ACTIVATOR
OF MEI-1 AND MEI-2 IN FEMALE MEIOTIC SPINDLE
FORMATION IN CAENORHABDITIS ELEGANS.
Chenggang Lu, Martin Srayko, Paul E. Mains
Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N
2T9
C. elegans female meiosis requires two meiotic-specific genes, mei-1 and mei-2. Loss of mei-1 or mei-2
function blocks female meiotic spindle formation while the subsequent mitotic cleavages are normal. In a
mei-1 gain-of-function mutant, however, the mitotic cleavages are disrupted after normal meiotic divisions.
Wild-type MEI-1 and MEI-2 localize to the meiotic, but not mitotic spindle. 1,2 However, MEI-1(gf) and
MEI-2 ectopically localize to mitotic spindles in mei-1(gf) embryos. mei-1 and mei-2 encode homologs of
p60 and p80 subunits of the sea urchin microtubule severing protein katanin, respectively, and MEI-1 and
MEI-2 together disassemble interphase microtubules in a HeLa cell system. 2 We propose that MEI-1 and
MEI-2 form a complex in meiosis and regulate meiotic spindle formation by katanin-like activities.
In a mei-1(gf) suppressor screen, we recovered three extragenic suppressors. One of them, sb26, was
found to be a missense mutation in a b -tubulin gene, tbb-2. tbb-2(sb26) also enhances a weak mei-2(lf)
allele. Together, these results suggest that tbb-2 is required for the function of mei-1 and mei-2. Antibody
staining shows that TBB-2 is widely expressed during worm development. Neither tbb-2 nor tbb-1
(another b -tubulin highly similar to tbb-2) RNAi has severe effects during early development. However,
tbb-2 and tbb-1 double RNAi results in 100% dead eggs indicating that they act redundantly during
embryogenesis. We are doing experiments to examine the interactions of MEI-1/MEI-2 with tbb-2 and
tbb-1.
1. Clark-Maguire, S. and Mains, P.E. (1994). J. Cell Biol. 126, 199-209.
2. Srayko, M., Buster, D.W., Bazirgan O.A., McNally, F.J., and Mains, P.E. (2000). Genes Dev. 14 (9).
195

West Coast Worm Meeting 2000
GLOBAL PROFILE OF GENE EXPRESSION DURING AGING
James Lund 1 , Pamela Larsen 2 , Pat Tedesco 3 , Thomas Johnson 3 ,
Stuart Kim 1
1Stanford University
2University of California Los Angeles
3Institute for Behavioral Genetics, University of Colorado, Boulder
Aging is a physiological phenomenon characteristic of metazoan life. As animals age, they suffer a broad
functional decline and an exponentially increasing probability of death. Many aspects of the aging
phenotype are present in animals from C. elegans to mammals, including a build-up of lipofuscin
deposits, mitochondrial DNA deletions, and degradation of organ function. The life span of an animal is
thought to be set by the balance between deleterious events and repair mechanisms. A number of C.
elegans mutants live significantly longer than wildtype, making C. elegans an attractive model for studies
of aging.
DNA microarrays can be used to profile the expression levels of a large number of genes in parallel.
Using our array, which contains a PCR product representing every C. elegans gene, we are profiling
expression patterns during the normal aging process. We isolated RNA from synchronized cultures of
sterile animals at various ages from young adult to old age. We hybridized this RNA to DNA microarrays,
and are now analyzing the data to identify groups of genes with expression levels which change as
reproduction ceases and the worms age. These expression profiles with be compared with the partial
profiles of aging mice and humans that have been published. The expression profile of normal aging will
be used as a reference to understand the expression changes underlying the extended life spans in
longevity mutants.
196

CONDITIONAL MUTATIONS AFFECTING MITOTIC SPINDLE
POSITIONING AND POLARITY IN THE C. ELEGANS EMBRYO
Rebecca Lyczak, Bruce Bowerman
West Coast Worm Meeting 2000
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403
Early polarity in the C. elegans embryo requires proper regulation of the cytoskeleton.
Cytoskeletal-dependent processes such as pronuclear migration, centrosome rotation, and spindle
positioning must be properly accomplished to ensure an asymmetric first cleavage. To investigate the role
of the cytoskeleton in regulating asymmetric cell division, we have undertaken a screen for temperature
sensitive embryonic lethal mutations affecting spindle positioning in the early embryo. We have identified
mutations in two classes that affect P0 spindle positioning: 1) misorientation of the spindle axis 2)
mispositioning of the spindle along the A-P axis.
We have identified several mutants that show a defect in pronuclear migration and P0 spindle orientation.
These include alleles of mel-26 and zyg-9 as well as at least three novel loci. In or346ts embryos, the
oocyte pronucleus does not always migrate to the posterior of the embryo before initiation of the first
mitotic spindle. The centrosome/nuclear complex remains in the posterior of the embryo and does not
rotate. Thus, the first mitotic spindle sets up transverse to the A-P axis. The misoriented spindle results in
a missegregation of P-granules and daughter cells of aberrant size and developmental potentials.
Terminally differentiated or346ts embryos also show striking patterning defects, often making extra
intestinal cells.
We have also identified at least two mutants in which the P0 spindle is mispositioned along the A-P axis.
In or282ts mutants, both pronuclei migrate toward the center of the embryo where they meet before
setting up a spindle along the A-P axis. This spindle varies in its position along the A-P axis resulting in a
randomization of cell size in the daughters. This mispositioning of the spindle is coupled with
mislocalization of P-granules and a loss of polarity in the daughter cells. Terminally differentiated or282ts
embryos show patterning defects, often making extra pharyngeal cells and lacking intestine. In or358ts
embryos, the P0 spindle is displaced too far posterior resulting in an excessively asymmetric first division.
Analysis of spindle positioning mutants should provide valuable insights into the link between
establishment of polarity and the cytoskeleton.
197

ROLE OF PDZ DOMAIN PROTEINS IN ESTABLISHING GUT
EPITHELIAL POLARITY
Kathleen E. Mach, Stuart K. Kim
West Coast Worm Meeting 2000
Department of Developmental Biology, Stanford University, Stanford, CA 94305
Epithelial cells are polarized cells that set up and maintain distinct basolateral and apical membrane
domains. The PDZ protein motif, originally recognized in the postsynaptic density protein PSD-95, the
Drosophila discs-large (Dlg), and the tight junction protein ZO-1, mediates protein-protein interactions and
is found in many proteins having a central role in localizing proteins to the basolateral membrane domain
of epithelial cells or to neuronal synapses. For example, our work has shown that three proteins with PDZ
domains (LIN-2, LIN-7 and LIN-10) are involved in basolateral localization of the LET-23 EGF receptor in
the vulval precursor cells. We are currently testing the hypothesis that other PDZ proteins may also be
involved in epithelial cell polarity. Analysis of the C. elegans genome revealed 58 open reading frames
predicted to encode PDZ domains, 51 of which represent genes that have not yet been characterized. To
examine the role of each of the PDZ proteins, the loss of function phenotype each gene is being
determined by RNAi. To date, the RNAi phenotype of over half the PDZ genes has been determined and
several of these genes have a defects in the polarity of the gut epithelia. These phenotypes fall into 2
distinct classes: 1. those where epithelial cell junctions form but cell organization is disrupted and 2. those
where cell junctions fail to form or are disrupted. Two genes in the latter group are the C. elegans
homologues of Drosophila Dlg (dlg-1) and scribble (scb-1). Early embryonic cell divisions are normal in
dlg-1(RNAi) and scb-1(RNAi) but embryos arrest at the two fold stage. MH27 antibody staining indicated
that the epithelial cell junctions begin to form in these animals but are not completed leading to
disconnected segments of junctional material.
198

West Coast Worm Meeting 2000
GENETIC ANALYSIS OF NMDA RECEPTOR EXPRESSION IN
C. ELEGANS
David M. Madsen, Chingju Lin, Penelope J. Brockie, Andres V. Maricq
Dept. of Biology, University of Utah, Salt Lake City, UT 84112
NMDA-type glutamate receptors are of great interest because of their roles in synaptic plasticity and
neuronal excitotoxicity. We have identified two genes, nmr-1 and nmr-2, that encode NMDA-type subunits
(see abstract by J. Mellem). Using the reporter molecule GFP, we have shown that nmr-1 and nmr-2 are
co-expressed in a small subset of interneurons that contribute to the locomotory control circuit (AVA,
AVD, AVE and PVC). Analysis of nmr-1 deletion mutants reveals defects in the timing of locomotion.
Despite their important roles in most nervous systems, we do not understand which genes are required
for the expression of NMDA receptors.
We performed a visual-based genetic screen for mutations that affect the development and differentiation
of NMDA expressing interneurons. We mutagenized a transgenic strain that expresses GFP under the
control of the nmr-1 promoter and screened ~20,000 haploid genomes for aberrant GFP expression. One
class of mutants showed a marked decrease of GFP expression in the PVC neuron. This neuron is still
present, suggesting that the mutation affects steps late in differentiation.
We identified this mutation as a nonsense mutation in ceh-14, a LIM-homeobox gene. Based on GFP
reporter constructs, ceh-14 expression is observed in several neurons, including PVC. In the ceh-14
mutant, expression of other glutamate receptor subunits (glr-1, glr-2, and nmr-2) is also decreased or
absent in PVC. We are currently testing for cell autonomous rescue using PVC specific promoters to drive
expression of ceh-14. Besides the defects in glutamate receptor expression, ceh-14 mutants exhibit a
mechanosensory defect. We hypothesize that this is due to diminished or loss of PVC function in the
locomotory control circuit. In addition, ceh-14 animals also exhibit a thermal avoidance defect (Tav) and a
phasmid dye-filling defect. Introduction of a wild-type copy of ceh-14 into transgenic worms rescued the
mutant phenotypes.
199

West Coast Worm Meeting 2000
LARGE SCALE REVERSE GENETIC APPROACH USING RNAI
Sarah Mahoney, Alex Phan, Mark Maxwell, Candace Swimmer,
Jonathan Heller, Brett Milash, Kate McKusick, Monique Nicoll
Exelixis, Inc. 170 Harbor Way, S. San Francisco, CA 94083-0511
One of our primary missions at Exelixis is the discovery of new drug targets for human pharmaceutical or
agrochemical research through the use of model organism genetics. To rapidly survey for genes that
function in disease-related pathways, we have undertaken several reverse genetic strategies, including
development of a genome wide RNAi library. First we data mine, which involves compiling predicted
cDNAs from GeneFinder in a database and sorting and categorizing them by predicted protein motifs.
Next, we employ an automated primer picking program created at Exelixis to generate primer pairs
against portions of the predicted coding sequence. PCR is then preformed on cDNA and the products are
cloned into a modified version of pCCM113 (kindly provided by C. Mello). Individual clones are
sequenced in an automated fashion and clones are validated by automatic blast via an inhouse database.
This has allowed us to correlate GeneFinder predictions with expression data. In our initial examination of
more than 1000 PCR reactions using cDNA as the template, we have observed that approximately 85 %
of the predicted genes are expressed.
In parallel to automating the cloning process, we are assessing the effectiveness of dsRNA delivery
methods. We initiated these experiments with a set of 30 genes and have compared injection, soaking,
and feeding. In addition, we have tested several parameters, including concentration dependence,
pooling effectiveness, and tissue specificity of RNAi. Our results indicate that RNAi is concentration
dependent, titratable, and that pooling of up to three dsRNAs gives us phenotypic results that are
comparable to RNAi of each gene individually. Initial RNAi soaking results from more than 100 predicted
genes suggests that a quarter of predicted genes have highly penetrant RNAi phenotypes in wild type
worms by dissection scope analysis.
200

West Coast Worm Meeting 2000
SEQUENCE CONFIRMATION OF 182 SNPS BETWEEN C.
ELEGANS N2 AND CB4856 STRAINS AND PLANS FOR
GENERATION OF 1000 NEW SNPS.
Penny Mapa, Kathryn Swan, Mike Ellis
Exelixis Inc., 170 Harbor Way, South San Francisco, CA 94080
Exelixis Inc. is a leader in model systems genetics and comparative genomics. As part of our C. elegans
mapping and cloning effort we have sequence confirmed 182 snps, evenly spaced throughout the
genome, between the N2 and CB4856 (Hawaiian) strains. These snps are a subset of the 1416 potential
snps initially identified by the Genome Sequencing center at the Washington University School of
Medicine (http://genome.wustl.edu/gsc/CEpolymorph/snp_chrom.shtml). We have successfully used
these genome wide, confirmed snps to map mutants. Additionally, to aid in fine scale mapping, we have
begun efforts aimed at identifying another 1000 new genome wide snps between these same strains. As
a service to the worm community we plan to release this information to the public.
201

BUILDING A DICTIONARY FOR C. ELEGANS PROMOTER
SEQUENCES
Steven McCarroll, Hao Li, Cori Bargmann
U.C. San Francisco
West Coast Worm Meeting 2000
We would like to understand how regulatory sequences encode the expression patterns of genes. The
availability of complete genomic sequence allows us to try to identify candidate transcriptional control
sequences based on statistical criteria. We have used a dictionary algorithm (Bussemaker, Li, and Siggia,
manuscript in preparation) to partition C. elegans promoter sequences into "words" -- discrete sequences
that are distributed statistically as if they represent a coherent functional unit. We have built a dictionary
for the upstream sequences of 850 C. elegans G-protein-coupled-receptor genes. When this dictionary is
used to partition these promoter sequences according to maximum-likelihood criteria, 2.1% of the
sequence is covered by words of length 8 or greater. Many of these words appear interesting according to
multiple criteria: (i) non-Poisson distribution across genes, suggesting a tendency to appear in clusters;
(ii) non-random distribution across positions, suggesting a preference for particular locations relative to
the transcriptional start site, and (iii) appearance in genes that have similar expression patterns. We are
using this dictionary in a number of ways:
(i) We have correlated the expression patterns of several dozen chemoreceptor genes to the distributions
of words across these genes, generating testable hypotheses about sequences that may confer
cell-specific gene regulation;
(ii) We are correlating gene-expression microarray data with the distribution of words across genes, to
identify words that correspond to genes whose expression is modulated in particular experiments;
(iii) We are testing candidate control sequences (words) in promoter-gfp fusion experiments, in which a
particular word is deleted from a promoter (to test the necessity of this sequence for wild-type gene
regulation) or added to a promoter (to test its sufficiency for conferring gene regulation).
202

West Coast Worm Meeting 2000
HIGH PRESSURE FREEZING METHODS FOR C. ELEGANS
EMBRYO ULTRASTRUCTURE AND EM IMMUNOLABELING
Kent L. McDonald 1 , Thomas Mueller-Reichert 2 , Akiko Tagawa 3 , Chad
A. Rappleye 3 , Raffi Aroian 3
1Electron Microscope Lab, UC Berkeley
2Max-Planck-Institute of Molecular Cell Biology and Genetics, Dresden, Germany
3Dept. of Biology, UC San Diego
Electron microscopic analysis of C. elegans has a long and distinguished history, from the early studies
reconstructing the nervous system to recent work by David Hall and his colleagues. However, attempts at
more routine EM studies of C. elegans have been hampered by the impermeability of the cuticle of the
worm and of the eggshell of embryos. These structures act as diffusion barriers, preventing the rapid
exchange of fixatives and other solutions that are used in typical EM processing protocols.
Our EM facility specializes in an alternative to conventional EM, high pressure freezing (HPF). In this
technique, 50 or more nematodes at a time are frozen under 2100 bars pressure in 10-20 milliseconds.
This method is useful for excellent preservation of ultrastructure to depths of 200 microns or more,
compared to 10 microns or so for most other freezing methods. Furthermore, this preservation is
independent of the impermeability of the eggshell or cuticle. HPF is particularly exciting for studying early
embryogenesis since the embryos inside hermaphrodites are well preserved. Thus, within each gravid
adult are a row of early embryos of various stages making it much easier than before to fix, find, orient,
and work with embryos. We are currently using this technique for studying early embryogenesis. Recent
work has suggested a new class of embryonic mutants important for early polarity, the polarity osmotic
mutants (see abstracts by Tagawa et al and by Rappleye et al). Our models predict that these genes are
involved in membrane trafficking. We are in the process of testing this at the ultrastructural level by
looking at the general morphology of the mutants and by studying the immunolocalization of one of the
proteins. We will present information of our technique of preservation, fixation, and embedding and some
preliminary results in the early embryo.
203

MOLECULAR IDENTIFICATION OF TRANSCRIPTIONAL
TARGETS OF THE DAF-16 WINGED HELIX TRANSCRIPTION
FACTOR
Joshua J. McElwee 1 , James H. Thomas 1,2
1Program in Molecular and Cellular Biology
2Department of Genetics, University of Washington
West Coast Worm Meeting 2000
Dauer arrest and longevity in C. elegans are controlled by an insulin-like signaling pathway transduced by
the winged helix transcription factor DAF-16. Mutations in several genes within this pathway (daf-2, age-1,
and pdk-1) result in constitutive dauer formation (Daf-c) and increased lifespan (Age) phenotypes.
Invariably, both the Daf-c and Age phenotypes of these mutants are suppressed by loss of function
mutations in daf-16. This suggests DAF-16 acts as the principal transcriptional output controlling both
diapause and lifespan governed by this pathway. We are attempting to identify both direct and indirect
transcriptional targets of DAF-16 utilizing several complementary molecular approaches. First, we are
performing in vitro binding site selection to isolate the DAF-16 DNA consensus sequence. Utilizing this
sequence, we will identify putative DAF-16 targets by genomic analysis and a candidate gene approach.
Second, we are performing direct selection for genomic sequences that are bound by this transcription
factor. Third, we are examining DAF-16 transcriptional outputs in vivo. Using cDNA arrays, we will assess
global RNA expression in various Daf mutants and double mutants. Presumably, these experiments will
allow us to begin to identify genes downstream of daf-16 involved in both dauer formation and regulating
lifespan.
204

FUNCTIONAL CONSERVATION OF C. ELEGANS UNC-30 AND
MOUSE PITX2 IN GABAERGIC NEURON SPECIFICATION
Jason McEwen, Yishi Jin
Department of Biology, University of California, Santa Cruz, CA. 95064
The C. elegans GABAergic nervous system contains 26 neurons of 5 types. The homeodomain protein
UNC-30 is required for the specification and function of the type-D motor neurons. In unc-30 mutants the
D neurons fail to express GABA, have axon guidance errors, form improper synapses and shrink on
themselves while trying to move backwards. It has previously been shown that UNC-30 directly activates
the expression of unc-25, the glutamic acid decarboxylase enzyme (GAD) and unc-47, the GABA
vesicular transporter, in these neurons (1).
The UNC-30 homeodomain is over 80% identical to those of the newly identified vertebrate Pitx family
and dPtx in Drosophila (2). Pitx proteins are expressed in many tissues and have been implicated in a
variety of developmental functions (2). Pitx2 is also widely expressed in the developing and adult central
nervous system, but its function there is not known. UNC-30 and Pitx2 can activate mammalian GAD67
expression in cultured neurons (Condie B, Pers. Com.), suggesting that Pitx2 and UNC-30 may have an
evolutionarily conserved role in GABAergic neuron specification.
We have shown that mouse Pitx2 can functionally replace UNC-30 in unc-30 mutant worms. Site-directed
mutagenesis experiments on the UNC-30 binding sites in the unc-25 promoter and the Pitx2
homeodomain support that Pitx2 mediated unc-25 activation depends on a homeodomain/UNC-30
binding site interaction. Like UNC-30, ectopic expression of Pitx2 under the control of heat shock
promoters activates unc-25 expression in other tissues (3). Our data supports that UNC-30 and Pitx2 are
likely functional homologues.
UNC-30 also regulates the expression of genes required for axon guidance and synapse formation but
these target genes have yet to be found. To address this we have generated modified UNC-30 proteins
using the activation domain of VP16 and the repressor domain of Engrailed. The modified UNC-30
proteins have shown corresponding changes in transgenic studies using the Punc-25::GFP reporter.
These constructs will be used to sensitize future screens for UNC-30 target genes.
References:
West Coast Worm Meeting 2000
1.
2. Eastman et al. (1999) J. Neuroscience 19(15), 6225-6234
3. Gage et al.(1999) Mamm. Gen. 10, 197-200
4. Jin et al. (1994) Nature Vol 372, 780-783
205

West Coast Worm Meeting 2000
GENES INVOLVED IN NICOTINIC NEUROTRANSMISSION IN
THE PHARYNX
Jim McKay 1,2 , David Raizen 3,4 , Leon Avery 1,5
1Department of Molecular Biology. University of Texas Southwestern Medical Center. Dallas, Texas
75390-6148
2jim@eatworms.swmed.edu
3Department of Neurology. University of Pennsylvania School of Medicine. Philadelphia, Pennsylvania
19104
4raizen@mail.med.upenn.edu
5leon@eatworms.swmed.edu
MC is the main excitatory motorneuron of the pharynx and is required for the large increase in pumping
rate in response to food. We identified eat-2 and eat-18 in genetic screens for worms incapable of rapid
pharyngeal pumping. Both eat-2 and eat-18 lack MC neurotransmission but have no other obvious
defects. eat-2 encodes a non-alpha nicotinic receptor subunit. It is expressed in pharyngeal muscle and is
localized to a region where we think the MC synapse is. We conclude that MC is cholinergic and acts
directly on pharyngeal muscle to stimulate pumping. We observed allele specific genetic interaction
between eat-2 and eat-18 indicating that the gene products physically interact. In an eat-18 mutant
background, EAT-2 is expressed and correctly localized but the channel is not functional. We are
attempting to clone eat-18 by transformation rescue. We have narrowed down to a 3 kb piece of genomic
DNA that can rescue eat-18 mutants and we are sequencing this region from two eat-18 mutant alleles to
help identify the coding region.
206

GENETIC ANALYSIS OF THE FUNCTIONS OF A
GSK-3SS HOMOLOG CALLED SGG-1 AND A
SS-TRCP/SLIMB HOMOLOG DURING C. ELEGANS
EMBRYOGENESIS
Marc Meneghini 1 , Greg Ellis 1 , Ann Schlesinger 2 , Bruce Bowerman 1
1Institute of Molecular Biology, University of Oregon, Eugene OR, 97403
2Whitehead Institute, Cambridge MA
During C. elegans embryogenesis, a four cell stage blastomere called P2 uses Wnt signaling to induce
anterior-posterior polarity in its sister blastomere EMS. In embryos defective for the function of Wnt
pathway components the posterior EMS daughter E, which normally produces endoderm, develops like
its anterior sister MS. We have previously reported the characterization of a C. elegans GSK-3ß homolog
called sgg-11, which shares this phenotype suggesting that sgg-1 acts positively with the Wnt pathway to
regulate a-p polarity in EMS. This was somewhat surprising because in other systems, GSK-3ß functions
to repress Wnt pathway outputs and is negatively regulated by Wnt signaling to relieve this repression.
We also reported that sgg-1 mutant embryos produce an ectopic E-like cell that is derived from C, a
daughter of P2. Further analysis has shown that the source of this ectopic endoderm is Cp, the posterior
daughter of C. Double mutant analysis shows that Wnt pathway components also are required for the
production of Cp-derived endoderm in sgg-1 embryos. This result suggests that Wnt signaling functions to
make Cp different from Ca and that sgg-1 is required in Cp to prevent it from adopting an E-like fate.
Furthermore, sgg-1 synergizes with the Wnt pathway in EMS indicating that sgg-1 and the Wnt pathway
have some non-overlapping functions. Rather than functioning with the Wnt pathway during polarity
induction, perhaps sgg-1 functions at the level of blastomere identity to make Cp and E, or perhaps EMS
and C, different from each other. Like the Wnt pathway, sgg-1 is required for the orientation of the mitotic
spindle in EMS1 suggesting an overlap with the Wnt pathway during induced polarity. We are also
studying the function of a ß-TRCP/slimb related gene which we call slm-1. ß-TRCP/slimb related proteins
are known to function with GSK-3ß to target proteins for proteolytic degradation. Interestingly, slm-1
embryos have many phenotypic similarities with sgg-1 embryos. We are performing more phenotypic
analyses to better understand the functions of sgg-1 and slm-1 during C. elegans embryogenesis.
1 Schlesinger et al., Genes & Dev. (1999)
West Coast Worm Meeting 2000
207

THE EFFECT OF NONIMMOBILIZERS ON C. ELEGANS
Laura B. Metz 1 , Mike Crowder 1,2
West Coast Worm Meeting 2000
1Department of Anesthesiology Washington University, St. Louis, MO
2Department of Molecular Biology/Pharmacology, Washington University, St. Louis, MO
The mechanism of action of volatile anesthetics (VAs) is still uncertain. The Meyer - Overton hypothesis
tries to correlate anesthetic potency directly to lipid solubility. Yet this theory does not explain why some
lipophilic compounds in many homologous series of anesthetics are devoid of any anesthetic potency. F6
and F8, so called nonimmobilizers, are two such compounds that should have anesthetic action but don’t.
Here, we test whether C. elegans is also unaffected by nonimmobilizers and whether mutants
hypersensitive to VAs are sensitized to nonimmobilizers. Behavioral assays including locomotion and
mating assays were all performed with concentrations of F6 and F8 at 3-4 times the EC 50 (concentration
when effect should be half-maximal) predicted by the Meyer-Overton hypothesis. The nonimmobilizers did
not significantly effect N2 in any behavioral assay, even after 24-hr exposure. Strains extremely
hypersensitive to bona fide VAs were tested with F6 and F8. At 4 times the EC 50, F8 had a slight effect
on ric-4(js20) while F6 showed none. Both F6 and F8 affected unc-64(js21) at those same high
concentrations. These data suggest that reduced neurotransmission somehow sensitize animals to these
drugs. To test whether F6 and F8 alter cholinergic neurotransmission as has been shown for true VAs, we
measured the effect of F6 and F8 on aldicarb sensitivity (which correlates with the level of cholinergic
neurotransmission). Neither F6 nor F8 had an effect on aldicarb sensitivity, and neither antagonized
VA-induced aldicarb resistance. Thus, as in vertebrate models, nonimmobilizers do not affect the
behavior of the wildtype C. elegans; however, the drugs do have some effect when synaptic transmitter
release is reduced.
208

West Coast Worm Meeting 2000
ISOLATION AND CHARACTERIZATION OF MUTATIONS
THAT ENHANCE LET-23(SA62GF) DURING VULVAL
DEVELOPMENT
Nadeem Moghal, Paul W. Sternberg
Dept. of Biology, California Institute of Technology. Pasadena, CA 91125
In C. elegans hermaphrodites, adoption of vulval fates by P5.p-P7.p is dependent on activation of the
LIN-3/LET-23 signaling pathway. This pathway is structurally and functionally related to EGF receptor/Ras
signaling pathways described in other systems. Although the major positive effectors of this pathway
including EGF(lin-3), the EGFR(let-23), GRB2(sem-5), SOS(let-341), Raf(lin-45), MEK(mek-2), and
MAPK(sur-1/mpk-1) have been identified, little is known regarding modulation or negative regulation of
this pathway in C. elegans or in any system.
To identify modulators and negative regulators of this pathway, a genetic screen was undertaken using
an allele of let-23, sa62, that encodes a ligand-independent activated receptor. In wildtype
hermaphrodites or animals harboring one copy of sa62, three Pn.p cells adopt vulval fates. However, in
the presence of two copies of sa62, on average, four cells acquire vulval fates. sa62 heterozygous
animals were mutagenized and screened for mutants with enhanced vulval induction. To date, at least
seven different loci have been isolated, and six have been assigned linkage to specific chromosomes.
The best characterized mutation, sy598, also enhances the activity of a reduction in function allele of
let-23, sy1, but has no effect on its own or in sensitized backgrounds harboring loss of function mutations
in the negative regulators sli-1 or gap-1, or a gain of function mutation in let-60. Laser microsurgery
experiments indicate sy598 does not regulate the production of LIN-3(EGF) in the somatic gonad. These
data suggest that sy598 may control LET-23 levels in the Pn.p cells. sy598 has been mapped to linkage
group IV, and its identity is being sought.
209

West Coast Worm Meeting 2000
THE TRAMPOLINE ASSAY: A NEW METHOD FOR
MEASURING THE STEP RESPONSE OF THE CHEMOTAXIS
MECHANISM IN C. ELEGANS.
Moravec, M.L, Cervantes, J., Lockery, S.R.
Institute of Neurosci., Univ of Oregon, Eugene, OR 97403
Chemotaxis in C. elegans involves a series of abrupt turns (pirouettes) triggered by movement down a
gradient of chemical attractant (Pierce-Shimomura, J.T., et al.,J. Neurosci. 19:9557-9569, 1999). Analysis
of the time series of concentration change experienced by a chemotaxing worm, together with its
pirouette record, suggests a three-stage model in which instantaneous attractant concentration is
differentiated, smoothed by low-pass filter, and thresholded by a sigmoidal function relating filter output to
pirouette probability. This model predicts that a sudden decrease in attractant concentration will produce
a sudden increase in pirouette probability. Moreover, the increase in probability should decay
approximately exponentially with a time constant that reflects the worm’s memory for concentration
changes in the recent past. To test these predictions, we have devised an apparatus that allows us to
stimulate an unteathered worm with a nearly instantaneous (step-wise) change in the concentration of
soluable attractants such as NaCl. The apparatus consists of a thin (10 mm) agarose film suspended over
a buffer-filled chamber, resembling trampoline placed over a swimming pool. The underside of the
agarose film contacts the surface of the buffer solution, while the top side of the film contacts the air. The
worm is placed on the top of the film and allowed to adapt to a buffer containing a high concentration of
attractant for 5 min. The chamber is then drained and quickly refilled with buffer containing a low
concentration of attractant. Preliminary results indicate that a step-wise decrease in attractant
concentration causes an immediate increase in pirouette probability, as predicted by the model. We plan
to use the step response to investigate the time course of the worm’s memory for concentration changes,
and how this memory is affected by mutations and neuronal ablations. Supported by NIMH MH51383,
and NSF IBN9458102,
210

IDENTIFICATION OF GENES REGULATING BODY LENGTH IN
THE DBL-1 PATHWAY BY DIFFERENTIAL HYBRIDIZATION
OF ARRAYED CDNAS
Kiyokazu Morita 1 , Makoto Mochii 1 , Yukiko Sugihara 1 , Satoru
Yoshida 1 , Yo Suzuki 2 , William B. Wood 2 , Yuji Kohara 3 , Naoto Ueno 1
1 Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology,
Okazaki 444-8585, JAPAN
2 Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, USA
3 National Institute of Genetics, Mishima 411-8540, JAPAN
dbl-1 has been shown to regulate C. elegans body length and male tail ray patterning 1, 2). To identify
downstream target genes of DBL-1 signaling, we screened arrayed cDNAs using differential hybridization
(HDF) analysis. C. elegans cDNAs representing 7,584 independent genes were arrayed on a nylon
membrane at a high density and hybridized with 33P-labeled DNA probes synthesized from mRNAs,
which were isolated from L3-stage worms. We compared signals from dbl-1(-) worms with those from N2
worms3), dbl-1(++) worms that carry multiple copies of a dbl-1 genomic fragment, or dbl-1(-); HSP::dbl-1
worms that express DBL-1 protein upon heat shock. Many genes positively or negatively regulated by the
dbl-1 signal were identified. dsRNAi experiments were performed with all the clones identified. Several
clones caused Sma or Lon phenotypes by dsRNAi. These results demonstrate that analysis with arrayed
cDNA combined with dsRNAi gene inactivation is a powerful approach to identify genes that are directly
or indirectly regulated by an extracellular signal.
1)K. Morita et al., (1999) Development 126, 1337-1347
2)Y. Suzuki et al., (1999) Development 126, 241-250
West Coast Worm Meeting 2000
3)M. Mochii et al., (1999) Proc. Natl. Acad. Sci. USA 96, 15020-15025
211

MUTATIONS IN THE EPHRIN MAB-26/EFN-4 CAUSE
DEFECTS IN CLOSURE OF THE GASTRULATION CLEFT AND
IN EPIDERMAL ENCLOSURE
Sarah L. Moseley, Andrew Chisholm
Department of Biology, UC Santa Cruz, CA 95064
West Coast Worm Meeting 2000
Signaling involving the Eph receptor tyrosine kinase VAB-1 and its ephrin ligand VAB-2/EFN-1 is required
for normal epidermal and neuronal morphogenesis. Our lab has shown that mab-26, identified by virtue of
its role in male tail morphogenesis, corresponds to another C. elegans ephrin ligand, efn-4 (see abstract
by Holcomb et al.).
mab-26/efn-4 null mutants display incompletely penetrant defects in epidermal morphogenesis.
Approximately 10% arrest during embryogenesis, and about 20% arrest as larvae. Surviving mab-26
adults do not display the head morphology defects (’Notched head’) characteristic of vab-1 or vab-2
mutants, but are instead typically defective in morphogenesis of the posterior body and tail epidermis.
Using 4-D microscopy we have found that mab-26 embryos, like vab-1 and vab-2 mutants, are defective
in the closure of the ventral gastrulation cleft by neuroblast movements, and in later enclosure of the
embryo by epidermal cells. Interestingly, almost all mab-26 embryos show delays in gastrulation cleft
closure, although in only some cases does this result in later morphogenetic defects. Thus, the defects of
mab-26 mutants are different from (although partly overlapping with) those of vab-1 and vab-2 mutants.
mab-26 mutations also display unexpected synthetic-lethal interactions with all vab-1 and vab-2 mutations
tested. Double mutants show completely penetrant morphogenetic defects, in striking contrast to the
incompletely penetrant vab-1 null phenotype. We are currently analyzing the phenotypes of these double
mutants.
The differences in mutant phenotypes and the synthetic lethal interactions are inconsistent with EFN-4
acting only as a ligand for VAB-1. If EFN-4 is not signaling through the VAB-1 receptor, how is it
functioning? To identify other genes required for mab-26 function we are screening for new mutations that
are synthetic-lethal with a weak vab-1 allele. Results of a pilot screen will be presented.
212

CELLULAR AND DEVELOPMENTAL EVENTS REQUIRED TO
GENERATE FUNCTIONAL MUSCLE IN C. ELEGANS.
K. Norman, S. Cordes, G. Mullen, P. Rahmani, T. Rogalski, D.
Moerman
Dept of Zoology, UBC
West Coast Worm Meeting 2000
In C. elegans a physical linkage between the myofilament lattice of the body wall muscle and the cuticle is
required to allow myofilament contraction to result in the movement of the animal. This complex linkage
system is composed of integrin containing adhesion-complexes within the body wall muscle cells that
anchor the myofibrils to the muscle cell membrane, a specialized basement membrane underlying the
muscle quadrant, and hemidesmosomal-like structures present in the epidermis. The epidermis has
specialized faces, one to anchor the epidermis to the basement membrane and the other to secrete and
anchor the cuticle. The nematode provides a very elegant system to study the interaction between the
body wall muscle and the epidermis during development. Through genetic and molecular approaches we
have identified (1) several components involved in assembly, localization, and maintenance of adhesion
complexes within muscle (the unc-52, unc-97, unc-112, dim-1 and spc-1 gene products), (2) components
that are involved in structuring the underlying basement membranes (the let-268 gene product), and (3)
others that function in regulating epidermal attachment structures (the unc-23 gene product). The role of
these molecules in muscle and epidermal development during C. elegans morphogenesis will be
described. For more information on unc-23 and unc-97 see posters by P. Rahmani and S. Cordes,
respectively.
213

West Coast Worm Meeting 2000
IS THE DAG KINASE DGK-1 AN EFFECTOR OF GO ALPHA
(GOA-1)?
Stephen Nurrish, Michael Dybbs, Joshua Kaplan
Molecular and Cell Biology, University of California, Berkeley, CA 94720
We (and others) have previously described two competing G-protein pathways acting in motor neurons to
either facilitate or inhibit synaptic transmission at neuromuscular junctions (NMJs) [1-4]. Facilitation of
release occurs via a pathway composed of a Gq alpha (EGL-30), a phospholipase C beta (EGL-8), and
the diacylglycerol (DAG) binding protein UNC-13 [2,4]. The Gq alpha pathway can be activated by
muscarinic agonists leading to the formation of the membrane bound second messenger DAG by EGL-8
and the subsequent recruitment of UNC-13 to sites of Acetylcholine release [2]. UNC-13 is essential for
neurotransmitter release and it’s enrichment at release sites correlates with an increase in
neurotransmitter release. Addition of serotonin agonists inhibits acetylcholine release at NMJs. Inhibition
of synaptic transmission by serotonin requires Go alpha (GOA-1) and a diacylglycerol kinase (DGK-1)
which phosphorylates DAG to Phosphatididic Acid which is unable to bind UNC-13. Serotonin antagonists
and goa-1 mutations result in the recruitment of UNC-13 to NMJs suggesting that the Gq and Go
pathways converge to regulate UNC-13 localization, most likely by regulating levels of DAG [1,2]. As yet
the Go signaling pathway is not well defined. Go may reduce DAG levels by activating DGK-1, by
negatively regulating the Gq signaling pathway, or by an as yet undefined pathway. We are currently
testing the first of these models. Co-expression of DGK-1 and activated Go alpha in human tissue culture
cells (HEK cells) has no effect on the kinase activity of DGK-1. This suggests that there is no direct
interaction between Go alpha and DGK-1. However, it is possible that Go alpha indirectly regulates
DGK-1 via intermediates that are not conserved in HEK cells. Therefore we have generated a rescuing
MYC tagged GFP::DGK-1 construct. The myc-DGK-1 protein can be immunoprecipitated from adult
animals and possesses DAG kinase activity. We are currently testing whether the DGK-1 DAG kinase
activity changes in response to levels of Go alpha signaling. We are also testing whether Go regulates the
subcellular localization of DGK-1.
1.
2. Nurrish et al.,(1999) Neuron:24 p231-242
3. Lackner et al.,(1999) Neuron:24 p335-346
4. Hajdu-Cronin et al.,(1999) Genes Dev 13: 1780-93
5. Miller et al.,(1999) Neuron 24: 323-33
214

TRANSFORMING NEMATODES INTO INSECTS:
UNDERSTANDING BT-RESISTANCE
Johanna O’Dell, Raffi Aroian
West Coast Worm Meeting 2000
University of California, San Diego, La Jolla, CA 92093
The utilization of transgenic crops expressing Bacillus thuringiensis (Bt) toxins is becoming a prevalent
alternative to chemical pesticides. Bt toxins have enormous potential as they are not harmful to
vertebrates, demonstrate a high degree of species-specific toxicity, and are a more environmentally
friendly pest control agent. Currently genetically engineered corn and cotton are being extensively used in
agriculture and express highly similar toxins. It is predicted that resistance to these toxins will develop
within the next 10 years.
We are interested in identifying the cellular machinery underlying Bt-toxicity and resistance and are using
the nematode Caenorhabditis elegans as a model system. The physiological mode of Bt action has been
elucidated from insect studies. Following ingestion by a susceptible animal, Bt toxin is solubilized and
activated by proteolytic processing. Active toxin interacts with receptors in the gut and becomes inserted
into the apical membrane creating a channel, ultimately leading to death. Despite our knowledge of this
process, we know very little about the molecular components required for toxin action.
Our laboratory has shown that C. elegans is susceptible to some Bt toxins and has successfully isolated
mutants resistant to one Bt toxin (see abstract by Marroquin et al). One of these mutants, called bre-2 for
Bacillus toxin resistant mutant, maps to the right arm of chromosome III. Three-factor mapping
experiments position bre-2 very close to dpy-18. We are performing transgenic rescue experiments and
additional mapping by polymorphisms to ascertain the molecular identity of this gene.
To determine whether our studies are directly applicable to understanding pest resistance to transgenic
crops we are attempting to make a C. elegans strain susceptible to Cry1Ac, the major Bt toxin used in
transgenic cotton. As toxin specificity is thought to result from binding to species-specific receptors, our
strategy involves the creation of transgenic worms that express the known Cry1Ac receptor,
aminopeptidase N (APN). If APN-expressing worms prove to be sensitive to Cry1Ac toxin, we will
determine whether the bre genes are required for a common pathway of Bt toxicity or for a Cry5B-specific
pathway.
215

THE CYTOSKELETAL PROTEIN ZK370.3 MAY CONTRIBUTE
TO OOCYTE DEVELOPMENT AND FERTILIZATION
Alex Parker, Ann M. Rose
West Coast Worm Meeting 2000
Department of Medical Genetics, University of British Columbia, 6174 University Blvd., Vancouver, B.C.,
V6T 1Z3, Canada
The cytoskeleton is a dynamic structure that facilitates many events within a cell. We are using the model
organism Caenorhabditis elegans to study a cytoskeletal gene in the context of animal development. The
C. elegans gene product, zk370.3, is conserved through evolution, from yeast to humans. Work in yeast
has determined a cellular role for this gene in the maintenance of the actin cytoskeleton, as well as in
endocytosis. However, very little is known about the role of this gene in the development of higher
organisms. We have used RNA interference to investigate the loss of function phenotype for this gene
product (protocol after Tavernarakis et al. 2000). Treated animals lay significantly more unfertilized
oocytes than controls. This experiment suggests that silencing of the endogenous zk370.3 gene product
results in developmental arrest prior to oocyte fertilization. We are working to determine where the defect
resides. Using a portion of the cDNA fused to GFP under control of the zk370.3 promoter we observed
expression in the proximal gonad, spermatheca and pharynx. Expression in the spermatheca is observed
when gametogenesis is occurring, at late larval stages and early adulthood. Pharyngeal expression can
first be observed in early larval stages, and is maintained through adulthood. We have raised a polyclonal
antibody to zk370.3 for use in immunofluorescence microscopy experiments. The localization of zk370.3
protein detected by the antibody is in agreement with our expression pattern analysis.
216

West Coast Worm Meeting 2000
OXIDANT STRESS RESPONSES IN C. ELEGANS
Farhang Payvar, Andrew DeMatteo, Tom Hazinski
Department of Pediatrics, Vanderbilt University Medical School, Nashville, TN, 37232
We are intrigued by the genetic events triggered with hyperoxia and how they might differ from oxidative
stress per se. Hyperoxia can influence gene expression by transcriptional (JCI 96:2083-2089, 1995)
and/or post-transcriptional mechanisms leading directly or indirectly to oxidant stress (OS). In C. elegans,
insulin-like signaling pathway regulates life span and sensitivity to OS. For example, animals with
mutations in DAF-2 (insulin-like receptor) or AGE-1 (PI3K) are relatively resistant to OS and have
extended life span; mutations in DAF-16 (fork head transcription factor) suppress the phenotypes of daf-2
and age-1 mutants, suggesting that DAF-16 is target of negative regulation by upstream AGE-1 and
DAF-2.
Two forms of OS-induction not previously explored in C. elegans were examined. The wild type (N2) &
mutant (daf-2, age-1, daf-16) animals were exposed to 3.5 days of sodium nitroprusside (NP), an OS
inducer, in normoxic (21 % oxygen) or hyperoxic (95% oxygen) environments. Survival rates were
measured, and the LD50 for NP treatment with and without hyperoxia was calculated.
Our results indicate that survival in wild-type animals is reduced by NP but not by hyperoxia. However,
hyperoxia increased the sensitivity to NP by ~ 3-fold. This increase in NP sensitivity suggests that the
genetic events mediating the effects of hyperoxia are, at least in part, distinct from those for NP in C.
elegans, and that there is cross-talk between hyperoxia and NP signaling pathways. Moreover, response
to NP, alone or in combination with hyperoxia is altered in several mutant C. elegans strains. Consistent
with the conventional DAF-16 pathway model, age-1 mutants are relatively NP-resistant, suggesting that
AGE-1 is involved in NP signaling. Unexpectedly, age-1 mutants are only ~ 17 % reduced in their
sensitivity to hyperoxia (+NP), pointing to alternate players/pathways for transmission of hyperoxic signal.
Surprisingly, daf-2 mutants are hypersensitive to NP & have nearly lost their NP + hyperoxia-response
phenotype. These findings are consistent with the notion that NP and hyperoxia signals are relayed via
DAF-2 to downstream molecules that are distinct from AGE-1.
217

West Coast Worm Meeting 2000
PHARYNGEAL PUMPING DEFECTS IN UNC-103 MUTANTS
Christina I. Petersen 1 , David J. Reiner 2 , Elizabeth M. Newton. 3 ,
James H. Thomas 3 , Jeffrey R. Balser 4
1 Dept. of Anesthesiology, Room 560B MRB II, Vanderbilt University, Nashville, TN 37232-6600
2 Howard Hughes Medical Institute and Dept. of Molecular And Cell Biology, 401 Barker Hall #3204,
University of California, Berkeley, CA 94720-3204
3 Dept. of Genetics, Box 357360, University of Washington, Seattle, WA 98195
4 Dept. of Anesthesiology, Room 560 MRB I, Vanderbilt University, Nashville, TN 37232-6600
Human ether-a-go-go (HERG) potassium channels play a critical role in cardiac repolarization and have
been genetically linked to an inherited arrhythmia, the long QT syndrome. unc-103 encodes the worm
ortholog of HERG with 70 % amino acid identity in the conserved transmembrane and pore regions.
unc-103 gain-of-function (gf) mutants display a variety of phenotypes reflecting reduced muscle
excitation. These phenotypes include defective egg-laying, paralysis and defects in defecation. Because
the worm pharynx displays many similarities to the mammalian heart, we examined unc-103 gf mutants
for defects in pharyngeal pumping. We find that these mutants display lengthy pauses in pharyngeal
pumping (as long as 7 seconds), reminiscent of cardiac bradydysrhythmias. Notably, in wildtype worms,
HERG-specific blockers such as dofetilide and d-sotalol slow the rate of pharyngeal pumping, but do not
induce pauses. Promoter-GFP fusion constructs reveal that unc-103 is expressed in I1, I2 and NSM
neurons, which innervate the pharynx. The molecular nature of the unc-103 gf mutant is an A to T change
at position 334 in the S6 domain, a region important for potassium channel gating. To understand the
diverse effects of the unc-103 gf mutation and HERG-specific blockers, we are undertaking an
electrophysiological characterization of the wild type and UNC-103 gf mutant using a mammalian
heterologous expression system. These studies may clarify whether complex ion channel gating effects
underlie differences between the unc-103 gf phenotype and the effects of HERG blockers in the pharynx.
Furthermore, comparing the electrophysiological properties of wt UNC-103 with HERG in our expression
system will also allow us to address structure/function differences using chimeric constructs of worm and
human channels.
218

A REQUIREMENT FOR C. ELEGANS RHO-BINDING KINASE
IN EARLY CLEAVAGE
Alisa J. Piekny, Paul E. Mains
West Coast Worm Meeting 2000
Genes and Development Research Group, Department of Biochemistry and Molecular Biology, University
of Calgary, Calgary, AB. CANADA T2N 4N1
Our lab is investigating genes that regulate the actin-mediated cell shape changes that drive C.elegans
embryonic elongation. let-502 encodes a Rho-dependent kinase and strong antimorphic mutations display
early larval arrest due to failed elongation 1 . We recently isolated hypomorphic homozygous viable let-502
mutations. Some of these alleles sufficiently decrease maternal expression to reveal a new, early
embryonic phenotype. The embryos either do not complete any cell cleavages or form cleavage furrows
that regress and result in multinucleated embryos. Some of these embryos also show defects in
asymmetry, some appear larger than wild-type embryos and this may indicate defects in oocyte
formation.
Consistent with a role in cleavage furrow formation, LET-502 localizes to the furrow during early cell
divisions. mel-11 encodes a myosin phosphatase and suppresses let-502 elongation defects 1,2 . A
hypomorphic mel-11 mutant also suppresses the cleavage defects caused by let-502 mutants suggesting
that mel-11 also may play a role in cytokinesis. However, these mel-11 mutant embryos do not show
cleavage defects on their own.
mlc-4 encodes a nonmuscle regulatory light chain and is a downstream target for both let-502 and mel-11
in embryonic elongation. mlc-4 previously has been shown to play a role in early cleavage 3 . Together,
these results suggest that the pathway regulating embryonic elongation may be similar to the pathway
regulating early cleavage. In higher eukaryotes nonmuscle myosin, Rho, RhoGEF and the Rho-binding
kinase effector, Citron-kinase, have been shown to regulate cytokinesis and cleavage furrow formation.
Since the predicted C. elegans homologue for Citron has no associated kinase domain, in C. elegans
LET-502 could be the kinase required for early cleavage. let-502’s role in cytokinesis is currently being
investigated using various molecular and genetic tools.
1. Wissmann, A., J. Ingles, J.D. McGhee and P.E. Mains, 1997. Genes Dev. 11:409-422.
2. Wissmann, A., J. Ingles and P.E. Mains, 1999. Develop. Biol. 209:111-127.
3. Shelton, C. A., J.C. Carter, G.C. Ellis and B. Bowerman, 1999. J. Cell. Biol. 146: 439-451.
219

West Coast Worm Meeting 2000
FUNCTION OF THE RECEPTOR TYROSINE KINASE
CAM-1/KIN-8 IN COORDINATED MOVEMENT
S. Poulson, D. Madsen, A.V. Maricq
Department of Biology, University of Utah, Salt Lake City UT 84112
Receptor Tyrosine Kinases (RTKs) are important for many types of cell-to-cell signaling. Many
developmental processes, including the development of synapses, are mediated by signal transduction
through RTKs. Recently, a Ror-like RTK, KIN-8, was identified in C. elegans. Forrester and Garriga
isolated a kin-8 (renamed cam-1) mutation and described the role of CAM-1 in normal migration of the
CAN neurons. Koga and Ohshima later addressed the daf-c phenotype of cam-1 mutants. Independently,
we had studied the expression pattern of kin-8/cam-1, found that it was expressed in many neurons as
well as muscles and muscle arms, and generated a deletion mutation (ak37) in the gene. The mutant
worms were severely uncoordinated, defective in mechanosensation, and showed a kinked head and
withered tail. We are interested in why these worms are so defective in locomotion.
Mechanosensory defects combined with locomotory defects suggested kin-8/cam-1 may be required for
the function of interneurons that subserve locomotion. Using nmr-1::GFP to visualize the command
interneurons (AVA, AVB, AVD, AVE, and PVC), we found that 85% of kin-8/cam-1(ak37) mutants exhibit
aberrant axon outgrowth in these interneurons. We are currently investigating whether kin-8/cam-1 is
required in the commnand interneurons for axon outgrowth. Uncoordination could also result from defects
in synaptic or muscle function. Using electrophysiological recording techniques, we plan to record
ligand-gated currents from identified neurons and muscles.
220

West Coast Worm Meeting 2000
THE AUTOSOMAL SEX SIGNAL IN C. ELEGANS?
Jennifer R. Powell, Barbara J. Meyer
HHMI and Department of Molecular and Cell Biology, University of California at Berkeley 94720.
In C. elegans, sex is determined by the ratio of X chromosomes to sets of autosomes. The low X:A ratio
in XO diploid worms permits high expression of the developmental switch gene xol-1, and hence male
development ensues. Conversely, a high X:A ratio in XX animals results in low xol-1 activity and
hermaphrodite development. The X portion of the sex signal is comprised of several X-signal elements
(XSE) that repress the activity of xol-1 in a dose-dependent manner. The nature of the autosomal
component of the sex signal is unknown, but presumably increases the activity of xol-1. It is possible that
discrete autosomal signal element genes (ASE) exist that are analogous to, but act in opposition to, the
XSEs. To test this hypothesis, we are performing a genetic screen to isolate loss-of-function mutations in
ASEs, if they exist.
This screen is based on the fact that the sex signal is cumulative; therefore, we expect loss-of-function
mutations in ASEs to rescue the XX-specific lethality caused by removing XSEs. Specifically, XX worms
homozygous for mutations in the two XSEs fox-1, an RNA-binding protein, and sex-1, a nuclear hormone
receptor, are completely inviable. We are screening for suppressor mutations that rescue this lethality;
these are candidate ASE mutations. The fox-1 sex-1 starting strain can be maintained as viable
hermaphrodites if these worms also overexpress sdc-2(+), a downstream gene in the pathway, from an
extrachromosomal array. Mutagenized worms that contain a suppressor mutation will no longer depend
on the array for viability, so we screen through F1, F2, and F3 generations to look for live
non-array-bearing hermaphrodites that potentially contain dominant, recessive, or maternal effect
suppressor mutations.
In initial rounds of mutagenesis, we have screened approximately 2000 haploid genomes and isolated 10
suppressor mutations. They include autosomal and X-linked mutations, and show a range of suppression
phenotypes. There are several classes of suppressors that could be recovered from this screen, including
ase (lf), xol-1 (lf), sdc-2 (gf), xse (gf), and fox-1 or sex-1 revertants. We are currently characterizing our
existing mutants and continuing to screen for additional suppressors.
221

West Coast Worm Meeting 2000
GOT THE BLUES? TRY ANOTHER GENETIC SCREEN!
Chad Rappleye 1 , Rebecca Lyczak 2 , Bruce Bowerman 2 , Raffi Aroian 1
1University of California, San Diego, La Jolla, CA 92093
2University of Oregon, Eugene, OR 97403
To better understand the process by which cell polarity is established, we are analyzing genes required
for the characteristic asymmetries of the early C. elegans embryo. Recently, we showed that the pod-1
locus represents a new class of polarity genes as mutation of pod loci results in osmosensitive embryos in
addition to loss of polarity (’pod’=polarity and osmotic defective). The identification of pod-2 by our lab
(see abstract by A. Tagawa) further underscores the importance of the pod class of mutants. The
osmosensitivity of pod mutants appears to result from permeability of the egg shell surrounding the
embryo. Understanding the connection between polarity establishment and egg shell formation should
begin to reveal the molecular mechanisms underlying cellular asymmetry.
To further identify the set of components responsible for this aspect of polarity establishment, we have
initiated a study of mutants that have osmosensitive phenotypes. Among previously identified osmotically
sensitive mutants, emb-8 and emb-30 mutants also exhibit the ’Pod’ phenotype. In these, as well as a
pod-1 null mutant, the polarity defect is not completely penetrant suggesting that multiple, parallel
pathways might contribute to overall cell polarity. However, analysis of double mutants between pod-1
and emb-8 or emb-30 indicate that these three function in a common genetic pathway.
Through two different genetic screens, one of which directly identifies osmosensitive embryos, we have
isolated a number of other mutations that represent new loci of the pod class. This collection includes
genes which when mutated cause near 100% penetrant loss of polarity. We are currently pursuing the
molecular identities of these loci which will enable us to refine our models for how cellular asymmetry is
controlled.
222

West Coast Worm Meeting 2000
IDENTIFICATION OF COMPONENTS OF THE MEIOTIC
MACHINERY IN C. ELEGANS
Kirthi Reddy, Monica Colaiacovo, Gillian Stanfield, Anne Villeneuve
Dept. of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305
Meiosis is the specialized form of cell division that diploid cells undergo to produce gametes with a
haploid chromosome number. During prophase of meiosis I, homologous chromosomes locate each other
(pair), associate tightly along their lengths (synapse), and undergo recombination. Failure to complete any
of these processes can result in improper chromosome segregation. We are interested in the molecular
mechanisms that are involved in chromosome pairing, synapsis and crossover formation. We have
initiated a functional genomics strategy to identify components of the C. elegans meiotic machinery (see
abstract by Colaiacovo et al). Microarray analysis has identified a set of genes that are upregulated in the
germline and are not differentially expressed in male and female germ cells (V. Reinke, S. Kim and
collaborators). We are screening a subset of these genes to identify those for which RNAi elicits defects
in meiosis.
We initially focused on genes encoding proteins with predicted coiled-coil domains, since several proteins
in this class have been implicated in chromosome structure and behavior. We have identified two genes,
F26D2.2 and F39H2.4, that appear to be involved in homologous chromosome synapsis. Both have RNAi
phenotypes characteristic of animals with severe chromosome segregation defects -- affected
hermaphrodites produce a high percentage of dead embryos, and among the few survivors, there is a
high incidence of males. Further, affected animals show achiasmate chromosomes late in meiotic
prophase, likely reflecting a failure to form crossovers. Earlier in prophase, when chromosomes should be
aligned and synapsed, affected animals exhibit extensive asynapsis. The chromatin morphology seen in
the affected animals is characteristic of sys-1 and sys-2 mutants, which are unable to stabilize
associations between homologs (see abstract by MacQueen and Villeneuve). Indeed, the sys-1(me17)
allele has a stop mutation in the F26D2.2 gene. No previously identified synapsis-defective mutants map
near F39H2.4, which we have designated sys-3.
Our RNAi screen for genes required for meiosis is ongoing. We will report on additional genes that we
identify on the basis of meiotic defects elicited by RNAi.
223

West Coast Worm Meeting 2000
NOVEL AND ATYPICAL RECEPTOR TYROSINE KINASES IN
MORPHOGENESIS.
David J. Reiner, Lewis Leng, Barbara J. Meyer
Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California,
Berkeley, CA 94720.
Most receptor tyrosine kinases (RTKs) fall into one of a number well-characterized growth factor receptor
families. However, in C. elegans we have used the genome sequence to identify four apparently novel
receptors and two atypical receptors with mammalian homologs. Over-expression of five of these six
genes results in variable, lumpy lethality, while RNAi of four of these genes results in visible phenotypes.
The RNAi causes mainly morphogenetic phenotypes. For example, RNAi for one of these genes causes
partially penetrant lethality due to extreme hypodermal deformities, and some escaping animals have
pronounced axon guidance and fasciculation defects. The atypical receptors are apparently nematode
homologs of the mammalian Discoidin Domain Receptors (DDRs), RTKs that function as collagen
receptors. The in vivo function of DDRs is unknown, and we hope to address this issue in C. elegans.
Candidate mutations have been identified for only one of the six receptors. T14E8.1 rescues the weakly
penetrant tail phenotypes of mab-19, but we were unable to find lesions corresponding to the two mab-19
alleles. A heat-shock promoter driven RNAi foldback construct of T14E8.1 causes moderate, incompletely
penetrant Mab phenotypes comparable to mab-19, plus a novel truncation of certain tail sensory rays. To
identify deletion mutations in these genes, we are currently constructing a large deletion library to be
screened with PCR. Future directions depend on the outcome of these screens.
224

West Coast Worm Meeting 2000
DIFFERENTIAL EFFECTS OF HEAT SHOCK AND COLD
SHOCK FOLLOWING MASSED AND DISTRIBUTED
LONG-TERM HABITUATION TRAINING IN C. ELEGANS
Jacqueline Rose 1 , Kenneth Eng 2 , Catharine Rankin 1
1Department of Psychology, University of British Columbia, Vancouver B.C.
2Neuroscience Graduate Program, University of British Columbia, Vancouver B.C.
A new en masse long-term habituation (LTH) training procedure was used to examine the effects of heat
and cold shock on LTH of the tap withdrawal response. In order to induce LTH a distributed training
protocol was used where groups of worms received four blocks of 20 taps separated by one-hour rest
periods and testing each worm individually 24 hours later. LTH following massed training was also
examined by administering one block of 120 taps to worm groups. Worms that received massed training
did not show LTH when tested 24 hours later. Temperature shock was administered by submerging
sealed worm-containing petri plates into either a 32° C (heat shock) or a 0° C bath for the first 40
minutes following training blocks: worms were kept at room temperature for the last 20 minutes. Worms in
the control condition underwent similar submersion during rest periods at 21° C (room temperature). It
was found that when heat shock is administered between distributed training blocks an attenuation of
LTH results. Predictably, heat shock after massed training did not produce LTH. Interestingly, when
worms receive distributed training blocks with cold shock between blocks, LTH of the tap withdrawal
response is at least retained and may be enhanced. Similar to before, LTH did not result from massed
training followed by cold shock. Twelve-hour retention has also been examined with distributed training
and similar findings resulted where LTH was induced after room-temperature submersion and cold shock
but not heat shock. Currently, studies are investigating if massed training results in LTH if measured 12
hours rather than 24 hours later. As well, the effects of heat shock and cold shock on 12-hour retention
following massed training are being examined. The results that have been reported thus far indicate that
differential mechanisms may underlie both LTH resulting from massed versus distributed training and the
effects of heat shock versus cold shock.
Research funded by the Natural Sciences and Engineering Research Council of Canada
225

A NEW EN MASSE TRAINING PROCEDURE TO STUDY
LONG-TERM HABITUATION IN C. ELEGANS
Jacqueline Rose, Catharine Rankin
West Coast Worm Meeting 2000
Department of Psychology, University of British Columbia, Vancouver B.C.
Past research examining long-term habituation (LTH) of the tap withdrawal response in C. elegans
required training individual worms with three blocks of 20 tap-trains separated by one hour rest periods
with test tap-trains delivered 24 hours later. This single worm method was time and labor intensive. In an
attempt to increase throughput, a between-groups design was implemented. Groups of worms were
trained at a 60 s interstimulus interval by receiving either 4 blocks of 20 taps with one-hour rest periods
(distributed) or 1 block of 80 taps (massed). Testing was conducted 24 hours after training with single
worms individually being given one block of 10 taps. Trained groups were compared to a control group
that received a single tap on training day and the same individual testing on day two. The results showed
that worms that received distributed training show significantly smaller responses compared to single-tap
controls. Worms that underwent massed training showed no difference in response magnitude compared
to the control group. Since LTH was only found following distributed training a follow-up experiment was
conducted where worms were given either one, two, three or four training blocks separated by one hour
rest periods and were tested individually 24 hours later. It was found that worms that received 4 training
blocks showed the greatest LTH while worms that received a single training block showed no LTH
demonstrating that an effect of accumulation of learning on LTH. Together these results effectively
replicate what has previously been found with the single worm training method showing this new protocol
can now be used to more effectively assess LTH in a variety of situations.
Research funded by the Natural Sciences and Engineering Research Council of Canada.
226

GLOBAL PATTERNS OF EXPRESSION PATTERNS IN
MUSCLE USING MRNA-TAGGING
Peter J. Roy, Stuart Kim
West Coast Worm Meeting 2000
Department of Developmental Biology, Stanford University, 279 Campus Drive, Stanford, CA 94305
Gene networks that control development must endow each unique cell with distinguishing properties.
Microarray technology along with the completed C. elegans genome now makes it possible to know these
gene networks in a comprehensive manner. Our microarrays contain PCR fragments representing nearly
every predicted C. elegans gene. By hybridizing labeled cDNA probes from different samples to the
microarray, one can simultaneously determine the expression differences between those two samples for
every gene. A limitation of current microarray technology is that RNA is isolated from whole worms, in
turn, making tissue specific expression patterns difficult to detect. We have developed a strategy to
circumvent this problem of identifying the mRNA profiles in specific tissues or cells by using an approach
we call "mRNA-tagging".
To isolate cell-specific mRNA, we are using previously characterized promoters to drive the expression of
epitope-tagged protein that binds mRNA in a non-biased fashion in a defined set of cells. By
immunoprecipitating the tagged mRNA-binding protein from whole animal lysates, cell-specific mRNA is
co-immunoprecipitated. The abundance of each mRNA species isolated from these distinct cells is then
assayed using microarrays.
To identify genes expressed in muscle, we are using the well-characterized promoter myo-3 (Okkema et
al., Genetics 135, 385-404) to drive the expression of the epitope-tagged mRNA-binding protein in body
wall muscle. To date, we have successfully and reproducibly co-immunoprecipitated muscle-specific
RNA, as assayed through both northern and microarray analysis. Control germ line and neuronal RNA
are not enriched. We are currently optimizing our mRNA-tagging protocol to identify gene expressed in
muscle, and are also extending our studies to identify neuronal and hypodermal genes.
227

West Coast Worm Meeting 2000
COOPERATION BETWEEN UNC-26/SYNAPTOJANIN AND
THE DYNAMIN-RELATED PROTEIN DRP-1 DURING
MITOCHONDRIAL DIVISION
Dan Rube 1 , Todd Harris 2 , Erik Jorgensen 2 , Alexander van der Bliek 1
1Department of Biological Chemistry, UCLA School of Medicine, P.O. Box 951737, Los Angeles, CA
90095-1737
2Department of Biology, University of Utah, 257 South 1400 East Salt Lake City, UT 84112-0840
Mitochondria often exist as a dynamic tubular network. Individual mitochondria frequently divide or fuse
with neighboring mitochondria in response to a variety of metabolic and cell division cues. Our laboratory
is interested in the mechanism of mitochondrial division and how this mechanism is related to
endocytosis. A first indication that these processes may be related came from our studies of
dynamin-related protein (DRP-1) in the nematode C. elegans. We discovered that DRP-1 is important for
the final stage of division of the mitochondrial outer membrane. This was surprising, because the protein
sequence is very similar to that of dynamin, which we know mediates an early stage of clathrin mediated
endocytosis. We hypothesize that division of the mitochondrial outer membrane is evolutionarily and
mechanistically related to endocytic vesicle formation. The very first endosymbiontic bacterium would
have retained parts of the endocytic machinery to divide the incipient mitochondrial membranes.
To confirm this hypothesis, we are looking for additional proteins that act both in endocytosis and in
division of the mitochondrial outer membrane. We are focusing on two well-known players in endocytic
vesicle formation, synaptojanin and endophilin, which might also be candidates for mitochondrial division.
We tested the effect of the synaptojanin mutant unc-26(e205) on mitochondrial morphology using a GFP
with a mitochondrial leader sequence under control of the myo-3 promoter. The mitochondria in C.
elegans body wall muscles appear as a few large clumps rather than as numerous regularly shaped
wild-type mitochondrial bodies. These clumps may arise from impaired mitochondrial division.
Furthermore, this phenotype appears to be partially rescued by overexpression of DRP-1. Our data thus
indicates that in C. elegans, synaptojanin itself plays a role in maintaining proper mitochondrial
morphology. Currently, we are seeking to determine whether the morphology defect of unc-26(e205)
mitochondria arises from impaired mitochondrial division or disruption of another mitochondrial process.
228

West Coast Worm Meeting 2000
CALCIUM DYNAMICS OF FERTILIZATION IN C. ELEGANS
Aravinthan D.T. Samuel 1 , Venkatesh N. Murthy 1 , Michael O.
Hengartner 2
1Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138.
2Cold Spring Harbor Laboratories, Cold Spring Harbor, New York 11724.
In all animals, fertilization generates a pattern of intracellular calcium dynamics within the oocyte that
constitutes an essential trigger for normal development. The spatiotemporal properties of the calcium
dynamics differ among animals, e.g. cnidarians, nemerteans, fish and frogs have single calcium
transients whereas annelids, ascidians, and mammals have multiple calcium oscillations. However, in all
animals, fertilization-induced calcium dynamics are mediated by release of internal calcium stores by
inositol 1,4,5-triphosphate (IP 3). Although little is known about the signaling pathway intervening
fertilization and the production of IP 3, features of the pathway are likely to be widely shared among
species [1]. Of the animals typically used to study fertilization-induced calcium dynamics, none is as
accessible to genetics and molecular biology as the model organism Caenorhabditis elegans. Motivated
by the experimental possibilities inherent in using such a well-established model organism to study
fertilization-induced calcium dynamics, we have characterized these dynamics in C. elegans. Owing to
the transparency of the nematode, we have been able to study the calcium signal in C. elegans
fertilization in vivo by monitoring the fluorescence of calcium indicator dyes that we introduce into the
cytosol of oocytes. In C. elegans, fertilization induces a single calcium transient that originates at the point
of sperm entry. This calcium elevation immediately spreads throughout the oocyte with an amplitude ~250
nM. The duration of this solitary calcium transient is ~6 min, after which the cytosolic calcium
concentration returns to that preceding fertilization. Among other effects, this calcium signal may trigger
the completion of meiosis and the formation of eggshell.
229

MUTANTS IN THERMOSENSORY NEURON SPECIFICATION
AND FUNCTION
John S. Satterlee, Piali Sengupta
West Coast Worm Meeting 2000
Department of Biology, Brandeis University, 415 South St. Waltham, MA 02454
The AFD neurons are required for thermosensation in C. elegans. This pair of ciliated sensory neurons
has a unique morphology and presumably expresses a specific suite of genes which confer
thermosensory function. For example, the promoters of both a nuclear hormone receptor homolog
(nhr-38) and a receptor guanylyl cyclase homolog (gcy-8) drive GFP expression specifically in this neural
pair. We are examining the mechanism by which the fate and functions of the AFD thermosensory
neurons are specified, by identifying genes that work in a cascade to direct expression of these
AFD-specific markers.
Two mutants with reduced expression of nhr-38::GFP in the AFD neurons were found to be allelic to tax-2
and tax-4. The tax-2/tax-4 genes encode subunits of a cGMP-gated channel which function in some
sensory neurons, including AFD. It is possible that the TAX-2/TAX-4 channel is required for
activity-dependent expression of some AFD specific genes. We have made promoter::GFP fusions to
other tax-4-like channels encoded by the worm genome and found that two of these genes are also
expressed in AFD, as well as a number of other sensory neurons.
We have identified mutations in two genes (2 alleles each) with reduced expression of gcy-8::GFP in the
AFD neurons. One of these (sns-6) has been mapped to a small region of LG IV. The other (sns-7) is
allelic to the previously identified thermotaxis mutant ttx-1. Single nucleotide polymorphisms have been
used to map ttx-1/sns-7 to a 130 kb region of LG V which encodes 9 predicted protein products.
Both sns-6 and ttx-1/sns-7 are thermotaxis defective, but are not defective in behaviors mediated by the
AWA, AWC, ASE, and ASH neurons. Mutations in both sns-6 and ttx-1/sns-7 suppress the
dauer-constitutive phenotype of daf-7 mutants at 25ûC.
We have isolated a mutant with a deletion of the gcy-8 guanylyl cyclase domain. This mutation has no
effect on gcy-8::GFP or nhr-38::GFP expression in AFD. Further characterization of this mutant is in
progress.
The molecular characterization of thermotaxis mutants should reveal how AFD function is specified and
may also shed light on how thermosensory signals are transduced.
230

VESICULAR GABA TRANSPORT IN C. ELEGANS REQUIRES
TWO PROTEINS UNC-47 AND UNC-46
Kim Schuske, Erik M. Jorgensen
West Coast Worm Meeting 2000
Department of Biology, University of Utah, Salt Lake City, UT 84112
For neurotransmitter to be released from a neuron, it must be transported into synaptic vesicles from the
cytoplasm where it is produced. Transport proteins are located in the vesicle membrane that bind and
load a specific neurotransmitter into the synaptic vesicle. Previously we cloned the gene unc-47, which is
defective in all GABA neurotransmission, and found that it encodes a transport protein that loads GABA
into synaptic vesicles. We now report that there is a second gene that is phenotypically defective in all
GABA neurotransmission, unc-46, which also appears to be required for loading of GABA into synaptic
vesicles. Overexpression of UNC-47 is capable of rescuing the unc-46 mutant phenotype. This suggests
that an increased level of vesicular GABA transporter (UNC-47) in the neuron can compensate for the
lack of UNC-46 protein. Therefore we believe that UNC-46 may be a modulator of vesicular GABA
transport.
To test this, we have cloned the unc-46 gene. The UNC-46 protein is novel and contains a single putative
transmembrane domain. unc-46 is expressed in GABA neurons and the protein is localized to synaptic
vesicles. UNC-47 protein is also localized to synaptic vesicles even in the absence of unc-46. However,
UNC-46 protein is mislocalized in the absence of unc-47. This suggests that UNC-47 and UNC-46
interact in the synaptic vesicle. The simplest model suggests that UNC-46 may be required for UNC-47
transport activity. Current experiments are aimed at determining whether UNC-46 and UNC-47 physically
interact, testing if UNC-46 can affect transport activity of GABA in cell culture, and identifiying a vertebrate
homolog of UNC-46.
231

West Coast Worm Meeting 2000
UTILIZING TWO APPROACHES, GENETIC AND GENOMIC,
TO IDENTIFY THE VESICULAR GLUTAMATE TRANSPORTER
Kim Schuske, Dan Williams, Erik M. Jorgensen
Department of Biology, University of Utah, Salt Lake City, UT 84112
Neurotransmitter is loaded into the synaptic vesicles by transport proteins present in the synaptic vesicle
membrane. Transport activity has been characterized for four distinct neurotransmitters in purified
synaptic vesicles from rat brain: GABA, glutamate, acetylcholine, and catecholamines. Of these, only the
vesicular glutamate transporter remains to be identified at the molecular level. We previously showed that
the unc-47 gene encodes the vesicular GABA transporter. Because GABA and glutamate are structurally
similar and their bioenergetics for transport into the vesicle are also similar, we proposed that the
vesicular glutamate transporter is related by sequence to UNC-47. There are fourteen proteins in the C.
elegans genome that have sequence similarity to UNC-47. We made GFP reporter constructs for twelve
of these genes, and identified two candidates that are expressed in glutamatergic neurons. We are
currently studying a gene knockout, made by the C. elegansgene knockout consortium, and GFP-tagged
protein localization for one candidate cevt6. However, preliminary data suggests that cevt6 is not the
vesicular glutamate transporter.
It is possible that the vesicular glutamate transporter does not resemble the vesicular GABA transporter
by sequence. For this reason we have undertaken a genetic screen for mutants that are likely to be
defective for glutamate neurotransmission. We are therefore looking for mutants resembling eat-4 since
they are defective for all known glutamate neurotransmission. These animals have an Osm phenotype.
We have screened for Osm mutants using a Mos1 transposon (from Drosophila mauritiana) with the goal
of identifying a mutant with a Mos1 insert in a transporter-like protein. We carried out a pilot screen and
identified three Osm mutants. One of these mutants contains a single transposon inserted into exon ten
of the eat-4 gene. The two other mutants are dye filling defective and are therefore not likely to encode
the vesicular glutamate transporter. An increase in the efficiency of Mos1 transposition should allow us to
do a saturation Osm screen in the future.
232

West Coast Worm Meeting 2000
ACTIN-DEPENDENT PROCESSES IN THE EARLY C.
ELEGANS EMBRYO REQUIRE THE PROFILIN GENE PFN-1,
THE FH GENE CYK-1, AND BEL-1
Aaron F. Severson 1 , Rebecca Lyczak 1 , David L. Baillie 2 , Bruce
Bowerman 1
1University of Oregon, Eugene, OR 97403 USA
2Simon Fraser University, Burnaby, B.C. CANADA V5A 1S6
The actin cytoskeleton is essential for many processes in the early C. elegans embryo, including ruffling
of the anterior cortex and pseudocleavage following fertilization, the asymmetric migration of pronuclei
and position of the first mitotic spindle, and cytokinesis. A worm profilin gene, called pfn-1, is required for
all of these processes. In pfn-1(RNAi) embryos, cortical ruffling and pseudocleavage are absent, the
pronuclei meet in the center of the embryo, the first mitotic spindle is centrally positioned, and a cleavage
furrow fails to form during cytokinesis. Profilin regulates the actin cytoskeleton by sequestering actin
monomers and preventing their polymerization into microfilaments. In addition, profilin can stimulate actin
assembly by converting actin into an assembly-competent GTP-bound state. In other systems, profilin can
bind to FH proteins, which may recruit profilin/actin complexes to sites of actin assembly. The C. elegans
FH protein CYK-1 is required for embryonic cytokinesis (Swan et al, 1998), and embryos produced by
worms mutant for a strong combination of cyk-1 alleles fail early in furrow ingression. We are currently
characterizing anti-PFN-1 antisera to examine PFN-1 localization in wild-type and CYK-1 mutant
embryos. In addition, we are examining actin, myosin, and CYK-1 localization in pfn-1(RNAi) embryos to
determine the effects of profilin inactivation on the actin cytoskeleton.
While PFN-1 may be required for organized contraction of the actin cytoskeleton, a second gene may
function to temporally and spatially restrict contractility in the embryo. In embryos mutant for a
temperature-sensitive, embryonic lethal allele of this gene, waves of contraction sweep across the early
embryo, resembling mobile cleavage furrows. We have named this gene bellydancer, or bel-1. To
determine if the mechanism that underlies these waves is similar to the contractile ring, we are staining
bel-1 mutant embryos with antibodies to actin, myosin, and CYK-1. In addition, we are mapping bel-1,
which we hope to clone by a combination of RNAi phenocopy and transgenic rescue.
233

LIN-12 POST-TRANSCRIPTIONAL DOWNREGULATION
DURING VPC SPECIFICATION
DD Shaye 1 , I Greenwald 2
West Coast Worm Meeting 2000
1Dept. of Genetics and Development, Columbia University, New York, NY 10032
2HHMI and Dept. of Biochemistry, Columbia University, New York, NY 10032
In wild-type hermaphrodites, each vulval precursor cell (VPC) adopts one of three distinct fates, 1°, 2° or
3°, in a precise and invariant spatial pattern (reviewed in 1). Analysis of mutants defective in vulval
development have suggested that at least three different signaling events are involved in the correct
specification of VPC fates. An inhibitory signal originates in the hypodermal syncytium. An inductive signal
originates in the gonadal anchor cell and allows VPCs to overcome the inhibitory signal from the
hypodermis. Finally, a lateral signal originates in the presumptive 1° cell and is received by the
presumptive 2° cells through LIN-12.
Studies using a lin-12::gfp fusion have shown that one output of the inductive signal is to influence LIN-12
protein accumulation (2). In wild-type hermaphrodites, all six VPCs initially express LIN-12::GFP.
However, LIN-12::GFP accumulation is reduced specifically in P6.p at the time of vulval induction,
although expression of a lin-12::lacZ reporter remains constant (3). The inductive signal seems to be
necessary and sufficient for this downregulation to occur. These results have led to the proposal that the
downregulation of LIN-12 in the presumptive 1° cell is at least one component of the mechanism by
which the inductive signal influences lateral signaling.
We are interested in elucidating the mechanism by which the inductive signal controls the downregulation
of LIN-12 in P6.p and whether this downregulation plays a role in correctly generating a lateral signal. In
order to answer these questions we are taking two approaches. First, we are examining the effects of
mutations in the inductive pathway and in other genes on LIN-12::GFP protein accumulation. Second, we
are determining the region of LIN-12 that targets LIN-12::GFP for downregulation in P6.p.
1) I. Greenwald, in C. elegans II. CSH Press, Cold Spring Harbor, NY, (1997).
2) D. Levitan and I. Greenwald, Development 125, 3101 (1998).
3) H. Wilkinson and I. Greenwald, Genetics 141, 513 (1995).
234

DISTINT AND REDUNDANT FUNCTIONS OF MU1 MEDIUM
CHAINS OF AP-1 CLATHRIN-ASSOCIATED PROTEIN
COMPLEX IN THE NEMATODE CAENORHABDITIS ELEGANS
Jaegal Shim, Junho Lee
West Coast Worm Meeting 2000
Department of Biology, Yonsei University, Seoul, Korea
The clathrin-associated protein complex I(AP-1 complex) in C. elegans is composed of four adaptor
proteins. Of the medium chains in the AP-1 complex of C. elegans, the first cloned is unc-101 and the
second chain cloned is apm-1. Also, according to a search of the C. elegans genome, unlike the medium
chains, only one each of the beta, gamma and sigma chains of the AP-1 complex exists in C. elegans.
These facts support the possibility that the two medium chains in C. elegans compose distinct AP-1
complex while sharing the other chains, similar to mammalian mu1a and mu1b. From the RNAi and
expression studies of AP-1 complex in C. elegans, we concluded that this hypothesis was correct. Since
the expression of unc-101 and apm-1 is ubiquitous throughout development, and the functions of unc-101
and apm-1 are redundant in embryogenesis and vulval development while becoming distinct during larval
development, we are interested in the distinct and similar functions of these chains as adaptor proteins.
Mediums of the AP complexes are generally known to play a part in cargo selection of clathrin-coated
vesicles, thus we have started experiments to elucidate their respective cargoes using the yeast
two-hybrid system. We screened about 400,000 colonies with full-length apm-1 cDNA as a bait, and
found several positive clones. One of them, F29G6.3A, was the same clone that Marc Vidal group had
found with unc-101 as a bait(Albertha J.M Walhout et al., Science ?????). We have also found several
novel proteins, and will present analyses on the candidate genes
235

West Coast Worm Meeting 2000
MOLECULAR MECHANISMS OF DAF-12 ACTION:
IDENTIFICATIONOF RESPONSE ELEMENTS AND
FUNCTIONAL ANALYSIS OF THE PROTEIN
Yuriy Shostak 1 , Adam Antebi 2 , Marc R. van Gilst 3 , Kieth R.
Yamamoto 1
1 Program in Biochemistry and Molecular Biology and Department of Cellular and Molecular
Pharmacology, UCSF, San Francisco, CA 94143-0450
2 Max-Planck-Institut fuer molekulare Genetik,D-14195 Berlin, Germany
3 Department of Cellular and Molecular Pharmacology, UCSF, San Francisco, CA 94143-0450
Dauer larva formation in Caenorhabditis elegans is regulated by the intracellular receptor daf-12, which
functions at the convergence of TGF-b, cGMP, and insulin-like signaling pathways. daf-12 has also been
implicated in the regulation of C. elegans lifespan and developmental age. Alleles of daf-12 with distinct
protein sequence alteration spartially uncouple its phenotypic effects.
As a step toward understanding the molecular biology of Daf-12 function, we identified specific DNA sites
in the C. elegans genome bound by the protein in vitro. We developed an in vitro selection and
amplification method that, using immobilized recombinant daf-12 DNA binding domain, yielded a series of
specific C. elegans genomic DNA fragments. We inserted some of these fragments into yeast reporter
plasmids and showed that Daf-12 selectively activated transcription of the reporter genes in
Saccharomyces cerevisiae. Hence, C. elegans DNA fragments bound specifically by Daf-12 in vitro
display Daf-12 response element activity in vivo.
Intracellular receptors are multidomain proteins with characteristic DNA binding, putative ligand binding,
and potentially ligand-controlled transcriptional regulatory domains. We used the heterologous yeast
expression system to investigate contributions of different Daf-12 regions to transcriptional regulation. We
found thatDaf-12 derivatives with truncated ligand binding domains are potent transcriptional activators,
and mapped an activation domain to the "hinge region" just downstream of the DNA binding domain.
In future work, we shall determine whether the identified response elements are linked to Daf-12regulated
target genes in C. elegans. Similarly, we shall examine the roles of the activation domain in Daf-12
biology and molecular function in the animal.
236

SEROTONIN-RESISTANT EGG-LAYING MUTANTS AND A
RECEPTOR KNOCKOUT IN PROGRESS
Stanley Shyn, William Schafer
UCSD, La Jolla, CA 92093
Serotonin (5HT) stimulates egg-laying in the nematode 1 , and we hypothesize that this occurs by
modulation of vulval muscle excitability from a quiescent mode to an active egg-laying state 2 . The
effectors used to accomplish this are largely unknown, so we are mapping and characterizing catalogued
as well as more recently isolated mutants (5 lines from our own lab) which are 5HT-resistant for
egg-laying. One such mutant is egl-24 (n572) 3 . Further characterization has confirmed its profound
5HT-resistance, but perhaps more intriguingly that, in the absence of any exogenous 5HT, its temporal
pattern of egg-laying is not grossly different from that of N2s. Preliminary data suggests its map interval
may be further narrowed to between -0.54 and -0.79 on LGIII, an interval spanned by 26 cosmids and a
portions of 2 YACs.
From a reverse-genetics approach, we are also preparing to knockout 5HT-Ce, the only known vulval
5HT receptor 4 . Among vertebrate classes, 5HT-Ce shows the most homology to the 5HT 1 subtype,
which typically inhibits adenylate cyclase 5 . This prompted us to look at worms mutant in acy-1, an
adenylate cyclase expressed in virtually all neurons and muscles, including the vulva 6 . nu329 and
pk450::Tc1, both only partial loss-of-function, demonstrated 5HT-resistance despite a baseline with
wildtype parameters. Whether cAMP stimulates or inhibits egg-laying is still unclear, but pharmacological
manipulations are planned to clarify the issue.
Finally, assuming 5HT-Ce inhibits adenylate cyclase, downstream G-protein candidates might include
goa-1 or gpa-7. Yet, functional goa-1 appears to play an inhibitory role in egg-laying 7,8 while testing in our
lab of gpa-7 worms did not uncover significant 5HT-resistance. Recently, the Plasterk lab surveyed the
complete family of genes encoding worm G-proteins 9 . One of these genes, gpa-14, is expressed in the
vulva and appears to be a novel G-protein whose downstream effectors are unknown. gpa-14 (pk347)
mutants were subsequently tested and found to be 5HT-resistant, suggesting that gpa-14 might be part of
a 5HT signalling pathway.
1 Science 216:1012
2 Neuron 21:203
3 Genetics 104:619
4 Tim Niacaris (Avery lab), pers comm
5 J Mol Neurosci 8:53
6 J Neurosci 18:2871
7 Science 267:1652
8 Science 267:1648
9 Nat Genetics 21:414
West Coast Worm Meeting 2000
237

A NOVEL GENETIC SCREEN FOR SYNAPTIC
TRANSMISSION GENES ACTING IN THE DIACYGLYCEROL
PATHWAY
Derek S. Sieburth, Wendy Cham, Josh M. Kaplan
UC Berkeley, MCB Dept , 361 LSA, Berkeley CA 94720
Acetylcholine release at the neuromuscular junction is modulated by diacylglycerol (DAG) levels which
are regulated by the opposing effects of two G protein pathways 1-4 . The Gqa pathway produces DAG
through the activation of the egl-8 phospholipase c (PLC) 1,4 , and the Gao pathway reduces DAG levels
possibly through diacylglycerol kinase, encoded by dgk-1 2 . DAG appears to modulate synaptic
acetylcholine release by recruiting UNC-13 to the synapse where it facilitates vesicle release 1,2 .
Mutations in genes acting in these two pathways have opposite effects on acetylcholine release as
assayed by the response to the paralytic effects of the acetylcholine esterase inhibitor, aldicarb 1,4 .
Mutations in the Gqa/PLC pathway result in decreased levels of DAG and resistance to Aldicarb (Ric),
while mutations in Goa/DGK-1 result in increased levels of DAG and hypersensitivity to aldicarb (Hic).
To identify additional components specifically acting the DAG pathway, we have screened for
suppressors of the Hic phenotype of dgk-1 null mutants. Suppressor mutations are likely to define
synaptic transmission genes involved in either DAG production or, like UNC-13 in the response to DAG,
or alternatively in genes acting in a parallel pathway that also modulates acetylcholine release. We have
identified 26 suppressors which each suppress the Hic phenotype of dgk-1 mutants to wild type or to Ric.
22 of these have wild type response to the acetylcholine receptor agonist, levamisole, indicating that they
define genes that are likely acting in the motor neurons. These 22 suppressors define at least eight
genes, including unc-13, an expected target of this screen. Seven suppressors are resistant or partially
resistant to the effects of an exogenously added DAG analogue, PMA, suggesting that they define DAG
targets. Several suppressors have weak or no obvious locomotion or Ric phenotypes in a dgk-1(+)
background, suggesting that they may have regulatory roles in vesicle release. At least two of the
suppressors appear to define new genes not previously implicated in acetylcholine release. Progress on
suppressor characterization will be reported.
1 Lackner et al., Neuron 24: 335-346 1999
2 Nurrish et al., Neuron 24: 231-242 1999
3 Hajdu-Cronin et al., Genes Dev 13: 1780-93 1999.
4 Miller et al., Neuron 24: 323-33 1999.
West Coast Worm Meeting 2000
238

EVIDENCE OF A MATE-FINDING CUE IN THE FREE-LIVING
SOIL NEMATODE C. ELEGANS
Jasper M. Simon, Paul W. Sternberg
HHMI and Caltech, Division of Biology
West Coast Worm Meeting 2000
Use of mate-finding cues is well documented throughout the nematode literature, but no assays for C.
elegans have come into common practice. Here we report two assays that suggest young adult males
can detect young adult hermaphrodites through some unidentified cue. Accumulation of 14 males (per
trial) to agar conditioned with 5 hermaphrodites (the muscle mutant unc-52 was used to construct a point
source), which were sequentially removed, suggests a cue given off by hermaphrodites and detected by
males [mean number of males observed in 1-cm diameter scoring circles shifted from 3.2+2.2 in control
trials (n=29) to 6.5+2.5 in conditioned-agar trials (n=28); p

UNDERSTANDING C27H5.1: FROM SEQUENCE TO SENSE
Jessica Smith, David Pilgrim
West Coast Worm Meeting 2000
CW 405 Biological Sciences Bldg., University of Alberta, Edmonton, AB, T6G 2E9
One of the goals of our lab is to determine the function of UNC-119, a protein involved in axon guidance
and outgrowth. In the course of this study we have chosen to look at C27H5.1, a weak homologue of
UNC-119 predicted by the C. elegans Genome Sequencing Project. Like UNC-119, C27H5.1 appears to
be expressed early in the developing embryo and pan-neuronally in all subsequent stages of
development, suggesting that it too plays a role in neural development. Unlike UNC-119, C27H5.1 shows
additional expression in pharyngeal and vulval muscles suggesting other roles for this protein.
Unfortunately there are no known mutations in C27H5.1. We are attempting to determine the phenotype
of a C27H5.1 knockout through both deletion screening and in vivo RNA interference.
While C27H5.1 is weakly similar to UNC-119, it is highly conserved (70% identity) with the delta subunit of
mammalian rod phosphodiesterase (PDE6d). This protein was first identified in the retina as a subunit of
rod phosphodiesterase (PDE). However, it has since been shown to be ubiquitously expressed and to
interact with a number of other proteins including Rab13, RPGR, and Arl2. These interactions have not
been fully characterized, but two general observations can be made. PDE6d solubilizes some proteins
and affects the cGMP or GTP binding properties of others. These data suggest that PDE6d plays a
regulatory role through interactions with other proteins. Interestingly, C27H5.1 interacts with and
solubilizes PDE in the same manner as PDE6d suggesting functional conservation between these two
proteins.
To determine the role of the C27H5.1 protein in C. elegans, we are in the process of performing a yeast
2-hybrid screen of a C. elegans cDNA library using C27H5.1 as bait. We are especially curious to see if
C27H5.1 interacts with proteins similar to those which interact with PDEd in mammals. As well, we are
producing antibodies against C27H5.1 which will allow us to further characterize the function and
expression of this protein.
240

GENETIC SCREENS FOR NOVEL COMPONENTS INVOLVED
IN BLASTOMERE ASYMMETRY IN THE EARLY C. ELEGANS
EMBRYO
Martha Soto, Craig C. Mello
West Coast Worm Meeting 2000
University of Massachusetts Medical Center, Worcester, MA 01605, USA
In the early C. elegans embryo asymmetric cell divisions result from the partitioning of factors and from
signaling between cells. At the four cell stage the EMS blastomere divides asymmetrically, leading to two
daughters, E and MS, with distinct cell fates. The E blastomere is the only source of gut in the embryo,
while MS makes pharynx and other tissues. This polarization requires a signal from the P2 blastomere to
EMS. In addition, rotation of the spindle of EMS results in only the E daughter contacting P2. Mutations
that disrupt this signal include homologs of WNT signaling factors. We have conducted forward genetic
screens to identify new factors that control the asymmetric divisions of EMS. To date, our screens have
identified many alleles of known WNT components, as well as a few novel genes.
We will discuss a novel mutation involved in regulating cleavage orientation. Several components
involved in spindle orientation have been identified by forward genetics in C. elegans, including the Par
genes and let-99, and by reverse genetics, including the small GTPase CDC42 as well as G proteins. In
our mutant, ne236, EMS divides left/right rather than anterior/posterior resulting in embryos in which both
E and MS are in contact with P2. This phenotype is more severe than the slight disruptions in spindle
rotation seen in other WNT mutations. ne236 embryos also have cell fate transformations, including
frequent ectopic gut and pharynx. Interestingly, ne236 interacts with components of WNT signaling,
enhancing partially penetrant gutless mutations. We are attempting to clone ne236, and testing models to
understand how cleavage orientation is regulated in asymmetric cell divisions.
241

West Coast Worm Meeting 2000
PAX BE WITH YOU - PATTERNING THE PHARYNX
Jeff Stevenson 1 , Andrew Chisholm 2 , Susan E. Mango 1
1Dept. of Oncological Sciences, Huntsman Cancer Institute Center for Children, University of Utah, Salt
Lake City, UT, 84112
2Dept. of Biology, UC Santa Cruz, Santa Cruz, CA, 95064
Formation of the pharynx consists of four phases: i) specification of pharyngeal precursors during early
embryogenesis, ii) assembly of these precursors into a pharynx primordium, iii) terminal differentiation of
the five pharyngeal cell types, and iv) morphogenesis of the pharynx into its adult form. Establishment of
the pharyngeal precursors requires the pha-4 gene, which encodes a transcription factor expressed in all
cells of the pharynx. In a pha-4 mutant background, no or few pharyngeal cells are generated.
How are five different pharyngeal cell types produced from an apparently homogeneous population of
precursor cells? And what is the role of pha-4 in this process, given its pan-pharyngeal expression
pattern? To address these questions we are studying how one pharyngeal cell type, the marginal cell, is
established during development. We have identified an early marker of marginal cells, PAX-9, a homolog
of the paired transcription factor. C. elegans pax-9::GFP is expressed in all nine marginal cells, as well as
three other pharyngeal cells. The involvement of transcription factors in most cell fate decisions suggests
that control of marginal cell specification lies at the transcriptional level. Hence, an understanding of pax-9
promoter elements may reveal the pathways that govern specification of distinct cell types within the
pharynx.
Deletion analysis of the pax-9 promoter shows that proper spatial and temporal expression of a
pax-9::GFP reporter requires only 125 bp of upstream sequence. Within this region, we have identified a
consensus PHA-4 binding site that alone is able to direct expression of a GFP reporter in nearly all cells
of the developing pharynx. Significantly, evidence suggests that PHA-4 binds the pax-9 promoter in vivo,
and that binding is not limited to marginal cells.
Our working model postulates that the binding of PHA-4 to this promoter endows organ (pharyngeal)
identity to pax-9 expression, while the binding of a second, as yet unidentified factor expressed only in
marginal cells, imparts cell type identity. We are presently scanning this region for cis-elements required
for the marginal cell specific expression of our reporter construct.
242

West Coast Worm Meeting 2000
THE EVOLUTION AND EXPRESSION OF FEM-2
Paul Stothard 1 , Dave Hansen 2 , Tamara Checkland 1 , Dave Pilgrim 1
1Department of Biological Sciences, University of Alberta, Edmonton, AB T6G 2E9
2Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110
FEM-2 is a protein phosphatase type 2C (PP2C) that regulates sex determination. We have recently
focused on three aspects of FEM-2 biology. First, why do sex determining proteins in general have an
accelerated rate of evolution? Second, how do mutations in other sex determining proteins affect the
expression pattern of FEM-2? Third, what do the mutant alleles tell us about the function of the protein?
To assess whether different selective pressures act on sex determining proteins we characterized the
orthologs of C. elegans PP2C genes from the related nematode C. remanei. Comparison of the C.
remanei sequences with their C. elegans orthologs indicates that FEM-2’s PP2C domain is much more
divergent than the same domain in the other proteins. PP2C sequences with no known sex determining
role were also isolated from the zebrafish, Danio rerio, and compared with their previously identified
mouse orthologs. The zebrafish/mouse PP2C domains are more conserved than FEM-2’s PP2C domain,
but less conserved than the other C. elegans/C. remanei PP2Cs. Experiments using transgenes suggest
that C. remanei and C. briggsae fem-2 are able to promote male somatic development but not sperm
production in C. elegans. To better understand how fem-2 regulates sexual development we have raised
antisera against the FEM-2 protein. The antisera can specifically detect FEM-2 on Western blots, and it
stains sexually dimorphic structures when used on whole animals. We are currently looking for
differences in staining between wild-type animals and animals that carry mutations in other sex
determining genes. Finally, studies of two temperature sensitive missense alleles (b245 and q117) are
underway. The b245 mutation is located in the PP2C domain while the q117 mutation is in the amino
terminus. These two alleles are being characterized in vivo as well as through in vitro protein studies.
243

West Coast Worm Meeting 2000
FORMING A GUT: THE VIEW FROM AN ELT AND TWO ODDS
Keith Strohmaier 1 , Morris Maduro 2 , Joel Rothman 2
1Biochemistry and Molecular Biology Program, University of California, Santa Barbara, CA
2MCDB Department, University of California, Santa Barbara, CA
In an effort to understand the genetic events that lead to the formation of the intestine, we are
investigating the network of regulatory factors expressed during gut development. Here, we describe our
studies of three such factors: the GATA-type transcription factor, ELT-7, and two ODD-SKIPPED-like
factors, ODD-1 (formerly EEL-1) and ODD-2.
elt-7::gfp is expressed continuously in the E lineage beginning at the 2E cell stage. This expression
pattern is identical to that of ELT-2, suggesting that these GATA factors may perform overlapping roles in
gut development. Thus, ELT-2 and -7 might act redundantly and immediately downstream of the END-1
and END-3 GATA factors, which redundantly specify E cell identity. However, elt-7(RNAi) embryos show
no discernible phenotype, and the degraded gut phenotype of the elt-2(0) mutation does not appear to be
enhanced by RNAi of elt-7. This contrasts with end-1/end-3 double RNAi, which results in a penetrant
loss of gut not seen with each RNAi alone. Ectopic elt-7 expressed from a heat-shock promoter results in
gut differentiation throughout the embryo, showing that ELT-7 is able to activate gut development.
Ian Hope’s expression screen identified odd-1 as a gut-specific gene. odd-2 was discovered through its
similarity to odd-1. Each encodes a 3 zinc finger protein similar to Drosophila ODD-SKIPPED family
members. odd-1::gfp is expressed in two phases. The first phase corresponds to E lineage expression
from the 4E cell stage through mid-elongation (about 1.5-fold). In the second phase, expression is
continuous throughout development after elongation in intestinal-rectal valve cells and in a decreasing
posterior to anterior gradient in the gut, with increased expression again in the four anterior (int1) cells.
Expression of odd-2::lacZ::gfp is similar to the second phase of odd-1::gfp expression, albeit at lower
levels and with somewhat altered kinetics. Both odd-1(0) and odd-2(RNAi) animals die as L1 larva, with
no obvious effect on gut development.
We are further characterizing the function of these genes to understand their action and regulatory
interrelationships during development of the intestine.
244

West Coast Worm Meeting 2000
C. ELEGANS HOMOLOGUE OF PROTEIN PHOSPHATASE 4
IS REQUIRED IN SPERMATOGENESIS
Eisuke Sumiyoshi, Asako Sugimoto, Masayuki Yamamoto
Dept. Biophys. Biochem., Grad. Schl. Sci., Univ. Tokyo, Japan
Protein phosphatase 4 (PP4) is a ser/thr protein phosphatase, which is known to localize to centrosomes.
Analysis using a fly mutant implicated that PP4 is required for the nucleation of microtubules during
mitosis. However, its function in other organisms is not yet understood. In C.elegans, two possible
homologues of PP4, namely Y75B8A.30 and Y49E10.3, have been identified by the genome project.
Here we report characterization of Y75B8A.30.
To understand the function of Y75B8A.30, we performed RNAi for it. Although the penetrance varied
among injections, RNAi for Y75B8A.30 at the highest efficiency caused 25% embryonic lethality at F1 and
nearly 100% embryonic lethality at F2.
Y75B8A.30 (RNAi) F2 embryos showed a multi polar spindle with two male pronuclei at 1-cell stage. We
suspected that an extra male pronucleus might result from a defect in spermatogenesis. To confirm this,
we examined the spermatogenesis of the F1 progeny. Observation with Nomarski optics and with DAPI
staining revealed that F1 spermatids often contained multiple nuclei. We also found that the nuclei of
secondary spermatocytes of F1 worms were not properly separated, indicating that Y75B8A.30 is
required for proper chromosome segregation in sperm meiosis. To see whether the multi polar spindle is
a result of the defect in sperm, F1 males were crossed to rde-1 hermaphrodites, which is resistant to
RNAi. 5/16 of cross progeny showed multi polar spindles at 1-cell stage, indicating that multi polar
spindles are indeed caused by the defect of sperm at least in part. Thus, we conclude that PP4 is
required for the segregation of chromosomes and centrosomes during spermatogenesis.
To see whether Y75B8A.30 function is required in embryogenesis, we crossed Y75B8A.30 (RNAi) F1
hermaphrodites with wild type male. Although wild type sperm were supplied in this cross, many cross
progenies died during embryogenesis, showing that Y75B8A.30 is required for embryogenesis. To know
the possible role of Y75B8A.30 in mitosis, we stained F2 embryos with anti-tubulin antibody and DAPI.
Some fraction of 1-cell embryos had condensed chromosomes at the metaphase plate, with microtubules
mislocalized around the nucleus, suggesting that Y75B8A.30 may be involved in the organization of
microtubules during mitosis.
245

TRANSCRIPTIONAL REGULATION OF THE TRYPTOPHAN
HYDROXYLASE GENE TPH-1
Ji Ying Sze
West Coast Worm Meeting 2000
Dept. of Anatomy and Neurobiology, Univ. of California, Irvine CA 92697
tph-1 encodes a tryptophan hydroxylase, the key enzyme for serotonin biosynthesis. When I was in Gary
Ruvkun’s lab, I collaborated with Martin Victor in Yang Shi’s lab and isolated a tph-1 deletion mutation,
tph-1(mg280). tph-1(mg280) shows no detectable serotonin based on anti-serotonin antibody staining
and exhibits several behavioral and metabolic defects (Sze et al., 2000). It has been proposed that in
mammals changes in the level of TPH mRNA correlate with long-term changes in the cellular serotonin
level and that the regulation of TPH mRNA tends to be "inducer"- and region-specific (Semple-Rowland et
al., 1996; Clark and Russo, 1997; Siuciak et al., 1998. Bethea, et al., 2000). Thus, the regulation of TPH
expression may be a key step by which the nervous system adjusts its long-term synaptic serotonin
levels.
To identify genes regulating tph-1, I have started a genetic screen for mutations that alter tph-1::gfp
expression in specific neurons. Thus far, mutations identified can be classified into three classes. (1) I
have previously reported that mutations in the POU-homeodomain transcription factor UNC-86 abolish
tph-1::gfp expression in NSM and HSN, but the expression in ADF is not affected. UNC-86 is expressed
in HSN and NSM, but not in ADF. (2) I have identified 18 mutations where tph-1::gfp expression in ADF is
reduced or eliminated. Laser ablation of ADF and ASI causes animals to form dauers (Bargmann and
Horvitz, 1993; Schackwitz et al., 1996) and the tph-1(mg280) mutation downregulates daf-7::gfp
expression and enhances daf-7(e1372) dauer formation at 15 o C. To test the role of ADF-produced
serotonin is important for a non-dauer growth, I am testing if these mutations enhance daf-7(e1372) dauer
formation at 15 o C. (3) I have identified one mutation that has tph-1::gfp expressed in an extra neuron.
These mutants provide me as useful means to characterize the role of serotonin produced in specific
neurons.
246

SEARCHING FOR NEW GENES INVOLVED IN DOSAGE
COMPENSATION
Chun Tsai, Barbara J. Meyer
West Coast Worm Meeting 2000
Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California,
Berkeley, CA 94720
In C. elegans dosage compensation equalizes expression of X-linked genes between males (XO) and
hermaphrodites (XX). This chromosome-wide regulatory process is essential for hermaphrodite
development. A biochemically defined protein complex containing DPY-26, DPY-27, DPY-28 and MIX-1
has been shown to associate with X chromosomes in a sex-specific fashion and is required for global
repression of hermaphrodite X-linked genes. MIX-1 is also required for proper segregation of mitotic
chromosomes. Mutations in mix-1 cause lethality in both sexes and therefore were missed from our
screens for sex-specific lethals. To identify other dosage compensation components that might have been
missed, we used the following observation: mutations in all known dosage compensation genes suppress
the sexual transformation caused by certain sex determination mutations in a dominant fashion, allowing
recessive lethal mutations to be identified by their dominant suppression effect. For example,
heterozygous mix-1 mutations can suppress the masculinization of XX animals caused by homozygous
sdc-3(Tra) mutations. The suppression screen is shown below.
sdc-3(Tra)/+ sdc-3(Tra) 99% Tra, 1% hermaphrodites
m/+; sdc-3(Tra)/+ m/+; sdc-3(Tra) 30-99% hermaphrodites isolate mutants
So far we have screened 12,000 haploid genomes and identified 9 mutations that cause moderate to
strong suppression: two are lethal mutations, two are maternal-effect lethal, one is a mix-1 allele, one is
an sdc-3 null allele, and three show no obvious phenotypes. The two lethal and one maternal-effect lethal
mutations have been mapped extensively and their map positions indicate that they are mutations in
three different genes that have not previously been implicated in dosage compensation.
Immunofluorescence studies showed that the two recessive lethal mutations disrupt the association of
DPY-26 and DPY-27 with the X chromosome. DAPI staining of dead embryos revealed abnormal DNA
morphology and multinucleated cells, suggesting an essential role in chromosome metabolism. These
observations suggest that the mutations isolated in the screen identify new genes that play a role in
dosage compensation as well as chromosome structure. Attempts are ongoing to clone the genes.
247

West Coast Worm Meeting 2000
CHARACTERIZING THE ROLE OF LET-99 IN SPINDLE
ORIENTATION
Meng-Fu Tsou, Adam Hayashi, Lesilee S. Rose
Section of Molecular and Cellular Biology, University of California, Davis, CA 95616 USA
Cell division plane, which is determined by the orientation of the mitotic spindle, is important for normal
development. The dynein-dynactin complex has been shown to be part of the mechanical force for
spindle orientation in C. elegans embryos. Cytoplasmic dynein and dynactin are also essential in a variety
of cell division processes in different organisms, including nuclear positioning and anaphase B spindle
pole separation, partially due to their role in connecting the astral microtubules and cell cortex.
The let-99 gene plays an important role in spindle orientation. We have cloned the let-99 gene and found
it encodes a novel protein that is found in the cytoplasm and at regions of cell-cell contacts in early
embryos. let-99 mutants show three major defects in cell division: first, abnormal spindle positioning in
both the AB and the P cell lineage, second, instability in position of the nuclear-centrosome complex at
prophase (nuclear rocking), and third, poor separation of the spindle poles at anaphase B. Double mutant
analysis indicates that the nuclear rocking activity seen in let-99 embryos requires the function of the
dynein-dynactin complex , suggesting that this behavior may be due to abnormal interaction between
astral microtubules and the cell cortex. However the nuclear rocking phenotype is not due to the
mislocalization of dynein-dynactin complex, because both the localization of DHC-1 (dynein heavy
chain-1) and DNC-1 (p150glued) appear to be normal in let-99 embryos. G protein beta subunit (GPB-1)
was also shown to be required in spindle orientation. Embryos in which the GPB-1 is maternally depleted
show similar phenotypes to that of let-99, suggesting that gpb-1 and let-99 may function in the same
biochemical pathway. Our preliminary result from let99 gpb-1(RNAi) double mutant analysis supports this
idea. The localization of LET-99 in gpb-1(RNAi) embryos appears to be normal, indicating that the
localization of LET-99 does not require the gpb-1 signaling pathway. We are currently exploring the
relationship among LET-99, dynein-dynactin complex and GPB-1 by a variety of techniques, in addition to
searching for LET-99 interacting proteins using the two-hybrid approach.
248

West Coast Worm Meeting 2000
CHARACTERIZATION AND CLONING OF THE MUSCLE
ACTIVATION GENE UNC-58
Monika Tzoneva, James H. Thomas
Dept. of Genetics, University of Washington, Seattle WA 98195
unc-58 was first identified by dominant mutations that cause hypercontracted body-wall and egg-laying
muscle in C. elegans. unc-58(dm) animals are rigidly paralyzed and egg-laying constitutive. Putative
loss-of-function unc-58 alleles have been isolated as revertants of the unc-58(dm) phenotype. These
alleles have no obvious phenotype on their own, suggesting that unc-58(dm) mutants result in an
inappropriately activated gene product. unc-58(dm) animals also frequently flip around their longitudinal
axis. Both the flipping and the hypercontarction phenotypes are partially rescued by the drug endosulfan,
best known as an antagonist of GABA-gated chloride channels (B. Wightman and G. Garriga, personal
communication; our unpublished data).
I have cloned unc-58 and found that it encodes a potassium channel of the TWIK family. TWIKs are
distinct from other potassium channels because they have four transmembrane domains (M1 to M4) and
two pore domains. Potassium-selective currents have been recorded from TWIKs in both mammals and
worms.
The three unc-58 gain-of-function mutations cluster in the C-terminal (cytoplasmic) part of the M4
transmembrane domain, which is equivalent to the S6 domain of the voltage-gated potassium channel
family. S6 is thought to form a channel gate that opens in response to voltage changes. The location of
the unc-58(dm) mutations suggests that its M4 helix may also form an activation gate, though the stimulus
that opens it is unknown.
The molecular identity of unc-58 raises several questions. First, how does an activated mutation in a
putative potassium channel lead to muscle hypercontraction? It is possible that the hypercontraction is
due to UNC-58 function in inhibitory motorneurons, while its slow movement may be due in part to its
function in other tissues. We will test this by determining the unc-58 site(s) of action. Second, how is the
unc-58(dm) phenotype rescued by the drug endosulfan? We will attempt to determine whether this rescue
is by direct channel block. Third, how does the UNC-58 channel contribute to overall membrane
excitability? It is not known whether or how UNC-58 activity is regulated and whether it acts alone or in
complex with other subunits. We have isolated extragenic suppressors of unc-58(dm), which may offer
insight into this question.
249

THE STRUCTURE/FUNCTION RELATIONSHIP OF CLK-1 IN
THE NEMATODE CAENORHABDITIS ELEGANS
Antonio Ubach, Siegfried Hekimi
Department of Biology, McGill University, Canada
West Coast Worm Meeting 2000
The lifespan of the nematode Caenorhabditis elegans is genetically determined by several classes of
genes. Our laboratory is particularly interested in the clk class of genes, which is composed of clk-1, clk-2,
clk-3, and gro-1. Mutations in these genes have been shown to extend lifespan and to deregulate several
developmental and behavioral processes. For example, clk-1 mutants exhibit an average slowing down of
cell division, pharyngeal pumping, and defecation rates relative to wild-type animals. These phenotypes
are maternally rescued, that is homozygous mutant animals coming from a heterozygous mother are
phenotypically wild type. clk-1 encodes a 187 amino acid mitochondrial protein that is composed of two
homologous TRC domains (TRC for Tandemly Repeated in CLK-1). Interestingly, the yeast homologue of
clk-1, COQ7, has been implicated in ubiquinone biosynthesis. Moreover, coq7 mutants are respiration
defective, yet the respiratory competence can be restored by the addition of exogenous ubiquinone. In
contrast, cellular respiration is only slightly affected in clk-1 mutant worms. However, C. elegans clk-1 as
well as the rat and the human homologues are capable of functionally complementing the Dcoq7 mutant.
This observation suggests that the biochemical function of clk-1 have been conserved throughout
evolution.
We have shown previously that a recombinant CLK-1::GFP fusion protein is capable of rescuing the clk-1
mutant phenotype when expressed from transgenic arrays. In the current study, we are taking advantage
of this observation to try to understand the structural requirements for CLK-1 function. In order to address
this, we are using a site-directed mutagenesis approach to generate several new CLK-1 mutant proteins.
We are analysing the effect these CLK-1 mutant proteins have on the general clk-1 phenotype. We are
also examining the subcellular distribution of the mutant fusion proteins in different genetic backgrounds,
such as the wild type, the clk-1 partial loss of function e2519, and the two clk-1 putative nulls qm30 and
qm51.
250

West Coast Worm Meeting 2000
CHARACTERIZATION OF TRANSCRIPTIONAL REGULATION
BY A CLASS OF MONOMERIC NUCLEAR RECEPTORS
FOUND IN C. ELEGANS
Marc R. Van Gilst, Keith R. Yamamoto
Cellular and Molecular Pharmacology, University of California-San Francisco, San Francisco, CA
94143-0450
Nuclear hormone receptors (NHRs) comprise a large family of metazoan transcription factors that
participate in numerous developmental and metabolic processes. NHRs commonly target their
transcriptional regulation to particular genes through interaction with DNA sequences called hormone
response elements (HREs) and the nature of the transcriptional regulation carried out by a NHR at these
genes is often determined by interaction with a small hydrophobic ligand.
C. elegans provides unique advantages over other animal models for studying the physiological
significance of several aspects of NHR function (i). Targeting of NHRs to promoters by HREs (ii)
Characterization of important regulatory domains of NHRs (iii) Interaction of NHRs with protein cofactors
and (iv) Interaction of NHRs with potential ligands.
We have focused our studies on the C. elegans homologs of a class of NHRs that can activate
transcription from AGGTCA DNA half-sites, a common binding motif for a mammalian sub-family of
monomeric nuclear receptors that includes ROR, TLX, ERR and SF-1. To this end, we have started our
investigations by utilizing the C. elegans receptor CHR3 (nhr-23). CHR3 is closely related to the
mammalian receptor ROR, with nearly 100% conservation in the DNA binding domain, and would be
expected to fall into the class of monomeric NHRs that we wish to study.
Using yeast and worm reporter assays, we have characterized the transcriptional regulatory properties of
CHR3. We found that CHR3 can activate transcription from a single AGGTCA half-site and activates
synergistically from multimerized half-sites. Transcriptional activation occurs in yeast in the absence of
any exogenous ligand. Similar to mammalian receptors, both the N and C terminal domains of CHR3
contain activation functions. Furthermore, CHR3 requires ATP-dependent chromatin remodeling
complexes for activation and can interact with a mammalian coactivator GRIP through a conserved
structural mechanism. We are currently expanding these systems to find and study other C. elegans
NHRs and to set up screens for the identification of potential protein cofactors and small-molecule
ligands.
251

West Coast Worm Meeting 2000
UNC-4 TARGETS ACR-5 AND DEL-1: ARE THEY
DETERMINANTS OF SYNAPTIC CHOICE?
Stephen E. Von Stetina, David M. Miller, III
Dept. of Cell Biology, Vanderbilt University , Nashville, TN 37232-2175
Mutations in the UNC-4 homeodomain transcription factor disrupt backward locomotion. EM
reconstruction of an unc-4 mutant revealed that the VA motor neurons fail to receive synapses from their
usual interneuron partners and instead accept inputs normally reserved for their sisters, the VB motor
neurons. Work done in this laboratory has shown that UNC-4 and the Groucho homolog UNC-37 function
together in the VA motor neurons to repress VB-specific genes. Two genes, del-1 (DEG/ENaC sodium
channel subunit) and acr-5 (alpha-like nictonic acetylcholine receptor subunit), are ectopically expressed
in the VA motor neurons in unc-4 and unc-37 mutants. As cell surface proteins and ion channel
components, ACR-5 and DEL-1 are attractive candidates for mediators of synaptic specificity. We have
now performed genetic experiments, however, that rule out a necessary role for either ACR-5 or DEL-1
in the specification of VB-type inputs. Deletion mutants of del-1 and acr-5, as well as the double mutant
(acr-5;del-1), do not perturb forward locomotion as would be expected for a mutation which disrupts
normal inputs to the VBs. Furthermore, assays designed to quantitate locomotion have detected no
differences between wild type and these mutants. Placement of acr-5 and del-1 mutants in an unc-4
background does not suppress the Unc-4 phenotype, suggesting that ACR-5 and DEL-1 are also not
required to promote VB-type inputs in VA motor neurons. A future goal is to ectopically express ACR-5
and DEL-1 in VA motor neurons to determine if these proteins are sufficient to cause miswiring. Also, we
will define the UNC-4 mediated repression domains in the acr-5 and del-1 promoters in an effort to
identify additional UNC-4 targets.
252

West Coast Worm Meeting 2000
NICOTINE ADAPTATION: A PROCESS INVOLVING
PKC-DEPENDANT REGULATION OF NACHR PROTEIN
LEVELS.
Laura Waggoner, Kari Dickonson, Daniel Poole, Bill Schafer
Department of Biology, UCSD, 9500 Gilman Dr., La Jolla, 92093
C. elegans is capable of undergoing adaptation to prolonged exposure to nicotine, through attenuation of
the activity of nicotinic acetylcholine receptors (nAChRs). We have discovered that in egg-laying behavior,
acute nicotine treatment results in stimulation of egg-laying, but long-term exposure inhibits further
stimulation by cholinergic agonists. Furthermore, we found that this adaptation process involves
UNC-29-containing nAChRs functioning in the vulval muscles. We were interested in determining whether
adaptation was due to a loss or attenuation of theseUNC-29-containing nAChRs; to address this, we
analyzed fluorescence levels of an UNC-29::GFP chimera in the vulval muscles before and after nicotine
treatment. Intriguingly, we found that nicotine treatment in fact decreased receptor protein levels in the
vulval muscles. In addition, we found that this regulation was independent of the unc-29 promoter and 3’
UTR. Lastly, we were interested in identifying other genes involved in this process, and we found that the
protein kinase C homolog encoded by tpa-1 was necessary for the observed decrease in nAChR protein
levels. Thus, it appears that nicotine treatment results in a PKC- dependant down-regulation of
UNC-29-containing nAChRs in the vulval muscles. Further screens are being performed to identify other
genes involved in this process.
253

West Coast Worm Meeting 2000
ANALYSIS OF GLR-7, GLR-5, IONOTROPIC GLUTAMATE
RECEPTOR SUBUNITS
Craig S. Walker, David M. Madsen, Penelope J. Brockie, Andres V.
Maricq
Department of Biology, University of Utah, Salt Lake City, UT 84112
Of the ten putative ionotropic glutamate receptor subunits identified in the worm, two non-NMDA subunits,
glr-5 and glr-7, show a very restricted expression pattern. When GLR-5 and GLR-7 are fused to GFP they
show expression in only a single cell, the interneuron RIA. RIA receives synaptic input from the neurons
AIY and AIZ. Previous studies have defined this group of cells to form part of the circuit that controls
thermotaxis.
To determine the possible roles of glr-5 and glr-7 in thermotaxis, we have generated deletion mutations in
both genes. These deletions remove a portion of the extracellular domain and the first three
transmembrane domains. In each case the mutations result in a functional null. Analysis of glr-5(ak57)
deletion mutants using thermotaxis assays is underway. glr-5(ak57) worms also appear to have a
diminished brood size, which is more severe at elevated temperatures.
In order to examine the role of RIA in processing thermal information we have generated transgenic
strains which express an activated form of the GLR-1 glutamate receptor subunit, GLR-1 (A/T), under
control of the glr-5 promoter. GLR-1 (A/T) causes constant depolarization when expressed in other cells
(see abstract by Zheng et al.). We are currently characterizing the thermotaxis behavior of these strains.
Any thermotaxic phenotype observed in glr-5(ak57) may be a result of the inability of RIA to receive
signals from the presynaptic cell AIY. Hobert et al. have demonstrated that the gene ttx-3 is expressed
only in AIY. Using ttx-3::VAMP::CFP and glr-5::GLR-5::YFP constructs we are looking at the presynaptic
and post-synaptic components of this synapse to determine if glr-5 could be mediating the signal from
AIY.
254

MICROARRAY ANALYSIS OF GENE EXPRESSION
PATTERNS IN DAUER LARVAE
John Wang, Stuart K. Kim
West Coast Worm Meeting 2000
Dept of Developmental Biology, Stanford University, Stanford CA
DNA microarrays containing about 95% of the genome can now be used to analyze gene expression
differences from nearly the entire genome. We are using the full-genome microarrays to profile gene
expression differences associated with the dauer larvae.
Under conditions of starvation or extreme crowding, C. elegans enter an alternative arrested stage, the
dauer larva. These larvae exhibit a unique morphology, an extended life span and resistance to
environmental stresses. The decision to form dauers is controlled by TGF-beta and insulin signaling
pathways.
The dauer larvae is an ideal system for microarray analysis. The dramatic morphological and
physiological changes that occur upon entrance into and exit from the dauer larvae suggest global
transcriptional regulation. In principle, by examining the gene expression differences associated with the
dauer larvae on the whole-genome level, we will be able to illuminate the complete set of genes that are
implicated for the dauer-specific attributes. In particular, these candidate genes would provide a
framework for understanding dauer-specific characteristics, such as altered energy metabolism, certain
aspects of aging and longevity, stress resistance, and the coordinated regulation and execution of events
necessary for a complex morphological change.
To identify the genes involved with dauer exit, we are performing DNA microarray experiments with RNA
isolated at different time points after addition of food to a pure dauer population. Each RNA sample is
compared to a common reference RNA, and multiple RNA samples are prepared for each time point. We
will identify those genes that are reproducibly altered during dauer exit and examine their kinetic profiles.
This analysis should reveal the temporal sequence of events that occur during dauer recovery at the
transcriptional level.
255

CHARACTERIZATION OF CAN CELL AND EXCRETORY
CANAL DEFECTS IN MIG-10(CT41) MUTANTS
Nicole Washington, Jim Manser
West Coast Worm Meeting 2000
Dept. of Biology, Harvey Mudd College, Claremont CA 91711
We have continued our characterization of mig-10 mutant defects using a ceh-23-gfp reporter strain
(kyIs5 IV, kindly provided by Jennifer Zallen) which allows scoring of CAN cell bodies and axons (see
Manser et al. WCWM 1998 abstracts, p.167). In the current studies, we have focused on the amber
(putative null) mig-10(ct41) allele.
Nearly 90% of CAN cell bodies (61/68) are significantly displaced anteriorward in mig-10(ct41);kyIs5
animals, compared to less than 8% (5/64) for the kyIs5 strain. Manser and Wood (1990) previously
reported a penetrance of 60% for CAN cell body misplacement in mig-10(ct41). The higher penetrance
observed using the gfp reporter probably reflects a synergy between mig-10 and ceh-23-gfp similar to that
reported by Forrester et al. (1997) for other CAN migration mutants.
A significant percentage of posteriorly directed CAN axons in mig-10(ct41);kyIs5 animals (23/67)
terminate in positions significantly anterior to their normal target region near the anus. In contrast, all
anteriorly directed CAN axons appear to extend to their normal target region in the head. This apparent
directional bias is somewhat surprising given that mig-10(ct41) disrupts both anteriorward and
posteriorward cell body migrations (Manser and Wood, 1990). One possibility is that the CAN axon
defects are an indirect result of the shortening of the posterior excretory canals observed in mig-10(ct41)
(Manser and Wood, 1990). Specifically, because CAN axons are closely associated anatomically with the
excretory canals (CAN=canal associated neuron), perhaps the canals serve as guidance cues for axon
outgrowth.
To investigate this possibility, we have scored both CAN axon and posterior excretory canal defects in the
same individuals. In more than 90% of mig-10(ct41);kyIs5 animals scored (28/31), the posteriorly directed
CAN axon extended significantly beyond the point of termination of the posterior excretory canal. This set
includes animals in which the posterior CAN axon extends to the anal region while the posterior excretory
canal terminates significantly anterior to the vulval region. Due to the generally severe truncation of the
posterior canals caused by mig-10(ct41), we have also observed animals in which the CAN cell body is
positioned posterior to the canal terminus. In such animals, anteriorly directed CAN axons were observed
to extend over regions of the anterior-posterior axis from which canals were missing. These observations
do not support the view that the excretory canals serve as guidance cues for CAN axon outgrowth.
256

RIC-7 ENCODES A NOVEL PRESYNAPTIC PROTEIN
REQUIRED FOR NEUROTRANSMISSION
Robby M. Weimer, Erik M. Jorgensen
University of Utah, Salt Lake City, UT 84112
West Coast Worm Meeting 2000
Regulated release of neurotransmitter from presynaptic terminals requires the coordinated function of
several presynaptic proteins. Our current knowledge of this process has come primarily from biochemical
analysis of synaptic proteins. We have taken a genetic approach in Caenorhabditis elegans to
complement these studies. Previously, we reported the identification of a novel protein, RIC-7, identified
genetically by screens for neurotransmission mutants. We now report that RIC-7 functions in neurons
and is localized to presynaptic terminals.
Mutations in ric-7 result in a pleiotropic neurotransmission phenotype. ric-7 animals lack enteric muscle
contractions during defecation and display a weak shrinker phenotype, indicating of a loss of GABAergic
function. In addition, ric-7 animals are resistant to the acetylcholinesterase inhibitor aldicarb, although
they remain sensitive to the acetylcholine receptor agonist levamisole indicating a presynaptic cholinergic
defect. These phenotypes are not due to defects in neuronal developmental since the overall structure of
the nervous system appears normal. This suggests that mutations in ric-7 disrupt a common presynaptic
step in chemical neurotransmission.
The ric-7 phenotype is rescued by an 18kb PCR fragment that contains a single open reading frame
encoding a predicted 694 amino acid protein with no similarities to known proteins or functional motifs.
Expression of the reporter gene green fluorescent protein (GFP) from the ric-7 promoter identifies a
predominantly neuronal expression pattern. RIC-7 GFP fusion protein is localized to synaptic terminals
independent of UNC-104 function. Furthermore, expression of RIC-7 solely in GABAergic neurons
rescues all GABA-specific phenotypes.
Together, these data suggest that ric-7 encodes a neuronal protein that is localized to and functions at
presynaptic terminals. In addition, ultrastructural data indicate that synaptic vesicle biogenesis is not
affected in a ric-7 mutant background. Thus, RIC-7 is likely to play a role in synaptic vesicle exocytosis.
Ongoing experiments are designed to identify RIC-7 interacting proteins and to determine the molecular
function of RIC-7 in neurotransmission.
257

West Coast Worm Meeting 2000
THE REQUIREMENT OF SYNAPTIC VESICLE LOADING FOR
SYNAPTIC VESICLE EXOCYTOSIS
Robby M. Weimer, Janet E. Richmond, Erik M. Jorgensen
Department of Biology, University of Utah, Salt Lake City, UT 84112-0840
Neurotransmitters are released from presynaptic terminals by the fusion of synaptic vesicles with the
plasma membrane. The amount of neurotransmitter in a vesicle causes a consistent amount of current,
called a quanta, in the postsynaptic cell. Studies have shown that quanta are invariant within a synapse.
This observation raises an interesting question. Does synaptic vesicle fusion require the proper loading
of neurotransmitter into the vesicle? We have characterized a mutant in C. elegans that will allow us to
address this question.
Loading of neurotransmitter into synaptic vesicles requires a vesicular neurotransmitter-specific
transporter and a driving force. Several studies have shown that the vacuolar H+ ATPase (vATPase)
generates the required driving force for loading. Previously, we reported the cloning of the C. elegans
homolog of the vacuolar ATPase B subunit, which we now refer to as vha-12 (vacuolar-type H+ ATPase).
A hypomorphic allele of vha-12 results in a pleiotropic neurotransmission phenotype which is consistent
with the know function of the vATPase in neurotransmission. However, animals homozygous for the
hypomorphic allele are not paralyzed. This suggests that neurotransmitter is still released from
presynaptic terminals in a vha-12 animal.
Two models could account for this observation (1) synaptic vesicles are fusing with the plasma membrane
with incomplete loading of neurotransmitter or (2) only completely loaded vesicles are competent to fuse
and the proportion of competent vesicles are decreased in a vha-12 mutant background. In order to
distinguish between these two models we are characterizing neurotransmitter release events in a vha-12
background by electrophysiologically recording from neuromuscular junctions.
258

West Coast Worm Meeting 2000
UNRAVELING THE BIOLOGICAL ROLE OF DMWD, A GENE
CLOSE TO THE UNSTABLE CTG-REPEAT IN THE MYOTONIC
DYSTROPHY LOCUS.
J. Westerlaken 1 , B. Wieringa 2 , P.E. Mains 1
1Department of Biochemistry & Molecular Biology, University of Calgary, Canada
2Department of Cell biology University of Nijmegen, the Netherlands
Myotonic Dystrophy (DM) is a frequent autosomal dominant disorder, caused by the expansion of an
unstable (CTG)n-repeat. The expanding repeat is located in the 3’-untranslated region of the DM protein
kinase gene (DMPK). The DMPKgene is located in a gene dense area, therefore it is unclear if it is
theonly gene involved in disease manifestation. Another possible candidate is the DMWD gene (formally
known as DMR-N9 in mouse, or gene 59 in human), which is located closely upstream of the DMPK
gene. The DMWD gene is mainly expressed in brain, testis and some smooth muscle tissues. In brain the
protein is located in many areas, but most prominently in places where there are synaptic glomeruli. In
testis the protein appears to be located in several distinct stages of spermatogenesis.
The DMWD gene is conserved during evolution and has homologs in plant, yeast and nematodes. To
futher understand the biological role of DMWD we started to investigate the C. elegans DMWD gene. In
C. elegans DMWD RNA is expressed in embryos and gravid adults. Preliminary results from RNAi studies
suggest that some progeny of the injected worms are sterile. We are further planning to do RNA in situ
hybridisation, GFP tagging of the protein and antibody staining experiments.
259

CALCIUM IMAGING OF THE DEFECATION RHYTHM IN C.
ELEGANS
Jeanna M. Wheeler, James H. Thomas
Department of Genetics, University of Washington, Seattle, WA 98195
Ultradian rhythms (those with a period shorter than 24 hours) have been shown to regulate a number of
biological processes, such as the vertebrate heartbeat and invertebrate swimming behavior. The
defecation cycle in C. elegans is an ultradian rhythm regulated by a clock-like mechanism (1). The cycle
has a 45 to 50 second periodicity that is insensitive to changes in temperature between 18° and 30° C. In
addition, the clock mechanism can keep time in the absence of the defecation motor program (when
animals leave their food source), indicating that the clock is distinct from the motor program. It has been
shown that the inositol trisphosphate (IP 3 ) receptor, a calcium channel that regulates cytoplasmic Ca 2+
concentration by release from intracellular stores, is required in the intestine for the defecation cycle (2).
Furthermore, calcium imaging with fura-2 revealed that periodic calcium spikes occur in the most
posterior intestinal cell, just preceding the initiation of each posterior body-wall muscle contraction (pBoc).
The pBoc begins at the posterior of the animal and proceeds anteriorly in a wave-like motion. There are
two simple possibilities for how the calcium spike signals to the posterior body-wall muscles: 1) Ca 2+ may
initiate secretion of a muscle activator only in the posterior intestine, which then propagates anteriorly
through the pseudocoelomic space or by muscle-to-muscle signaling, or 2) the Ca 2+ spike may be
propagated anteriorly through the intestinal cells themselves, initiating signaling to adjacent muscle cells
along the way. In order to distinguish between these possibilities, I am constructing a strain that
expresses fluorescent indicators for Ca 2+ ("cameleons") in the intestine. This strain will be used to
characterize the defecation cycle in wild type worms, as well as in various mutant strains with cycle
defects.
1. D.W. Liu and J.H. Thomas, 1994. J Neurosci 14: 1953-1962.
2. P. DalSanto et al, 1999. Cell 98: 757-767.
West Coast Worm Meeting 2000
260

ESTABLISHING THE LEFT/RIGHT ASYMMETRY OF Q
NEUROBLAST POLARISATION AND MIGRATION IN C.
ELEGANS
Lisa Williams, Lee Honigberg, Cynthia Kenyon
University of California, San Francisco
West Coast Worm Meeting 2000
The QL and QR neuroblasts are born in bilaterally symmetric positions along the A-P body axis in C.
elegans (QL on the left and QR on the right). Shortly after hatching, the two cells undergo characteristic,
stereotypical L/R asymmetric migrations. QL extends a long process to the posterior and its nucleus then
migrates into this projection. Following this migration, the Hox gene mab-5 is expressed in QL and its
descendants remain in the posterior. mab-5 expression is both necessary and sufficient for Q
descendants to remain in the posterior. Conversely, QR extends a process to the anterior and
subsequently migrates anteriorly. mab-5 is not expressed in QR, hence its descendants continue to
migrate anteriorly. Each cell moves to a well-defined final position.
In unc-40 or dpy-19 mutants, polarisation of both Q cells is randomised and the Q nuclei fail to undergo
their migrations. This randomisation of Q polarisation is coupled with a randomisation of mab-5
expression. Either cell, QL or QR, can express mab-5, and this expression then determines the fate of
that cell’s descendants.
In order to gain a more complete picture of how the initial L/R asymmetry of the Q cells is established, we
are studying 11 new mutants from an extensive Q cell migration screen (Queelim Ch’ng and Mary Sym,
unpublished results). These mutants fall into 4 complementation groups and have similar phenotypes to
unc-40 and dpy-19, and are therefore good candidates for new genes in this pathway. Work is underway
to characterise, map and clone these new genes. qid --1 (Q Is Defective, 8 alleles) maps to a 180kb
region on the left arm of chromosome III, and is rescued by injection of a cosmid pool covering this
region. qid-2 (1 allele) maps to the right arm of chromosome X, and further mapping is underway.
Mapping and further characterisation are also planned for qid-3 (1 allele) and qid-4 (1 allele), although
these mutants have less penetrant Q phenotypes and appear to be more pleiotropic.
261

A SCREEN FOR CELL MIGRATION AND AXON OUTGROWTH
MUTANTS
Jim Withee, Gian Garriga
West Coast Worm Meeting 2000
Molecular and Cellular Biology, University of California, Berkeley
Proper development of the nervous system requires that individual neurons and their processes arrive at
precise coordinates to form the correct pattern of connectivity. Directed migration of cells and growth
cones involves recognition of environmental cues by receptor proteins and the translation of these cues
into growth or movement to precise destinations. Thus, a primary goal of developmental neurobiology is
the identification and characterization of directional cues, their cognate receptors and the intracellular
proteins that interpret these cues.
The left and right CAN cells are born in the head and migrate posterior to the gonad primordium. After
migration is complete, the CAN cells each extend a single, unbranched axon anterior to the base of the
nerve ring and posterior to the lumbar ganglia. Worms expressing GFP from the ceh-23 promoter exhibit
fluorescence from a subset of neurons including the CAN cell (Wang et al, 1993, Cell 74). We are utilizing
a ceh-23::GFP expressing strain and fluorescence microscopy to screen directly for mutations that disrupt
proper migration and axon outgrowth of the CANs. To date, we have identified at least five
complementation groups that display defective cell migration and axon outgrowth. The mutants exhibit a
wide variety of CAN axon defects ranging from simple truncation to extensive ectopic outgrowth and
branching. We are in the process of mapping and characterizing the mutations as we screen for additional
mutants. Because a large portion of cell and axon guidance mechanisms appear to be conserved from C.
elegans to vertebrates, it is likely that some of the genes identified in this screen will play a conserved role
in nervous system development.
262

West Coast Worm Meeting 2000
MAPPING AND CHARACTERIZATION OF HAD-1, AN HSN
AXON GUIDANCE GENE
Lianna Wong, Jim Rader, Gian Garriga
Molecular and Cell Biology, University of California, Berkeley, CA 94720-3204
To understand how growth cones are guided to their synaptic targets, we are studying the outgrowth of
the HSN axons. The HSNs are a bilaterally symmetric pair of serotonergic motor neurons that innervate
the vulval muscles and stimulate egg laying in C. elegans. The mature HSN axon morphology can be
visualized using gmIs1, an integrated arrestin::GFP reporter that illuminates the HSN cell bodies and
axons. In wild-type animals, each HSN axon extends ventrally to the ipsilateral ventral nerve cord (VNC)
tract, turns anteriorly along it, then skirts dorsolaterally around the vulva, elaborating varicosities and
small branches that innervate the vulval muscles. The left and right HSN axons then continue their
anteriorly-directed extensions in their respective, parallel axon tracts until they reach the nerve ring in the
head.
Our lab performed a genetic screen for mutants defective in HSN axon guidance. Two of the mutants
isolated, gm188 and gm204, display grossly abnormal HSN axon outgrowth patterns, consisting of circling
and looping of the axons around the vulva, elaboration of excessively long branches, and multiple
crossovers of the axons between the left and right VNC tracts. In some mutant animals, the HSN axons
stop before reaching the nerve ring; in others, the HSN axons turn posteriorly and migrate back to or
beyond the vulva. The gm188 and gm204 mutations both map to LG V and fail to complement one
another. We have tentatively named the gene defined by these mutations as had-1 (HSN axon defective).
Three-factor mapping places had-1 between sma-1 and him-5. There are a number of overlapping
deficiencies in the region; nDf42 and yDf12 both uncover had-1, while arDf1 does not, placing had-1
between the right breakpoint of arDf1 and him-5, a region of less than one map unit. We have injected
had-1 mutants with cosmids in this region, but thus far have not achieved rescue of the HSN phenotype.
To more precisely define the position of had-1 in this interval, we are presently mapping had-1 relative to
cloned genes and Tc1 polymorphisms.
In addition to mapping and cloning had-1, we are characterizing the HSN axon defects of had-1 mutants
in animals lacking vulval cells and sex muscles to ascertain what roles these tissues play in the Had-1
HSN axon phenotype.
263

West Coast Worm Meeting 2000
RECOGNITION OF X-CHROMOSOME-ENRICHED DNA
ELEMENTS BY DOSAGE COMPENSATION PROTEINS
Tammy F. Wu 1 , Jason D. Lieb 2 , Barbara J. Meyer 1
1Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California,
Berkeley, CA, 94720
2Howard Hughes Medical Institute and Department of Biochemistry, Stanford University Medical Center,
Stanford, CA 94305
In C. elegans X-chromosome dosage compensation is accomplished by repressing gene expression from
both hermaphrodite X chromosomes to equal expression from the single male X. This essential process is
carried out through the action of a large protein complex that localizes to X starting around the 50-cell
stage in XX, but not XO, embryos.
To study how dosage compensation proteins come to be localized to X chromosomes, we are
investigating whether dosage compensation proteins recognize specific cis-acting elements. Previously,
dosage compensation proteins were shown to localize to extrachromosomal arrays bearing specific
regions of the promoter of the autosomal her-1 gene, whose expression is required for male development.
Thus, one approach to the problem of X recognition is to test candidate elements for their ability to be
recognized by dosage compensation proteins. To find such candidates, we have searched the genome
for sequence elements that occur more often on X than on autosomes. Sixteen such elements were
identified, and were found to occur in a wide variety of distributions throughout the X chromosome. These
candidates are being tested for recognition by dosage compensation proteins on arrays in vivo, as
previously described (Carmi et al., Nature 1998, and Dawes et al., Science 1999). Because the sequence
context of these elements may be important for recognition, we are using PCR to amplify products
containing unique surrounding sequence, as well as the candidate element itself.
Two PCR products tested have yielded promising results. Interestingly, the sequences tested have a
roughly 250 base pair region in common, which is itself enriched on the X chromosome. We are currently
investigating the significance of these sequences.
264

West Coast Worm Meeting 2000
RAC-LIKE GTPASES AND CELL MIGRATION
Yi-Chun Wu, Li-Chun Cheng, Nei-Yin Weng, Ting-Wen Cheng
Zoology Department, National Taiwan University, No 1 Roosevelt Road Sec4, Taipei 10617, Taiwan,
Republic of China
Cytoskeletal rearrangement and cell polarization are important for fundamental cellular processes, such
as cell migration and the phagocytosis of apoptotic cells. We are interested in how migration cues and
apoptotic signals are interpreted by the receiving cells and subsequently affect their cytoskeletal
rearrangement during cell migration and cell-corpse-phagocytosis in Caenorhabditis elegans.
At least two C. elegans Rac-like GTPases have been shown to be important for cell migration. MIG-2 is
necessary for the migration of Q cells, Q cell descendents and coelomocytes (1). CED-10 is important for
the migration of distal tip cells as well as the engulfment of apoptotic cells (2). To test if these two
GTPases may function redundantly in the migration of other cells, we have constructed ced-10;mig-2
double mutants and are currently analyzing the mutant phenotype. It has been proposed that ced-10 may
function downstream of ced-2 and ced-5 during cell-corpse-engulfment (2). We have also constructed
ced-2; mig-2 and ced-5; mig-2 double mutants. Results will be presented at the meeting.
1. Zipkin et al., Cell, 90, 883-894 (1997)
2. Reddien and Horvitz, Nature Cell Biology, 2, 131-136 (2000)
265

TWO NEW GENES REGULATING NEUROBLAST MIGRATION
IN C. ELEGANS
Lucie Yang, Mary Sym, Queelim Ch’ng, Cynthia Kenyon
Box 0448, 513 Parnassus, Dept. of Biochemistry, San Francisco, CA 94143-0448
In C. elegans, the Q cells are bilaterally symmetric neuroblasts present in the posterior body region of the
worm at hatching. During the first larval stage, the Q cells divide and migrate. QR and its descendents
migrate anteriorly whereas QL and its descendents migrate posteriorly. Several genes that regulate the
anterior migrations of QR and its descendents have been identified. These include: 1) lin-39, a homeobox
gene required in QR and its descendents for migration (Wang B. et al, 1993; Clark S. et al, 1993); 2)
mig-13, a novel transmembrane protein required outside of QR and its descendents for migration (Sym M
et al, 1999); and 3) egl-20, a Wnt homolog expressed in cells in the tail region (Whangbo J.. and Kenyon
C, 1999). Two mutant screens (Mary Sym and Queelim Ch’ng) were conducted to identify additional
genes that regulate the migration of QR and its descendents.
From these screens, mutations in two new genes were identified that cause certain cells in the QR
lineage to stop migrating prematurely. These two genes seem likely to be involved in guidance rather than
in providing cells with the ability to migrate because, in these mutants, other cells sometimes migrate in
the wrong direction. Current efforts are directed toward cloning these two genes and determining how
these genes regulate the migration of QR and its descendents together with genes previously known to
regulate these migrations.
References:
West Coast Worm Meeting 2000
Clark SG, Chisholm AD, and Horvitz HR. 1993. Control of Cell Fates in the Central Body Region of C.
elegans by the Homeobox Gene lin-39. Cell 74: 43-55.
Sym M, Robinson N, Kenyon C. 1999. mig-13 Positions Migrating Cells Along Anteroposterior Body Axis
of C. elegans. Cell 98: 26-36.
Wang BB, Muller-Immergluck MM, Austin, J, Robinson, NT, Chisholm, A, Kenyon, C. A Homeotic Gene
Cluster Patterns the Anteroposterior Body Axis of C.. elegans. Cell 74: 29-42.
Whangbo J, Kenyon, C. A Wnt Signaling System that Specifies Two Patterns of Cell Migration in C.
elegans. Molecular Cell 4: 851-858.
266

West Coast Worm Meeting 2000
IDENTIFICATION AND CHARACTERIZATION OF TELOMERE
BINDING PROTEINS IN THE NEMATODE C. ELEGANS
Su Young Yi, Seunghyun Kim, Junho Lee
Department of Biology, Yonsei University, Seoul 120-749
Telomeres are multifunctional elements that shield chromosome ends from degradation and end-to-end
fusions, prevent activation of DNA damage checkpoints, and modulates the maintenance of telomeric
DNA by telomerase. Telomeric DNA has been found to interact with proteins in many organisms. For
example, hTRF1, a human telomeric-repeat binding factor, is involved in the telomere length regulation.
We wanted to identify and elucidate functions of telomere binding proteins in the nematode C. elegans.
Telomeric repeat of C. elegans consists of a long stretch of (TTAGGC)n.
1. We have isolated Ceh-37, a novel homeotic protein in C. elegans, as double-stranded telomere binding
factor by yeast one-hybrid screening. We found that Ceh-37 specifically binds C. elegans telomere in
vitro. We found that the N-terminal domain and the homeotic domain is sufficient for telomere binding. We
also found that Ceh-37 binds the telomere as dimers, and that the N-terminal is required for the
dimerization. Ceh-37 is expressed not in all cells, but in subsets of cells. Over-expression of Ceh-37
caused death in late larval stages.
2. We have identified a protein (CeST-BP) in C. elegans embryonic nuclear extract that specifically binds
single-stranded telomere sequences by gel mobility shift assay. We found that the two Ts and Gs of the
telomere sequence were necessary to efficiently bind the C. elegans single-stranded telomere binding
protein (CeST-BP). CeST-BP did not efficiently bind with repeated RNA sequences of UUAGGC, thus we
concluded that CeST-BP bound specifically with DNA only. We also found that CeST-BP needs at least
2.5 repeats of (GGCTTA) sequence for binding. CeST-BP was sensitive to salt concentrations, and
insensitive to RNase treatment. The size of CeST-BP was found to be about 40 kD in South-Western
hybridization. We plan to identify CeST-BP by purification using DNA-streptavidin sepharose affinity
chromatography and MALDI-TOF protein sequence search. Subsequently, studies of the functions of
CeST-BP in vivo will follow.
267

West Coast Worm Meeting 2000
MOLECULAR ANALYSIS OF THE DOSAGE COMPENSATION
GENE DPY-21
Stephanie Yonker, Edith Cookson, Barbara J. Meyer
Howard Hughes Medical Institute and Department of Molecular and Cell Biology, University of California,
Berkeley, CA 94720
Dosage compensation in Caenorhabditis elegans equalizes X chromosome expression between XO
males and XX hermaphrodites. DPY-21 is one of several proteins required for proper dosage
compensation. However, DPY-21 is unique among known dosage compensation proteins. While other
dosage compensation mutations cause maternal-effect lethality and affect only XX animals, dpy-21
mutations cause only modest lethality and affect both XX and XO animals. Furthermore, different assays
of X-linked gene expression have shown that dpy-21 mutations cause both elevation and reduction in
X-linked gene expression in XO animals. We have cloned dpy-21 by injecting single-stranded RNA from
candidate ESTs that map to a YAC close to dpy-21. Two positive ESTs were found to represent the same
gene and were used to assemble the dpy-21 mRNA sequence in conjunction with 5’ and 3’ RACE
products. To verify that we had identified dpy-21, we sequenced six dpy-21 alleles and found DNA lesions
within the predicted coding sequence. Three of the sequenced dpy-21 alleles contain premature STOP
codons. The 5610 nucleotide dpy-21 transcript, which is both SL1 and SL2 spliced, encodes a novel 1651
amino acid protein. While there is no sequence similarity to known proteins, putative homologues are
present in Drosophila melanogaster and human genomes. Preliminary immunoflourescence experiments
with DPY-21 antibodies indicate that DPY-21 localizes to the nucleus.
268

A SEARCH FOR LETHAL SYNAPTIC FUNCTION MUTANTS
USING A SENSITIZED BACKGROUND
Karen Yook, Erik Jorgensen
West Coast Worm Meeting 2000
Dept. of Biology, University of Utah, Salt Lake City, UT 84112 USA
We are interested in identifying genes essential for neurotransmitter release. However mutations in
genes essential for neurotransmission can result in L1 lethality, as observed with animals homozygous for
null mutations in cha-1, the biosynthetic enzyme for acetylcholine. In the course of characterizing
synaptic function mutants, we noticed that mutations in separate synaptic function loci do not complement
one another when heterozygous, that is, they exhibit nonallelic noncomplementation. In an in-depth
analysis of nonallelic noncomplementation among synaptic function alleles, we demonstrated that
nonallelic noncomplementation occurs between lethal synaptic function alleles and hypomorphic alleles at
other loci (manuscript in preparation). Therefore, in order to uncover synaptic function loci which have
been mutated to lethality, we adopted a nonallelic noncomplementation strategy to screen for mutations
that interact with a hypomorphic allele of unc-13. Specifically, we looked for double heterozygotes (mut/+;
unc-13(n2813)/+) which decrease neurotransmitter release. Although the noncomplementation
interaction between synaptic function loci is robust, we increased the sensitivity of our screening assay
with an additional background mutation in unc-29, a subunit of the acetylcholine receptor. Therefore we
carried out a screen for genes whose functions are effected by mutations in unc-13 and unc-29. Initially,
we screened 2800 EMS mutagenized genomes in this sensitized background and uncovered 117
potential synaptic function loci of which 37 exhibited an uncoordinated phenotype on its own and another
12 exhibited L1 lethality. Characterization of the uncoordinated mutants identified mutant alleles of
unc-26, unc-2, aex-3, ric-4, unc-38 all synaptic function loci known to be involved in neurotransmission.
In addition, we uncovered a novel locus. These results suggest that the sensitized background is a good
strategy for identifying the components of the synaptic transmission machinery. More recently, we used
the ENU mutagen and screened through 3900 genomes. This screen has turned up 134 potential
synaptic function mutations; 22 of these mutations are associated with a lethal phenotype, of which 13
exhibit L1 lethality. We are currently pursuing the identity of the lethal loci.
269

IDENTIFICATION OF DOWNSTREAM TARGET GENES IN
DAF-2 PATHWAY
Hui Yu, Pamela L. Larsen
West Coast Worm Meeting 2000
Molecular Biology Program and Division of Biogerontology, University of Southern California, Los
Angeles, CA 90089
In a search for downstream genes in the daf-2 insulin-like receptor signaling pathway, nine genes were
identified by differential display PCR and verified by Northern analysis to be differentially expressed in
daf-2 mutants compared to N2 animals. The genes discovered have been named dao for daf-2 pathway
over- and under-expressed genes. Three dao genes, dao-1, dao-8 and dao-9, are homologs of an
intracellular transducer FK-506 binding protein. These three genes were down-regulated in daf-2(m41)
animals and this effect in part relies upon daf-16. Among the other six genes, dao-2, dao-3 and dao-4
were positively regulated by daf-2 signaling, while the opposite pattern was observed for dao-5, dao-6
and dao-7. dao-2, dao-4 and dao-6 encode novel proteins. By homology, dao-3 is probably a
methylenetetrahydrofolate dehydrogenase. The predicated DAO-5 protein has many phosphorylation site
repeats and showed 38% identity to xNopp180, a nuclear localization sequence-binding protein shuttling
between the cytoplasm and the nucleus. dao-7 contains a zinc finger region similar to mammalian ZFP36
protein and thereby is a potential transcription factor. Distinct expression patterns and molecular identities
of these new downstream targets in the daf-2 pathway indicates that daf-2 signaling is a complicated
signaling network involved in multiple cellular processes.
270

FATE SPECIFICATION IN MALE P(9-11).P EQUIVALENCE
GROUP
Hui Yu, Paul W. Sternberg
West Coast Worm Meeting 2000
HHMI and Division of Biology, California Institute of Technology, Pasadena, CA 91125
C. elegans P(9-11).p cells in male pre-anal ganglion (PAG) equivalence group have three potential fates,
1° , 2° , and 3° . In wild-type males, P10.p and P11.p adopt the 2° and 1° respectively, divide to generate
distinct subsets of cells. P9.p (3° fate), which is undivided, fuses with hyp7. osm-6::gfp is specifically
expressed in two hook neurons HOA and HOB derived from the 2° lineage. To understand the fate
specification in P(9-11).p cells, we constructed a series of strains carrying the osm-6::gfp marker with
mutations in lin-12, lin-15 and mab-5 genes. Instead of normal one pair of GFP expressing cells, lin-12(gf)
mutants showed 2-3 pairs of GFP expression characteristic of the HOA and HOB neurons, indicating a
transformation to the 2° fate in P9.p and P11.p cells. In lin-15(lf) mutants, P9.p could divide and a second
pair of GFP expression was observed anteriorly sometimes because of a P9.p Æ P10.p fate
transformation. The results suggested that the lin-12 activity is sufficient to promote the 2° fate and lin-15,
a negative regulator of let-23 -ras pathway, is required for the 3° fate. The roles of mab-5 and let-23/ras
are under investigation. A genetic screen for other candidates involved in P(9-11).p fate determination is
also in progress.
271

West Coast Worm Meeting 2000
LOSS OF A DYNAMIN RELATED PROTEIN MGM-1 CAUSES
EXCESSIVE MITOCHONDRIAL FRAGMENTATION
Mauro Zappaterra, Alexander van der Bliek
Department of Biological Chemistry, UCLA School of Medicine, P.O. Box 951737, Los Angeles, CA
90095-1737
Our lab studies the functions of dynamin and dynamin related proteins. These proteins form a small family
of large GTP-binding proteins. We have recently shown that the C. elegans dynamin related protein
DRP-1, is involved in the scission of the mitochondrial outer membrane (1). Since little is known about
mitochondrial division and mitochondrial morphology in animal cells, we are investigating the mechanisms
that regulate these processes.
MGM-1 is another member of the dynamin family present in animal cells. As of now we know that this
protein is present in yeast, C. elegans, and humans. Unlike other members of the dynamin family, MGM-1
has an N-terminal mitochondrial leader sequence. MGM-1 is also more closely related to bacterial
dynamin-like proteins than it is to dynamin or DRP-1, suggesting that this protein has followed a different
evolutionary path. We speculate that MGM-1 was introduced into eukaryotic cells by the progenitors of
mitochondria. Expression studies using ß-galactosidase under the control of the mgm-1 promoter show
high levels of expression in intestines, body wall muscles, and neurons. These high levels might reflect
high metabolic rates in those tissues, because the MGM-1 expression pattern is similar to that of another
dynamin-related protein, DRP-1, which is also important for mitochondrial maintenance. Mgm-1 RNAi
drastically slows the growth rate and approximately doubles the life span of C. elegans. We show that
excessive fragmentation of mitochondria is induced by RNAi and mgm-1 antisense cDNA. We present
evidence that MGM-1 is localized to the mitochondrial matrix where it might regulate the morphology of
the mitochondrial inner membrane.
1. Labrousse, A.M., Zappaterra, M.D., Rube, D.A., and van der Bliek, A.M. Molecular Cell 4: 815-826.
1999.
272

West Coast Worm Meeting 2000
ISOLATION AND PHENOTYPIC ANALYSIS OF SYD-7
Mei Zhen 1,2 , Nikki Alvarez 1 , Yishi Jin 1
1Department of Biology, 327 Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064
2zhen@darwin.ucsc.edu
Using a synaptic vesicle-tagged GFP marker, Punc-25-SNB-1::GFP, that is localized to the presynaptic
terminals of VD and DD motoneurons, we isolated two recessive loss-of-function mutations, ju43 and
ju58, that define the syd-7 locus. In wild-type animals, the Punc-25-SNB-1::GFP marker is expressed as
discrete fluorescent puncta spaced evenly along the ventral and dorsal sides of the animal. In syd-7
mutants some of the fluorescent puncta are absent in various regions, while the remaining puncta appear
normal in morphology and spacing. 90% of ju43 and ju58 animals lose SNB-1::GFP puncta in places
corresponding to the presynaptic regions of VD1 and VD2 neurons. 50% of ju58 animals also lack
SNB-1::GFP puncta in presynaptic regions of different DD neurons. Despite of the defects in GFP marker,
the locomotion of syd-7 animals is indistinguishable from wild-type animals. Absence of SNB-1::GFP
puncta can be caused by defects in either cell fate or neural differentiation. To distinguish these
possibilities we are examining the axonal morphology of D neurons and the expression of cell-type
specific markers in syd-7 animals.
We mapped syd-7 to chromosome V, in a small region overlapped by yDf12 and arDf1. This tentatively
places syd-7 between eat-6 and him-5. Currently we are further fine-mapping syd-7 and trying to rescue
syd-7 with cosmids in the region. We are also in the progress of testing for the genetic interactions
between syd-7 and genes known to be involved in the development of the nervous system.
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West Coast Worm Meeting 2000
A SCREEN TO IDENTIFY GENES THAT REGULATE THE
ACTIVITY OF THE C. ELEGANS GLUTAMATE RECEPTOR
GLR-1.
Yi Zheng, Heng Xie, Pene J. Brockie, Andres V. Maricq
Department of Biology, University of Utah, Salt Lake City, Utah 84112
Efficient synaptic transmission relies on an organized distribution of neurotransmitter receptors.
Activity-dependent changes in synaptic strength are believed to be crucial for information processing and
for the refinement of neuronal network during development. The presence of silent synapses and their
activation indicates rapid and activity-dependent changes in the number of glutamate receptors at
excitatory glutamatergic synapses. However, the molecular mechanisms underlying the regulation of the
density of receptors at synapses are unclear.
We have previously shown that the C. elegans glutamate receptor subunit GLR-1 is expressed in the
interneuronal circuitry that is required for normal locomotion. To perturb the function of this circuit, we
engineered into GLR-1 the A/T amino acid change first identified in the d2 glutamate receptor of the
mutant Lurcher mouse. Transgenic worms (akIs9) that express this mutant glutamate receptor show a
striking change in locomotion where they rapidly alternate between forward and backward movement,
presumably secondary to the constitutive depolarization of the command interneurons by the GLR-1(A/T)
channel. In contrast, worms that have a null mutation in GLR-1 (ky176) move about the same as wild-type
animals.
The dramatic difference between ky176 and akIs9 worms provides an opportunity to identify genes that
regulate glutamate receptor density in C.elegans. Mutations that decrease the membrane density of
GLR-1(A/T) should suppress the hyper-reversal phenotype of akIs9 worms and make them move more
like ky176 animals. We mutagenized the akIs9 strain and screened ~25,000 haploid genomes for
mutations that suppress the locomotory phenotype. We have isolated 17 candidate mutants that suppress
the hyper-reversal movement and we are in the process of characterizing them. Amongst these mutants,
we expect to find mutations that affect the insertion or stability of glutamate receptors at the synapse.
274

West Coast Worm Meeting 2000
A RESOURCE FOR C. ELEGANS MICROARRAYS
Stuart K. Kim, Min Jiang, Kyle Duke
Department of Developmental Biology and Genetics, Stanford University Medical Center, Stanford, CA
We are entering a new age in molecular genetics in which we can use sequence data from genome
projects to dissect cell, developmental and disease pathways more completely and more sensitively than
ever before. One key functional genomics approach is to use DNA microarrays to define changes in gene
expression patterns during development and during the onset of disease. DNA microarrays could be used
to identify genes that are regulated by specific transcription factors, by specific cell signaling pathways, by
expression of homologs of human disease genes in transgenic animals or by addition of various
pharmaceutical drugs.
We are currently producing DNA microarrays that contain every gene in the C. elegans genome. Our goal
is to develop C. elegans microarray technology, and to make this technology available to all other C.
elegans labs. We will provide these microarrays to any C. elegans lab by hybridizing their RNA samples
to the full genome microarrays. The results are deposited in the Stanford Microarray Database, and can
be accessed over the web. We have already performed about 200 microarray hybridizations using the full
genome microarrays, in collaboration with 27 different academic C. elegans labs. We are also helping to
provide needed tools and reagents so that other labs can establish their own microarraying facilities.
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