C. elegans Development, Cell Biology, & Gene Expression Meeting ...

PROGRAM & ABSTRACTS 

C. elegans Development, Cell Biology, & 

Gene Expression Meeting 2012 

Thursday, June 7 – Sunday, June 10, 2012 

University of Wisconsin-Madison 

Memorial Union 

800 Langdon Street 

Madison, Wisconsin 53706 

Meeting Organizers 

E. Jane Hubbard: Skirball Institute, NYU School of Medicine (jane.hubbard@med.nyu.edu) 

Jeremy Nance: Skirball Institute, NYU School of Medicine (jeremy.nance@med.nyu.edu) 

Ahna Skop: University of Wisconsin-Madison (skop@wisc.edu) 

Martha Soto: Robert Wood Johnson Medical School, UMDNJ (sotomc@umdnj.edu) 

2012 Organizing Committee 

Jon Audya, U. Wisconsin, Madison (USA) 

Zhirong Bao, Sloan-Kettering Institute (USA) 

Ryan Baugh, Duke U. (USA) 

Rafal Ciosk, Friedrich Miescher Institute for Biomedical Research, (Switzerland) 

Monica Colaiacovo, Harvard U. (USA) 

Monica Gotta, U. de Geneve (Switzerland) 

Alla Grishok, Columbia U. (USA) 

Caroline Goutte, Amherst College, (USA) 

Kristen Hagstrom, U. Mass Medical Center (USA) 

Max Heiman, Harvard Medical School (USA) 

Steve L’Hernault, Emory U. (USA) 

Valerie Reinke, Yale U. (USA) 

Lesilee Rose, U.C. Davis (USA) 

Rick Roy, McGill U., Quebec (Canada) 

Jennifer Schisa, Central Michigan U. (USA) 

Asako Sugimoto, Tohuko U., Sendai (Japan) 

Xiaochen Wang, NIBS, Bejing (China) 

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C. elegans Development, Cell Biology, & Gene 

Expression Meeting 


Conference Program 

Thursday, June 07, 2012 

12 noon–7:30 pm Registration Check-In Annex Room 

12 noon Poster Set up Great Hall, Reception Room, and Main Lounge 

5:00–7:00 pm Opening Reception Tripp Commons 

7:00 Opening Remarks 

7:00–9:00 pm Platform Session #1 Union Theater 

Morphogenesis I and Polarity 

Chairs: Lesilee Rose and Asako Sugimoto 

7:15 Keynote: Ken Kemphues 

Three pathways to polarity maintenance 

7:45 Jessica L Feldman (Lab: Priess) 

A role for the centrosome and PAR-3 in the hand-off of 

microtubule organizing center function during epithelial 

polarization 

8:00 Yelena Y Bernadskaya (Lab: Soto) 

Three Axonal Guidance Pathways Help Polarize the Actin 

Cytoskeleton During Embryonic Epidermal Cell Migration 

8:15 Jessica Shivas (Lab: Skop) 

Arp2/3 mediates early endosome dynamics that participate in 

the maintenance of polarity in C. elegans 

8:30 Hongjie Zhang (Lab: Gobel) 

Clathrin/AP-1 cooperate with sphingolipids to regulate apical 

polarity and lumen formation during C. elegans tubulogenesis 

8:45 Vijaykumar S Meli (Lab: Frand) 

The Fibrillin-like fbn-1 Gene Regulates Epithelial Stem Cell and 

ECM Dynamics in Molts 

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9:00–11:00 pm Poster Session #1 & Refreshments Great Hall, Reception Room, 

and Main Lounge 

(ODD number posters present) 

vi 

Great Hall & Reception Room (4th floor) 

Cell Biology 51 83 

Cell cycle and cytokinesis 84 92 

Cell Death 93 103 

Cell Fate 104 121 

Gene Regulation 122 145 

Germline 146 184 

Main Lounge (2nd floor) 

Morphogenesis 185 211 

New Technologies 212 220 

Polarity 221 231 

Sex Determination 232 234 

Friday, June 08, 2012 

7:00 am Registration continues Annex Room 

7:30–9:00 am Breakfast Buffet Inn Wisconsin 

9:00–10:45 am Platform Session #2 Union Theater 

Morphogenesis II & Cell Death 

Chairs: Max Heiman and Caroline Goutte 

9:00 Keynote: Shai Shaham (Lab: Shaham) 

A New C. elegans Cell Death Program: Implications for 

Neurodegeneration and Cancer 

9:45 Yan Zhang (Lab: Wang) 

C. elegans NRF-5 Regulates Cell Corpse Engulfment By 

Mediating PS Appearance On Phagocytes 

10:00 Sasha De HeBoldnau (Lab: Braeckman) 

Globin 12 of Caenorhabditis elegans Regulates the p38 and JNK 

MAPK Pathways through Redox Signaling to Control Germline 

Apoptosis 

10:15 Michael Hurwitz (Lab: Hurwitz) 

sli-1 Cbl Inhibits the Engulfment of Apoptotic Cells 

10:30 Matthias K Morf (Lab: Hajnal) 

MADD-2 Negatively Regulates Anchor Cell Invasion 

10:45 Vida Praitis (Lab: Praitis) 

The C. elegans Hailey-Hailey Disease Homolog pmr-1 is Essential 

for Cell Migration During Gastrulation 

11:00–11:15 am Refreshment Break Union Theater Lobby

11:15 am–1:00 pm Platform Session #3 Union Theater 

Germline I and Gametogenesis 

Chairs: Steve L’Hernault and Rafal Ciosk 

11:15 Keynote: David Greenstein (Lab: Greenstein) 

Control of Oocyte Meiotic Maturation: Links to Germ Cell 

Proliferation and Global Control of the Oogenic Program 

12:00 Kari Messina (Lab: Shakes) 

Regulators of MSP Assembly and Dynamics in C. elegans 

Spermatocytes 

12:15 Gunasekaran Singaravelu (Lab: Singson) 

The sperm surface localization of the TRP-3/SPE-41 

Ca2+ permeable channel depends on SPE-38 function in 

Caenorhabditis elegans 

12:30 Jun Takayama (Lab: Onami) 

Timely Generation of the Fertilization Calcium Wave by a 

Sperm TRP Channel 

12 :45 Simona Rosu (Lab: Villeneuve) 

Regulation of Meiotic DSB Formation in C. elegans 

1:00–2:30 pm Luncheon Buffet Inn Wisconsin 


Cell Cycle and Cell Biology 

Chairs: Jon Audhya and Richard Roy 

2:30 Keynote: Karen Oegema (Lab: Oegema) 

Title: TBD 

3:15 Marie Delattre (Lab: Delattre) 

Evolution of spindle shape and motion in one-cell stage 

nematode embryos 

3:30 Jill M Schumacher (Lab: Schumacher) 

The Tousled-like Kinase TLK-1 is a Component of the Outer 

Kinetochore and Potentiates Mitotic Spindle Dynamics in the 

Early C. elegans Embryo 

3:45 Asako Sugimoto (Lab: Sugimoto) 

Identification of unconventional components of the γ-tubulin 

complex in C. elegans 

4:00 Elsa Kress (Lab: Gotta) 

The Cdc48/p97 cofactor UBXN-2 and its orthologues p47/p37 

control centrosome maturation in prophase via Aurora A 

4:15–4:30 pm Refreshment Break Union Theater Lobby 

4:30 Anjon Audhya (Lab: Audhya) 

Regulation of COPII subunit recruitment to ER exit sites 

4:45 Joshua N Bembenek (Lab: Chan) 

Condensin I: A New Component of the Abscission Checkpoint 

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5:00 Matyas Gorjanacz (Lab: Mattaj) 

LEM-4 Coordinates Mitotic Signaling on BAF to Enable its 

Essential Function in Nuclear Envelope Formation 

5:15 Ismar Kovacevic (Lab: Cram) 

Filamin is Required to Initiate Calcium Signaling and Maintain 

F-actin Organization in the Spermatheca 

5:30–7:00 pm Dinner Buffet Inn Wisconsin 

7:00–9:00 pm Poster Session #2 & Refreshments Great Hall, Reception Room, 


(EVEN number posters present) 

viii 

Great Hall & Reception Room (4th floor) 

Cell Biology 51 83 

Cell cycle and cytokinesis 84 92 

Cell Death 93 103 

Cell Fate 104 121 

Gene Regulation 122 145 

Germline 146 184 

Main Lounge (2nd floor) 

Morphogenesis 185 211 

New Technologies 212 220 

Polarity 221 231 

Sex Determination 232 234 

9:00–11:30 pm Late Night Poster Session Great Hall, Reception Room, 


Open Viewing (All numbered posters present) 

Saturday, June 09, 2012 

7:00 am Registration Continues Annex Room 

7:30–9:00 am Breakfast Buffet Inn Wisconsin 

9:00 am–12:00 noon Platform Session #5 Union Theater 

Germline II, Meiosis, and Sex Determination/Dimorphism 

Chairs: Monica Colaiácovo and Jennifer Schisa 

9:00 Keynote: Monica Colaiácovo (Lab: Colaiácovo) 

Germline maintenance and meiosis: mechanistic insights from C. 

elegans 

9:30 Aaron Kershner (Lab: Kimble) 

Identification of Direct GLP-1/Notch Targets that Regulate 

Germline Stem Cells 

9:45 Rafal Ciosk (Lab: Ciosk) 

Genome-wide Analysis of GLD-1 Mediated mRNA Regulation 

Uncovers a Role in mRNA Storage

10:00 E. Jane Albert Hubbard (Lab: Hubbard) 

In the C. elegans Germ Line, S6K promotes Cell Cycle 

Progression and the Proliferative Fate and mediates the Effects 

of Diet 

10:50–10:30 am Refreshment Break Union Theater Lobby 

10:30 Mara Schvarzstein (Lab: Villeneuve) 

Chromosome and centrosome inheritance in meiosis 

10:45 Daniel Cortes Estrada (Lab: McNalley) 

Non-random Segregation of Unpaired X Chromosomes in C. 

elegans Female Meiosis (asbt. # 152) 

11:00 Anna K Allen (Lab: Golden) 

Role of the Inhibitory Kinase WEE-1.3 in Regulating the Meiotic 

Cell Cycle and Fertility in C. Elegans 

11:15 Michael J. White VanGompel (Lab: Rose) 

The Torsin Homolog OOC-5 is Required for Normal 

Nucleoporin Localization 

11:30 Matthew Berkseth (Lab: Zarkower) 

Identification of Direct Targets of the Caenorhabditis 

elegans Global Sexual Regulator TRA-1 by Chromatin 

Immunoprecipitation 

11:45 Te-Wen Lo (Lab: Meyer) 

Evolution of Caenorhabditis Dosage Compensation 

12:00–2:00 pm Luncheon Buffet (posters down by 2:00 pm) Inn Wisconsin 

2:30–4:00 pm Workshops Union Theater 

4:00–4:30 pm Refreshment Break Union Theater Lobby 


Gene Regulation 

Chairs: Valerie Reinke and Ryan Baugh 

Introduction: Alla Grishok 

4:30 Keynote: Craig Mello (Lab: Mello) 

RNAi and Immortality: Recognition of Self/non-Self RNA in the 

C. elegans Germline 

5:15 Gyorgyi Csankovszki (Lab: Csankovszki) 

The onset of dosage compensation is linked to the loss of 

developmental plasticity 

5:30 David J Katz (Lab: Katz) 

The Histone Demethylase SPR-5 and the Histone 

Methyltransferase MET-2 Comprise a Novel Epigenetic 

Reprogramming Switch 

5:45 Shouhong Guang (Lab: Guang) 

Nuclear RNAi mediates silencing of repetitive sequences in C. 


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6:00 Xiao-Dong Yang (Lab: Lin) 

Dimerization of γCatenin/WRM-1 Allows Intermolecular 

Autophosphorylation of LIT-1 in the Activation Loop 

6:15 Morris Maduro (Lab: Maduro) 

Organ defects in adults resulting from threshold blastomere 

specification 

7:00–9:30 pm Banquet Union South 

9:30–Midnight Dance Union South 

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Sunday, June 10, 2012 


Cell Fate and Emerging Technologies 

Chairs: Monica Gotta and Zhirong Bao 

9:00 am Keynote: Julie Ahringer (Lab: Ahringer) 

Title: TBD 

9:45 Hillel Kugler (Lab: Kugler) 

Modeling germline population dynamics 

10:00 Julia L Moore (Lab: Bao) 

Dev-scape: An intuitive tool for automated phenotyping with 

single cell resolution 

10:15 Abigail Cabunoc (Lab: Stein) 

WormBase 2012: Website Redesign 

11:00 Scott Robertson (Lab: Lin) 

DSL-2 Mediates a Notch Signal From EMS Descendant(s) to 

ABp Descendants 

11:15 Jennifer A Schumacher Tucker (Lab: Chuang) 

Intercellular Calcium Signaling in a Gap Junction Cell Network 

Establishes Left-Right Asymmetric Neuronal Fates 

11:30 Colin Maxwell (Lab: Baugh) 

Nutritional control of mRNA isoform expression during 

developmental arrest and recovery in C. elegans 

11:45 David J Reiner (Lab: Reiner) 

Ras and its Effector RalGEF Both Perform Dual, Antagonistic 

Functions during C. elegans Vulval Patterning 

12:00 Allison L Abbott (Lab: Abbott) 

The microRNA miR-786 is Required for Rhythmic Calcium 

Wave Initiation in the C. elegans Intestine 

12:30–2:00 pm Luncheon Buffet Inn Wisconsin

TABLE OF CONTENTS 

Thursday, June 07, 2012 - 7:00–9:00 pm 

Platform Session #1 - Union Theater 

Morphogenesis I and Polarity 

Abstracts 1 - 6 

Chairs: Lesilee Rose and Asako Sugimoto 

1 Keynote: Three pathways to polarity maintenance 

Ken Kemphues 

2 A role for the centrosome and PAR-3 in the hand-off of microtubule 

organizing center function during epithelial polarization 

Jessica Feldman, James Priess 

3 Three Axonal Guidance Pathways Help Polarize the Actin Cytoskeleton 

During Embryonic Epidermal Cell Migration 

Yelena Bernadskaya, Andre Wallace, Jillian Nguyen, William Mohler, Martha Soto 

4 Arp2/3 mediates early endosome dynamics that participate in the 

maintenance of polarity in C. elegans 

Jessica Shivas, Ahna Skop 

5 Clathrin/AP-1 cooperate with sphingolipids to regulate apical polarity 

and lumen formation during C. elegans tubulogenesis 

Hongjie Zhang, Ahlee Kim, Nessy Abraham, Liakot Khan, David Hall, John Fleming, Verena 

Gobel 

6 The Fibrillin-like fbn-1 Gene Regulates Epithelial Stem Cell and ECM 

Dynamics in Molts 

Vijaykumar Meli, Alison Frand 

Friday, June 08, 2012 - 9:00–10:45 am 


Morphogenesis II & Cell Death 


Chairs: Max Heiman and Caroline Goutte 

7 Keynote: A New C. elegans Cell Death Program: Implications for 


Shai Shaham 

8 C. elegans NRF-5 Regulates Cell Corpse Engulfment By Mediating PS 

Appearance On Phagocytes 

Yan Zhang, Haibin Wang, Xiaochen Wang 

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9 Globin 12 of Caenorhabditis elegans Regulates the p38 and JNK MAPK 

Pathways through Redox Signaling to Control Germline Apoptosis 

Sasha De Henau, Lesley Tilleman, Francesca Germani, Caroline Vlaeminck, Jacques 

Vanfleteren, Luc Moens, Sylvia Dewilde, Bart Braeckman 

10 sli-1 Cbl Inhibits the Engulfment of Apoptotic Cells 

Courtney Anderson, Shan Zhou, Emma Sawin, Bob Horvitz, Michael Hurwitz 

11 MADD-2 Negatively Regulates Anchor Cell Invasion 

Matthias Morf, Ivo Rimann, Mariam Alexander, Peter Roy, Alex Hajnal 

12 The C. elegans Hailey-Hailey Disease Homolog pmr-1 is Essential for 

Cell Migration During Gastrulation 

Vida Praitis, Rebecca Mandt, Leah Imlay, Charlotte Feddersen, Alexander Sullivan-Wilson, 

Tyson Stock, Walter Liszewski, Adityarup Chakravorty, Dae Gon Ha, Angela Schacht, 

Michael Miller, Lensa Yohannes, Juliet Mushi, Zelealem Yilma, Sarah Kniss, Jeff Simske 

xii 

Friday, June 08, 2012 - 11:15 am–1:00 pm 


Germline I and Gametogenesis 


Chairs: Steve L’Hernault and Rafal Ciosk 

13 Keynote: Control of Oocyte Meiotic Maturation: Links to Germ Cell 

Proliferation and Global Control of the Oogenic Program 

David Greenstein 

14 Regulators of MSP Assembly and Dynamics in C. elegans Spermatocytes 

Kari Messina, Marc Presler, Leah Towarnicky, Diane Shakes 

15 The sperm surface localization of the TRP-3/SPE-41 Ca2+ permeable 

channel depends on SPE-38 function in Caenorhabditis elegans 

Gunasekaran Singaravelu, Indrani Chatterjee, Sina Rahimi, Marina Druzhinina, Lijun 

Kang, Shawn Xu, Andrew Singson 

16 Timely Generation of the Fertilization Calcium Wave by a Sperm TRP 

Channel 

Jun Takayama, Shuichi Onami 

17 Regulation of Meiotic DSB Formation in C. elegans 

Simona Rosu, Anne Villeneuve

18 Keynote: Title: TBD 

Karen Oegema 

Friday, June 08, 2012 - 2:30–5:30 pm 


Cell Cycle and Cell Biology 


Chairs: Jon Audhya and Richard Roy 

19 Evolution of spindle shape and motion in one-cell stage nematode 

embryos 

Aurore-Cecile Valfort, Soizic Riche, Reza Farhadifair, Daniel Needleman, Marie Delattre 

20 The Tousled-like Kinase TLK-1 is a Component of the Outer 

Kinetochore and Potentiates Mitotic Spindle Dynamics in the Early C. 

elegans Embryo 

Jessica De Orbeta, Jason Ford, Gary Deyter, Tokiko Furuta, Jill Schumacher 

21 Identification of unconventional components of the γ-tubulin complex 

in C.elegans 

Nami Haruta, Eisuke Sumiyoshi, Yu Honda, Masahiro Terasawa, Mika Toya, Asako 

Sugimoto 

22 The Cdc48/p97 cofactor UBXN-2 and its orthologues p47/p37 control 

centrosome maturation in prophase via Aurora A 

Elsa Kress, Francoise Schwager, Rene Holtackers, Esther Zanin, Francois Prodon, Jonas 

Seiler, Annika Eiteneuer, Asako Sugimoto, Hemmo Meyer, Patrick Meraldi, Monica Gotta 

23 Regulation of COPII subunit recruitment to ER exit sites 

Kristen Witte, Amber Schuh, Jan Hegermann, Ali Sarkeshik, Jonathan Mayers, Katrin 

Schwarze, John Yates III, Stefan Eimer, Anjon Audhya 

24 Condensin I: A New Component of the Abscission Checkpoint 

Joshua Bembenek, Koen Verbrugghe, Gyorgyi Csankovszki, Raymond Chan 

25 LEM-4 Coordinates Mitotic Signaling on BAF to Enable its Essential 

Function in Nuclear Envelope Formation 

Matyas Gorjanacz, Claudio Asencio, Iain Davidson, Rachel Santarella-Mellwig, Geraldine 

Seydoux , Iain Mattaj 

26 Filamin is Required to Initiate Calcium Signaling and Maintain F-actin 

Organization in the Spermatheca 

Ismar Kovacevic, Erin Cram 

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Saturday, June 09, 2012 - 9:00 am–12:00 noon 


Germline II, Meiosis, and Sex Determination/Dimorphism 


Chairs: Monica Colaiácovo and Jennifer Schisa 

27 Keynote: Germline maintenance and meiosis: mechanistic insights from 

C. elegans 

Monica Colaiácovo 

28 Identification of Direct GLP-1/Notch Targets that Regulate Germline 

Stem Cells 

Aaron Kershner, Heaji Shin, Judith Kimble 

29 Genome-wide Analysis of GLD-1 Mediated mRNA Regulation Uncovers 

a Role in mRNA Storage 

Claudia Scheckel, Dimos Gaidatzis, Jane Wright, Rafal Ciosk 

30 In the C. elegans Germ Line, S6K promotes Cell Cycle Progression and 

the Proliferative Fate and mediates the Effects of Diet 

Dorota Korta, Debasmita Roy, Simon Tuck, E. Jane Albert Hubbard 

31 Chromosome and centrosome inheritance in meiosis 

Mara Schvarzstein, Anne Villeneuve 

32 Role of the Inhibitory Kinase WEE-1.3 in Regulating the Meiotic Cell 

Cycle and Fertility in C. Elegans 

Anna Allen, Jessica Nesmith, Andy Golden 

33 The Torsin Homolog OOC-5 is Required for Normal Nucleoporin 

Localization 

Michael White VanGompel, Lesilee Rose 

34 Identification of Direct Targets of the Caenorhabditis elegans Global 

Sexual Regulator TRA-1 by Chromatin Immunoprecipitation 

Matthew Berkseth, Kohta Ikegami, Jason Lieb, David Zarkower 

35 Evolution of Caenorhabditis Dosage Compensation 

Te-Wen Lo, Caitlin Schartner, Catherine Pickle, Barbara Meyer

Saturday, June 09, 2012 - 4:30–6:30 pm 




Chairs: Valerie Reinke and Ryan Baugh 

36 Keynote: RNAi and Immortality: Recognition of Self/non-Self RNA in 

the C. elegans Germline 

Craig Mello 

37 The onset of dosage compensation is linked to the loss of 


Laura Custer, Gyorgyi Csankovszki 

38 The Histone Demethylase SPR-5 and the Histone Methyltransferase 

MET-2 Comprise a Novel Epigenetic Reprogramming Switch 

Shana Kerr, Chelsey Chandler, Joshua Francis, Erica Mills, David Katz 

39 Nuclear RNAi mediates silencing of repetitive sequences in C. elegans 

Fei Xu, Xufei Zhou, Hui Mao, Jiaojiao Ji, Shouhong Guang 

40 Dimerization of γCatenin/WRM-1 Allows Intermolecular 


Xiao-Dong Yang, Scott Robertson , Rueyling Lin 

41 Organ defects in adults resulting from threshold blastomere 

specification 

Morris Maduro, Gina Broitman-Maduro, Leila Magistrado, Shruthi Satish 

Sunday, June 10, 2012 - 9:00–12:30 pm 


Cell Fate and Emerging Technologies 


Chairs: Monica Gotta and Zhirong Bao 

42 Keynote: Title: TBD 

Julie Ahringer 

43 Modeling germline population dynamics 

Hillel Kugler, E. Jane Albert Hubbard 

44 Dev-scape: An intuitive tool for automated phenotyping with single cell 

resolution 

Julia Moore, Zhuo Du, Anthony Santella, Christian Pohl, Zhirong Bao 

xv

45 WormBase 2012: Website Redesign 

Abigail Cabunoc, Norie de la Cruz, Adrian Duong, Maher Kassim, Xiaoqi Shi, Todd Harris, 

Lincoln Stein 

46 DSL-2 Mediates a Notch Signal From EMS Descendant(s) to ABp 

Descendants 

Scott Robertson, Jessica Medina, Rueyling Lin 

47 Intercellular Calcium Signaling in a Gap Junction Cell Network 


Jennifer Schumacher Tucker, Chieh Chang, Chiou-Fen Chuang 

48 Nutritional control of mRNA isoform expression during developmental 

arrest and recovery in C. elegans 

Colin Maxwell, Igor Antoshechkin, Nicole Kurhanewicz, Jason Belsky, L. Ryan Baugh 

49 Ras and its Effector RalGEF Both Perform Dual, Antagonistic Functions 

during C. elegans Vulval Patterning 

Kimberly Monahan, Rebecca Whitehurst, Tanya Zand, Channing Der, David Reiner 

50 The microRNA miR-786 is Required for Rhythmic Calcium Wave 

Initiation in the C. elegans Intestine 

Benedict Kemp, Erik Allman, Lois Immerman, Megan Mohnen, Maureen Peters, Keith 

Nehrke, Allison Abbott 

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Poster Topic 

Cell Biology 


51 GLO-2 is a BLOC-1 Subunit that Functions in Gut Granule Biogenesis 

Alec Barrett, Olivia Foster, Annalise Vine, Greg Hermann 

52 The Conventional Kinesin-1/UNC-116 Acts in PHB Phasmid Neurons 

to Mediate Proper Cell Body Position 

Ben Barsi-Rhyne, Kristine Miller, Chris Vargas, Miri VanHoven 

53 Genetic Interaction and Structure/Function Studies of MEL-28, a 

Protein Required for Nuclear Envelope Function and Chromosome 

Segregation 

Anita Fernandez, Carly Bock, Allison Lai, Emily Mis, Fabio Piano 

54 Oocyte Meiotic Spindle Assembly in C. elegans 

Amy Connolly, Sara Christensen, Valerie Osterberg, Josh Lowry, John Yochem, Bruce 

Bowerman 

55 Identifying Proteins that Interact with the Serine/Threonine Kinase 

UNC-82 in Muscle Cells 

Christopher Duchesneau, April Reedy, Hiroshi Qadota, Guy Benian, Pamela Hoppe

56 A LET-23 localization and expression screen identifies a novel 

mechanism of EGFR regulation through Ezrin/Radixin/Moesin proteins 

Juan Escobar Restrepo, Peter Gutierrez, Andrea Haag, Alessandra Buhler, Christina 

Herrmann, Maeva Langouet, David Kradolfer, Erika Frohli, Attila Stetak, Alex Hajnal 

57 Growth of Muscle Adhesion Complexes During Postembryonic 

Development 

Brandon Fields, Nate Szewczyk, Lewis Jacobson 

58 CDK-1 inhibits meiotic spindle shortening and dynein-dependent 

spindle rotation in C. elegans 

Jonathan Flynn, Marina Ellefson, Francis McNally 

59 The C. elegans Uterine Seam Cell: a Model for Studying Nuclear 

Migration and Cell Outgrowth 

Srimoyee Ghosh, Paul Sternberg 

60 Cadherin FMI-1 Maintains the Structure of the PVD Mechanosensory 

Neurons 

Julie Grimm, Benjamin Podbilewicz 

61 Two Functional Domains in C. elegans Glypican LON-2 Can 

Independently Inhibit DBL-1 Growth Factor Signaling but Require 

Accessory Moieties 

Suparna Bageshwar, Tina Gumienny 

62 Mutational Analysis of Residues Required for Activation the UNC-82 

Serine-Threonine Kinase 

Jason Kintzele, Pamela Hoppe 

63 Genetic Analysis of Calcium Regulation in the C. elegans Intestine 

Jocelyn Laboy, Kenneth Norman 

64 The Tubulin Deglutamylase CCPP-6 Functions Exclusively in Ciliated 

Dopaminergic Neurons in C. elegans 

Ethan Landes, Brendan O’Flaherty, Elizabeth De Stasio, Peter Swoboda, Brian Piasecki 

65 Protein Sequences Within the UNC-82 S/T Kinase that Affect 

Subcellular Localization in Pharyngeal Muscle 

Latrisha Lane, Chiyen Wong, Caitlyn Carter, Pamela Hoppe 

66 Characterization of vh45, a Candidate Regulator of Early to Late 

Endosomal Maturation 

Fiona Law, Shang Xiang, Christian Rocheleau 

67 cil-5 Mediates Ciliary Receptor Localization and Sensory Function in C. 


Kara Braunreiter, Greg Fischer, Casey Gabrhel, Jamie Lyman Gingerich 

xvii

68 Neuroligin has Cell-autonomous and Non-autonomous Functions in C. 


Jacob Manjarrez, Greg Mullen, Ellie Mathews, Jerrod Hunter, Jim Rand 

69 Genetic and Molecular Dissection of Novel Pathways Required for 

Nuclear Migration in the Model System C. elegans. 

Yu-Tai Chang, Shaun Murphy, Jonathan Kuhn, Minh Ngo, Daniel Starr 

70 FLN-1/filamin is required for spermathecal contractility 

Jose Orozco, Ismar Kovacevic, Erin Cram 

71 Isolation of Mutations that alter Nile Red Staining in C. elegans 

Stephanie Burge, Anthony Otsuka 

72 Epithelial Dynamics During the G1-to-G2 Pore Cell Swap in the 

Excretory System 

Jean Parry, Amanda Zacharias, Hasreet Gill, John Murray, Meera Sundaram 

73 The Arp2/3 activator WAVE/SCAR Promotes Clathrin Mediated 

Endocytosis in the Polarized C. elegans Intestinal Epithelia 

Falshruti Patel, Martha Soto 

74 Visualizing Dynamics of Meiotic Prophase Chromosome Structures 

Divya Pattabiraman, Marc Presler, Grace Chen, Anne Villeneuve 

75 CRL2/LRR-1 E3-Ligase Prevents Progression Through Meiotic Prophase 

in the Adult C. elegans Germline 

Julien Burger, Jorge Merlet, Nicolas Tavernier , Benedicte Richaudeau, Asja Moerkamp, 

Rafal Ciosk, Bruce Bowerman, Lionel Pintard 

76 Regulated Nucleocytoplasmic Shuttling of SPAT-1/BORA Coordinates 

CDK-1 and PLK-1 Activation For Proper Mitotic Entry in the Early C. 


Nicolas Tavernier , Anna Noatynska, Julien Burger, Costanza Panbianco, Jorge Merlet, 

Benedicte Richaudeau, Emmanuelle Courtois, Thibaud Leger, Monica Gotta, Lionel Pintard 

77 PPFR-1 Phosphatase 4 subunit is a regulator of MEI-1/Katanin activity 

during meiosis that is rapidly targeted for degradation by CRL-3/MEL- 

26 E3-ligase in the transition to mitosis in C. elegans 

Jose-Eduardo Gomes, Benedicte Richaudeau, Etienne Formstecher, Paul Mains, Lionel 

Pintard 

78 A Genetic Analysis of the Axon Guidance of the C. elegans Pharyngeal 

Neuron M1 

Osama Refai, Evvi Rollins, Patrcia Rhos, Jeb Gaudet 

79 Using C. elegans to Explore the Role of Presenilin in Calcium Signaling 

Shaarika Sarasija, Kenneth Norman 

xviii

80 Novel Roles For A Cell Adhesion Protein DYF-7 In C. elegans Body Size 

Determination 

Robbie Schultz, Tina Gumienny 

81 DAF-16 Promotes Developmental Growth in Response to Persistent 

Somatic DNA Damage 

Michael Muller, Maria Ermolaeva, Laia Castells-Roca, Peter Frommolt, Sebastian Greiss, 

Jennifer Schneider, Bjorn Schumacher 

82 Purification and Characterization of Glyceraldehyde-3-Phosphate 

Dehydrogenase from Caenorhabditis elegans 

Valeria S. Valbuena, Megan Gautier, Justin Spengler, M. Banks Greenberg, M. Leigh 

Cowart, Katherine Walstrom 

83 Three axonal guidance pathways differentially signal to the regulators 

of the actin cytoskeleton during axonal migration 

Andre Wallace, Yelena Bernadskaya, Martha Soto 

Poster Topic 

Cell cycle and cytokinesis 


84 Microtubules and Fertilization: The MEI-1/Katanin mediated 

cytoskeletal transition from meiosis to mitosis in the developing 

embryo 

Sarah Beard, Paul Mains 

85 Understanding Proteasomal Regulation of SZY-20 in the Centrosome 

Assembly Pathway 

Michael Bobian, Mi Hye Song 

86 Mitotic spindle proteomics reveals conserved Caenorhabditis elegans 

proteins potentially necessary for cytokinesis 

Mary Kate Bonner, Daniel Poole, Tao Xu, Ali Sarkeshik, John Yates III, Ahna Skop 

87 Non-random Segregation of Unpaired X Chromosomes in C. elegans 

Female Meiosis 

Daniel Cortes Estrada, Francis McNally 

88 Parallel mechanisms promote RhoA activation during polarization and 

cytokinesis in the early C. elegans embryo 

Yu Chung Tse, Michael Werner, Katrina Longhini, Jean-Claude Labbe, Bob Goldstein, 

Michael Glotzer 

89 ATX-2, the C. elegans ortholog of ataxin 2, is necessary for cytokinesis. 

Megan Gnazzo, Ahna Skop 

xix

90 Identification and Characterization of mel-15 as a New Paternal-effect 

Lethal Mutant in C. elegans 

Aimee Jaramillo-Lambert, Kathryn Stein, Andy Golden 

91 RNA-binding Proteins ATX-2/PAB-1 Regulate Centrosome Assembly 

and Size 

Sarah Mets, Kelly Haynes, Eric Vertin, Dongyan Zhang, Mi Hye Song 

92 ubc-25 encodes a conserved ubiquitin-conjugating enzyme that is 

required for developmentally controlled cell cycle quiescence 

David Tobin, Sarah Roy, Mako Saito 

xx 

Poster Topic 

Cell Death 


93 NAD salvage biosynthesis and programmed cell death; a new model for 

investigating cell death mechanisms 

Matt Crook, Wendy Hanna-Rose 

94 The Possible Role of Autophagic Cell Death in the Regulation of 

Excitotoxicity in C. elegans 

John Del Rosario, Itzhak Mano 

95 Genes Required for Cell Shedding, a Caspase-Independent Mechanism 

of Programmed Cell Elimination 

Dan Denning, Bob Horvitz 

96 Investigating the pro-apoptotic function of ced-9 

Kaitlin Driscoll, Peter Reddien, Brad Hersh, Bob Horvitz 

97 SPTF-3 SP1 and PIG-1 MELK Function in Distinct Pathways to 

Promote M4 Neuron Cell-Type Specific Programmed Cell Death 

Takashi Hirose , Bob Horvitz 

98 Using HITS-CLIP to study mRNA targets of RNA-binding proteins 

involved in germ cell apoptosis in C. elegans 

Martin Keller, Deni Subasic, Kishore Shivendra, Michaela Zavolan, Micheal Hengartner 

99 Utilization of Alternative mRNAs for CED-4/Apaf-1 During Germ Cell 

Apoptosis 

J. Kaitlin Morrison, Brett Keiper 

100 A Small-Molecule Screen Identifies a Linker Cell Death Inhibitor 

Andrew Schwendeman, Shai Shaham 

101 Wave Regulatory Complex Genes Are Involved in the Engulfment of 

Apoptotic Cells 

Elena Simionato, Michael Hurwitz

102 In Search of Genes that Regulate Germ Cell Apoptosis in C. elegans 

Angel Villanueva-Chimal , Carlos Silva-Garcia , Laura Lascarez-Lagunas, Rosa Navarro 

103 let-70, an E2 Ubiquitin-Conjugating Enzyme, Promotes the Non- 

Apoptotic Death of the Linker Cell 

Jennifer Zuckerman 

Poster Topic 

Cell Fate 


104 Elucidating the let-7 Independent Role of lin-28 

Jennifer Alaimo, Bhaskar Vadla, Kevin Kemper, Eric Moss 

105 Regulation and function of SYS-1/beta-catenin during hypodermal 

stem cell divisions 

Austin Baldwin, Bryan Phillips 

106 Germline Expressed GLP-1 Regulates Embryonic Endoderm 

Specification 

Ahmed Elewa, Takao Ishidate, Sandra Vergara, Tae-Ho Shin, Masaki Shirayama, Craig 

Mello 

107 Investigating the Role of SEM-4/SALL in Development of the 

Postembryonic Mesoderm 

Vikas Ghai, Chenxi Tian, Jun Liu 

108 A Screening To Find Suppressors Of The Wnt Pathway 

Eva Gomez-Orte, Begona Ezcurra, Beatriz Saenz-Narciso, Juan Cabello 

109 MEX-5 regulates mRNA stability during germ cell development and 

asymmetric cell division 

Manoel Prouteau, Gilles Udin, Monica Gotta 

110 A Screen for Mislocalization and Misexpression of LET-23 EGF 

Receptor during Vulval Development 

Andrea Haag, Juan Escobar Restrepo, Alex Hajnal 

111 A Role of the LIN-12/Notch Signaling Pathway in Diversifying the Non- 

Striated Egg-Laying Muscles in C. elegans 

Jared Hale, Carolyn George, Nirav Amin, Zachary Via, Leila Toulabi, Jun Liu 

112 UNC-62/Meis and CEH-20/Pbx proteins work together to control 

asymmetric cell divisions during C. elegans development by regulating 

WRM-1/γ-catenin localisation 

Samantha Hughes, Charles Brabin, Alison Woollard 

xxi

113 The Ras-ERK/MAPK Regulatory Network Controls Dedifferentiation In 

Caenorhabditis elegans Germline 

Dong Seok Cha, Udaya Sree Datla, Sarah Hollis, Judith Kimble, Myon-Hee Lee 

114 A sma-9 Suppressor Screen to Identify New Players in the BMP-like 

Sma/Mab Pathway in C. elegans 

Lindsey Szymczak, Katharine Constas, Arielle Schaeffer, Sinthu Ranjan, Saad Kubba, 

Emad Alam, Dennis Liu, Chenxi Tian, Herong Shi, Jun Liu 

115 Further evidence for the importance of the MED-1 and -2 GATA 

factors in endoderm specification 

Morris Maduro, Gina Broitman-Maduro, Shruthi Satish 

116 Regulation and function of nhr-67/tailless in uterus development 

George McClung, Lauren Pioppo, Jenny Hall, Rachel Dordal, Catherine Ezzio, Evan 

Fletcher, Amanda Gavin, Sheila Clever, Bruce Wightman 

117 Does lin-46 Tip the Balance of hbl-1 Activity in the Succession of 

Hypodermal Blast Fates? 

Eric Moss, Kevin Kemper, Bhaskar Vadla 

118 Post-transcriptional Regulation of Maternally-supplied Wnt Ligand 

During Early Embryogenesis 

Marieke Oldenbroek, Scott Robertson, Tugba Guven-Ozkan, Rueyling Lin 

119 Abstract withdrawn 

120 Regulation of LET-23 EGFR signaling and trafficking by a putative Arf1- 

GEF 

Olga Skorobogata, Christian Rocheleau 

121 Examining the Fate of Centrosomally Uncoupled SYS-1/Beta-catenin 

to Explore Spindle-Independent Roles of the Centrosome during 

Asymmetric Cell Divisions 

Setu Vora, Bryan Phillips 

xxii 

Poster Topic 



122 Function and evolution of the diverged NR2E nuclear receptors nhr-111 

and nhr-239 

Emily Bayer, G. Michael Baer, Christopher Alvaro, Katherine Weber, Ramzy Burns, Michael 

Lilly, Anvi Patel, Benjamin Perlman, Sheila Clever, Bruce Wightman 

123 Redefining POP-1 Binding Sites in C. elegans 

Chandan Bhambhani, Ken Cadigan

124 In vivo Regulation of the Alternative Splicing of the Pro- and Anti- 

Apoptotic Gene ced-4 

Anna Corrionero, Bob Horvitz 

125 Identifying HLH-8/Twist Homodimer Target Genes 

Nirupama Singh, Peng Wang, Ann Corsi 

126 Understanding the Role of Overlapping MicroRNA Networks During 

Nematode Development 

Jeanyoung Jo, Kimberly Breving, Kenya Madric, Aurora Esquela-Kerscher 

127 Intracellular Trafficking and Endocytic Regulation of the DBL-1/BMPlike 

pathway in C. elegans 

Ryan Gleason, Adenrele Akintobi, Ying Li, Barth Grant, Richard Padgett 

128 Identification and characterization of targets of the REF-1 family 

member, HLH-25 

Raymarie Gomez, Han-ting Chou, Casonya Johnson 

129 The Mediator Subunit CDK-8 Negatively Regulates EGFR-Ras-MAPK 

in Vulva Development 

Jennifer Grants, Stefan Taubert 

130 A Lipid-Binding Protein that Modifies cGMP Signaling is Required for 

Host Odor Sensing and Body Morphology in Pristionchus pacificus 

Ray Hong, Jessica Cinkornpumin, Dona Roonalika Wisidagama, Veronika Rapoport 

131 Elucidating The Role of Genetic Redundancy In The Wnt Signaling 

Pathway In Regulating Q Neuroblast Migration 

Ni Ji, Teije Middelkoop, Hendrik Korswagen, Alexander van Oudenaarden 

132 Can the Rate of Transcription be Quantitatively Determined in 

Relation to Transcription Factor Binding Affinity? 

Brett Lancaster, James McGhee 

133 Regulated Splicing of the Cholinergic Gene Locus 

Ellie Mathews, Greg Mullen, Jim Rand 

134 Short Capped RNAs and Nuclear Run-On Reveal Pol II Pausing and 

Backtracking in C. elegans 

Colin Maxwell, William Kruesi, Nicole Kurhanewicz, Leighton Core, Colin Waters, Igor 

Antoshechkin, John Lis, Barbara Meyer, L. Ryan Baugh 

135 The mRNA Splicing Regulator SPK-1 Is Required for Cell Polarity in 

One-Cell C. elegans Embryos 

Martin Mikl, Carrie Cowan 

xxiii

136 The Transcriptional Repressor Protein CTBP-1 Regulates the 

Differentiation of DA Motor Neurons 

Hannah Nicholas, Duygu Yucel, Estelle Llamosas, Anna Reid, Aaron Lun, Sashi Kant, 

Merlin Crossley 

137 The Role of C. elegans bHLH-29 Transcription Factor in Stress 

Response 

Thanh Quach, Casonya Johnson 

138 Loss of the ubiquitin-specific protease usp-48 allows for direct 

conversion of a somatic tissue into neurons in Caenorhabditis elegans 

Dylan Rahe, Tulsi Patel, Oliver Hobert 

139 Chromatin Structure and Genome Stability in C. elegans 

Valerie Robert, Cedric Rakotomalala, Cecile Bedet, Florence Couteau, Monique Zetka, 

Francesca Palladino 

140 A New Attempt to Elicit an RNAi Phenotype with the LIMhomeodomain 

Transcription Factor LIM-7 

Laura Vallier, John Coppola 

141 The Histone Demethylase UTX-1 Is Essential for Normal 

Development, Independently of Its Enzymatic Activity 

Julien Vandamme, Lisa Salcini 

142 A Conserved SBP-1/Phosphatidylcholine Feedback Circuit Regulates 

Lipogenesis in Metazoans 

Amy Walker, Rene Jacobs, Jenny Watts, Veerle Rottiers, Lorissa Niebergall, Anders Naar 

143 HLH-29, REF-1 family protein functions in the spermatheca 

Ana White, Casonya Johnson 

144 Promoter analysis of the GATA type transcription factor ELT-2 

Tobias Wiesenfahrt, Jannette Berg, James McGhee 

145 Genetic Screen for Novel Repair Genes Implicated in UV-induced DNA 

Damage Response 

Stefanie Wolters, Bjoern Schumacher 

xxiv 

Poster Topic 

Germline 


146 The eIF4E-binding protein IFET-1 is a broad-scale translational 

repressor and is required for normal P granule ultrastructure 

Madhu Sengupta, Lloyd Low, Joseph Patterson, Traude Beilharz, Jennifer Schisa, Peter 

Boag

147 Spindle assembly checkpoint proteins monitor synapsis during meiosis 

in C. elegans 

Tisha Bohr, Piero Lamelza, Needhi Bhalla 

148 A global genomic survey of genes that mediate LKB1/PAR-4dependent 

germline stem cell quiescence in C. elegans 

Rita Chaouni, Richard Roy 

149 VPR-1, a VAPB homolog required for germ line proliferation and 

differentiation 

Pauline Cottee, Jack Vibbert, Sung Min Han, Michael Miller 

150 Paternal Mitochondria Elimination From the Germline in C. elegans 

Embryos 

Dominika Bienkowska, Sylvain Bertho, Carrie Cowan 

151 CACN-1 is required for gonad and germline development 

Hiba Tannoury, Erin Cram 

152 HIS-35, a histone H2A variant that differs from canonical H2A by one 

amino acid, functions in fertility 

Francisco Guerrero, Rodrigo Estrada, Meghann Shorrock, Margaret Jow, Diana Chu 

153 SNF-10, an SLC6 transporter required for sperm activation by C. 

elegans males 

Kristin Fenker, Angela Hansen, Conrad Chong, Molly Jud, Gillian Stanfield 

154 Putative protamines, SPCH-1/2/3, localize to mature sperm chromatin 

and may play a role in fertility 

Jennifer Gilbert, Dana Byrd, Diana Chu 

155 Sperm Vs Sperm: Determining the Cellular Basis of Sperm 

Competition 

Jody Hansen, Daniela Chavez, Gillian Stanfield 

156 Evaluating the Role of the V-ATPase B Subunit Utilizing C.elegans 

Sperm 

Melissa Henderson, Elizabeth Gleason, Ying Long, Taylor Walsh, Emily Wang, Steven 

L’Hernault 

157 The RNA binding protein TIA-1.2 is essential for fertility in C. elegans 

Gabriela Huelgas Morales, Carlos Silva Garcia, Rosa Navarro Gonzalez 

158 Germline Hexosamine Pathway Synthesis of UDP-GlcNAc is Regulated 

by SUP-46 

Wendy Johnston, Aldis Krizus, Arun Ramani, Andrew Fraser, James Dennis 

159 Role of Notch re-localization in establishing germline stem cell 

quiescence in C. elegans dauer larvae 

Pratik Kadekar, Nathan Navidzadeh, Patrick Narbonne, Emily Wendland, Richard Roy 

xxv

160 Protein synthesis regulation in the germline: eIF4 factors promote 

selective mRNA translation for meiosis, differentiation, maturation or 

apoptosis. 

Melissa Henderson, Jacob Subash, Vince Contreras, Anren Song, Sara Labella, Andrew 

Friday, Monique Zetka, Robert Rhoads, Brett Keiper 

161 P-TEFb—Independent Phosphorylation of RNA Polymerase II CTD- 

Ser2 in the C. elegans Germline 

Elizabeth Bowman, Bill Kelly 

162 sacy-1 Links Somatic Control of Oocyte Meiotic Maturation, Germline 

Sex Determination, and Gamete Maintenance 

Seongseop Kim, J. Amaranath Govindan, Zheng Jin Tu, David Greenstein 

163 Investigating the Role of SMC-5/6 in Preventing Germline Genomic 

Rearrangement 

Killeen Kirkconnell, Dane Session, Raymond Chan 

164 The let-479 Gene Encodes a Homolog of SPE-42 and is Required for C. 

elegans Fertilization 

Tim Kroft, Luke Wilson, Lindsey Magnuson, Gabe Fall 

165 Spindle Assembly Checkpoint Plays a Role In DNA-damage-induced 

Cell Cycle Arrest In C. elegans Male Germ Line 

Katherine Lawrence, JoAnne Engebrecht 

166 Investigating the Role of Membrane Trafficking in Temperature- 

Sensitive Lethal Mutants with Defects in both Gonad Development and 

Embryonic Eggshell Production 

Josh Lowry, Amy Connolly, John Yochem, Bruce Bowerman 

167 Genome destabilization and checkpoint activation during cell cycle 

reentry of the primordial germ cells Z2 and Z3 

Ash Williams, Brendan Kramer, Matthew Michael 

168 Sensory Regulation of the C. elegans Germ Line through TGF-γ- 

Dependent Signaling in the Niche 

Diana Dalfo, David Michaelson, E Albert Hubbard 

169 In Vitro Analysis of C. elegans H2A Variants 

Ahmad Nabhan, Geeta Narlikar, Diana Chu 

170 ZHP-3 Regulates Meiotic Chromosome Dynamics 

Christian Nelson, Cate Paschal, Needhi Bhalla 

171 Distinct roles for FBF-1 and FBF-2 in silencing meiotic mRNAs 

Alexandre Paix, Ekaterina Voronina, Geraldine Seydoux 

xxvi

172 Natural Variants of C. elegans Demonstrate Defects in Both Sperm 

Function and Oogenesis at Elevated Temperatures 

Lisa Petrella, Susan Strome 

173 Exploring Novel Features of Gametogenesis in a Non-C. elegans Clade 

Kathryn Rehain, Zechariah Dillingham, Ethan Winter, Diane Shakes 

174 Nutritional Control of Germline Stem Cells 

Hannah Seidel, Judith Kimble 

175 Characterization of SYGL-1, A Novel Regulator of Germline Stem 

Cells 

Heaji Shin, Aaron Kershner, Judith Kimble 

176 Uncovering the Role of Condensin I during C. elegans Meiosis 

Margarita Sifuentes, Joshua Bembenek, Karishma Collette, Gyorgyi Csankovszki 

177 The metazoan gene akirin is required for synaptonemal complex 

disassembly and bivalent structure during Caenorhabditis elegans 

meiosis 

Amy Clemons, Heather Brockway, Yizhi Yin, Yaron Butterfield, Steven Jones, Monica 

Colaiacovo, Sarit Smolikove 

178 Chromatin Regulation in the Meiotic Germ Line 

Matthew Snyder, Xia Xu, Eleanor Maine 

179 Global Control of the Oogenic Program by Components of OMA-1 

Ribonucleoprotein Particles 

Caroline Spike, Donna Coetzee, David Greenstein 

180 Early and Late Roles for Gonadal Innexins: Germ Cell Proliferation and 

Meiotic Maturation 

Todd Starich, David Hall, David Greenstein 

181 Oocyte-to-embryo Transition: a Screen for mbk-2 Suppressors 

Yuemeng Wang, Harold Smith, Kevin O’Connell, Geraldine Seydoux 

182 A Functional RNAi Screen Identifies Regulators of RNP Granule 

Assembly in Aging Oocytes 

Megan Wood, Kevin Gorman, Joseph Patterson, Jennifer Schisa 

183 A Novel Function of MRE-11 in Caenorhabditis elegans 

Yizhi Yin, Sarit Smolikove 

184 Illuminating the Formation and Regulation of Meiotic Crossovers with 

GFP:COSA-1 

Karl Zawadzki, Rayka Yokoo, Anne Villeneuve 

xxvii

xxviii 

Poster Topic 

Morphogenesis 


185 exc-2 and Maintenance of Tube Structure of the Excretory Canals 

Hikmat Al-Hashimi, Matthew Buechner 

186 C. elegans nuclear hormone receptor, nhr-25 regulates vulval terminal 

cell properties and migrations during development 

Nagagireesh Bojanala, Marek Jindra, Masako Asahina 

187 Characterizing regulators of the C. elegans cytoskeleton 

Benjamin Chan, Simon Rocheleau, Paul Mains 

188 The Morphological and Functional Alterations of the Anal Depressor 

Muscle in Male C.elegans 

Xin Chen, L. Rene Garcia 

189 TMD-1 / Tropomodulin Regulates Intestinal and Excretory Cell 


Rachel Walker, Corey Hoffman, Elisabeth Cox-Paulson 

190 Roles Of Heparan Sulfate Proteoglycans In Embryonic Morphogenesis 

Katsufumi Dejima, Suk-Ryool Kang , Andrew Chisholm 

191 C. elegans body size is regulated by TGF-γ signalling in multiple tissues. 

Aidan Dineen, Jeb Gaudet 

192 Functional Dissection of SAX-7, a Homologue of Human L1CAM in C. 

elegans Dendritic Branch Formation 

Xintong Dong, Oliver Liu, Kang Shen 

193 ani-1 is required for morphogenesis of C. elegans embryos and functions 

in parallel to the rho-1 pathway. 

Nellie Fotopoulos, Yun Chen, Alisa Piekny 

194 A Genome-Wide RNAi Screen to Identify New Components of a 

Muscle-To-Epidermis Mechanotransduction Pathway Essential for 

Embryonic Elongation 

Christelle Gally, Agnes Aubry, Michel Labouesse 

195 The EXC-1 RAS-Domain Protein Mediates Vesicle Movement in the 

Excretory Canals 

Kelly Grussendorf, Brendan Mattingly, Alex Salem, Matthew Buechner 

196 A Screen For Genes Controlling Vulval Morphogenesis 

Qiutan Yang, Matthias Morf, Sarfarazhussain Farooqui , Juan Escobar, Alex Hajnal

197 LEP-2/Makorin Promotes let-7 microRNA-mediated Terminal 

Differentiation in Male Tail Tip Morphogenesis 

R Antonio Herrera, Karin Kiontke, Samuel Ahn, David Fitch 

198 pix-1 Generates a Gradient of Contraction Forces in Hypodermal Cells 

of Elongating Embryos in Caenorhabditis elegans 

Sharon Harel, Emmanuel Martin, Bernard Nkengfac, Karim Hamiche, Mathieu Neault, 

Sarah Jenna 

199 Analysis of the Role of ENU-3 in Axon Outgrowth and Guidance in C. 


Callista Yee, Karmen Lam, Anna Bosanac, Marie Killeen 

200 Identifying Regulators of Gonadal Development in C. elegans by Cellspecific 

Transcriptional Profiling 

Mary Kroetz, David Zarkower 

201 Caenorhabditis elegans DNA-2 Helicase/Endonuclease Plays A Vital 

Role In Maintaining Genome Stability, Morphogenesis, And Life Span 

Myon-Hee Lee, Sarah Hollis, Bum Ho Yoo, Keith Nykamp 

202 The Role of LIN-3 During Morphogenesis of the Dorsal Lumen in the 

Vulva 

Louisa Mueller, Matthias Morf, Alex Hajnal 

203 Somatic gonad precursor migration in C. elegans 

Monica Rohrschneider, Jeremy Nance 

204 VAB-9 and Vertebrate Orthologue TM4SF10 Cooperate with Adherens 

Junction Proteins and Actomyosin to Regulate Epithelial Polarity and 

Morphogenesis 

Jeff Simske 

205 The C. elegans DM domain genes dmd-3 and mab-3 function during the 

late stages of male gonad development 

Michele Smith, Alyssa Herrmann, Emily Kivlehan, Lauren Whipple, Douglas Portman, D. 

Adam Mason 

206 Analysis of Non-Muscle Myosin II During Dorsal Intercalation in 


Elise Walck-Shannon, Jeff Hardin 

207 Establishing Caenorhabditis elegans as a Model for Neural Tube Defects 

Bridget Waller, Kassi Crocker, Timothy Walston 

208 Anillin is required for Epidermal Morphogenesis during C. elegans 

Embryogenesis 

Denise Wernike, Alisa Piekny 

xxix

209 What Causes Partial Penetrance of a Developmental Phenotype? 

Claire Williams, Maxwell Heiman 

210 MIG-10 interacts with ABI-1 to induce asymmetric outgrowthpromoting 

activity in response to guidance cues 

Yan Xu, Christopher Quinn 

211 Molecular characterization of maternally malformed 3 (mal-3) 

Yemima Budirahardja, Thang Doan, Ronen Zaidel Bar 

xxx 

Poster Topic 

New Technologies 


212 A Semi-Automated Pipeline for the Identification of Novel Mutants 

with Cell Number Defects 

Peter Appleford, Alison Woollard 

213 A Novel Fluorescence-Based Method to Visualize Protein-Protein 

Interactions in Living Caenorhabditis elegans 

Han Ting Chou, Casonya Johnson 

214 Spectrum: Building Pathways to Biomedical Research Careers for Girls 

and Women of Color 

Diana Chu, Rebecca Garcia, Kimberly Tanner 

215 Establishing and using a modified NGM (ENGM) to culture an 

manipulate the entomopathogenic nematode, Heterorhabditis 

bacteriophora 

Zsofia Csanadi, Abate Birhan Addise, Anita Alexa, Barnabas Jenes, Zsofia Banfalvi, Andrea 

Mathe-Fodor, Katalin Belafi-Bako, Andras Fodor 

216 A MultiSite Gateway®-Compatible Three-Fragment Vector 

Construction Kit Using Galactose Selection 

Iskra Katic, Wolfgang Maier 

217 Screening for C. elegans Mutants with Subtle Phenotypes with 

Microfluidics and Computer Vision 

Adriana San-Miguel, Matthew Crane, Peri Kurshan, Kang Shen, Hang Lu 

218 Two Novel Staining Protocols Resolve Caenorhabditis elegans Cuticular 

Structures For Live Imaging And Transmission Electron Microscopy 

Robbie Schultz, E. Ann Ellis, Tina Gumienny 

219 Improving the Sensitivity and Selectivity of Mutation Identification by 

Next-Generation Sequencing 

Sijung Yun, Michael Krause, Harold Smith

220 Worm Proteins Overtake Biochemistry Lab to Inspire Inquiry 

Katherine Walstrom 

Poster Topic 

Polarity 


221 Understanding temporal and spatial features of polarity establishment 

Simon Blanchoud, Felix Naef, Pierre Gonczy 

222 PAR proteins regulate the localization of LET-99 during asymmetric 

division 

Eugenel Espiritu, Jui-Ching Wu, Lesilee Rose 

223 On the Role of RGA-3/4 in Foci Formation of NMY-2 in C. elegans 

Masashi Fujita, Shuichi Onami 

224 Isolation, Identification, and Characterization of Free-Living 

Nematodes 

Lauren Leister, Alan Massouh, Alexis Plaga, Ramon Carreno, Danielle Hamill 

225 A Dominant Mutation in a C. elegans Splicing Factor Results in 

Reversed AP Polarity in the Early Embryo 

Reza Keikhaee, Bruce Nash, John Yochem, Bruce Bowerman 

226 Identifying Mechanisms of Contact-Mediated Cell Polarization 

Diana Klompstra, Dorian Anderson, Jeremy Nance 

227 ER Compartmentalisation and the Regulation of Polarity in the C. 

elegans Embryos 

Zuo Yen Lee, Monica Gotta, Yves Barral 

228 A Cullin-5-RING Ubiquitin Ligase Regulates Asymmetric Cell Division 

in Early C.elegans Embryos 

Anne Pacquelet, Emeline Daniel, Gregoire Michaux 

229 Evolution of GPR Regulation in the Control of Spindle Positioning for 

Two Cænorhabditis Species Embryos 

Soizic Riche, Francoise Argoul, Melissa Zouak, Alain Arneodo, Jacques Pecreaux, Marie 

Delattre 

230 Coupling Centrosome Position And Cortical Polarity 

Sabina Sanegre, Carrie Cowan 

231 GLD-3(S) Contributes to PIE-1 Asymmetry in Zygotes 

Jarrett Smith, Geraldine Seydoux 

xxxi

xxxii 

Poster Topic 

Sex Determination 


232 Phosphorylation State of a Tob/BTG Protein, FOG-3, Regulates 

Initiation and Maintenance of the Caenorhabditis elegans Sperm Fate 

Program 

Myon-Hee Lee, Kyung Won Kim, Clinton Morgan, Dyan Morgan, Judith Kimble 

233 Molecular Analyses of FOG-1 and FOG-3, Terminal Regulators of the 

Sperm/Oocyte Cell Fate Decision 

Daniel Noble, Scott Aoki, Marco Ortiz Sanchez, Kyung Won Kim, Judith Kimble 

234 RNA-Seq Analysis of Germline Sex Reprogramming 

Elena Sorokin, Judith Kimble

Three pathways to polarity maintenance 

Ken Kemphues 

Cornell University 

Contact: kjk1@cornell.edu 

Lab: Kemphues 

Keynote 1 

1

A role for the centrosome and PAR-3 in the hand-off of microtubule 

organizing center function during epithelial polarization 

Jessica Feldman, James Priess 

Fred Hutchinson Cancer Research Center, Seattle, WA, USA 

The centrosome is the major microtubule organizing center (MTOC) in dividing cells 

and in many post-mitotic, differentiated cells. In other cell types, however, MTOC function 

is reassigned from the centrosome to non-centrosomal sites. Here, we analyze how MTOC 

function is reassigned to the apical membrane of C. elegans intestinal cells. After the terminal 

intestinal cell division, the centrosomes and nuclei move near the future apical membranes, 

and the postmitotic centrosomes lose all, or nearly all, of their associated microtubules. We 

show that microtubule-nucleating proteins such as γ-tubulin and CeGrip-1 that are centrosome 

components in dividing cells become localized to the apical membrane, which becomes 

highly enriched in microtubules. Our results suggest that centrosomes are critical to specify 

the apical membrane as the new MTOC. First, γ-tubulin appears to redistribute directly from 

the migrating centrosome onto the lateral, then apical membrane. Second, γ-tubulin fails to 

accumulate apically in wild-type cells following laser ablation of the centrosome. We show 

that centrosomes localize apically by first moving toward lateral foci of the conserved polarity 

proteins PAR-3 and PAR-6, and then move together with these foci toward the future apical 

surface. Embryos lacking PAR-3 fail to localize their centrosomes apically, and have aberrant 

localization of γ-tubulin and CeGrip-1. These data suggest that PAR proteins contribute to 

apical polarity in part by determining centrosome position and that the reassignment of MTOC 

function from centrosomes to the apical membrane is associated with a physical hand-off of 

nucleators of microtubule assembly. 

Contact: jlfeldma@fhcrc.org 

Lab: Priess 

2 

Platform Session #1 - Morphogenesis I and Polarity

Three Axonal Guidance Pathways Help Polarize the Actin 

Cytoskeleton During Embryonic Epidermal Cell Migration 

Yelena Bernadskaya1 , Andre Wallace1 , Jillian Nguyen1 , William Mohler2 , Martha 

Soto1 1 2 UMDNJ/RWJMS, Piscataway, NJ, USA, University of Connecticut, 

Farmington, CT, USA 

Migrating cells must integrate multiple guidance cues to direct their movements during 

embryonic development. Some of the best-studied regulators of cell migration and growth are 

the UNC-6/netrin, SLT-1/slit and VAB-2/Ephrin guidance cues and their receptors, UNC-40/ 

DCC, SAX-3/Robo and VAB-1/Eph. However, the mechanisms that interpret these signals 

downstream of the receptors and reorganize the actin cytoskeleton accordingly are not well 

understood. Using live imaging of F-actin in developing embryos we show three guidance 

receptors, UNC-40/DCC, SAX-3/Robo and VAB-1/Eph, differentially regulate the subcellular 

polarization and abundance of F-actin in migrating epidermal cells. Interestingly, our data 

suggests that high levels of F-actin are not essential for directed migration but that correct 

polarization of remaining F-actin is. Using genetic and molecular techniques we find that the 

three guidance receptors affect the localization of the WAVE/SCAR complex and its activator 

CED-10/Rac1, thus regulating formation of branched actin networks in the embryonic epidermis. 

Loss of any of these receptors results in defects in epidermal morphogenesis similar to those 

observed in the ced-10 and wve-1 mutants. Our results suggest that proper membrane 

recruitment and activation of CED-10/Rac1 and of WAVE/SCAR result in polarized F-actin that 

permits polarized movements and suggest how multiple guidance cues can result in distinct 

changes in actin nucleation during morphogenesis. 

Contact: bernadye@umdnj.edu 

Lab: Soto 

Platform Session #1 - Morphogenesis I and Polarity 

3

Arp2/3 mediates early endosome dynamics that participate in the 

maintenance of polarity in C. elegans 

Jessica Shivas, Ahna Skop 


The widely conserved Arp2/3 complex is crucial for the formation of branched actin networks. 

These networks play important roles in a variety of cellular processes, including endocytosis. 

In C. elegans, the actin cytoskeleton has been characterized in its role in the establishment of 

PAR asymmetry and cytokinesis. However, the contributions of actin to maintaining polarity, 

prior to the onset of mitosis, remain unclear. Endocytic recycling has been reported to function 

as an important mechanism in the dynamic stabilization of cellular polarity. We previously 

reported a role for the C. elegans ortholog of dynamin, DYN-1, in the stabilization of PAR 

asymmetry during maintenance phase through its participation in spatially and temporally 

regulated endocytosis. We now provide evidence that depletion of the Arp2 subunit of the Arp2/3 

complex, ARX-2, disrupts the cortical formation and localization of short actin filaments and 

foci that are normally present during polarity maintenance phase. We also observe defects in 

the organization and dynamics of endocytic regulators and polarity proteins during this time. 

We detect actin in association with the early endosome and endosomes are significantly larger 

upon disruption of ARX-2 levels. Finally, we detect aberrant accumulations of cytoplasmic 

PAR-6 in association with the enlarged early endosomes for prolonged periods of time when 

ARX-2 levels are reduced. This is observed when PAR-6 occupies a slightly smaller cortical 

area, suggesting a disruption in the endocytic dynamics associated with PAR-6. We propose 

a mechanism in which Arp2/3 regulates actin dynamics at the early endosome that promote 

rapid recycling of internalized polarity cues during polarity maintenance phase. 

Contact: shivas@wisc.edu 

Lab: Skop 

4 


Clathrin/AP-1 cooperate with sphingolipids to regulate apical polarity 

and lumen formation during C. elegans tubulogenesis 

Hongjie Zhang 1 , Ahlee Kim 1 , Nessy Abraham 1 , Liakot Khan 1 , David Hall 2 , John 

Fleming 1 , Verena Gobel 1 

1 Massachusetts General Hospital/Harvard Medical School, Boston, MA, 

USA, 2 Albert Einstein College of Medicine, Bronx, NY, USA 

Biological tubes are composed of polarized epithelial cells with apical membranes building 

the central lumen and basolateral membranes contacting adjacent cells and the extracellular 

matrix. We carried out a genome-wide morphological RNAi screen that examined the requirement 

of lethal genes for tube/lumen formation, using animals engineered with ERM-1::GFP-labeled 

apical/lumenal membranes. This screen identified a distinctive intestinal phenotype where 

the contiguous central lumen was transformed into multiple ectopic laterals lumens. Further 

analysis revealed that multiple-lumen formation was caused by a conversion of apicobasal 

polarity, with displacement of apical membrane components to the lateral membrane and/or 

cytoplasm and de novo formation of microvilli at lateral membranes. This polarity conversion 

appeared to occur independent of prior junction assembly defects, compatible with a trafficking 

defect disrupting the directional targeting of membrane components or polarity regulators. 

Among other molecules, the loss of several unrelated fatty-acid- and sphingolipid(SL)biosynthetic 

enzymes was found to cause this phenotype. Follow-up biosynthetic pathway 

screens identified membrane glycosphingolipids (GSLs) as the underlying lipid compound, 

mediating the function of these enzymes. GSLs are presumed raft components that reside 

on vesicle membranes and on lumenal plasma membranes. They have a documented 

apical sorting function in mammalian cell lines, although have not yet been shown to define 

membrane domain identities in vivo. The loss of CHC-1, the clathrin heavy chain, and of 

several subunits of the clathrin AP-1 adaptor also caused a polarity/ectopic lumen phenotype, 

supporting the notion of an underlying trafficking defect. Clathrin, however, has a well-defined 

role in endocytosis, but its regulation of plasma-membrane-directed transport is thought to be 

limited to the basolateral membrane. Here, we demonstrate that CHC-1/AP-1 cooperate with 

SL-biosynthetic enzymes in apical sorting. We show that GFP::CHC-1 and BODIPY-ceramide 

vesicles associate perinuclearly and assemble asymmetrically at polarized plasma membrane 

domains, in a codependent and AP-1-dependent manner. Based on these findings, we propose 

a trafficking pathway for apical membrane polarity in tubulogenesis that implies: (1) a clathrin/ 

AP-1 function on an apically-directed transport route; and (2) the convergence of this route 

with a sphingolipid-dependent apical trafficking path. 

Contact: hzhang14@partners.org 

Lab: Gobel 

Platform Session #1 - Morphogenesis I and Polarity 

5

The Fibrillin-like fbn-1 Gene Regulates Epithelial Stem Cell and ECM 

Dynamics in Molts 

Vijaykumar Meli, Alison Frand 

University of California Los Angeles, Los Angeles, California, USA 

The molting cycle involves the periodic removal and deposition of extracellular matrices 

(ECM). The stem cell-like lateral seam cells contribute to the production of new matrices 

during the molts, but undergo asymmetric divisions early in every larval stage. In addition, 

successive transitions between seam cell temporal fates coincide with the molts. However, the 

molecular mechanisms that coordinate ECM and stem cell dynamics during the molts are not 

yet understood. Here, we describe FBN-1, a protein that is similar to human fibrillins, which 

are the major components of ECM fibers defective in Marfan Syndrome and other inherited 

disorders of skin and connective tissue. The fbn-1 gene emerged from a full-genome, RNAibased 

screen for larvae unable to fully shed cuticles; fbn-1(tm290) mutants also exhibit molting 

defects. A transcriptional fbn-1::gfp-pest fusion gene is transiently but reiteratively expressed 

in the hypoderm during every molt. In addition, the expression of multiple splice variants of 

fbn-1 suggests substantial post-transcriptional gene regulation. To better define the function 

of fbn-1, we characterized the status of the cuticle in fbn-1 mutants, using cell and molecular 

biological approaches including TEM. A functional COL 19::GFP fusion protein was improperly 

deposited and disorganized in cuticles of fbn-1(lf) adults, and structural cuticle abnormalities 

were detected by TEM. Consistent with these findings, rearrangements in the actin cytoskeleton 

of the hypodermis were not obvious in fbn-1 mutants undergoing the fourth molt, but were 

readily detected in wild-type animals stained with Rh-phalloidin. Further, the lateral seam 

cells were detected using standard markers for the cell nuclei and margins. At the L4-to-adult 

transition, some seam cells failed to fuse or exit the cell cycle in approximately 35% of fbn-1(-) 

animals. The axis of seam cell division was also abnormal in fbn-1(RNAi) animals, suggesting 

de-regulation of the Wnt signaling pathway. Indeed, genetic analyses confirmed that mutations 

that affect the Wnt or other conserved cell-ECM signaling pathways modify the phenotypes of 

fbn-1(-) larvae. Taken together, our findings indicate that FBN-1 polymers likely serve as both 

structural and instructive components of matrices remodeled during the molts. We propose 

that the certain activities of FBN-1 macromolecules orchestrate stem cell and ECM dynamics 

in larval development. 

Contact: vmeli@mednet.ucla.edu 

Lab: Frand 

6 


A New C. elegans Cell Death Program: Implications for 


Shai Shaham 

Rockefeller (USA) 

Death is a vital developmental cell fate required to sculpt organs, eliminate harmful cells, 

and counter cell division. Apoptosis, an extensively studied cell death process, requires 

caspase protease activation, and is accompanied by chromatin compaction and cytoplasmic 

shrinkage. Mice lacking apoptotic effectors survive to adulthood, a surprising result given 

the prevalence of cell death during murine development. Thus, non-apoptotic cell death may 

play key roles in animal development. Genes dedicated to non-apoptotic developmental cell 

death have not been previously described. We study the programmed death of the linker cell, 

which leads gonadal elongation in Caenorhabditis elegans males. Strikingly, the linker cell dies 

independently of caspases and other apoptotic effectors. Moreover, dying linker cells display 

non-apoptotic ultrastructural features including nuclear envelope crenellation, uncondensed 

chromatin, and organelle swelling. We uncovered a novel program, unleashed within the 

linker cell to promote its demise. One component, PQN-41- a polyglutamine-repeat protein, 

promotes and is expressed at the onset of death. Regulators and co-expressed effectors have 

also been identified. Dying linker cells bear ultrastructural similarities to dying cells in normal 

vertebrate development and to degenerating cells in polyglutamine-induced diseases. Our 

results may, therefore, provide in-roads to understanding non-apoptotic cell death in metazoan 

development and disease. 

Contact: Shai.Shaham@rockefeller.edu 

Lab: Shaham 

Keynote 2 

7

C. elegans NRF-5 Regulates Cell Corpse Engulfment By Mediating PS 

Appearance On Phagocytes 

Yan Zhang, Haibin Wang, Xiaochen Wang 

National Institute of Biological Sciences, Beijing, China 

Phagocytosis of apoptotic cells is crucial for tissue remodeling, suppression of inflammation, 

and regulation of immune responses. Phosphatidylserine (PS), which is confined to the inner 

leaflet of plasma membrane in living cells, is exposed on the surface of apoptotic cells, thus 

serving as an “eat me” signal for engulfment. How PS is externalized and recognized is 

not well understood. We recently identified C. elegans TTR-52 as an extracellular bridging 

molecule which links PS on apoptotic cells with the CED-1 receptor on phagocytes. However, 

whether additional extracellular proteins are involved in recognizing apoptotic cells remains 

to be determined. 

In this study, we identified NRF-5, a secreted lipid transfer/LPS-binding family protein, as 

a novel regulator of cell corpse engulfment. The NRF-5 protein is expressed in and secreted 

from body wall muscle cells and clusters around apoptotic cells. We found that recognition of 

cell corpses by NRF-5 is disrupted in ced-7(lf) mutants but not altered in tat-1(lf) mutants which 

cause ectopic exposure of PS on living cell surfaces. As loss of tat-1 results in appearance 

of TTR-52 on the surface of both dying and living cells, NRF-5 and TTR-52 may recognize 

apoptotic cells in different manners. We observed that PS, which is externalized to the outer 

leaflet of plasma membranes in apoptotic cells, is also detected on the surface of engulfing 

cells. Loss of NRF-5 function completely blocks PS appearance on engulfing cells, a phenotype 

observed in both ced-7(lf) and ttr-52(lf) mutants. Our data suggest that NRF-5 may function 

together with CED-7 and TTR-52 to mediate PS appearance on phagocytes, and thus promotes 

cell corpse engulfment. 

Contact: yanzhang@nibs.ac.cn 

Lab: Wang 

8 

Platform Session #2 Morphogenesis II and Cell Death

Globin 12 of Caenorhabditis elegans Regulates the p38 and JNK 

MAPK Pathways through Redox Signaling to Control Germline 

Apoptosis 

Sasha De Henau1 , Lesley Tilleman2 , Francesca Germani2 , Caroline Vlaeminck1 , 

Jacques Vanfleteren1 , Luc Moens2 , Sylvia Dewilde2 , Bart Braeckman1 1 2 Ghent University, Ghent, Belgium, University of Antwerp, Antwerp, 

Belgium 

Redox signaling is present in a wide range of cell biological processes, including cell 

proliferation, cell differentiation, cell migration, and apoptosis. This form of signaling is tightly 

controlled, compartmentalized and tissue-specific. Herein, we show that a globin of C. elegans, 

globin-12 (GLB-12), functions through redox signaling and that it regulates multiple aspects of 

oogenesis, including germline apoptosis. 

The broad role of GLB-12 in oogenesis is demonstrated by the effects of glb-12 RNAi; it 

causes severely reduced fecundity, smaller gonads, increased levels of germline apoptosis, 

and several defects during oocyte development. A translational reporter shows that GLB-12 is 

membrane-bound and present in the distal gonadal sheath cells, the spermatheca-uterine valve 

and the uterus. By focusing on the increase in germline apoptosis, we found that GLB-12 signals 

through the JNK and p38 MAPK pathways; when one or both of these pathways is eliminated, 

the increase in germline apoptosis following glb-12 RNAi is no longer present. This finding is 

further supported by Western blot data, which shows that GLB-12 negatively regulates the p38 

MAPK pathway. Furthermore, we demonstrate that the p38 MAPK pathway is specifically active 

in the distal part of the germline, overlapping the same region where GLB-12 is present. 

Our biochemical analysis of GLB-12 made clear that this globin functions in redox signaling. 

Unlike most other globins, GLB-12 cannot bind oxygen; instead, it will actively convert oxygen 

to superoxide by electron transfer. The relatively unstable superoxide can in vivo be converted 

into hydrogen peroxide by superoxide dismutases (SODs). This hydrogen peroxide can then 

act as a biological messenger in redox signaling. When we applied glb-12 RNAi in mutants for 

the five C. elegans sod genes, we found that fecundity is further reduced in the intracellular 

SOD-1 mutant and restored to almost normal levels in the extracellular SOD-4 mutant. This 

suggests that these two SODs modulate the redox signaling pathway that is used by GLB-12. 

Translational reporters for SOD-1 and SOD-4 confirm their presence in, or around, the gonadal 

sheath. Furthermore, we also show that GLB-12, SOD-1 and SOD-4 work together to regulate 

p38 MAPK activity and germline apoptosis levels. 

Based on our results, we present a model in which GLB-12 is part of a redox signaling pathway 

that is modulated by an intracellular and extracellular SOD. This pathway regulates p38 and JNK 

MAPK activity, and, consequently, germline apoptosis levels. 

Contact: sasha.dehenau@ugent.be 

Lab: Braeckman 

Platform Session #2 Morphogenesis II and Cell Death 

9

sli-1 Cbl Inhibits the Engulfment of Apoptotic Cells 

Courtney Anderson1 , Shan Zhou1 , Emma Sawin1 , Bob Horvitz2 , Michael Hurwitz1 1 2 Yale University School of Medicine, New Haven, CT, USA, MIT, Cambridge, 

MA, USA 

The engulfment of apoptotic cells is required for normal metazoan development and 

tissue remodeling. In C. elegans, two parallel and partially redundant conserved pathways 

act in cell-corpse engulfment. One pathway includes the adaptor protein CED-2 CrkII and the 

small GTPase CED-10 Rac, and acts to rearrange the cytoskeleton of the engulfing cell. The 

other pathway includes the receptor tyrosine kinase CED-1 and might recruit membranes to 

extend the surface of the engulfing cell. Loss-of-function (lf) mutations in these genes cause 

the persistence of unengulfed cell corpses. 

Cbl, the mammalian homolog of the C. elegans signaling protein SLI-1, interacts with CrkL 

and Rac and modulates the actin cytoskeleton. Mutation of Cbl contributes to a wide range of 

human cancers. SLI-1 contains three domains, an N-terminal tyrosine kinase binding domain, 

a RING finger domain and a C-terminal proline-rich domain. SLI-1 inhibits LET-23 EGFR/LET- 

60 Ras signaling by ubiquitinating LET-23 via its RING finger domain. 

We found that sli-1(lf) suppresses the engulfment defects of ced-1 pathway null mutants 

but not of ced-10 Rac pathway mutants, suggesting that sli-1 acts either upstream of the 

ced-10 Rac pathway, in parallel to both pathways or downstream of the ced-1 pathway. The 

ced-10 Rac pathway is also required for proper migration of the distal tip cells (DTCs) during 

the development of the C. elegans gonad. sli-1(lf) partially restores normal DTC migration 

in ced-10 Rac pathway null mutants. Thus, SLI-1 does not act by inhibiting the CED-10 Rac 

pathway and is unlikely to be downstream of the CED-1 pathway since the CED-1 pathway is 

not involved in DTC migration. Mutation of another inhibitor of engulfment, the tyrosine kinase 

gene abl-1, in combination with sli-1(lf) enhanced the effect on both engulfment and DTC 

migration, demonstrating that the two genes act independently of each other. 

Experiments using sli-1 transgene constructs lacking specific domains of sli-1 show that 

only constructs lacking the N-terminus fail to rescue the sli-1 DTC migration defect completely, 

suggesting that the role of SLI-1 in these processes requires the tyrosine kinase binding domain 

but is at least partially ubiquitin ligase-independent. Consistent with this finding, modulation 

of LET-60 Ras signaling had no effect on engulfment. We propose that SLI-1 opposes the 

engulfment of apoptotic cells via a pathway that is distinct from the two known engulfment 

pathways using a mechanism not yet identified for SLI-1 in worms. 

Contact: Michael.hurwitz@yale.edu 

Lab: Hurwitz 

10 


MADD-2 Negatively Regulates Anchor Cell Invasion 

Matthias Morf1,2 , Ivo Rimann1 , Mariam Alexander3 , Peter Roy3 , Alex Hajnal1 1Institute of Molecular Life Sciences, University of Zurich, Zurich, 

Switzerland, 2Molecular Life Sciences PhD program, Uni ETH Zurich, 

Switzerland, 3Department of Molecular Genetics, The Terrence Connelly 

Centre for Cellular and Biomolecular Research, University of Toronto, 

Toronto, Canada 

Cell invasion is a tightly regulated process, during which cells cross tissue borders. 

Uncontrolled invasion can lead to metastatic cancer growth. During C. elegans larval 

development, a specialized cell in the somatic gonad called anchor cell (AC) breaks two 

basal laminae and then invades the adjacent vulval tissue to form a connection between the 

developing vulva and uterus. Multiple signals from the vulval cells and the ventral nerve cord 

regulate AC invasion. How the AC can integrate these signals is largely unknown. 

We have identified MADD-2, a conserved RING finger and E3 ubiquitin ligase protein, as a 

regulator of AC invasion. madd-2 has independently been identified by both the P. Roy lab as a 

gene acting downstream of the UNC-40 Netrin receptor and controlling muscle arm extensions 

and by the Bargmann & Tessier-Lavigne groups that showed a similar role of madd-2 in axon 

branching. The human madd-2 homologue, Mid1, is mutated in most cases of Opitz syndrome, 

a disease characterized by ventral midline defects. 

Our analysis of basal laminae breaching in madd-2 mutants demonstrates that AC invasion 

is delayed, but to a lesser extend than in unc-6 mutants. Surprisingly, madd-2(lf) partially 

rescues the unc-6(lf) AC invasion defects, suggesting that during AC invasion madd-2 is 

epistatic to unc-6. Additional analyses of AC shape, polarity and dynamics demonstrate a loss 

of proper AC orientation and the formation of highly dynamic ectopic protrusions in madd-2(lf) 

mutants, indicating a loss of directed invasion. We thus tested whether madd-2(lf) could allow 

AC invasion in the absence of any guidance cues. To eliminate all guidance cues for the AC, 

we ablated the VPCs in madd-2(lf); unc-6(lf) double mutants and checked for signs of basal 

laminae breaching. While the AC detached from the basal laminae that remained intact in VPC 

ablated unc-6(lf) single mutants, we observed AC attachment and initial signs of basal lamina 

breaching but no invasion in VPC ablated madd-2(lf); unc-6(lf) double mutants. 

We propose that madd-2 prevents undirected AC invasion, thus allowing the AC to respond 

to guidance cues from the ventral nerve cord and the vulval cells. In summary, madd-2 is the 

first negative regulator of AC invasion identified. 

Contact: matthias.morf@imls.uzh.ch 

Lab: Hajnal 

Platform Session #2 Morphogenesis II and Cell Death 

11

The C. elegans Hailey-Hailey Disease Homolog pmr-1 is Essential for 

Cell Migration During Gastrulation 

Vida Praitis 1 , Rebecca Mandt 1 , Leah Imlay 1 , Charlotte Feddersen 1 , Alexander 

Sullivan-Wilson 1 , Tyson Stock 1 , Walter Liszewski 1 , Adityarup Chakravorty 1 , Dae 

Gon Ha 1 , Angela Schacht 1 , Michael Miller 1 , Lensa Yohannes 1 , Juliet Mushi 1 , 

Zelealem Yilma 1 , Sarah Kniss 2 , Jeff Simske 3 

1 Grinnell College, Grinnell, IA, USA, 2 U. of Chicago, Chicago, IL USA, 

3 Rammelkamp Ctr, Cleveland, OH, USA 

Hailey-Hailey disease or benign familial pemphigus (MIM# 169600) is a semi-dominant 

human disease marked by severe skin lesions and blistering, thought to be the result of 

altered cell adhesion in keratinocytes. The disease is caused by mutations in ATP2C1/ 

SPCA1, a Ca2+/Mn2+ ATPase that localizes to the golgi where it acts in protein processing, 

metal homeostasis, and Ca2+ signaling. Our laboratory has been characterizing the role of 

PMR-1, the C. elegans homolog of ATP2C1. We first identified alleles of pmr-1 in two genetic 

screens designed to identify conditional alleles of genes required during morphogenesis. 

Subsequent mapping, complementation, and sequencing analysis confirmed that the identified 

strains carried mutations in pmr-1. All four alleles of pmr-1 are temperature-sensitive, showing 

complete embryonic lethality at 25C, but with increased viability at lower temperatures. Embryos 

homozygous for pmr-1 loss-of-function alleles die with variable terminal phenotypes, including 

enclosure failures, head ruptures, body morphogenesis defects and pharynx unattached 

phenotypes, defects that look superficially similar to the human cell adhesion phenotypes. 

Analysis using GFP expression constructs, as well as antisera staining experiments with a 

variety of cell fate markers, indicate cell fates are normal in Pmr-1 embryos. Similarly, cell 

lineaging analysis using StarryNite and Acetree software indicates cell division timing is normal. 

However, using these same tools, and taking advantage of temperature-shift analysis, we 

were able to identify specific cells that become mis-positioned in Pmr-1 embryos during midgastrulation. 

Our analysis indicates that while ingression is normal, cells that migrate along 

the surface of the embryo, including ventral neuroblasts, C-derived blastomeres, and anterior 

cells that give rise to hypodermis and ring ganglia, exhibit significantly reduced rates of cell 

migration compared to controls. It is these failed migrations that cause the later enclosure and 

morphogenesis defects. To better understand the molecular pathways responsible for the cell 

migration defects we have begun gene interaction experiments. Our analysis indicates that the 

embryonic lethality caused by pmr-1 loss-of-function alleles can be partially suppressed by a 

gain-of-function allele of the inositol-sensitive Ca++ channel gene itr-1. The most parsimonious 

explanation for these data is that pmr-1 and itr-1 act in the same Ca++ signaling pathway 

during cell migration. 

Contact: praitis@grinnell.edu 

Lab: Praitis 

12 


Control of Oocyte Meiotic Maturation: Links to Germ Cell Proliferation 

and Global Control of the Oogenic Program 

David Greenstein 

University of Minnesota, Minneapolis, MN, USA 

In sexually reproducing animals, oocytes arrest at diplotene or diakinesis and resume 

meiosis (meiotic maturation) in response to hormones. Chromosome segregation errors in 

female meiosis I are the leading cause of human birth defects; age-related changes in the 

hormonal environment of the ovary are a suggested cause. C. elegans serves as an incisive 

genetic model for studying the control of oocyte meiotic maturation by hormonal signaling and 

soma-germline interactions. The meiotic maturation processes in C. elegans and mammals 

share a number of molecular and biological similarities. Major sperm protein (MSP) and 

luteinizing hormone (LH), though unrelated in sequence, both trigger meiotic resumption 

using somatic Gαs-adenylate cyclase-PKA pathways and soma-to-germline gap-junctional 

communication. At a molecular level, the oocyte responses apparently involve the control of 

conserved protein kinase pathways and post-transcriptional gene regulation in the oocyte. At 

a cellular level, the responses include cortical cytoskeletal rearrangement, nuclear envelope 

breakdown, assembly of the acentriolar meiotic spindle, chromosome segregation, and likely 

changes important for fertilization and the oocyte-to-embryo transition. In my talk I will discuss 

our efforts to define oocyte meiotic maturation control mechanisms from signal reception 

to oocyte response. I will focus on recent results that establish links between the control of 

meiotic maturation and mechanisms needed for germ cell proliferation and the global control 

of the oogenic program. 

Contact: green959@umn.edu 

Lab: Greenstein 

Keynote 3 

13

Regulators of MSP Assembly and Dynamics in C. elegans 

Spermatocytes 

Kari Messina, Marc Presler, Leah Towarnicky, Diane Shakes 

College of William and Mary, Williamsburg, VA, USA 

In crawling spermatozoa, the major sperm protein (MSP) plays two key roles: as a 

cytoskeletal protein, its polymerization/depolymerization dynamics drive pseudopod motility, 

and as an extracellular signaling molecule, it triggers both oocyte maturation and ovulation. 

However during spermatogenesis, all transcription and most translation of this highly abundant 

protein (40% of the soluble protein in haploid sperm) occurs prior to the meiotic divisions. Thus 

in developing spermatocytes, MSP is sequestered into transient organelles called fibrous bodies 

(FBs) which both prevent MSP from interfering with chromosome segregation and cytokinesis 

and subsequently facilitate its segregation away from residual bodies and into spermatids. While 

biochemical studies have illuminated key details regarding the polymerization of MSP within 

pseudopods, the only factor previously known to be essential for the initial assembly of MSP 

into FBs was SPE-6, a spermatogenesis-specific member of the casein kinase I superfamily 

(Varkey et al., 1993; Muhlrad and Ward, 2002). 

Our laboratory has now identified SPE-7 as a key player in FB assembly. spe-7(mn252) 

spermatocytes fail to assemble FBs and exhibit subsequent cell cycle and cytokinesis defects. 

An anti-SPE-7 antibody reveals that wildtype SPE-7 dynamically localizes to FBs, segregates 

to budding spermatids, and then becomes undetectable in mature, haploid spermatids. We are 

currently analyzing SPE-7 dynamics in a variety of spermatogenesis-defective (Spe) mutants to 

understand its role in FB assembly and disassembly. Intriguingly, SPE-7 assembles into FB-like 

structures in the absence of spe-6, suggesting a model in which SPE-7 functions to nucleate 

FB assembly with kinase activity by SPE-6 required for MSP addition. On the other hand, the 

disappearance of SPE-7 does not coincide with FB disassembly; in gsp-3/4 mutant spermatids, 

MSP remains persistently locked in FB-like structures yet the timely disappearance of SPE-7 

remains unaffected. Notably, SPE-7 is stabilized in mutants in which individual spermatids fail 

to separate from the central residual body, suggesting a link to the segregation and separation 

events of the budding division and perhaps to the subcellular relocalization of SPE-6, which 

also occurs at this time. 

Contact: klprice@email.wm.edu 

Lab: Shakes 

14 

Platform Session #3 - Germline I and Gametogenesis

The sperm surface localization of the TRP-3/SPE-41 Ca2+ permeable 

channel depends on SPE-38 function in Caenorhabditis elegans 

Gunasekaran Singaravelu1 , Indrani Chatterjee1 , Sina Rahimi1 , Marina 

Druzhinina1 , Lijun Kang2 , Shawn Xu2 , Andrew Singson1 1 2 Waksman Institute, Piscataway, NJ, USA, University of Michigan, Ann 

Arbor, MI, USA 

Despite undergoing normal development and acquiring normal morphology and motility, 

mutations in spe-38 or trp-3/spe-41 cause identical phenotypes in Caenorhabditis elegans 

– mutant sperm fail to fertilize oocytesdespite direct contact. SPE-38 is a novel, four-pass 

transmembrane protein and TRP-3/SPE-41 is a Ca2+ permeable channel. Localization of 

both of these proteins is confined to the membranous organelles (MOs) in undifferentiated 

spermatids. In mature spermatozoa, SPE-38 is localized to the pseudopod and TRP-3/SPE-41 

is localized to the whole plasma membrane. Here we show that the dynamic redistribution of 

TRP-3/SPE-41 from MOs to the plasma membrane is dependent on SPE-38. In spe-38 mutant 

spermatozoa, TRP-3/SPE-41 is trapped within the MOs and fails to reach cell surface despite 

MO fusion with the plasma membrane. Split-ubiquitin yeast-two-hybrid analyses revealed that 

the cell surface localization of TRP-3/SPE-41 is likely regulated by SPE-38 through a direct 

protein-protein interaction mechanism. We have identified sequences that influence the physical 

interaction between SPE-38 and TRP-3/SPE-41, and show that these sequences in SPE-38 

are required for fertility in transgenic animals. We identified additional proteins that can bind 

with either SPE-38 or TRP-3/SPE-41 and a novel protein that can bind with both SPE-38 and 

TRP-3. Despite the mislocalization of TRP-3/SPE-41 in spe-38 mutant spermatozoa, ionomycin 

or thapsigargin induced influx of Ca2+ remains unperturbed. This work reveals a new paradigm 

for the regulated surface localization of a Ca2+ channel. 

Contact: guna@waksman.rutgers.edu 

Lab: Singson 

Platform Session #3 - Germline I and Gametogenesis 

15

Timely Generation of the Fertilization Calcium Wave by a Sperm TRP 

Channel 

Jun Takayama, Shuichi Onami 

RIKEN Quantitative Biology Center, Kobe, Japan 

Fertilization calcium wave is a universal trigger for the egg activation that converts the 

egg to the embryo. Although egg activation occurs in a timely coordinated fashion among 

other processes, the mechanism that ensures the on-time generation of the calcium wave is 

unknown. Here we show that the timely generation of the calcium wave is controlled by TRP- 

3, a sperm-specific calcium-permeable channel in C. elegans. 

First, we found that fertilization generates a biphasic calcium wave in the oocyte: the fast 

local wave and the slow global wave. The fast local wave emerged and disappeared near the 

sperm entry point, whereas the slow global wave traveled from that point to the opposite pole. 

The calcium response started at the moment of sperm entry, which was visualized either by a 

sudden bulge of the oocyte cytoplasm or by a dark “patch” appeared in the oocyte membrane 

labeled by GFP-PH. 

Next, we examined the calcium response of mutants defective in sperm or oocyte function. 

spe-9 mutants, whose sperm cannot enter into the oocyte, showed no calcium response. Egg 

activation factor mutant spe-11, whose sperm cannot activate the egg but can enter into the 

oocyte, showed a wildtype-like biphasic wave. Although most of the mutants of trp-3 cannot 

enter into the oocyte, we found that trp-3(sy693) escapers that entered into the oocyte showed 

no local wave but a wildtype-like global wave with delayed onset. Local calcium elevation could 

trigger the simultaneous generation of the global wave in computer simulation that assumes 

that the oocyte has calcium-induced calcium release (CICR) machinery. Approximately half of 

the embryos fertilized by trp-3 escapers did not hatch. On the other hand, egg factor mutant 

egg-3(tm1191), whose oocyte fails to become activated, showed a reduced amplitude in the 

global wave. 

These results suggest that (i) triggering factor(s) for the calcium wave and SPE-11 are 

independently transmitted from sperm and (ii) TRP-3 is required both for the generation of the 

local wave and the timely generation of the global wave. The global wave may be generated 

either spontaneously with delayed onset or in response to the local calcium elevation, as is the 

case with IP3 receptors that are stimulated by IP3. Therefore the local calcium wave mediated 

by sperm TRP-3 may guarantee the on-time generation of the calcium wave probably to 

coordinate with other processes during the oocyte-to-embryo transition. 

Contact: jtakayama@riken.jp 

Lab: Onami 

16 

Platform Session #3 - Germline I and Gametogenesis

Regulation of Meiotic DSB Formation in C. elegans 

Simona Rosu, Anne Villeneuve 

Stanford University, Stanford, CA 

Crossovers (COs) between the DNA molecules of homologous chromosomes provide the 

basis of physical links (chiasmata) that ensure proper segregation of homologs at the meiosis 

I division. Crossover recombination is initiated by formation of double strand DNA breaks 

(DSBs) by the SPO-11 protein. 

We identified a new gene (F26H11.6, defined by the me96 mutation) involved in promoting 

DSB formation during C. elegans meiosis. me96 mutant worms exhibit a defect in chiasma 

formation that becomes progressively more severe with age. Immunofluorescence experiments 

suggest the defect is a result of reduced DSBs; furthermore, providing exogenous breaks 

by irradiation rescues chiasma formation. The F26H11.6 protein localizes to chromatin in 

transition zone and early pachytene nuclei, corresponding to the presumed timing of DSB 

formation during meiotic prophase. This localization suggests that F26H11.6 may help create 

a chromatin environment that is competent for SPO-11 dependent DSB formation. F26H11.6 

localization is dependent on CHK-2, a kinase required for early meiotic events including pairing, 

chromosome mobilization, and DSB formation. Furthermore, F26H11.6 localization is prolonged 

in mutants lacking synaptonemal complex (SYP) proteins (which exhibit persistent chromosome 

mobilization and elevated levels of DSB-dependent recombination intermediates) and in some 

mutants impaired in downstream steps of CO formation. These and other data suggest that 

F26H11.6 localization serves as a marker for DSB competence, providing a visual readout for 

the operation of feedback mechanisms that coordinate competence for DSB formation with 

the organizational state of the chromosomes and that couple cessation of DSB formation with 

the formation of CO-competent recombination intermediates. 

Contact: srosu@stanford.edu 

Lab: Villeneuve 

Platform Session #3 - Germline I and Gametogenesis 

17

Title: TBD 

Karen Oegema 

University of California - San Diego 

Contact: koegema@ucsd.edu 

Lab: Oegema 

18 

Keynote 4

Evolution of spindle shape and motion in one-cell stage nematode 

embryos 

Aurore-Cecile Valfort1 , Soizic Riche1 , Reza Farhadifar2 , Daniel Needleman2 , 

Marie Delattre1 1 2 LBMC, CNRS-University of Lyon1, Lyon, France, Harvard University, 

SEAS, MCB, Cambridge, MA, USA 

Although genomic, molecular, development and phenotypic evolution have been largely 

explored; there is little understanding of how cell biological processes evolve. Due to their 

fundamental role at the basis of developmental processes, cellular functions are highly 

constrained and are often thought to rely on very conserved underlying mechanisms. However, 

constraints on the final phenotype could hide a diversity of underlying mechanisms that remain 

cryptic, yet contributing to the flexibility of cellular processes. Most nematode embryos undergo 

a first asymmetric division that gives rise to a small posterior cell containing germ cell fate. 

We used this unique feature to ask 1) whether this strong phenotypic conservation reflects 

different underlying mechanisms of asymmetric spindle positioning, 2) which steps are the 

most constrained, 3) which steps can vary and allow the diversification of solutions retained 

in the course of evolution. 

To address these questions we recorded the first embryonic division of ~60 different 

nematode species (3 different strains per species, 15 embryos per strain) by DIC microscopy 

and found a large diversity of intermediate phenotypes such as nuclei centration, spindle 

position, centrosome size, spindle motion, spindle elongation, etc. We developed an automated 

DIC image analysis, enabling us to quantity the differences in thousands of embryos from 

hundreds of strains. Importantly, differences are also found on a microevolutionnary time 

scale, between species of the Caenorahbditis genus. Parameter measurement is underway 

and will be mapped on the well-known phylogeny to deduct the evolutionary trend of spindle 

shape and motion changes. 

In a subset of interesting species, in depth analysis will be done to identify parameters 

changes. We will present our detailed comparison between C. elegans and C. briggsae 

embryos. Through a combination of molecular and biophysical approaches we uncovered 

a new conserved mechanism in the control of anaphase spindle oscillations and identified 

interspecific changes in the control of the G-protein regulator GPR. 

Contact: marie.delattre@ens-lyon.fr 

Lab: Delattre 

Platform Session #4 - Cell Cycle and Cell Biology 

19

The Tousled-like Kinase TLK-1 is a Component of the Outer 

Kinetochore and Potentiates Mitotic Spindle Dynamics in the Early C. 


Jessica De Orbeta, Jason Ford, Gary Deyter, Tokiko Furuta, Jill Schumacher 

University of Texas MD Anderson Cancer Center 

Tousled-like kinases are highly conserved and have been implicated in chromatin 

remodeling, transcription, and DNA replication and repair. We discovered that C. elegans 

TLK-1 also has an essential role in mitosis as a substrate and activator of the Aurora B 

kinase AIR-2. AIR-2 phosphorylates TLK-1 at S634 and while TLK-1 is highly expressed in 

interphase nuclei, a phospho-specific antibody revealed that pTLK-1(S634) is localized to 

kinetochores, as is a GFP-TLK-1 transgenic protein. To discern the functional role of TLK-1 at 

kinetochores, we interrogated the placement of TLK-1 in the kinetochore assembly hierarchy. 

This analysis revealed that TLK-1 is localized to the outer kinetochore downstream of BUB-1, 

and is independent of the NDC-80 or RZZ kinetochore complexes. Examination of BUB-1dependent 

kinetochore proteins revealed that TLK-1 kinetochore localization is disrupted in 

hcp-1/cenp-f(RNAi) embryos but is not affected by loss of the highly related HCP-2 protein, or 

the CENP-F-dependent proteins LIS-1 and CLS-2/CLASP. Instead, our results revealed that 

TLK-1 is required for the kinetochore localization of both LIS-1 and CLS-2. While neither CLS-2 

nor TLK-1-depletion results in severe mitotic defects in early embryos, embryos co-depleted 

for TLK-1 and CLS-2 display gross errors in chromosome congression and segregation, 

suggesting that these proteins are collaborating to enable functional kinetochore-microtubule 

attachments. We recently identified a second role for TLK-1 in mitotic spindle dynamics in 

the early C. elegans embryo. Live imaging of GFP-labeled embryos treated with control or 

tlk-1(RNAi) revealed that spindle rotation is significantly delayed in TLK-1-depleted one-cell 

embryos. While nuclear envelope breakdown (NEB), and chromosome condensation and 

congression occurred with similar kinetics, the nuclear/centrosome complex did not rotate 

until after NEB and congression in tlk-1(RNAi) embryos, resulting in metaphase spindles that 

were perpendicular to the anterior-posterior axis. Despite this delay, the spindles of the TLK- 

1-deficient embryos completed rotation by mid-anaphase, essentially undergoing a very fast, 

albeit late rotation. Additional live imaging revealed that significantly fewer microtubules reach 

the cell cortex in one-cell tlk-1(RNAi) embryos, suggesting that TLK-1 potentiates microtubule 

dynamics in the early C. elegans embryo. 

Contact: jschumac@mdanderson.org 

Lab: Schumacher 

20 

Platform Session #4 - Cell Cycle and Cell Biology

Identification of unconventional components of the γ-tubulin complex 

in C. elegans 

Nami Haruta1 , Eisuke Sumiyoshi1 , Yu Honda1 , Masahiro Terasawa2 , Mika Toya2 , 

Asako Sugimoto1,2 1Graduate School of Life Sciences, Tohoku University, Sendai, Japan, 

2Laboratory for Developmental Genomics, RIKEN Center for Developmental 

Biology,Kobe, Japan 

The γ-tubulin complexes (γTuC) play an important role in microtubule nucleation. Many 

organisms have two types of γTuCs,the γ-tubulin small complex (γTuSC) and the γ-tubulin 

ring complex (γTuRC).The γTuSC consists of γ-tubulin and two other components (GCP2and 

GCP3 in mammals), and γTuSCs and several additional components(such as GCP4~6) 

form γTuRCs that have higher polymerization activity than γTuSCs. In C.elegans, while the 

γTuSC components are known (γ-tubulin/TBG-1,GCP2/GIP-1, GCP3/GIP-2), γTuRC-specific 

components have not been identified in the genome. 

Here, we report the identification of two candidate proteins for γTuC components. These 

two proteins, tentatively named GTAP-1 and GTAP-2, were co-immunoprecipitated with 

FLAG-tagged γ-tubulin from C. elegans embryonic extracts. Although both proteins did not 

have detectable homologies with γTuRC-specific components of other organisms, yeast 

two-hybrid analysis showed the physical interaction between GTAP-2 and one of the γTuSC 

components, GIP-2, and sucrose gradient centrifugation showed that both GTAP-1 and-2 were 

cofractionated with γ-tubulin. Live imaging of GFP-tagged GTAP-1 and -2 in C. elegans embryo 

revealed that both proteins colocalized with γ-tubulin at centrosomes throughout cell cycle, 

and this centrosomal localization is dependent on γ-tubulin. On the other hand, RNAi depletion 

of GTAP-1 and/or -2 caused ~70% decrease of γ-tubulin at centrosomes, but surprisingly the 

amount of microtubules was unaffected and the RNAi-treated embryos were viable. Under the 

condition in which the amount of centrosomal γ-tubulin was reduced, depletion of GTAP-1/2 

decreased centrosomal microtubules. Taken together, we propose that GTAP-1 and GTAP-2 

are novel components of the γTuC, and that they contribute to the recruitment of γ-tubulin to 

centrosomes. Additionally, our results imply that in normal conditions only a minor fraction of 

the γTuC at centrosomes is used for microtubule nucleation. 

Contact: asugimoto@m.tohoku.ac.jp 

Lab: Sugimoto 


21

The Cdc48/p97 cofactor UBXN-2 and its orthologues p47/p37 control 

centrosome maturation in prophase via Aurora A 

Elsa Kress1 , Francoise Schwager1 , Rene Holtackers2 , Esther Zanin3 , Francois 

Prodon1 , Jonas Seiler4 , Annika Eiteneuer4 , Asako Sugimoto5 , Hemmo Meyer4 , 

Patrick Meraldi2 , Monica Gotta1 1Faculty of Medicine, University of Geneva, Geneva, Switzerland, 

2Eidgenossische Technische Hochschule (ETH), Zurich, Switzerland, 

3Ludwig Institute for Cancer Research, University of California, San Diego, 

USA, 4Faculty of Biology, Universitat Duisburg-Essen, Essen, Germany, 

5Graduate School of Life Sciences, Tohoku University, Sendai, Japan. 

Tight temporal regulation of mitotic events and the spatial coordination of the cleavage 

plane with the axis of chromosome segregation are essential prerequisites for a successful 

cell division. During asymmetric cell divisions, the axis of chromosome segregation must also 

be aligned with the axis of polarity to allow proper segregation of cell fate determinants. This is 

achieved by orienting the mitotic spindle along the axis of polarity. Here we show that premature 

centrosome maturation results in defects in spindle orientation and aberrant asymmetric cell 

division. 

We find that depletion of UBXN-2, a substrate adapter of the AAA ATPase cdc48/ 

p97, prevents alignment of the mitotic spindle with the polarity axis in C.elegans. Our data 

indicate that UBXN-2 limits Aurora A accumulation at centrosomes during early prophase. In 

ubxn-2(RNAi) embryos, Aurora A is recruited earlier than in wild type, centrosomes display 

enhanced accumulation of microtubule dynamics regulators and an increased growth rate of 

astral microtubules. Furthermore UBXN-2 and AIR-1 co-immunoprecipitate from embryonic 

extracts suggesting a close association of these molecules. The spindle defects observed in 

ubxn-2(RNAi) embryos are partially rescued by co-depletion of AIR-1. Therefore, we postulate 

that UBXN-2 regulates AIR-1 levels at centrosomes to couple centrosome maturation timing 

with mitotic progression. This pathway is conserved as we further find in human cells, that 

the two orthologues of UBXN-2, p37 and p47 regulate the timing of centrosome separation in 

prophase, by limiting the centrosomal recruitment of Aurora A. 

Contact: elsa.kress@unige.ch 

Lab: Gotta 

22 


Regulation of COPII subunit recruitment to ER exit sites 

Kristen Witte1 , Amber Schuh1 , Jan Hegermann2 , Ali Sarkeshik3 , Jonathan 

Mayers1 , Katrin Schwarze2 , John Yates III3 , Stefan Eimer2 , Anjon Audhya1 1 2 University of Wisconsin-Madison, Madison, WI, USA, European 

Neuroscience Institute and Center for Molecular Physiology of the Brain 

(CMPB), 37077 Goettingen, Germany, 3The Scripps Research Institute, La 

Jolla, California 92037, USA 

Export of proteins from the endoplasmic reticulum (ER) in COPII-coated vesicles occurs at 

defined sites, which contain the scaffolding protein SEC-16. Although SEC-16 has been shown 

to interact with multiple COPII subunits to mediate vesicle biogenesis at the ER, mechanisms 

by which COPII recruitment is regulated remain poorly defined. Using both functional genomics 

and biochemical approaches, we identify a new, conserved SEC-16-interacting protein named 

TFG-1 that is required for COPII subunit accumulation at ER exit sites. Consistent with this 

finding, depletion of TFG-1 inhibits secretion of multiple cargoes from the ER. Furthermore, 

using immuno-gold EM techniques, we demonstrate that TFG-1 localizes to a matrix between 

ER exit sites and the Golgi. We hypothesize that a TFG-1 matrix may serve as a molecular 

sink, which helps to retain COPII components locally and facilitate efficient vesicle egress 

from the ER. 

Contact: audhya@wisc.du 

Lab: Audhya 


23

Condensin I: A New Component of the Abscission Checkpoint 

Joshua Bembenek, Koen Verbrugghe, Gyorgyi Csankovszki, Raymond Chan 

University of Michigan, Ann Arbor, MI, USA 

During cell division, chromosomes must clear the path of the cleavage furrow before the 

onset of cytokinesis. If chromatin obstructs the furrow, it can be damaged and furrow regression 

can occur causing mutations and aneuploidy, defects common in cancer. The abscission 

checkpoint stabilizes the cleavage furrow in the presence of chromatin obstructions, thus 

preventing the cleavage furrow from regressing and allowing more time for resolving chromatin 

obstructions (Steigemann et al., Cell, 2009). Whether cells with chromatin obstructions in a 

developing embryo can suppress cytokinesis failure has not been determined. This is especially 

important because conventional checkpoints that minimize chromosome segregation defects 

are sometimes attenuated during early embryonic divisions. To investigate this, we inactivated 

several genes essential for segregation in C. elegans embryos. We found that the P0 blastomere 

robustly suppresses furrow regression following depletion of condensin, cohesin, HCP-3 

(CENP-A) and TOP-2 (topoisomerase II). The robustness of this suppression is reduced in AB 

and P1 blastomeres and when condensin is depleted. This response correlates with activation 

of AIR-2 (Aurora B) at the spindle midzone, which is needed for the abscission checkpoint in 

other systems. Condensin I, but not condensin II, localizes to the spindle midzone in anaphase 

and to the midbody during cytokinesis. Interestingly, condensin I shows prominent accumulation 

in the spindle midzone and midbody region in the presence of chromatin bridges, in a SPD-1 

and AIR-2 dependent manner. We postulate that condensin I is required for either sensing or 

responding to chromatin obstructions to prevent cleavage furrow regression. 

Contact: bembenek@umich.edu 

Lab: Chan 

24 


LEM-4 Coordinates Mitotic Signaling on BAF to Enable its Essential 

Function in Nuclear Envelope Formation 

Matyas Gorjanacz1 , Claudio Asencio1 , Iain Davidson1 , Rachel Santarella- 

Mellwig1 , Geraldine Seydoux 2 , Iain Mattaj1 1 2 European Molecular Biology Laboratory, Heidelberg, Germany, Howard 

Hughes Medical Institute, Johns Hopkins University School of Medicine, 

Baltimore, USA 

The nucleus is the most complex eukaryotic organelle. Its structure is defined by the 

nuclear envelope (NE); composed of the NE membranes, nuclear pore complexes and in 

metazoa the nuclear lamina. In higher eukaryotes NE disassembles and reassembles during 

every cell division in order to allow faithful segregation of condensed sister chromatids. These 

mitotic events are driven by spatiotemporally controlled reversible phosphorylation of key 

molecules. While multiple phosphorylation events have been already described to drive NE 

disassembly, it is almost entirely unknown how dephosphorylation is regulated to enable its 

reassembly. By screening Caenorhabditis elegans strains harboring temperature sensitive 

embryonic lethal mutations we have identified lem-4 as a mitotic regulator that is required for 

the function of both a mitotic kinase and a phosphatase that act on BAF, an essential factor of 

nuclear assembly. We found that during mitotic exit LEM-4 is required for dephosphorylation 

of BAF by simultaneously inhibiting BAF’s mitotic kinase, VRK-1, and by stimulating a protein 

phosphatase 2A (PP2A) complex that can dephosphorylate BAF. By coordinating VRK-1 and 

PP2A mediated signaling on BAF, LEM-4 enables postmitotic NE reformation in a function that 

is conserved from worm to humans. 

Contact: gorjanac@embl.de 

Lab: Mattaj 


25

Filamin is Required to Initiate Calcium Signaling and Maintain F-actin 

Organization in the Spermatheca 

Ismar Kovacevic, Erin Cram 

Northeastern University, Boston, MA, USA 

Mechanosensation at the cellular and tissue levels is critical for normal development and 

organ function. We are using the C. elegans spermatheca as a model system to study how cells 

sense mechanical forces in vivo. The spermatheca is a simple myoepithelial tube that naturally 

experiences cycles of stretching and constriction. Ovulated oocytes stretch the spermatheca, 

and trigger directional constriction to propel embryos into the uterus. We identified the C. 

elegans filamin ortholog FLN-1 as being required for normal spermathecal transit. Filamin is a 

stretch-sensitive structural and signaling scaffold that binds F-actin, transmembrane receptors, 

and a variety of intracellular signaling proteins. FLN-1 is expressed in spermathecal and 

uterine cells, colocalizes with F-actin, and is required to maintain the actin cytoskeleton in the 

spermatheca and uterus. Filamin-deficient animals accumulate embryos in the spermatheca, 

and consequently lay damaged eggs and exhibit reduced brood sizes. PLC-1/phospholipase 

C-ε is also required for the exit of embryos from the spermatheca, and analysis of double 

mutant animals suggests that PLC-1 and FLN-1 act in the same pathway. Because PLC-1 

is thought to be upstream of intracellular calcium release, we used GCaMP—a genetically 

encoded calcium indicator—to image calcium during ovulation and spermathecal transit. 

Entry of an oocyte into the spermatheca initiates a distinctive series of calcium oscillations. 

The calcium transients originate at the distal end of the spermatheca and travel towards 

the uterus, in the same direction as ovulated oocytes. This suggests the calcium waves are 

controlling the directional constriction of the spermatheca. Loss of FLN-1 results in delayed 

onset of calcium signaling, followed by abnormal calcium oscillations. As expected, loss of 

PLC-1 entirely eliminates calcium signaling in the spermatheca. Gain-of-function mutations in 

the ITR-1/IP3 receptor enhance calcium release in the spermatheca, and partially rescue the 

brood size defect of filamin-deficient animals. We hypothesize that filamin is required in the 

spermatheca to maintain the cytoskeleton, respond to increased tension, and initiate calcium 

oscillations via the phosphatidylinositol pathway. Current work is focused on understanding 

the dual roles of filamin as a signaling and structural scaffold, as well as uncovering other 

components of the pathway. 

Contact: ismar.k@husky.neu.edu 

Lab: Cram 

26 


Germline maintenance and meiosis: mechanistic insights from C. 


Monica Colaiácovo 

Harvard Medical School 

Contact: mcolaiacovo@genetics.med.harvard.edu 

Lab: Colaiácovo 

Keynote 5 

27

Identification of Direct GLP-1/Notch Targets that Regulate Germline 

Stem Cells 

Aaron Kershner1 , Heaji Shin1 , Judith Kimble1,2 1 2 University of Wisconsin-Madison, Madison, WI, USA, Howard Hughes 

Medical Institute, University of Wisconsin-Madison, Madison, WI, USA 

GLP-1/Notch signaling maintains germline stem cells (GSCs): its loss drives germ cells 

from mitosis to meiosis at any stage of development and in either sex. Downstream of GLP- 

1/Notch, two PUF proteins, FBF-1 and FBF-2 (collectively FBF), are also required for GSC 

maintenance, but only in L4s and adults. Thus, GLP-1/Notch must act through other factors 

in addition to FBF. We reasoned that such other factors might be targets of both GLP-1/Notch 

and FBF, and therefore investigated 14 genes common to lists of likely GLP-1/Notch (1) and 

FBF targets (2). Interestingly, simultaneous loss of two of these genes had the glp-1 null 

phenotype: single lst-1 (lateral signaling target) or sygl-1 (synthetic Glp) mutants had virtually 

normal germlines, but lst-1 sygl-1 double mutants made only 4-8 germ cells that differentiated 

into sperm (also see Shin et al abstract). Moreover, lst-1 and sygl-1 were also required for 

GSC maintenance in males and adults. Thus, lst-1 and sygl-1 are redundantly required for 

GSC maintenance throughout development, and their deletion mimics glp-1 loss. LST-1 protein 

harbors one Nanos-like zinc finger motif, suggesting a role in RNA regulation, but SYGL-1 has 

no recognizable motifs. To gain clues to function, we placed lst-1 sygl-1 in the genetic pathway 

of GSC control and examined their expression. Epistasis placed lst-1 and sygl-1 downstream 

or parallel to GLP-1/Notch and upstream of the GLD meiotic entry regulators. In situs revealed 

abundant lst-1 and sygl-1 mRNAs in the distal germline where GSCs reside, but not more 

proximally until oogenesis. Consistent with the idea that they are GLP-1/Notch targets, lst-1 and 

sygl-1 mRNA distal expression depended on active GLP-1/Notch signaling. Moreover, removal 

of LAG-1 binding sites from the sygl-1 promoter abolished distal but not proximal expression. 

Therefore, lst-1 and sygl-1 are likely bona fide targets ofGLP-1/Notch signaling in the distal 

germline. Epitope-tagged LST-1 protein localized to the cytoplasm. Based on its cytoplasmic 

location and its putative zinc finger, we suggest that LST-1, and possibly SYGL-1, have a key 

role in RNA regulation. Most importantly, this work forges an essential link between GLP-1/ 

Notch signaling and its direct targets in the GSC self-renewal pathway. 

(1) Yoo and Greenwald (2004), Science 303:637-8; (2) Kershner and Kimble (2010), PNAS 107:3936-41. 

Contact: akershner@wisc.edu 

Lab: Kimble 

28 

Platform Session #5 - Germline II, Meiosis and Sex Determination/Dimorphism

Genome-wide Analysis of GLD-1 Mediated mRNA Regulation 

Uncovers a Role in mRNA Storage 

Claudia Scheckel, Dimos Gaidatzis, Jane Wright, Rafal Ciosk 

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland 

Translational repression is often accompanied by mRNA degradation. In contrast, many 

mRNAs in germ cells and neurons are ‘stored’ in the cytoplasm in a repressed but stable 

form. Unlike repression, the stabilization of these mRNAs is little understood. A key player 

in C. elegans germ cell development is the STAR domain protein GLD-1. By genome-wide 

analysis of mRNA regulation in the germ line, we observed that GLD-1 has a widespread role in 

repressing translation but, importantly, also in stabilizing a sub-population of its mRNA targets. 

Additionally, these mRNAs appear to be stabilized by the DDX6-like RNA helicase CGH-1, which 

is a conserved component of germ granules and processing bodies. Because many GLD-1 

and CGH-1 stabilized mRNAs encode factors important for the oocyte-to-embryo transition 

(OET), our findings suggest that the regulation by GLD-1 and CGH-1 serves two purposes. 

Firstly, GLD-1 dependent repression prevents precocious translation of OET-promoting mRNAs. 

Secondly, GLD-1 and CGH-1 dependent stabilization ensures that these mRNAs are sufficiently 

abundant for robust translation when activated during OET. In the absence of this protective 

mechanism, the accumulation of OET-promoting mRNAs, and consequently the oocyte-toembryo 

transition, might be compromised. 

Contact: rafal.ciosk@fmi.ch 

Lab: Ciosk 

Platform Session #5 - Germline II, Meiosis and Sex Determination/Dimorphism 

29

In the C. elegans Germ Line, S6K promotes Cell Cycle Progression 

and the Proliferative Fate and mediates the Effects of Diet 

Dorota Korta1 , Debasmita Roy1 , Simon Tuck2 , E. Jane Albert Hubbard1 1 2 New York University, New York (NY), USA, Umea University, Umea, 

Sweden 

Proper coordination of cell cycle progression with the balance between proliferative and 

differentiated fates is crucial for normal development in all organisms. In addition, an organism’s 

nutritional environment can also influence development. However, mechanisms underlying 

this coordination remain poorly understood. We use C. elegans germline development as a 

model to understand the molecular basis for this coordination. Our recent results implicate 

several highly conserved signaling pathways in this regulation, including the Insulin (Michaelson 

et al., 2010), Target of Rapamycin (TOR) (Korta et al. 2012), and TGFβ (Dalfó et al., 2012) 

pathways, together with the previously known role of the Notch pathway (see Hansen and 

Schedl, 2006; Kimble and Crittenden, 2007 for reviews). Recently, we found that rsks-1/ 

p70S6-Kinase (S6K), a direct substrate of the TOR/RAPTOR complex, is required for the 

proper accumulation of proliferative germ cells (“progenitors”) during larval development. 

This accumulation is important for establishing an optimal adult germline progenitor pool. 

This role for rsks-1 is germline-autonomous and requires a conserved TOR phosphorylation 

site. We found a similar but more severe defect upon germline-depletion of let-363/TOR or 

daf-15/RAPTOR. In other organisms, the TOR/RAPTOR complex also positively regulates 

eIF4E. Consistent with a conserved pathway in worms, we found that ife-1/eIF4E is also 

required for optimal expansion of the larval progenitor pool and that loss of both rsks-1 and 

ife-1 together reduces the germline progenitor pool more severely than either single mutant, 

similar to the depletion of TOR. Further, we found that rsks-1/S6K both promotes overall cell 

cycle progression and inhibits larval germline progenitor differentiation, and its germline role 

is genetically distinct from its influence on lifespan. Finally, we showed that rsks-1 activity is 

an important mediator of the effects of diet, especially amino acids, on the expansion of the 

larval germline progenitor pool . Our current work explores differences and similarities between 

the roles of TOR/ RAPTOR, ife-1 and rsks-1, and the mechanisms by which these pathways 

regulate cell cycle, differentiation and response to diet. 

Contact: jane.hubbard@med.nyu.edu 

Lab: Hubbard 

30 


Chromosome and centrosome inheritance in meiosis 

Mara Schvarzstein, Anne Villeneuve 

Stanford University 

Successful embryonic development requires that at fertilization each gamete provide 

complementary components to the zygote. In C. elegans, the sperm contribute both a haploid 

genome and a pair of engaged centrioles. Correct centriole organization during male meiosis is 

critical to ensure normal bipolar mitotic spindle in the zygote. We identify a new role for meiosis 

specific HORMA domain proteins in regulating centriole dynamics during spermatocyte meiosis. 

During male meiosis centrioles normally undergo two rounds of duplication, resulting in haploid 

sperm each containing a single tightly engaged centriole pair. In horma mutants, we observe 

inappropriate separation of centrioles during meiosis II, resulting in separated centrioles in 

sperm. Further, an extra pair of centrosomes is detected in a subset of zygotes, presumably 

reflecting a single additional round of centriole duplication that was enabled by precocious 

centriole separation. We showed previously that HORMAs HTP-1/2 prevent premature loss of 

sister chromatid cohesion in the meiotic divisions by preventing removal of cohesin complexes 

containing the meiosis-specific subunit REC-8. We find that rec-8 spermatocytes have similar 

inappropriate centriole separation phenotypes to those observed in htp-1/2 mutants. Our 

mutant analysis also implicates separase and shugoshin in centriole separation. Our findings 

are consistent with HORMA proteins preventing centriole disengagement by antagonizing 

separase-dependent cohesin removal. These findings suggest that the same specialized 

meiotic mechanisms that evolved to prevent premature release of sister chromatid cohesion 

also function to inhibit centriole separation, thereby ensuring that the zygote inherits the 

appropriate complement of chromosomes and centrioles. 

Although premature separation of centrioles in the horma spermatocyte results in a subset 

of one-cell embryos having multipolar spindles, centriole separation is not sufficient to trigger 

multipolar spindle formation in the dividing mutant spermatocytes. Instead we found that 

chromosome structure is important for normal spindle organization in spermatocyte meiosis. 

Our analysis of meiotic mutant spermatocytes, uncovered that a subset of these mutants 

exhibited multipolar spindles. Presence or absence of aberrant spindles correlated with 

ability of chromosomes to biorient at meiosis I. Our analyses imply that the presence of a few 

chromosomes proficient for biorientation at meiosis I ensure the formation of bipolar spindles. 

Contact: maras1@stanford.edu 



31

Role of the Inhibitory Kinase WEE-1.3 in Regulating the Meiotic Cell 

Cycle and Fertility in C. Elegans 

Anna Allen, Jessica Nesmith, Andy Golden 

National Institutes of Health, Bethesda, MD, USA 

Meiosis is a specialized cell cycle by which the haploid gametes, oocytes and sperm, are 

produced. It is of crucial importance for sexual reproduction and human health, as defects 

during the meiotic divisions have serious deleterious outcomes such as infertility, spontaneous 

miscarriages, birth defects, and tumorigenesis. Meiosis is controlled via dueling regulatory 

phosphorylation events on the cyclin-dependent kinase (Cdk1) component of maturation 

promoting factor (MPF). The Wee1/Myt1 family of kinases provides inhibitory phosphorylations 

that keep MPF inactive, halting the meiotic cell cycle until it is stimulated to resume and 

coordinate oocyte maturation with fertilization. We have previously shown in C. elegans that 

depletion of the Myt1 ortholog WEE-1.3 causes precocious oocyte maturation and a very 

penetrant infertility phenotype. To further investigate the function of WEE-1.3 during oocyte 

maturation, we generated WEE-1.3 fluorescently tagged transgenic fusion lines and found 

that the WEE-1.3 protein exhibited a perinuclear expression pattern throughout the germline 

and developing embryo. By quantitative PCR we demonstrated that WEE-1.3-depleted 

germlines, containing precocious oocytes, have begun to transcribe embryonic genes and 

exhibit inappropriate expression of proteins normally limited to fertilized eggs. In addition, we 

performed an RNAi suppressor screen of the infertile phenotype exhibited upon WEE-1.3 

depletion to identify novel factors that when co-depleted with WEE-1.3 restore fertility to the 

animals. We screened ~1900 essential genes and identified 150 that are suppressors of the 

WEE-1.3 depletion phenotype. Currently we are investigating the mechanism of how one 

suppressor ETR-1, an RNA-binding protein with human homologs implicated in disease, is 

functioning. Notably, our data supports a novel role for ETR-1 in germline development and/ 

or function. Many of the genes identified in this screen have the potential to be important, 

previously unknown, players in both the meiotic and mitotic cell cycles due to their interaction 

with a known cell cycle inhibitor. These studies are providing valuable input not only into meiotic 

maturation, but also into how the cell cycle is appropriately regulated and potential ways to 

bring an abnormal cell cycle back under control. 

Contact: allenanna@mail.nih.gov 

Lab: Golden 

32 


The Torsin Homolog OOC-5 is Required for Normal Nucleoporin 

Localization 

Michael White VanGompel, Lesilee Rose 

UC Davis, Davis, CA, USA 

The AAA+ ATPase OOC-5 is required for the re-establishment of polarity and spindle 

rotation in the P1 cell of 2-cell embryos. OOC-5 localizes to the endoplasmic reticulum (ER) 

and contiguous nuclear envelope, and is a homolog of the human DYT1 gene encoding the 

TorsinA protein. Mutations in DYT1 lead to early onset dystonia, a neuromuscular disease 

that usually presents during adolescence. Recent work has identified nuclear envelope (NE) 

proteins as interacting partners of TorsinA, including the outer nuclear envelope KASH protein 

Nesprin-3. What the precise role of TorsinA at the NE is remains unclear. Interestingly, it has 

been shown that in C. elegans the depletion of certain nucleoporins, components of nuclear 

pore complexes (NPCs), leads to an ooc-5 like phenotype. This suggests a link between 

Torsins and nuclear pores in the worm. Using antibody staining and GFP reporter strains 

we found defects in nucleoporin localization in ooc-5 worms, though lamin appears normal. 

Nucleoporin localization is apparently normal in the mitotic zone of mutant gonads. Clustering of 

nucleoporins occurs in the transition zone, where germ cells enter meiosis, and persists through 

all stages of meiosis. Interestingly, mislocalization of the outer nuclear envelope KASH protein 

ZYG-12 is not present until pachytene, after the transition zone, and is less severe than NPC 

defects. We are currently investigating whether NPC defects precede ZYG-12 defects in ooc-5 

worms, and whether nuclear exclusion is affected. Nucleoporin clustering is also apparent in 

ooc-5 intestinal cells, indicating that ooc-5-dependent localization of NPC components is not 

germ-cell specific. Furthermore, EM analysis of ooc-5 oocytes shows blebbing of the nuclear 

envelope similar to that reported in torsinA mutant mice. Our results suggest that OOC-5 plays 

an important role in nuclear pore biology, a function which is likely to be broadly conserved. 

Contact: mjwvangompel@ucdavis.edu 

Lab: Rose 


33

Identification of Direct Targets of the Caenorhabditis elegans Global 

Sexual Regulator TRA-1 by Chromatin Immunoprecipitation 

Matthew Berkseth1 , Kohta Ikegami2 , Jason Lieb2 , David Zarkower1 1 2 University of Minnesota, Minneapolis, MN, USA, University of North 

Carolina, Chapel Hill, NC, USA 

The nematode Caenorhabditis elegans naturally occurs as two highly dimorphic sexes, the 

XX hermaphrodite and the XO male. Sex is determined by a genetic pathway culminating in 

the transcription factor TRA-1, the worm homologue of vertebrate GLI proteins. Null mutations 

in tra-1 result in hermaphrodite-to-male sex reversal, indicating that TRA-1 and its downstream 

targets are responsible for generating all sexual dimorphism in the worm. However only a few 

direct TRA-1 targets have been described, and additional biologically important targets likely 

remain to be identified. 

To identify TRA-1 target genes throughout the C. elegans genome, we have performed 

chromatin immunoprecipitations using an affinity-purified rabbit polyclonal TRA-1 antibody 

followed by deep sequencing (ChIP-seq). We have identified ~400 TRA-1 binding sites in C. 

elegans with this approach. This list includes most of the previously identified TRA-1 binding 

sites and is significantly enriched for close matches to the TRA-1 consensus binding sequence. 

We have performed ChIP-seq experiments in L2, L3, and young adult animals, and found that 

TRA-1 binding at some sites varies across developmental time. We have also performed TRA-1 

ChIP-seq in mutant animals lacking a germline and identified ~40 peaks significantly reduced 

in germline-less animals, suggesting they may be bound by TRA-1 only in the germline. We 

have also performed ChIP-seq on L3 stage C. briggsae, and identified ~50 TRA-1 binding sites 

in this species, several of which have been conserved in C. elegans, suggesting that that the 

regulation of nearby genes by TRA-1 is likely to be functionally important. 

To examine what role putative TRA-1 targets may play in sexual development, we have 

generated reporters for many genes adjacent to TRA-1 binding sites, and identified several 

that are expressed in a largely male-specific manner. We are in the process of ablating TRA-1 

binding sites in these reporters to determine whether their male-specific expression patterns 

are controlled directly by TRA-1. Surprisingly, several targets are genes known to function 

upstream of TRA-1 in the global sex determination pathway, including xol-1, fem-3, and sup- 

26, suggesting that one function of TRA-1 is to feed back onto the sex determination pathway 

to reinforce the sex determination decision. 

Contact: berk0136@umn.edu 

Lab: Zarkower 

34 


Evolution of Caenorhabditis Dosage Compensation 

Te-Wen Lo, Caitlin Schartner, Catherine Pickle, Barbara Meyer 

HHMI/UC Berkeley, Berkeley, CA, USA 

Comparative studies have shown remarkable divergence in the conservation of developmental 

mechanisms. Strategies to determine sexual fate and to compensate X-chromosome dosage 

between sexes have evolved particularly rapidly: mammals, flies, and worms use fundamentally 

different methods. Understanding such rapidly changing processes requires comparisons 

over shorter evolutionary time-scales, such as between C. briggsae and C.elegans (15-30 

Myr). Comparison of sex determination and dosage compensation across nematode species 

using heritable, targeted mutagenesis protocols we developed has shown that key features 

of the dosage compensation complex (DCC) and the genetic pathway that coordinates sex 

determination and dosage compensation are conserved. Despite conservation of the DCC 

and its regulatory hierarchy, the mechanisms for targeting the DCC to X chromosomes have 

diverged. The cis-acting DNA recruitment elements on X (rex) and their motifs that attract the 

DCC are distinct. C. elegans rex sites ported to C. briggsae fail to bind the C. briggsae DCC. 

The reciprocal also holds: C.briggsae rex sites ported into C.elegans fail to bind the C. elegans 

DCC. Also, C. briggsae rex sites lack the X-enriched C. elegans DNA motifs pivotal for DCC 

recruitment. The divergence of DCC binding sites between C. elegans and C. briggsae prompted 

us to explore X targeting in C. sp.9, which is closer to C. briggsae than to C. elegans. C. sp. 

9 proteins homologous to DCC subunits of both C. briggsae and C. elegans co-localize on X 

chromosomes of C. sp. 9 hermaphrodites and C. briggsae/C. sp. 9 hybrid hermaphrodites. 

On-going ChIP-seq experiments will reveal the level of divergence in X targeting mechanisms. 

Dosage compensation provides a unique opportunity to study the co-evolution of regulator 

proteins and their binding sites. The evolution of DCC binding sites followed a different pattern 

from that of binding sites for conserved regulatory proteins that control many unrelated cellular 

processes. For multi-functional proteins few significant changes have occurred in their DNA 

binding domains and cognate DNA binding motifs. In contrast, DCC complexes, which lack 

the constraints of multiple functions, exhibit robust divergence in binding sites. 

To facilitate our evolutionary studies, we are optimizing site-directed mutagenesis for other 

nematode species and devising protocols for integration of homologous DNA. The most recent 

success has been P. pacificus. 

Contact: te-wen.lo@berkeley.edu 

Lab: Meyer 


35

RNAi and Immortality: Recognition of Self/non-Self RNA in the C. 

elegans Germline 

Craig Mello 

University of Massachusetts Medical School, HHMI 

Contact: craig.mello@umassmed.edu 

Lab: Mello 

36 

Keynote 6

The onset of dosage compensation is linked to the loss of 


Laura Custer, Gyorgyi Csankovszki 


Dosage compensation is a specialized gene regulatory process to equalize X chromosome 

gene expression between sexes. The dosage compensation complex (DCC) localizes to 

both X chromosomes of hermaphrodites, which leads to a two-fold downregulation of gene 

expression. In adult somatic tissues this is accompanied by DCC-dependent depletion of 

histone H4 lysine 16 acetylation (H4K16ac), a mark of active transcription, and enrichment of 

H4K20 monomethylation (H4K20me1), a repressive mark, on the X chromosomes. The DCC 

first accumulates on the X chromosomes at the 30-cell stage, coinciding with the developmental 

transition from plasticity to differentiation. We found that dosage compensated X chromosomes 

acquire their distinguishing chromatin marks with different kinetics. H4K20me1 becomes 

X-enriched at the comma stage, several cell cycles after DCC accumulation, suggesting that 

it is a late mark in the dosage compensation process. By contrast, H4K16ac is depleted on 

the X chromosomes even before the onset of dosage compensation. As opposed to later 

in development, this early depletion of acetylation does not depend on the presence of the 

DCC. Instead, depletion requires the activities of MES-2 (a subunit of a complex similar to 

the Polycomb Repressive Complex), as well as MES-4, and is observed in both males and 

hermaphrodites, perhaps as a consequence of germline silencing of the X chromosomes. 

The MES proteins are also required for the timely loss of developmental plasticity, and mes 

mutant embryos exhibit a delay in this transition. Consistent with the hypothesis that the 

onset of dosage compensation is linked to differentiation, DCC localization and H4K20me1 

accumulation on the X chromosomes are delayed in mes mutant hermaphrodite embryos. We 

propose that as embryonic blastomeres lose their developmental plasticity, the X chromosomes 

in hermaphrodites transition from a MES protein-regulated state to DCC-mediated repression. 

Contact: gyorgyi@umich.edu 

Lab: Csankovszki 

Platform Session #6 - Gene Regulation 

37

The Histone Demethylase SPR-5 and the Histone Methyltransferase 

MET-2 Comprise a Novel Epigenetic Reprogramming Switch 

Shana Kerr, Chelsey Chandler, Joshua Francis, Erica Mills, David Katz 

Emory University, Emory University 

Extensive epigenetic reprogramming is required at fertilization to re-establish a developmental 

ground state, but the mechanism of this reprogramming is poorly understood. We previously 

demonstrated that mutation of spr-5, the C. elegans ortholog of the histone demethylase 

LSD1/KDM1, results in progressive sterility over generations due to the transgenerational 

accumulation of the histone modification H3K4me2. Thus H3K4me2 can serve as a stable 

epigenetic transcriptional memory, and erasure of H3K4me2 by SPR-5 in the germline prevents 

the inappropriate transgenerational transmission of this memory. Intriguingly, mutation of met- 

2, which encodes an H3K9 methyltransferase similar to SETDB1, also results in progressive 

sterility over generations. As H3K4me2 and K3K9me are thought to have opposing effects 

on transcription, these similarities are consistent with a role for MET-2 in reinforcing SPR- 

5 mediated erasure of H3K4me2 through the addition of H3K9me. In order to investigate 

this possibility, we created spr-5;met-2 double mutant worms. spr-5;met-2 mutants exhibit a 

synthetic maternal effect sterile phenotype with germline defects that are reminiscent of late 

generation spr-5 worms. In addition, spr-5;met-2 mutants have an accumulation of H3K4me2 

at SPR-5 germline targets that is well beyond that of the single mutants alone and this increase 

in H3K4me2 correlates with huge increases in the expression of these genes. These data 

suggest that erasure of the active histone modification (H3K4me2) is coupled to the acquisition 

of a repressive histone modification (H3K9me2) during reprogramming at fertilization and that 

this novel epigenetic reprogramming mechanism is critical to undergo the cell fate transition 

from gametes to the embryo. 

Contact: djkatz@emory.edu 

Lab: Katz 

38 

Platform Session #6 - Gene Regulation

Nuclear RNAi mediates silencing of repetitive sequences in C. elegans 

Fei Xu, Xufei Zhou, Hui Mao, Jiaojiao Ji, Shouhong Guang 

Univ. of Sci. & Tech. of China 

Nuclear RNAi (Nrde) pathway has been shown to inhibit transcription elongation and mediate 

heritable gene silencing in C.elegans. The endogenous function of Nrde pathway remains 

unclear. We investigated the genetic requirements of RNAi-induced off-target gene silencing 

and surprisingly identified that the nrde mutants are resistant to off-target gene silencing. 

>dpy-13 is a collagen gene, which belongs to a large gene family that contains more than 

150 members with high sequence similarity. dpy-13(e458) mutant lacks most of the coding 

region, likely being a null mutation. dpy-13(e458) animals exhibit a dumpy phenotype, with 

a length roughly half of wild type N2 animals. eri(-) and ergo-1(-) animals exhibit enhanced 

sensitivity to RNAi. Feeding eri(-) and ergo-1(-) animals targeting the dpy-13 sequence elicits 

a phenotype which is extremely more severe (worm-ball-like) than dpy-13(e458). This finding 

indicates that dsRNA targeting the dpy-13 gene is able to trigger an off-target silencing effect. 

We examined the genetic requirements for this off-target effect. Interestingly, nrde-3, but 

not its secondary Argonaute paralogues in C. elegans, is critical for this process,suggesting 

that the Nrde pathway preferentially silences repetitive sequence elements. Consistent 

with this, nrde genes are required to silence transgenes which form repetitive arrays. We 

examined the endogenous small RNAs bound small RNAs bound to NRDE-3. Unlike the other 

two secondary Argonaute proteins CSR-1 and WAGO-1, most of the NRDE-3 targets share 

extensive sequence similarity. 

The functionality of Nrde pathway in silencing repetitive sequences is discussed. 

Contact: sguang@ustc.edu.cn 

Lab: Guang 


39

Dimerization of βCatenin/WRM-1 Allows Intermolecular 


Xiao-Dong Yang, Scott Robertson , Rueyling Lin 

UT Southwestern Medical Center, Dallas, TX, USA 

Activation of Wnt target genes in C. elegans embryos requires the TCF protein, POP-1, 

and a coactivating β-catenin, SYS-1. In Wnt responsive cells, nuclear POP-1 is lowered as a 

result of nuclear export, a process requiring phosphorylation of POP-1 by the conserved MAP 

kinase LIT-1. We have shown previously that the diverged β-catenin /WRM-1 binds to both 

POP-1 and LIT-1, functioning as a substrate-binding subunit for the LIT-1 kinase. In addition, 

WRM-1 is required for LIT-1 kinase activity, independent of its substrate-binding capability. 

The molecular mechanism by which WRM-1 activates LIT-1 remains unknown. Despite being 

highly conserved, the mammalian homolog NLK differs from LIT-1 in that it can be activated 

when expressed by itself. It has been shown that NLK undergoes homodimerization, which 

is essential for intermolecular autophosphorylation of T286 in the activation loop and kinase 

activity. We show that, unlike NLK, LIT-1 does not oligomerize effectively, nor does it undergo 

intermolecular autophosphorylation when expressed by itself. Coexpression with WRM-1 

resulted in LIT-1 self association and phosphorylation at T220, which corresponds to T286 in 

NLK. We identify the domain required for both LIT-1 self association and activation to be the 

C-terminal 150 amino acids of WRM-1, a domain distinct from that responsible for binding to 

LIT-1 (aa’s 1-150) or POP-1 (ARM repeats 3-5). The C-terminal domain of WRM-1 is predicted 

to contain two coiled-coil motifs, motifs that often mediate protein-protein interactions. Using 

artificial coiled-coil motifs that can oligomerize, we show that substituting the C-terminal domain 

of WRM-1 with a dimmerization motif restores the ability of WRM-1 to activate LIT-1. More 

importantly, fusing an oligomerization motif to full-length LIT-1 results in LIT-1 activation in the 

absence of WRM-1. Together, our results demonstrate a molecular mechanism by which WRM- 

1 activates LIT-1. WRM-1 dimerizes via its C-terminal coiled-coil motifs, resulting in multiple 

molecules of LIT-1, bound to the N-terminal domain of WRM-1, to be juxtaposed, allowing 

intermolecular phosphorylation. The resultant phosphorylation of LIT-1 in the activation loop 

leads to kinase activity and POP-1 phosphorylation. 

Contact: xiaodong.yang@utsouthwestern.edu 

Lab: Lin 

40 

Platform Session #6 - Gene Regulation

Organ defects in adults resulting from threshold blastomere 

specification 

Morris Maduro, Gina Broitman-Maduro, Leila Magistrado, Shruthi Satish 

University of California, Riverside, Riverside, CA, USA 

The embryonic E cell generates the C. elegans gut. E specification results from the 

transient expression of end-1 and end-3 in the early E lineage, which results in activation of 

elt-2 and elt-7, which is maintained by positive autoregulation. A recent work (Raj et al., 2010) 

examined the correlation of expression of end-1 and end-3 with activation of elt-2 in a skn-1 

mutant background, in which specification of endoderm occurs in approximately 20-30% of 

embryos. The results suggested that activation of elt-2 occurs when a threshold of end-1 and 

end-3 expression has been reached. What is not known is whether successful activation of 

elt-2 by a threshold amount of the ends is sufficient for normal endoderm development, and 

whether or not animals that have ‘just barely specified E’ develop otherwise normally due to 

the positive autoregulation of elt-2, and the ability of embryos to buffer early deficits in end-1 

and/or end-3 expression. These questions have been difficult to address experimentally, as 

knockdown of the upstream regulators that affect activation of the ends (skn-1, pop-1, med- 

1/2) results in arrested embryos due to their essential roles in other lineages; the existing 

null mutants in end-1 and end-3 have only subtle defects individually (95-100% of embryos 

make gut), and double end-1 end-3 mutants completely fail to make any endoderm at all; and 

overexpression of elt-2 is known to be sufficient to make endoderm, but this does not reflect 

threshold activation by the ends. By inserting single-copy transgenes in which the MED-1,2 

binding sites have been mutated, we have generated strains in which activation of only end-1 

and/or end-3 are compromised, resulting in a spectrum of endoderm specification phenotypes 

arising from otherwise isogenic animals. Many embryos contain no gut, some have a few or 

several gut cells, and others make a relatively normal gut of 20 or more gut cells. Adults derived 

from the latter have phenotypes that can be attributed to abnormalities in intestine differentiation, 

such as accumulation of significantly more lipids. We have performed RNA-Seq of intact L4s 

and dissected adult intestines in these strains and identified differences with wild-type. Our 

results suggest that threshold activation of elt-2 by itself is not sufficient for normal endoderm 

differentiation, even when an apparently normal intestine is made, and that the adult intestine 

can carry a memory of incomplete specification by the ends. 

Contact: mmaduro@ucr.edu 

Lab: Maduro 


41

Title: TBD 

Julie Ahringer 

Gurdon Institute, University of Cambridge 

Contact: j.ahringer@gurdon.cam.ac.uk 

Lab: Ahringer 

42 

Keynote 7

Modeling germline population dynamics 

Hillel Kugler1 , E. Jane Albert Hubbard2 1 2 Microsoft Research Cambridge, New York University School of Medicine, 

Skirball Institute, Kimmel Stem Cell Center 

In recently published work [1] we constructed a dynamic computer model of the C. elegans 

germ cell population and used it to analyze the interplay between Notch signaling, cell cycle 

control, and anatomical constraints. Our analyses of model simulations provided predictions 

that were validated by laboratory studies. For example, we showed that germ cell proliferation 

rate during larval stages can influence the differentiation pattern in the adult. 

In more recent work, we have extended the previous model and have overcome certain 

limitations. For example we can now capture the three dimensional structure of the gonad 

and represent cells “pushing’’ each other during division. We have developed new general 

web-based tools to make the model accessible and amenable to extension and in-silico 

experimentation. The development of these tools is based on the premise that while a modelbuilding/model-testing 

cycle is part and parcel of experimental biology that could be facilitated 

by dynamic computer-based models, many existing computational methods and tools are 

not accessible to experimental biologists (e.g., large sets of differential equations or complex 

software code). We are building these tools with the hope that they will be amenable to modeling 

many aspects of C. elegans development. 

[1] Y. Setty, D. Dalfo, D.Z. Korta, E.J. A. Hubbard, and H. Kugler (2012) A model of stem cell population 

dynamics: in-silico analysis and in-vivo validation. Development 139: 47-56. 

Contact: hkugler@microsoft.com 

Lab: Kugler 

Platform Session #7 - Cell fate and New Technologies 

43

Dev-scape: An intuitive tool for automated phenotyping with single 

cell resolution 

Julia Moore1 , Zhuo Du1 , Anthony Santella1 , Christian Pohl2 , Zhirong Bao1 1 2 Sloan-Kettering Institute, New York, NY, USA, Frankfurt Institute for 

Molecular Life Sciences, Frankfurt, Germany 

Advances in imaging technology have provided an experimental platform in which dynamic 

biological processes can be observed at high spatial and temporal resolution. These advances 

have facilitated studies directly observing the progression of C. elegans embryogenesis at 

the single cell level. New computational tools are needed to efficiently identify subtle but 

statistically significant abnormalities in this high resolution data. We addressed this problem 

by constructing an automated phenotyping pipeline to format, analyze and display the data in 

intuitive and biologically meaningful forms. 

Dev-scape takes advantage of C. elegans’ invariant lineage to carefully characterize each 

cell’s wild type behavior. 50 wild type embryos were used to define statistical distributions of 

each cell’s proliferation, differentiation and morphogenesis. Mutant and RNAi treated embryos 

are measured and compared to the wild type distributions to quantify the statistical significance 

of any abnormalities. The raw data and corresponding significance values are displayed in 

multiple ways to elucidate complex phenotypes. Dev-scape provides 1) unprecedented insights 

into the variability in normal development and 2) the ability to detect aberrant behavior in 

single cells. By pairing Dev-scape with whole genome RNAi libraries, we can investigate the 

landscape of possible perturbations of embryogenesis. 

Contact: moorej@mskcc.org 

Lab: Bao 

44 

Platform Session #7 - Cell fate and New Technologies

WormBase 2012: Website Redesign 

Abigail Cabunoc, Norie de la Cruz, Adrian Duong, Maher Kassim, Xiaoqi Shi, 

Todd Harris, Lincoln Stein 

Ontario Institute for Cancer Research, Toronto, Canada 

WormBase (www.wormbase.org) has served the ever growing needs of the nematode 

research community since 2000. Initially created as a resource for the C.elegans genome and 

its biology, WormBase now includes a wider variety of data from 15 related species. This growth 

in data is mirrored by increased website use. In response to the constantly growing data and 

user traffic, the WormBase web team has redesigned both the underlying architecture and 

user interface of the website. In this interactive presentation, we will review the new WormBase 

website, walk through basic and advanced tasks using the new interface, and examine several 

features which take advantage of the nematode research community. 

Contact: abigail.cabunoc@oicr.on.ca 

Lab: Stein 


45

DSL-2 Mediates a Notch Signal From EMS Descendant(s) to ABp 

Descendants 

Scott Robertson, Jessica Medina, Rueyling Lin 

UT Southwestern Medical Center, Dallas, Texas, USA 

Specification of pharyngeal precursors during embryogenesis requires multiple Notchmediated 

cell-cell interactions. In the 12-cell embryo, the MS blastomere contacts two of the 

four ABa descendants and induces them to produce pharyngeal muscle later. At this stage, 

all ABa- and ABp-derived blastomeres express the Notch receptor, GLP-1, and some ABp 

descendants contact MS. However, no pharyngeal muscle is made from ABp-derived cells. 

It has been shown that a Notch interaction occurring in the 4-cell stage between P2 and ABp 

renders ABp descendants unresponsive to the subsequent Notch signal from MS. The Notch 

ligand responsible for the interaction in 4-cell is APX-1, but that in MS is not known. apx-1 

mutant embryos produce induced pharynx from both ABa and ABp descendants. We generated 

a transcriptional GFP reporter that expresses in all Notch responsive early blastomeres, in 

a manner dependent on GLP-1. We noted a high level of GFP expressed in two of the four 

ABp descendants, ABprp and ABplp, in 12-cell embryos. At this stage, ABprp and ABplp, but 

not their siblings are in close contact with EMS descendants. We show that the high level of 

GFP in ABprp and ABplp is dependent on the EMS-specific transcription factor, SKN-1. Via 

microarray analyses, we identified two SKN-1-dependent Notch ligands, DSL-1 and DSL-2, 

expressing in the 12-cell embryo. dsl-2, but not dsl-1, depletion reduces the GFP in ABprp 

and ABplp to a level similar to other ABp descendants. Furthermore, depletion of dsl-2 in 

apx-1(-) mutant embryos resulted in a decrease of pharyngeal tissue. We showed that the 

pharyngeal marker, PHA-4 is only abolished in ABp-derived, but not ABa-derived cells in apx- 

1;dsl-2(RNAi) embryos. In summary, we have uncovered a Notch/DSL-2-mediated signal from 

the EMS descendant(s) to ABp descendants at the 12-cell stage that is distinct from the one 

inducing the anterior pharynx. Our results show that the P2-to-ABp Notch signal does not 

render ABp unresponsive to subsequent Notch signaling. Instead, it alters the way in which 

ABp descendants respond to subsequent Notch signaling. 

Contact: scott.robertson@utsouthwestern.edu 

Lab: Lin 

46 


Intercellular Calcium Signaling in a Gap Junction Cell Network 


Jennifer Schumacher Tucker, Chieh Chang, Chiou-Fen Chuang 

Cincinnati Children’s Hospital Research Foundation, Cincinnati, OH, USA 

Many highly specialized cells must derive from a limited number of progenitors during 

nervous system development. An example of neuronal diversification is establishment of 

asymmetric gene expression across the left-right (L-R) axis, which occurs in the C. elegans 

AWC chemosensory neuron pair. Expression of the odorant receptor str-2 is random: either the 

right or left AWC expresses str-2 to become AWCON , and the contralateral cell becomes AWCOFF . 

Intercellular communication between the AWCs and 34 other neurons through the NSY-5/innexin 

gap junction network is required for asymmetric str-2 expression, but the mechanism by which 

NSY-5 mediates communication is unclear. Ca2+ is a good candidate signal because it moves 

through gap junctions in other contexts, and Ca2+ signaling cascade components are required 

autonomously in AWC to promote AWCOFF . However, the role of Ca2+ flux in non-AWCs has 

not been investigated. To test the hypothesis that Ca2+ mediates intercellular communication 

by traveling through NSY-5 gap junctions, we expressed genetically encoded calcium buffer 

proteins and tissue-specific nsy-5 RNAi under the control of various promoters in the NSY-5 

network. Using genetic mosaic analysis to modify intracellular Ca2+ or NSY-5 levels in specific 

cells, we show that Ca2+ within non-AWCs requires NSY-5 to influence str-2 expression, 

suggesting that Ca2+ may be the signal that passes through NSY-5 gap junctions. Our results 

reveal that the AWC neuron pair relies on a balance of autonomous and non-autonomous Ca2+ inputs to diversify cell fates. 

Contact: jennifer.tucker@cchmc.org 

Lab: Chuang 


47

Nutritional control of mRNA isoform expression during developmental 

arrest and recovery in C. elegans 

Colin Maxwell 1 , Igor Antoshechkin 2 , Nicole Kurhanewicz 3 , Jason Belsky 1 , L. Ryan 

Baugh 1 

1 Duke University, Durham (NC), USA, 2 California Institute of Technology, 

Pasadena (CA), USA, 3 University of North Carolina, Chapel Hill (NC), USA 

Nutrient availability profoundly influences gene expression. Many animal genes encode 

multiple transcript isoforms, yet the effect of nutrient availability on transcript isoform expression 

has not been studied in genome-wide fashion. When C. elegans larvae hatch without food they 

arrest development in the first larval stage (L1 arrest). Starved larvae can survive L1 arrest 

for weeks, but growth and post-embryonic development are rapidly initiated in response to 

feeding. We used RNA-seq to characterize the transcriptome during L1 arrest and over time 

after feeding. 27% of detectable protein coding genes were differentially expressed during 

recovery from L1 arrest, with the majority of changes initiating within the first hour, demonstrating 

widespread, acute effects of nutrient availability on gene expression. We used two independent 

approaches to track expression of individual exons and mRNA isoforms, and we connected 

changes in expression to functional consequences by mining a variety of databases. These 

two approaches identified an overlapping set of genes with alternative isoform expression, and 

they converged on common functional patterns. Genes affecting mRNA splicing and translation 

are regulated by alternative isoform expression, revealing post-transcriptional consequences 

of nutrient availability on gene regulation. We also found that phosphorylation sites are often 

alternatively expressed, revealing a common mode by which alternative isoform expression 

modifies protein function and signal transduction. Our results detail rich changes in C. elegans 

gene expression as larvae initiate growth and post-embryonic development, and they provide 

an excellent resource for ongoing investigation of transcriptional regulation and developmental 

physiology. 

Contact: cs.maxwell@gmail.com 

Lab: Baugh 

48 


Ras and its Effector RalGEF Both Perform Dual, Antagonistic 

Functions during C. elegans Vulval Patterning 

Kimberly Monahan, Rebecca Whitehurst, Tanya Zand, Channing Der, David 

Reiner 

University of North Carolina, Chapel Hill 

The Ras small GTPase oncoprotein interacts with a spectrum of functionally diverse effectors 

to promote cancer growth. How effector utilization is dynamically regulated to achieve different 

cellular consequences remains poorly understood. This is a critical question since many anti- 

Ras drug discovery efforts focus on inhibition of a single effector, Raf, and clinical results 

suggest that multidrug therapies will be frequently required to avoid drug resistance or increase 

treatment efficacy. Developmental fate patterning of the C. elegans vulva and Drosophila R7 

photoreceptor are classic model systems for studying Ras signaling. EGF induces the six 

C. elegans epithelial vulval precursor cells (VPCs) to form a highly reproducible 3°-3°-2°- 

1°-2°-3° pattern of fates; 1° and 2° cells pursue specialized vulval development programs 

while 3° cells remain unspecialized epithelial cells. EGFR activates Ras and the canonical 

Raf-MEK-ERK effector pathway to regulate transcription-dependent induction of a single 1 

cell, which subsequently produces DSL ligands that laterally induce Notch-dependent 2 fate 

in the two neighboring VPCs. Multiple signaling mechanisms are thought to mediate mutual 

antagonism between 1° and 2° fates. We described recently that Ras switches effectors during 

vulval patterning by, in presumptive 2° cells, engaging the noncanonical effector RalGEF. Ras 

thereby mediates an EGF pro-2° signal in support of the Notch pro-2° signal, and provides the 

molecular mechanism for interpretation of an EGF patterning gradient. RalGEF activates the 

Ral small GTPase to promote 2° fate through an unknown downstream pathway. We find that 

RalGEF has an additional, Ral-independent function that antagonizes its Ral-dependent pro- 

2° function. Previous mammalian cell studies suggest that RalGEF can scaffold PDK and Akt 

as a component of the PI3K phospholipid-signaling cascade. We find that PI3K antagonizes 

2° fate in a RalGEF-dependent manner, consistent with RalGEF functioning as a PDK-Akt 

scaffold. Furthermore, the PI3K cascade inhibits FoxO transcription factor activation, and our 

results suggest that FoxO promotes 2° fate. We therefore hypothesize that RalGEF toggles 

between activating Ral to drive 2° fate and scaffolding the PI3K cascade to inhibit 2° fate, and 

that these two RalGEF pathways compete to control FoxO pro-2° activity. Perhaps Ras and 

RalGEF dual signaling reinforces initial cell fate patterning and increases developmental fidelity. 

Contact: dreiner@med.unc.edu 

Lab: Reiner 


49

The microRNA miR-786 is Required for Rhythmic Calcium Wave 

Initiation in the C. elegans Intestine 

Benedict Kemp 1 , Erik Allman 2 , Lois Immerman 3 , Megan Mohnen 1 , Maureen 

Peters 3 , Keith Nehrke 2 , Allison Abbott 1 

1 Marquette University, Milwaukee, (WI), USA, 2 University of Rochester 

School of Medicine and Dentistry, Rochester, (NY), USA, 3 Oberlin College, 

Oberlin, (OH), USA 

Rhythmic behaviors are ubiquitous phenomena in animals. The best studied rhythmic 

behavior in C. elegans is defecation, which involves three coordinated muscle contractions 

every ~50 sec. The execution of the defecation motor program depends on intercellular 

calcium waves that initiate in the posterior intestine. Thus, the posterior intestine functions as 

the pacemaker for this rhythmic behavior. However, the molecular mechanism for pacemaker 

activity is not well understood. We found that the microRNA mir-786 is necessary for the 

supremacy of the posterior cell in the rhythmic initiation of calcium waves. Loss of mir-786 

results in long arrhythmic defecation cycles (Miska et al., 2007) with calcium wave initiation 

often observed in the non-posterior regions of the intestine. These calcium waves often fail to 

trigger a full defecation motor program. Genetic data indicates that mir-786 functions upstream 

of IP 3R dependent calcium release. mir-786 is expressed in the posterior-most ring of intestinal 

cells, int9. We identify elo-2, as a likely direct target for miR-786 in the posterior intestine. elo-2 

encodes a fatty acid elongase previously demonstrated to regulate defecation cycling (Kniazeva 

et al., 2003). We propose that miR-786 regulates lipid composition in the int9 posterior cells 

thereby functioning to establish or maintain pacemaker activity for this rhythmic behavior. 

Kniazeva, M.,Sieber, M., Mccauley, S., Zhang, K., Watts, J. L., and Han, M. (2003). Genetics 163, 159- 

169. 

Miska, E. A., Alvarez-Saavedra, E., Abbott, A. L., Lau, N.C., Hellman, A. B., McGonagle, S. M., Bartel, D. 

P., Ambros, V. R., and Horvitz, H. R. (2007). PLoS Genet 3,e215. 

Contact: allison.abbott@marquette.edu 

Lab: Abbott 

50 


GLO-2 is a BLOC-1 Subunit that Functions in Gut Granule Biogenesis 

Alec Barrett, Olivia Foster, Annalise Vine, Greg Hermann 

Lewis and Clark College, Portland, OR, USA 

Caenorhabditis elegans intestinal cells are characterized by the presence of gut granules, 

lysosome-related storage organelles that contain autofluorescent and birefringent material. Gut 

granule formation requires the activity of evolutionarily conserved genes that when disrupted 

result in the loss and/or mislocalization of birefringent material into the embryonic intestinal 

lumen (the glo phenotype). Here we present our phenotypic and molecular analysis of glo-2, 

which encodes a small cytoplasmically localized protein that is orthologous to mammalian 

Pallidin. Pallidin functions as part of the 8-subunit containing BLOC-1 complex in trafficking 

to lysosome related organelles and defects in BLOC-1 activity result in the human disease 

Hermansky-Pudlak Syndrome. Seven BLOC-1 subunit homologues exist in C. elegans and we 

show that they likely form a complex and function together in gut granule biogenesis. Our studies 

of intracellular trafficking are consistent with BLOC-1 acting in parallel to the AP-3 mediated 

pathway to gut granules. Notably, loss of BLOC-1 activity has only subtle effects on trafficking 

to conventional lysosomes. We find that glo-2(-) is partially suppressed by overexpression 

of RAB-7, suggesting overlap in trafficking pathways to lysosome-related and conventional 

lysosomal organelles, which co-exist in C. elegans intestinal cells. Phenotypic analysis and 

genetic interactions point to a role of RAB-7 in facilitating the movement of gut granule cargo 

from the conventional endolysosomal trafficking pathway to gut granules. We present results 

of a genetic enhancer screen to identify factors that function with, and parallel to, BLOC-1. 

Contact: apbarrett@lclark.edu 

Lab: Hermann 

Poster Topic: Cell Biology 

51

The Conventional Kinesin-1/UNC-116 Acts in PHB Phasmid Neurons 

to Mediate Proper Cell Body Position 

Ben Barsi-Rhyne, Kristine Miller, Chris Vargas, Miri VanHoven 

San Jose State University, San Jose,(CA), USA 

Cell migration in the nervous system is vital for developing proper nervous tissue structure 

and defects in this process have been implicated in mental retardation. Cell migration is an 

active process that begins with a migration promoting signal that leads to cell polarization 

and extension of membrane protrusions at the leading edge. Many pathways have been 

found to play a role in this process, but the downstream molecular mechanisms have yet 

to be thoroughly characterized. UNC-116/Kinesin-1 has been previously been shown to be 

required for many processes including trafficking of synaptic vesicle components to the active 

zone and growth cone migration. We have found that UNC-116/Kinesin-1 also plays a role in 

maintaining neuronal cell body position. To understand the role of UNC-116/Kinesin-1 and its 

potential pathway members in this process, we study the PHB phasmid neurons in the lumbar 

ganglion of Caenorhabditis elegans. Interestingly this class of neurons is born in approximately 

its final position. However, using a combination of dye filling and cell-specific expression of 

the mCherry fluorophore, we have found that UNC-116/Kinesin-1 is required to maintain the 

anterior-posterior position of the PHB cell body throughout development. In unc-116/kinesin-1 

mutant animals, at least one PHB cell body is frequently found anterior of the anal valve. 

In addition, the number of animals with the mutant phenotype increases progressively with 

developmental stage from approximately 15% in L1s to 65% in adult animals. Furthermore, cell 

specific rescue experiments indicate that UNC-116/Kinesin-1 functions cell autonomously to 

mediate this process. Our preliminary results suggest that this process occurs partially through 

an UNC-6/Netrin attractive signal. In addition, we have found that KLC-2, one of two kinesinlight-chains, 

also plays a role in this process. Interestingly, our preliminary data indicates that 

this defect may be specific for the UNC-116/Kinesin-1, as OSM-3/ Kinesin-2 and several other 

kinesin-like proteins including KLP-11, ZEN-4, VAB-8, and UNC-104 do not have severe defects 

in this process. We believe that these results suggest a novel role for UNC-116/Kinesin-1 in 

maintenance of PHB cell body position. To further elucidate the function of UNC-116/kinesin-1 

in this process, we will continue to test additional potential pathway members. 

Contact: ben.barsirhyne@gmail.com 

Lab: VanHoven 

52 

Poster Topic: Cell Biology

Genetic Interaction and Structure/Function Studies of MEL-28, a 

Protein Required for Nuclear Envelope Function and Chromosome 

Segregation 

Anita Fernandez2 , Carly Bock2 , Allison Lai2 , Emily Mis3 , Fabio Piano1 1 2 New York University, New York, (NY), USA, Fairfield University, Fairfield, 

(CT), USA, 3Yale University, New Haven. (CT), USA 

We have been studying mel-28/elys, a gene required both for the structural integrity of the 

nuclear envelope during interphase and the proper behavior of the chromatin during mitosis. 

MEL-28/ELYS is a large conserved protein that shuttles between the nuclear envelope and 

the kinetochore during the cell cycle. We performed a mel-28 genetic interaction screen and 

identified ~50 genes that cause novel phenotypes when RNAi-depleted in a mel-28 mutant 

background. We found genes that encode proteins required for translation, components of 

dynein and its regulators, and nucleoporins. To determine which domains of MEL-28 are 

required for its function we are executing a structure/function approach. We have found that 

the N-terminal AT-hook domains are required for MEL-28 function, but that the conserved 

coiled-coil domain is not. 

Contact: carlybock14@gmail.edu 

Lab: Piano 


53

Oocyte Meiotic Spindle Assembly in C. elegans 

Amy Connolly, Sara Christensen, Valerie Osterberg, Josh Lowry, John Yochem, 

Bruce Bowerman 

Institue of Molecular Biology, University of Oregon 

In contrast to the centrosome-based assembly of mitotic spindles, how acentrosomal 

oocytes assemble bipolar meiotic spindles in the absence of centrosomes remains poorly 

understood. With its powerful genetics and transparent anatomy, Caenorhabditis elegans is a 

powerful model system for investigating this fundamental process. We can analyze the dynamics 

of oocyte meiotic spindle assembly with live cell imaging using GFP and mCherry fusions 

to meiotic spindle proteins. Using these tools, we are investigating how the meiotic spindle 

assembly genes mei-1, klp-18 and aspm-1 interact to produce a bipolar meiotic spindle. We are 

also investigating two new temperature-sensitive mutants with meiotic spindle defects, called 

or1092ts and or1292ts. Mutant one-cell or1092ts zygotes have multiple maternal pronuclei 

after the completion of the oocyte meiotic cell divisions, but mitotic cell divisions appear 

roughly normal. Complementation tests show or1092ts is not an allele of mei-1, mei-2, klp-18, 

or aspm-1. Based on live cell imaging, the or1092ts meiotic spindleis a disorganized array of 

microtubules with unorganized chromosomes and fails to extrude a polar body. The or1292ts 

mutant exhibits defects in both meiotic and mitotic spindle function. The meiotic spindle often 

fails to segregate chromosomes properly, as evident by the anaphase bridges detected during 

meiosis and the presence of multiple maternal pronuclei during the one cell stage. During the 

one-cell stage in or1292 mutant zygotes, pronuclear migration is abnormal and while the mitotic 

spindle aligns along the long axis it fails to move toward the posterior pole. In addition, we have 

observed intriguing defects in the regulation of microtubule dynamics in or1292ts mutants. 

Dense arrays of microtubules appear throughout the cortex during meiosis, and cytoplasmic 

microtubules appear throughout the anterior end of the embryo during pronuclear migration. We 

are currently using whole genome sequencing methods to determine the mutation site in both 

or1092ts and or1292ts. We have so far learned that or1092ts maps approximately between 

10.5 and 12 Mb on Linkage group III; or1292ts maps roughly between 2 and 4 Mb on Linkage 

group III. Once we identify the causal mutations, we will further investigate the requirements 

for the affected genes during meiotic spindle assembly, and the role of the or1292ts locus in 

regulating microtubule dynamics. 

Contact: amyc@uoregon.edu 

Lab: Bowerman 

54 


Identifying Proteins that Interact with the Serine/Threonine Kinase 

UNC-82 in Muscle Cells 

Christopher Duchesneau2 , April Reedy2 , Hiroshi Qadota1 , Guy Benian1 , Pamela 

Hoppe2 1 2 Emory University, Atlanta, Georgia, U.S.A., Western Michigan University, 

Kalamazoo, Michigan, U.S.A. 

We are interested in the mechanisms underlying myosin filament organization in striated 

muscle. UNC-82 is a serine/threonine kinase orthologous to human ARK5 (NUAK1) and 

SNARK (NUAK2). Mutations in the unc-82 gene cause defects in thick filament organization in 

striated muscle cells (Waterston et al. 1980). A full-length UNC-82::GFP fusion protein rescues 

this phenotype and localizes at or near the M-line, which is the thick filament attachment 

structure. Defects in the localization of the thick filament proteins myosin and paramyosin as 

well as the M-line component UNC-89/obscurin are observed in all unc-82(e1323) null mutant 

worms by the three-fold stage of embryogenesis (Hoppe et al. 2010). To better understand 

the role of unc-82, we have analyzed the distribution of components representing different 

“complexes” at the M-line, which contains many structural and probably signaling components. 

We found that membrane-proximal proteins such as UNC-112/kindlin were unaffected while 

membrane-distal proteins such as UNC-98, a thick filament component, showed large abnormal 

accumulations similar to those observed with the M-line component UNC-89/obscurin. These 

results indicate that UNC-82 activity is required for the proper organization of membrane-distal 

M-line components and suggest that UNC-82 targets an M-line or thick filament protein. To 

test which proteins may interact with UNC-82 directly, adults with ectopically localized myosin, 

paramyosin, and UNC-82::GFP were examined using antibody staining to determine which other 

proteins were recruited to the ectopic accumulations of these three proteins. Colocalization has 

been observed between UNC-82::GFP, myosin, paramyosin, and UNC-98, which is a potential 

chaperone required for the incorporation of paramyosin into thick filaments (Miller et al. 2008). 

A full-length paramyosin::GFP fusion expressed through heat shock in adults localizes to the 

thick filaments in wild-type adults, but fails to completely incorporate into filaments in unc-82 

null mutants. Instead, much of the paramyosin is localized in numerous aberrant accumulations 

within the cytoplasm. Taken together, these results suggest that UNC-82 physically interacts, 

either directly or indirectly, with one or more of these three proteins: myosin, paramyosin, 

and UNC-98. These closely associated proteins may be targets of UNC-82 kinase activity, 

regulators of UNC-82, or possibly effectors of UNC-82 function. 

Contact: christopher.d.duchesneau@wmich.edu 

Lab: Hoppe 


55

A LET-23 localization and expression screen identifies a novel 

mechanism of EGFR regulation through Ezrin/Radixin/Moesin 

proteins 

Juan Escobar Restrepo1 , Peter Gutierrez1 , Andrea Haag1 , Alessandra Buhler1 , 

Christina Herrmann1 , Maeva Langouet1 , David Kradolfer2,1 , Erika Frohli1 , Attila 

Stetak3,1 , Alex Hajnal1 1Institute of Molecular Life Sciences. University of Zurich, Zurich, 

Switzerland, 2Swedish University of Agricultural Sciences. Uppsala 

BioCenter. Sweden, 3University of Basel, Basel, Switzerland 

C. elegans let-23 encodes the sole member of the ErbB family of receptors and is required 

for the formation of the hermaphrodite vulva. LET-23 is expressed at the plasma membrane of 

the six epithelial Vulval Precursor Cells (VPC) where is retained at the baso-lateral membrane 

by interacting with its C-terminal PDZ binding motif to a ternary complex formed by the PDZ 

proteins LIN-2, LIN-7 and LIN-10. Baso-lateral localization of LET-23 in the VPCs is required 

for efficient interaction with LIN-3 EGF secreted by the anchor cell (AC) in the somatic gonad. 

We have screened an RNAi library of all genes exhibiting a Protruding Vulva (Pvl) phenotype 

for defects in receptor localization and/or expression using a functional LET-23::GFP reporter 

(poster A.Haag et. al). We have identified ERM-1, the homologue of mammalian Ezrin, Radixin 

and Moesin proteins that link plasma membrane proteins to the actin cytoskeleton, as a negative 

regulator of the EGFR/RAS/MAPK pathway possibly by sequestering and stabilizing LET-23 

in an inactive compartment at or near to the baso-lateral plasma membrane of the VPCs. The 

following lines of evidence support our model: (1) erm-1 (lf) or RNAi treatment against erm-1 

causes a reduction in the baso-lateral LET-23::GFP signal. (2) erm-1(lf) suppresses the Vulvaless 

phenotype in reduction-of-function mutations in the LET-23/LET-60/MPK-1 pathway and enhances 

the Multivulva phenotype in a gain-of-function mutation in let-60 ras. (3) An ERM-1::mCherry 

translational reporter co-localizes with LET-23::GFP at the baso-lateral plasma membrane of the 

VPCs. (4) Recombinant ERM-1::GST interacts with LET-23 from worm extracts. The interaction 

is independent of LIN-7 or the PDZ binding motif of LET-23, suggesting that ERM-1 interacts 

with LET-23 through a complex distinct from the LIN-2/LIN-7/LIN-10 complex. (5) Fluorescence 

recovery after photobleaching with erm-1(lf) mutants showed a significantly faster recovery of 

basal LET-23::GFP compared to the wild-type. Taken together, our results indicate that ERM-1 

inhibits the internalization of LET-23::GFP from the baso-lateral plasma membrane and/or the 

lateral diffusion within the plasma membrane. We propose that ERM-1 retains a fraction of LET- 

23 in an inactive compartment, thereby competing with the activating LET-23/LIN-2/LIN-7/LIN-10 

complex. ERM-1 may act as a buffer to prevent the immediate activation of the entire pool of 

baso-lateral LET-23 by LIN-3 and thus allow a prolonged LET-23 signal 

Contact: juan.escobar@imls.uzh.ch 

Lab: Hajnal 

56 


Growth of Muscle Adhesion Complexes During Postembryonic 


Brandon Fields1 , Nate Szewczyk2 , Lewis Jacobson1 1Department of Biological Sciences, University of Pittsburgh, Pittsburgh, 

PA 15260 USA, 2MRC/Arthritis Research UK Centre for Musculoskeletal 

Ageing Research, University of Nottingham, Derby, DE22 3DT UK 

The integrin-containing transmembrane attachment complexes of C. elegans are highly 

homologous to human focal adhesion complexes. In C. elegans, these form muscle-muscle 

attachments and anchor muscle contractile fibers to the hypodermis (dense bodies). Knockdown 

via mutation (reduces protein function) of either of two genes (unc-112 or unc-52) or RNAi 

(reduces amount of normal protein) of any one of fourteen genes encoding members of this 

complex provokes protein degradation in muscle cytosol and a variety of structural defects. 

In an unc-112ts mutant, paralysis occurs within 24h after shift of adults to nonpermissive 

temperature. Worms heterozygous for the unc-112ts mutation move at rates comparable to wild 

type, showing that the mutant protein does not “poison” adhesion complexes. Furthermore, 

acute RNAi treatment of adults causes sarcomere disruption and soluble protein degradation 

in 24h. Taken together, these observations imply a continuing requirement for new dense 

body proteins to maintain structural integrity. Does this reflect addition of new dense bodies, 

or protein accretion to existing dense bodies? Confocal microscopy of an unc-95::gfp strain 

and morphometric analysis were used to show that the number of dense bodies per muscle 

cell remains constant as the worm nearly doubles in length from L4 to mid-adulthood, while 

the mean size of each dense body increases. This implies that dense bodies are dynamic 

structures to which new proteins are added during postembryonic development and growth. 

Contact: fields.bdf@gmail.com 

Lab: Jacobson 


57

CDK-1 inhibits meiotic spindle shortening and dynein-dependent 

spindle rotation in C. elegans 

Jonathan Flynn, Marina Ellefson, Francis McNally 

University of California, Davis 

In animals, the female meiotic spindle is positioned at the egg cortex in a perpendicular 

orientation to facilitate the disposal of half of the chromosomes into a polar body. In 

Caenorhabditis elegans, the metaphase spindle lies parallel to the cortex, dynein is dispersed 

on the spindle, and the dynein activators ASPM-1 and LIN-5 are concentrated at spindle poles. 

Anaphase-promoting complex (APC) activation results in dynein accumulation at spindle poles 

and dynein-dependent rotation of one spindle pole to the cortex, resulting in perpendicular 

orientation. To test whether the APC initiates spindle rotation through cyclin B–CDK-1 

inactivation, separase activation, or degradation of an unknown dynein inhibitor, CDK-1 was 

inhibited with purvalanol A in metaphase-I–arrested, APC-depleted embryos. CDK-1 inhibition 

resulted in the accumulation of dynein at spindle poles and dynein-dependent spindle rotation 

without chromosome separation. These results suggest that CDK-1 blocks rotation by inhibiting 

dynein association with microtubules and with LIN-5–ASPM-1 at meiotic spindle poles and 

that the APC promotes spindle rotation by inhibiting CDK-1. 

Contact: flynn.jonathan@gmail.com 

Lab: McNally 

58 


The C. elegans Uterine Seam Cell: a Model for Studying Nuclear 

Migration and Cell Outgrowth 

Srimoyee Ghosh, Paul Sternberg 

California Institute of Technology, Pasadena, CA, USA 

Defects in nuclear migration and cellular outgrowth are hallmarks of degenerative disease. 

Determining the molecular mechanisms that govern proper nuclear migration and cellular 

outgrowth may shed light on the pathologies of these diseases. We hope to identify new 

complexes that are involved in these processes by studying a cell where both nuclear migration 

and cellular outgrowth are occurring -- the uterine seam cell. The uterine seam cell (UTSE) 

connects the uterus to the body wall. It is a syncytium composed of nine nuclei that move 

outward in a bidirectional manner. The UTSE cell body stretches outward faster than its nuclei 

move, indicating that these two processes are distinct from one another. We are using a twoprong 

approach to identify the molecular mechanisms necessary for proper nuclear migration 

and cell outgrowth in UTSE development. Initially, we looked for cues from other parts of the 

uterus that influence UTSE behavior. Using ablation experiments we saw that two types of 

epithelial cells that line the uterine lumen, uterine toroid 1 and uterine toroid 2, are necessary 

for proper UTSE development. We also used a candidate screen to identify genes involved in 

UTSE nuclear migration and cell outgrowth. Not surprisingly, the KASH protein UNC-83, the 

SUN protein UNC-84, and its corresponding nuclear anchoring protein, ANC-1, are necessary 

for proper UTSE nuclear migration. Genes involved in extracellular matrix formation, such as 

the alpha integrin INA-1, and the laminin EPI-1 also play a role. Based upon expression studies 

we tested astacins, a class of zinc metalloproteases with no known function in migration or 

outgrowth. Two of these astacins, NAS-22 and NAS-21, had effects on UTSE development. 

We are currently determining the mechanism by which these genes ensure proper UTSE 

nuclear migration and cell outgrowth. 

Contact: sghosh@caltech.edu 

Lab: Sternberg 


59

Cadherin FMI-1 Maintains the Structure of the PVD Mechanosensory 

Neurons 

Julie Grimm, Benjamin Podbilewicz 

Technion Institute of Technology, Haifa, Israel 

C. elegans arborized sensory neuron--the PVD--is complex but highly stereotyped. It 

must innervate a large area without leaving gaps that could endanger the survival of the 

nematode. On the other hand, the dendrites should not be overly elaborate or hindered by 

unnecessary connections. The process of growth, retraction and overall maintenance during 

the development and maturation of the PVD may provide insight into how more complex 

neuronal circuits function. Recently, the seven transmembrane cadherin FMI-1 was implicated 

in PVD morphology (Oren-Suissa, M. , PhD thesis, 2011). FMI-1 is known to be important for 

follower axon navigation in C. elegans (Steimel et. al, 2010) as well as synapse morphology 

(Najarro et. al, 2012). It was found that mutants lacking the extracellular domain of FMI-1 

showed defects in follower axon navigation. However, the intracellular domain was crucial for 

pioneer axon migration, which suggests dual or even multiple functions for the cadherin. FMI-1’s 

Drosophila ortholog, flamingo (fmi-1), was also found to be crucial for neuronal development 

(Kimura et. al, 2006). Kimura et. al found that fmi-1 null mutants exhibited extensive dendritic 

branching--but only in their sensory neurons. Our findings suggest that FMI-1 may have 

the opposite effect in the PVD of C. elegans. Work on the null allele tm306 has shown that 

without FMI-1 the PVD is severely retarded in growth. It appears that this phenotype becomes 

more pronounced with age and may even result from excessive retraction or degeneration of 

branches; however this aspect needs further investigation. Our aim is to understand the role 

of FMI-1 in PVD development and maintenance, and also determine if and how it interacts 

with other known PVD modifying proteins. Live imaging of PVD development during different 

life stages and recovery from dendritic injury will help elucidate the importance of FMI-1 in 

neuronal maintenance. Furthermore, using the various alleles now available to us, as well as 

fmi-1 constructs containing the full gene as well as engineered constructs missing various 

domains, we can understand not only if fmi-1 is required, but also which aspects are required 

for what steps of dendritic maintenance. Finally, and to merge this work with previous studies 

of the lab, we plant to evaluate which pathway FMI-1 functions in: in the same pathway as 

dendritic sculptors identified in our lab EFF-1 or NHR-25, or in parallel? 

Contact: julie@tx.technion.ac.il 

Lab: Podbilewicz 

60 


Two Functional Domains in C. elegans Glypican LON-2 Can 

Independently Inhibit DBL-1 Growth Factor Signaling but Require 

Accessory Moieties 

Suparna Bageshwar, Tina Gumienny 

Texas A&M Health Science Center College of Medicine, College Station 

(TX), USA 

Glypicans are GPI-linked proteoglycans with regulatory roles in several intercellular signaling 

pathways. How their structural complexity specifies function, including regulating Transforming 

Growth Factor-β (TGF-β) signaling, is a significant, but unresolved question relevant to both 

developmental and disease biology. We have previously established that C. elegans glypican 

LON-2 negatively regulates body size signaling by DBL-1, a TGF-β superfamily member, 

and binds TGF-β members (Gumienny et al., Current Biology (2007) 17(2): 159-164). We 

examined the functional requirements for glypican regulation of body length by DBL-1, a C. 

elegans TGF-β superfamily member. 

We provide evidence that two parts of C. elegans glypican LON-2 can independently 

inhibit DBL-1 signaling in vivo: the N-terminal furin protease product and the heparan-sulfated 

C-terminal region. While these two parts are each sufficient for LON-2 activity, furin cleavage of 

LON-2 into two parts is not required for LON-2 to inhibit DBL-1 signaling. While the C-terminal 

protease product is dispensable for LON-2 minimal core protein activity, it does affect the 

localization of LON-2. The glycosyl-phosphatidylinositol (GPI) membrane anchor is also not 

absolutely required for LON-2 core protein activity, but is required for the heparan-sulfated 

C-terminus to function. 

Furthermore, we show that an RGD protein-protein interaction motif in the LON-2 N-terminal 

domain is necessary for LON-2 core protein activity. Our work supports a model that LON-2 

inhibits TGF-β signaling by acting as a scaffold for growth factor and an RGD-binding protein. 

In the context of the native LON-2 glypican, the N-terminal protein core and heparan sulfate 

side chains may together specify growth factor regulatory activity, facilitated by the GPI anchor 

and the RGD protein-protein interaction accessory moieties. 

Contact: gumienny@medicine.tamhsc.edu 

Lab: Gumienny 


61

Mutational Analysis of Residues Required for Activation the UNC-82 

Serine-Threonine Kinase 

Jason Kintzele, Pamela Hoppe 

Western Michigan University, Kalamazoo, Michigan, United States 

C. elegans UNC-82 is a member of the AMPK related kinase family (AMPK-RK), which 

contains at least 12 genes related to AMPK (reviewed by Bright, 2009). Although AMPK was 

originally identified as a kinase that monitored cellular energy levels, other members of the 

AMPK-RK family have several other roles in the cell, including establishment of cell polarity, 

cytoskeletal organization, and neuronal pathfinding (Kemphues et al. 1988, Kim et al. 2010, 

Chartier et al. 2011). The C. elegans gene unc-82 is required for myosin organization in the 

striated body wall muscles (Hoppe et al. 2010). The vertebrate orthologs of C. elegans UNC-82 

are the AMPK-RK family members ARK-5/NUAK1 and SNARK/NUAK2, which in human cell 

lines have been implicated in cancer cell survival, cellular stress responses, cellular motility 

and metabolic disorders (Suzuki et al. 2004, Yamamoto et al. 2008, Legembre et al. 2004, 

Tsuchihara et al. 2008). The role of these proteins in normal development and physiology 

is unclear. In cell lines, both have been found to localize to the nucleus and to regulate the 

cytoskeleton (Kuga et al. 2008, Zagórska et al. 2010). Biochemical studies of the human 

enzymes (Lizcano et al 2004) suggest that most AMPK-RKs are activated by the upstream 

kinase LKB1 (the ortholog of C. elegans PAR-4). However, data from other cell lines suggest that 

an NDR kinase and or the AKT/PKB kinase may also be involved (Suzuki et al. 2005, Lizcano 

et al 2004). We are using C. elegans as a model system to determine the mechanism(s) of 

activation of kinase catalytic activity and to identify possible downstream targets of the UNC- 

82/ARK-5/SNARK proteins. In current experiments, we have targeted conserved residues 

that have been implicated in regulation of kinase activity in vertebrate systems and are testing 

their requirement in proper patterning of myosin in body-wall muscle. The easily scoreable 

myosin disorganization phenotype of the unc-82 gene will also allow us to screen for possible 

upstream kinases required for UNC-82 activation. 

Contact: jason.a.kintzele@wmich.edu 

Lab: Hoppe 

62 


Genetic Analysis of Calcium Regulation in the C. elegans Intestine 

Jocelyn Laboy, Kenneth Norman 

Albany Medical College, Albany NY USA 

Defecation in the nematode Caenorhabditis elegans is a readily observable ultradian 

behavioral rhythm that occurs once every 40-50 s and is mediated by rhythmic calcium 

oscillations in the intestinal epithelium. For this behavior, calcium release from the endoplasmic 

reticulum into the cell cytosol is dependent on the inositol-1,4,5-triphosphate receptor (IP3R). 

One proposed mechanism of action through which this occurs in the C. elegans intestine 

involves the PLC-gamma (PLC-3) mediated cleavage of phosphatidylinositol-4,5-bisphosphate 

(PIP2) into two products, diacylglycerol and IP3. Cleavage of PIP2 activates TRPM-family 

calcium channel, GON-2, and allows calcium ions into the cell (1). This calcium signal and the 

other cleavage product of PIP2, IP3, trigger the opening of IP3Rs to create a robust calcium 

transient. To further understand the mechanisms underlying intestinal calcium oscillations, we 

are investigating the role of two other mutants in this behavior, vav-1 and kqt-3. vav-1 encodes 

a conserved Rho/Rac-family guanine nucleotide exchange factor. VAV-1 is expressed in the 

C. elegans intestine, and the null mutant exhibits calcium related behavioral defects, such as 

lengthened defecation cycle period similar to plc-3 and gon-2 mutants. kqt-3 encodes a KCNQfamily 

potassium channel that is also expressed in the intestine and the null mutation results 

in an altered defecation cycle similar to plc-3 and gon-2. Using genetic and cell biological 

analyses, we are examining the hypothesis that kqt-3, vav-1, plc-3 and gon-2 act in a common 

signaling pathway to mediate IP3R calcium transients. Thus far, our preliminary analysis 

indicates that plc-3 and gon-2 act in a common pathway, which is consistent with a previous 

study (1); however, kqt-3 appears to act in a parallel pathway to regulate calcium oscillations 

in the intestinal epithelium. Since signaling pathways are well conserved, these studies should 

provide insight into the mechanisms underlying IP3R mediated calcium oscillations. 

1. Xing J, Strange K., Am J Physiol Cell Physiol. 2010 Feb;298(2):C274-82. 

Contact: laboyj@mail.amc.edu 

Lab: Norman 


63

The Tubulin Deglutamylase CCPP-6 Functions Exclusively in Ciliated 

Dopaminergic Neurons in C. elegans 

Ethan Landes1 , Brendan O’Flaherty1 , Elizabeth De Stasio1 , Peter Swoboda2 , 

Brian Piasecki1 1 2 Lawrence University, Appleton, (WI), USA, Karolinska Institute, Huddinge, 

Sweden 

Cilia are microtubule-based organelles that protrude from the cell surfaces of most 

eukaryotic cells. These complex organelles are utilized in a variety of sensory and motilitybased 

processes, including olfaction, light perception, and fluid propulsion. In Caenorhabditis 

elegans, cilia are found exclusively at the terminal ends of the dendritic processes of 60 

neurons in an adult hermaphrodite. These ciliated sensory neurons (CSNs) are completely 

non-motile and are utilized in a variety of behavioral processes including chemosensation, 

mechanosensation, and thermosensation. Recently, the tubulin modifying protein CCPP-1 has 

been implicated in the ciliogenic pathway of C. elegans (Curr Biol. 21: 1685-1694. 2011). CCPP- 

1 and its paralog, CCPP-6, both function in the deglutamylation of a-tubulin, a posttranslational 

modification that affects the velocity of kinesin-II along ciliary microtubules. We are currently 

characterizing the ccpp-6 gene in C. elegans. A ccpp-6 gene to GFP translational fusion 

construct is expressed exclusively in cephalic neurons (CEMs), a class of CSNs that have 

been implicated in dopaminergic signaling in C. elegans.CCPP-6::GFP localizes to the cilium, 

dendrite, axon, and neuronal cell body but is excluded from the nucleus of these cells. We 

intend to identify how this gene affects the behavior of nematodes and the role that CCPP-6 

plays in dopaminergic signaling. 

Contact: ethan.e.landes@lawrence.edu 

Lab: Piasecki 

64 


Protein Sequences Within the UNC-82 S/T Kinase that Affect 

Subcellular Localization in Pharyngeal Muscle 

Latrisha Lane, Chiyen Wong, Caitlyn Carter, Pamela Hoppe 

Western Michigan University, Kalamazoo, Michigan, USA 

We are interested in the mechanisms underlying the establishment and maintenance of 

the contractile apparatus in muscle cells. In previous work we identified the UNC-82 serine/ 

threonine kinase, which is required for the organization of the myosin filaments and some 

M-line components of body-wall muscle. The human orthologs of UNC-82 are NUAK1/ARK5 

and NUAK2/SNARK. In body-wall muscle, which is similar to skeletal muscle in vertebrates, 

UNC-82::GFP is located at or near the M-line, which is the site where myosin filaments attach 

(Hoppe et al., 2010). We are currently investigating the function of the UNC-82 kinase in 

pharyngeal muscle cells, which resemble cardiac muscle. In the pharynx muscle of an otherwise 

wild-type strain, UNC-82::GFP is detectable only near the apical plasma membrane, which 

is adjacent to the cuticle-lined pharyngeal lumen. Since these cells are single-sarcomere 

muscles that contain radially-arranged actin and myosin filaments, the UNC-82::GFP is distant 

from the myosin filaments, which lie in the central region of the cell. To investigate the protein 

sequences required for UNC-82 localization in pharyngeal muscle, we generated GFP fusion 

constructs driven by the pharynx-muscle-specific myo-2 promoter. A GFP fusion that contained 

only the N-terminal region including the kinase domain showed diffuse cytoplasmic localization. 

In contrast, a fusion containing the remaining ~1300 C-terminal amino acids localized to the 

nucleus. Point mutation of conserved catalytic residues within the kinase domain of a full-length 

construct resulted in UNC-82::GFP appearing in radially-arranged filamentous structures. These 

data suggest that a full-length, catalytically active protein is required for protein localization 

in pharyngeal muscle cells. The regulatory pathway of the UNC-82 ortholog NUAK1 in some 

human cell lines involves both phosphorylation in the kinase domain as well as phosphorylation 

of a threonine residue well outside the kinase domain in a putative Akt site. In C. elegans, the 

putative Akt site is located in an alternatively spliced exon that is included in transcripts made 

in pharyngeal muscle. We hypothesize that the regulatory pathway of NUAK 1 is conserved 

in UNC-82, and that activation of UNC-82 kinase activity requires phosphorylation at this site. 

To test this, we are currently performing site-directed mutagenesis of the threonine within the 

phosphorylation motif to test if mutant constructs have altered subcellular localization similar 

to that observed with kinase-dead mutant constructs. 

Contact: latrisha.s.lane@wmich.edu 

Lab: Hoppe 


65

Characterization of vh45, a Candidate Regulator of Early to Late 

Endosomal Maturation 

Fiona Law, Shang Xiang, Christian Rocheleau 

McGill University, Montreal, Canada 

Diseases such as cancer, metabolic disorders and neuronal degeneration can originate from 

endocytic trafficking defects whereby improper management of cargoes, such as transmembrane 

receptors, result in prolonged activation of signaling pathways or toxic accumulation of 

aggregates. To understand how these defects arise, functions of the components controlling 

endocytic trafficking need to be characterized and clarified. 

As internalized cargo progresses along the endocytic pathway, Rab GTPase proteins 

associate with the enclosing vesicular membrane. These proteins alternate between inactivated 

GDP and activated GTP bound forms through the actions of Guanine nucleotide Exchange 

Factors (GEFs) and GTPase Activating Proteins (GAPs). Rab GTPases provide directionality 

to endocytic traffic and recruit effectors for mediating downstream processes. A vesicle 

carrying cargo destined for degradation requires Rab5 to be exchanged for Rab7 GTPase as 

it matures from an early to late endosome. This Rab conversion involves a multistep process 

where Rab5-GTP first recruits Rab7 GEF to activate Rab7, and Rab7-GTP then recruits a 

GAP to inactivate Rab5. Yet how this event is regulated and the mechanisms necessary for 

this event to occur are not fully understood. Using C. elegans, our lab has identified TBC-2 

as a regulator of RAB-5/RAB-7 conversion. From biochemical and genetic studies, we found 

that TBC-2 is a RAB-5 GAP and that it requires RAB-7 to localize onto endocytic membranes. 

Results suggest that RAB-7 recruits TBC-2 to inactivate RAB-5 and therefore facilitates the 

RAB-5/RAB-7 conversion. Loss of tbc-2 function or expression of constitutively active RAB-5 

result in the formation of large RAB-7 positive endosomes in intestinal cells. 

To find regulators of TBC-2 function, a forward genetic screen was conducted to isolate 

mutations that exhibit a tbc-2-like endosomal phenotype. One mutant, named vh45, displays 

large GFP::RAB-7 positive vesicles in intestinal cells. Data from complementation tests indicate 

that vh45 is not a mutation in tbc-2, but represents a new candidate regulator of endosomal 

maturation. I aim to determine which gene is affected by the vh45 mutation using a combination 

of whole-genome-sequencing and SNP mapping. I will further characterize the vh45 large 

vesicular phenotype through genetic and cell biological approaches to determine how vh45 

disrupts endosomal maturation and whether it regulates TBC-2 localization or function. 

Contact: fiona.law@mail.mcgill.ca 

Lab: Rocheleau 

66 


cil-5 Mediates Ciliary Receptor Localization and Sensory Function in 


Kara Braunreiter 1 , Greg Fischer 2 , Casey Gabrhel 1 , Jamie Lyman Gingerich 1 

1 University of Wisconsin-Eau Claire, Eau Claire, WI, U.S.A., 2 University of 

Wisconsin-Madison, Madison, WI 

Primary (non-motile) cilia act as sensory antennae enabling cells to perceive the extracellular 

environment and respond appropriately. We are investigating the role of cil-5, a gene originally 

identified by its role in ciliary localization of the male-specific PKD-2 receptor. In addition to 

the PKD-2::GFP mislocalization observed in males, both male and hermaphrodite cil-5(my13) 

mutant C. elegans exhibit defective uptake of lipophilic dyes in head and tail neurons. These 

phenotypes suggest that cilium structure and function may be affected. Both sensitivity to 

chemicals and regulation of fat storage have previously been shown to be dependent on 

functional cilia. In chemotaxis assays, cil-5 mutants show hypersensitivity to some, but not all, 

volatile chemicals. In addition, analysis of intestinal fat droplets suggests that cil-5 mutants do 

not properly regulate fat storage. We are currently examining the integrity of the sheath cells 

which support ciliated neurons, using a whole genome sequencing approach to clone cil-5, and 

employing RNAinterference to identify additional factors involved in ciliary receptor localization. 

Contact: lymangjs@uwec.edu 

Lab: Lyman Gingerich 


67

Neuroligin has Cell-autonomous and Non-autonomous Functions in 


Jacob Manjarrez, Greg Mullen, Ellie Mathews, Jerrod Hunter, Jim Rand 

Oklahoma Medical Research Foundation, Oklahoma City, OK 

Neuroligins are postsynaptic adhesion proteins originally identified by their binding to 

presynaptic neurexins. Studies suggest that neuroligins function primarily in the maturation 

and/or maintenance of synapses. There are four neuroligin genes in humans, and mutations 

in the genes encoding neuroligin-3 and neuroligin-4 are associated with autism spectrum 

disorders (ASDs) in some families. We had previously examined the expression, localization and 

biological functions of neuroligin in Caenorhabditis elegans. C. elegans has a single neuroligin 

gene (nlg-1), and we had shown that nlg-1 null mutants are viable and are not deficient in 

any major motor functions. However, they are defective in a subset of sensory behaviors and 

sensory processing. nlg-1 mutants are also hypersensitive to oxidative stress (i.e., exposure 

to paraquat); this is an unexpected phenotype for a synaptic mutant. Like many other stresssensitive 

mutants, nlg-1 mutants also have a reduced lifespan and an increased level of 

oxidative protein damage (Hunter et al., 2010). All of these mutant phenotypes are rescued 

by transgenic expression of mammalian neuroligin (human neuroligin-4 or rat neuroligin-1). 

The C. elegans and mammalian neuroligins, therefore, appear to be functionally equivalent 

(including having the ability to prevent or counteract oxidative stress). 

NLG-1 is normally expressed in ~20% of C. elegans neurons, including the pair of AIY 

interneurons. AIY cells receive direct synaptic input from different types of sensory neurons 

(e.g., chemosensory, thermosensory, nociceptive), and have been shown to play an important 

role in integration of sensory information. We find that expressing NLG-1 only in the AIY 

interneurons is sufficient to rescue all of the sensory deficits as well as the elevated oxidative 

stress present in nlg-1 mutants. However, we find that expressing NLG-1 ectopically in the 

AWA or AFD sensory neurons or the RIA or RIM interneurons (neurons which do not normally 

express this protein) can also rescue some mutant phenotypes. It is both noteworthy and 

surprising that expression of NLG-1 in only the two AIY neurons is sufficient to rescue all of 

the mutant phenotypes we examined. Equally noteworthy and surprising is the phenotypic 

rescue observed when the only NLG-1 in the animal is expressed ectopically in AWA or RIM 

cells that normally do not express NLG-1 - clearly a cell-non-autonomous effect. 

Supported by the Simons Foundation and Autism Speaks. 

Contact: Jacob-Manjarrez@omrf.org 

Lab: Rand 

68 


Genetic and Molecular Dissection of Novel Pathways Required for 

Nuclear Migration in the Model System C. elegans. 

Yu-Tai Chang, Shaun Murphy, Jonathan Kuhn, Minh Ngo, Daniel Starr 

University of California, Davis, University of California, Davis 

Moving the nucleus to an intracellular location facilitates many cell and developmental 

processes including mitotic and meiotic cell divisions, fertilization, cell migration, differentiation, 

and establishment of cellular polarity. The (Linker ofNucleoskeleton and Cy to skeleton) 

complexof SUN (UNC-84) and KASH (UNC-83) nuclear envelope proteins are involved in 

conserved mechanisms of nuclear migration. However, many nuclear migration events rely on 

independent mechanisms. To investigate novel mechanisms of nuclear migration, we utilize the 

behavior of larval P-cell nuclei in C. elegans. Failure in nuclear migration leads to P-cell death 

resulting in uncoordinated (Unc) and egg-laying defective(Egl) animals missing P-cell derived 

lineages. Null mutations in unc-83 or unc-84 inhibit nuclear migration by disrupting interactions 

between the nucleoskeleton and the cytoskeleton at 25°C, but at 15°C, P-cell nuclear migration 

occurs similar to wild-type. We therefore hypothesize that additional pathway(s) function 

synthetically to the unc-83/unc-84pathway to move P-cell nuclei at 15°C. To test our hypothesis, 

we isolated eightemu (enhancer of the nuclear migration defect of unc-83 or unc-84) alleles. 

Compared to unc-84null animals, emu; unc-84 double mutants had significantly fewer UNC- 

47::GFP-positive GABA neurons (that were derived from P-cell lineages) at all temperatures. 

Using whole-genome sequencing, we have determined that the yc20 allele is a lesion in toca-1. 

We also found that fln-2 is an emu gene. toca-1(RNAi) and toca-1(tm2056) phenocopied yc20. 

Moreover, the P-cell-specific rescue by the p hlh-3::toca-1::gfptransgene suggested that TOCA-1 

functions to move nuclei in a cell-autonomous manner. TOCA-1 and FLN-2 have both been 

shown to be involved in actindynamics. Thus, we are currently examining actin organization in 

toca-1 and fln-2 mutant P-cells. In summary, TOCA-1 and FLN-2 function in a novel pathway 

for nuclearmigration. 

Contact: spmurphy@ucdavis.edu 

Lab: Starr 


69

FLN-1/filamin is required for spermathecal contractility 

Jose Orozco, Ismar Kovacevic, Erin Cram 


The ability of tissues to sense and respond to mechanical forces is critical in development and 

normal physiology. We use the C. elegans spermatheca as a model to study mechanosensation 

in vivo. The spermatheca is a simple contractile tube that stretches to accommodate oocytes 

following ovulation. Following the entry of an oocyte, the spermatheca constricts in the distal 

to proximal direction to propel the fertilized oocyte into the uterus. The spermathecal cells are 

characterized by circumferential F-actin filaments, which play a critical role during constriction 

of the spermatheca. Previous work by our group has shown that FLN-1/filamin is required for 

maintenance of the F-actin cytoskeleton and for normal spermathecal constriction. Loss of 

FLN-1 results in a progressive disorganization of the F-actin cytoskeleton, suggesting that 

FLN-1 is required to reinforce the F-actin cytoskeleton. FLN-1 is required to initiate calcium 

signaling in the spermatheca and may play a structural role during spermathecal constriction. 

Calcium release in contractile cells stimulates myosin contractility. NMY-1/non-muscle myosin 

appears to be the only myosin expressed in the spermatheca, suggesting that NMY-1 is the main 

force generator. Indeed, nmy-1 depletion via RNAi, like fln-1 depletion, results in spermathecal 

constriction defects, and an abnormal spermatheca-uterine valve. NMY-1 contractility is 

controlled by the phosphorylation state of MLC-4/myosin light chain. MLC-4 activity is negatively 

regulated by MEL-11/myosin light chain phosphatase. mel-11(RNAi) results in a robust and 

striking spermathecal rupture phenotype due to hyperconstriction of the spermatheca around 

the embryo. The mel-11(RNAi) rupture phenotype is suppressed by loss of FLN-1. Ongoing 

work is focused on understanding how calcium signaling impinges on the myosin regulatory 

network, and whether filamin serves a structural and/or signaling role in the spermatheca. 

Contact: orozco.jos@husky.neu.edu 

Lab: Cram 

70 


Isolation of Mutations that alter Nile Red Staining in C. elegans 

Stephanie Burge1 , Anthony Otsuka2,1 1 2 Illinois State University, Normal, IL, USA, University of Hawaii at Hilo, Hilo, 

HI, USA 

There has been considerable interest in changes in gene activity that alter Nile red staining 

(Asrafi et al. Nature, 421:268-271, 2003). Some of these genes are involved in insulin regulation 

and lipid accumulation. We employed standard ethylmethane sulfonate mutagenesis to identify 

mutants that alter Nile red staining. Snip-SNP mapping was used to position several mutations 

on the genetic and physical maps. Further characterization was conducted based on the Nile 

red phenotype, light scattering phenotype, confocal microscopy, and thin layer chromatographic 

analysis of lipids. Optical sectioning by confocal microscopy revealed different sizes and 

patterns of lipid droplets in the mutants. Thin layer chromatography of lipids from the mutants 

demonstrated altered lipid profiles. In studies on longevity, one mutant showed a small, but 

significant, increase in life span. Because of the power of C. elegans genetics and the available 

molecular tools, this system is well suited to the study of lipid accumulation. 

Contact: ajotsuka@hawaii.edu 

Lab: Otssuka 


71

Epithelial Dynamics During the G1-to-G2 Pore Cell Swap in the 

Excretory System 

Jean Parry, Amanda Zacharias, Hasreet Gill, John Murray, Meera Sundaram 

University of Pennsylvania, Philadelphia, PA, US 

Epithelial cells from the epidermis, rectum, and excretory system have all been shown 

to dedifferentiate and migrate away to form neurons during the course of normal C. elegans 

development (Jarriault et al., 2008, Sulston and Horvitz, 1977). These events resemble classic 

epithelial-to-mesenchymal transition (EMT) and provide simple models for investigating the 

genetic control of epithelial junction dynamics and cell fate plasticity. The excretory system 

is comprised of three unicellular epithelial tubes connected in tandem, the canal cell, duct 

cell, and pore cell. The excretory pore cell is initially formed by the G1 cell during embryonic 

development. During mid L1, the G1cell will lose its epithelial characteristics and migrate 

towards the head, eventually dividing to produce two neuronal daughters. As G1 withdraws, 

it is replaced by the neighboring G2 cell. This process involves loss of the G1 autocellular 

and intercellular junctions, and remodeling of duct cell junctions to connect to a new partner. 

Incredibly, this transition occurs several hours after the excretory system has begun functioning. 

Newly discovered markers for the excretory system allowed us to observe this programmed 

EMT-like event in vivo. By fluorescently tagging the duct and pore cytoplasm (dct-5p::mCherry), 

junctions (AJM-1::GFP), and canal and duct lumen (VHA-5::GFP), we can perform live imaging 

of the G1/G2 swap. This imaging has revealed a highly stereotyped sequence of events that 

occur with sharp temporal precision over the course of a single hour in development. A forward 

mutagenesis screen in our lab was recently performed to identify prospective mutants in which 

the G1 cell does not withdraw from the excretory system or in which other aspects of the pore 

swap go awry, giving us a molecular entryway into this dynamic process. 

Contact: jparry@mail.med.upenn.edu 

Lab: Sundaram 

72 


The Arp2/3 activator WAVE/SCAR Promotes Clathrin Mediated 

Endocytosis in the Polarized C. elegans Intestinal Epithelia 

Falshruti Patel, Martha Soto 

UMDNJ-RWJMS 

Cells must internalize proteins and other molecules from their surfaces to survive. Studies 

in single-celled yeasts demonstrate the essential role of the branched actin nucleator, Arp2/3, 

and its activating nucleation promoting factors (NPFs) in the process of invagination from 

the cell surface through Clathrin-Mediated Endocytosis (CME). However, some mammalian 

studies have disputed the role of F-actin and Arp2/3 in CME in multicellular organisms. We 

investigated the role of Arp2/3 during endocytosis in C. elegans, a multicellular organism with 

polarized epithelia. The Arp2/3 activator, WAVE/SCAR, is essential for C. elegans embryonic 

morphogenesis, which was attributed to its ability to promote cellular protrusions. However, 

depletion of WAVE/SCAR alters junctional maturation, suggesting processes beyond protrusion 

formation are disrupted. We have shown that loss of the WAVE complex components lead 

to progressive defects in intestinal lumen morphogenesis and altered distribution of Apical 

Junction proteins, which suggested a role for WAVE in maintenance of polarity. We show 

here that loss of WAVE complex components severely disrupts the distribution of Transferrin 

Receptor, a protein that is internalized via CME. We find that the WAVE complex components 

and proteins involved in CME are mutually dependent for proper enrichment at the apical region 

of the C. elegans intestine. Consistent with these observations of WAVE/SCAR’s role in CME, 

one of the components of WAVE complex, GEX-3, interacts with dynamin in yeast-two hybrid 

studies. Further, the TOCA/F-BAR endocytosis proteins biochemically interact with the WAVE/ 

SCAR complex in mammals and C. elegans. We propose that WAVE-Arp2/3 dependent actin 

nucleation promotes CME at the apical intestinal epithelium and that altered CME contributes 

to the apical morphogenesis defects of WAVE mutants. 

Contact: patelfb@umdnj.edu 

Lab: Soto 


73

Visualizing Dynamics of Meiotic Prophase Chromosome Structures 

Divya Pattabiraman1 , Marc Presler2 , Grace Chen1 , Anne Villeneuve1 1 2 Stanford University, Stanford, California, USA, Harvard University, 

Cambridge, Massachusetts, USA 

The synaptonemal complex (SC) is a highly-ordered proteinaceous structure that assembles 

at the interface between aligned homologous chromosome pairs during meiotic prophase. 

Although EM images of SCs give the impression of a rigid, scaffold-like structure, recent 

studies suggest that the SC may be much more dynamic than previously appreciated. We 

are investigating the dynamics of the SC structure using FRAP (fluorescence recovery after 

photobleaching) to visualize exchange of SC components within assembled SCs during meiotic 

prophase. The C. elegans system is particularly well-suited for this analysis, as the dispersal of 

pachytene chromosomes around the periphery of the nucleus, surrounding a centrally located 

nucleolus, makes it possible to bleach a portion of the SCs within a given nucleus while leaving 

the remainder of the SCs unbleached, providing both internal controls and imaging landmarks. 

Further, several different nuclei can be bleached and monitored in a single experiment. We 

have developed a protocol for FRAP analysis in pachytene nuclei of intact worms, using a 

strain expressing a functional GFP-tagged version of SYP-3, a component of the SC central 

region. Using this approach, we have revealed a previously hidden dynamics of the SC 

structure. We detect significant recovery of GFP::SYP-3 within 10 minutes after photobleaching, 

and recovery approaches a maximal value by 1- 1.5 h. In many experiments, recovery in 

the bleached portion of a partially bleached nucleus occurs concomitant with diminishing of 

signal in the unbleached portion of the same nucleus (relative to adjacent unbleached control 

nuclei), implying exchange of subunits between SCs. The observed time scale of recovery is 

slower than that seen for an oocyte nucleoplasmic protein (on the order of a few seconds) or 

for microtubules in the first mitotic spindle (on the order of a few minutes). However, it is much 

faster than that observed for components of the nuclear pore scaffold, which do not turnover 

in differentiated post-mitotic cells. Moreover, the observed rate of subunit exchange raises the 

possibility that the SCs may undergo complete turnover of their subunits during the duration of 

the pachytene stage. Thus, this structure has the potential to undergo substantial remodeling 

and reorganizing in response to different ongoing events of meiotic prophase. 

Contact: divyapr@stanford.edu 


74 


CRL2/LRR-1 E3-Ligase Prevents Progression Through Meiotic 

Prophase in the Adult C. elegans Germline 

Julien Burger 1 , Jorge Merlet 1 , Nicolas Tavernier 1 , Benedicte Richaudeau 1 , Asja 

Moerkamp 2 , Rafal Ciosk 2 , Bruce Bowerman 3 , Lionel Pintard 1 

1 Institut Jacques Monod, CNRS, Paris, France, 2 Friedrich Miesher Institute 

For Biochemical Research, Basel, Switzerland, 3 Institut of Molecular 

Biology, University of oregon, Eugene, US 

Precise control of the transition from self-renewal to terminal differentiation in stem cells 

is critical to maintain a balance between cell populations: an excess of stem cell self-renewal 

can lead to tumourigenesis, whereas an excess of differentiation can deplete the stem-cell 

pool. In the adult Caenorhabditis elegans germline, Notch signals emanate from the somatic 

distal tip cell to maintain germline stem cells (GSCs) in a proliferative state by repressing the 

expression of meiotic promoting factors. In this study, we show that the ubiquitin-proteolytic 

system act synergistically with the Notch pathway to prevent meiotic entry. Using a novel 

temperature-sensitive allele of the cul-2 gene, we found that the CUL-2 RING E3 ubiquitin 

ligase in combination with the Leucine Rich Repeat 1 substrate recognition subunit (CRL2/ 

LRR-1) negatively regulates the transition from the mitotic zone of the germline to the meiotic 

programme of chromosome pairing, synapsis, and recombination. 

Contact: pintard.lionel@ijm.univ-paris-diderot.fr 

Lab: Pintard 


75

Regulated Nucleocytoplasmic Shuttling of SPAT-1/BORA Coordinates 

CDK-1 and PLK-1 Activation For Proper Mitotic Entry in the Early C. 


Nicolas Tavernier 1 , Anna Noatynska 2 , Julien Burger 1 , Costanza Panbianco 2 , 

Jorge Merlet 1 , Benedicte Richaudeau 1 , Emmanuelle Courtois 1 , Thibaud Leger 1 , 

Monica Gotta 2 , Lionel Pintard 1 

1 Institut Jacques Monod CNRS, Paris , 2 CMU, University of Geneva 

Acquisition of lineage-specific cell cycle duration is critical for metazoan development. In 

early C. elegans two-cell stage embryos, the anterior AB blastomere divides systematically 

before the posterior P1 blastomere and this asynchrony of cell division appears critical for 

proper embryonic development. Previous work established that asymmetric localization of 

the polo-like kinase PLK-1 promotes precocious mitotic entry in AB but it remains unclear 

how PLK-1 is regulated. Here we identify a positive feedback loop that coordinates PLK-1 

and CDK-1 that involves tight regulation of the PLK-1 activator SPAT-1/Bora. We show that 

SPAT-1 is a nucleocytoplasmic shuttling protein containing functional nuclear localization 

(NLS) and nuclear export (NES) sequences. CDK-1 phosphorylates SPAT-1 presumably in 

the nucleus on multiple phosphorylation sites including a polo-docking site (S251), which is 

adjacent to the nuclear localization signal (NLS). Phosphorylation of S251 residue has two 

functions: first it orients the shuttling of SPAT-1 towards the cytoplasm by inhibiting the NLS 

activity and second, it contributes to PLK-1 activation by promoting the interaction between 

SPAT-1 and PLK-1. Once activated in the cytoplasm, PLK-1 reinforces CDK-1 activation. In 

addition, PLK-1 phosphorylates and targets SPAT-1 for degradation, possibly to terminate the 

positive feedback loop and to recycle PLK-1. We propose that multisite phosphorylation of 

SPAT-1 might set the threshold of mitotic entry, and contribute to the robustness of cell cycle 

timing regulation in the early embryo. 


Lab: PINTARD 

76 


PPFR-1 Phosphatase 4 subunit is a regulator of MEI-1/Katanin activity 

during meiosis that is rapidly targeted for degradation by CRL-3/MEL- 

26 E3-ligase in the transition to mitosis in C. elegans 

Jose-Eduardo Gomes 1 , Benedicte Richaudeau 1 , Etienne Formstecher 2 , Paul 

Mains 3 , Lionel Pintard 1 

1 Institut Jacques Monod, CNRS, Paris, France, 2 HYBRIGENICS SA, Paris, 

France, 3 Departments of Biochemistry & Molecular Biology/Medical 

Genetics University of Calgary, Canada 

Protein phosphorylation by kinases is one of the most widespread forms of post-translational 

modification in eukaryotes. Owing to the action of protein phosphatases, phosphorylation 

can be reversed. Whereas protein Kinases and their phosphorylation targets have received 

much attention, comparatively much less is known about the role and regulation of protein 

phosphatases. Here we present a thorough analysis of the function and regulation of PPFR-1, 

a regulatory subunit of a trimeric Protein Phosphatase 4 (PP4) complex during the meiosis to 

mitosis transition in C. elegans. We show that PPFR-1 positively regulates the microtubulesevering 

activity of the MEI-1/MEI-2 Katanin complex during meiosis. PPFR-1 dephosphorylates 

MEI-1 and thereby activates the complex, which facilitates the disassembly of the meiotic 

spindle during anaphase and proper extrusion of polar bodies. Importantly, like its target MEI- 

1, PPFR-1 is degraded by the ubiquitin-proteolytic system after meiosis. PPFR-1 specifically 

interacts with MEL-26, the substrate recognition subunit of the CRL-3/MEL-26 E3-ligase and 

like MEI-1, accumulates at centrosomes during mitosis in mel-26(-) embryos. We conclude 

that CRL3/MEL-26 degrades both MEI-1 and its activating PPase presumably to ensure spatial 

regulation of the microtubule-severing activity of the katanin complex and its rapid inactivation 

during the meiosis-to-mitosis transition. 


Lab: Pintard 


77

A Genetic Analysis of the Axon Guidance of the C. elegans 

Pharyngeal Neuron M1 

Osama Refai, Evvi Rollins, Patrcia Rhos, Jeb Gaudet 

University of Calgary, Calgary, Alberta, Canada 

The guidance of axons to their correct targets within an organ is a critical step in 

development. During the pharyngeal development, the M1 motorneuron establishes an axon 

that spans the whole organ and encounter its different tissues. Thus, M1 is most likely receiving 

guidance and interacting with its neighbour cells e.g. muscles and glands. Electron micrographs 

show that the M1 axon bundles with the g1P gland projection through the anterior half of the 

pharynx (i.e. the procorpus). Ablation of glands results in defects of the M1 trajectory at the 

procorpus, suggesting that g1P is necessary for M1 guidance. Growth cone defective mutants 

e.g. unc-51, unc-119, unc-115 and unc-34 showed defects similar to these that was observed 

after killing the gland. Whereas, genes of the major guidance pathways e.g. unc-6, sax-3, 

vab-1, spm-1 and smp-2 appear to play a minor role in guidance of the M1 axon. To identify 

novel mutations that may affect M1 axon migration, we screened 5000 hapliod genomes in a 

forward genetic screen. We isolated 12 mutants with abnormal morphology of the M1 neuron 

including alleles for known genes such as unc-51 and rpm-1. Interestingly, we didn’t observe 

any guidance defect at the M1 trajectory within the isthmus, suggesting that this part of the 

axon is established in a non-guidance manner. Taken together, our results present a model 

where the M1 axon outgrowth involves two phases: a growth cone-independent phase in the 

isthmus, which is followed by a growth cone-dependent phase through the procorpus. 

Contact: omrefai@ucalgary.ca 

Lab: Gaudet 

78 


Using C. elegans to Explore the Role of Presenilin in Calcium 

Signaling 

Shaarika Sarasija, Kenneth Norman 

Albany Medical College, Albany, NY, USA 

Mutations in the genes encoding Presenilin-1 (PS1) and Presenilin-2 (PS2) occur in 

most early onset Familial Alzheimer’s Disease (FAD), a rare form AD. Despite the fact that 

altered presenilin activity has been known to have a role in Alzheimer’s disease pathology, 

the functional consequences of mutations in presenilins are controversial and hence not fully 

understood. In fact, mutations in presenilins have been implicated in such diverse functions 

as altered processing of beta-amyloid precursor protein, Notch signaling, calcium entry and 

calcium removal from the cytoplasm. Thus, the role of presenilins in Alzheimer’s disease has 

remained elusive. The classic hallmarks of Alzheimer’s disease pathology are the formation of 

amyloid plaques and neurofibrillary tangles. However, it is thought that altered cellular events, 

like unbalanced calcium signaling precedes the formation of these pathological markers. 

Importantly, the dysregulation of intracellular calcium signaling can lead to excitotoxicity and 

cell degeneration. To identify mechanisms that regulate intracellular calcium signaling, we are 

utilizingC.elegans to understand the role presenilins play in calcium regulation. The C. elegans 

genes sel-12 and hop-1 encode transmembrane domain proteins orthologous to human 

presenilins. We are interested in investigating whether mutations in sel-12 and/or hop-1 can 

alter calcium homeostasis in C. elegans. Thus far we have found that the sel-12 null mutant, 

ty11, is hypersensitive to the muscle cell acetylcholine receptor agonist, levamisole, and the 

acetylcholine esterase inhibitor, aldicarb, suggesting that the muscle of the sel-12 mutant is 

hyper-excitable. Additionally, we have found that the mitochondria in the muscle of sel-12 

mutants are structurally disrupted. Mitochondria act as a significant cytosolic calcium buffer 

in cells and mitochondrial calcium overload can lead to their disruption. Furthermore, our 

preliminary data points to the rescue of the hypersensitivity of the ty11 mutant to levamisole 

when it is introduced into a ryanodine null mutant, unc-68 background, which further supports 

a role of SEL-12/presenilin in calcium regulation. To directly investigate calcium signaling in 

the muscle of sel-12 mutants, we will employ optogenetic tools to measure calcium transients 

upon muscle stimulation. Since signaling mechanisms are well conserved, using C. elegans 

as a model system to unravel the role presenilins have in calcium signaling should provide 

insight into the pathological conditions that arise in AD. 

Contact: sarasis@mail.amc.edu 

Lab: Norman 


79

Novel Roles For A Cell Adhesion Protein DYF-7 In C. elegans Body 

Size Determination 

Robbie Schultz, Tina Gumienny 

Molecular and Cellular Medicine, Texas A&M Health Science Center, College 

Station, TX, USA 

Cell adhesion is critical for all multicellular organisms. While gross disruption of cell contacts 

is lethal, mild or tissue-specific cell adhesion defects can lead to developmental disorders 

and contribute to diseases, including deafness and schizophrenia. One class of cell adhesion 

proteins is the zona pellucida (ZP)-domain proteins, a family of extracellular molecules defined 

by their ZP domain, a protein polymerization motif. My project’s goal is to establish C. elegans 

ZP-domain family member DYF-7 as a model to understand the molecular and cellular action 

of ZP-domain proteins during postnatal development. DYF-7 is a critical anchoring protein for 

adhering dendrites of sensory neurons during embryonic development [1]. Loss of dyf-7 function 

is non-lethal and leads not only to sensory perception defects, as the affected neuronal cells do 

not contact the environment properly, but also body size defects [2]. Body size development is 

strictly regulated in C. elegans through genetic, structural, and environmental components. The 

best studied of these genetic regulators is the TGF-β DBL-1 pathway. While previous studies 

have shown that DYF-7 is involved in neural tip anchoring, explaining its sensing defect, DYF- 

7’s role in body size development remains unexplored. 

We found dyf-7 acts post-embryonically to regulate body size. dyf-7 is expressed in larvae 

and adults. To determine the mode of dyf-7’s regulation of body size, we performed epistasis 

analyses between dyf-7 and dbl-1 pathway members. Double mutants are significantly smaller 

than either single mutant, indicating dyf-7 regulates body length at least partially independent 

from the dbl-1 TGF-β pathway. However, we also showed that expression of a DBL-1 pathway 

reporter was altered in animals lacking endogenous dyf-7, suggesting that dyf-7 regulates dbl-1 

pathway signaling. In addition to a role in DBL-1 signaling, we found that loss of dyf-7 affects 

the organization of the cuticle, a defect that could be caused by mild cell adhesion defects 

in the cells secreting the cuticle and could also contribute to the small body size phenotype. 

These results indicate that the cell adhesion protein DYF-7 regulates body size development 

post-embryonically through integration of independent and dependent mechanisms, including 

TGF-β DBL-1 pathway signaling, cuticle organization, and environmental sensation. 

1. Heiman, M. and Shaham, S. (2009). Cell 137, 344-355. 

2. Starich, T., et al. (1995). Genetics 139, 171-188. 

Contact: Schultz@medicine.tamhsc.edu 

Lab: Gumienny 

80 


DAF-16 Promotes Developmental Growth in Response to Persistent 

Somatic DNA Damage 

Michael Muller, Maria Ermolaeva, Laia Castells-Roca, Peter Frommolt, Sebastian 

Greiss, Jennifer Schneider, Bjorn Schumacher 

Cologne Excellence Cluster for Cellular Stress Responses in Aging- 

Associated Diseases (CECAD), Institute for Genetics, University of 

Cologne, Germany 

Congenital defects in genome maintenance systems cause complex disease phenotypes 

characterized by developmental failure, cancer susceptibility and premature aging. In contrast to 

well-characterized cellular DNA damage checkpoint mechanisms, it remains poorly understood 

how DNA damage responses affect organismal development and maintain functionality of 

tissues when DNA damage gradually accumulates with aging. Here we show that transcriptioncoupled 

repair defects that in human Cockayne syndrome patients lead to developmental 

growth defects and progeria, specifically impair somatic development upon UV damage in C. 

elegans. DNA repair proficient animals, in contrast, transiently arrest development. Employing 

comprehensive gene expression analysis we identified a network of insulin-like growth factor 

signalling (IIS) genes that responds to DNA damage during C. elegans development. We 

show that the FoxO transcription factor DAF-16 is activated in response to DNA damage 

during development while the DNA damage responsiveness of DAF-16 declines with aging. 

We demonstrate that DAF-16 alleviates DNA damage induced developmental arrest through 

differential activation of downstream target genes that contrasts its established role in the 

starvation response, and even in the absence of DNA repair promotes developmental growth 

and enhances somatic tissue functionality. We propose that IIS mediates developmental DNA 

damage responses and that DAF-16 activity enables developmental progression amid persistent 

DNA lesions and promotes tissue maintenance through enhanced tolerance of DNA damage 

that accumulates with aging. 

Contact: bjoern.schumacher@uni-koeln.de 



81

Purification and Characterization of Glyceraldehyde-3-Phosphate 

Dehydrogenase from Caenorhabditis elegans 

Valeria S. Valbuena, Megan Gautier, Justin Spengler, M. Banks Greenberg, M. 

Leigh Cowart, Katherine Walstrom 

New College of Florida, Sarasota, FL 

Glyceraldehyde-3-Phosphate Dehydrogenase (GPD) is the glycolytic enzyme that adds 

inorganic phosphate to its substrate so that net ATP production is possible later in the glycolysis 

pathway. C. elegans has four gpd genes. The genes gpd-1 and gpd-4 are nearly identical 

and mainly expressed in embryos, while the homologous gpd-2 and gpd-3 are expressed 

in postembryonic worms (Huang et al., 1989, JMB 206, 411). The postembryonic genes are 

involved in protection from anoxia (Mendenhall et al., 2006, Genetics 174, 1173) and are 

upregulated in dauers and long-lived daf-2 mutants (McElwee et al. 2006, Mech. Age. Dev. 127, 

922). In this project, high yields of worm extracts were achieved by large-scale worm production 

in egg plates. GPD was purified from mixed populations of C. elegans using a new protocol 

that included gel filtration and Blue Sepharose CL-6B affinity chromatography. In comparison 

to the previous methods described by Yarbrough and Hetch (JBC 259, 14711, 1984), our 

purification resulted in a higher yield of enzyme. Based on the Yarbrough and Hetch results, 

we expect that our GPD sample consists mainly of the adult GPD-2 and GPD-3 enzymes. 

SDS-PAGE results showed that the affinity column fractions contained several bands, but 

control enzyme assays did not indicate the presence of contaminating activity. When stored at 

2 °C, the partially purified enzyme retained its activity for over a week. The reaction conditions 

were optimized, and a pH near 8.5 was a critical condition for maximum GPD activity with the 

glyceraldehyde-3-phosphate (G3P) substrate. Kinetic assays with varying concentrations of 

G3P and NAD+ were performed, and the Km values were 0.3 mM and ~1 mM, respectively. 

* This project was funded by grants from the NCF Council of Academic Affairs and 

the NCF Dubois-Felsmann Student Research and Travel Endowment. 

Contact: valeria.valbuena@ncf.edu 

Lab: Walstrom 

82 


Three axonal guidance pathways differentially signal to the regulators 

of the actin cytoskeleton during axonal migration 

Andre Wallace, Yelena Bernadskaya, Martha Soto 

RWJMS-UMDNJ 

Neuronal development is controlled by multiple guidance cues which are responsible for 

orchestrating the directed growth and migration of axons and growth cones. In C. elegans, 

it is known that UNC-40/DCC, SAX-3/Robo and VAB-1/Eph are three of the main receptors 

governing this process but it is still not clear how they regulate the actin cytoskeleton. Recent 

work from our lab has shown that during embryonic morphogenesis, these axonal guidance 

receptors function to modulate the actin cytoskeleton through effects on the WAVE/SCAR 

complex. Subsequently, we hypothesize that during neuronal development, directed growth 

and migration of axons and growth cones are dependent on proper reorganization of the 

actin cytoskeleton by the WAVE/SCAR complex. To test this hypothesis, we employed gainof-function 

mutations of these three axonal guidance receptors which are commonly used to 

identify downstream targets of these pathways. We examined how mutations of the WAVE/ 

SCAR complex affect axonal migration and cell body morphology of the AVM mechanosensory 

neuron. Depletion of WAVE proteins led to suppressed AVM defects in the SAX-3 gain-offunction 

mutant. On the other hand, AVM ventral migration defects in the myr::unc-40 gainof-function 

were enhanced with the depletion of WAVE/SCAR proteins. Finally, depletion of 

WAVE/SCAR components had no effect on AVM defects in the VAB-1 gain-of-function mutation. 

These results suggest that SAX-3 signals through the WAVE/SCAR complex during neuronal 

development. In addition, our results propose that UNC-40 may be functioning in other WAVE/ 

SCAR-independent pathways to regulate neuronal development. Overall, these results provide 

a platform on which we can study how axonal guidance cues function in reorganization of the 

actin cytoskeleton during neuronal development. 

Contact: wallacag@umdnj.edu 

Lab: Soto 


83

Microtubules and Fertilization: The MEI-1/Katanin mediated 

cytoskeletal transition from meiosis to mitosis in the developing 

embryo 

Sarah Beard, Paul Mains 

The University of Calgary, Calgary. AB, Canada 

During embryonic development, dramatic changes of the C. elegans cytoskeleton occur 

in the transition from meiosis to mitosis requiring precise regulation of molecules specific to 

each type of spindle. Defects in microtubule organization during development can result in 

tissue pathologies, aneuploidy or even cancer. The microtubule severing complex, MEI-1, is 

required in meiosis to keep the spindle small but is inactivated prior to mitosis. This inhibition of 

MEI-1 during mitosis is dependent on the MEL-26/CUL-3 E3 ubiquitin ligase complex targeting 

MEI-1 for degradation prior to mitosis. The first aim of the project is to measure anti-MEI-1 

staining levels in several mutant strains to determine how known genes function relative to 

one another. We aim to establish a standardized method to measure antibody staining levels 

from images of the embryos. Another pathway, involving the anaphase promoting complex 

(APC) and the MBK-2/DYRK kinase, has been found to promote mitotic MEI-1 degradation 

in parallel to MEL-26 mediated degradation of MEI-1. We wish to decipher whether APC and 

MBK-2 act in parallel or sequentially relative to one other in this process. We are also interested 

in deciphering the exact role of CUL-2, another E3 ubiquitin ligase, that is previously known 

to prevent MEL-26 from accumulating during meiosis. Making double mutants should resolve 

whether CUL-2 is also the missing ligase for MBK-2 mediated MEI-1 degradation functioning 

in parallel to the MEL-26/CUL-3 pathway, or if CUL-2 acts sequentially as an upstream 

activator of MEL-26/CUL-3. The second aim of the project is to continue investigating potential 

regulators of the cytoskeleton in the transition from meiosis to mitosis. We will conduct targeted 

RNAi screens for missing components of the pathway such as kinases, ubiquitin ligases 

and substrate adaptors. For example, FEM-1, a substrate adaptor for CUL-2 E3 ubiquitin 

ligase involved in sex determination, could be potential candidate for MEI-1 regulation during 

embryonic development. This project will assist in decoding the key regulatory molecules of 

the developmental remodeling of the cytoskeleton and progressively work our way back to the 

initial triggers of the pathway at fertilization. 

Contact: sarahmbeard@gmail.com 

Lab: Mains 

84 

Poster Topic: Cell cycle and cytokinesis

Understanding Proteasomal Regulation of SZY-20 in the Centrosome 

Assembly Pathway 

Michael Bobian, Mi Hye Song 

Michigan Technological University, Houghton, MI, USA 

The centrosome is a critical mediator in the animal cell cycle and serves as the primary 

microtubule-organizing center. During the cell cycle, the centrosome orchestrates microtubule 

dynamics, and forms mitotic bipolar spindles that are critical for accurate chromosome 

segregation. In C. elegans, SZY-20 is a suppressor of ZYG-1, a functional homolog to the 

human kinase Plk4. SZY-20 plays a critical role in regulating centrosome size and duplication. 

This protein localizes both at the centrosome and in the cytoplasm in a cell cycle dependent 

manner. Centrosomal levels of SZY-20 are highest during prometaphase and metaphase. Loss 

of SZY-20 leads to increased centrosome size, abnormal cell divisions, a failure of polar body 

extrusion, cytokinesis failure, shortened metaphase spindles, and detached centrosomes from 

the nuclear envelope (Song et al., 2008). To further elucidate the role of SZY-20 in regulating 

centrosome assembly and cell cycle, we utilized proteomics to identify proteins complexed 

with SZY-20. Among them, we have identified proteasome components that are reproducibly 

pulled-down with SZY-20. Interestingly, the proteasome has been shown to function at the 

centrosome to regulate the cell cycle (Didier et al., 2007). Here, we seek to understand how 

the proteasome functions in association with SZY-20 to regulate centrosome assembly. We 

hypothesize that SZY-20 is negatively regulated by the proteasome. Since centrosomal 

levels of SZY-20 are antiphasic to those of proteasomal component levels during the cell 

cycle progression, the proteasome might influence SZY-20 levels to control centrosome size 

and assembly. We have begun to characterize the genetic interaction between SZY-20 and 

proteasomal components by RNAi-knockdown and confocal microscopy. 

Contact: mrbobian@mtu.edu 

Lab: Song 

Poster Topic: Cell cycle and cytokinesis 

85

Mitotic spindle proteomics reveals conserved Caenorhabditis elegans 

proteins potentially necessary for cytokinesis 

Mary Kate Bonner1 , Daniel Poole1 , Tao Xu2 , Ali Sarkeshik2 , John Yates III2 , Ahna 

Skop1 1 2 University of Wisconsin-Madison, Madison, WI, USA, Scripps Research 

Institute, La Jolla, CA, USA 

Cytokinesis is an important and fundamental process in the development of all organisms. 

The factors that establish the cleavage furrow have remained mysterious and have eluded 

many for over 130 years. In order to identify factors required for early steps in cytokinesis, 

mitotic spindles from synchronized Chinese Hamster Ovary (CHO) cells were isolated. Proteins 

enriched from isolated metaphase-enriched spindles were identified by multidimensional protein 

identification technology (MudPIT) in collaboration with the Yates Lab at Scripps. We identified 

1155 proteins associated with the mitotic spindle at one or more peptide hit (Bonner et al., 

2011). Comparison of our data to the previously published CHO midbody proteome (Skop 

et al., 2004) revealed 314 proteins in common and 841 proteins unique to the CHO spindle 

associated proteins. Gene Ontology (GO) analysis revealed that 27% of the spindle proteins 

were associated with membrane, microtubules, actin cytoskeleton or unknown classes. To 

identify factors necessary for the membrane-cytoskeleton remodeling during cytokinesis, we 

are currently screening the orthologs in Caenorhabditis elegans using feeding RNAi. We have 

identified cognate C. elegans genes that correspond to 71% of the CHO candidate proteins. 

Of these candidates, 30% were not assigned a phenotype in previous C. elegans cell division 

screens. Of the C. elegans candidate genes that had been identified in previous screens, 65% 

of these proteins had been attributed with an EMB phenotype. We are currently screening 

candidate genes by feeding RNAi. To do this, we are assaying for embryonic lethality and 

multi-nucleate phenotypes in early embryos. We will present the results from these screens. 

Contact: mbonner@wisc.edu 

Lab: Skop 

86 


Non-random Segregation of Unpaired X Chromosomes in C. elegans 

Female Meiosis 

Daniel Cortes Estrada, Francis McNally 

UC Davis, Davis CA 

Attachment between homologous chromosomes during meiosis I is essential for accurate 

segregation of chromosomes. Surprisingly, humans with three X chromosomes (triploX) have 

normal fertility and give birth predominantly to children with a normal chromosome complement. 

If the unpaired X segregated randomly at meiosis I, triploX mothers would produce 50% triploX 

daughters. The low frequency of triploX offspring observed, around 11-14% of daughters, 

suggests that female meiosis possesses a mechanism that prevents the inheritance of the 

unpaired X chromosome. In the C. elegans him-8 mutant, which possesses two unpaired X 

chromosomes at meiosis I, a similar situation occurs. If the two univalent X chromosomes 

segregated randomly at meiosis I, him-8 mutants should produce 25% XO male, 50% XX 

hermaphrodite and 25% XXX progeny. Instead Hodgkin et al., reported 38% XO male, 56% 

XX hermaphrodite and 8% XXX progeny, suggesting that female meiosis in C.elegans also 

possesses a mechanism of selective removal of unpaired X chromosomes. We are currently 

testing the hypothesis that univalent X chromosomes are deposited into the first polar body with 

high frequency. Using live imaging and fixed immunofluorescence, we found that 95% of him-8 

metaphase I spindles have 7 chromosomes whereas 100% of wild-type metaphase I spindles 

had 6 chromosomes; demonstrating that in him-8 worms both univalent X chromosomes 

are still present by metaphase I. At metaphase II, 100% of embryos had 6 chromosomes 

whereas 40% of him-8 metaphase II spindles had 5 chromosomes, 55% had 6 chromosomes 

and 5% had 7 chromosomes. These numbers suggest that univalent chromosomes are lost 

between metaphase I and metaphase II. 88% of him-8 anaphase I spindles had 1 or 2 lagging 

chromosomes whereas only 2% of wild-type anaphase I spindles had lagging chromosomes. 

Quantifying the fates of lagging chromosomes reveals that 65% of these are expelled into 

the polar bodyand 35% are retained in the embryo. Our results are consistent with a model in 

which univalent X chromosomes biorient at metaphase I but lag at anaphase I because cohesin 

between sister chromatids is not cleaved. The delayed, poleward movement of these bioriented 

univalents is biased toward the polar body end of the spindle. Preliminary data suggests that 

the contractile ring may be involved in resolution of lagging chromosomes through its normal 

polar body formation activity. We are currently testing the involvement of the contractile ring 

in biased resolution of lagging chromosomes. 

Contact: dbcortes@ucdavis.edu 

Lab: McNally 


87

Parallel mechanisms promote RhoA activation during polarization and 

cytokinesis in the early C. elegans embryo 

Yu Chung Tse 1 , Michael Werner 1 , Katrina Longhini 1 , Jean-Claude Labbe 2 , Bob 

Goldstein 3 , Michael Glotzer 1 

1 University of Chicago, Chicago, IL, USA, 2 Universite de Montreal, Montreal, 

Canada, 3 University of North Carolina at Chapel Hill, Chapel Hill, NC, USA 

The GTPase RhoA is a central regulator of cellular contractility in a wide variety of 

biological processes. During these events, RhoA is activated by guanine nucleotide exchange 

factors (GEFs). These molecules are highly regulated to ensure that RhoA activation occurs 

at the proper time and place. During cytokinesis, RhoA is activated by the RhoGEF ECT-2. 

In human cells, ECT-2 activity requires its association with CYK-4, which is a component of 

the centralspindlin complex. In contrast, in C. elegans embryos, not all ECT-2 dependent 

functions during cytokinesis require CYK-4. Here, we identify a novel protein, NOP-1, that 

functions in parallel to CYK-4 to promote RhoA activation. We use mutations in nop-1 and 

cyk-4 to dissect cytokinesis and cell polarization. NOP-1 makes a significant, albeit largely 

redundant, contribution to cytokinesis. In contrast, NOP-1 is required for RhoA activation during 

the establishment phase of polarization. 

Contact: mglotzer@uchicago.edu 

Lab: Glotzer 

88 


ATX-2, the C. elegans ortholog of ataxin 2, is necessary for 

cytokinesis. 

Megan Gnazzo, Ahna Skop 


Mutations in ataxin-2 give rise to the devastating neurodegenerative disease spinocerebellar 

ataxia type 2 (SCA2). In SCA2 an increased expansion of a CAG repeat encoding a polyglutamine 

tract in ataxin-2 is observed. The human gene ataxin-2 has also been implicated in an increased 

risk for amyotrophic lateral sclerosis (ALS). The reason by which the mutations in ataxin-2 lead 

to neurodegeneration are unknown, and the cellular functions of ataxin-2 remain unclear. Our 

lab identified the ataxin-2 gene from isolated mammalian midbodies and the corresponding 

C. elegans ortholog, ATX-2, displayed defects in cytokinesis (Skop et al, 2004). To determine 

why ATX-2 leads to cytokinesis defects, we are characterizing its role in the early C. elegans 

embryo. Bioinformatic analysis revealed that ATX-2 is very highly conserved. ATX-2 contains 

several RNA binding motifs suggesting a role for ATX-2 in the control of mRNA translation. 

Local control of mRNA translation has been proposed as a mechanism for regulating synapse 

plasticity. We hypothesize that ATX-2 may play a role in mediating the local translation of RNAs 

found in the midbody during cytokinesis. We have identified four ATX-2 isoforms and would 

like to know how these isoforms function throughout embryonic development. We are currently 

constructing GFP constructs to two of the identified isoforms (full-length and C-terminal) to 

determine the localization of these constructs in the early embryo. Live imaging analysis has 

revealed defects in both meiotic and mitotic cytokinesis. Here, the second polar body often 

fails to be extruded and during the first division late failures in cytokinesis are observed. We 

will present our current analysis of ATX-2 function in cytokinesis. 

Contact: gnazzo@wisc.edu 

Lab: Skop 


89

Identification and Characterization of mel-15 as a New Paternal-effect 

Lethal Mutant in C. elegans 

Aimee Jaramillo-Lambert, Kathryn Stein, Andy Golden 

LBG/NIDDK/NIH 

During fertilization the oocyte and the sperm fuse, restoring the somatic chromosome 

number and initiating zygote development. Oocytes and sperm are both products of meiosis, 

however, they are highly differentiated with distinct characteristics unique to their roles in 

fertilization and early embryogenesis. Oocytes are large, sedentary cells that provide a haploid 

genome and large stockpiles of RNA and proteins necessary for early embryonic cell divisions 

until zygotic transcription is initiated. Sperm are small, motile cells streamlined for fertilization. 

Sperm are generally thought of as only providing a haploid genome, but sperm also supply 

centrosomes and the signal for the initiation of the embryonic program. There is evidence that 

sperm contain other factors required for embryogenesis. Absence of these paternally provided 

components results in embryonic lethality (paternal-effect embryonic-lethal). In C. elegans 

only a single paternal-effect lethal (PEL) gene has been characterized, spe-11. SPE-11 is a 

novel cytoplasmic protein supplied by the sperm. The absence of functional SPE-11 results 

in embryonic failure at early stages. Although important, the molecular details of the sperm’s 

contribution to early embryogenesis remain largely unknown. We are interested in identifying 

and characterizing other potential paternal-effect candidates. Previous genetic screens identified 

embryonic lethal mutants that could be rescued by wild-type males indicating a sperm defect. 

Currently, we are characterizing one of these mutants, mel-15. Initial analysis confirms that 

sperm from mel-15 mutant males produce dead embryos, even when fertilizing wild-type 

oocytes. In addition, preliminary characterization indicates that mel-15 male sperm lack DNA 

and may have aberrant tail development. We are currently in the process of determining the 

molecular identity of mel-15. 

Contact: jaramillolamban@mail.nih.gov 

Lab: Golden 

90 


RNA-binding Proteins ATX-2/PAB-1 Regulate Centrosome Assembly 

and Size 

Sarah Mets, Kelly Haynes, Eric Vertin, Dongyan Zhang, Mi Hye Song 

Michigan Technological University, Houghton, MI, USA 

Centrosomes are critical sites for controlling microtubule dynamics, and exhibit dynamic 

changes in size, promptly responding to changing cellular demands during the cell cycle. 

As cells progress to mitosis, centrosomes recruit more PCM (maturation) and nucleate 

more microtubules to form bipolar spindles. The szy-20 gene encodes a novel centrosomeassociated 

RNA-binding protein that negatively regulates ZYG-1. szy-20 mutants possess 

enlarged centrosomes which lead to abnormal microtubule processes and embryonic lethality. 

Thus, SZY-20 limits centrosome size by negatively regulating the recruitment of centrosome 

components. SZY-20 contains putative RNA-binding domains; mutating these domains perturbs 

RNA-binding by SZY-20 in vitro and its capacity to regulate centrosome size in vivo. It has been 

shown that a number of RNA-binding proteins associate with centrosomes and microtubules, 

and that they function to assemble mitotic spindles. 

To further understand the roles of SZY-20 and RNA-binding proteins in the regulation of 

centrosome assembly and size, We used proteomics to identify factors complexed with SZY-20, 

and identified other known RNA-binding proteins. By RNAi knockdown, we found that many 

SZY-20 interacting factors affect cell cycle and centrosome behavior. Some of these factors 

also exhibit strong genetic interactions with szy-20 and/or zyg-1. We are currently characterizing 

RNA-binding proteins (ATX-2 and PAB-1) using the range of genetic, cell biological, biochemical 

and optical approaches, to understand how this RNA-binding protein complex coordinates with 

SZY-20 and ZYG-1 to achieve proper centrosome assembly and size. 

Contact: mhsong@mtu.edu 

Lab: Song 


91

ubc-25 encodes a conserved ubiquitin-conjugating enzyme that is 

required for developmentally controlled cell cycle quiescence 

David Tobin 1 , Sarah Roy 1 , Mako Saito 1,2 

1 Dartmouth Medical School, Hanover, NH, USA, 2 Norris Cotton Cancer 

Center, Lebanon, NH, USA 

Temporal control of cell cycle regulation is crucial in developing multi-cellular organisms, 

however the mechanisms that coordinate this process are largely unknown. Forward and 

reverse genetic screens in our lab identified genes necessary for the quiescence of vulva 

precursor cells (VPCs) in C. elegans, which temporarily exit the cell cycle at their generation 

in L1 until resuming divisions in L3. Notably, this screen identified the cdc-14 phosphatase as 

a novel regulator of quiescence in several cell lineages, contrary to its requirement for mitosis 

in yeast. Over 100 genes were identified our screens, most of which have not been previously 

implicated in regulating cell cycle quiescence. 

The screen identified ubc-25, a gene encoding a conserved ubiquitin-conjugating enzyme, 

as a positive regulator of VPC quiescence. ubc-25(ok1732) mutants display hyper-proliferation 

of intestinal nuclei, indicating its requirement for quiescence of intestinal nuclei. Hyperproliferation 

of ubc-25(ok1732) intestinal nuclei is enhanced by cdc-14(he141) or fzr-1(ku298) 

mutant alleles, or by lin-35 RNAi, suggesting that ubc-25 acts in a distinct genetic pathway 

from these known negative regulators of G1/S. In contrast, RNAi of the SCF component cul-1 

produces excess intestinal nuclei, which is not enhanced by the ubc-25(ok1732) mutation, 

suggesting that ubc-25 and cul-1 act in a linear genetic pathway. Loss of ubc-25 activity partially 

restores intestinal nuclear divisions in cyd-1(he112) mutant animals, and is further rescued when 

combined with RNAi of either cdc-14 or lin-35. These data are consistent with the hypothesis 

that cyd-1 promotes cell cycle entry through inhibiting parallel negative regulators of G1/S 

such as lin-35, cdc-14, and ubc-25. 

Our genetic evidence places ubc-25 in the SCF mediated pathway to control G1/S. We 

propose that ubc-25 is involved in degrading CYE-1. Consistent with this hypothesis, RNAi 

of ubc-25 restores intestinal nuclear divisions in cye-1(eh10) null mutants, which may occur 

through stabilization of maternally contributed CYE-1 protein. We currently are using western 

blot analysis to directly test the role of ubc-25 in CYE-1 stability, and generating UBC-25::GFP 

expressing strains to determine its localization during development and cell division. 

Contact: david.v.tobin@gmail.com 

Lab: Saito 

92 


NAD salvage biosynthesis and programmed cell death; a new model 

for investigating cell death mechanisms 

Matt Crook, Wendy Hanna-Rose 

The Pennsylvania State University, University Park, (PA), USA 

We have developed a new model for studying programmed cell deaths in response to 

insult in C. elegans. PNC-1 converts nicotinamide (NAM) to nicotinic acid (NA) and is the first 

enzyme in the salvage pathway for biosynthesis of NAD+. In pnc-1 mutants two cell types, uv1 

and OLQ, die in response to distinct insults and using mechanisms distinct from each other 

and from mec-4-induced necrosis. The four uv1 cells, at the uterine-vulval junction, die with a 

typical necrotic morphology in response to excess NAM, a by-product of NAD+ consumption 

that is cleared by PNC-1 activity. In stark contrast to mec-4d induced touch cell necrosis, uv1 

death is unaffected by gross manipulation of cytoplasmic Ca2+ concentration or knockdown 

of genes involved in autophagy and nutrient sensing. uv1 cell death is, however, rescued by 

overexpression of LIN-3 or overactivation of its receptor LET-23, which are also responsible 

for uv1 specification. let-60/ Ras signaling is necessary but not sufficient for this rescue and 

IP3 production is neither required nor sufficient for rescue. This suggests that let-23 signals 

via another, as yet unidentified, pathway to promote survival of the uv1 cells, an avenue 

under further investigation. In contrast to the uv1 cell death model, NAM accumulation is not 

a sufficient insult to cause death of the OLQ cells, which are mechanosensory cells involved 

in head withdrawal and foraging. OLQ cell death is also not rescued by NA, suggesting that 

both PNC-1 product depletion (and the subsequent effects on NAD production) and substrate 

accumulation contribute to the insult that kills the OLQ cells. OLQ cell death increases when 

animals are grown on dead food, an undefined yet nutritionally poor growth medium that has 

previously been shown to exacerbate phenotypes caused by loss of NAD biosynthesis. Our 

data suggests a role for Ca2+ signaling, autophagy and nutrient sensing in OLQ cell death, but 

in a very different way to their roles in mec-4d induced touch cell necrosis. Thus, it is becoming 

clearer that not only is necrosis very much a controlled form of cell death, but that there are 

a wide range of insults that triggers the necrotic cell death program and variety of underlying 

mechanisms by which it is carried out. We believe that our pnc-1 model presents a powerful 

and complimentary system to further increase our understanding of genetically programmed 

cell death, its causes and its execution. 

Contact: mxc83@psu.edu 

Lab: Hanna-Rose 

Poster Topic: Cell Death 

93

The Possible Role of Autophagic Cell Death in the Regulation of 

Excitotoxicity in C. elegans 

John Del Rosario, Itzhak Mano 

Physiology, Pharmacology & Neuroscience, Sophie Davis Biomedical 

School, City College, City University of New York, New York, NY, USA 

Stroke is a leading cause of death in the United States. One of the main causes of stroke is a 

blockage of blood supply to the brain. The ensuing lack of oxygen triggers a neurodegenerative 

cascade in a process called brain ischemia. The excitatory neurotransmitter L-Glutamate (Glu) 

is normally expelled out from the synaptic cleft by the Glu Transporters (GluTs). The malfunction 

of GluTs due to a shortage in energy causes Glu to accumulate in the synapses to abnormal 

levels and over stimulates the Glu receptors (GluRs) on the post-synaptic cell, leading to the 

degeneration of the post-synaptic neuron in a process called excitotoxicity. We use a model 

of excitotoxicity in the nematode Caenorhabditis elegans by knocking-out the GluT gene glt-3 

in a sensitive background. Recent reports suggest that autophagy, an evolutionary conserved 

cell death process, is involved in related forms of neurodegeneration. We now examine the 

possible role of autophagy in excitotoxic neurodegeneration and elaborate on its mechanism. 

We have two main focuses: 1) we are testing if autophagy takes place by monitoring it 

through an autophagy fluorescent flag; 2) we are determining the probable role of autophagy 

in excitotoxic neurodegeneration by using mutations that block autophagy such as bec-1 and 

unc-51(master-regulatory genes for autophagy).We are following the effect of autophagy-related 

mutation on the extent of neurodegeneration in our model of excitotoxicity. Understanding the 

molecular cascade of excitotoxicity and the potential involvement of autophagy in this process 

in nematodes might help us suggest protective strategies to reduce brain damage caused by 

brain ischemia. 

Contact: jdelros00@ccny.cuny.edu 

Lab: Mano 

94 

Poster Topic: Cell Death

Genes Required for Cell Shedding, a Caspase-Independent 

Mechanism of Programmed Cell Elimination 

Dan Denning, Bob Horvitz 

HHMI, Dept. Biology, MIT, Cambridge, MA 02139 USA 

Programmed cell death plays critical roles in metazoan development and in the removal of 

damaged, infected or cancerous cells. Although most developmental cell deaths in C. elegans 

require the CED-3 caspase, some cells die in mutants completely lacking ced-3 function. We 

have determined the identities of eight cells that can be eliminated via extrusion (or shedding) 

from ced-3 mutant embryos. In wild-type embryos, these cells undergo ced-3-mediated 

apoptosis followed by engulfment. Thus, the canonical programmed cell death pathway and 

cell shedding function redundantly to ensure the elimination of a subset of cells fated to die. 

One of the cells that can be shed from ced-3 embryos is ABplpappap, the sister cell of which 

produces the RMEV neuron and the excretory cell. We predicted that ABplpappap might survive 

and adopt the fate of its sister cell in animals defective in both canonical programmed cell death 

and cell shedding. To identify factors required for cell shedding, we screened mutagenized 

ced-3 animals for ectopic excretory cells, using the transgenic reporter Ppgp-12::gfp to facilitate 

visualization of excretory-like cells. Three of our isolates with ectopic excretory cells contain 

mutations in pig-1, which encodes an AMPK-related serine-threonine kinase. A null mutation 

of pig-1 reduces the number of shed cells in ced-3 embryos by 75%, indicating that pig-1 is 

required generally for the generation of shed cells. Most mammalian AMPK-related kinases 

are activated via phosphorylation by the LKB1:STRAD:MO25 tumor suppressor complex. 

Inactivation of par-4/LKB1, strd-1/STRAD or mop-25.1 and mop-25.2 (paralogs of MO25) also 

blocks cell shedding in ced-3 animals. Additionally, the conserved T-Loop threonine (T169) 

of PIG-1, the predicted phosphorylation target of PAR-4/LKB1, is required for PIG-1 function, 

suggesting that the PAR-4 complex directly activates PIG-1 to regulate cell shedding. 

We are currently using SNP mapping and whole-genome DNA sequence determination 

to identify the genes mutated in other mutant strains with ectopic excretory cells. Through 

biochemical, genetic and cell biological experiments, we will determine how these genes 

cooperate with pig-1. Our goal is a mechanistic understanding of the cell shedding process. 

Contact: denningd@mit.edu 

Lab: Horvitz 


95

Investigating the pro-apoptotic function of ced-9 

Kaitlin Driscoll, Peter Reddien, Brad Hersh, Bob Horvitz 

Massachusetts Institute of Technology, Cambridge, MA 

Programmed cell death is a fundamental process that is required for proper development 

and tissue homeostasis in many organisms. Genetic analyses of C. elegans led to the discovery 

of the core components of the apoptosis pathway. One component, ced-9, is known to have 

an anti-apoptotic function, as ced-9(null) animals are maternal-effect lethal due to massive 

amounts of cell death and gain-of-function mutations in ced-9 prevent normal programmed 

cell deaths from occurring. However, ced-9 also has a pro-apoptotic function, which has not 

been well characterized. This function was discovered because weak ced-3 loss-of-function 

animals have more extra cells in a ced-9(null) background, indicating that decreasing ced-9 

function can decrease cell death. 

In a genetic screen for mutations that enhance a defect in programmed cell death of weak 

ced-3(n2472) mutants, we recovered a ced-9(n3377) allele that not only enhances the ced-3 

cell-death defect but also has a recessive cell-death defect on its own. Evidence suggests 

that ced-9(n3377) has a loss of ced-9 killing function rather than a gain of protective function. 

First, ced-9(n3377) confers a recessive increase in cell survival, which is different from the 

dominant gain of protective function ced-9(n1950) allele. Second, ced-9(n3377) acts different 

from the gain-of-function allele in relation to CED-4 localization. CED-9 normally localizes 

to mitochondria, where it binds CED-4 and prevents CED-4 from activating CED-3. Upon 

EGL-1 binding to CED-9, CED-4 is released and localizes to the perinuclear membrane. In 

ced-9 gain-of-function animals CED-4 is localized to mitochondria, even when EGL-1 is overexpressed. 

By contrast, in ced-9(n3377); ced-3(n2427) animals CED-4 is localized to the 

perinuclear membrane, as it is in ced-9(null);ced-3(n2427) animals. This finding is consistent 

with the increase in cell survival in ced-9(n3377) animals being caused by a loss of ced-9 

killing function rather than a slight gain of protective function. 

Currently we are performing genetic screens to obtain additional loss of ced-9 killing function 

alleles and to identify suppressors of the ced-9(n3377) cell-death defect. These screens might 

identify genes that regulate the ced-9 killing function as well as unknown components of the 

cell-death pathway. We are also using molecular and biochemical techniques with ced-9(n3377) 

to evaluate if the ced-9 pro-apoptotic function is mediated through the core apoptotic pathway 

components and/or its regulation of mitochondrial fusion and fission. Ideally, these experiments 

will lead to a better understanding of the apoptosis pathway and possibly novel therapeutic 

targets for diseases caused by misregulation of programmed cell death. 

Contact: kbd@mit.edu 

Lab: Horvitz 

96 


SPTF-3 SP1 and PIG-1 MELK Function in Distinct Pathways to 

Promote M4 Neuron Cell-Type Specific Programmed Cell Death 

Takashi Hirose , Bob Horvitz 

Dept. Biology, MIT, Cambridge, MA 02139 USA 

In C. elegans, 131 somatic cells undergo programmed cell death during wild-type 

hermaphrodite development. While genes that cause programmed cell death have been well 

studied, less is known about how a particular cell is specified to survive or to die by programmed 

cell death. To identify pathways involved in cell-type specific programmed cell death, we 

screened for mutations that cause a defect in the death of the sister of the pharyngeal M4 

motor neuron. The M4 neuron is generated during embryonic development and survives to 

regulate muscle contraction in the pharynx, while the M4 sister dies by programmed cell death. 

Using genetic screens, we identified seven genes required for M4 sister cell death: ceh-32, 

ceh-34, eya-1, sptf-3, pig-1, gcn-1 and abcf-3. Here we describe our studies of the SP1 family 

transcription factor SPTF-3 and the AMPA-related protein kinase PIG-1. Reduction of sptf-3 

function decreases expression of the pro-apoptotic BH3-only gene egl-1 in the M4 sister and 

does not enhance a defect in M4 sister cell death in ced-9 null mutants. By contrast, a loss 

of pig-1 function does not affect egl-1 expression in the M4 sister and enhances a defect in 

M4 sister cell death in ced-9 null mutants. Also, sptf-3; pig-1 double mutants have a stronger 

defect in M4 sister cell death than do either of the single mutants. These results indicate that 

sptf-3 acts through the canonical cell-death execution pathway, while pig-1 acts in a distinct 

pathway in the regulation of M4 sister cell death. 

We previously reported that the C. elegans Six family homeodomain protein CEH-34 and 

the Eyes absent homolog EYA-1 promote the death of the M4 sister through the transcriptional 

activation of egl-1 (Hirose et al., PNAS 107, 15479-15484, 2010). An sptf-3 deletion does not 

affect ceh-34 or eya-1 expression in the M4 sister. This result suggests that sptf-3 acts in a 

distinct pathway from that of ceh-34 and eya-1 to promote egl-1 expression in the M4 sister. 

Our findings indicate that M4 sister cell death is regulated by at least three different 

pathways, in which 1) ceh-34 and eya-1 promote egl-1 expression, 2) sptf-3 promotes egl- 

1 expression via a pathway distinct from that of ceh-34 and eya-1, and 3) pig-1 functions 

independently of the canonical cell-death execution pathway. 

Contact: thirose@mit.edu 

Lab: Horvitz 


97

Using HITS-CLIP to study mRNA targets of RNA-binding proteins 

involved in germ cell apoptosis in C. elegans 

Martin Keller 1,3 , Deni Subasic 1,3 , Kishore Shivendra 2 , Michaela Zavolan 2 , Micheal 

Hengartner 1 

1 Institute of Molecular Life Science, University of Zurich, Switzerland, 

2 Biozentrum, University of Basel, Switzerland, 3 Molecular Life Science PhD 

Programm, Life Science Zurich Graduate School, ETH/University of Zurich, 

Switzerland 

Post-transcriptional control of mRNAs by RNA-binding proteins (RBPs) has a prominent 

role in the regulation of gene expression. RBPs interact with mRNAs to control their biogenesis, 

splicing, transport, localization, translation and stability. Defects in such regulation can lead to 

a wide range of human diseases from neurological disorders to cancer. Many RBPs are known 

to regulate apoptosis in the adult C. elegans germline. How these RBPs control apoptosis 

is however largely unknown. To address this question, we set out to establish a method to 

identify the mRNA targets apoptosis-associated RBPs. In a proof of principle experiment, we 

selected GLD-1, as translational regulator already has many known targets, including CEP-1, 

the C. elegans homologue p53 tumor suppressor. Using HITS-CLIP (cross-linking and RBP 

immunoprecipitation coupled with high-throughput sequencing) we could identify many of 

these known targets and the known binding motif of GLD-1 demonstrating the value of our 

method. Interestingly, we found that GLD-1 strongly binds to several sites in the 3’UTR of its 

own mRNA, suggesting that GLD-1 undergo auto regulation. Moreover, mRNAs for many 

of the other germline apoptosis RBPs (CGH-1, CPB-3, DAZ-1 and GLA-3) were also bound 

by GLD-1, hinting at the possible existence of an RBP regulon that orchestrates germ cell 

apoptosis. We now plan to apply our method on the complete set of RBPs that regulate germ 

cell apoptosis in order to identify the pathway that links germline apoptosis RBPs to the core 

apoptosis machinery. 

Contact: martin.keller@imls.uzh.ch 

Lab: Hengartner 

98 


Utilization of Alternative mRNAs for CED-4/Apaf-1 During Germ Cell 

Apoptosis 

J. Kaitlin Morrison, Brett Keiper 

Brody School of Medicine at East Carolina University 

Germ cell apoptosis is the process by which superfluous oocyte progenetor cells are 

eliminated by committing themselves to die via signaling through the cell death (ced) signaling 

pathway. Nearly half of all germ cells in the C. elegans gonad are fated for death before reaching 

maturity. These cells are believed to act as “nurse cells” providing cytoplasmic components 

needed by their sibling cells. During apoptosis changes in protein synthesis occur upon 

activation of caspases that cleave the translation initiation factor, eIF4G, which is involved in 

the cap dependent recruitment of mRNA to the ribosome. Our study focuses on the relative 

contribution of the C. elegans eIF4G (IFG-1) cap dependent and independent isoforms (p170 

and p130) to shifts in the protein synthesis mechanism and the selection of germ cells to die. 

Specifically, we are assessing the effect of such mechanisms on the translation of ced-4 mRNA 

variants. In addition to previously known splice variants of ced-4, ced-4L and ced-4S that have 

opposing apoptotic activities we have identified several alternative ced-4 message variants by 

RT-PCR and RNase Protection mapping. The distribution of the message variants and their 

translational efficiency was assayed in wildtype worms and worms depleted of either IFG-1 

p170, CED-9, CED-3 or the germ line protein GLA-3. Our findings suggest a physiological 

link between translational control by IFG-1 and the expression of CED-4 to induce germ cell 

apoptosis. 

Contact: morrisonju09@students.ecu.edu 

Lab: Keiper 


99

A Small-Molecule Screen Identifies a Linker Cell Death Inhibitor 

Andrew Schwendeman, Shai Shaham 

Rockefeller University, New York, NY, USA 

Programmed cell death plays a central role in animal development and disease. Although 

apoptosis is the best characterized cell death mechanism, it does not seem to account for all 

vertebrate programmed cell death. Our lab has described molecular and morphological features 

of the death of the C. elegans male-specific linker cell at the L4-adult transition (Abraham et 

al., 2007; Blum et al., 2012). These studies show that linker cell death is independent of all 

known apoptotic genes, and is accompanied by ultrastructural characteristics distinct from 

those of apoptotic cells. This novel morphological signature, including uncondensed chromatin, 

nuclear envelope crenellation, and swollen mitochondria and endoplasmic reticulum, have 

been described in dying cells of the developing vertebrate nervous system and in pathologies 

such as Huntington’s disease. To isolate additional regulators of linker cell death, and to test 

conservation with vertebrate cell-death processes, we developed a high-throughput smallmolecule 

screen to identify inhibitors of linker cell death. Animals carrying a mig-24::GFP 

reporter transgene, expressed specifically in the linker cell, are grown on E. coli OP50-seeded 

plates until the L4 stage, washed, and incubated in S Basal buffer with candidate compounds in 

384-well plates. After animals have become adults, a fluorescence cytometer is used to count 

surviving linker cells in each well. From a pilot screen of 1269 compounds, we identified a 

single small molecule that inhibits linker cell death without obvious pleiotropies. The compound 

has an IC50 in the nanomolar range, and its characteristics and method of action are under 

investigation. We aim to screen larger libraries to identify additional inhibitors. 

Contact: aschwendem@rockefeller.edu 

Lab: Shaham 

100 


Wave Regulatory Complex Genes Are Involved in the Engulfment of 

Apoptotic Cells 

Elena Simionato, Michael Hurwitz 

Yale University, School of Medicine, New Haven, CT, USA 

The engulfment of apoptotic cells is a conserved process involving the cytoskeletal 

rearrangement of one cell to surround another cell. This process is mediated by two parallel 

and partially redundant signalling pathways (CED-1, CED-6, CED-7, DYN-1 and CED-2, 

CED-5, CED-10, CED-12 and ABI-1). CED-1, 6 and 7 activate the GTPase DYN-1 Dynamin, a 

regulator of membrane dynamics. CED-2, 5 and 12 activate the small GTPase CED-10 Rac, a 

regulator of the actin cytoskeleton. ABI-1 is also a cytoskeletal regulator found in several protein 

complexes in mammals. One such complex is the Wave regulatory complex (WRC), which 

mediates actin polymerization in response to activation by Rac signaling. The WRC genes in 

worms are abi-1, wve-1, gex-2, gex-3 and nuo-3. Prior work has suggested that these genes 

are also involved in engulfment (Soto et al., 2002; Patel et al., 2008). Because all WRC genes 

are essential to the development of the animal, we tested if wve-1 and gex-2 are involved 

in engulfment using partial knockdown by feeding RNAi. We analyzed the effects of wve-1 

and gex-2 RNAi in strains that were already partially defective in dyn-1 and ced-10 pathway 

genes to look for engulfment defects. The partial knockdown of wve-1 and gex-2 enhance 

the engulfment defects of all genes tested. To further analyze the role of wve-1 and gex-2, 

we tested their effects on distal tip cell (DTC) migration during gonad development, a process 

regulated by the ced-10 pathway. wve-1 and gex-2 RNAi enhanced the DTC migration defect 

of all ced-10 Rac pathway genes, suggesting that wve-1 and gex-2 act downstream of or in 

parallel to CED-10 Rac pathway in this process. We also tested the effect of wve-1 and gex-2 

RNAi on actin dynamics during engulfment in strains defective in dyn-1 and ced-10 pathway. 

wve-1 and gex-2 RNAi led to a decrease in the number of cell corpses surrounded by actin, 

consistent with their known effects on the actin cytoskeletal rearrangement. Since RNAi of all 

WRC genes tested enhanced the engulfment defects of both known engulfment pathways, 

the WRC might act in parallel to both pathways. Alternatively, the WRC could be downstream 

of CED-10 but also activated by other pathways in parallel to the CED-10 pathway. In either 

model, our data imply that proteins independent of CED-10 activate the WRC. To date no 

activators of the WRC have been found independent of Rac proteins. We are now working to 

identify new CED-10 Rac-independent regulators of the WRC. 

Contact: elena.simionato@yale.edu 

Lab: Hurwitz 


101

In Search of Genes that Regulate Germ Cell Apoptosis in C. elegans 

Angel Villanueva-Chimal , Carlos Silva-Garcia , Laura Lascarez-Lagunas, Rosa 

Navarro 

Departamento de Biologia Celular y Desarrollo, Instituto de Fisiologia 

Celular, Universidad Nacional Autonoma de Mexico. Mexico. 

Apoptosis is a common feature of metazoan germ line. In C. elegans, fifty percent of germ 

cells are eliminated by apoptosis during oogenesis (physiological apoptosis). Higher levels of 

germ line apoptosis can be triggered by stress conditions such as DNA damage, starvation, 

heat shock, oxidative and osmotic stress. DNA damaged-induced apoptosis requires the p53 

protein and the DNA damage machinery repair. On the other hand, heat shock, oxidative 

and osmotic stresses increase germ cell apoptosis in C. elegans by the MAPKK pathway, 

and through an EGL-1 and CEP-1 independent mechanism. The mechanisms that regulate 

physiological apoptosis and starvation-induced germ cell apoptosis are still unknown. We 

are currently searching genes that control physiological apoptosis and/or starvation-induced 

germ cell apoptosis. We tested six DNA or RNA binding protein coding genes that change 

their expression levels when animals are starved for 6 h. We found that under normal growing 

conditions high levels of germ line apoptosis are detected when C54G4.6, C28H8.9, T23F6.4, 

M03C11.8 and C27A12.2 are silenced. Additionally, we found a regular response to starvationinduced 

germ cell apoptosis when F56D2.6, C54G4.6, T23F6.4, M03C11.8 were silenced. On 

the other hand, C28H8.9(RNAi) and C27A12.2(RNAi) animals did not respond to starvationinduced 

germ cell apoptosis. C28H8.9(RNAi) animals showed less oocytes per gonad than 

control animals and C27A12.2(RNAi) animals showed early embryonic and growing defects, 

oocyte accumulation, alterations in the transition of mitosis to meiosis and 40% less offspring 

than control animals. 

Contact: leunammevill@gmail.com 

Lab: Navarro 

102 


let-70, an E2 Ubiquitin-Conjugating Enzyme, Promotes the Non- 

Apoptotic Death of the Linker Cell 

Jennifer Zuckerman 

The Rockefeller University, New York, NY, USA 

Cell death is essential for animal development.Though apoptosis occurs during vertebrate 

development,evidence suggests it may not be the only operative cell-death process.The 

C.elegans linker cell is a male-specific cell that guides gonad formation.It lives for two days and 

dies to allow fusion of the vas deferens to the cloaca.Our studies showed that the linker cell 

dies independently of all known C.elegans cell-death genes.Further,dying linker cells exhibit 

non-apoptotic features including nuclear-membrane invagination, decondensed chromatin 

and swelling of organelles- features characteristic of non-apoptotic developmental cell death 

in the vertebrate nervous system and of neurodegeneration promoted by polyQ expansions, 

as in Huntington’s disease. 

An RNAi screen for genes promoting linker cell death uncovered the ubiquitin-conjugating 

enzyme let-70.Wild-type males fed E.coli expressing let-70 dsRNA show a strong block in linker 

cell death.The final stage of linker cell migration is blocked, but is genetically separable from 

death.Linker cell-only RNAi using rde-1 rescue blocks death but not migration, supporting the 

idea that these processes are independent and that let-70 functions cell-autonomously to kill. 

let-70::GFP fusions are expressed in the linker cell as the cell dies.Similar expression is 

seen with PQN-41,a polyQ protein important for linker cell death(Blum et al., 2012),suggesting 

that a transcriptional program regulates its timing. pqn-41 expression is controlled by the SEK- 

1 MAPKK.Epistasis studies show that let-70 RNAi in concert with a sek-1 mutation does not 

result in additive survival.Also, let-70::GFP is not expressed in sek-1(ag1)mutants,suggesting 

that sek-1 operates upstream of let-70.Similar results were obtained with RNAi against the 

MAPK scaffold tir-1,and the MLL complex genes swd-2.2 and set-16.By contrast,let-70::GFP 

expression remains high after RNAi of the nuclear hormone receptor nhr-67,and joint RNAi of 

nhr-67/let-70 produces additive survival,suggesting these genes act in parallel. 

To identify LET-70 targets,we screened for E3s required for linker cell death. We found that 

RNAi against the seven-in-absentia homolog siah-1 or the ring-box rbx-1 blocked linker cell 

death in 10% of animals.siah-1(tm1968);rbx-1(RNAi)animals show a 30% survival,indicating 

that these genes play partially-redundant roles.Furthermore,linker cell-only RNAi of proteasome 

subunits blocks linker cell death.Our studies suggest an important role for protein degradation 

pathways in linker cell death. 

Contact: jzuckerman@rockefeller.edu 

Lab: Shaham 


103

Elucidating the let-7 Independent Role of lin-28 

Jennifer Alaimo, Bhaskar Vadla, Kevin Kemper, Eric Moss 

UMDNJ-Graduate School of Biomedical Sciences, Stratford, New Jersey, 

United States of America 

lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, 

it is a component of the heterochronic gene pathway that governs larval developmental timing, 

while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. 

LIN-28 is an RNA binding protein that can directly inhibit let-7 microRNA processing by a 

novel mechanism that is conserved from worms to humans. We found that C. elegans LIN-28 

protein can interact with four distinct let-7 family pre-microRNAs, but in vivo, can inhibit the 

premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or 

its relatives for its characteristic promotion of second larval stage cell fates. We have shown 

that lin-28 acts in two steps: first, the let-7-independent positive regulation of hbl-1 through its 

3’UTR to control L2 stage-specific cell fates; and second, a let-7-dependent step that controls 

subsequent fates via repression of lin-41. The transcription factor encoded by hbl-1 is known 

to be regulated by at least three let-7 family members, and is thought to be the most proximal 

regulator of the succession of L2 to L3 cell fates. However, in addition to the eight potential 

let-7 family binding sites, there are also two potential lin-4 sites in hbl-1’s 3’UTR. The role of 

these sites in hbl-1 regulation is currently unknown. Present work seeks to elucidate if lin- 

28’s positive regulation of hbl-1 is direct through binding of the hbl-1 mRNA or indirect via a 

mechanism involving the lin-4 family of microRNAs. 

Contact: tsialije@umdnj.edu 

Lab: Moss 

104 

Poster Topic: Cell Fate

Regulation and function of SYS-1/beta-catenin during hypodermal 

stem cell divisions 

Austin Baldwin, Bryan Phillips 

University of Iowa 

The stem cell-like asymmetric divisions of the epithelial seam cells are controlled by the 

Wnt/beta-catenin Asymmetry (WBA) signaling pathway. The activity of this pathway results 

in the anterior daughter fusing to hyp7 (WBA independent fate) and the posterior daughter 

retaining the seam cell fate (WBA dependent fate). POP-1/TCF is exported from the posterior 

nucleus by a known mechanism involving WRM-1/beta-catenin and LIT-1/nemo-like kinase. 

SYS-1/beta-catenin is regulated postranslationally by the WBA pathway in other tissues, but 

little is known of SYS-1 expression or regulation in the seam stem cells. To investigate the 

mechanism of seam cell specification, we analyzed SYS-1 localization in wild type larvae 

and manipulated SYS-1 levels during seam cell division. We show that SYS-1 displays 

dynamic localization during seam cell division. SYS-1 localizes in rapid sequence to the cell 

cortex, midbody, and both centrosomes in the dividing mother cell. After cytokinesis, SYS-1 is 

undetectable in the unsignaled hypodermal daughter, but accumulates in the nucleus of the 

signaled seam cell daughter. The SYS-1 localization pattern is under the control of upstream 

WBA pathway members. The resultant reciprocally asymmetric pattern of low SYS-1/high 

POP-1 in the anterior daughter nucleus and high SYS-1/low POP-1 in the posterior daughter 

nucleus is consistent with a role in Wnt target gene transactivation. We also find that elevating 

SYS-1 levels in both daughters at the time of seam cell division induces duplication of the 

stem cell fate at the expense of the hypodermal fate. These data position SYS-1 as a target 

of Wnt regulation during seam stem cell specification and suggest that SYS-1, like canonical 

beta-catenin, plays a conserved role in maintaining stem cell populations. 

Contact: austin-t-baldwin@uiowa.edu 

Lab: Phillips 

Poster Topic: Cell Fate 

105

Germline Expressed GLP-1 Regulates Embryonic Endoderm 

Specification 

Ahmed Elewa1 , Takao Ishidate1 , Sandra Vergara1 , Tae-Ho Shin2 , Masaki 

Shirayama1 , Craig Mello1 1 2 UMass Medical School, Worcester, MA, USA, Baylor College of Medicine, 

Huston, TX, USA 

Mutations in the CCCH zinc finger gene pos-1 and RNAi of the KH domain gene gld-1 

result in identical maternal-effect embryonic lethal phenotypes involving the misspecification 

of several embryonic cell fates: including transformation of ABp to ABa, failure to specify 

endoderm and failure to specify germ-line cell fates. POS-1 and GLD-1 have been shown to 

function together to suppress translation of the GLP-1 mRNA in posterior sister cells at the 

4-cell stage of embryogenesis. However, GLP-1, a transmembrane receptor related to Notch, 

is not required for endoderm specification, and its mis-expression in early pos-1 embryos has 

not been linked to the loss of endoderm fate. We were therefore surprised to find the mutation 

glp-1( as a temperature sensitive suppressor of the pos-1 endoderm defect. 

glp-1( exhibits an amino-acid substitution (G1031D) in the 4th (of 7) ankyrin repeats. 

Several other glp-1ts alleles exhibit amino acid substitutions in the same ankyrin repeat region 

(including e2141, bn18 and q231). We found that all of these apparent loss of function ts alleles 

also suppress the endoderm defect of pos-1(zu148).Interestingly, the temperature sensitivity 

point for endoderm restoration occurs in the gonad hours prior to fertilization suggesting that 

glp-1 activity in the distal gonad can interfere with endoderm specification in pos-1 mutants 

much later in the early embryo. 

This temporal displacement of suppression is reminiscent of that described between efl-1 

and pos-1 (Page B et al Mol. Cell March 2001 and Tenlen J et al. Genetics December 2006). 

EFL-1 is a homologue of vertebrate E2F transcription factor and mediates the expression of 

a host of targets in the C. elegans germline (Chi W & Reinke V Development 2006). Coupled 

with our previous finding that translation regulator gld-3 suppresses the endoderm defect of 

pos-1 and gld-1, we are exploring a model where glp-1 and efl-1 activity in the distal gonad 

promotes the expression of a gut antagonizer dependent on GLD-3 for its translation and 

repressed by GLD-1 and POS-1 in wild-type embryos to permit gut development. 

Contact: ahmed.elewa@umassmed.edu 

Lab: Mello 

106 


Investigating the Role of SEM-4/SALL in Development of the 

Postembryonic Mesoderm 

Vikas Ghai, Chenxi Tian, Jun Liu 

Cornell University, Ithaca, NY, USA 

The C. elegans postembryonic mesoderm lineage, the M lineage, is a unique system 

that provides a high degree of temporal and cellular resolution for studying development of 

mesodermal tissues. The M lineage is derived from a single pluripotent precursor cell, the 

M mesoblast, which undergoes two waves of proliferation and differentiation to produce 32 

cells. These cells include 14 bodywall muscles, two coelomocytes (dorsally-derived), and two 

multi-potent sex myoblasts (SMs; ventrally-derived) that will give-rise to 16 vulval and uterine 

muscles. Using this system, we have elucidated the mechanisms of several highly conserved 

transcription factors and signaling pathways in mesoderm development. These include a 

rolefor LET-381/FoxF and CEH-34/Six2 in coelomocyte specification (Amin et al.,2010), a 

role for SEM-2/SoxC in promoting the proliferative SM fate (Tian etal., 2011), and input from 

BMP, Notch, and Wnt pathways (Foehr et al., 2006; Greenwald et al.,1983; Foehr and Liu, 

2008; Amin et al. 2009). 

Interestingly, SEM-4, the sole C. elegans member of the Spalt/SALL family of C2H2 zinc 

finger transcription factors is required for both dorsal and ventral cell fates in the M-lineage, 

as both coelomocytes and SMs are transformed to BWM cells in sem-4 mutants (Basson and 

Horvitz, 1996). Here we investigate the role of SEM-4 during the development of the M-lineage. 

sem-4 is expressed in a pattern in the M-lineage overlapping with both LET-381 and SEM-2. 

The M-lineage expression of sem-2 does not change in sem-4(n1378)mutants, nor does the 

expression of sem-4 in sem-2(n1343) mutants. This suggests that while sem-2 and sem-4 do 

not regulate each other’s expression, they probably either directly interact with each other or 

regulate the same set of target genes. We are currently testing these possibilities. We are also 

performing experiments between sem-4 and let-381,as well as other genes in the M-lineage 

to determine their regulatory relationships. 

Contact: vghai@cornell.edu 

Lab: Liu 


107

A Screening To Find Suppressors Of The Wnt Pathway 

Eva Gomez-Orte2 , Begona Ezcurra1 , Beatriz Saenz-Narciso1 , Juan Cabello1 1 2 CIBIR, FUNDACION RIOJA SALUD, Center for Biomedica Research of La 

Rioja (CIBIR), Logrono, Spain 

The Wnt pathway typically has been defined as a pathway involved in fate specification. 

However, in addition to this function, the Wnt pathway is involved in other processes such as 

cell migration, engulfment of apoptotic corpses or proper mitotic spindle orientation. Our lab 

works in understanding how this pathway is regulated and how different signals are integrated 

to produce a coordinated response. 

We have started a screening to find genetic suppressors of mutants in different components 

of the Wnt pathway. Towards this aim, we have generated a strain lit-1 (t1512); wIs84 (pJM66 

elt-2::GFP::LacZ, pRF4 rol-6(su1006dm)) that carries an integrated intestinal GFP marker and 

a Temperature Sensitive mutation in lit-1. LIT-1 is a Nemo-like kinase that phosphorilates POP- 

1, the transcription factor of the Wnt pathway. In the absence of LIT-1, the unphosphorilated 

POP-1 remains in the nucleus and avoid the expression of the endoderm specific genes. Thus, 

at 15 C, lit-1 (t1512); wIs84 is viable and has a fluorescent intestine; whereas at 25 C produces 

only dead embryos without intestine and hence without any fluorescence. 

We have performed a pilot EMS screening to find lit-1(t1512) TS worms that at 25 C, were 

able to produce embryos with intestine (GFP positive). After mutagenize 100000 haploid 

genomes, we have found two independent lit-1 (t1512) strains that develop intestine. These 

strains are currently being outcrossed. The nature of the suppressor mutation will be determined 

by deep sequencing. We expect to find regulators downstream or in parallel to LIT-1. 

Contact: egogmj@yahoo.es 

Lab: Cabello 

108 


MEX-5 regulates mRNA stability during germ cell development and 

asymmetric cell division 

Manoel Prouteau, Gilles Udin, Monica Gotta 

CMU, University of Geneva, Geneva, Switzerland 

Establishment and maintenance of cell polarity are essential for many biological processes 

such as asymmetric cell division, proliferation, differentiation, and morphogenesis. In the C. 

elegans embryo the conserved PAR proteins regulate the cytoplasmic asymmetric localization 

of CCCH tandem zinc finger proteins (CCCH-proteins), which specify somatic and germ cell 

fates. Two redundant CCCH-proteins, MEX-5 and MEX-6 (referred to as MEX-5/6), accumulate 

in the anterior half of the one-cell embryo and are important to regulate polarity and cell fate 

specification. In yeast and mammalian cells, CCCH proteins bind specific mRNAs and promote 

their degradation. This activity depends on their ability to shuttle in and out of the nucleus. 

Interestingly, in mex-6;mex-5 mutant embryos, the enrichment of certain mRNAs (called 

class II mRNAs) in the germline precursors is lost and these mRNAs are found in all cells. 

We therefore investigated whether MEX-5/6 regulate polarity processes also by controlling 

class II mRNA stability. We find that MEX-5 is in a complex with proteins of the mRNA decay 

machinery in the embryo. Consistent with a role in mRNA decay, the total levels of class II 

mRNAs are increased in mex-5 mutants. In addition, we show that MEX-5, as the yeast and 

mammalian homologues, is shuttling in the nuclei of somatic cells. In the germline lineage, 

where MEX-5 must be kept inactive to avoid mRNA degradation, nuclear shuttling is inhibited by 

PAR-1 phosphorylation. Taken together our data suggest a model in which MEX-5 contributes 

to germline development by controlling the segregation of mRNAs in the P lineage via their 

degradation in somatic blastomeres. 

Contact: monica.gotta@unige.ch 

Lab: Gotta 


109

A Screen for Mislocalization and Misexpression of LET-23 EGF 

Receptor during Vulval Development 

Andrea Haag, Juan Escobar Restrepo, Alex Hajnal 

University of Zurich, Zurich, Switzerland 

In polarized epithelial cells, the apical and basolateral membranes are composed of distinct 

proteins and lipids that provide specific functions. The mammalian epidermal growth factor 

receptor (EGFR), a member of the ErbB family of receptors, is mainly localized to the basolateral 

cell membrane (Kuwada et al., 1998). Mislocalization of mammalian ErB family members to 

the apical surface can de-regulate signaling by the receptor and result in disease (Du et al., 

1995). Similarly, the C. elegans EGFR homolog LET-23 is targeted to the basolateral plasma 

membrane in vulval precursor cells. Vulval development is impaired if LET-23 is mislocalized. 

Previously, a ternary complex consisting of LIN-7, LIN-2 and LIN-10 has been shown to play 

an important role in the retention of LET-23 on the basolateral surface (Kaech et al., 1998). 

Nonetheless, the exact mechanism of LET-23 localization and the control of the receptor 

dynamics remain poorly understood. To identify new regulators of LET-23 localization, we 

performed an RNAi feeding screen using a functional LET-23::GFP translational reporter. We 

analyzed over 700 RNAi clones that are known to cause a protruding vulva (Pvl) phenotype. 

By evaluating LET-23::GFP expression at different developmental stages, we were able to 

identify several genes regulating LET-23 localization or expression in the VPCs and their 

descendants. To investigate if receptor mislocalization alters LET-23 signaling, we performed 

RNAi against selected candidates in a sensitized let-60 ras(gf) background. By this approach, 

we have so far identified three genes that negatively regulate RAS/MAPK signaling and control 

LET-23 localization. To confirm the RNAi results, we are currently analyzing LET-23::GFP 

localization in the corresponding mutant strains. Further experiments aim at investigating how 

these candidate genes control LET-23 localization and/or expression. 

Kuwada SK, Lund KA, Li XF, Cliften P, Amsler K, Opresko LK, Wiley HS (1998) Differential 

signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells. Am J 

Physiol. 275; C1419-28 

Du J, Wilson PD (1995) Abnormal polarization of EGF receptors and autocrine stimulation 

of cyst epithelial growth in human ADPKD. Am J Physiol. 269; C487-95 

Kaech SM, Whitfield CW, Kim SK (1998) The LIN-2/LIN-7/LIN-10 complex mediates 

basolateral membrane localization of the C. elegans EGF receptor LET-23 in vulval epithelial 

cells. Cell. 94; 761-71 

Contact: andrea.haag@imls.uzh.ch 

Lab: Hajnal 

110 


A Role of the LIN-12/Notch Signaling Pathway in Diversifying the Non- 

Striated Egg-Laying Muscles in C. elegans 

Jared Hale, Carolyn George, Nirav Amin, Zachary Via, Leila Toulabi, Jun Liu 

Department of Molecular Biology and Genetics, Cornell University, Ithaca, 

NY, 14853 

Our lab aims to understand the mechanisms underlying the diversification of the 

postembryonic mesoderm lineage, which arises during embryogenesis from a pluripotent cell 

known as the M mesoblast. During postembryonic development in C. elegans, the M mesoblast 

divides to generate fourteen striated body-wall muscles, two coelomocytes, and two sex 

myoblasts (SMs). The SMs further divide and differentiate into sixteen non-striated egg-laying 

muscles: four each of type I and type II vulval muscles and uterine muscles, respectively. While 

the MyoD family of transcription factors has been shown to play an evolutionarily conserved 

role in specifying the striated/skeletal muscles, relatively little is known about how different 

types of non-striated/smooth muscles are specified. In an RNAi screen for transcription factors 

important for muscle development, we found that RNAi knockdown of lag-1, which encodes a 

component of the LIN-12/Notch pathway, led to the production of extra type I vulval muscles. 

Similar phenotypes were also observed in animals with reduced functions of the Notch receptor 

LIN-12 and its ligand LAG-2. The extra type I vulval muscles in animals with reduced LIN-12/ 

Notch signaling did not appear to be a result of extra SMs or extra SM proliferation, rather a 

fate transformation from type II vulval muscles to type I vulval muscles. Consistent with this, 

we observed nuclear localization of the functional LIN-12::GFP in the undifferentiated type 

II vulval muscles. Thus LIN-12/Notch signaling is used repeatedly in the M lineage, first for 

promoting the ventrally-derived SM fate (Greenwald et al., 1983; Foehr and Liu, 2008), and 

second for specifying the type II vulval muscles in diversifying the SM lineage. 

Contact: jjh278@cornell.edu 

Lab: Liu 


111

UNC-62/Meis and CEH-20/Pbx proteins work together to control 

asymmetric cell divisions during C. elegans development by 

regulating WRM-1/β-catenin localisation 

Samantha Hughes, Charles Brabin, Alison Woollard 

Oxford University, Oxford 

During larval development, stem cell-like seam cells undergo asymmetric divisions producing 

an anterior daughter cell that differentiates by fusing with the hypodermal syncytium and a 

posterior daughter that retains the seam fate and has the ability to undergo further proliferation. 

As a result of a genome wide RNAi screen, we identified two transcription factor genes, ceh-20/ 

Pbx and unc-62/Meis, that are required for these asymmetric cell fate decisions. Animals that 

lack ceh-20 and/or unc-62 display severe seam cell hyperplasia that is absolutely dependent 

upon the function of rnt-1 and bro-1. Intriguingly the hyperplasia is largely restricted to the 

anterior H lineages of the seam. Lineage analysis reveals that the hyperplasia is the result of 

complete symmetrisation of asymmetric divisions towards the posterior proliferative fate. We 

observed the dynamic nature of CEH-20 localisation whose nuclear distribution is regulated 

by UNC-62. In addition, we found that the distribution of WRM-1/β-catenin is perturbed in the 

absence of CEH-20 suggesting a molecular mechanism by which ceh-20/unc-62 may control 

the establishment of seam division asymmetry. 

Contact: samantha.hughes@bioch.ox.ac.uk 

Lab: Woollard 

112 


The Ras-ERK/MAPK Regulatory Network Controls Dedifferentiation In 

Caenorhabditis elegans Germline 

Dong Seok Cha1 , Udaya Sree Datla1 , Sarah Hollis1 , Judith Kimble2,3 , Myon-Hee 

Lee1,4 1Brody School of Medicine at East Carolina University, Greenville, NC, USA, 

2 3 4 HHMI, University of Wisconsin-Madison, Madison, WI, USA, Lineberger 

Comprehensive Cancer Center, University of North Carolina-Chapel Hill, 

Chapel Hill, NC, USA. 

How a committed cell can be reverted to an undifferentiated state is a central question in 

stem cell biology. This biological event, called dedifferentiation, is capable of replacing stem cells 

as they get aged or damaged. Tremendous progress has been made, but the mechanisms are 

poorly understood. Here we demonstrate that activation of Ras-ERK/MAPK signaling promotes 

cellular dedifferentiation in the Caenorhabiditis elegans germline. To activate signaling, we 

removed two negative regulators, the PUF-8 RNA-binding protein and the LIP-1 dual specificity 

phosphatase. Removal of these two regulators caused spermatocytes to dedifferentiate and 

begin mitotic divisions. Interestingly, reduction of Ras-ERK/MAPK signaling, either by mutation 

or chemical inhibition, blocked the initiation of dedifferentiation. By RNAi screening, we identified 

RSKN-1/P90RSK as a downstream effector of MPK-1/ERK and as critical for dedifferentiation: 

rskn-1 RNAi suppressed dedifferentiation of spermatocytes and induced meiotic cell divisions. 

Because these regulators are all broadly conserved, we suggest that similarly molecular circuitry 

may control cellular dedifferentiation in other organisms, including humans. 

Contact: leemy@ecu.edu 

Lab: Lee 


113

A sma-9 Suppressor Screen to Identify New Players in the BMP-like 

Sma/Mab Pathway in C. elegans 

Lindsey Szymczak, Katharine Constas, Arielle Schaeffer, Sinthu Ranjan, Saad 

Kubba, Emad Alam, Dennis Liu, Chenxi Tian, Herong Shi, Jun Liu 

Cornell University, Ithaca, USA 

The bone morphogenetic protein (BMP) pathway plays essential roles in multiple 

developmental processes during metazoan development. In C. elegans, the BMP-like Sma/Mab 

pathway regulates body size and male tail patterning. We have previously shown that the Sma/ 

Mab pathway also regulates mesoderm development. In particular, mutations in the zinc finger 

containing protein SMA-9 cause a dorsal to ventral fate transformation in the postembryonic 

mesodermal M lineage. This M lineage phenotype of sma-9 mutants can be suppressed by 

mutations in the Sma/Mab pathway, suggesting that SMA-9 antagonizes the function of Sma/ 

Mab signaling in patterning the M lineage (Foehr et al., 2006). The suppression of the sma- 

9 M lineage phenotype by Sma/Mab pathway mutants appears specific, as mutations in the 

TGFbeta-like dauer pathway or mutations affecting body size without affecting the Sma/Mab 

pathway do not suppress the sma-9 M lineage defect. Through the suppressor screen, we 

have identified a positive modulator of the Sma/Mab pathway named DRAG-1, which is a 

membrane-associated protein that belongs to the RGM (repulsive guidance molecule) family 

(Tian et al., 2010). 

Motivated by our findings described above, we carried out a large-scale sma-9 suppressor 

screen for additional players in the Sma/Mab pathway. By clonally screening through 5300 

haploid genomes, we identified thirty seven single-locus, recessive sma-9 suppressor mutations. 

Complementation tests showed that these include two alleles of sma-2, three alleles of sma-3, 

one allele each of sma-4, sma-6, daf-4, dbl-1 and lon-1. The remaining mutations define at least 

eleven complementation groups. Through whole genome sequencing, RNAi and transgenic 

rescue experiments, we have identified the corresponding gene for one of the complementation 

groups. Molecular genetic studies of this gene suggest that it encodes a trans-membrane 

protein that functions as a positive modulator of Sma/Mab signaling. Current research aims to 

decipher the molecular mechanism on how this gene product modulates Sma/Mab signaling. 

Contact: JL53@cornell.edu 

Lab: Liu 

114 


Further evidence for the importance of the MED-1 and -2 GATA factors 

in endoderm specification 

Morris Maduro, Gina Broitman-Maduro, Shruthi Satish 

University of California, Riverside, Riverside, CA, USA 

The E cell clonally generates the C. elegans intestine. A feed-forward transcription factor 

cascade involving SKN-1, MED-1,2 and END-1,3 drives specification of E in the pre-gastrulation 

embryo. We have previously reported that RNAi of med-1,2 results in some 50% of embryos 

lacking endoderm. A strain that is homozygous for putative null alleles of med-1 and med-2 

also results in ~50% of gut(-) embryos when the mothers carry irDp1, a modified version of 

sDp3 that carries unc-32(+), unc-119::YFP and med-1(+). Curiously, when mothers of med- 

1,2(-) embryos are heterozygous for one (or both) of the med genes, the resultant med-1,2(-) 

embryos fail to make gut only ~15% of the time. We have shown using in situ hybridization 

that there are maternal med transcripts in the germlines of wild-type hermaphrodites, but not 

irDp1(+) hermaphrodites, suggesting that a maternal contribution of the meds explains the 

difference in penetrance of the gut defect. Others have suggested that the unc-32 transgene, 

and not abrogation of a maternal contribution, is responsible for the difference, and that the 

higher RNAi phenotype results from non-specific knockdown of other genes. Using transgene 

reporters for gut fate, we find that unc-32 sequences in irDp1 have no detectable effect on 

whether or not gut cells contain gut granules in med-1,2(-) embryos. We further assessed 

the contribution of the MEDs to endoderm specification by removing the GTATACYYY MED 

binding sites from the end-1 and end-3 genes. We inserted single copies of wild-type and 

MEDsite-less end-1 and end-3 genes into other genomic contexts by MosSCI and microparticle 

bombardment. We introduced these into a double mutant end-1,3 background or a strain 

carrying the deficiency itDf2, which deletes end-1 and end-3 (as well as many other genes). 

We find that when the MED sites are mutated in end-1 and end-3, 30%-50% embryos fail 

to make intestine. This is significantly higher than the 15% that is seen due to zygotic loss 

of the meds alone, and consistent with our prior reports using med-1; med-2; irDp1 mothers 

and RNAi. We also find that adults derived from embryos in which the end genes lack MED 

sites have defects associated with gut differentiation (see other abstract by Maduro et al.). 

Our results add to our prior findings that the MEDs play a significant role in the specification 

of endoderm in C. elegans. 

Contact: mmaduro@ucr.edu 

Lab: Maduro 


115

Regulation and function of nhr-67/tailless in uterus development 

George McClung, Lauren Pioppo, Jenny Hall, Rachel Dordal, Catherine Ezzio, 

Evan Fletcher, Amanda Gavin, Sheila Clever, Bruce Wightman 

Muhlenberg College, Allentown, PA USA 

The tailless family of nuclear receptors is highly conserved among animals. The C. elegans 

tailless ortholog, nhr-67, is expressed in a dynamic pattern in pre-uterine cells. nhr-67 is 

initially expressed in the 4 pre-VU cells during the L2 stage, and subsequently upregulated in 

the anchor cell (AC), in response to the lin-12/lag-2 reciprocal signaling system. During the 

L3 stage, nhr-67 expression is maintained at high levels in the AC and at low levels in the six 

π cells whose twelve progeny form cells of the adult ventral uterus. nhr-67 is also expressed 

in the male LC and in neurons and functions in these cells have been defined by the Hobert 

and Sternberg labs. 

Homozygous deletions of nhr-67 generally cause developmental arrest in embryogenesis 

or after hatching at L1, with tail morphology defects. Mutants homozygous for hypomorphic 

nhr-67 promoter mutations that were identified by Bernard Lakowski’s laboratory, are viable, 

but defective for the development of the π cells and the AC. nhr-67(lf) mutations are epistatic to 

lin12(gf) mutations in the π cells, indicating that lin-12 activity depends on nhr-67. Expression 

of lin-12::gfp in all four pre-VU cells and later in VU cells depends on nhr-67, indicating that 

nhr-67 is an important regulator of lin-12 and therefore VU response to lag-2 signal. nhr-67 is 

also required for expression of zmp-1 and lag-2 in the AC. Taken together, these data define 

an important regulatory role for nhr-67 in both AC and VU development (Verghese et al., 2011, 

Dev. Biol. 356:516). 

Phenotypic similarities suggest that egl-43 and hlh-2 might function upstream or in parallel 

with nhr-67 to regulate AC development. The nhr-67 promoter mutations define a 276bp region 

that is important for nhr-67 function in uterine development. Deletion of this region results in a 

loss of nhr-67 expression in pre-VU, AC, and VU cells. We have collaborated with the Walhout 

Lab to identify potential upstream transcription factors that may bind to six evolutionarilyconserved 

candidate cis-acting elements in the 276bp region. Two of the six elements are 

perfect matches to E boxes that are predicted to bind HLH-2/HLH-4 and/or HLH-2/HLH-10 

heterodimers. We are evaluating several candidates using one-hybrid and EMSA approaches. 

This work was supported by a grant from the NSF. 

Contact: GM237053@gws2.muhlenberg.edu 

Lab: Wightman 

116 


Does lin-46 Tip the Balance of hbl-1 Activity in the Succession of 

Hypodermal Blast Fates? 

Eric Moss, Kevin Kemper, Bhaskar Vadla 

UMDNJ-GSBS, Stratford, NJ, USA 

In the regulation of larval hypodermal cell fates, the heterochronic genes lin-28 and lin-46 

appear to act oppositely (positively and negatively, respectively) on hbl-1. HBL-1 is a zincfinger 

transcription factor of the Ikaros family and seems to be the most direct effector of the 

L2 seam cell lineage pattern, which is characterized by a symmetric division followed by an 

asymmetric one that produces a hyp7 syncytial nucleus and a blast cell. 

lin-28 is well known to block let-7 accumulation, but let-7 and its target lin-41 are not involved 

in the regulation of seam cell fates in the L2 (see abstract by J. Alaimo). Through lin-46 we 

expect to learn more about how lin-28 and hbl-1 work. 

lin-46 null alleles completely suppress lin-28 null alleles restoring the normal cell lineage 

succession throughout the animal. LIN-46 protein is similar to proteins of bacteria and 

eukaryotes involved in the biosynthesis of an enzyme cofactor, but it seems to have been 

exapted for a role in the heterochronic pathway. Although LIN-46 retains the overall folding 

and structure of its homologs, the certain critical residues and surface charges have changed. 

Somewhere on LIN-46 is a surface that binds specifically to a important pair of C-terminal 

zinc fingers of HBL-1. In all other Ikaros family members, these zinc fingers are for homo- and 

hetero-dimerization. HBL-1 is unique among Ikaros proteins for not homodimerizing. 

So we have several questions to answer: Is the sole purpose of HBL-1’s C-terminal zinc 

fingers to permit negative regulation by LIN-46? Do these zinc fingers bind another Ikaros 

family member or some other protein necessary for HBL-1 activity? Does LIN-46 block this 

binding or does it simply drag HBL-1 off to the trash? So far we have evidence that HBL-1 

uses these zinc fingers for another purpose. 

Finally, lin-46 shows oscillating expression at both the protein and RNA levels slightly out 

of phase with the major oscillating gene in the heterochronic pathway, lin-42. lin-46 expression 

peaks just prior to the molt, suggesting a burst of HBL-1 inhibitory activity a few hours before 

cell fates are executed. If lin-46 is missing, hbl-1 appears to resist down-regulation for at least 

one more stage. Therefore, the role of lin-46 in the pathway may be to ensure repression of 

hbl-1 activity at a critical time and the “ticking over” of cell fates in proper succession. 

Contact: mosseg@umdnj.edu 

Lab: Moss 


117

Post-transcriptional Regulation of Maternally-supplied Wnt Ligand 

During Early Embryogenesis 

Marieke Oldenbroek1 , Scott Robertson1 , Tugba Guven-Ozkan2 , Rueyling Lin1 1 2 UTsouthwestern, Dallas, Texas, USA, Scripps Research Institute, Jupiter, 

Florida, USA 

During C. elegans embryogenesis, blastomere fate specification requires critical cell-cell 

interactions that are under precise spatiotemporal regulation. The mRNA for most components 

of signaling pathways in embryos are provided maternally and distributed uniformly. Temporal 

and spatial expression patterns of most maternally supplied proteins are regulated through the 

3’ UTR of their mRNA. One Wnt signaling pathway in early embryos specifies the endoderm 

precursor and orientates division axes. While the signaling cells for these Wnt-mediated 

interactions have been demonstrated by genetic analyses and chimeric experiments, the 

expression pattern of the Wnt ligand, MOM-2, has not been determined. We show here that 

a reporter containing GFP fused to H2B and the mom-2 3’UTR is expressed precisely in 

known Wnt signaling cells. The expression is first detected in the germline blastomere, P2, 

in 4-cell embryos, and continues in descendants of P2. Through in vitro RNA binding assays 

and genetic analysis, we identified proteins that bind to the mom-2 3’ UTR and regulate its 

expression. Repression of mom-2 in 1- and 2-cell embryos is dependent on two RNA binding 

proteins, MEX-3 and SPN-4. In 4-cell embryos, MEX-3 becomes localized primarily in somatic 

blastomeres where it continues to repress mom-2. In the germline blastomere P2, SPN-4 is 

present at a high level but does not repress mom-2 expression. Expression of MOM-2 in P2 

requires two germline-blastomere-specific RNA-binding proteins, PIE-1 and MEX-1, which both 

outcompete SPN-4 for binding to the mom-2 3’ UTR, thereby alleviating its repressive effect in 

P2. Repression of mom-2 in oocytes depends on two other RNA binding proteins, OMA-1 and 

OMA-2. After fertilization, OMA-1 and OMA-2 are degraded, partly due to their phosphorylation 

by GSK-3. Failure to degrade OMA proteins results in a phenotype similar to mom-2 mutant 

embryos. GSK-3 is well established as a negative regulator in the canonical Wnt pathway in 

flies and vertebrates. However, previous genetic studies reveal a positive role for GSK-3 in C. 

elegans embryos. Our results show this discrepancy to be due to a negative feedback loop, 

in which reduced GSK-3 activity leads to OMA protein persistence and ectopic repression of 

MOM-2 expression. Overall, our findings show the importance of translational regulation in 

restricting the expression of the C. elegans Wnt ligand MOM-2. 

Contact: marieke.oldenbroek@utsouthwestern.edu 

Lab: Lin 

118 


Abstract withdrawn. 

Contact: riddle@lifesci.ucsb.edu 

Lab: Rothman 


119

Regulation of LET-23 EGFR signaling and trafficking by a putative 

Arf1-GEF 

Olga Skorobogata, Christian Rocheleau 

McGill University, Montreal, QC, Canada 

Epidermal Growth Factor Receptor (EGFR)/Ras/Mitogen Activated Protein Kinase (MAPK) 

signaling regulates cell proliferation, migration and apoptosis and misactivation can lead to 

cancer. An important mechanism of signal downregulation involves EGFR endocytosis and 

trafficking to the lysosome. 

In C. elegans a highly conserved LET-23 EGFR signaling pathway is required for vulval cell 

fate specification.The LIN-2 CASK/ LIN-7 Veli/ LIN-10 Mint11 complex is required for basolateral 

localization of LET-23 in the Vulval Precursor Cells (VPCs). Mutations in lin-2 result in a strong 

Vulvaless (Vul) phenotype due to the apical mislocalization of LET-23. 

To identify new regulators of LET-23 signaling and trafficking, we conducted a forward 

genetic screen for essential suppressors of lin-2(e1309). One of the strong suppressor mutants 

identified, vh4, is partial embryonic lethal, has secretory defects in several tissues and enlarged 

lysosomal compartments. We used SNP mapping with whole-genome sequencing, as well as 

RNAi and genetic complementation, to identify vh4 as a hypomorphic allele of agef-1. AGEF-1 

is homologous to the human BIG1/2Arf Guanine nucleotide exchange factors that localize to 

the trans-Golgi and endosomes. The vh4 mutation leads to substitution of a conserved Glu to 

Lys within a conserved domain of unknown function. Consistent with AGEF-1 being a Class 

I and II Arf GEF, a deletion mutant of Class I Arf, arf-1.2(ok796), was able to suppress the 

lin-2 Vul phenotype, moreover RNAi-mediated depletion of ARF-3, a Class II Arf, in arf-1.2; 

lin-2 animals resulted in even stronger suppression. Genetic epistasis experiments suggest 

that agef-1 might function at the level of LET-23 EGFR. Thus AGEF-1, ARF-1.2, and ARF-3 

might promote the degradation or antagonize signaling of LET-23 EGFR in the VPCs. We 

hypothesize that AGEF-1 might either regulate the secretion of a negative regulator of LET-23 

EGFR or trafficking of lysosomal enzymes required for efficient degradation of LET-23 EGFR. 

< 

Contact: christian.rocheleau@mcgill.ca 

Lab: Rocheleau 

120 


Examining the Fate of Centrosomally Uncoupled SYS-1/Beta-catenin 

to Explore Spindle-Independent Roles of the Centrosome during 

Asymmetric Cell Divisions 

Setu Vora, Bryan Phillips 

University of Iowa 

A specialized Wnt/β-catenin signaling pathway is responsible for carrying out reiterated 

asymmetric cell divisions, allowing cells of the developing embryo and larva to navigate 

through a multitude of lineages. Asymmetric divisions regulated by this pathway give rise to 

daughter cells that exhibit differential activity of Wnt signaling components, allowing them 

to take on distinct transcriptional profiles. SYS-1/β-catenin is a major transcriptional effector 

of this pathway and is expressed at higher levels in the signaled daughter cell after a given 

Wnt-regulated asymmetric division. SYS-1 is subject to strict post-translational regulation and 

shows dynamic patterns of subcellular localization during the cell cycle. We are studying how 

subcellular localization of SYS-1 contributes to its proper regulation. In a number of different 

systems, β-catenin localizes to the centrosomes and has previously been implicated to function 

in microtubule outgrowth and centriole splitting. Similarly, SYS-1 localizes to the pericentriolar 

material (PCM) of the centrosomes during C. elegans cell divisions but is dispensable for proper 

spindle formation and orientation. Thus, the functional or regulatory significance of SYS-1 

centrosomal localization is not known. We have used a two-step screening process to identify 

SYS-1 centrosomal regulators. First, a split-ubiquitin based yeast two hybrid screen with a SYS- 

1 bait was used to identify SYS-1 interactors. Second, RNAi knockdown of interactors identified 

specific factors required for SYS-1 centrosomal localization. As these treatments uncouple 

SYS-1 from the centrosomes, we can now evaluate the regulatory status of centrosomally 

localized SYS-1 and examine the resulting effects on SYS-1 expression and target cell fates. 

Because the centrosome has been implicated in protein processing and degradation during 

mitosis, we are using this experimental system to explore a potential spindle-independent role 

of the centrosome in housing and distributing regulatory molecules such as β-catenin during 

asymmetric cell divisions. 

Contact: setu-vora@uiowa.edu 

Lab: Phillips 


121

Function and evolution of the diverged NR2E nuclear receptors nhr- 

111 and nhr-239 

Emily Bayer, G. Michael Baer, Christopher Alvaro, Katherine Weber, Ramzy 

Burns, Michael Lilly, Anvi Patel, Benjamin Perlman, Sheila Clever, Bruce 

Wightman 

Muhlenberg College, Allentown, PA, USA 

The NR2E subclass of nuclear receptors is conserved from cnidarians to vertebrates. 

Phylogenetic analysis identifies at least three major clades: the NR2E1/2 clade (nhr-67 and 

tailless), the NR2E3/5 clade (fax-1 and PNR), and a new clade that includes nhr-239 of 

Caenorhabditis, the HR83 nuclear receptors of insects, and an ortholog in non-vertebrate 

deuterostomes. Therefore, the nhr-239 clade appears to have an ancient origin, but has been 

lost from vertebrates. An additional NR2E gene, nhr-111, has a fax-1-like ligand-binding domain 

(LBD), but so far appears only in C. elegans and C. brenneri genomes, indicating that it is a 

relatively recent evolutionary elaboration within Caenorhabditis. We performed swaps among 

the LBDs of NR2E genes, demonstrating that NHR-111 and NHR-67 could function in place 

of the FAX-1 LBD. This prompted us to test a simple deletion of the FAX-1 LBD; it too could 

provide robust function. These experiments suggest that the LBD is not required for at least 

some functions of an evolutionarily-conserved C. elegans NR—and by extension that fax-1 

activity is not necessarily ligand-dependent. 

The ok2526 deletion removes 5’ flanking DNA and the first exon of nhr-239, plus the last 

exon of the neighboring feh-1 gene. We have found that this deletion generates a fusion 

transcript between feh-1 and nhr-239, which probably produces no functional NHR-239 

product. ok2526 homozygotes display a behavioral phenotype that appears to be related to 

that of npr-1; they avoid lawns of OP50 bacteria often congregating a short distance outside 

of the lawn, creating a “halo.” This phenotype is not observed on UV-killed lawns, suggesting 

that it is not a simple physical response to bacteria. nhr-239 is expressed at low levels in two 

to three pairs of head neurons, at least one of which appears to be of a sensory type and is 

located in the vicinity of URX. 

The ok519 deletion of the nhr-111 gene produces no detectable nhr-111 transcript, but 

does not cause an obvious morphological or behavioral phenotype. nhr-111 is a major node in 

high-throughput one-hybrid analysis of neuronal and metabolic promoters, an observation that 

seems surprising for a relatively recent evolutionary elaboration. We have found that nhr-111 

is expressed broadly in most nematode tissues. We are currently testing possible functions 

for nhr-111 in regulation of genes that function in metabolism. 

This work is supported by a grant from the NSF. 

Contact: EB246457@gws1.muhlenberg.edu 

Lab: Wightman 

122 

Poster Topic: Gene Regulation

Redefining POP-1 Binding Sites in C. elegans 

Chandan Bhambhani, Ken Cadigan 


We are deciphering the rules for DNA binding used by POP-1, a member of the T-cell 

factor (TCF) family of transcription factors and a major mediator of Wnt/β-catenin signaling in 

C. elegans. While all TCFs contain a High Mobility Group (HMG) domain that can bind DNA 

specifically, most invertebrate and some vertebrate TCFs also possess another DNA binding 

domain termed the C-Clamp. In Drosophila, the C-Clamp is known to bind motifs known as 

Helper sites, which are found in the vicinity of functional HMG binding sites and are essential 

components of the cis-regulatory Wnt Response Elements (WREs). POP-1 contains both the 

HMG and C-clamp domains, and so to better understand the architectural make-up of worm 

WREs, we are exploring the role of Helper sites in this organism. Potential Helper sites were 

found near the functional HMG sites in the WREs of Wnt targets ceh-22, psa-3 and end-1. We 

find that Helper sites are essential for Wnt induced expression of ceh-22 in the distal tip cells of 

the somatic gonad. In vitro, Helper sites are required for binding of POP-1 to all three WREs, 

and their importance is being tested for psa-3 and end-1 in vivo. Our data thus far indicate a 

bias in the spacing and orientation of functional HMG and Helper sites, which has enabled 

us to identify additional putative WREs in a computational search. Our study highlights the 

importance of Helper sites in defining functional POP-1 binding sites, and a better understanding 

of how POP-1 locates Wnt targets should facilitate further in silico identification of new WREs 

in the genome. 

Contact: bchandan@umich.edu 

Lab: Cadigan 

Poster Topic: Gene Regulation 

123

In vivo Regulation of the Alternative Splicing of the Pro- and Anti- 

Apoptotic Gene ced-4 

Anna Corrionero, Bob Horvitz 

HHMI, Dept. Biology, MIT, Cambridge, MA 02139 USA 

The processing of pre-mRNAs by alternative splicing provides a cell with the ability to 

generate multiple mRNAs from a single gene. Despite many efforts to study this process, 

the regulation of alternative splicing in vivo and in a tissue- or developmental stage-specific 

manner as well as the functional implications of alternative splicing are not well understood. 

The C. elegans CED-4 protein promotes the activation of the caspase CED-3 and is essential 

for canonical programmed cell death (PCD). However, the ced-4 transcript is alternatively 

spliced, giving rise to two different isoforms with antagonistic functions generated by use of 

alternative 3’ splice sites (ss) in exon 4. The main isoform, CED-4S, is pro-apoptotic, while 

CED-4L is anti-apoptotic. ced-4 is the only apoptotic gene known to be alternatively spliced in 

C. elegans. How ced-4 alternative splicing is regulated is largely unknown. 

To study the regulation of ced-4 alternative splicing in vivo we have generated fluorescent 

reporters so that expression of CED-4L will give rise to GFP, while expression of CED-4S will 

give rise to RFP. These reporters showed a higher levels of expression of the ced-4S isoform, 

consistent with the alternative splicing levels observed for the endogenous gene. A deletion 

analysis of the reporters indicated the presence of two sequences important for the regulation 

of ced-4 splicing. One, located upstream of the exon 4L 3’ss, contains possible binding sites 

for the Fox-1 family members FOX-1 and ASD-1 and for the muscle-specific splicing factor 

SUP-12. These proteins might prevent the recognition of exon 4L 3’ss. Deletion of the FOX- 

1/ASD-1 but not of the SUP-12 binding site weakly increased CED-4L expression. However, 

single mutants of fox-1, asd-1 or sup-12 did not modify the ced-4 isoform ratio and did not 

exhibit PCD defects in the anterior pharynx or ventral cord. 

We are now testing the effects of double mutants of these genes as well as of other genes 

involved in alternative splicing, such those that encode the Serine/Arginine-rich proteins 

implicated in cell survival. We are also trying to identify factor/s that bind upstream of exon 

4 using biochemistry. In addition, we are performing a genetic screen for mutants with an 

increased CED-4L signal. 

We hope that by studying ced-4 alternative splicing using our fluorescent reporter system 

we will identify not only factors involved in alternative splicing but also modulators of the 

apoptotic pathway itself. 

Contact: annacs@mit.edu 

Lab: Horvitz 

124 


Identifying HLH-8/Twist Homodimer Target Genes 

Nirupama Singh, Peng Wang, Ann Corsi 

The Catholic University of America, Washington, DC, USA 

The basic helix-loop-helix (bHLH) transcription factor, Twist, plays an important role in 

mesoderm development. The bHLH factors influence transcription by binding to a consensus 

DNA sequence called an E box as either a homodimer or a heterodimer. In C. elegans, the 

Twist homolog HLH-8 is required for a subset of mesodermal development, including M lineage 

patterning and differentiation of vulval and enteric muscles. The only known heterodimeric 

partner for HLH-8 is the more broadly expressed bHLH factor HLH-2/Daughterless protein. 

Previous work in our laboratory has shown that distinct HLH-8-containing dimers function in C. 

elegans development. For example, HLH-8 homodimers play a major role in the undifferentiated 

M lineage cells whereas HLH-8/HLH-2 heterodimers function in differentiated cells derived 

from the M lineage, the vulval muscles. In order to understand the unique functions of these 

dimers in development, we wanted to identify a collection of target genes that are regulated 

by each dimer. To identify target genes of HLH-8 homodimers, we overexpressed the protein 

and looked for genes that were upregulated using Affymetrix Oligonucleotide microarrays. 

We screened though the most promising candidates by making gfp reporters and looking for 

mesodermal expression. This approach led to 5 genes whose expression was coincident with 

HLH-8. These reporters were also examined in hlh-8 mutants to validate that their expression 

depended on HLH-8 and examined in hlh-2 mutants since HLH-8 homodimer candidates 

should not depend on HLH-2 for expression. Based on these criteria, we identified one gene 

that is expressed in vulval muscles and depends on HLH-8 but not HLH-2 for expression. The 

promoter of this gene was examined by making 5’ deletion constructs and by site-directed 

mutagenesis. These approaches led to the identification of a single palindromic E box that is 

responsible for vulval muscle expression and makes the prediction that this site may be found 

in other HLH-8 homodimer target genes. 

Contact: corsi@cua.edu 

Lab: Corsi 


125

Understanding the Role of Overlapping MicroRNA Networks During 

Nematode Development 

Jeanyoung Jo, Kimberly Breving, Kenya Madric, Aurora Esquela-Kerscher 

Eastern Virginia Medical School, Norfolk, Virginia, USA 

Developmental genetics has aided our understanding of the intricate regulatory programs 

that rapidly turn a fertilized egg into a fully-grown organism. MicroRNAs (miRNAs) constitute 

a major class of conserved ~22 nucleotide non-coding RNAs that play pivotal roles in 

developmental gene regulation. They are found to control a wide-range of processes in the 

embryo such as cellular differentiation, proliferation, and apoptosis. However, few physiologically 

relevant and developmentally important miRNA targets have been identified and in silico 

miRNA target predictions are often error-prone and unreliable. Work with the simple but 

genetically powerful Caenorhabditis elegans (C. elegans) nematode worm allows us to screen 

for biologically relevant miRNA-target interactions in vivo that direct essential events during 

development and are potentially conserved in mammalian systems. We have focused on the 

role of the lin-4 and let-7 miRNA families during the formation of the gonad and egg-laying 

structures (vulva) in worms. This study sought to determine if members within a miRNA family 

are biologically distinct due to their unique temporal and spatial expression patterns or if 

they are really functionally different despite possessing identical “miRNA seeds”. Our results 

indicated that 1) closely related miRNAs members of the lin-4 family could not functionally 

compensate for one another using an in vivo lin-4 loss-of-function rescue assay while 2) certain 

members of the let-7 family are functionally redundant with non-homologous lin-4 miRNAs. 

Specifically, our deletion studies revealed that the poorly characterized lin-4 homologue, miR- 

237, and the let-7 family members, miR-48 and miR-84 function in an overlapping network 

likely with chromatin remodeling genes to direct cell cycle progression in the germline as well 

as vulva morphogenesis. We are currently using unbiased RNAi suppressor screens to identify 

physiologically relevant miRNA targets and investigate if these miRNA-target interactions are 

conserved in mammalian cells. This dual system approach will provide novel insights into the 

biological roles of miRNAs during human development and disease. 

Funding: This work is supported by EVMS start-up funds and a grant from the Thomas F. 

and Kate Miller Jeffress Memorial Trust (to A.E-K.). 

Contact: kerschae@evms.edu 

Lab: Esquela-Kerscher 

126 


Intracellular Trafficking and Endocytic Regulation of the DBL-1/BMPlike 

pathway in C. elegans 

Ryan Gleason 1 , Adenrele Akintobi 2 , Ying Li 1 , Barth Grant 2 , Richard Padgett 1 

1 Waksman Institute, Rutgers University, 2 Rutgers University 

Endocytosis has long been known as simply a way to internalize and traffic nutrients and 

membrane associated molecules that cannot pass the plasma membrane of the cell. Recent 

evidence has led to an emerging model where endocytosis orchestrates the topological 

landscape of signal transduction pathways throughout the cell by regulating the availability 

and trafficking (degradation versus recycling) of transmembrane signaling proteins. Using C. 

elegans as a model to study the endocytic trafficking of two TGFβ transmembrane receptors, 

DAF-4 and SMA-6, we have identified various endocytic pathways that regulate receptor 

internalization, availability, and recycling. Interestingly, these two receptors function in the 

same signal transduction pathway, but are recycled disparately. This work was prompted by 

the identification of sma-10, which was discovered through a genetic screen in the Padgett 

Lab, and which we show also affects the trafficking of the type I and type II receptors, SMA-6 

and DAF-4. Experiments were done to distinguish between a role in receptor secretion and/ 

or in internalization of the receptors. We show that sma-10 does not act in secretion of the 

receptors but acts after receptor internalization. Further data will be presented to show how 

TGFβ receptors traffic in C. elegans and what role sma-10 plays in this process. 

Contact: rygleason@me.com 

Lab: Padgett 


127

Identification and characterization of targets of the REF-1 family 

member, HLH-25 

Raymarie Gomez, Han-ting Chou, Casonya Johnson 

Georgia State University 

Proteins in the REF-1 family are distinguished by the presence of two basic helix-loop helix 

domains. HLH-25 is one of six members of this family, and though the gene encoding hlh-25 

is known to be expressed during embryonic development in response to Notch signaling, 

there is no genetic or molecular data about the role of this family member in embryonic or 

post-embryonic development. Recently, our laboratory completed a microarray analysis of 

gene expression changes in hlh-25 mutants compared to wild-type animals. This analysis 

has uncovered putative roles for HLH-25 in regulating genes required for cell division and cell 

cycle progression. The objective of the research presented here is to further characterize the 

role of HLH-25 in these processes. My aims are first to validate twenty HLH-25 target genes 

by monitoring changes in their expression in wild-type and hlh-25 mutant animals. Here, I 

report on my efforts to generate transcriptional fusions for four of the targets and to isolate 

and characterize transgenic lines for each reporter construct. 

Contact: rgomezvazquez1@student.gsu.edu 

Lab: Johnson 

128 


The Mediator Subunit CDK-8 Negatively Regulates EGFR-Ras-MAPK 

in Vulva Development 

Jennifer Grants, Stefan Taubert 

University of British Columbia, Vancouver, Canada 

The Mediator complex is a conserved coregulator of eukaryotic transcription. Certain 

Mediator subunits are required for transcription of all protein coding genes, whereas others 

regulate specific gene programs. Cyclin dependent kinase 8 (CDK-8) is one Mediator subunit 

that exerts gene-specific regulation. To identify genes and programs regulated by CDK-8, we 

performed microarray analysis comparing cdk-8 null mutants and wild-type worms. Intriguingly, 

CDK-8 regulates gene targets in common with LIN-35/pRB, a regulator of C. elegans vulva 

development that negatively affects signaling through an EGFR-Ras-MAPK pathway. We found 

that, like LIN-35, CDK-8 also regulates vulva development, as cdk-8 null mutants display a 

low-penetrance multivulva (Muv) phenotype. Furthermore, null mutation of a negative regulator 

of the EGFR-Ras-MAPK pathway enhances the Muv phenotype of cdk-8 mutants. Thus, CDK- 

8 controls vulva development via negative regulation of an EGFR-Ras-MAPK pathway. As 

another Mediator subunit, SUR-2, is a critical positive regulator of transcription downstream 

of the EGFR-Ras-MAPK pathway, I will explore the possibility that antagonism between the 

SUR-2 and CDK-8 Mediator subunits may fine-tune the output of an important developmental 

signaling cascade. 

Contact: jgrants@cmmt.ubc.ca 

Lab: Taubert 


129

A Lipid-Binding Protein that Modifies cGMP Signaling is Required for 

Host Odor Sensing and Body Morphology in Pristionchus pacificus 

Ray Hong, Jessica Cinkornpumin, Dona Roonalika Wisidagama, Veronika 

Rapoport 

California State University Northridge, Northridge (CA) 

In Pristionchus pacificus, the cGMP dependent protein kinase Ppa-EGL-4 is an important 

genetic modifier of insect pheromone reception. Upregulation of Ppa-egl-4 transcripts depends 

on intracellular cGMP levels and is highly variable among wild isolates from diverse host 

ranges. In particular, chemoattraction to the Oriental beetle sex pheromone is very strong in 

the Washington isolate (PS1843) but is elicited in the reference isolate California (PS312) only 

after a brief exogenous cGMP treatment. To obtain additional cGMP signaling factors involved in 

insect pheromone attraction, we performed forward genetic screens in P. pacificus for mutants 

that do not show attraction toward the oriental beetle pheromone (Z-7-tetradecen-2-one). 

Out of the four Oriental Beetle pheromone Insensitive mutants (obi’s) that we have isolated, 

the obi-1 allele showed the strongest chemosensory phenotype specific for lack of attraction 

to ZTDO. In addition to chemosensation, obi-1 acts upstream and parallel to Ppa-egl-4 in 

regulating pharyngeal pumping and locomotion. We identified the molecular lesion of obi-1 and 

found a nonsense mutation in an uncharacterized ORF encoding a protein with a lipid-binding 

motif. The transcriptional reporter shows obi-1p::gfp expression in specialized hypodermal cells, 

including the seam and putative duct cells. More importantly, obi-1p::gfp is also expressed in 

the amphid sheath cells, the glial cells responsible for forming enclosed compartments around 

the chemosensory amphid neurons. We further analyzed the obi-1 ortholog expression in C. 

elegans and found overlapping expression patterns with obi-1 in P. pacificus, but lacking in 

amphid sheath expression. We hypothesize that OBI-1 proteins are secreted and interacting 

with chemosensory neurons in the fluid-filled amphid compartments to modulate cGMP 

signaling during chemosensation. OBI-1 belongs to a small, mostly uncharacterized, but highly 

conserved group of lipid-binding proteins found in both C. elegans and P. pacificus as well as 

most metazoans. 

Contact: ray.hong@csun.edu 

Lab: Hong 

130 


Elucidating The Role of Genetic Redundancy In The Wnt Signaling 

Pathway In Regulating Q Neuroblast Migration 

Ni Ji1 , Teije Middelkoop2 , Hendrik Korswagen2 , Alexander van Oudenaarden1 1 2 Massachusetts Institute of Technology, Cambridge (MA), USA, Hubrecht 

Institute, Utrecht (Utrecht), The Netherlands 

The intriguing observation that functionally redundant genes are highly conserved despite 

little selective pressure has puzzled biologists for a long time. To explore the emergent function 

of genetic redundancy, we used the C. elegans Qneuroblasts to examine how multiple Wnt 

receptors function together to regulate mab-5/Hox, a key Wnt target gene necessary and 

sufficient to drive posterior migration. To identify the contribution of each receptor to mab- 

5activation, we used single molecule Fluorescent In Situ Hybridization(smFISH) to profile the 

expression of Wnt receptor and target genes in a series of wild-type and Wnt signaling mutant 

strains. Specifically, we found three Frizzled-type receptors (mig-1/Fz, lin-17/Fz, and mom-5/ 

Fz) to be specifically expressed in QL, two of which (mig-1 and lin-17) exhibited strong but 

opposite correlation with mab-5. In single and compound Frizzledmutants, we found mab-5 

expression to be progressively reduced and increasingly variable. The variability in mab-5 

expression correlated strongly with the partially penetrant migration phenotype, consistent with 

our recent report (Raj et al., 2010). Surprisingly, we found that mutations in either the Frizzled 

receptors, mab-5 or the Wnt ligand egl-20, can induce changes in the expression of the (nonmutated) 

receptors. Applying a mathematical inference algorithm to our gene expression data, 

we arrived at amost-probable network topology that features two negative and one positive 

egl-20-dependent feedback loops targeting, respectively, mig-1, mom-5 and lin-17. Network 

modeling predicts a division of labor among the three receptors between the initialization 

and maintenance phase of mab-5 activation. In an ongoing effort, we express Q cell specific 

Frizzled transgenes to assess downstream consequences when feedback regulation on the 

receptors is perturbed. 

Reference: Raj A etal. (2010) Variability in gene expression underlies incomplete penetrance. 

Nature 463,913: 913-918. 

Contact: seaurchinjinni@gmail.com 

Lab: van Oudenaarden 


131

Can the Rate of Transcription be Quantitatively Determined in Relation 

to Transcription Factor Binding Affinity? 

Brett Lancaster, James McGhee 

University of Calgary, Calgary, Alberta, Canada 

Transcription factors (TFs) are proteins that bind to DNA, usually at a sequence motif

Regulated Splicing of the Cholinergic Gene Locus 

Ellie Mathews, Greg Mullen, Jim Rand 

Oklahoma Medical Research Foundation, Oklahoma City, OK, USA 

Acetylcholine (ACh) is a major neurotransmitter in both vertebrate and invertebrate nervous 

systems. A single conserved locus encodes both the ACh biosynthetic enzyme (ChAT; cha- 

1) and the vesicular ACh transporter (VAChT; unc-17) proteins. The VAChT coding region is 

contained within the first intron of the cha-1 gene, and alternative splicing gives rise to separate 

ChAT and VAChT transcripts. We identified two sets of inverted repeat sequences (designated 

R1 and R2) in the non-coding sequences flanking the unc-17 coding region, which form the 

basis for a model of “hairpin-mediated” alternative splicing of unc-17 and cha-1 transcripts. To 

test the role(s) of these inverted repeat sequences, we engineered a dual reporter in which 

the unc-17 coding region was replaced with green fluorescent protein (GFP) and the cha-1 

coding region with a red florescent protein (“wCherry”). The resulting construct contains all of 

the conserved non-coding sequences in the regions flanking unc-17 and cha-1, and transgenic 

animals containing this construct correctly express both GFP and wCherry in the appropriate 

neurons. We have shown that both R1 and R2 repeats are necessary for normal expression of 

wCherry (cha-1). We found that the R1 hairpin structure, but not the precise R1 sequence, was 

important for downstream splicing. In contrast, both the R2 sequence and the hairpin structure 

were important for splicing. Analysis of unc-17 deletions (p1156 and md1447) identified a cryptic 

R1-pairing sequence, designated R’. We believe that R1/R1’ pairing is sufficient for adequate 

splicing of cha-1 mRNA, and we are currently testing this assumption. We have also analyzed 

the cha-1 - unc-17 genomic regions from five Caenorhabditis species. There is considerable 

species-to-species variation in the sequences, yet in each case, the R1 sequences from each 

species are much better matches to each other than they are to any other species. We also 

found structural counterparts of the R2 sequences in the Drosophila genome, and counterparts 

of the R1 sequences in the mouse and rat genomes. We therefore believe that such “hairpinmediated” 

alternative splicing of VAChT and ChAT transcripts may be a general feature of 

cholinergic regulation. Supported by NIH grant R21 NS072923. 

Contact: mathewse@omrf.org 

Lab: Rand 


133

Short Capped RNAs and Nuclear Run-On Reveal Pol II Pausing and 

Backtracking in C. elegans 

Colin Maxwell 1 , William Kruesi 2 , Nicole Kurhanewicz 5 , Leighton Core 3 , Colin 

Waters 3 , Igor Antoshechkin 4 , John Lis 3 , Barbara Meyer 2 , L. Ryan Baugh 1 

1 Duke University, Durham (NC), USA, 2 University of California at Berkeley, 

Berkeley (CA), USA, 3 Cornell University, Ithaca (NY), USA, 4 California 

Institute of Technology, Pasadena (CA), USA, 5 University of North Carolina, 

Chapel Hill (NC), USA 

When C. elegans larvae hatch in the absence of food they enter a state of development 

arrest (L1 arrest). Starved larvae respond rapidly to feeding, quickly initiating growth and postembryonic 

development. We used ChIP-seq to show that Pol II is poised at the 5’ ends of growth 

and development genes during L1 arrest (Baugh et al, Science 2009). Pol II binding patterns 

suggest that elongation is under nutritional control, with the polymerase increasing elongation 

at many genes during immediate recovery from arrest. mRNA abundance also increases 

disproportionately for these genes. However, from these experiments it is impossible to know 

the post-recruitment point of regulation; ie, pre-initiation or elongation complex. We therefore 

used an RNA-seq protocol specific for short, capped RNAs to determine if we could detect the 

5’ end of nascent transcripts as hallmarks of a paused elongation complex. We conducted a 

series of controls to confirm the specificity of our protocol, and we analyzed Drosophila S2 cells 

to compare with published results. We complemented this approach with global nuclear run-on 

(GRO-seq). Our analysis also benefits from mRNA-seq data for mature transcripts during L1 

arrest and recovery as well as published Pol II ChIP-seq. We detect short, capped RNAs from 

the 5’ end of approximately 2,000 protein coding genes in L1 larvae. Pol II accumulation is also 

observed in the promoter-proximal region of these genes, and GRO-seq confirms that Pol II is 

paused on these genes. 3’ sequencing reveals a short, capped RNA size distribution similar 

to what has been reported for Drosophila and mammals. Analysis of a TFIIS mutant alters the 

size distribution, consistent with a model that involves TFIIS- facilitated cleavage of the 3’ end 

of the nascent transcript and backtracking to relieve pausing. Our results clearly demonstrate 

that Pol II pausing occurs in C. elegans larvae and that it is mechanistically related to pausing 

in other systems. This is particularly noteworthy in light of the absence of NELF homologs and 

the presence of 5’ trans-splicing in the worm. 

Contact: cs.maxwell@gmail.com 

Lab: Baugh 

134 


The mRNA Splicing Regulator SPK-1 Is Required for Cell Polarity in 

One-Cell C. elegans Embryos 

Martin Mikl, Carrie Cowan 

IMP Vienna, Austria 

Cell polarity is a prerequisite for asymmetric division, which gives rise to daughter cells with 

different developmental fates. In one-cell C. elegans embryos, cell polarity comprises distinct 

cortical domains of actomyosin contractility and PAR proteins. Polarity establishment is initiated 

by a centrosome-dependent cue. In an RNAi screen for polarity establishment defects we 

identified one gene, spk-1, that appeared to have normal centrosome assembly and only mildly 

reduced cortical activity but nonetheless failed to establish correct PAR protein localization. 

Consistent with defects in PAR polarity, spk-1(RNAi) embryos divided symmetrically to give 

rise to equivalent daughter cells. 

SPK-1 is a kinase targeting the SR protein family of mRNA splicing factors and thereby 

potentially influences splice site selection. A transcriptome-wide analysis of RNA from WT 

and spk-1(RNAi) worms by Illumina sequencing did not show a general splicing defect, 

but misregulation of transcript levels in a small number of genes. By analyzing splice form 

abundance of candidate polarity mediators in SPK-1 depleted worms, we identified a splicing 

change in the par-5 3’UTR that leads to a reduction in PAR-5 protein levels in the embryo. 

par-5(RNAi) embryos showed similar polarity establishment defects to spk-1(RNAi) embryos, 

namely a reduced and instable PAR-2 domain. Thus SPK-1 may facilitate polarity establishment 

by regulating PAR-5, which in turn controls PAR-2 availability and maintenance at the cortex. 

Contact: martin.mikl@imp.ac.at 

Lab: Cowan 


135

The Transcriptional Repressor Protein CTBP-1 Regulates the 

Differentiation of DA Motor Neurons 

Hannah Nicholas1 , Duygu Yucel1 , Estelle Llamosas1 , Anna Reid1 , Aaron Lun1 , 

Sashi Kant1 , Merlin Crossley2 1 2 University of Sydney, University of New South Wales, Sydney, NSW, 

Australia 

The C-terminal binding proteins are a group of transcriptional co-repressor proteins that 

are conserved throughout the animal kingdom. Nematodes have a single CtBP gene, called 

CTBP-1, which we have found to be predominantly expressed in the nervous system of C. 

elegans. In ctbp-1 mutants, motor neurons of the DA class fail to express the important marker of 

terminal differentiation unc-4. This is of interest since mutation of zag-1, which encodes a critical 

neuronal transcription factor, leads to a similar defect in DA motor neurons. We discovered 

through yeast two-hybrid screens that ZAG-1 physically interacts with CTBP-1. These results 

suggest that ZAG-1 regulates neuronal gene expression at least in part by recruiting CTBP-1 

as an essential co-repressor in vivo. 

Mammalian members of the CtBP family are recruited to promoters through interactions 

with DNA-bound transcription factors that contain amino acid motifs of the form PXDLS, and 

CTBP-1 is similarly able to interact with PXDLS-containing transcription factors, such as ZAG- 

1. Interestingly, we have found that the C. elegans CTBP-1 protein also contains intrinsic DNA 

binding capacity the form of a THAP domain. Following in vitro site-selection experiments, we 

have used the CisOrtho program1 to identify promoters that contain putative CTBP-1-THAP 

binding sites, representing candidate CTBP-1 target genes. With reference to both our own 

and published2 microarray datasets comparing transcripts from wild type animals with those 

from ctbp-1 mutants, and to expression pattern data, we have defined a sub-set of these as 

likely in vivo targets of CTBP-1-mediated repression. 

Given the reported role of CTBP-1 in the regulation of lifespan and stress resistance2 , our 

identification of CTBP-1 target genes will make an important contribution to understanding the 

function of this transcriptional regulator in a range of contexts. 

1. Bigelow HR, Wenick AS, Wong A, Hobert O. 2004. BMC Bioinformatics 5: 27 

2. Chen S, Whetstine JR, Ghosh S, Hanover JA, Gali RR, et al. 2009. Proc Natl Acad Sci 

U S A 106: 1496-501 

Contact: hannah.nicholas@sydney.edu.au 

Lab: Nicholas 

136 


The Role of C. elegans bHLH-29 Transcription Factor in Stress 

Response 

Thanh Quach, Casonya Johnson 

Georgia State University, Atlanta, GA, USA 

Iron is an essential element that involves in many biological processes such as oxygen 

transport, electron transport, mitochondrial energy production, DNA synthesis, and heme 

synthesis. In excess, iron can become toxic due to free radicals generation when reacting with 

oxygen through Fenton reaction. Thus, ferritin plays an important role in iron homeostasis in 

both prokaryotes and eukaryotes. The basic helix-loop-helix transcription factor, HLH-29, a 

member of the REF-1 family, is found to affect the expression of the ferritin genes ftn-1 and ftn- 

2. Our focus is to determine the role of HLH-29 transcription factor in the iron homeostasis and 

stress response through the regulation of ftn-1. We are also interested in how ftn-1 is regulated 

by HLH-29. We have confirmed with RT-qPCR that the ftn-1 is affected by hlh-29 knockout. To 

determine if excess iron would cause toxicity and thereby shortens the C. elegans life span, 

we exposed the worms to ferric ammonium citrate. However, no change in the life span was 

observed. We have also tried to depleted the iron with a metal chelator, 2,2’-dipyridyl, and 

observed if the development was affected. Our preliminary result was inconclusive. 

Contact: tquach1@student.gsu.edu 

Lab: Johnson 


137

Loss of the ubiquitin-specific protease usp-48 allows for direct 

conversion of a somatic tissue into neurons in Caenorhabditis 


Dylan Rahe, Tulsi Patel, Oliver Hobert 

Columbia University 

Multicellular organisms begin as a single cell, executing an intricate choreography of 

genetic and epigenetic regulation to eventually become a complete organism. As pluripotent 

cells divide and specialize, their potential to become different cell types becomes increasingly 

restricted. The conversion of one cell fate to another, either in a normal context (development) 

or in an artificial environment (cellular reprogramming), is dependent on cellular context and 

involves both genetic and epigenetic changes. Fate-specifying transcription factors are not 

only regulated by proper spatiotemporal expression, but also negative cues which are thought 

to be mediated at the chromatin level. This has been evidenced by the limited and contextdependent 

ability for key fate-specifying transcription factors such as MyoD to induce specific 

cell fates in other cell types. To understand this context dependency and approach a mechanistic 

understanding of these negative cues during development, we use an assay in which a single 

terminal-fate-specifying transcription factor, CHE-1, is overexpressed by heatshock in late- 

or post-developmental stages. In wild-type animals, very few cells adopt CHE-1-dependent 

fates upon ectopic induction, consistent with previous data; however, we have recently shown 

that the absence of lin-53, a component of many chromatin-remodeling complexes, results 

in efficient and direct conversion of mitotic germ cells to a CHE-1-dependent fate. To extend 

these studies, we undertook a forward genetic screen for mutants in which direct conversion of 

tissues is observed upon ubiquitous CHE-1 induction. In one such mutant, ot674, we observe 

the expression of CHE-1-dependent fate markers in hypodermal tissue of all developmental 

stages. ot674 is an early stop in the gene usp-48. usp-48 encodes a ubiquitin-specific protease, 

which shares homology with the human gene USP48 and the yeast UBP15, all of which have 

no known function. Here we present initial characterization of the usp-48 mutant and the 

reprogramming phenotype it exhibits. 

Contact: dpr2113@columbia.edu 

Lab: Hobert 

138 


Chromatin Structure and Genome Stability in C. elegans 

Valerie Robert1 , Cedric Rakotomalala1 , Cecile Bedet1 , Florence Couteau2 , 

Monique Zetka2 , Francesca Palladino1 1 2 CNRS UMR5239, ENS-Lyon, Lyon, France, Departement of Biology, McGill 

University, Montreal, Canada 

Methylation of histone H3 lysine 4 (H3K4me), a mark associated with gene activation, is 

mediated by SET1 and the related mixed lineage leukemia (MLL) histone methyltransferases 

(HMTs) across species. Caenorhabditis elegans contains one SET1 protein, SET-2, and one 

MLL-like protein, SET-16. 

In a previous study we demonstrated that SET-2 is required for both di-and tri-methyation 

of H3K4 in the germline. Loss of SET-2 results in progressive sterility over several generations, 

suggesting an important function for H3K4 methylation in the maintenance of a functional 

germ line. 

In this poster, we will present data from genetics and irradiation assays showing that SET-2 

is essential for genome stability and might be involved in double-strand break repair (DSBR). 

To further investigate a putative link between chromatin structure and DSBR in the C. 

elegans germline, we examined the IR sensitivity of strains affected for two additional chromatin 

marks, H3K9 or H3K36 methylation. Interestingly, preliminary results indicate that the H3K36 

methyltransferase MET-1 may be required for DSBR. 

Contact: valerie.robert@ens-lyon.fr 

Lab: Palladino 


139

A New Attempt to Elicit an RNAi Phenotype with the LIMhomeodomain 

Transcription Factor LIM-7 

Laura Vallier, John Coppola 

Hofstra University 

The LIM-homeodomain family of transcription factors are required for a multitude of 

developmental processes and family members have been noted to contribute in T-cell leukemia 

and breast cancer. Proteins within this family are expressed in C. elegans in neuronal tissues 

and in the gonadal sheath and other somatic non-neuronal tissue types. The LIM-7 LIMhomeodomain 

protein is an Islet ortholog and is expressed in numerous neurons, the gonadal 

sheath, as well as other cells that are probably muscular in origin. An in-frame deletion of the 

lim-7 gene results in L1 larval lethality; prior to death mutant animals exhibit pleiotropy: they are 

uncoordinated and ~50% have unattached pharynges as the two most prevalent phenotypes. 

Rescue of the deletion mutation using extragenic arrays results in sterile hermaphrodites with 

a few non-viable embryos and loose sperm in the body cavity. Despite a lethal phenotype, 

numerous attempts to elicit a double-stranded RNA (dsRNA) interference phenotype have 

not resulted in robust differences from controls. Recently, a new sensitized background was 

developed to assist in the transport of dsRNA into neuronal tissues, which are notoriously 

resistant to the effect of dsRNA interference. In this system, the SID-1 transmembrane protein, 

which is essential for systemic RNAi (Winston et al 2002), is placed under the pan-neuronal 

promoter unc-119 (Punc-119sid-1) to allow increased transport of dsRNA into the neurons (Calixto 

et al 2010). Since lim-7 is expressed in many neurons we hypothesized that the lack of RNAi 

phenotype may lie in failure to allow dsRNA to enter neuronal cells efficiently. Using the sid-1 

technology we are testing various aspects of the lim-7 phenotype via dsRNAi to ascertain if 

lack of a phenotype using dsRNAi in previous attempts was due to the expression of LIM-7 

within neurons. Early results indicate a slower growth in Punc-119sid-1 animals treated with lim-7 

dsRNAi than in the controls. 

Contact: biolgv@hofstra.edu 

Lab: Vallier 

140 


The Histone Demethylase UTX-1 Is Essential for Normal Development, 

Independently of Its Enzymatic Activity 

Julien Vandamme, Lisa Salcini 

BRIC - University of Copenhagen, Denmark 

Eukaryotic DNA is packaged within the nucleus through its association with histone proteins 

to form chromatin. Histones are subject to a wide variety of post-translational modifications, 

these epigenetic modifications influence gene expression and provide a unique mechanism 

for fine-tuning cellular differentiation and development in multicellular organisms. In particular, 

histone lysine methylation (mono-, di- and tri- states) is a dynamic epigenetic mark, playing 

fundamental roles in chromatin organization and function. Proteins of the Jumonji family are 

able to demethylate lysines via their JmjC domain and are conserved from yeast to humans. 

Here we report on the biological functions of the JmjC-containing protein UTX-1, the C. elegans 

homologue of mammalian UTX, a histone demethylase specific for di- and tri-methylated lysine 

27 of histone H3 (H3K27me2/3). We demonstrate that utx-1 is an essential gene that is required 

for correct embryonic and postembryonic development. Consistent with its homology to UTX, 

UTX-1 regulates global levels of H3K27me2/3. Surprisingly, we found that the catalytic activity 

is not required for the developmental function of this protein. Biochemical analysis identified 

UTX-1 as a component of a complex that includes: SET-16 (a histone methyltransferase for 

H3K4), PIS-1, F21H12.1, ASH-2 and WDR-5. This complex is identical to the one described 

in mammals. Genetic analysis indicates that the defects associated with loss of UTX-1 are 

likely mediated by compromised SET-16/UTX-1 complex activity. Taken together, these 

results demonstrate that UTX-1 is required for many aspects of nematode development but, 

unexpectedly, this function is independent of its enzymatic activity. 

Contact: julien.vandamme@bric.ku.dk 

Lab: Salcini 


141

A Conserved SBP-1/Phosphatidylcholine Feedback Circuit Regulates 

Lipogenesis in Metazoans 

Amy Walker3 , Rene Jacobs4 , Jenny Watts1 , Veerle Rottiers2 , Lorissa Niebergall3 , 

Anders Naar2 1 2 Washington State University, Pullman, WA, Harvard Medical School, 

Boston, 3UMASS Medical School Worcester, MA USA, 4University of Alberta, 

Edmonton, Canada 

Transcriptional regulation may be affected by developmental cues, signal transduction or 

stress response pathways. A growing body of evidence also suggests that some transcription 

factors are affected by metabolic cues to coordinate gene expression with nutrient availability. 

The SREBP family of transcription factors regulate genes for generating fatty acids and 

phospholipid in metazoans and cholesterol synthesis in vertebrates. These factors are inhibited 

by cholesterol in vertebrates, however the regulatory feedback mechanisms for lipogenic 

genes have been less clear. Using C. elegans and mammalian models, we have found that 

SBP-1 and mammalian SREBP-1 control the expression of genes in the 1-carbon cycle, which 

produces methyl groups necessary for protein and phospholipid methylation. In addition, lack 

of phospholipid methylation initiates a regulatory cascade that results in increased SBP-1/ 

SREBP-1 activity. Up-regulation of SBP-1/SREBP-1 activity when methylation capacity or 

phospholipid synthesis is diminished results in increased lipogenesis and may be relevant to 

the development of fatty liver in mammals. Finally, our discovery that SREBP transcription 

factors are linked to metabolic pathways controlling methylation opens additional avenues in 

understanding how gene regulation is linked to metabolic control. 

Contact: amy.walker@umassmed.edu 

Lab: Walker 

142 


HLH-29, REF-1 family protein functions in the spermatheca 

Ana White, Casonya Johnson 

GSU 

HLH-29, a member of the REF-1 family of transcription factors (TFs), is a basic helix-loophelix 

(bHLH) protein that contains two bHLH domains, domain A and domain B. HLH-29 is 

expressed throughout development and life of the animal. Loss of hlh-29 and its paralog hlh-28 

produces various phenotypes which includes sever ovulation defects. Genetic data indicates 

that hlh-29 functions in the distal spermatheca valve and in the spermatheca-uterine valve in 

the IP3 signaling pathway to regulate ovulation. 

bHLH TFs are known to form homodimers and heterodimers. In order to find binding 

partners for HLH-29 our lab has performed a yeast-two-hybrid screening using the C. elegans 

transcription factor library. Results identified FKH-6 as a protein that interacts with HLH-29 

domain A. Far-western and pull-down assays confirmed HLH-29/FKH-6 interactions. 

Comparing expression profiles of each reporter gene, HLH-29::gfp was found to be 

expressed in the developing spermatheca of L4 stage animals and in the adult animals. 

Whereas FKH-6 is expressed in precursors of the somatic gonad, the Z1/Z4 cells during the 

L1 stage, and then expressed again in the spermatheca of early L3 to adult stage animals. 

These profiles suggest that HLH-29 interacts with FKH-6 in the L4 or in the adult animals. We 

propose that they regulate genes required in the L4 stage for spermatheca development or at 

a later stage for proper ovulation and fertilization. 

To further establish molecular interactions in vitro and in vivo our goals are 1) identify 

downstream targets of HLH-29/FKH-6 using electrophoresis mobility shift assays (EMSA) and 

RT-qPCR; 2) to correlate morphological changes in single and double mutant animals. One 

candidate gene is a nuclear hormone receptor NR4A human homolog of C.elegans nhr-6 which 

causes similar loss of function phenotypes as hlh-29 mutants, including ovulation defects, 

abnormal egg morphology, oocyte fragmentation, emo (endomitoticoocytes), and spermathecal 

exit and entrance defects. Our previous data also suggest that nhr-6 transcriptional activity is 

affected by the loss of HLH-29. More recently we identified regulatory binding sequences for 

FKH-6 and HLH-29 in both the promoter region and the regulatory sequences within intron 5. 

Additionally, our preliminary EMSA show that FKH-6 can bind to the regulatory region of nhr-6, 

and that HLH-29 can supershift this complex. Finally, our morphological studies suggest that, 

unlike FKH-6, HLH-29 does not affect morphology of the somatic gonad. 

Contact: ana.white@att.net 

Lab: Johnson 


143

Promoter analysis of the GATA type transcription factor ELT-2 

Tobias Wiesenfahrt, Jannette Berg, James McGhee 

University of Calgary, University of Calgary 

It has been suggested that the GATA type transcription factor ELT-2 is the major regulator 

of transcription in the C. elegans intestine after endoderm specification, both embryonically and 

post embryonically. Rothman and Maduro have shown that the redundant GATA factors END-1 

and END-3 are necessary for endoderm specification. Ectopic expression of END-1 or END-3 

can initiate ectopic expression of ELT-2, suggesting that END-1 and END-3 can activate elt-2 

expression in the earliest endoderm lineage. Previous experiments also showed that ELT-2 can 

bind to its own promoter in vivo. To understand the molecular details of how elt-2 transcription 

is initiated during embryonic development and is maintained thereafter, we are analyzing the 

promoter region of elt-2 in C. elegans. Comparison of 5 kb upstream sequences of the elt-2 

gene from 4 different Caenorhabditis species revealed three conserved regions (CRI-CRIII). 

Deletion series as well as analysis of reporter constructs containing different combinations of 

the CRs suggested that CRI contains the basal promoter and CRIII contains the main enhancer 

of elt-2. The function of CRII is not yet clear. To find potential binding sites for END-1, END-3 

and ELT-2, we searched for GATA sites within the CRs of the 5kb upstream region of elt-2. 

We identified 3, 3 and 4 conserved GATA sites within CRI, CRII and CRIII respectively. Band 

shift assays showed that END-1 and ELT-2 can bind to at least one and all four GATA sites 

within CRIII in vitro respectively. This suggests that END-1 (END-3 has not yet been tested) 

can activate elt-2 expression directly. Mutating the GATA sites within CRIII individually and 

in different combinations, suggested that every CRIII GATA site contributes positively to elt-2 

expression. Reporter expression was absent after mutating all GATA sites within CRIII and CRI, 

suggesting that elt-2 regulation is exclusively dependent on GATA factors. To test if ELT-2 can 

drive intestinal specification and differentiation in the absence of END-1/END-3, we expressed 

elt-2 under control of the end-1 and end-3 promoters in the end-1/end-3 double mutant (kindly 

provided by Morris Maduro). Indeed, the end-1p::elt-2 construct is able to rescue the end-1/ 

end-3 double mutant with reasonable penetrance, showing that the endoderm differentiation 

factor ELT-2 can drive endoderm specification and further supporting the hypothesis that ELT-2 

is involved in the regulation of every gene expressed in the intestine. 

Contact: tobias2278@gmx.de 

Lab: McGhee 

144 


Genetic Screen for Novel Repair Genes Implicated in UV-induced DNA 

Damage Response 

Stefanie Wolters, Bjoern Schumacher 

CECAD at the institut of genetics, Cologne, Germany 

Ultraviolet radiation of the sunlight represents an environmental carcinogen, which can lead 

to helix-distorting lesions in the genome and therefore cause cancer. To cope with this damaging 

influence cells developed a multistep “cut and patch” mechanism to repair the impaired DNA. 

The Nucleotide Excision Repair (NER) removes helix distorting lesions and is divided into two 

branches, which differ in damage recognition: while initiation factors of Global-Genome-NER 

(GG-NER) scan the whole genome for damage, Transcription Coupled Repair (TCR) initiates 

repair when RNA polymerase II encounters a lesion during transcription. 

The NER pathway is highly conserved between mammals and C. elegans. We identified 

UV hypersensitivity phenotypes of known C. elegans NER mutants distinct for GG-NER und 

TCR. While mutations in TCR lead to developmental arrest at L1 larval stage, defects in GG- 

NER lead to germ line arrest and sterility upon UV radiation of L1 larvae. We took advantage 

of these phenotypes to design a random mutagenesis based screening strategy. 

By performing this screening method we identified five different mutants that are 

hypersensitive to UV light. The respective mutations were mapped to the C. elegans genome 

via SNP mapping technique and whole genome sequencing was performed in order to identify 

the impaired genes. Non-complementation analysis revealed that mutations in structural 

maintenance of chromosomes-5 (smc-5) lead to the UV-sensitivity phenotype in two of the five 

mutants. Previous studies, which were performed mainly in yeast, showed that smc-5 plays a 

role in homologous recombination repair and might be necessary for DNA structure as well. In 

this work we analyze the role of smc-5 in repair of UV-B induced DNA damage and its interplay 

with NER by genetic analysis, biochemistry and immunohistochemistry. 

Contact: wolterss@uni-koeln.de 



145

The eIF4E-binding protein IFET-1 is a broad-scale translational 

repressor and is required for normal P granule ultrastructure 

Madhu Sengupta1 , Lloyd Low1 , Joseph Patterson2 , Traude Beilharz1 , Jennifer 

Schisa2 , Peter Boag1 1 2 Monash University, Melbourne, Victoria, Australia, Central Michigan 

University, Mount Pleasant, MI 

P granules are large cytoplasmic ribonucleoprotein complexes that associate with the 

nuclear pore complexes on developing germ cells. We have identified that the C. elegans 

homologue of the eIF4E-transporter, IFET-1, is required for normal P granule formation and 

translational regulation of many germ cell mRNAs. Ultrastructural analysis using Transmission 

electron microscopy (TEM) of ifet-1 null animals indicates that the electron dense “crest” and 

“base” of P granules are abnormally formed, suggesting that RNA may not be concentrated 

in the granule normally. In addition, although P granules are similar in size to wild-type, there 

is a significant reduction in the number of nuclear pores associated with P granules in ifet-1 

null animals. In the absence of IFET-1, the P granule components CGH-1 and CAR-1 fail to 

localise normally and instead are diffusely spread throughout the cytoplasm. When grown at 

25oC, ~25% ifet-1 null animals have a masculinised gonad containing only sperm, and this 

phenotype is enhanced when the general translational inhibitors CGH-1, CAR-1 or PATR-1 

are knocked down by RNAi. We also found that IFET-1 is required for translational repression 

in the distal gonad for 7 out 7 GFP-tagged germline reporter constructs tested. Orthologues of 

IFET-1, CGH-1, CAR-1 and PATR-1, play a crucial role in decapping-mediated mRNA decay in 

yeast and mammalian cells, where they repress translation as a first committed step towards 

mRNA decay. We propose that IFET-1, CGH-1, CAR-1 and PATR-1 function collectively as 

broad-scale translational inhibitors that repress some maternal mRNAs by forming a repressive 

complex on mRNA in P granules. 

Contact: peter.boag@monash.edu 

Lab: Boag 

146 

Poster Topic: Germline

Spindle assembly checkpoint proteins monitor synapsis during 

meiosis in C. elegans 

Tisha Bohr, Piero Lamelza, Needhi Bhalla 

University California Santa Cruz, Santa Cruz (CA), USA 

In order to achieve proper meiotic chromosome segregation, homologous chromosomes 

must pair and synapse in prophase I to promote crossover recombination. Improper chromosome 

segregation can lead to aneuploidy, which is associated with miscarriages, birth defects and 

tumorigenesis. Cell cycle checkpoints ensure accurate chromosome segregation by monitoring 

key events during cell division. In C. elegans, the synapsis checkpoint monitors synapsis of 

homologous chromosomes and triggers cell death in the event of asynapsis. The synapsis 

checkpoint requires cis-acting sites near the end of each chromosome, termed Pairing Centers 

(PCs), for activation. PCs promote pairing and synapsis by establishing transient connections 

with the cytoplasmic microtubule network via attachment to the nuclear envelope, but how 

they activate the synapsis checkpoint is currently unknown. The mitotic spindle assembly 

checkpoint also uses cis-acting sites, centromeres, as platforms for checkpoint activation to 

monitor microtubule attachments and tension at kinetochores. I will show that components 

of the spindle assembly checkpoint are also required for the synapsis checkpoint during C. 

elegans meiosis. Moreover, two components, Mad2 and Zwilch, localize to prophase I nuclei 

of the germline. These data support a model in which spindle assembly checkpoint proteins 

localize to pairing centers to monitor tension between homologues and/or attachment to 

microtubules in order to satisfy the synapsis checkpoint. These and future experiments will 

not only dissect how the synapsis checkpoint works in C. elegans but will also provide insight 

into conserved mechanisms that monitor chromosome behavior during cell division to maintain 

genomic integrity. 

Contact: tbohr@ucsc.edu 

Lab: Bhalla 

Poster Topic: Germline 

147

A global genomic survey of genes that mediate LKB1/PAR-4dependent 

germline stem cell quiescence in C. elegans 

Rita Chaouni, Richard Roy 

Department of Biology, DBRI, McGill University, Montreal, Quebec, Canada 

Upon encountering harsh environmental conditions, Caenorhabditis elegans larvae are able 

to alter their developmental program and enter the dauer diapause, an alternative developmental 

stage that enables larvae to endure long periods of starvation and stress. During this arrested 

state, the germline stem cells, which normally divide during reproductive development, halt 

their proliferation and are consequently rendered quiescent. Previous work has revealed that 

LKB1/par-4 and AMPK/aak-2 cooperate under such nutrient-deficient conditions in order to 

mediate this germline stem cell quiescence. The knockdown of either one of these genes 

causes aberrant germline hyperplasia in dauer larvae, while the inactivation of LKB1/par-4 in 

AMPK/aak-2 null mutants causes an enhanced hyperplasia phenotype. Thus, although LKB1/ 

par-4 is known to regulate AMPK/aak-2 in C. elegans, our genetic analyses suggest that it is 

unlikely that AMPK/aak-2 is the sole mediator of germline stem cell quiescence downstream 

of LKB1/par-4. 

LKB1/par-4 is a tumor suppressor protein kinase that is implicated in the rare, autosomal 

dominant disease Peutz-Jeghers syndrome (PJS). In order to better understand its function in 

tumorigenesis, we characterized its role in regulating cellular quiescence in developmentally 

arrested larvae using a genome-wide RNA interference-based screen to identify suppressors 

of PAR-4-mediated germline hyperplasia. We identified several genes whose loss-of-function 

was found to rescue the germline hyperplasia observed in par-4 dauer larvae, suggesting that 

their expression is misregulated in the absence of LKB1/par-4. Future endeavors include the 

characterization of key candidates, many of which impinge on the actin cytoskeleton and its 

regulation. Further understanding of the function of these genes will provide additional insight 

as to how LKB1/PAR-4 blocks tumorous growth by regulating cell cycle quiescence. 

Contact: rita.chaouni@mail.mcgill.ca 

Lab: Roy 

148 


VPR-1, a VAPB homolog required for germ line proliferation and 

differentiation 

Pauline Cottee, Jack Vibbert, Sung Min Han, Michael Miller 

University of Alabama at Birmingham, Birmingham, Alabama, USA 

The Major Sperm Protein (MSP) is an important protein for sperm motility and oocyte 

maturation. Secreted from motile sperm, MSP binds to the Eph receptor VAB-1 and other 

unknown receptors expressed on oocytes and sheath cells to induce oocyte maturation 

and ovulation. The MSP domain is an evolutionary conserved motif and is linked to the 

neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and late-onset spinal muscular 

atrophy (Nishimura et al., 2004). A point mutation (P56S) within the MSP domain of VAPB/ 

ALS8 gene causes reduced VAPB expression in humans and ALS mouse models, indicating 

VAPB plays a role in the pathogenesis of this disease. Previous studies have shown that the 

VAPB-MSP domain is cleaved and secreted, and acts as a ligand for Eph receptors. Further, 

the P56S point mutation causes VAPB proteins to aggregate and is failed to be secreted (Tsuda 

et al., 2008; Han et al., 2012). VPR-1 is the worm VAPB homologue. Loss of this protein in the 

worm recapitulates many of the pathologies observed in ALS patients, including mitochondrial 

dysfunction and lipid metabolism defects. Further, VPR-1 null mutant animals are sterile due 

to germ cells failing to proliferate and differentiate. Germ line proliferation and differentiation 

defects in VPR-1 null mutant animals were rescued with extrachromosomal arrays of fosmid 

DNA containing the complete vpr-1 genomic locus, indicating that VPR-1 has a role in germ 

line development. Examination of the distal tip cells of VPR-1 null mutant animals has shown 

that these cells were enlarged and have shorter processes in comparison those of wild-type 

animals. Additionally, visualization of the sheath cells in VPR-1 mutant hermaphrodites showed 

severe defects in these cells. Using a sheath cell driven CED-1::GFP transgene, we have 

also observed an increase in apoptosis in the germ line of vrp-1 mutant animals. Silencing of 

selected genes involved in the physiological and DNA-damage induced apoptosis pathways 

indicate that the increase in apoptosis in VPR-1 mutant animals is of a physiological basis 

rather than from DNA damage. My current goal is to use mosaic analysis to understand where 

VPR-1 functions in regulating germ line and somatic gonad development. 

Contact: Pcottee@uab.edu 

Lab: Miller 


149

Paternal Mitochondria Elimination From the Germline in C. elegans 

Embryos 

Dominika Bienkowska, Sylvain Bertho, Carrie Cowan 

I.M.P., Vienna, Austria 

An almost universal feature of sexual reproduction is the strict uniparental inheritance of 

mitochondria in the zygote. While several theories have been postulated to account for why 

mitochondria should be inherited from only one parent, there is sparse evidence in support of 

these ideas. In C. elegans, paternal mitochondria are delivered to the oocyte during fertilization 

but are gradually degraded in the embryo through autophagy. Using long-term time-lapse 

imaging of paternal mitochondria during embryogenesis, we find that the germline lineage 

preferentially eliminates paternal mitochondria compared to the soma. Already in the first 

asymmetric cell division of the embryo P0, PAR polarity promotes segregation of paternal 

mitochondria away from the germline. Germline elimination of paternal mitochondria appears 

to be completed by division of P3. In many embryos, some paternal mitochondria are present 

in the soma as late as the 100-cell stage, suggesting paternal mitochondria may not be harmful 

to the organism per se but rather specifically prevented from entering the germline. We are now 

using genetics to identify the mechanisms downstream of PAR polarity that control germlinespecific 

elimination of paternal mitochondria and to identify the effects of aberrant paternal 

mitochondria inheritance on germline development. 

Contact: cowan@imp.ac.at 

Lab: Cowan 

150 


CACN-1 is required for gonad and germline development 

Hiba Tannoury, Erin Cram 


CACN-1 is a well conserved protein of unknown molecular function, required in C. elegans 

for larval development including DTC migration, somatic gonad and germline development 

and fertility. CACN-1 is expressed throughout the hermaphrodite reproductive system, and 

observations in somatic gonad marker strains treated with cacn-1 RNAi show that cacn-1 is 

required for the development of the somatic gonad. Observations in rrf-1 mutant animals, 

in which the somatic (but not germline) RNAi response is defective, suggest that cacn-1 is 

required in the germline for fertility. C. elegans, a self-fertile hermaphrodite, produces sperm 

late in larval development before switching to oocyte production in adulthood. This switch is 

regulated by a set of RNA binding proteins and splicing factors downstream of the germline 

sex determination gene fem-3. In cacn-1 RNAi treated animals, copious sperm, but few, if 

any, oocytes are produced. Genetic interaction studies indicate that cacn-1 normally functions 

upstream of the sperm-to-oocyte differentiation decision pathway by repressing the fem-3, 

fog-1, and fog-3 male-fate promoting genes. Transcriptome sequencing data analysis shows 

upregulation of terminal male-fate differentiation factors, fog-1 and fog-3, in cacn-1 depleted 

animals. Continued expression of FOG-1 and FOG-3 likely disrupts the germ cell decision to 

switch to production of oocytes. Therefore, CACN-1 functions similarly to the known spermto-oocyte 

regulatory RNA binding proteins/splicing factors to negatively regulate the male fate 

promoting genes of the fem-3 pathway. 

Contact: e.cram@neu.edu 

Lab: Cram 


151

HIS-35, a histone H2A variant that differs from canonical H2A by one 

amino acid, functions in fertility 

Francisco Guerrero, Rodrigo Estrada, Meghann Shorrock, Margaret Jow, Diana 

Chu 

San Francisco State University, San Francisco, CA, U.S. 

Histone variants are incorporated during germ cell development to execute transcriptional 

programs that specify cell fate. HIS-35, a histone H2A variant that we have found via proteomic 

analysis, is enriched on sperm chromatin in comparison to embryo chromatin. Because HIS-35 

differs by only 1 amino acid from canonical S-phase histone H2A, it is unclear when or why 

HIS-35 is incorporated during spermatogenesis. We hypothesize that HIS-35 incorporation 

during sperm formation regulates germ cell differentiation. Consistent with this, we found 

his-35(tm1328) deletion mutant hermaphrodites only produce 49% of the total progeny of N2 

control hermaphrodites and lay few unfertilized oocytes or dead embryos. They also exhibit slow 

growth and small size. Cytological analysis of his-35(tm1328) mutant male and hermaphrodite 

germlines reveal incompletely penetrant defects in germ cell formation, including reduced 

numbers of developing germ cells and occasional defects in chromosome segregation. This 

suggests HIS-35 incorporation is important for optimal germ cell formation. Because of the high 

degree of similarity to H2A, HIS-35 is resistant to traditional immunochemistry techniques, thus 

we are creating transgenes to investigate HIS-35 incorporation during germ cell development. 

We have constructed GFP::HIS-35 to follow HIS-35 incorporation during specific stages 

of spermatogenesis. We are also constructing HIS-35 fused to DNA methyltransferase to 

help us track HIS-35 incorporation throughout the genome. Defining HIS-35 expression and 

localization, as well as the genes it interacts with, will elucidate mechanisms for how HIS-35 

functions in fertility. 

Contact: rodrigo_estrada@sbcglobal.net 

Lab: Chu 

152 


SNF-10, an SLC6 transporter required for sperm activation by C. 

elegans males 

Kristin Fenker, Angela Hansen, Conrad Chong, Molly Jud, Gillian Stanfield 

University of Utah, Salt Lake City, UT, USA 

A key step of spermatogenesis is the acquisition of cellular motility. In nematodes, sperm 

move by crawling, and they become motile during a regulated process termed sperm activation 

in which subcellular rearrangements lead to formation of a pseudopod and relocalization of 

proteins required for fertilization to the cell surface. In C. elegans, sperm activation is regulated 

differentially in males and hermaphrodites to promote the two sexes’ distinct requirements for 

reproductive success. Male sperm activation is regulated by a serine protease, TRY-5, which 

is transferred during mating in seminal fluid to couple the onset of male sperm motility to their 

entrance into the female reproductive tract. The coupling of these events is important, as early 

sperm activation leads to male infertility, while a delay in achieving motility could cause sperm 

to be displaced from the hermaphrodite’s reproductive tract. Protease treatment of sperm in 

vitro induces their activation, suggesting TRY-5 may act directly on targets on the sperm plasma 

membrane. To search for such targets, we performed a genetic screen for factors required for 

sperm activation by males and obtained several alleles of the solute carrier 6 (SLC6) family 

plasma membrane transporter snf-10. Like try-5, snf-10 is not required for fertility and acts in 

parallel to the spe-8 group hermaphrodite sperm activation genes. However, unlike try-5, snf- 

10 is not required for males to transfer activating factor, and its function is required in sperm. 

snf-10 mutant sperm fail to activate in response to protease in vitro or TRY-5 in vivo, but they 

can activate upon treatment with another known activator, the weak base triethanolamine. 

We currently are analyzing the localization of SNF-10 in sperm cells, and preliminary results 

suggest that it is present on the plasma membrane. Taken together, this plasma membrane 

localization and the fact that it functions downstream of try-5 make SNF-10 a strong candidate 

for the target of TRY-5 cleavage. 

Contact: kristin.fenker@utah.edu 

Lab: Stanfield 


153

Putative protamines, SPCH-1/2/3, localize to mature sperm chromatin 

and may play a role in fertility 

Jennifer Gilbert, Dana Byrd, Diana Chu 

San Francisco State University, San Francisco, (CA), USA 

During spermatogenesis, chromatin becomes highly compacted to ensure the efficient 

delivery of DNA to the oocyte. Compaction of sperm chromatin in most animals is facilitated by 

deposition of small nuclear basic proteins (SNBPs) called protamines, which bind in the major 

groove of DNA to allow bending of the DNA. While protamine incorporation to compact sperm 

chromatin is well conserved, the high variability of protamine gene and protein sequences across 

phyla has complicated the identification of these proteins across species. We hypothesize that 

C. elegans have SNBPs that share similar molecular features and functions as protamines. 

As such, we expect them to be small, highly basic proteins enriched in sperm chromatin, and 

localize to DNA during late stages of spermatogenesis. To identify such proteins, a proteomic 

approach was taken. We identified three nearly-identical proteins, SPCH-1/2/3 that were 

abundant in sperm chromatin samples and not found in embryo chromatin. SPCH-1/2/3 are 

only 22kD and they have a predicted isoelectric point of ~13.7. Also consistent with the amino 

acid content of SNBPs, SPCH-1/2/3 consist of a high percent of arginine (28%) and serine 

(29%) residues. Immunostaining using an antibody that recognizes all three SPCH proteins 

shows that SPCH-1/2/3 localize to DNA in late stages of spermatogenesis and around adult 

sperm. Immediately after fertilization, SPCH-1/2/3 mark the paternal pronuclei and then are 

removed as the sperm pronucleus decondenses. Using proteomic analysis of acid solubilized 

sperm chromatin, we find that SPCH-1/2/3 are highly phosphorylated. Interestingly, major 

sites of phosphorylation are found on amino acids that differentiate SPCH-1 from SPCH-2 and 

SPCH-3. Due to the importance of protamines in sperm DNA compaction, we anticipate that 

loss of SPCH function could lead to fertility defects. In fact, our preliminary progeny counts of 

single SPCH mutants suggest elimination of SPCH function reduces fertility. Thus, SPCH-1/2/3 

appear to function as protamines and may play an important role in male fertility. 

Contact: jenji@mail.sfsu.edu 

Lab: Chu 

154 


Sperm Vs Sperm: Determining the Cellular Basis of Sperm 

Competition 

Jody Hansen, Daniela Chavez, Gillian Stanfield 

University of Utah 

In C. elegans, male sperm must compete with hermaphrodite self sperm to fertilize 

oocytes. Male sperm precedence, the differential fertilization success of male sperm over 

hermaphrodite self sperm, is nearly absolute and relies on intrinsic differences between male 

and hermaphrodite sperm. Sperm motility, but not fertilization competence, is required for 

male precedence. Thus, C. elegans sperm competition provides a robust system to study 

cell competition in the context of migrating cells. Male spermatid size is significantly larger 

than that of hermaphrodites, and this larger size is correlated with faster crawling speeds and 

preferential residence in the spermathecae, where fertilization occurs. We seek to understand 

the cellular and molecular mechanisms contributing to male sperm precedence. By identifying 

sperm-specific gene products required for this process, we will improve our understanding of 

sperm migration and how it relates to cell competition. We have identified a mutant, me69, that 

displays reduced male sperm precedence. In addition, me69 mutant male sperm accumulate 

slowly in the spermathecae as compared to those of the wild type, suggesting a defect in some 

aspect of motility or directional migration. However, me69 mutant hermaphrodites have normal 

brood sizes, providing evidence that mutant sperm retain functional motility. Notably, me69 

mutant spermatids are the same size as wild-type spermatids, suggesting that mechanisms 

in addition to size contribute to male sperm precedence. Our current goal is to identify the 

me69 gene and to determine whether the me69 defect is due to changes in adhesion, cell 

signaling, or other factors affecting cell migration. Ultimately, identifying the cellular basis of 

the me69 defect also will differentiate among models of sperm competition. Currently, we have 

preliminary data identifying a candidate gene for me69, and we are building strains to test for 

rescue and determine protein expression. We also are characterizing the me69 migration defect 

by tracking wild-type and mutant sperm as they travel in the hermaphrodite reproductive tract. 

Contact: jody.hansen@utah.edu 

Lab: Stanfield 


155

Evaluating the Role of the V-ATPase B Subunit Utilizing C.elegans 

Sperm 

Melissa Henderson, Elizabeth Gleason, Ying Long, Taylor Walsh, Emily Wang, 

Steven L’Hernault 

Emory University, Atlanta, GA, USA 

Secretory vesicles are used during spermatogenesis to deliver proteins to the cell surface 

prior to sperm-egg fusion. Many of these proteins are essential for fertilization to take place. 

In C.elegans the membranous organelles (MOs) fulfill the role of these important secretory 

vesicles. As MOs mature, they undergo acidification, which is similar to what occurs in the 

acrosome of mammalian sperm. Our recent publication demonstrated that the acidification on 

MOs results from the presence of the V-ATPase complex and this process can be disrupted 

with the drug bafilomycin. C. elegans encodes two V-ATPase subunits, vha-12 and spe-5. 

vha-12 is located on the X chromosome and transcriptionally silent during spermatogenesis 

leaving spe-5, which is located on chromosome I, as the only B subunit utilized in sperm. The 

B subunit has two distinct roles in the V-ATPase complex, the hydrolysis of ATP and the binding 

of actin, which occurs at a highly conserved actin-binding site. We are evaluating these roles 

by further analysis of several separation of function spe-5 mutants, including a transgenic 

MosSci line in which actin binding has been abolished. vha-12 expression is required in many 

somatic cells, so the existence of spe-5 allows analysis the sperm V-ATPase mutants in sterile, 

but otherwise viable, animals. 

Contact: melissa.henderson@emory.edu 

Lab: L’Hernault 

156 


The RNA binding protein TIA-1.2 is essential for fertility in C. elegans 

Gabriela Huelgas Morales, Carlos Silva Garcia, Rosa Navarro Gonzalez 

Cellular Physiology Institute UNAM, Mexico City, Mexico 

RNA binding proteins, such as TIA-1 and TIAR, regulate RNA at different levels in a variety 

of organisms. In the nucleus, these proteins participate in alternative splicing while in the 

cytoplasm they regulate mRNA stability and/or translation rate. Along with these functions, 

under stress conditions, TIA-1/TIAR aggregate to form stress granules in a reversible manner 

to inhibit translation and protect mRNAs in harmful conditions. In C. elegans, the lack of one 

of the three homologs of TIA-1, C18A3.5, leads to several germ line defects, including sterility. 

This fact is interesting since Tia-1(-/-) and Tiar(-/-) knockout mice show a high percentage of 

sterility and embryonic lethality as well, by a mechanism that remains unknown. 

Our aim is to study the phenotype of the tia-1.2 mutant focusing in its germ line defects, and 

specifically to understand TIA-1.2’s role in oogenesis, ovulation and fertility. tia-1.2 mutants are 

temperature sensitive, showing p-vulva and sterility at 25°C. By Nomarski and fluorescence 

microscopy we observed that tia-1.2 (tm361) have a smaller distal region along with shorter 

mitosis and larger pachytene regions, suggesting that there could be a misregulation on this 

mechanism. Oocytes seem to go through all maturation steps, but later on, mutant worms 

show an EMO phenotype and oocytes accumulate in the uterus. Based on this phenotype, we 

are currently testing several candidate genes that could be regulated by TIA-1.2. 

Contact: ghuelgas@email.ifc.unam.mx 

Lab: Navarro Gonzalez 


157

Germline Hexosamine Pathway Synthesis of UDP-GlcNAc is 

Regulated by SUP-46 

Wendy Johnston1 , Aldis Krizus1 , Arun Ramani2 , Andrew Fraser2 , James Dennis1 1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, 

Canada, 2Department of Molecular Genetics, University of Toronto, Toronto, 

Canada 

Eukaryotic cells are encased in a sugar coating, in the form of secreted and membraneassociated 

glycoconjugates. Extracellular glycoconjugates are essential for cell-cell interactions, 

and play critical roles in modulating cell signaling. The hexosamine pathway synthesizes UDP- 

GlcNAc, an essential building block for N- and O-glycan-modified glycoproteins, proteoglycans 

and sugar polymers including chitin. Chitin and chitin-binding glycoproteins in the eggshell 

and extraembryonic matrix (EEM) are required for multiple events in the C. elegans oocyteto-embryo 

transition, including generation of a polyspermy barrier at fertilization, high fidelity 

meiotic chromosome segregation, polar body extrusion, normal MBK-2 localization, and A-P 

polarization (reviewed in Johnston and Dennis, 2012, Genesis). Therefore, the C. elegans 

zygote and surrounding eggshell/EEM provide an excellent model system for investigating the 

role of extracellular matrix glycoconjugates in early developmental events. 

gna-2 and gna-1 encode a key enzyme in the hexosamine pathway. In a gna-2(qa705) 

suppressor screen, we isolated 4 alleles of sup-46. Suppression of gna-2(qa705) maternal effect 

embryonic lethality depends on GNA-1 for production of UDP-GlcNAc. SUP-46 is required for 

normal hatchling number, particularly at high temperature (26oC) where sup-46 mutant brood 

sizes are ~25% of control. Genome-wide profiling of transcripts in sup-46(qa710) by RNA-Seq 

demonstrates increased abundance of gna-1. Moreover, sup-46(qa710, 708) have elevated 

levels of GNA-1::GFP in the germline. Together, these results identify SUP-46 as a regulator of 

GNA-1 in the hexosamine pathway synthesis of UDP-GlcNAc. Current studies are underway 

to determine the mechanism by which SUP-46 controls gna-1 transcript level, and to examine 

the role of SUP-46 and GNA-1 in stress resistance. 

Supported by a CIHR grant. 

Contact: johnston@lunenfeld.ca 

Lab: Dennis 

158 


Role of Notch re-localization in establishing germline stem cell 

quiescence in C. elegans dauer larvae 

Pratik Kadekar, Nathan Navidzadeh, Patrick Narbonne, Emily Wendland, Richard 

Roy 

Department of Biology, DBRI, McGill University, Montreal, Quebec, Canada 

Stem cells are regulated and maintained by signals that emanate from their specific 

environmental niche. Similarly in C. elegans germline, the somatic distal tip cells (DTCs) 

located at the extremities of the gonad, form a niche for the germline stem cells (GSCs) and 

regulate their proliferation. GSCs express the Notch receptor/GLP-1 on their membrane and 

is activated by the Notch ligand/LAG-2 expressed in the DTCs. This activation instructs the 

GSCs to undergo mitosis while inhibiting them from executing the meiotic pathway. Interestingly, 

the Notch ligand/LAG-2, is expressed and is active in the quiescent C. elegans dauer germ 

line. This suggests that in dauers, mitotic quiescence is regulated at some point downstream 

of receptor activation. We have found that over the course of dauer diapause, the Notch 

receptor/GLP-1 undergoes subcellular re-localization from the membrane to the rachis. In the 

hyperplasic dauer germline of an AMPK or PAR-4 mutant, GLP-1 is still present around the 

membrane of the GSCs and fails to re-localize to the rachis. This re-localization of the Notch 

receptor may therefore contribute to the appropriate establishment of mitotic arrest in the 

GSCs. If AMPK or PAR-4 are compromised, the Notch receptor/GLP-1 does not appropriately 

re-localize and may therefore be associated with the observed germline hyperplasia in these 

mutants. We are currently trying to unravel the link between LKB1/AMPK signaling and this 

novel Notch response. 

Contact: pratik.kadekar@mail.mcgill.ca 

Lab: Roy 


159

Protein synthesis regulation in the germline: eIF4 factors promote 

selective mRNA translation for meiosis, differentiation, maturation or 

apoptosis. 

Melissa Henderson1 , Jacob Subash1 , Vince Contreras1 , Anren Song2 , Sara 

Labella3 , Andrew Friday1 , Monique Zetka3 , Robert Rhoads2 , Brett Keiper1 1Brody School of Medicine at East Carolina University, Greenville, NC 

27834, 2LSU Health Sciences Center, Shreveport, LA 71130, 3McGill University, Montreal, Quebec, Canada 

Translational control of mRNAs represents the most important mode of gene regulation in 

animal germ cells, and many specific examples have been described. Only recently has the role 

of translation initiation factors (eIFs) in such translational control been appreciated. Our labs 

are uncovering mRNAs specifically regulated by individual isoforms of eIF4E and eIF4G, two 

subunits of the cap-binding initiation complex, in C. elegans germ cells. Deficiency in individual 

isoforms of eIF4E (e.g. IFE-1, IFE-2, IFE-3) or eIF4G (IFG-1) changes the fate of oocyte and/or 

sperm differentiation, resulting in blocked maturation steps, inefficient meiotic recombination, 

gamete fate switching, or induced germ cell apoptosis. Each reduces fertility, but in surprisingly 

unique ways depending upon the translation factor type. Altering the balance of IFG-1 p170/ 

p130 isoforms triggers the physiological apoptotic cascade by inducing cap-independent 

synthesis of CED-4, the worm Apaf-1 homolog. For two IFE isoforms we have identified mRNAs 

that uniquely require that isoform for efficient translation. These mRNAs encode proteins of 

critical function in oocyte and/or spermatocyte differentiation. IFE-1 is required for late stage 

oocytes to efficiently translate pos-1, pal-1, mex-1, and oma-1 mRNAs. Spermatocytes lacking 

IFE-1 fail in the final budding/cytokinesis step, accumulating as multinucleated secondary 

spermatocytes. Translation of msh-4 and msh-5 mRNAs, on the other hand, requires IFE-2 

to synthesize proteins for meiotic crossover, allowing those cells to complete proper meiotic 

chromosome segregation. Neither eIF4E deficiency results in loss of global protein synthetic 

activity nor general growth capacity of the gonad. Thus, germ cell translation initiation factors 

appear to drive mRNA selection for specific developmental functions. The evidence supports 

a positive regulatory network of eIF4E-eIF4G-mediated translational control directing gamete 

differentiation/survival and cell death. 

Contact: keiperb@ecu.edu 

Lab: Keiper 

160 


P-TEFb—Independent Phosphorylation of RNA Polymerase II CTD- 

Ser2 in the C. elegans Germline 

Elizabeth Bowman, Bill Kelly 

Emory University, Atlanta, GA, USA 

Multiple phosphorylation events targeting the C terminal domain (CTD) of the catalytic 

subunit accompany the progression of RNA Polymerase II (Pol II) through different stages of 

transcription. For example, the elongation stage is associated with phosphorylation of Serine 2 

(Ser2P) of the Pol II CTD. This phospho-epitope has long been considered to be the product of 

the P TEFb (CDK 9/Cyclin T) complex in metazoans. Surprisingly, we have found that Ser2P in 

the C. elegans germ line occurs independently of the P TEFb complex. Instead, the appearance 

of Ser2P is fully dependent on another CTD Ser2 kinase/cyclin complex, CDK 12/Cyclin K. 

Whereas CDK 9/Cyclin T knockdown results in complete loss of Ser2-P in somatic lineages, 

substantial Ser2P is still present in the germ line at all stages. In contrast, CDK 12 and/or Cyclin 

K knock down results in only a partial Ser2P decrease in somatic nuclei, but complete loss of 

Ser2P in germ cells at all developmental stages. In addition, we find that CDK 12/Cyclin K, 

rather than P TEFb, appears to be the Pol II Ser2 kinase complex regulated by the maternal 

transcriptional repressor, PIE 1, in the nascent embryonic germ line. Transgenic analyses 

suggest that although CDK-9, CDK-12, and Cyclin K are ubiquitously expressed, Cyclin T (the 

partner of CDK-9) appears to have reduced expression in the germ line. Interestingly, the Pol 

II elongation regulator and known target of CDK-9, DSIF, is also expressed in all lineages. 

The striking prominence of P TEFb-independent Pol II CTD phosphorylation in the germline 

suggests that there may be basic, separable differences between transcriptional processes 

operating in the germline cycle, and in those engaged in differentiating somatic lineages. 

Contact: bkelly@emory.edu 

Lab: Kelly 


161

sacy-1 Links Somatic Control of Oocyte Meiotic Maturation, Germline 

Sex Determination, and Gamete Maintenance 

Seongseop Kim, J. Amaranath Govindan, Zheng Jin Tu, David Greenstein 


All described MSP-dependent meiotic maturation events in the germline require the 

activity of the Gas-adenylate cyclase-protein kinase A pathway in the gonadal sheath cells. We 

conducted genetic screens for mutations that suppress the sterility of acy-4 adenylate cyclase 

null mutations. We identified 66 suppressor of adenylate cyclase (sacy) mutations that define 

at least 17 genes. Using a combination of whole-genome sequencing and positional cloning, 

we identified the molecular identities of eight sacy genes, which include sacy-1, twk-1, uev- 

1, tom-1, pde-6, spr-2, spr-4, and spr-5. Our screen appears to have recovered regulators 

of meiotic maturation that function in both the germline (sacy-1 and spr-5) and the gonadal 

sheath cells (twk-1). sacy-1, spr-5, and twk-1 function downstream of kin-1/pka and appear 

to affect fertility cumulatively. 

Here we introduce sacy-1 which, in addition to negatively regulating meiotic maturation, 

functions in the hermaphrodite sperm-oocyte switch, and is required for gamete maintenance. 

sacy-1 encodes a highly conserved DEAD-box helicase orthologous to the Drosophila 

translational regulator Abstrakt. A rescuing GFP::SACY-1 fusion is expressed in most or 

all cells, and is localized to the nucleus and cytoplasm. We isolated three viable and fertile 

hypomorphic missense alleles of sacy-1 as acy-4 suppressors. Surprisingly, we found that 

these sacy-1 mutant alleles could suppress germline feminization and self-sterility caused 

by null mutations in fog-2. A maternal wild-type copy of sacy-1 antagonizes the suppression 

of fog-2 feminization. sacy-1 functions upstream of fem-3 in the germline sex-determination 

pathway, possibly at the level of tra-2. A sacy-1 null allele is sterile in both sexes and exhibits 

a gamete degeneration phenotype–sperm and oocytes appear to undergo necrotic cell death. 

The sacy-1 null gamete degeneration phenotype is independent of ced-3 and ced-4, but partially 

suppressed by a mutation in UNC-68 ryanodine receptor. Interestingly, germline feminization 

markedly delays oocyte degeneration in sacy-1 null mutants. sacy-1 null females ovulate 

large numbers of unfertilized oocytes, indicating that sacy-1 is a strong negative regulator of 

meiotic maturation. Genetic epistasis analysis indicates that sacy-1 likely functions upstream 

of oma-1 and oma-2, which are redundantly required for meiotic maturation. SACY-1 might 

function widely in translational regulation in the germline. 

Contact: kimx1285@umn.edu 


162 


Investigating the Role of SMC-5/6 in Preventing Germline Genomic 

Rearrangement 

Killeen Kirkconnell, Dane Session, Raymond Chan 


De novo germline mutations can lead to spontaneous abortion and genetic defects in 

humans. A frequently observed class of germline mutation is genomic rearrangement, which 

includes deletions and duplications (Lupski, 1997). Study of recurrent genomic rearrangements 

in human sperm found evidence suggesting rearrangements can originate from aberrant 

inter-homolog and intra-chromatid homologous recombination (Turner, 2008). Surprisingly, 

rearrangements from inter-sister recombination are estimated to be extremely rare, which 

may indicate that inter-sister recombination normally acts to suppress germline genomic 

rearrangement. We have previously shown that the C. elegans smc-5 and smc-6 mutants had 

defects in meiotic homologous recombination repair, likely in sister-chromatid recombination 

(Bickel, 2010). Homozygous smc-5 and smc-6 mutants also exhibit a mortal germline phenotype 

and become sterile over several generations, which suggests an accumulation of germline 

mutations. 

This study addresses whether the smc-5 and smc-6 mutants accumulate a higher 

frequency of germline mutations, and determines the types of mutations occurring. We 

adapted a phenotypic assay to identify spontaneous germline mutations at three genetic 

loci in the smc-5 smc-6 mutant background. The unc-93(e1500) mutation produces a toxic 

protein which confers uncoordinated movement. An unc-93(e1500) worm can revert to 

wildtype movement with mutations that disrupt the expression of the mutant UNC-93 protein 

or two ancillary proteins, SUP-9 and SUP-10 (Greenwald,1980). We found that the smc-5 

smc-6 mutant has an approximately 93-fold higher reversion rate than wildtype worms. The 

reversion phenotype is heritable, so these reversion events are the result of germline mutations. 

Genetic complementation tests and PCR analysis are performed to determine which of the 

three genes contains the mutation responsible for the reversion. Our preliminary results 

confirm that the majority of reversions are due to disruption of unc-93, sup-9, or sup-10. More 

importantly, PCR analysis of the mutated genes indicates a bias towards deletion events, with 

many removing an entire gene. These data support the prediction that smc-5 and smc-6 are 

important for preventing chromosome structural variations. We are mapping and sequencing 

the rearrangement breakpoints, and we plan to conduct a genome-wide analysis of de novo 

rearrangements in the smc-5 smc-6 mutant. 

Contact: killeenk@umich.edu 

Lab: Chan 


163

The let-479 Gene Encodes a Homolog of SPE-42 and is Required for 

C. elegans Fertilization 

Tim Kroft, Luke Wilson, Lindsey Magnuson, Gabe Fall 

University of Minnesota Duluth, Duluth, MN, USA 

We analyzed our collection of spermatogenesis defective (spe) mutants for late acting spe-9 

class mutants that are likely to be involved in sperm-egg recognition, binding, or fusion during 

fertilization. Both males and hermaphrodites homozygous for eb137 mutations produce sperm 

that appear normal but fail to fertilize oocytes. Sperm persist in the spermathecae of eb137 

mutant hermaphrodites and the hermaphrodites produce large numbers of oocytes, suggesting 

that sperm activation and major sperm protein signaling to the developing oocytes and somatic 

gonad are not compromised. Following mating, eb137 male-derived sperm migrate to the 

hermaphrodite spermathecae and compete with hermaphrodite sperm despite their inability 

to fertilize oocytes. Complementation testing with known LG V spe genes demonstrated that 

eb137 is an allele of let-479. Positional cloning, transgenic rescue, and sequence analysis 

revealed that let-479 encodes a homolog of the SPE-42 protein, which we previously showed 

is also required for C. elegans fertilization. LET-479 is predicted to be a 6-pass sperm plasma 

membrane protein with membrane topology identical to that of SPE-42. Like SPE-42, LET-479 

contains 3 domains of interest: a large extracellular hydrophilic domain between transmembrane 

spans 3 and 4 containing 6 conserved cysteine residues, a DC-STAMP domain that includes 

transmembrane spans 5 and 6, and a C-terminal cytoplasmic RING domain. The presence of 

two proteins that are so similar, function in the same process, yet are completely non-redundant 

raises intriguing questions regarding the roles LET-479 and SPE-42 play during fertilization. The 

presence of SPE-42 and LET-479 homologs in all species that use bona fide sperm and eggs 

for fertilization suggests what we learn in the C. elegans system will be useful in understanding 

the mechanism of fertilization in other species, including humans. 

Contact: tkroft@gmail.com 

Lab: Kroft 

164 


Spindle Assembly Checkpoint Plays a Role In DNA-damage-induced 

Cell Cycle Arrest In C. elegans Male Germ Line 

Katherine Lawrence, JoAnne Engebrecht 

University of California Davis, Davis, CA, USA 

Persistent DNA damage in germline stem cells leads to embryonic lethality, progeny 

inviability or germline tumors. Consequently, cells closely monitor genomic integrity and can 

delay their progress through the cell cycle so that repair precedes division. In C. elegans, 

genotoxic perturbations to proliferative cells in the distal tip of the gonad activate checkpoints 

that initiate a cell cycle arrest. When this arrest is in response to stalled replication forks induced 

by hydroxyurea (HU), it is characterized by enlarged nuclei and can be visualized cytologically. 

HU damage is sensed by the C. elegans homolog of ATR, a PI3-related protein kinase, 

and launches a signaling cascade that results in a G1/S phase arrest. The signal transducers 

and downstream effectors of this DNA-damage-response (DDR) pathway have been studied 

extensively in hermaphrodites, but have not been investigated fully in males. While RNAi 

knockdown of several of these genes disrupts checkpoint output in hermaphrodites, the same 

treatment does not prevent HU-induced arrest in males. Our preliminary results strongly 

suggest that not all components of the DDR are essential for male cell-cycle arrest in response 

to stalled replication forks. 

We next investigated functional redundancy between the DDR and the spindle assembly 

checkpoint (SAC), which is most often associated with regulating kinetochore attachment 

to spindles during prometaphase/metaphase of mitosis and meiosis. We found that RNAi 

knockdown of several SAC components alone did not affect HU-induced cell-cycle arrest in 

males; however, knockdown of both ATR and SAC resulted in a failure to arrest in the presence 

of HU. This result suggests that, in males, the DDR and SAC work together to elicit arrest in 

the presence of stalled forks. To analyze this differentially regulated HU-induced arrest, we 

identified markers that characterize the stages of the cell cycle. Preliminary data suggests 

that the SAC, like the DDR, mediates an S phase arrest not predictive of its expected role as 

an inhibitor of cdc20 at metaphase. Future work aims to understand this novel role for SAC 

components and investigate the mechanisms used by the SAC to induce an S phase arrest. 

Contact: kslawrence@ucdavis.edu 

Lab: Engebrecht 


165

Investigating the Role of Membrane Trafficking in Temperature- 

Sensitive Lethal Mutants with Defects in both Gonad Development 

and Embryonic Eggshell Production 

Josh Lowry, Amy Connolly, John Yochem, Bruce Bowerman 

Institute of Molecular Biology, University of Oregon, Eugene OR, USA 

Membrane trafficking plays a major role in gonad development and embryogenesis in C. 

elegans. For example, signaling between the Distal Tip cells and early germline cells relies on 

both secretory and endocytic functions. Eggshell components are delivered to the surface of 

fertilized zygotes through secretory pathways as well. In the course of a recent mutant screen, 

we have identified temperature-sensitive, embryonic lethal mutants with eggshell defects that, 

when shifted to 26?C at the L1 stage, also have gonad development defects as adults. Those 

with >50% adult sterility make up the Osm/Ste class of mutants, comprised of 39 strains. We 

hypothesize that the two phenotypes reflect different gene requirements at different stages of 

development, and that the causal mutations affect genes involved in membrane trafficking. 

To determine if these mutants do have defects in membrane trafficking, we have taken a 

high-throughput genomics approach, employing the method of simultaneous whole-genome 

sequencing and SNP mapping1, to identify the affected genes in these mutants. To do this, 

we outcross our mutant strains to the Hawaiian polymorphic strain CB4856. We then isolate 

~50 F2 progeny homozygous for the mutation and allow them to reproduce. These populations 

are then pooled, using approximately equivalent numbers of individuals from each population, 

and genomic DNA is extracted and prepared for sequencing on an Illumina HiSeq 2000. The 

short-read alignment program MAQgene is used to both align the resultant data to the C. 

elegans reference genome and identify SNPs from each of the parent strains. Plotting the ratio 

of Hawaiian SNP reads to total read depth allows us to map the mutations to intervals ranging 

from ~1-3Mbp in length. We can then search through the sequencing pileup in the mapped 

interval to identify candidate mutations. 

We have performed a pilot study and obtained high resolution SNP mapping data for 

8 Osm/Ste mutants and are now compiling a list of candidate mutations for each strain. In 

addition, we are also preparing the other 31 strains for sequencing and analysis. This work 

will provide us with a toolkit we can use to understand the molecular basis for the phenotypes 

we have observed. 

References: Doitsidou, M. et al. C. elegans mutant identification with a one-step wholegenome-sequencing 

and SNP mapping strategy. PLoS One 5(11): e15435 (2010). 

Contact: jlowry@uoregon.edu 

Lab: Bowerman 

166 


Genome destabilization and checkpoint activation during cell cycle 

reentry of the primordial germ cells Z2 and Z3 

Ash Williams, Brendan Kramer, Matthew Michael 

University of Southern California, Los Angeles, CA, USA 

The primordial germ cells Z2/Z3 are born during early embryogenesis. After completing 

S phase, Z2/Z3 arrest their cell cycles at prophase and remain arrested for the remainder 

of embryogenesis. During this period of prophase arrest, RNA pol II transcription is largely 

quiescent. Upon L1 hatching, and only if nutrients are present, Z2/Z3 activate RNA pol II 

transcription and reenter the cell cycle. We have been studying the events that occur within 

Z2/Z3 during cell cycle reentry. We find that the genome is transiently destabilized soon after 

feeding, at the same time that RNA pol II transcription is activated, and that this destabilization 

event activates the cell cycle checkpoint protein CHK-1. After repair of DNA damage, CHK-1 is 

deactivated, and the cells proceed into mitosis. Importantly, this cycle of genome damage and 

repair does not occur in somatic nuclei. We have also observed that RNAi-mediated depletion 

of CHK-1 causes Z2/Z3 to enter mitosis precociously, about one hour ahead of schedule, and 

they do so with unrepaired DNA damage. Taken together, our results identify a germ line-specific 

genome destabilization event that is monitored by CHK-1 and that is part of the normal cell 

cycle reentry process for Z2/Z3. The results of more recent experiments designed to uncover 

the molecular basis for the genome destabilization event will also be reported at the meeting. 

Contact: mattm@usc.edu 

Lab: Michael 


167

Sensory Regulation of the C. elegans Germ Line through TGF-β- 

Dependent Signaling in the Niche 

Diana Dalfo, David Michaelson, E. Jane Albert Hubbard 

New York University School of Medicine, New York, NY, USA 

Germline progenitors accumulate during larval development to form an adult pool from 

which gametes are produced. We found that, independent of its roles in the dauer decision 

and lifespan, TGF-β modulates the balance of proliferation versus differentiation in the larval C. 

elegans germ line in response to sensory cues that report population density (dauer pheromone) 

and food abundance. TGF-β ligand-producing ASI sensory neurons are required for TGFβ-mediated 

germ cell accumulation, and the TGF-β receptor and downstream transcription 

complex act in the distal tip cell, the germline stem cell niche. Our results implicate TGF-β 

neuroendocrine signaling as a mediator between environmental cues, sensory neurons and 

the stem cell niche to influence the balance of proliferation and differentiation of the germline 

stem/progenitor pool. 

Contact: mole333@gmail.com 

Lab: Hubbard 

168 


In Vitro Analysis of C. elegans H2A Variants 

Ahmad Nabhan 1 , Geeta Narlikar 2 , Diana Chu 1 

1 San Francisco State University, San Francisco, CA, USA, 2 University of 

California, San Francisco, San Francisco, CA USA 

The introduction of histone variants enables eukaryotic cells to regulate access to DNA. 

How histone variants act to alter chromatin structure and perform their function remains a 

mystery. Studies have implicated variants in a plethora of activities, for example, HTZ-1 plays 

a role in gene regulation and repair. Also in some organisms, including C. elegans, sperm 

specific histone variants are incorporated during global transcriptional repression. In C. elegans, 

HTZ-1 and HTAS-1 are the only H2A variants showing significant variation from H2A (59% 

and 51% identical, respectively). HTZ-1 is enriched in the embryo while HTAS-1 is only found 

in sperm. The disparity these two proteins exhibit in localization is reflected structurally: HTZ- 

1 has an extended C-terminus while HTAS-1 has an extended N terminus. This leads to an 

interesting question: what type of chromatin structure do histone variants enriched in vastly 

different environments create? To understand this, we will assess how HTZ-1 and HTAS-1 

alter chromatin structure to modulate accessibility to genetic material. We hypothesize the two 

variants alter chromatin structure to meet the transcriptional needs of the cell. Therefore we 

expect HTZ-1 increase accessibility to genetic material and HTAS-1 to decreases accessibility. 

To this end, we have expressed recombinant HTZ-1 and HTAS-1 along with C. elegans 

H2A H2B, H3 and H4 in E. coli. Proteins were purified using size exclusion and ion exchange 

chromatography. They were then combined in equa-molar amounts to create canonical and 

variant nucleosome core particles (NCPs). The stability of these core particles, which is 

inversely proportional to accessibility, will be measured as a function of ionic concentration 

using electrophoresis. We have also labeled H2A and its variants with Cy5, which were refolded 

with Cy3 labeled DNA, which will be used in our FRET experiments to gain a quantitative 

understanding of the influence variants have on stability of the nucleosome. Another factor 

affecting chromatin accessibility, reversible nucleosome unwrapping, will also be monitored 

using restriction enzyme activity (REA) (Widom 1991). In vitro analysis of C. elegans H2A 

subtypes will reveal the intrinsic properties responsible for their funcion in vivo. 

Contact: A nab 

Lab: Chu 


169

ZHP-3 Regulates Meiotic Chromosome Dynamics 

Christian Nelson, Cate Paschal, Needhi Bhalla 

University of California Santa Cruz, Santa Cruz, CA, USA 

During meiosis, homologs must pair with their unique partner and undergo crossover 

recombination, forming physical linkages that hold chromosomes together until the first meiotic 

division. Once homologs have recombined, chromosomes must be restructured to promote 

attachment of homologs to opposite spindle poles and to ensure proper segregation. In C. 

elegans, ZHP-3 is required for both genetic exchange as well as the accompanying largescale 

changes in chromosome structure during meiotic prophase. ZHP-3 contains a RING 

finger domain and purified ZHP-3 possesses auto-ubiquitination activity in vitro, suggesting 

that ZHP-3 is a ubiquitin ligase and that it may ubiquitinate target substrates to accomplish 

its tasks. Moreover, the localization of ZHP-3 during meiotic prophase is highly dynamic, 

suggesting it is heavily regulated. My project aims to identify meiotic substrates of ZHP-3 

taking both a candidate biochemical approach as well as an unbiased genetic approach. 

Additionally, purified ZHP-3 will be analyzed via mass spectrometry to identify co-purifying 

proteins, potential regulators, and post-translational modifications. Preliminary data shows that 

ZHP-3 is a MAP kinase target in vitro and that MAP kinase is required for changes in ZHP-3 

localization, suggesting that phosphorylation may act to temporally target ZHP-3 to unique 

substrates as meiotic prophase progresses. Combined, these data will help us to elucidate how 

ZHP-3 coordinates genetic recombination with large-scale chromosome structure changes to 

ensure proper meiotic chromosome segregation. 

Contact: chrinels@ucsc.edu 

Lab: Bhalla 

170 


Distinct roles for FBF-1 and FBF-2 in silencing meiotic mRNAs 

Alexandre Paix, Ekaterina Voronina, Geraldine Seydoux 

Johns Hopkins University School of Medicine, Baltimore, MD, USA 

Many genes in the C. elegans germline are regulated by post-transcriptional mechanisms 

acting through 3’ UTR sequences. We are interested in how this regulation manifests itself at 

the level of RNA stability, transport, translation and/or localization. To address this question, 

we have examined the sub-cellular localization of mRNAs in the germline, particularly meiotic 

mRNAs that are silenced in the distal (mitotic) region by FBF-1 and FBF-2 (gld-1, him-3, htp- 

1/2). We have found that meiotic RNAs are distributed in a low distal/high proximal gradient in 

the mitotic zone, as reported previously for gld-1 (Jones et al., 1996). This pattern is unaffected 

in fbf-2 mutants. In contrast, in fbf-1 mutants, meiotic RNAs accumulate with FBF-2 in large 

aggregates in the rachis of the mitotic zone. In fbf-1 fbf-2 double mutants, meiotic mRNAs 

are uniformly distributed throughout the mitotic zone and translated. These findings suggest 

that FBF-1 and FBF-2 silence meiotic mRNAs by different mechanisms, and support a role 

for FBF-1 in RNA transport or degradation. 

Contact: apaix1@jhmi.edu 

Lab: Seydoux 


171

Natural Variants of C. elegans Demonstrate Defects in Both Sperm 

Function and Oogenesis at Elevated Temperatures 

Lisa Petrella, Susan Strome 

UC Santa Cruz, Santa Cruz 

The temperature sensitivity of fertility is conserved from nematodes through mammals 

and is generally correlated with a loss of sperm function. In order to better understand the 

mechanisms that underlie high temperature sterility in worms, we are analyzing fertility and 

germline organization at elevated temperature in 15 different wild-type isolates of C. elegans 

from around the world. Previous studies in C. briggsae and Drosophila showed that strains 

originating from temperate latitudes lose fertility at a lower temperature than strains originating 

from tropical latitudes. We determined the fertility of temperate and tropical strains of C. elegans 

and observed no correlation between latitude of strain origin and fertility at high temperature. We 

observed a wide distribution of population fertility among wild-type isolates at 27°C, ranging from 

7% to 56%. In order to better understand the causes of high temperature sterility, temperature 

shift experiments were performed. Males up-shifted to high temperature as L4/young adults 

maintain fertility, while males raised at high temperature lose fertility. Sterile animals contain 

a wild-type-appearing germ line with mature spermatids. Down-shifting males raised at 

high temperature does not restore fertility. These male results differ from those observed in 

Drosophila and suggest that in C. elegans spermatogenesis is irreversibly impaired in males 

that develop at high temperature. Mating and down-shift experiments with hermaphrodites 

were performed to investigate the relative contributions of spermatogenic and oogenic defects 

to high temperature loss of fertility. We identified isolates that demonstrate predominantly 

spermatogenic defects, strains that show a mixture of spermatogenic and oogenic defects, 

and one strain that shows predominantly oogenic defects. Interestingly, among strains, the 

ability to maintain hermaphrodite sperm function at hightemperature does not correlate with 

the ability to maintain male sperm functionat high temperature. Our studies have uncovered 

unexpectedly high variation in both the loss of fertility and underlying problems with sperm 

function in natural variants of C. elegans at high temperature. One variant provides a novel 

example of oogenic defects underlying loss of fertility. These variants can now be used to 

investigate the molecular mechanisms that underlie the buffering of fertility in the face of 

environmental change. 

Contact: lpetrell@ucsc.edu 

Lab: Strome 

172 


Exploring Novel Features of Gametogenesis in a Non-C. elegans 

Clade 

Kathryn Rehain, Zechariah Dillingham, Ethan Winter, Diane Shakes 

College of William and Mary, Williamsburg, VA, USA 

Several features of the C. elegans germline make it an excellent and somewhat simplified 

system for studying gametogenesis: 1) the germline can be observed directly through the 

transparent body wall, 2) the entire linear timeline of gametogenesis can be observed within 

individual gonads, and 3) mitotically dividing germ cells have the capacity to develop into 

either oocytes or spermatocytes. However as other nematode species are equally well suited 

for comparative studies of gametogenesis, we have begun to characterize gametogenesis 

in a Rhabditid clade that phylogenetically includes both Rhabditella axei and Rhabditis. sp. 

SB347, species with diverse reproductive modes, and all of which can be easily cultivated in 

the lab (Kiontke and Fitch, 2005; Felix, 2004; Shakes et al. 2011). In comparison to C. elegans, 

nematodes within this clade have gonads with many fewer cells. More specifically, both the 

distal mitotic region and the pachytene regions are not only organized differently than in C. 

elegans, but are, in comparison, highly reduced in both size and cell number. In the proximal 

gonad, the developing oocytes have extremely unusual chromatin morphology which proves 

to correlate with the presence of extremely large nucleoli. One interesting feature of this clade 

is that the males exhibit a wide range of sperm sizes and their spermatocytes exhibit diverse 

patterns of meiotic and partitioning divisions which, in some cases, lead to the differential 

survival of their X and non-X-bearing sperm (Shakes et al., 2011; this study). In summary, 

analysis of gametogenesis within this non C. elegans clade is revealing “natural variations on 

a theme” which have important implications not only for both our general understanding of 

nematode gametogenesis but also for modifications in the program of spermatogenesis that 

may underlie the evolution of diverse reproductive modes. 

Contact: knrehain@email.wm.edu 

Lab: Shakes 


173

Nutritional Control of Germline Stem Cells 

Hannah Seidel, Judith Kimble 

Howard Hughes Medical Institute, University of Wisconsin-Madison, 

Madison, WI, USA 

The germline of C. elegans represents a tractable model for studying how nutritional 

cues regulate stem cell behavior. Previous work showed that food availability influences the 

proliferation of undifferentiated germ cells in the larval germline; this response is mediated in 

part by insulin/IGF-like signaling (Dev. 2010 137:671-80) and TGF-β signaling (Curr. Biol. 2012 

Epub Apr 5). Likewise, in oogenic hermaphrodites, nutrient deprivation causes germlines to 

shrink, and this shrinkage is reversible upon re-feeding (Science 2009 326:954-8, PLoS One 

2011 6: e28074). Here we show that proliferation of adult germline stem cells (GSCs) requires 

the presence of food: Under fed conditions, GSCs divide continuously, only ceasing division 

as they enter the meiotic cell cycle; upon food removal, however, GSCs stop dividing and 

become quiescent. This quiescence can last for several days, with cell division only resuming 

upon the reintroduction of food. In addition, we find that food availability may also influence 

the effect of GLP-1/Notch signaling. Under fed conditions, GLP-1 signaling from the niche 

maintains GSCs in an undifferentiated state, and loss of this signal causes all germ cells to 

differentiate. By contrast, under some conditions, GSCs do not enter the meiotic cell cycle 

when starved glp-1(q224ts) mutants are shifted to restrictive temperature. One interpretation 

of this result is that GLP-1 is dispensable for maintenance of the quiescent GSCs typical of 

starved animals; however other interpretations exist. We are currently investigating possible 

regulators of starvation-induced quiescence and stem cell maintenance. 

Contact: hsseidel@wisc.edu 

Lab: Kimble 

174 


Characterization of SYGL-1, A Novel Regulator of Germline Stem Cells 

Heaji Shin1 , Aaron Kershner1 , Judith Kimble1,2 1Department of Biochemistry, University of Wisconsin-Madison, Madison, 

WI, USA, 2Howard Hughes Medical Institute, University of Wisconsin- 

Madison, Madison, WI, USA 

C. elegans germline stem cells (GSCs) are maintained by GLP-1/Notch signaling from the 

stem cell niche. We have identified two putative Notch target genes, lst-1 (lateral signaling 

target) and sygl-1 (synthetic germline proliferation defective), that act redundantly to maintain 

GSCs throughout development and in both sexes (see Kershner, Shin and Kimble abstract). 

Here we focus on characterization of sygl-1, which had not been studied previously. The sygl- 

1 locus encodes a single transcript (T27F6.4) that is predicted to generate a protein of 206 

amino acids length. Standard programs (NCBI conserved domain search, Pfam 26.0, Phyre2) 

revealed no predicted folded domains or motifs in the SYGL-1 amino acid sequence. The 

sygl-1 locus is found in closely-related nematodes (C. japonica, C. remanei, C. briggsae, and 

C. brenneri), but is not broadly conserved. A sygl-1 deletion mutant (tm5040) removes 720bp, 

including the first exon and part of the first intron. The sygl-1 deletion mutant is homozygous 

viable, but possesses a smaller than normal mitotic region, consistent with the idea that sygl-1 

is required to maintain the normal number of germ cells within the mitotic region. To understand 

the molecular function of SYGL-1, we are in the process of making an epitope-tagged SYGL-1 

transgene and screening for SYGL-1 partner proteins. 

Contact: hshin38@wisc.edu 

Lab: Kimble 


175

Uncovering the Role of Condensin I during C. elegans Meiosis 

Margarita Sifuentes, Joshua Bembenek, Karishma Collette, Gyorgyi Csankovszki 

University of Michigan, Ann Arbor, Michigan 

Condensin complexes are key determinants of higher-order chromatin structure during 

meiosis and mitosis in eukaryotes. However the different roles of condensins I and II in 

meiosis are poorly understood and have yet to be elucidated in eukaryotes. Interestingly, 

analysis of condensin I in C. elegans, demonstrates it localizes to a ring shaped domain 

between chromosomes at the midbivalent during metaphase and to the meiotic spindle 

between separating chromosomes during anaphase. Other proteins such as the Chromosomal 

Passenger Complex, BUB-1, KLP-19, HCP-1/2, and CLS-2 also localize to the midbivalent, 

suggesting that condensin I may function with proteins at the ring shaped domain to promote 

chromosome orientation, alignment, and separation. 

Depletion of condensin I by RNAi interference leads to gross meiotic spindle defects and 

abnormal chromosome organization. Our data suggests that condensin I supports orientation 

and movement of chromosomes throughout meiosis. Additionally, condensin I depletion 

prevents successful chromosome resolution at anaphase. 

Future studies will determine how condensin I functions together with other proteins at the 

midbivalent to influence chromosomal orientation during meiotic congression and segregation 

in C. elegans. 

Contact: marqidez@umich.edu 

Lab: Csankovszki 

176 


The metazoan gene akirin is required for synaptonemal complex 

disassembly and bivalent structure during Caenorhabditis elegans 

meiosis 

Amy Clemons1 , Heather Brockway1 , Yizhi Yin1 , Yaron Butterfield2 , Steven Jones2 , 

Monica Colaiacovo3 , Sarit Smolikove1 1 2 3 University of Iowa, British Columbia Cancer Research Centre, Harvard 

Medical School 

During meiotic prophase I homologous chromosomes associate via the synaptonemal 

complex (SC). This structure is essential for proper crossover formation and therefore 

required for accurate chromosome segregation during meiosis. At the late stages of meiotic 

prophase I, following the formation of crossovers, the SC disassemble concurrently with the 

remodeling of the newly formed bivalent. It is expected that both events are crucial for proper 

meiotic chromosome segregation. However, little direct evidence supports this claim and the 

mechanisms controlling SC disassembly remain unclear. Here we identify akir-1 as a novel 

gene involved in these key events of meiotic prophase I in Caenorhabditis elegans. AKIR-1 

is a conserved protein in metazoans that lacks any known function in meiosis. We show that 

akir-1 mutants exhibit aberrant meiotic division accompanied by severe meiotic defects in late 

prophase I. These defects include improper disassembly of the SC and aberrant restructuring 

of the bivalent. Although initial steps of SC disassembly (asymmetric disassembly) progress 

normally, resolution of chromosome arms is severely impaired. This includes defects specific 

to central region proteins of the SC, that are still capable of bridging homologs in the absence 

of crossovers (spo-11 mutant background). Earlier meiotic events, such as homologous 

pairing, RAD-51 loading and crossover formation, are not affected in the akir-1 mutants. Our 

studies places AKIR-1 downstream from any other protein known to affect SC disassembly. 

Furthermore, defects in arm resolution lead to structural abnormalities of the bivalent. These 

are accompanied by failure to compact the bivalent, which is independent of the condensin 

complex. Overall our data indicates that akir-1 is involved in promoting proper bivalent formation 

by the timely disassembly of the SC and its proper restructuring, leading to sharpening of our 

understanding of these fundamental meiotic events. 

Contact: sarit-smolikove@uiowa.edu 

Lab: Smolikove 


177

Chromatin Regulation in the Meiotic Germ Line 

Matthew Snyder, Xia Xu, Eleanor Maine 

Syracuse University, Syracuse, NY, USA 

Meiotic silencing is a conserved phenomenon targeting unpaired chromosomes and 

chromosomal regions during prophase of meiosis I. Meiotic silencing in animals typically occurs 

at the chromatin level and involves accumulation of histone modifications thought to promote 

a closed chromatin configuration. This chromatin structure may contribute to transcriptional 

repression and meiotic chromosomal events such as chromosome disjunction (Bean et al 

2004, Jaramillo-Lambert and Engebrecht 2010). During meiosis in C. elegans, non-synapsed 

chromosomes are enriched for H3K9me2 relative to synapsed chromosomes (Kelly et al. 

2002; Bean et al. 2004). Such non-synapsed chromosomes include the male X, homologous 

chromosomes that fail to synapse due to mutation, and chromosomal translocations/ 

duplications. The pattern of H3K9me2 accumulation during meiosis depends on activity of a 

small RNA pathway (Maine et al. 2005; She et al. 2009), which may direct activity of the histone 

methyltransferase, MET-2, responsible for germline H3K9me2 (Bessler et al. 2010). Taking 

a combined biochemical/genetic approach, we are identifying additional factors important for 

regulating H3K9me2 distribution during meiosis. We will describe our latest findings. 

Contact: mpsnyd01@syr.edu 

Lab: Maine 

178 


Global Control of the Oogenic Program by Components of OMA-1 

Ribonucleoprotein Particles 

Caroline Spike, Donna Coetzee, David Greenstein 


The oocytes of most sexually reproducing animals arrest at diplotene or diakinesis and 

resume meiosis (meiotic maturation) in response to hormones. The defining feature of meiotic 

maturation is M-phase entry triggered by CDK/cyclin B. In wild-type C. elegans, M-phase entry is 

spatially restricted to the –1 oocyte by mechanisms that remain to be defined. Meiotic maturation 

requires the presence of sperm, Gas-adenylate cyclase-PKA signaling in the gonadal sheath 

cells, and germline function of two Tis11-like CCCH zinc-finger proteins, OMA-1 and OMA- 

2. To elucidate the mechanisms by which the OMA proteins promote meiotic maturation, we 

purified OMA ribonucleoprotein particles (OMA RNPs) from oocytes and identified associated 

proteins and mRNAs using mass spectrometry and RIP-Chip, respectively. To distinguish core 

OMA RNP components from those tethered by RNA, we also purified OMA complexes after 

treatment with RNase A. Many protein and mRNA components of OMA RNPs are critically 

important regulators of the oogenic program. OMA RNPs contain a large number of germlineexpressed 

RNA-binding proteins including translational activators and repressors. Core OMA 

RNP components include multiple subunits of the GLD-2 poly(A) polymerase and CCR4/NOT1 

deadenylase complexes and many RNA-binding proteins, including, MEX-3, MEX-1, and LIN- 

41. OMA RNP components that appear to be tethered primarily via RNA associations include 

the P-body proteins CGH-1/p54 and CAR-1/Rap55. 

To examine the role of OMA RNP components in translational regulation, we generated 

3’UTR-reporter constructs for several mRNA components of OMA RNPs. OMA-1/2 repress the 

translation of most tested targets in oocytes. Depletion of core OMA RNP components identified 

the NHL-family protein LIN-41 as a regulator of at least two OMA targets. Strikingly lin-41 is 

required for normal oogenesis and meiotic progression. In lin-41 null mutants, pachytenestage 

female germ cells fail to progress to diplotene, and instead enter M-phase. By contrast, 

oma-1; oma-2 mutant oocytes never enter M-phase. Interestingly, the OMA RNP components 

GLD-2 and LIN-41 exhibit epistasis and mutual suppression: double mutant oocytes arrest 

with a chromosome configuration resembling pachytene. We hypothesize that the OMA RNP 

complex promotes meiotic maturation in the –1 oocyte by balancing and spatially restricting 

the repression and activation of critical mRNA targets. 

Contact: spike001@umn.edu 



179

Early and Late Roles for Gonadal Innexins: Germ Cell Proliferation 

and Meiotic Maturation 

Todd Starich1 , David Hall2 , David Greenstein1 1Department of Genetics, Cell Biology and Development, University of 

Minnesota, Minneapolis, MN 55455, 2Albert Einstein College of Medicine, 

Yeshiva University, Bronx, NY 10461 

The germline gap junction innexins inx-14 and inx-22 are negative regulators of meiotic 

maturation that act downstream of Gαs. In addition, inx-14(0) animals are sterile, producing 

few germ cells. We have further investigated germline innexins and determined functional 

relationships between inx-8 and inx-9 in the somatic sheath and DTC, and inx-14, inx-21 and 

inx-22 in the germline; we have found that innexins have an early role in germ cell proliferation, 

as well as later roles including meiotic maturation. 

Through use of INX-8, -14, -21 and -22 specific antibodies, and INX-8, -9 and -14 GFP 

fusions, we have established a complex co-dependency for localization of innexin proteins to 

gap junction-like puncta. Within the germline, INX-14 requires either INX-21 or INX-22 to localize 

to puncta; INX-21 and INX-22 reciprocally require INX-14 to localize. A reciprocal dependency 

also exists between germline innexins and INX-8 and -9 for localization. inx-14(0) animals 

produce few germ cells; this phenotype is mimicked in inx-22(0) inx-21(RNAi-injected) animals, 

supporting dependence of INX-14 on INX-21 or INX-22 for function. This phenotype is also 

mimicked in inx-8 inx-9 double mutants (see below), which avg.

Oocyte-to-embryo Transition: a Screen for mbk-2 Suppressors 

Yuemeng Wang1 , Harold Smith2 , Kevin O’Connell2 , Geraldine Seydoux1 1Johns Hopkins University School of Medicine, Baltimore, MD, USA, 

2National Institutes of Health, Bethesda, MD, USA 

MBK-2 is a kinase required for the oocyte-to-embryo transition. MBK-2 is activated during 

the transition and phosphorylates oocyte proteins whose stability or activity must be modified 

in zygotes. To identify new factors that function with MBK-2 during the transition, we have 

conducted a screen for suppressors of dd5, a temperature sensitive mutation in the kinase 

domain of MBK-2 (Quintin et al, 2003). We isolated 21 suppressors in a screen of more than 

one million genomes. We are using whole genome sequencing after outcrossing to a Hawaiian 

strain to map and identify the suppressors (Doitsidou et al, 2010). So far we have found that 

four suppressors are intragenic, and four map to genes implicated in cell cycle regulation 

(emb-30, fzy-1, plk-1, such-1). We will present our progress at the meeting. 

Contact: ywang125@jhmi.edu 

Lab: Seydoux 


181

A Functional RNAi Screen Identifies Regulators of RNP Granule 

Assembly in Aging Oocytes 

Megan Wood, Kevin Gorman, Joseph Patterson, Jennifer Schisa 

Central Michigan University, Mount Pleasant, MI, US 

In many animal species, oocytes arrest in meiosis until they are fertilized. It is well 

established that fertility diminishes as oocytes age, and several cytological studies have 

reported changes in the cytoplasm of aging, arrested oocytes. In this study our goal is to 

better understand the regulation and function of large ribonucleoprotein (RNP) granules that 

assemble in the germlines of Caenorhabditis nematodes that are either stressed, or in which 

ovulation is arrested due to old age or an absence of sperm. The large RNP granules are 

hypothesized to regulate mRNA stability or translation in arrested or stressed oocytes when 

fertilization is delayed (Jud et al., 2008). 

We have performed a targeted, functional RNAi screen to identify genes that are required 

for the assembly of RNP granules in meiotically-arrested oocytes, and we have identified over 

100 genes that are necessary for the RNA-binding protein MEX-3 to assemble efficiently into 

large granules. Preliminary analyses suggest the largest gene classes of the screen positives 

are: RNA-binding proteins that localize to the RNP granules including cgh-1, car-1, puf-5, and 

pie-1; MSP signaling pathway members including inx-14, ceh-18, and goa-1; RNAi regulators 

including dcr-1 and csr-1; nuclear pore complex components including npp-9, npp-10, and ran-4; 

and microtubule-related proteins including tbb-1, tbb-2, and dhc-1. Interestingly, in yeast and 

mammalian cells, disruption of microtubules stimulates the formation of P bodies (processing 

bodies), while such disruption prevents the assembly of stress granules. With the discovery 

of these novel regulators of RNP granule assembly, we are now testing our hypothesis for 

their function. In several cases, when the normal assembly of RNP granules is prevented, 

we observe that fertility is decreased, supporting the hypothesis that RNP granules may help 

maintain the integrity of oocytes when fertilization is delayed. We are currently determining if 

RNA stability is diminished or translation of maternal mRNAs is de-repressed in oocytes when 

RNP granule assembly is defective. 

Contact: meganpwood@yahoo.com 

Lab: Schisa 

182 


A Novel Function of MRE-11 in Caenorhabditis elegans 

Yizhi Yin, Sarit Smolikove 

Univ.of Iowa,Iowa,IA, USA 

Accurate chromosome segregation in meiosis requires interhomolog crossovers (COs) 

generated by repair of programmed meiotic double-strand DNA breaks (DSBs) via homologous 

recombination (HR) pathway. Mre11 is one member of the MRX (Mre11, Rad50, Xrs2/Nbs1) 

complex that is essential for the formation of interhomolog COs. Studies in yeast indicate the 

MRX complex plays dual roles in COs formation, the formation of DSBs and the resection 

of DSBs. However, in Caenorhabditis elegans the evidence for MRX complex’s role in DSBs 

resection is limited. The currently known mutants of genes in MRX complex are null, showing 

absence of chiasma, indicating lack of meiotic DSBs. Here we report the first separation of 

function allele of mre-11 in C. elegans, mre-11(iow1). This allele shows defects specific to 

resection. mre-11 mutants exhibit a phenotype of chromosomal fragmentation and aggregation 

at the diakinesis stage of meiotic prophase I. This is accompanied by 100% of embryonic 

lethality and high level of germline apoptosis. As expected, mre-11(iow1) is epistatic to mre- 

11 null and msh-5 mutants, and acts downstream of spo-11. RPA-1 and RAD-51 are the 

single stranded DNA (ssDNA) binding proteins in C. elegans, which sequentially associate to 

ssDNA following resection. In mre-11(iow1) mutants, the loading of RPA-1 onto the meiotic 

chromosomes is severely decreased , while RAD-51 does not load to meiotic chromosomes. 

These results suggest failure to resect meiotic DSBs to form ssDNA required for formation of 

COs via HR pathway in mre-11(iow1) mutants. However, since the HR pathway is blocked in 

mre-11(iow1) mutants, meiotic DSBs can only be repaired by other DNA repair pathways that are 

not usually used in meiosis. One of these pathways is the non-homologous end joining (NHEJ) 

pathway. Blocking the NHEJ pathway by the addition of the cKu-80 (a gene essential for NHEJ 

pathway) null mutation to the mre-11(iow1) genetic background, partially rescues mre-11(iow1) 

phenotypes (measured by RAD-51 loading, chromosomal aggregation and fragmentation, 

embryonic lethality and germline apoptosis). These results suggest NHEJ partially attributes 

to the mutant phenotypes observed in mre-11(iow1) and plays a role in repairing the meiotic 

DSBs in mre-11(iow1) mutants. Compared to wild type worms, mre-11(iow1) mutants do not 

load RAD-51 on DSBs induced by γ-irradiation from onset of prophase I to early-mid pachytene 

and show a reduced RAD-51 localization in other stages. This indicates that the resection 

function of MRE-11 is mainly required in early to mid meiotic prophase and is replaced in the 

other stages by an alternative nuclease. Overall our analysis of our unique mutant establish a 

role or MRE-11 in resection of DSBs, restricted to a subset of meiotic stages, and in blocking 

the NHEJ pathway in these nuclei. MRE-11 therefore plays a central role in controlling modes 

of DSB rapier in meiosis through is function in DSB resection. 

Contact: yizhi-yin@uiowa.edu 

Lab: Smolikove 


183

Illuminating the Formation and Regulation of Meiotic Crossovers with 

GFP:COSA-1 

Karl Zawadzki, Rayka Yokoo, Anne Villeneuve 

Stanford University, Stanford, USA 

Faithful chromosome segregation during meiosis I requires crossover (CO) recombination 

events that form the basis of temporary links between homologous chromosomes. Rather than 

creating many COs, most organisms create only a small number of widely spaced COs while 

ensuring that each pair of homologs receives at least one. This regulation of CO number and 

placement is collectively termed “crossover control” but the underlying mechanisms are poorly 

understood. In C. elegans CO control is particularly robust, with each homolog pair receiving 

one CO. We are investigating the formation and regulation of meiotic COs, building on our 

discovery of COSA-1 (Crossover Site Associated), a conserved CO protein that is required 

for the formation of COs and localizes to CO sites during the late pachytene and diplotene 

stages of meiotic prophase. A functional GFP::COSA-1 fusion protein serves as a robust in 

vivo reporter for the CO control system, as GFP::COSA-1 reliably localizes to six foci per 

nucleus (i.e. one focus for each homolog pair), even in the context of a large excess of DSBs. 

These and other data suggest that GFP::COSA-1 foci represent a more reliable surrogate 

than COs per se for the events that are being distributed by the interference mechanism. Thus 

to investigate the basis of CO control, we are conducting a genetic screen for mutations that 

alter the number of GFP::COSA-1 foci. We have isolated over a dozen mutants with defects in 

different meiotic processes and are further characterizing those mutants that may be relevant 

to understanding CO control. 

Contact: zawadzki@stanford.edu 


184 


exc-2 and Maintenance of Tube Structure of the Excretory Canals 

Hikmat Al-Hashimi, Matthew Buechner 

University of Kansas, Lawrence, (KS), USA 

Long, narrow, single-celled tubes are found in many tissues in our body, such as capillaries 

and the Schwann cells that wrap neurons with a myelin sheath. The excretory canal cell provides 

a model for investigating the formation and maintenance of narrow tubes. We study nine exc 

genes, mutations of which allow the excretory canals to swell into fluid-filled cysts. The position 

of all these genes have been narrowed, but a few remain to be cloned. I am currently cloning 

the exc-2 gene. This locus was shown previously to be located on the X chromosome between 

mec-2 and dpy-8, a region of ~425 Kb that contains ~170 genes. We have microinjected 12 

fosmids that cover the majority of this region; though the mutant has not been rescued yet, 

we have narrowed the locus to ~145KB. 

Additionally, in order to identify new genes that affect the maintenance of the tube structure 

of the excretory canal, we plan to carry out an RNAi screening via feeding. exc mutations are 

viable, but other mutations that affect structure of the canal lumen (such as erm-1 and cdc- 

42) have lethal effects in other tissues. In order to overcome the possible extra-canal lethality 

of candidate genes, we have constructed transgenic worms that should be RNAi-sensitive 

within the excretory cell and not in other tissues, by adapting the method used by the Chalfie 

lab (Calixto et al., Nature Methods ‘10) to knock down genes in the touch neurons. We have 

integrated a construct that drives expression of sid-1 to high levels in the canal through the 

use of the vha-1 promoter, in a sid-1 (RNAi-deficient) mutant. Preliminary tests show loss of 

RNAi effects for a hypodermally expressed gene (dpy-8), and we are currently confirming the 

effects of knockdowns of exc and other genes on both canal and extra-canal phenotypes. 

Contact: hikmat@ku.edu 

Lab: Buechner 

Poster Topic: Morphogenesis 

185

C. elegans nuclear hormone receptor, nhr-25 regulates vulval terminal 

cell properties and migrations during development 

Nagagireesh Bojanala, Marek Jindra, Masako Asahina 

Biology Centre, ASCR/Univ. South Bohemia, Ceske Budejovice, Czech 

Republic 

C. elegans vulva has been intensively studied for cell fate specifications and morphogenesis 

during development and a number of signaling pathways are involved in this process. The 

mature vulva is composed of 22 cells formed from the daughters of 3 vulva precursor cells 

[VPCs, P(5-7).p] that undergo three rounds of cell divisions to differentiate into seven distinct 

cell types, Vul A to F and the proper migration of these cells is essential to form functional vulva. 

Previous studies have shown the role of nhr-25 (ftz-f1/sf-1 homolog) in vulval cell inductions 

and morphogenesis, yet its role in cell migration has not been fully explored. We followed the 

terminal differentiation properties of Vul A-F and their migrations in nhr-25(lf) animals. L2-nhr- 

25(RNAi) was used in this study to bypass the initial VPC induction defects. Defects during 

the short range migrations of Pn.pxx cells were seen in our nhr-25(RNAi) animals; P6.pxx 

(1°) and P5/7.pxx (2°) cells failed to initiate their dorsal and lateral migrations respectively. 

At the time of vulva fate execution process, the presumptive Vul E and Vul C cells changed 

their division axis from transverse (T) to longitudinal (L). In addition, the outer most cell types 

Vul A, B1 and B2 often failed to reach the vulva midline. Similar defects have been reported 

when semaphorin and Rac/Rho pathway gene functions were compromised. Interestingly, 

SMP-1::GFP expression was affected in nhr-25 (RNAi) animals and the genetic interaction 

between nhr-25 and smp-1(ev715) or plx-1(ev724) revealed that nhr-25 works in parallel to the 

semaphorin pathway. We also observed abnormal toroidal fusions within Vul E/F, A/B1/B2 and 

B1/B2 in nhr-25(RNAi) animals. Altered expressions of terminal vulval differentiation markers 

indicate that non-homologous cells may adopt homologous fate in nhr-25 (lf) background and 

later fused with each other. In summary, NHR-25 regulates vulva morphogenesis at multiple 

levels; at the initial induction, at the terminal differentiation of Vul A-F cell types and at the 

time of migration of vulval cells where NHR-25 co-operates with semaphorin pathway. Since 

we observed the genetic interaction between nhr-25 and semaphorin pathway also in the 

epidermal seam cells, the co-operation of these pathways may be universal. Supported by 

204/09/H058 and Z60220518. 

Contact: gireesh@paru.cas.cz 

Lab: Asahina 

186 

Poster Topic: Morphogenesis

Characterizing regulators of the C. elegans cytoskeleton 

Benjamin Chan, Simon Rocheleau, Paul Mains 

Genes and Development Research Group, University of Calgary, Calgary, 

AB, Canada 

All eukaryotic organisms begin as a roughly spherical ball of cells, but the final shape of 

a species is a precise and tightly regulated process. In Caenorhabditis elegans, a smooth 

muscle-like contraction of an actin/myosin network mediates the elongation of a worm embryo 

from a ball of cells into a long, thin worm. Here we will continue to characterize a gene known 

to regulate this process, and discuss a novel gene which may be also involved. Previous work 

has shown that non-muscle myosin is able to generate contractile force through two redundant 

pathways in C. elegans. Phosphorylation of myosin light chain (MLC-4) activates non-muscle 

myosins NMY-1/2 which drives contraction. In contrast, dephosphorylation of MLC-4 is regulated 

by MEL-11/myosin phosphatase. In one pathway, the small GTPase RHO-1 activates LET-502/ 

Rho-binding kinase which inhibits MEL-11, halting contraction. In a second parallel pathway, 

FEM-2/protein phosphatase 2c is involved in regulating contraction. In a suppressor screen 

of mel-11, an allele of a Rho GEF (guanine exchange factor) rhgf-2 was identified. Previous 

work has shown that rhgf-2 genetically acts upstream of let-502 and in parallel to fem-2. In 

addition, RHGF-2 is believed to act as a GEF for RHO-1. However, the cellular localization 

of RHGF-2 has yet to be determined, and our current work will use a RHGF-2 antibody to 

address this question. In our previous suppressor screen of mel-11, an allele of a novel gene, 

sb89, was also isolated. We will continue to characterize this gene through traditional mapping 

experiments and whole genome sequencing. 

Contact: bgchan@ucalgary.ca 

Lab: Mains 


187

The Morphological and Functional Alterations of the Anal Depressor 

Muscle in Male C.elegans 

Xin Chen 1 , L. Rene Garcia 1,2 

1 Department of Biology Texas A&M University College Station TX , 2 Howard 

Hughes Medical Institute 

The anal depressor is a sexually dimorphic muscle in C. elegans. It is an H-shaped cell 

in hermaphrodites and larvae males and alters its shape and function significantly during 

L4 male development. In adult males, the anal depressor functions as an auxiliary spicule 

protractor muscle whereas in hermaphrodites and larvae males it participates in defecation. 

We are interested in exploring the mechanism through which the anal depressor alters its 

morphology and function. 

First, we observed the morphological change of the anal depressor throughout the larval and 

adult stages using fluorescent microscopy. We found that from L1 to L3 stage the myofilaments 

contained within the anal depressor run dorsal-ventrally and from L2 stage relative to the 

hermaphrodite, the male anal depressor does not increase in size. At early L4, a ventral slit 

forms at the anal depressor, demarcating the ventral attachment into anterior and posterior 

regions. During mid L4 stage the anterior region elongates dorsal-anteriorly to attach to the 

developing dorsal protractor muscle, whereas the posterior region remains attached to the 

rectum. The myofilaments contained within the anterior attachment begin to reorient to run 

anterior-posteriorly. From late L4 until adulthood, the myofilaments contained within the posterior 

region atrophies making the central region of the anal depressor extremely thin. 

Questions arise as to whether the morphogenesis responds to intrinsic or extrinsic signals, 

or a combination of both. Extrinsic signals might come from the cells that are adjacent to the 

anal depressor. To test this hypothesis, we laser-ablated muscle, neuronal and epithelial 

precursor cells and monitored anal depressor development. We found that the M cell ablation, 

which eliminated all the sex muscles, resulted in anterior movement defects and abnormal 

morphology of the anal depressor in the adult male. 

We performed EMS mutagenesis and identified three mutant lines where the males had 

a normal larval, but abnormal adult anal depressor.Two mutant lines have severe defects in 

spicule morphogenesis whereas the third line has a subtle alteration in the position of the 

spicules. In the third mutant line, the adult anal depressor took the late L4 morphology with 

the posterior myofilaments failed to reorganize anterior-posteriorly. 

Contact: xchen@bio.tamu.edu 

Lab: Garcia 

188 


TMD-1 / Tropomodulin Regulates Intestinal and Excretory Cell 


Rachel Walker, Corey Hoffman, Elisabeth Cox-Paulson 

SUNY College at Geneseo, Geneseo, NY 

Tropomodulins are actin-binding proteins that regulate the stability of the slow-growing 

ends of actin filaments. C. elegans has a tropomodulin homolog, TMD-1/UNC-94 that encodes 

two isoforms with functional and sequence similarity to vertebrate tropomodulins. TMD-1 is 

involved in body wall muscle development (1,2), regulation of junctional actin in hypodermal 

cells (3), and we have also found that it is needed for proper morphology of the intestine and 

excretory cell. In the intestine, TMD-1 localizes to the terminal web, which is an actin and 

intermediate filament rich structure that underlies the apical, luminal membrane. Loss of tmd- 

1 function results in flattened morphology of the intestinal lumen and a reduction in luminal 

volume. In worms homozygous for the strong loss-of-function allele, tmd-1(tm724), the terminal 

web is thinner, the concentration of F-actin is reduced, and the intermediate filament protein, 

IFB-2, shows a slightly abnormal distribution. This points to a role for TMD-1 in regulating the 

ultrastructure of the terminal web. Additionally, tmd-1(tm724) mutants exhibit an overabundance 

of gut granules, indicating a potential defect in vesicle trafficking. In addition to intestinal 

abnormalities, tmd-1(tm724) mutants have excretory cell defects; specifically the canals fail 

to extend anteriorly and posteriorly during development. Improper structure of the intestine 

and excretory cell may account for the larval lethality, accumulation of fluid, and slow growth 

exhibited by tmd-1(tm724) mutants. Together, the data supports a new role for a tropomodulin 

in the development of tubular epithelial tissues. 

1. Yamashiro et al. (2008) J. Cell Sci. 121: 3867-77. 

2. Stevensen et al. (2007) J. Mol. Bio. 374: 936-50. 

3. Cox-Paulson et al. (2012) in revision at Current Biology. 

Contact: coxe@geneseo.edu 

Lab: Cox-Paulson 


189

Roles Of Heparan Sulfate Proteoglycans In Embryonic Morphogenesis 

Katsufumi Dejima, Suk-Ryool Kang , Andrew Chisholm 

University of California, San Diego, La Jolla, CA, USA 

Heparan sulfate proteoglycans (HSPGs) are cell surface or extracellular matrix molecules 

that are comprised of a core protein modified with heparan sulfate (HS), a negatively charged 

linear polysaccharide. HSPGs have been known to be associated with various biological 

processes such as growth factor signaling and cell adhesion. In C. elegans, mutations in genes 

involved in HSPG synthesis lead to embryonic lethality with morphogenetic defects, including 

defective ventral enclosure (Refs. 1,2). However, the cellular roles of specific HSPGs during 

early embryogenesis prior to ventral enclosure are still unclear. To define the specific roles of 

HSPGs in early development we are taking several approaches. First, we are examining the 

expression pattern of HSPGs in early embryonic development. Immunostaining of embryos 

with the HSPG side chain antibody 3G10 reveals widespread cell surface expression. We 

are testing whether this expression pattern reflects early embryonic expression of the known 

core proteins such as syndecan/SDN-1 or glypican/GPN-1. Second, we are characterizing 

the phenotypes of mutants with severe defects in HS synthesis (rib-1, rib-2, hst-1) using 

semi-automated cell lineage analysis (ref. 3). While such HS synthesis mutants display fully 

penetrant early embryonic lethality, animals lacking multiple known HSPG core proteins are 

semi-viable, suggesting the existence of additional as-yet uncharacterized essential HSPGs in 

the embryo. We are biochemically purifying total embryonic HSPGs in the hopes of identifying 

the complete complement of C. elegans HSPGs. 

1. Hudson ML et al. 2006. Dev Biol 294: 352-65. 

2. Kitagawa H et al. 2007. J Biol Chem 282: 8533-44. 

3. Giurumescu CA et al. 2011. Worm Breeder’s Gazette 18 No. 4 p5 

Contact: kdejima@ucsd.edu 

Lab: Chisholm 

190 


C. elegans body size is regulated by TGF-β signalling in multiple 

tissues. 

Aidan Dineen, Jeb Gaudet 

University of Calgary, Calgary, (Alberta), Canada 

The coordination of organ size and body size to mediate proportional growth is an interesting 

biological problem. In C. elegans , body size is partially regulated by a TGF-β signaling pathway 

that also functions in male tail development and is therefore termed the Sma/Mab pathway 

(for Small and Male abnormal). Loss of function mutations in pathway genes such as the 

ligand dbl-1 or downstream receptor regulated Smad (R-Smad) sma-3 result in decreased 

post-embryonic growth, with adult sma mutants only achieving ~60-70% the body size of wild 

type animals. This small phenotype is due to a decrease in cell size, rather than cell number. 

Previous work has demonstrated that the Sma/Mab pathway can function cell-autonomously 

in the hypodermis to positively regulate body size. However, many of the components of this 

signalling pathway are expressed in additional organs such as the intestine and pharynx, raising 

the question of a possible role for this pathway in these organs. We compared the pharynx 

size of sma-3 mutants to those of dpy mutants, a class of mutants that have small body sizes 

due to cuticle defects, not the Sma/Mab pathway. We found that while sma and dpy mutants 

have a similar reduction in body lengths, the pharynx size of sma mutants was significantly 

reduced compared to dpy mutants. This result indicates that Sma/Mab pathway signalling is 

required for normal growth of the pharynx in addition to its previously described role in body size 

regulation in the hypodermis. We further show that contrary to previous models, the Sma/Mab 

pathway functions in multiple tissues to control body size. In particular, we find that pharyngeal 

expression of the R-Smad protein SMA-3 is sufficient to partially rescue both pharynx size 

and body size defects of sma-3 mutants. These results suggest that the Sma/Mab signalling 

pathway can function non cell-autonomously to regulate cell size and therefore body size. In 

addition, we find that rescue of sma-3 mutants improves as the number of sma-3 expressing 

tissues is increased (e.g. expression of sma-3 in pharyngeal muscles, marginal cells and the 

hypodermis provides better rescue than expression in any tissue alone). Overall, our results 

suggest a model where TGF-β signalling in multiple tissues activates one or more downstream 

secreted signals that act non cell-autonomously to regulate body size in C. elegans. 

Contact: aedineen@ucalgary.ca 

Lab: Gaudet 


191

Functional Dissection of SAX-7, a Homologue of Human L1CAM in C. 

elegans Dendritic Branch Formation 

Xintong Dong, Oliver Liu, Kang Shen 

Stanford University, Stanford (CA), US 

Dendritic morphology is critical for neuronal information processing. The location, shape and 

size of dendritic arbors determine what signals a neuron receives and how they are integrated 

and further transmitted. However, the mechanisms by which neurons acquire elaborate typespecific 

dendritic arbors have not been completely elucidated. In particular, the interaction 

between dendritic branches and its growth substrate is poorly understood. Unlike most neurons 

in the nematode Caenorhabditis elegans that are simple in morphology, the mechanosensory 

neuron PVD forms elaborate branches that envelope the body of the worm. The architecture 

of these branches is stereotypical and beautifully organized, following general principles of 

dendritic development such as tiling and self-avoidance, thus providing a strong tool to study 

genetic regulation of dendritic branching. Immunoglobin superfamily cell adhesion molecules 

(IgCAMs) have been implicated in many important aspects of neurogenesis including axon 

guidance, cell migration and synapse formation. This study focused on SAX-7, a homologue 

of human L1CAM. We have shown that loss of SAX-7 results in truncated 1° branches, 

disorganized 2° branches and complete loss of 3° or 4° branches in PVD. SAX-7 functions in 

hypodermal cells and subcellularly localizes around hypodermal-SEAM cell junctions close to 

1° branches and sublateral regions where 3° branches are formed. Forward genetic screen has 

revealed a putative receptor for SAX-7 which functions autonomously in PVD. These results 

suggest a role of interaction between cell surface molecules and pre-patterned extracellular 

cues in directing dendritic branch development. 

Contact: xdong1@stanford.edu 

Lab: Shen 

192 


ani-1 is required for morphogenesis of C. elegans embryos and 

functions in parallel to the rho-1 pathway. 

Nellie Fotopoulos, Yun Chen, Alisa Piekny 

Concordia University, Montreal (Quebec), Canada 

Morphogenesis describes the changes in cell shape and movement that give rise to 

tissues and is crucial for the development of all metazoans. These changes are driven by 

the actomyosin cytoskeleton. Cytokinesis, the final stage of mitosis, also requires the cell to 

undergo extensive cell shape changes to form two daughter cells. This is controlled by RhoA, 

which regulates the polymerization of actin and activation of myosin to build and ingress the 

contractile ring. Anillin is a component of the contractile ring, which maintains stable furrow 

ingression by scaffolding RhoA, myosin and actin filaments. Some of the core regulators of 

cytokinesis also regulate epidermal cell shape changes during C. elegans morphogenesis, 

but roles for anillin in morphogenesis have not yet been described. Of the three C. elegans 

anillin homologues, ANI-1 has the highest overall homology to human anillin. ani-1 regulates 

myosin localization and asymmetric furrow ingression during cytokinesis and polar body 

extrusion during meiosis. We hypothesize that ani-1 may also regulate the cytoskeletal-driven 

cell shape changes and movements for morphogenetic events. We found that ani-1 depletion 

leads to a range of phenotypes, including ventrally-ruptured embryos and lumpy dumpy larva 

that could indicate problems in ventral enclosure or elongation. This requirement for ani-1 is 

not strictly maternal since rde-1(ani-1 RNAi) worms outcrossed to N2 also displayed lumpy 

dumpy larva. Time-lapse imaging AJM-1::GFP (ani-1 RNAi) embryos revealed both ventral 

enclosure failure due to failed migration and fusion of ventral epidermal cells, and elongation 

phenotypes due to failed seam cell shape changes. ani-1 RNAi enhanced alleles of genes 

known to regulate elongation (rho-1, mlc-4, mel-11, let-502, rhgf-2and nmy-1), suggesting that 

ani-1 functions in parallel to the rho-1-mediated actin-myosin seam cell shape changes that 

drive elongation. Furthermore, time-lapse imaging of the enhanced nmy-1(sb113); ani-1 RNAi 

phenotype revealed ventral enclosure defects. Collectively, these findings support roles for 

ani-1 during ventral enclosure and we are currently investigating genetic interactions between 

ani-1 and known regulators of ventral enclosure. 

Contact: nelliefotopo@gmail.com 

Lab: Piekny 


193

A Genome-Wide RNAi Screen to Identify New Components of a 

Muscle-To-Epidermis Mechanotransduction Pathway Essential for 

Embryonic Elongation 

Christelle Gally, Agnes Aubry, Michel Labouesse 

IGBMC, Strasbourg, France 

C. elegans embryonic elongation is controlled by myosin II in the epidermis and muscle 

contractions. Indeed, loss of muscle activity results in elongation arrest at the 2-fold stage 

(Pat phenotype) for a reason that has long remained unclear. We recently unraveled a 

mechanotransduction pathway between muscles and epidermis that accounts to a large extent 

for the Pat phenotype (Nature, 471, 99-103, 2011). Specifically, during the second phase of 

embryonic elongation, muscle contractions trigger a remodeling of hemidesmosomes (HDs) 

in the epidermis through the activation of the serine/threonine p21-activated kinase PAK-1. 

HDs connect the apical and basal sides of the epidermis through intermediate filaments. They 

ensure epidermis integrity and anchor it to the extracellular matrix. We showed that PAK-1 is 

a HD component whose kinase activity controls intermediate filament phosphorylation and 

their anchoring to HDs. PAK-1 seems to relay activity of the adaptor protein GIT-1, with which 

it forms a highly conserved complex together with the RacGEF protein PIX-1 at the HDs. 

GIT-1 appears to be the most upstream target of the mechanotransduction pathway since its 

localization to the HDs is maintained by muscle contraction. However, since git-1(tm1962) 

mutants do not induce a Pat-like embryonic arrest, a prediction is that muscle contraction 

also triggers a second parallel pathway, probably to activate myosin II. In order to identify this 

putative parallel pathway, we have recently undertaken a genome-wide RNAi screen in the 

synthetic git-1(tm1962) mutant background. We are looking for enhancers that might lead to 

a Pat-like elongation arrest. As an approach, we adapted the protocol developed by the lab 

of Andrew Fraser (Nat Protoc, 1, 1617-20, 2006), where we use 96-well worm liquid cultures 

looking for late embryonic arrests. We will present our preliminary findings. 

To better understand the link between HDs biogenesis and embryonic elongation, we are 

also developing new tools to visualize different components of the myosin II pathway and of 

the HDs. One of them is VAB-10A, the homolog of human plectin. vab-10 is a 27kb gene that 

encodes several isoforms. VAB-10A is localized to the HDs. We developed a genomic fusion 

with the GFP to follow dynamics of VAB-10A localization during muscle contraction. We think 

that this new fluorescent tool will help us to complete our view of the mechanotransduction 

pathway that controls embryonic elongation. 

Contact: gally@igbmc.fr 

Lab: Labouesse 

194 


The EXC-1 RAS-Domain Protein Mediates Vesicle Movement in the 

Excretory Canals 

Kelly Grussendorf, Brendan Mattingly, Alex Salem, Matthew Buechner 

University of kansas, Lawrence, (KS), USA 

Tubulogenesis involves formation of tubule shape and diameter along both the apical 

(lumenal) and basal sides. Once formed, the lumen diameter must be maintained as the 

animal moves and grows. The single-cell excretory canal cell provides a simple model to study 

these processes. The cell is located near the terminal bulb of the pharynx, and extends two 

hollow processes to the left and right lateral side of the worm, where they bifurcate and extend 

anteriorly and posteriorly to form an H-shaped structure. A set of nine exc genes maintain the 

narrow diameter of the canal apical surface. Mutations in these genes allow formation of fluidfilled 

cysts in the lumen of the canal. 

The Exc-1 loss-of-function (lof) phenotype shows cysts in the canals that are often located 

at the ends of the canal. These cysts vary in size and number, from cysts not much wider 

than normal lumen to cysts expanded to the entire diameter of the worm. We have cloned the 

exc-1 gene, which encodes a homologue of the RAS GTPase family, specifically the family 

of Interferon-Inducible GTPases (IIGP). This protein is expressed in the canals, and also in 

the amphid sheath, a glial structure that surrounds the amphid neuron sensory endings. exc- 

1 (lof) mutants show accumulation of recycling endosome marker EEA-1, and concomitant 

attenuation of recycling endosome marker RME-1 within the excretory canals, a phenomenon 

also seen for exc-5 mutants. 

Overexpression of exc-1 forms a tubule with a normal apical surface but defective in the 

formation of the basal surface. Epistasis experiments suggest that EXC-1 acts downstream 

of the EXC-9 LIM domain protein, and upstream of the EXC-5 guanine exchange factor. In 

addition, EXC-1 and EXC-9 bind directly to each other, as indicated via yeast two-hybrid assay. 

Our results suggest that these proteins function together to allow efficient movement from early 

endosomes to recycling endosomes. We are conducting further assays to assess binding of 

EXC-1 to other possible target proteins. 

Contact: grusseke@ku.edu 

Lab: Buechner 


195

A Screen For Genes Controlling Vulval Morphogenesis 

Qiutan Yang, Matthias Morf, Sarfarazhussain Farooqui , Juan Escobar, Alex 

Hajnal 

Institute of molecule life science, University of Zurich, Zurich, Switzerland 

The C. elegans egg-laying organ, the vulva, is an outstanding system to investigate the 

principles of organogenesis. Studies of vulval induction have led to a detailed molecular model 

of vulval fate specification, which is based on the concerted action of the conserved EGFR/ 

RAS/MAPK, NOTCH and WNT signaling pathways. However, the molecular mechanisms 

governing vulval morphogenesis are largely unknown. 

After the last of three rounds of cell divisions, the 22 vulval cells undergo morphogenesis, 

which involves several distinct aspects: (1) The formation of a lumen through invagination of 

the vulval cells, (2) the circumferential extension of the cells towards the vulval midline, (3) the 

formation of homotypic contacts and fusion between contralateral partner cells, which results 

in the formation of seven syncytial toroids, (4) the contraction of the ventral lumen, (5) the 

expansion of the dorsal lumen through invasion of the anchor cell (AC) invasion, and (6) the 

eversion of the vulval tissue. 

In order to systematically identify genes required for vulval morphogenesis, we are 

performing a RNA interference (RNAi) screen of all genes, which have been reported to exhibit a 

protruding vulva (Pvl) phenotype when mutated or upon RNAi treatment. Since a Pvl phenotype 

can be caused by defects at any stage of vulval development, we are using the AJM-1::GFP 

reporter to label the apical junctions, which allows us to examine the number and shape of 

the toroids, the size of the vulval lumen and to observe the cell fusions. In our screen, we are 

scoring the morphology of the vulva at the “Christmas tree” stage in mid L4 larvae, after the 

toroids have been formed but before vulval eversion begins. Since most of the genes affecting 

cell fate specification have been previously identified, we are concentrating on candidate genes 

that do not alter the cell fates or vulval lineage but act at a later stage during fate execution. 

We will present our classification of the different morphogenesis phenotypes observed so far 

and further explore the roles of promising candidates genes during vulval morphogenesis. 

Contact: alex.hajnal@imls.uzh.ch 

Lab: Hajnal 

196 


LEP-2/Makorin Promotes let-7 microRNA-mediated Terminal 

Differentiation in Male Tail Tip Morphogenesis 

R Antonio Herrera, Karin Kiontke, Samuel Ahn, David Fitch 

New York University, New York, (NY), US 

In C. elegans, heterochronic genes regulate when stage-specific events occur during larval 

development. They interact in a pathway to ultimately schedule terminal differentiation at the 

last larval stage (L4). In males, heterochronic genes control when a sex-specific terminal 

differentiation program, tail tip morphogenesis (TTM), occurs. During TTM the tail tip cells 

(hyp8-11) change their larval cone-shape by cell fusion, retraction, and migration to produce 

the rounded dome found in adults. Heterochronic genes that specify L4 fates (lin-41 and let-7) 

schedule TTM to start at middle L4. When lost, lin-41 and let-7 cause TTM to occur earlier (in 

L3) or later (in adults), resulting in adult tail tip phenotypes that are over-retracted or unretracted 

and leptoderan-like (Lep), respectively. We found a new heterochronic gene, lep-2, which 

is required for TTM to occur during L4. With comparative genomic hybridization on a lep-2 

deletion mutant, we identified lep-2 as the sole C. elegans Makorin (Mkrn). Mkrns are ancient 

eukaryotic genes which have conserved motifs; a RING domain flanked by four C3H-zinc 

fingers. However, the functional role of Mkrns during development is not well known. We found 

that lep-2 mutant males retain the larval tail tip into adulthood and shift the expression of the 

TTM master regulator, dmd-3, later than expected. lep-2 animals exhibit other developmentaldelay 

phenotypes: they fail to exit the larval molting cycle or produce an “adult” cuticle and, in 

males, the Lep phenotype is suppressed after passage through the dauer larvae stage—all 

characteristics of a mutation in a heterochronic gene. Through epistasis analysis, we have 

determined that lep-2 resides in the heterochronic pathway downstream of lin-14 to promote 

let-7. In lep-2 mutants we observe elevated levels of heterochronic gene reporters that are 

downregulated prior to and during TTM (lin-28 & lin-41). Our genetic data suggest that the 

function of lep-2 is to negatively regulate lin-28, the let-7 repressor. Also, LIN-28 protein levels 

are elevated in lep-2 mutants. LEP-2/Mkrn, LIN-28 and let-7 are highly conserved genes that 

regulate differentiation in mammals in a manner consistent with C. elegans heterochronic 

development. This suggests that an ancient Mkrn function may be to promote let-7 during 

differentiation across eukaryotes. 

Contact: antonio.herrera@nyu.edu 

Lab: Fitch 


197

pix-1 Generates a Gradient of Contraction Forces in Hypodermal Cells 

of Elongating Embryos in Caenorhabditis elegans 

Sharon Harel, Emmanuel Martin, Bernard Nkengfac, Karim Hamiche, Mathieu 

Neault, Sarah Jenna 

UQAM, Montreal, Quebec, Canada 

Early stage of elongation is driven by the contraction of circumferential actin filaments (CAFs) 

in lateral hypodermal cells where myosin-light chains (MLC-4 and MLC-5) are phosphorylated 

by the Rho GTPases effectors LET-502, MRCK-1 and PAK-1. These kinases are antagonized 

by the MLC phosphatase MEL-11 which is active in ventral and dorsal hypodermal cells and 

inactive in the lateral cells were most of the contraction force occurs. The regulators of MLC 

phosphorylation are organized in two parallel pathways the let-502/mel-11/mrck-1 and the 

pak-1 pathways. We identified the Rac- and Cdc-42-GEF, pix-1, as a new component of the 

pak-1 pathway. We showed that pix-1controls early and late stages of elongation in parallel of 

let-502 and mel-11 and in parallel or upstream of the GTPases rac-2 and cdc-42. We also show 

that pix-1 activity during early elongation establishes a gradient of contraction forces along the 

anterior-posterior and the dorsal-ventral axes of the embryo. These contraction gradients are 

required to insure the appropriate morphology and elongation of the larvae. 

Contact: jenna.sarah@uqam.ca 

Lab: Jenna 

198 


Analysis of the Role of ENU-3 in Axon Outgrowth and Guidance in C. 


Callista Yee1 , Karmen Lam2 , Anna Bosanac1 , Marie Killeen1 1 2 Ryerson University, Toronto, York University, 4700 Keele St., Toronto, 

Ontario 

During development, many cells including neurons migrate from their places of birth to their 

final destinations along defined and usually invariant pathways. Development of a properly 

patterned and functional nervous system relies on many guidance cues including Netrin/UNC- 

6, Slit/SLT-1 and the Wnts that guide migrating axons to their final correct destinations and 

allow them to synapse with the correct targets. There are receptors for each of these cues 

expressed on the growth cones at the tips of the migrating axons in chordates and in many 

metazoa. C. elegans has proven to be a good model for analysis of these proteins due to its 

simple nervous system consisting of 302 neurons in the hermaphrodite that can be visualized 

in vivo using appropriate markers. 

We conducted a genetic enhancer screen in an unc-5(e53)background to find mutations that 

enhanced the axon guidance defects of the DA and DB classes of motor neurons and found 

five independent mutants. Mutations in enu-3 had very weak motor axon guidance defects 

and enhanced the short range migration defects of the motor neurons in the hypomorphic 

strain unc-5(e152). The mutations enhanced the motor axon outgrowth defects in worm strains 

lacking either functional Netrin/UNC-6 or its receptor UNC-5 (Yee et al., 2011). Strains lacking 

functional UNC-40 were not significantly affected in motor neuron outgrowth or guidance by 

mutations in enu-3. It is likely that the motor neuron axon outgrowth defects observed in the 

absence of UNC-5 are due to the presence of functional UNC-40. The involvement of UNC- 

40 and ENU-3 in motor axon outgrowth the absence of UNC-5 has been further investigated. 

ENU-3 (H04D03.1) is a novel putative trans-membrane protein of unknown function with 

four close homologues in the C. elegans genome, all larger than ENU-3. All five proteins have 

putative signal peptides and are predicted to be trans-membrane proteins. ENU-3::GFP was 

expressed throughout the nervous system, particularly along the ventral cord. We found that 

enu-3(tm4519) had no significant defects in the migrations of the touch receptor neurons but 

enhanced the defects of an unc-40 mutant strain in an UNC-6 dependent manner. The defects 

observed suggest that the axons may not be properly adherent to the surfaces over which they 

migrate. Our current research is directed towards understanding the nature of the interactions 

between ENU-3 and UNC-40. 

Contact: mkilleen@ryerson.ca 

Lab: Killeen 


199

Identifying Regulators of Gonadal Development in C. elegans by Cellspecific 

Transcriptional Profiling 

Mary Kroetz, David Zarkower 


The gonad of C. elegans originates in the embryo as a four-cell primordium composed 

of two somatic precursor cells (Z1 and Z4) that flank two germ line precursor cells (Z2 and 

Z3). The gonad primordium is morphologically identical in the two sexes, but soon after the 

animal completes embryogenesis it begins to develop via one of two distinct sex-specific 

programs of organogenesis. Despite the extensive sexual dimorphism and previously defined 

cell lineages of the gonad, the genetic pathways that direct the development of this organ, 

including the sex-specific development, remain largely unknown. The overall aim of this work 

is to define the genetic networks that regulate gonadal development in both sexes. To identify 

early gonadal regulators, we used cell-specific transcriptional profiling of Z1/Z4 during mid-L1 

larval development, just prior to the first division of Z1/Z4 when gonadogenesis begins and 

the first sex-specific differences of the gonad arise. We used a Z1/Z4-specific gfp reporter 

to isolate these cells in hermaphrodites by FACS and profiled transcripts by RNA-seq. Of 

the eight transcripts that are known to be enriched in Z1/Z4 during L1, all of them showed 

Z1/Z4-enrichment. Among the 200 most enriched transcripts in Z1/Z4, we identified several 

unannotated transcripts that are highly specific to Z1/Z4. A number of the Z1/Z4-enriched 

transcripts have subsequently been validated by reporter analysis, confirming the effectiveness 

of this approach. We are determining loss of function phenotypes by RNAi depletion and mutant 

analysis. Work is currently underway to identify male-specific Z1/Z4-enriched transcripts from 

fully masculinized XX-pseudomales. Comparisons of male vs hermaphrodite Z1/Z4-enriched 

transcripts will help identify transcripts important for sex-specific gonadal development. 

Contact: kroet006@umn.edu 

Lab: Zarkower 

200 


Caenorhabditis elegans DNA-2 Helicase/Endonuclease Plays A Vital 

Role In Maintaining Genome Stability, Morphogenesis, And Life Span 

Myon-Hee Lee1,2 , Sarah Hollis1 , Bum Ho Yoo3 , Keith Nykamp4 1Brody School of Medicine at East Carolina University, Greenville, NC, USA, 

2Lineberger Comprehensive Cancer Center, University of North Carolina- 

Chapel Hill, Chapel Hill, NC, USA, 3Department of Biochemistry, Yonsei 

University, South Korea, 4Center for Molecular Diagnostics and BioBanking, 

Prevention Genetics, Marshfield, WI, USA 

In eukaryotes, highly conserved Dna2 helicase/endonuclease proteins are involved in DNA 

replication, DNA double-strand break repair, telomere regulation, and mitochondrial function. 

The Dna2 protein assists Fen1 (Flap structure-specific endonuclease 1) protein in the maturation 

of Okazaki fragments. In yeast, Dna2 is absolutely essential for viability, whereas Fen1 is not. In 

C. elegans, however, CRN-1 (a Fen1 homolog) is essential, but Dna2 is not. Here we explored 

the biological function of C. elegans Dna2 (Cedna-2) in multiple developmental processes. 

We find that Cedna-2 contributes to embryonic viability, the morphogenesis of both late-stage 

embryos and male sensory rays, and normal life span. Our results support a model whereby 

CeDNA-2 minimizes genetic defects and maintains genome integrity during cell division and 

DNA replication. These finding may provide insight into the role of Dna2 in other multi-cellular 

organisms, including humans, and could have important implications for development and 

treatment of human conditions linked to the accumulation of genetic defects, such as cancer 

or aging. 

References: 

Lee et al. (2011) Caenorhabditis elegans DNA-2 helicase/endonuclease plays a vital role in maintaining 

genome stability, morphogenesis, and life span. Biochem Biophys Res Commun., 407(3): 495-500. 

Lee et al.(2003) Caenorhabditis elegans dna-2 is involved in DNA repair and is essential for germ-line 

development. FEBS Lett., 555(2): 250-256. 

Lee et al.(2003) Dna2 requirementfor normal reproduction of Caenorhabditis elegans is temperaturedependent. 

Mol Cells,15(1): 81-86. 


Lab: Lee 


201

The Role of LIN-3 During Morphogenesis of the Dorsal Lumen in the 

Vulva 

Louisa Mueller, Matthias Morf, Alex Hajnal 

Institute of Molecular Life Science, Zurich, Switzerland 

The hermaphrodite vulva is an excellent organ to identify and study the molecular 

mechanisms controlling tissue morphogenesis during development. Vulval development is 

initiated by the anchor cell (AC) in the somatic gonad, which secretes LIN-3 EGF and induces 

the vulval cell fates in three of the six adjacent vulval precursor cells. After vulval induction, 

the AC breaches two basal laminae and invades in-between the innermost 1°-fated VPC 

descendants (the VulF cells). AC invasion is important for proper morphogenesis of the dorsal 

lumen formed by the VulF toroids [1] and to establish the uterine-vulval connection. During 

vulval morphogenesis, LIN-3 is secreted from the VulF cells to specify the uv1 fate [2]. Here, we 

investigated another function of LIN-3 produced by VulF during dorsal lumen morphogenesis. 

Vulva-specific lin-3 RNAi using an rde-1(lf) mutant expressing rde-1(wt) in the Pn.p cells 

prevented the expansion of the dorsal lumen by the AC. A similar defect in dorsal lumen 

morphogenesis was observed in egl-38(lf) mutants that do not express LIN-3 in VulF cells 

[1,3]. Moreover, egl-38(lf) mutants displayed defects in AC polarization. Based on these results, 

we propose that LIN-3 expressed by the VulF cells controls dorsal lumen morphogenesis by 

polarizing the AC and thus enabling it to migrate in between the VulF cells and expand the 

dorsal lumen. 

[1] Estes, K. A. and Hanna-Rose, W. (2009). Dev Biol 328, 297-304. 

[2] Chang, C., Newman, A. P. and Sternberg, P.W. (1999). Curr Biol 9, 237-46. 

[3] Rajakumar, V. and Chamberlin, H. M. (2007). Dev Biol 301, 240-53. 

Contact: louisa.mueller@imls.uzh.ch 

Lab: Hajnal 

202 


Somatic gonad precursor migration in C. elegans 

Monica Rohrschneider, Jeremy Nance 

New York University School of Medicine, New York, NY, USA 

In many organisms, the somatic gonad precursor cells (SGPs) and the primordial germ 

cells (PGCs) are born at a distance from one another, and must migrate in order to coalesce 

and form the primordial gonad. In C. elegans, the SGPs migrate nearly half the length of the 

embryo to reach the PGCs. This migration is critical, as the SGPs are required for survival and 

proliferation of the germ cells. However, little is known about what drives the SGPs to migrate, 

and what triggers them to stop. 

As a first step in addressing these questions, we constructed fluorescent transgenic strains 

to characterize the migration of the SGPs and their interactions with neighboring cells. We 

observed three distinct phases of SGP migration—first the SGPs migrated posteriorly along 

the edge of the endoderm. When they reached the PGCs, the SGPs extended a single long 

projection around the posterior of the PGCs. And finally the SGPs wrapped completely around 

the PGCs. 

We used genetic transformation of the PGCs and of endoderm cells to test the hypothesis 

that these cells are required for the three phases of SGP migration. Surprisingly, SGP posterior 

migration was grossly normal in mes-1 mutants which lack PGCs, and in end-1 end-3 mutants, 

which lack endoderm, suggesting that PGCs and endoderm do not provide a long-range 

attractive cue to the SGPs. However, SGPs extended longer and more disorganized projections 

in mes-1 mutants, and continued to extend projections long after wild-type SGPs had wrapped 

around the PGCs, suggesting that PGCs direct SGP wrapping. In end-1 end-3 mutants, SGP 

projections and wrapping were partially disrupted, suggesting that endoderm development or 

morphogenesis may be required for normal SGP wrapping of PGCs. 

Because the SGPs migrate posteriorly between the endoderm and mesoderm cell layers, 

and it had previously been reported that basement membrane forms between the germ layers, 

we depleted laminin to investigate the role of the basement membrane in SGP migration. In 

lam-1(RNAi) embryos, the SGPs migrate farther posteriorly, while still extending relatively 

normal projections and eventually wrapping around the PGCs. Therefore, basement membrane 

may be required for the SGPs to stop migrating when they reach the PGCs. Our analysis of 

SGP migration provides a foundation for identifying the molecular mechanisms that promote 

each step of primordial gonad assembly. 

Contact: Monica.Rohrschneider@med.nyu.edu 

Lab: Nance 


203

VAB-9 and Vertebrate Orthologue TM4SF10 Cooperate with Adherens 

Junction Proteins and Actomyosin to Regulate Epithelial Polarity and 

Morphogenesis 

Jeff Simske 

Rammelkamp Center, Cleveland (OH), USA 

Regulation of morphogensis and cell polarity requires the coordinated interaction between 

the actomyosin contractile apparatus and cellular junctions. VAB-9 is an adherens junction 

protein belonging to the claudin/PMP22/EMP family of tetraspan integral membrane proteins. 

vab-9 mutants have variable defects in elongation. Elongation requires coordinated contraction 

of actomyosin cables in the embryonic epidermis following enclosure. Non-muscle myosin 

activity is regulated in part by phosphorylation and activation of the regulatory light chain by Rho 

kinase LET-502 and de-phosphorylation and inactivation by the phosphatase MEL-11. Prior to 

elongation, MEL-11 is present in the cytoplasm of epidermal cells, but becomes localized to cell 

junctions as elongation proceeds, suggesting that preventing MEL-11 inhibition of actomyosin 

contractility requires junctional re-localization (Piekney and Mains, 2003). VAB-9, MEL-11 and 

activated myosin light chain proteins all co-localize at the cell junctions of enclosing epidermal 

cells and remain at seam cell junctions during elongation. Co-localization between MEL-11 

and activated myosin light chain at junctions suggests regulation of myosin activity by MEL- 

11 is complex and highly dynamic. In vab-9 mutants, MEL-11 fails to localize to cell junctions, 

indicating that VAB-9 promotes elongation through MEL-11 sequestration at junctions. In 

vab-9 animals activated myosin light chain levels appear lower in the cytosol, possibly due to 

inactivation by mislocalized MEL-11. vab-9 mutations suppress reduction-of-function mel-11 

enclosure defects and sterility; similarly, mel-11 activity is required for VAB-9 localization, and 

altering actomyosin activity modifies vab-9 phenotypes. The VAB-9 vertebrate orthologue 

TM4SF10 was examined in MDCK cells to determine whether function is conserved and to 

characterize TM4SF10 protein complexes. Since vab-9 can be functionally replaced by a GFPtagged 

version of the vertebrate protein TM4SF10, it is likely that at least some functions are 

conserved. In MDCK cells, TM4SF10-GFP co-localizes with and co-IPs with adherens junction 

proteins. Overexpression of TM4SF10-GFP results in delay in reformation of cell junctions 

following calcium switch, reduced apical surface area, altered cell polarity, disorganized cell 

junctions and altered F-actin organization. SiRNA inactivation appears to generally result in 

the opposite phenotypes. Direct interaction between VAB-9/TM4SF10 and actomyosin proteins 

has not been demonstrated in either system. 

Contact: jsimske@metrohealth.org 

Lab: Simske 

204 


The C. elegans DM domain genes dmd-3 and mab-3 function during 

the late stages of male gonad development 

Michele Smith1 , Alyssa Herrmann1 , Emily Kivlehan1 , Lauren Whipple1 , Douglas 

Portman2 , D. Adam Mason1,2 1 2 Siena College, Loudonville, (NY), 12211, USA, University of Rochester, 

Rochester, (NY) USA 

The development of an organism from a fertilized egg into a fully formed adult is a spectacularly 

complex process. Complexity is further compounded by the fact that, in most animal species, the 

sex-determination pathway must modify developmental pathways in order to generate two sexually 

dimorphic body forms. Previous studies have demonstrated that DM-domain transcription factors 

control sex-specific development in diverse animal phyla, suggesting that these genes were part of 

a core sexual differentiation pathway in the common ancestor of all eumetozoans. We have been 

examining the function of two C. elegans DM-domain transcription factors, DMD-3 and MAB-3, in 

directing the development of male-specific structures. We have previously observed that, DMD-3 

and MAB-3 play a central role in guiding the male-specific remodeling of the L4 tail that generates 

the sexually dimorphic blunt-ended adult male tail. 

The expression patterns of dmd-3 and mab-3 reporter transgenes suggested that these genes 

also play a role in directing male gonad development. Beginning in mid-L3 males, both dmd-3 and 

mab-3 are expressed in the male-specific linker cell, a cell that functions to lead the male gonad 

from the mid-body down to the hindgut. In addition, in early L4 males dmd-3 expression commences 

in a subset of vas deferens cells that lie directly behind the migrating linker cell. Finally, dmd-3 is 

expressed male-specifically in the hindgut cells that eventually engulf the linker cell in mid-L4 males. 

Consistent with this expression pattern, mab-3 ; dmd-3double mutant males show distinct defects 

in linker cell migration. We observe that the linker cell fails complete its migration down to the hindgut 

in some mab-3; dmd-3 double mutant L4 males. In addition, double mutant linker cells exhibit an 

aberrant morphology during migration. Specifically, we observe that a high percentage of double 

mutant linker cells project out long cellular processes in both the anterior and posterior direction 

during the final leg of their migration in early L4 males. This occasionally culminates in pieces of the 

linker cell becoming detached from the main cell body. dmd-3single mutant males exhibit a similar, 

but less severe defect in linker cell migration, while linker cell migration in mab-3 mutants appears 

indistinguishable from wild type. Earlier stages of linker cell migration appear normal in the single and 

double mutant males. In addition, we find that linker cells that do complete migration fail to be engulfed 

properly by the hindgut cells in mab-3 ; dmd-3 late-L4 males, resulting in the persistence of the linker 

cell into adulthood. The failure of the linker cell to reach and/or be engulfed by the hindgut should 

result in a gonad that does not properly connect to the cloaca. Together, these results demonstrate 

that DMD-3 and MAB-3 function together to direct the late stages of male gonad development. 

Contact: me21smit@siena.edu 

Lab: Mason 


205

Analysis of Non-Muscle Myosin II During Dorsal Intercalation in 


Elise Walck-Shannon, Jeff Hardin 


Cell intercalation is a morphogenetic movement that is used throughout animal development 

to shorten a tissue along one axis and extend it along the orthogonal axis. Dorsal intercalation 

within the C. elegans embryonic epidermis is a simple model to study cell intercalation. During 

dorsal intercalation, two rows of ten epidermal cells converge into one row of twenty cells. 

Non-muscle myosin II is required for cell intercalation in multiple systems; however, its role 

during these processes is not well understood. In C.elegans there are at least two non-muscle 

myosin II isoforms expressed during embryonic development that differ in their heavy chains, 

which are encoded by nmy-1 and nmy-2. I find that non-muscle myosin II has isoform-specific 

requirements immediately after dorsal cell division that are essential for dorsal intercalation 

to complete in C. elegans. Loss of function for nmy-2 alone yields misshapen cells that still 

increase in length, while loss of both nmy-1 and nmy-2 function yields misshapen cells that do 

not elongate. As dorsal cells acquire their normal polarized morphology shortly after division, 

NMY-2::GFP filaments accumulate inthe apical cortex and show periodic changes in intensity 

that are inversely correlated to cell area. Together, this suggests that non-muscle myosin II has 

a role early in intercalation. In future studies, I plan to study both the regulation of contractile 

activity by the phosphorylation of myosin regulatory light chain and the regulation of non-muscle 

myosin II localization. Determining the normal regulation and function of non-muscle myosin II 

during cell intercalation could have broad implications for both its misregulation during disease 

and its role during normal development. 

Contact: walck@wisc.edu 

Lab: Hardin 

206 


Establishing Caenorhabditis elegans as a Model for Neural Tube 

Defects 

Bridget Waller, Kassi Crocker, Timothy Walston 

Truman State University 

The exact causes of most neural tube defects (NTDs) remain unknown. The disabling birth 

defect spina bifida, however, may result from a combination of genetic and environmental risk 

factors, including alcohol consumption early in pregnancy. Morphogenesis in Caenorhabditis 

elegans involves similar cell movements to what is seen in the vertebrate neural tube and 

many similar molecular contributions. The ease of embryonic study makes C. elegans a 

tractable model to understand the mechanisms that affect cell migration. Therefore, the goal 

of this project is to establish C. elegans as a model for NTDs through studying the embryonic 

defects that result from alcohol exposure. In this experiment, C. elegans embryos exposed in 

utero to 300 mM alcohol, equivalent to a BAC of 0.075, experienced a lethality rate of 47.25% 

(n=3200). Current work is classifying the stage of failed embryogenesis and the morphogenetic 

movements that are sensitive to exposure to alcohol. 

Contact: tdwalston@truman.edu 

Lab: Walston 


207

Anillin is required for Epidermal Morphogenesis during C. elegans 

Embryogenesis 

Denise Wernike, Alisa Piekny 

Concordia University, Montreal 

In the early embryo, anillin (ANI-1) serves as a scaffold for actin, myosin and membranebinding 

septins during cytokinesis. Cytokinesis is the process when one mother cell gives 

rise to two genetically identical daughter cells. It requires the rearrangement of cytoskeletal 

components to form an actin-myosin contractile ring, which ingresses to pinch in the cytoplasm 

of the cell. Anillin crosslinks and controls the dynamics of contractile ring components to ensure 

that the ring closure occurs with high fidelity. Cytokinesis can be interpreted as a form of cell 

shape change comparable to morphogenetic events that take place during tissue formation in 

C. elegans embryogenesis. Epidermal cells that undergo these events must rearrange their 

cytoskeleton, using actin-myosin filaments for cell shape changes and migrations to enclose 

the embryo and to drive its shape change. However, it is not known if anillin fulfills functions 

outside of cell division, particularly during C. elegans embryonic morphogenesis. We recently 

determined that anillin is maternally and zygotically required throughout embryogenesis, and 

RNAi-depleted embryos display phenotypes consistent with roles for ani-1 in ventral enclosure 

and elongation. Live-imaging of ani-1 RNAi embryos during ventral enclosure revealed that 

epidermal cells failed to meet at the ventral midline. In order to further explore a role for ani-1 

in ventral enclosure, genetic epistasis experiments were performed with known regulators of 

cell adhesion. Adherens junctions are believed to play a pivotal role during C. elegans ventral 

enclosure, where they tightly seal the migrating ventral cells along the ventral midline. Adherens 

junctions consist of an extracellular domain, termed cadherin, that connects adjacent epithelial 

cells to each other, and an intracellular region that directly links to the actin cytoskeleton through 

its interaction with α-catenin and β-catenin. We found that hypomorphic alleles of cadherin and 

catenin enhanced anillin loss-of-function phenotypes. In addition, overexpression of α-catenin 

significantly alleviated anillin phenotypes, suggesting that anillin and α-catenin are in the same 

pathway. Further experiments are being performed to elucidate the relationship between anillin 

and components of the cell adhesion machinery. 

Contact: denise.wernike@gmail.com 

Lab: Piekny 

208 


What Causes Partial Penetrance of a Developmental Phenotype? 

Claire Williams1,2 , Maxwell Heiman1,2 1 2 Children’s Hospital, Boston, MA, USA, Harvard Medical School, Boston, 

MA, USA 

Partial penetrance is a poorly understood phenomenon that hinders the accurate prediction 

of phenotype from genotype. From first principles, partial penetrance implies a variable activity 

and a thresholding mechanism that partitions that activity into an “all or none” phenotype. To 

address the question of which activities may be variable and subject to a threshold, we have 

turned to an example in C. elegans neurodevelopment. Amphid sensory neuron dendrites are 

normally anchored at the nose tip by DYF-7, an extracellular matrix protein with a zona pellucida 

domain. Individual animals bearing a partially penetrant dyf-7 allele, dyf-7(ns117), exhibit one 

of two phenotypes, either short or full-length dendrites, even within isogenic populations raised 

in a shared environment. This partial penetrance suggests that, in these mutants, a limiting 

activity in dendrite development is subject to stochastic variability. To determine if this limiting 

activity is that of dyf-7(ns117) itself, we increased the dosage of dyf-7(ns117) in an otherwise 

dyf-7-null genetic background. Indeed, higher dosages of dyf-7(ns117) led to a decrease in 

penetrance of the abnormal phenotype, implying that some aspect of dyf-7(ns117) expression is 

the limiting, variable activity. This variability could arise at the level of transcription, translation, 

or protein processing and trafficking: DYF-7 normally undergoes extracellular proteolytic 

cleavage to assemble a “cap” at the dendrite tip. To visualize DYF-7 processing and trafficking 

in vivo, we tagged DYF-7 with an extracellular GFP and a cytoplasmic mCherry and observed 

differential localization of the two tags, consistent with proteolytic cleavage. The extracellular 

tag assembled in caps at dendrite tips while the cytoplasmic tag spread diffusely throughout 

cell membranes. In contrast, the extracellular and cytoplasmic tags on DYF-7(ns117) showed 

extensive overlap and were both present throughout cell membranes, suggesting that defective 

protein processing underlies the partial penetrance of this phenotype. Previous examples of 

partial penetrance have involved transcriptional variability and a thresholding mechanism 

based on positive feedback loops in gene regulatory networks. Thus, dyf-7(ns117) presents a 

counterexample in which variability in protein processing is somehow thresholded to produce 

an “all or none” developmental phenotype. 

Contact: cwilliams@genetics.med.harvard.edu 

Lab: Heiman 


209

MIG-10 interacts with ABI-1 to induce asymmetric outgrowthpromoting 

activity in response to guidance cues 

Yan Xu, Christopher Quinn 

University of Wisconsin Milwaukee 

Actin regulatory proteins have been implicated in the control of growth cone morphology. 

However, little is known about how these proteins are spatially organized to orchestrate the 

directional response to axon guidance cues. A key to understanding this process may be 

provided by the asymmetric accumulation of the MIG-10 (lamellipodin) cytoplasmic scaffolding 

protein in response to guidance cues. However, the mechanism that links MIG-10 to actin 

polymerization is not known. Using an RNAi screen in C. elegans, we identified the actin 

regulatory protein ABI-1 (Abi1) as a partner for MIG-10. We find that MIG-10 binds to the SH3 

domain of ABI-1 and dosage sensitive genetic interactions indicate that MIG-10, ABI-1 and 

WVE-1 function together to mediate axon guidance. Analysis of double mutants shows that 

these proteins function in both the attractive response to UNC-6 (netrin) and the repulsive 

response to SLT-1 (slit). Epistasis analysis reveals that ABI-1 and WVE-1 function downstream 

of MIG-10 to mediate its outgrowth-promoting activity. Moreover, experiments with cultured 

mammalian cells suggest that the interaction between MIG-10 and ABI-1 mediates a conserved 

mechanism that promotes formation of lamellipodia. Together, these observations indicate that 

ABI-1 and WVE-1 mediate the outgrowth-promoting activity of MIG-10 to spatially direct axon 

growth in response to the UNC-6 and SLT-1 guidance cues. 

Contact: xu2@uwm.edu 

Lab: Quinn 

210 


Molecular characterization of maternally malformed 3 (mal-3) 

Yemima Budirahardja1 , Thang Doan1 , Ronen Zaidel Bar1,2 1 2 Mechanobiology Institute Singapore, Singapore, Department of 

Bioengineering, National University of Singapore, Singapore 

During C. elegans morphogenesis, the ovoid-shaped embryo elongates four fold into a 

worm-shaped larva before hatching. Defects in elongation result in larval/adult body shape 

defects or developmental arrest. Genetic screens for maternal effect morphologically abnormal 

worms are likely to uncover genes required during embryonic morphogenesis. We are using 

whole genome sequencing to identify the affected genes in several mutant strains isolated by 

Hekimi et. al. in 1995 in such a screen [1]. One of the strains, MQ466 (mal-3), shows specific 

defects in elongation. It is temperature sensitive with close to 5% embryonic lethality (Emb) 

at 15°C, over 50% Emb at 20°C, and approximately 90% Emb at 25°C. Time-lapse Nomarski 

imaging at 20°C showed the majority of Emb is due to elongation arrest, concomitant with 

the formation of vacuoles within the embryo. Visualizing the junctional protein alpha-catenin/ 

HMP-1 in mutant embryos revealed improper cell alignment during ventral enclosure and what 

appears like uneven pulling forces perpendicular to the seam cells during elongation. Whole 

genome sequencing suggests mal-3 most likely results from a single missense mutation in 

T09B4.1, a mannosyltransferase involved in the synthesis of glycosylphosphatidylinositol 

(GPI). Attachment of a GPI-anchor is a post-translational modification leading to anchoring of a 

protein to the outer leaflet of the plasma membrane. Recently, GPI synthesis was shown to be 

essential for germline development in the worm [2], but its role in morphogenesis is currently 

unknown. Among the candidate GPI-anchored proteins in C. elegans are several cell adhesion 

and signaling proteins. Ongoing work is aimed at validating the molecular nature of mal-3 and 

at identifying the presumed GPI-anchored proteins whose function is critical for elongation. 

References: 

[1] Hekimi, S., Boutis, P., and Lakowski, B. (1995). Viable maternal-effect mutations that affect the 

development of the nematode Caenorhabditis elegans. Genetics 141, 1351-1364. 

[2] Murata, D., Nomura, K.H., Dejima, K., Mizuguchi, S., Kawasaki, N., Matsuishi-Nakajima, Y., Ito, S., 

Gengyo-Ando, K., Kage-Nakadai, E., Mitani, S., et al. (2012). GPI-anchor synthesis is indispensable for 

the germline development of the nematode Caenorhabditis elegans. Mol Biol Cell 23, 982-995. 

Contact: biezbr@nus.edu.sg 

Lab: Zaidel-Bar 


211

A Semi-Automated Pipeline for the Identification of Novel Mutants 

with Cell Number Defects 

Peter Appleford, Alison Woollard 

University of Oxford, Oxford, UK 

C. elegans RNAi screens provide a rapid and convenient way to identify novel genes 

involved in cell and developmental processes. However, not all tissues are amenable to 

silencing by RNAi and the knock down effect itself is both transient and variable. With the 

advent of cheaper whole-genome resequencing, traditional forward genetic screens are once 

again becoming a more attractive proposition. 

Although the mapping of new alleles by Whole Genome Sequencing (WGS) has been 

demonstrated to work by at least two similar methodologies, the initial screening for interesting 

phenotypes still represents a significant bottleneck in terms of the labour and time required. 

Our lab is interested in the mechanisms controlling the balance between proliferation and 

differentiation in the stem-like seam cell lineage. We have used the Biosorter (essentially, a 

large particle flow cytometer) developed by Union Biometrica to test whether such a platform is 

suitable for counting seam cells in the worm, utilising expression of the seam-specific scm::gfp 

reporter. As proof of principle, we initially showed that the Biosorter is capable of scoring the 

expected 16 peaks corresponding to seam nuclei present in late L4 hermaphrodites and that 

worms with aberrantly high numbers of seam nuclei are also correctly identified. 

We have now conducted three pilot screens with the Biosorter and have isolated at least 12 

mutant strains which exhibit a range of defects in seam cell development, including seam cell 

hyperplasia and seam cell spacing abnormalities. We are currently preparing mutant strains 

for sequence analysis and we will present our progress at the meeting. 

Contact: peter.appleford@bioch.ox.ac.uk 

Lab: Woollard 

212 

Poster Topic: New Technologies

A Novel Fluorescence-Based Method to Visualize Protein-Protein 

Interactions in Living Caenorhabditis elegans 

Han Ting Chou, Casonya Johnson 

GSU, Atlanta, Georgia, USA 

Molecular and genetic studies have shown that proteins work in multimeric complexes to 

direct cellular events, and that transcription factors, in particular, can use selective interactions 

to differentially regulate gene expression. Existing methods for detecting these interactions are 

limited in the information that they provide, usually because the method of detection is most 

appropriate for studies done in cell culture or in single-celled organisms. Here we developed 

a transformative method to detect transient interactions among transcription factors within 

the nuclei of the cells of living animals. The transparent nematode Caenorhabditis elegans 

was used as the model organism and fluorescence imaging was used to detect interaction 

between a transcription factor tethered to the nuclear membrane and a fluorescently-labeled 

partner. Experiments were carried to study interactions of the REF-1 family proteins of the basic 

helix-loop-helix (bHLH) transcription factor superfamily involved in a wide range of biological 

processes. Interactions between HLH-29 and its dimerization partners were monitored during 

the normal life-cycle of wild-type hermaphrodites. This novel technique of capturing dynamic 

protein-protein interactions complements genome-wide studies of transcriptional networks 

that have been critical for the mechanistic and systems-wide studies being performed today. 

Contact: hanting@yahoo.com 

Lab: Johnson 

Poster Topic: New Technologies 

213

Spectrum: Building Pathways to Biomedical Research Careers for 

Girls and Women of Color 

Diana Chu, Rebecca Garcia, Kimberly Tanner 

San Francisco State University, San Francisco, CA, USA 

While progress has been made in encouraging girls in science, women of color are still 

largely absent from the biomedical research community and few materials or models currently 

exist that are designed specifically to attract girls of color tothese careers. The Science 

Education Partnership and Assessment Laboratory (SEPAL) in the Department of Biology 

at San Francisco State University (SFSU) has developed the Spectrum effort to address the 

dearth of women of color in biology. Through Spectrum, biomedical scientists who are women 

of color – including SFSU undergraduate students, Masters students, alumni in local doctoral 

and biotechnology positions, and faculty – and middle and high school students and teachers 

collaborate to: 1) co-sponsor after-school science clubs targeted at girls of color in high needs 

public schools, 2) develop a mentoring community of women of color trainees in biomedical 

research, 3) develop a series of video biographies that highlight the research programs of 

women of color biomedical researchers and scientific trainees, and 4) partner with the local 

and national Expanding Your Horizons organizations to disseminate Spectrum activities. During 

its initial three years, Spectrum engaged 279 middle and high school girls (46% Latina, 12% 

African American, 21% Asian, 13% Unknown, 8%White) across five club sites providing ~20 

hours of academic enrichment inbiomedical science for each girl, including two field trips to 

the laboratories of SFSU women of color biologists. Evaluation data shows increases in the 

percentage of participating girls who agree with the following statements: 1) I have heard a 

woman scientist talk about how she became a scientist (pre: 50%, post: 92%), 2) I have heard 

a woman scientist talk about why she likes science (pre:61%, post: 95%), and 3) I have met a 

woman scientist who is like me (pre: 33%, post: 62%). Spectrum is supported by the National 

Center for Research Resources and the Division of Program Coordination, Planning, and 

Strategic Initiatives of the National Institutes of Health through R25RR024307, Supplement 

R25RR024307-05S, and Supplement R25RR024307-03S1. 

Contact: chud@sfsu.edu 

Lab: Chu 

214 


Establishing and using a modified NGM (ENGM) to culture an 

manipulate the entomopathogenic nematode, Heterorhabditis 

bacteriophora 

Zsofia Csanadi1 , Abate Birhan Addise2 , Anita Alexa3 , Barnabas Jenes4 , Zsofia 

Banfalvi4 , Andrea Mathe-Fodor5 , Katalin Belafi-Bako1 , Andras Fodor2 1Research Institute of Bioengineering, Membrane Technology and 

Energetics H-8200, Veszprem, (Egyetem), Hungary, 2Institute of Animal 

Science & Breeding, University of Pannonia, Keszthely, Deak F., Hugary, 

3Department of Biochemistry, Eotvos Lorand University, Budapest, 

Pazmany, Hugary, 4Biotechnology Research Center, Godollo, Szent- 

Gyorgyi, Hungary, 5Molecular and Cellular Imaging Center, Ohio State 

University, Wooster, (OH), United States 

Entomopathogenic nematode (EPN) species belonging to Heterorhabditis and Steinernema 

genera similarly to C. elegans are feeding on bacteria. But they can grow only on their own 

symbionts, Xenorhabdus and Photorhabdus bacteria, respectively. NGM is not an appropriate 

medium for neither of the symbiotic partners. 

In order to make reliable genetics on EPN species we developed a new solid media called 

ENGM (Entomopathogenic Nematode Growth Media) on which both H. bacteriophora and C. 

elegans develop normally. They are visible on the plate under stereomicroscope throughout 

their life cycles. We compromised the advantages of NGM and Woots agar media, and tested 

different ingredients both qualitatively and quantitatively. 

A proof of the usefulness of the new (ENGM) media is that we managed produce RNAi 

phenocopies from both C. elegans and H. bacteriophora by feeding them with the appropriate 

construction. In case of H. bacteriophora it was a Photorhabdus strain transformed with C. 

elegans dpy-3 feeding construction. 

We have started a project to produce and breed transgenic H. bacteriophora expressing 

tps1 gene from the yeast. We found that the Gene Booster technique is amenable for producing 

transgenic Heterorhabditis expressing govern by heat-inducible (hsp2) promoter governed 

yeast tps-1 cloned into the appropriate Fire vector. The phenotype is high osmotic tolerance. 

Contact: csanadi@almos.uni-pannon.hu 

Lab: Belafi-Bako 


215

A MultiSite Gateway®-Compatible Three-Fragment Vector 

Construction Kit Using Galactose Selection 

Iskra Katic, Wolfgang Maier 

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland 

Invitrogen’s Gateway system, which uses recombinational cloning through phage lambda 

integrase, offers speed and reliability in creating molecular constructs. MultiSite Gateway 

enables modular design of expression vectors containing promoters, genes, and 3’UTRs in a 

sequence- and orientation-specific manner. The efficiency of Gateway cloning relies strongly 

on a negative selection against the ccdB death gene, which kills cells transformed with nonrecombined 

educt vector. 

We and others have observed occasional mutations in the ccdB counter-selection cassette 

causing many false positive clones in subsequent Gateway reactions. We have therefore 

replaced the ccdB cassette with a galactokinase (galK) cassette, which, in principle, allows for 

both positive and negative selection. This system offers several advantages over conventional 

Gateway cloning using ccdB selection, including easy maintenance of donor and destination 

vectors. We have created a MultiSite Gateway-compatible vector construction kit, which also 

contains MosSCI and MosSCI-biotic destination vectors. 

Contact: iskra.katic@fmi.ch 

Lab: N/A 

216 


Screening for C. elegans Mutants with Subtle Phenotypes with 

Microfluidics and Computer Vision 

Adriana San-Miguel1 , Matthew Crane1 , Peri Kurshan2 , Kang Shen2 , Hang Lu1 1 2 Georgia Institute of Technology, Atlanta, (GA), USA, Stanford University, 

Stanford, (CA), USA 

Genetic screens in C. elegans have led to understanding the function of many relevant 

genes by detecting animals with interesting phenotypes. The identification of mutants with 

significantly altered phenotypes, easily identifiable by simple visual inspection, has reached a 

saturation point. Aside from the difficulty of typical screens that require manual handling and 

inspection of a very large number of animals, the current challenge lies on the identification 

of mutants with very subtle phenotypes difficult to identify by eye. Performing screens based 

on fluorescent reporters of very small features, such as synapses, present an exceptionally 

difficult scenario. 

The challenges include extracting quantitative information regarding synapse size, intensity 

and distribution from images where lipid droplets and gutauto-florescence is present. While 

human eyes can distinguish between some of these, they are not sensitive to slight differences 

in size, intensity or size distribution of such small features. Here, we use computer vision 

algorithms to objectively quantify relevant information from a mutagenized animal population 

and thus, identify mutants with subtle phenotypes. 

Additionally, we utilize microfluidic devices as a platform for automated worm imaging, 

handling and sorting. The developed microfluidic device has several advantages over devices 

previously used for worm imaging: it is a robust single layer device which can be easily fabricated 

without the need of alignment or incorporation of valves in a second layer. Moreover, the device 

has been designed to orient the worms in a dorsal-down position with the purpose of imaging 

the posterior dorsal area in all worms, where synapses are located. Computer algorithms allow 

the identification of worms which are in the right orientation and are suitable for imaging, as 

well as those which have a slightly altered phenotype and are sorted as mutants. 

Integrating microfluidics and computer vision we have generated a platform for automated 

high-throughput imaging and sorting of thousands of worms based on quantitative synapserelated 

features. This method enables imaging, phenotyping and sorting about 100 times 

faster than manual handling. Not only does this method allow performing genetic screens in 

a simple, automated and fast manner, but it also provides a platform for discovery of mutants 

which would otherwise be overlooked in a typical manual screen. 

Contact: asanmiguel@gatech.edu 

Lab: Lu 


217

Two Novel Staining Protocols Resolve Caenorhabditis elegans 

Cuticular Structures For Live Imaging And Transmission Electron 

Microscopy 

Robbie Schultz 1 , E. Ann Ellis 2 , Tina Gumienny 1 

1 Molecular and Cellular Medicine, Texas A&M Health Science Center, 

College Station, TX, USA, 2 Microscopy and Imaging Center, Texas A&M 

University, College Station, TX, USA 

The C. elegans cuticle is a transparent exoskeleton that surrounds the animal, protecting 

the organism from the environment and facilitating locomotion. The cuticle is composed of 

several layers, including multiple inner collagen layers and an outer lipid layer. Ultrastructure 

patterns the cuticle, where alae form longitudinal ridges that run the length of the animal and 

annuli form circumferential ridges around the animal. While this ultrastructure is of interest 

to many researchers using C. elegans, it is not easily distinguished using standard methods. 

We have developed two techniques to visualize C. elegans cuticle structures: staining living 

organisms using a vital lipophilic dye, DiI (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine 

perchlorate), for compound or confocal microscopy studies, and staining and fixing tissues 

with malachite green for transmission electron microscopy (TEM) studies. 

For live animal imaging, DiI fluorescently stains the outermost lipid layer. This stain resolves 

both annuli and alae (when present) [1]. Also, finer structures of the adult male tail, including 

rays and the fan, are highlighted upon staining with DiI [1]. 

For TEM studies of adult C. elegans, malachite green binds to the lipid layer that surrounds 

the animal, including the outer layer of the alae. Malachite green also preserves and differentially 

stains the inner layers of cuticle, distinguishing the cortical, medial, and basal layers. 

We demonstrate DiI and malachite green dyes are useful tools to resolve the structure of 

the C. elegans cuticle for correlative studies using compound/confocal and TEM microscopy, 

respectively. 

Reference 

[1] R. D. Schultz, T. L. Gumienny, JoVE (2012) 59: e3362. 

Contact: Schultz@medicine.tamhsc.edu 

Lab: Gumienny 

218 


Improving the Sensitivity and Selectivity of Mutation Identification by 

Next-Generation Sequencing 

Sijung Yun, Michael Krause, Harold Smith 

NIDDK/NIH, Bethesda, MD 

Next-generation sequencing provides a rapid and powerful means for identifying 

mutations on a genome-wide scale. Advances in sequencing technology have sparked similar 

improvements in software for sequence analysis. We have developed a modular analysis 

pipeline for mutation identification that uses a combination of publicly available tools: BFAST 

(for alignment), SAMTools (for mutation calling), and ANNOVAR (for annotation). We compared 

our results to those obtained using MAQGene, the standard analysis pipeline for C. elegans 

mutation identification. We observed a large degree of overlap between the sets of singlenucleotide 

polymorphisms (SNPs) identified by the two pipelines. For SNPs common to both 

pipelines, validation by Sanger sequencing indicated a high degree of accuracy. Similar 

analysis of the SNPs unique to each pipeline revealed a high false-positive rate for both, as 

well as a modest false-negative rate for MAQGene. We defined additional criteria that allow 

us to discriminate false-positive from true-positive SNP calls. We also validated a number of 

small insertions and deletions (indels) that were not detected using MAQGene. Our pipeline 

provides advantages for mutation identification in terms of sensitivity (by recovering SNPs 

and small indels that were previously missed) as well as selectivity (by limiting the number of 

false-positive SNP calls). 

Contact: smithhe2@niddk.nih.gov 

Lab: Krause 


219

Worm Proteins Overtake Biochemistry Lab to Inspire Inquiry 

Katherine Walstrom 

New College of Florida, Sarasota, FL 

During Spring 2012, I changed my Biochemistry Lab course from a mostly “cookbook” lab 

course to an inquiry-based course. I chose lactate dehydrogenase (LDH-1) and four C. elegans 

predicted alcohol dehydrogenases as enzymes the students could study. Each group chose 

an enzyme, and they subcloned the cDNA into a protein expression vector and purified the 

enzyme during the first half of the course. During the rest of the course, the students proposed 

research projects to perform with their enzyme (if it had detectable activity) or with a similar, 

purchased enzyme. All seven groups proposed very different research projects, and some 

were more sophisticated than others. Most of the projects could eventually be developed 

into an undergraduate thesis project, which is required for all students at our institution. The 

projects addressed two main topics. One group of projects involved site-directed mutagenesis 

to change the substrate specificity of LDH-1. The other projects involved removing the zinc 

ion(s) in the alcohol dehydrogenases and replacing them with other metal ions. Enzyme kinetics 

experiments will be performed with the original and modified enzymes. The experiments we 

performed could easily be adapted to other lab courses or for other undergraduate research 

projects. I am also taking suggestions for enzymes to study next year! The most successful 

projects will involve enzymes with fewer than ~500 amino acids and with a predicted enzyme 

activity that can be detected using UV-VIS spectroscopy. 

Contact: kwalstrom@gmail.com 

Lab: Walstrom 

220 


Understanding temporal and spatial features of polarity establishment 

Simon Blanchoud1 , Felix Naef2 , Pierre Gonczy1 1 2 EPFL SV ISREC, Lausanne, Switzerland, EPFL SV IBI, Lausanne, 

Switzerland 

Even though polarity establishment in the one-cell stage C. elegans embryo has been 

studied qualitatively using forward genetic and RNAi-based functional genomics, how polarity 

components interact in space and time remains poorly understood. This is due in part to 

the lack of automated methods to gather quantitative information with subcellular precision. 

Consequently, data quantification is often performed manually, which is repetitive, inefficient 

and prevents the precise and consistent analysis of large datasets. 

To circumvent this limitation, we developed a multi-channel image analysis software 

coupled with a reference coordinate system that we termed ASSET (for Algorithm for the 

Segmentation and the Standardization of C. elegans Time-lapse recordings). By automatizing 

the segmentation, our algorithm enables us to combine the great spatial and temporal resolution 

achieved in live recordings with an efficient computational pipeline, permitting the fast and 

coherent processing of a large number of recordings. Consequently, ASSET provides an 

adequate platform for image-based automated quantifications of dynamical processes. 

We now use ASSET to precisely measure fluorescence intensities from time-lapse recordings 

of PAR fusion proteins, starting with the posterior GFP-PAR-2 fusion protein. Combined with 

an effective mathematical model [Goehring et al., Science, 2011], these recordings allow 

us to quantify precisely key spatio-temporal features of polarity establishment. Of particular 

importance, we can derive in this manner values for the parameters governing the mutual 

inhibition of the anterior and posterior polarity complexes, yielding important insights on the 

underlying molecular mechanisms. 

Contact: simon.blanchoud@epfl.ch 

Lab: Gönczy 

Poster Topic: Polarity 

221

PAR proteins regulate the localization of LET-99 during asymmetric 

division 

Eugenel Espiritu, Jui-Ching Wu, Lesilee Rose 

University of California, Davis, CA, USA 

Mitotic spindle positioning is essential for asymmetric divisions, where the spindle must be 

aligned with the axis of cell polarity. In many systems, PAR polarity proteins establish polarization 

of the cell and regulate spindle movements via a complex including Gα, GPR and LIN-5. We 

previously showed that LET-99 is a key regulator of GPR asymmetry in C. elegans one-cell 

embryos. LET-99 is asymmetrically localized at the cortex in a lateral-posterior band pattern, 

where it inhibits GPR localization. Analysis of LET-99 localization in mutant backgrounds 

showed that PAR-3 inhibits cortical LET-99 at the anterior cortex, while a gradient of PAR-1 

inhibits LET-99 at the posterior-most cortex. In addition, PAR-1, a Ser/Thr kinase, was found to 

associate with LET-99 in vitro. To gain further insight in the mechanism of LET-99 localization, 

we tested LET-99 for association with the C. elegans 14-3-3 protein, PAR-5. In other systems, 

phosphorylation of targets by PAR-1 and the PAR-3 associated kinase PKC-3 generates 

binding sites for 14-3-3 proteins, which alters the targets’ localization. We found that PAR-5 

bound to His-LET-99 in wild-type embryo extracts, but PAR-5 binding was greatly diminished 

in extracts from par-1(RNAi) embryos. Computer predictions for 14-3-3 binding sites followed 

by yeast-two hybrid (Y2H) assays identified two LET-99 serine residues essential for PAR-5 

binding. To determine the in vivo relevance of these sites, we introduced S-to-A mutations 

into an otherwise full-length rescuing LET-99 transgene (LET-99-AA). When transferred into a 

let-99 deletion mutant background, the transgene-encoded LET-99-AA protein mislocalized to 

the entire posterior cortex of the one-cell embryo, similar to what was observed for LET-99 in 

par-1 mutant embryos. These and other results support the model that PAR-5 binds to LET-99 

to prevent association with the posterior-most cortex, and that this interaction is regulated by 

phosphorylation of LET-99 by PAR-1. To begin to determine how LET-99 localization is restricted 

from the anterior cortex, we analyzed LET-99 after depletion of anterior PAR components. 

We found that PAR-3 is not sufficient for normal LET-99 localization, but rather the PAR-3 

associating proteins, PAR-6 and PKC-3, restrict LET-99 localization from the anterior. In the 

future, we will test the hypothesis that PAR-1 and PKC-3 directly phosphorylate LET-99 using 

in vitro kinase assays. 

Contact: eugespiritu@ucdavis.edu 

Lab: Rose 

222 

Poster Topic: Polarity

On the Role of RGA-3/4 in Foci Formation of NMY-2 in C. elegans 

Masashi Fujita, Shuichi Onami 

RIKEN Quantitative Biology Center, Kobe, Japan 

Cortical actomyosin network often forms dense foci in animal embryos. It is unknown 

what mechanism underlies these non-uniform distributions. In vitro studies have reported that 

reconstituted actomyosin network can form aggregates without help of additional biochemical 

regulation. However, it is unclear whether the same mechanism governs foci formation in 

embryos. During the polarity establishment phase of one-cell C. elegans embryos, nonmuscle 

myosin NMY-2 forms many foci at the cell cortex, and its contractility is regulated by small 

GTPase RHO-1, RhoGAP RGA-3/4, and RhoGEF ECT-2. 

Here we propose a hypothesis that de novo formation of NMY-2 foci is controlled by 

RhoGAP-mediated lateral inhibition. We observed that mCherry::RGA-3 has foci-like distribution 

at the cell cortex and colocalize with NMY-2::GFP during the polarity establishment phase. 

Based on this observation, we constructed a mathematical model, in which Rho diffuse laterally 

on the membrane surface. Foci were modeled to have RhoGAP activity, which would make Rho 

predominantly the GDP-bound inactive form in the neighborhood of pre-existing foci. Computer 

simulation of this model successfully generated alternate patterns of active Rho and RhoGAP. 

We will perform knockdown experiments and compare the results with theoretical predictions. 

Contact: m-fujita@riken.jp 

Lab: Onami 


223

Isolation, Identification, and Characterization of Free-Living 

Nematodes 

Lauren Leister, Alan Massouh, Alexis Plaga, Ramon Carreno, Danielle Hamill 

Ohio Wesleyan University 

Our lab is interested in cell division, polarity establishment, and other early developmental 

processes. Nematodes, which include the rhabditids, are among the most widespread phyla 

of animals, and Caenorhabditis elegans is the best studied of the rhabditids. We seek to 

understand the similarities and differences between C. elegans and other rhabditids. In this 

study, we traveled to southern Florida to collect rhabditid nematodes for further characterization. 

Free-living rhabditids are often found in the soil, and many of these are known to spend part of 

their life cycle in association with millipedes; for this reason, both soil samples and millipedes 

were collected. We were able to culture 27 isolates using reagents and techniques commonly 

applied to C. elegans. We are using a combination of phenotypic and molecular techniques to 

characterize and identify these worms. We have photographed and measured the worms, taken 

time-lapse videos of their embryos, and used immunofluorescence microscopy to visualize 

sub-cellular components including microtubules and DNA. We have observed differences in the 

overall size of the worms, in tail morphologies, in the gonads, and in embryonic development. 

Furthermore we have isolated genomic DNA from each strain, amplified the 18S rRNA gene 

using PCR, and sequenced the products. These sequences were compared to each other 

and to sequences from public databases. We believe that the worms we collected represent 

five species, three of which are from the genus Oscheius, and some of which may represent 

previously undescribed species. Additional phenotypic and DNA sequence analysis will be 

needed to confirm this. With respect to early embryonic development, the rhabditids we isolated 

share some similarities with C. elegans, but there are intriguing differences as well. There are 

double-nuclei in the blastomeres of one group of worms. We have also noticed differences 

in polarity, represented by variability in the meeting point of the sperm and egg pronuclei, the 

relative sizes of the cells at the two-cell stage, and the timing and orientation of cell division at 

the two-cell stage. Essential elements of early polarity in C. elegans are not observed in some 

of these rhabditid nematodes. We believe that comparative studies like this will not only help 

us better understand an important phylum of animals, but they will also help us to understand 

cell division and developmental patterns more generally. 

Contact: drhamill@owu.edu 

Lab: Hamill 

224 


A Dominant Mutation in a C. elegans Splicing Factor Results in 

Reversed AP Polarity in the Early Embryo 

Reza Keikhaee, Bruce Nash, John Yochem, Bruce Bowerman 

Institute of Molecular Biology, University of Oregon, Eugene, Oregon, USA 

Cell polarity is a fundamental property of most cells and is critical to generate cell diversity 

during development. While many of the molecules required for anterior-posterior (AP) cell 

polarity are conserved across animals, the mechanisms that establish it remain unclear. 

We have identified a semi-dominant, temperature-sensitive (ts), embryonic-lethal allele of 

the C. elegans ortholog of human SF3a66 called or430ts. SF3a66 is one of three subunits 

composing SF3a (splicing factor 3a) that is involved in the processing of pre-mRNA, but recent 

studies have shown that SF3a66 may also act as a microtubule binding and bundling protein 

independent of its RNA splicing function. About one half of the embryos produced by or430ts/ 

or430ts worms exhibit a remarkable reversal of AP cell polarity at the one-cell stage when 

raised at the restrictive temperature. Using a combination of visible marker mapping and whole 

genome sequencing, we found that or430ts maps to LG IV near the C. elegans ortholog of 

SF3a66 (which we have named repo-1 for reversed polarity), and we identified a mis-sense 

mutation in this gene. Reducing repo-1 function with RNAi results in significant reduction in 

the penetrance of polarity reversal; hence we concluded that the repo-1 mis-sense mutation 

is responsible for this phenotype. In addition to the polarity reversal, we also have observed 

several microtubule related defects in or430ts one-cell zygotes, consistent with a possible 

role for a REPO-1 microtubule binding activity. As a partial reversal of polarity has previously 

been observed in mutants that arrest in Meiosis I, we examined meiotic spindle assembly but 

did not observe defects in spindle structure or cell cycle progression. Nevertheless, we have 

been able to partially eliminate the or430ts polarity reversal by reducing the function of unc- 

116 and lin5 using RNAi, which was shown previously to result in a failure of the oocyte meotic 

spindle to move to the cortex. We then hypothesized that a reduction in PKC-3 activity, due 

to splicing defects in or430ts mutants,might make these mutant embryos sensitive to polarity 

reversal by a normal oocyte meiotic spindle. Consistent with this hypothesis, we have found 

that reducing the gene dosage of pkc-3 in or430ts mutant results in an increased frequency 

of polarity reversal. We conclude that one important role for anteriorly enriched PKC-3 is to 

prevent oocyte meiotic spindle microtubules from establishing a reversed posterior pole. 

Contact: keikhaee@uoregon.edu 

Lab: Bowerman 


225

Identifying Mechanisms of Contact-Mediated Cell Polarization 

Diana Klompstra, Dorian Anderson, Jeremy Nance 

Skirball Institute, NYU School of Medicine , New York, NY 

During gastrulation, cells move to a position in the embryo that is appropriate for the type 

of tissue or organ that they will form. The directional movements of gastrulation are facilitated 

by a polarity that early embryonic cells acquire that allows them to asymmetrically localize 

cytoskeletal components. The polarity of early embryonic cells is determined by cell-cell 

contacts, which restrict the PAR polarity proteins PAR-3, PAR-6, and PKC-3 to contact-free 

surfaces. The goal of my project is to determine how cell contacts induce the PAR protein 

asymmetries that polarize early embryonic cells, preparing them for gastrulation. 

We previously identified the RhoGAP protein PAC-1 as an upstream regulator that is 

required to exclude PAR proteins from contacted surfaces of early embryonic cells. PAC-1 

itself is recruited specifically to sites of cell contact, and directs PAR protein asymmetries by 

inhibiting the Rho GTPase CDC-42. How PAC-1is able to sense where contacts are located 

and localize specifically to these sites is unknown. We identified an N-terminal fragment of 

PAC-1 that is sufficient for localization to cell contacts, and showed that localization of this 

fragment depends on HMR-1/E-cadherin. We show that HMR-1recruitment to cell contacts 

depends on the presence of HMR-1 in the adjacent cell, suggesting that HMR-1 homotypic 

interactions recruit or stabilize the protein at contact sites. Finally, we show catenins that 

interact with the HMR-1 cytoplasmic tail function redundantly to recruit the PAC-1 N-terminal 

domain. In contrast to the PAC-1 N-terminus, full-length PAC-1 can localize to cell contacts 

when HMR-1/E-cadherin is removed, indicating that a redundant signal functions with HMR- 

1/E-cadherin to recruit PAC-1 to contacts. These findings provide insights into how a polarity 

regulator is spatially segregated to a subdomain of the cortex to polarize cells. 

Contact: diana.klompstra@med.nyu.edu 

Lab: Nance 

226 


ER Compartmentalisation and the Regulation of Polarity in the C. 

elegans Embryos 

Zuo Yen Lee 1 , Monica Gotta 2 , Yves Barral 1 

1 Institut fur Biochemie, ETH Zurich, Zurich, Switzerland, 2 Centre Medical 

Universitaire, University of Geneva, Geneva, Switzerland 

Diffusion barriers are used to compartmentalise different domains in polarised and 

specialised cells, such as budding yeast, primary cilium, spermatozoa and neurons. They are 

important in maintaining the structure and the functions of the cells. In budding yeast, lateral 

diffusion barriers have been identified at the bud neck in the plasma, the nuclear envelope and 

the endoplasmic reticulum (ER) membranes. As an example, the lateral diffusion barrier in the 

nuclear envelope promotes asymmetric distribution of aging factors by retaining them in the 

mother cells while creating young buds. The C. elegans embryos are highly polarised entities, 

particularly in their first division where polarity markers and fate determinants are asymmetrically 

distributed. During the establishment of polarity, the cytoplasmic proteins MEX-5 and PIE-1 

are enriched exclusively in the anterior and the posterior of the embryo, respectively. The 

mechanisms of MEX-5 maintenance in the anterior domain are not well-understood. In wild 

type embryos, it was found that the ER was reorganised and redistributed asymmetrically after 

the first division with anterior enrichment. Additionally, the dynamics of MEX-5 was found to be 

decreased in the anterior domain. Therefore, we hypothesise that MEX-5 could be retained 

by an organelle in the cell and our interest is to investigate if the ER barrier is conserved in 

the C. elegans embryo, the molecular nature of such barrier and subsequently how it may 

contribute to the regulation of polarity in the embryos. By using photobleaching technique, the 

data showed restricted diffusion of the membrane protein SP12::GFP between the anterior 

and posterior ER domains. In contrast, the luminal protein KDEL::GFP diffused rapidly. The 

results showed that the ER in the C. elegans embryo is compartmentalised and potentially 

contribute as a mechanism to the polarity maintenance in the embryo. 

Contact: zuoyen.lee@bc.biol.ethz.ch 

Lab: Barral 


227

A Cullin-5-RING Ubiquitin Ligase Regulates Asymmetric Cell Division 

in Early C.elegans Embryos 

Anne Pacquelet, Emeline Daniel, Gregoire Michaux 

Institut de Genetique et de Developpement de Rennes, Rennes, France 

The early C. elegans embryo undergoes several rounds of asymmetric divisions in the P 

lineage, which are essential to generate different embryonic cell types, including germline cells. 

These asymmetric divisions depend on the establishment of a polarity axis - defined by the 

asymmetric localization of the PAR proteins – prior to mitosis. They also require proper spindle 

positioning along this polarity axis as well as the asymmetric inheritance of cell fate determinants 

during mitosis. We are interested in understanding the role of protein degradation and in 

particular of Cullin-RING ubiquitin ligases (CRLs) in these processes. CRLs are multisubunit 

complexes containing a cullin family protein, a RING domain containing protein, RBX-1 or 

RBX-2, as well as a substrate recognition module. We are currently investigating the role of 

Cullin-5-RING ubiquitin ligase complexes (CRL5) whose functions are so far poorly understood. 

We found that cul-5 mutants strongly enhance the embryonic lethality due to partial par-2 

and par-4 loss of function. Notably, par-2;cul-5 embryos divide similarly to par-2 embryos at 

the one-cell stage but show enhanced defects in P1 spindle orientation at the two-cell stage, 

suggesting that CUL-5 may have a specific role in regulating polarity in P1 cells. Surprisingly, 

while par-4 embryos divide asymmetrically, cul-5par-4 mutants divide symmetrically at the onecell 

stage, giving rise to two equally sized cells. However, these embryos do not have strong 

defects in PAR protein polarity. Ongoing experiments will help us understanding whether the 

symmetric division observed in cul-5par-4 embryos is due to improper mitotic spindle and/or 

cytokinesis furrow positioning. Importantly, we found that loss of the RING domain containing 

protein RBX-2 gives rise to similar phenotypes as loss of CUL-5. Moreover, we are currently 

investigating which substrate specific adaptor is working together with CUL-5 in the regulation 

of asymmetric divisions. 

Altogether, our results indicate that a Cullin-5-RING ubiquitin ligase contributes to the 

asymmetric divisions of the P lineage, by regulating both the size asymmetry of the first 

embryonic division and the orientation of the mitotic spindle during P1 division. These results 

thereby uncover new mechanisms involved in regulating asymmetric divisions as well as so 

far unknown functions of Cullin-5-RING ubiquitin ligases. 

Contact: anne.pacquelet@univ-rennes1.fr 

Lab: Michaux 

228 


Evolution of GPR Regulation in the Control of Spindle Positioning for 

Two Cænorhabditis Species Embryos 

Soizic Riche1 , Francoise Argoul2 , Melissa Zouak1 , Alain Arneodo2 , Jacques 

Pecreaux3 , Marie Delattre1 1 2 Laboratory of Molecular Biology of the Cell, Lyon, France, Physic 

Laboratory, Lyon France, 3Institute of Genetics and Developmental biology 

of Rennes, Rennes, France 

Asymmetric cell division is a fundamental mechanism relying on proper mitotic spindle 

positioning. In C. elegans one-cell embryos, it gives rise to two daughter cells of unequal size 

and fate. After fertilization and pronuclei meeting at the posterior side of the cell, the nucleuscentrosome 

complex (NCC complex) migrates back to the cell center. This centrally located 

spindle is then displaced toward the posterior pole during anaphase. While it is displaced, the 

spindle undergoes transverse oscillations that are more pronounced for the posterior than the 

anterior pole. These movements are known to be controlled by pulling forces acting on astral 

microtubules and the number and the molecular nature of motors have been characterized: a 

complex made of Gα proteins, linked to GPR (a GoLoco containing protein), LIN-5 (the Numa 

homolog) and Dynein is thought to be anchored at the cortex and activated at the onset of 

mitosis to pull on the spindle during anaphase. 

Our comparative analysis between C. elegans and C. briggsae embryos shows that a same 

division is achieved through different movements of pronuclei and spindle. We found that the 

pronuclei migrate further towards the anterior cell side in a Gα/GPR/LIN-5 dependent manner. 

Furthermore, anaphase spindle oscillations are delayed, lower in amplitude, and shorter in 

duration in C. briggsae. Through a combination of microtubule laser destruction, mutant analysis 

and mathematical modelling, we revealed the existence of a conserved positional switch for 

oscillations superimposed on the time control for spindle positioning. This switch is linked to 

the localisation of GPR crescent at the posterior cell side, which is conserved between species. 

However, we uncovered a differential localisation of GPR at the anterior cortex of embryos 

between species, suggesting evolutionary changes in GPR regulation. Importantly, GPR 

proteins share only 67% of similarity between C. elegans and C. briggsae. To gain insights 

into GPR regulation, we performed gene replacement experiment of gpr between species. 

Our preliminary data suggest that the differential localisation of GPR could be explained by 

differences in the protein sequences. Experiments are underway to narrow down the important 

residues responsible for conserved and divergent GPR functions. 

Contact: soizic.riche@ens-lyon.fr 

Lab: Delattre 


229

Coupling Centrosome Position And Cortical Polarity 

Sabina Sanegre, Carrie Cowan 

Institute of Molecular Pathology, Vienna, Austria 

In one-cell C. elegans embryos, centrosomes play a key role in polarity establishment. 

Coincident with the initiation of polarity, paternally contributed centrioles begin to recruit 

pericentriolar material (PCM). Delays in PCM assembly delay polarization, and depletion of core 

PCM components, such as the structural protein SPD-5, prevent polarization. The molecular 

mechanism by which centrosomes control polarity establishment, however, is still unknown. The 

centrosomal kinase AIR-1 has been shown to be required for correct polarization of one-cell 

embryos. We find that AIR-1 has two functions in polarity establishment: it inhibits spontaneous 

polarization, and it coordinates the site of polarity establishment with the position of the 

centrosomes. In embryos depleted of AIR-1, spontaneous polarization generated a functional 

anterior-posterior polarity axis, including proper segregation of cortical and cytoplasmic fate 

determinants, suggesting that AIR-1 may not be required for polarity itself but rather for 

positioning the polarity axis in response to centrosome position. Despite AIR-1’s established 

role in centrosome maturation, AIR-1 depletion had no effect on the initial recruitment of PCM, 

further supporting that AIR-1 acts downstream of PCM assembly to control polarity. AIR-1’s 

centrosomal localization appears to be mediated by direct interaction with SPD-5. Thus SPD-5dependent 

recruitment of AIR-1 to centrosomes integrates the temporal and spatial information 

provided by PCM assembly with downstream signals that establish polarity. 

Contact: sabina.sanegre@imp.ac.at 

Lab: Cowan 

230 


GLD-3(S) Contributes to PIE-1 Asymmetry in Zygotes 

Jarrett Smith, Geraldine Seydoux 

Johns Hopkins School of Medicine 

PIE-1is a germ cell fate determinant that is asymmetrically segregated to the posterior 

of the zygote before the first division. In a screen for temperature-sensitive lethal mutations, 

Yingsong Hao identified three alleles of gld-3 that delayPIE-1 asymmetry in the zygote (Hao, 

Y.,2005 - Ph. D. Thesis, Johns Hopkins U.). gld-3 codes for two isoforms GLD-3S and GLD- 

3L with different carboxy-termini (Eckmann et al.,2004). GLD-3S and GLD-3L share five KH 

domains and a region that interacts with the poly-A polymerase GLD-2. GLD-3L also contains 

an FBF-binding site (Eckmannet al., 2004). We sequenced the allele ax202 and found thatthis 

mutation maps to the carboxy-terminus of GLD-3S. gld-3S(RNAi),but not gld-3L(RNAi), lead 

to embryonic lethality. Yeast two-hybrid experiments showed that PIE-1 binds preferentially to 

GLD-3S, and that this interaction is attenuated by ax202. These results suggest that GLD-3S 

could play a direct role in promoting PIE-1 asymmetry in zygotes. 

Contact: jsmit314@jhmi.edu 

Lab: Seydoux 


231

Phosphorylation State of a Tob/BTG Protein, FOG-3, Regulates 

Initiation and Maintenance of the Caenorhabditis elegans Sperm Fate 

Program 

Myon-Hee Lee1,2 , Kyung Won Kim2 , Clinton Morgan2 , Dyan Morgan2 , Judith 

Kimble2 1Brody School of Medicine at East Carolina University, Greenville, NC, USA, 

2University of Wisconsin-Madison, Madison, WI, USA 

FOG-3, the single Caenorhabditis elegans Tob/BTG protein, directs germ cells to adopt 

the sperm fate at the expense of oogenesis. Importantly, FOG-3 activity must be maintained 

for the continued production of sperm that is typical of the male sex. Vertebrate Tob protein 

shave antiproliferative activity and ERK phosphorylation of Tob proteins has been proposed to 

abrogate “antiproliferative” activity. Here we investigate FOG-3 phosphorylation and its effect 

on sperm fate specification. We found both phosphorylated and unphosphorylated forms of 

FOG-3 in nematodes. We then interrogated the role of FOG-3 phosphorylation in sperm fate 

specification. Specifically, we assayed FOG-3 transgenes for rescue of a fog-3 null mutant. 

Wild-type FOG-3 rescued both initiation and maintenance of sperm fate specification. A FOG- 

3 mutant with its four consensus ERK phosphorylation sites substituted to alanines, called 

FOG-3(4A), rescued partially: sperm were made transiently but not continuously in both 

sexes. A different FOG-3 mutant with its sites substituted to glutamates, called FOG-3(4E), 

had no rescuing activity on its own, but together with FOG-3(4A) rescue was complete. Thus, 

when FOG-3(4A) and FOG-3(4E) were both introduced into the same animals, sperm fate 

specification was not only initiated but also maintained, resulting incontinuous spermatogenesis 

in males. Our findings suggest that unphosphorylated FOG-3 initiates the sperm fate program 

and that phosphorylated FOG-3 maintains that program for continued sperm production typical 

of males. We discuss implications of our results for Tob/BTG proteins in vertebrates. 


Lab: Kimble 

232 

Poster Topic: Sex Determination

Molecular Analyses of FOG-1 and FOG-3, Terminal Regulators of the 

Sperm/Oocyte Cell Fate Decision 

Daniel Noble1 , Scott Aoki2 , Marco Ortiz Sanchez3,1 , Kyung Won Kim1 , Judith 

Kimble1,2 1 2 University of Wisconsin-Madison, Madison, WI, USA, Howard Hughes 

Medical Institute, University of Wisconsin-Madison, Madison, WI, USA, 

3Universidad Nacional Autonoma de Mexico, Cuernavaca, Mexico 

Signaling from somatic cells regulates germline sex determination in all animals tested 

(worms, flies, vertebrates), but the germ cell response is best understood in nematodes. C. 

elegans uses a divergent hedgehog signaling pathway to control sexual differentiation in both 

somatic and germ cells (1). Within germ cells, the fog-1 and fog-3 genes are essential for 

sperm fate specification in both sexes and are terminal regulators of the sperm/oocyte fate 

decision (2). Both FOG-1 and FOG-3 proteins are implicated in mRNA regulation: FOG-1 is an 

RNA-binding protein of the cytoplasmic polyadenylation element binding (CPEB) class, while 

FOG-3 is a putative Tob/BTG protein (2),which functions in vertebrates as an adapter within 

an RNA regulatory complex. We previously reported the generation of a functional epitopetagged 

fog-3::FLAG transgene (3). We have now also generated a functional epitope-tagged 

FLAG::fog-1 transgene. Both fog-3::FLAG and FLAG::fog-1 are maintained as rescuing 

transgenes in strains that lack the corresponding endogenous gene. Our current work focuses 

on identification of interacting proteins as well as associated mRNAs for both FOG proteins. 

Our preliminary results suggest that FOG-1 and FOG-3 may interact with each other and 

control an overlapping set of mRNAs. 

(1) Zarkower, D. (2006) Somatic sex determination, WormBook; (2) Ellis, R. and Schedl, T. (2007) Sex 

determination in the germ line, WormBook; (3) Lee, M.-H. et al (2011) PNAS 108, 9125-9130. 

Contact: dcnoble@wisc.edu 

Lab: Kimble 

Poster Topic: Sex Determination 

233

RNA-Seq Analysis of Germline Sex Reprogramming 

Elena Sorokin1 , Judith Kimble1,2 1 2 University of Wisconsin-Madison, Madison, WI, USA, Howard Hughes 

Medical Institute, University of Wisconsin-Madison, Madison, WI, USA 

The molecular basis of germ cell fate specification as sperm or oocyte remains poorly 

understood. Whereas cell fates in somatic tissues are typically regulated transcriptionally, 

only post-transcriptional regulators have emerged as key for sperm/oocyte specification (e.g. 

FOG-1, FOG-3: see abstract by Noble et al). We hypothesized that, if transcriptional factors 

are terminal regulators of the sperm/oocyte decision, they must not be not tractable genetically, 

either due to redundancy or pleiotropy. We therefore took a completely different approach, 

which takes advantage of our recent discovery that U0126, a MEK kinase inhibitor, transforms 

a puf-8; lip-1 masculinized germline to produce functional oocytes instead of sperm (Morgan et 

al., 2010). More recently we have learned that MAPK activity is lowered within 15 minutes of 

U0126 treatment, and reprogramming occurs within hours of drug treatment (C. Morgan and 

J. Kimble, unpublished). Here we sequenced mRNAs isolated from whole animals during an 

18-hr time course of chemical treatment. Data were obtained from U0126-treated or DMSO 

vehicle-treated animals of three distinct genotypes: puf-8; lip-1, which begin spermatogenic 

but reprogram their germlines to oogenesis after U0126 treatment; N2, which are oogenic 

with and without drug; and puf-8; fbf-1, which are spermatogenic with and without drug. We 

are analyzing our data for changes in mRNA abundance and changes in mRNA isoforms. Our 

preliminary results reveal a strong drug response in all three strains, but few changes specific 

to reprogramming. We are now testing the few genes changed for effect on germline sex 

determination in sensitized mutant backgrounds. Our progress will be reported at the meeting. 

Morgan, C.T., Lee, M.-H., Kimble, J., 2010. Chemical reprogramming of Caenorhabditis 

elegans germ cell fate. Nat Chem Biol 6, 102-4. 

Contact: sorokin@wisc.edu 

Lab: Kimble 

234 

Poster Topic: Sex Determination

A 

Abbott, Allison L .............................. 50 

Abraham, Nessy ............................... 5 

Addise, Abate Birhan .................... 215 

Ahn, Samuel ................................. 197 

Ahringer, Julie ................................. 42 

Akintobi, Adenrele M..................... 127 

Alaimo, Jennifer ............................ 104 

Alam, Emad .................................. 114 

Alexa, Anita................................... 215 

Alexander, Mariam.......................... 11 

Al-Hashimi, Hikmat ....................... 185 

Allen, Anna K .................................. 32 

Allman, Erik .................................... 50 

Alvaro, Christopher ....................... 122 

Amin, Nirav M ................................111 

Anderson, Courtney........................ 10 

Anderson, Dorian.......................... 226 

Antoshechkin, Igor .................. 48, 134 

Aoki, Scott .................................... 233 

Appleford, Peter J ......................... 212 

Argoul, Francoise ......................... 229 

Arneodo, Alain .............................. 229 

Asahina, Masako .......................... 186 

Asencio, Claudio............................. 25 

Aubry, Agnes ................................ 194 

Audhya, Anjon ................................ 23 

B 

Baer, G. Michael ........................... 122 

Bageshwar, Suparna ...................... 61 

Baldwin, Austin T .......................... 105 

Banfalvi, Zsofia ............................. 215 

Bao, Zhirong ................................... 44 

Barral, Yves .................................. 227 

Barrett, Alec .................................... 51 

Barsi-Rhyne, Ben ........................... 52 

Baugh, L. Ryan ....................... 48, 134 

AUTHOR INDEX 

Bayer, Emily.................................. 122 

Beard, Sarah M .............................. 84 

Bedet, Cecile ................................ 139 

Beilharz, Traude H ........................ 146 

Belafi-Bako, Katalin ...................... 215 

Belsky, Jason A ............................... 48 

Bembenek, Joshua N ............. 24, 176 

Benian, Guy M ................................ 55 

Berg, Jannette .............................. 144 

Berkseth, Matthew .......................... 34 

Bernadskaya, Yelena Y .............. 3, 83 

Bertho, Sylvain ............................. 150 

Bhalla, Needhi ...................... 147, 170 

Bhambhani, Chandan ................... 123 

Bienkowska, Dominika ................. 150 

Blanchoud, Simon ........................ 221 

Boag, Peter R ............................... 146 

Bobian, Michael R .......................... 85 

Bock, Carly ..................................... 53 

Bohr, Tisha.................................... 147 

Bojanala, Nagagireesh ................. 186 

Bonner, Mary Kate .......................... 86 

Bosanac, Anna ............................. 199 

Bowerman, Bruce ..... 54, 75, 166, 225 

Bowman, Elizabeth ....................... 161 

Brabin, Charles ............................. 112 

Braeckman, Bart P ........................... 9 

Braunreiter, Kara ............................ 67 

Breving, Kimberly ......................... 126 

Brockway, Heather ....................... 177 

Broitman-Maduro, Gina .......... 41, 115 

Budirahardja, Yemima .................. 211 

Buechner, Matthew ............... 185, 195 

Buhler, Alessandra.......................... 56 

Burge, Stephanie A ......................... 71 

Burger, Julien............................ 75, 76 

Burns, Ramzy ............................... 122 

Butterfield, Yaron .......................... 177 

Byrd, Dana.................................... 154 

235

C 

Cabello, Juan................................ 108 

Cabunoc, Abigail............................. 45 

Cadigan, Ken ................................ 123 

Carreno, Ramon A ........................ 224 

Carter, Caitlyn ................................. 65 

Castells-Roca, Laia ........................ 81 

Cha, Dong Seok ........................... 113 

Chakravorty, Adityarup ................... 12 

Chan, Benjamin G ........................ 187 

Chan, Raymond C .................. 24, 163 

Chandler, Chelsey N....................... 38 

Chang, Chieh.................................. 47 

Chang, Yu-Tai ................................. 69 

Chaouni, Rita ................................ 148 

Chatterjee, Indrani .......................... 15 

Chavez, Daniela ........................... 155 

Chen, Grace ................................... 74 

Chen, Xin ...................................... 188 

Chen, Yun ..................................... 193 

Chisholm, Andrew D ..................... 190 

Chong, Conrad ............................. 153 

Chou, Han Ting............................. 213 

Chou, Han-ting ............................. 128 

Christensen, Sara ........................... 54 

Chu, Diana............ 152, 154, 169, 214 

Chuang, Chiou-Fen ........................ 47 

Cinkornpumin, Jessica K .............. 130 

Ciosk, Rafal .............................. 29, 75 

Clemons, Amy .............................. 177 

Clever, Sheila ....................... 116, 122 

Coetzee, Donna............................ 179 

Colaiacovo, Monica ................ 27, 177 

Collette, Karishma ........................ 176 

Connolly, Amy ......................... 54, 166 

Constas, Katharine ....................... 114 

Contreras, Vince ........................... 160 

Coppola, John .............................. 140 

Core, Leighton J ........................... 134 

Corrionero, Anna .......................... 124 

Corsi, Ann K.................................. 125 

236 

Cortes Estrada, Daniel B ................ 87 

Cottee, Pauline A .......................... 149 

Courtois, Emmanuelle .................... 76 

Couteau, Florence ........................ 139 

Cowan, Carrie R ........... 135, 150, 230 

Cowart, M. Leigh ............................ 82 

Cox-Paulson, Elisabeth A ............. 189 

Cram, Erin J ...................... 26, 70, 151 

Crane, Matthew M ........................ 217 

Crocker, Kassi .............................. 207 

Crook, Matt ..................................... 93 

Crossley, Merlin ............................ 136 

Csanadi, Zsofia............................. 215 

Csankovszki, Gyorgyi ....... 24, 37, 176 

Custer, Laura M .............................. 37 

D 

Dalfo, Diana .................................. 168 

Daniel, Emeline ............................ 228 

Datla, Udaya Sree ........................ 113 

Davidson, Iain F.............................. 25 

De Henau, Sasha ............................. 9 

de la Cruz, Norie............................. 45 

De Orbeta, Jessica ......................... 20 

De Stasio, Elizabeth ....................... 64 

Degema, Karen .............................. 18 

Dejima, Katsufumi ........................ 190 

Del Rosario, John S........................ 94 

Delattre, Marie ........................ 19, 229 

Denning, Dan.................................. 95 

Dennis, James W ......................... 158 

Der, Channing J .............................. 49 

Dewilde, Sylvia ................................. 9 

Deyter, Gary M. .............................. 20 

Dillingham, Zechariah ................... 173 

Dineen, Aidan ............................... 191 

Doan, Thang ................................. 211 

Dong, Xintong ............................... 192 

Dordal, Rachel .............................. 116 

Driscoll, Kaitlin ................................ 96 

Druzhinina, Marina ......................... 15

Du, Zhuo ......................................... 44 

Duchesneau, Christopher D ........... 55 

Duong, Adrian ................................. 45 

E 

Eimer, Stefan .................................. 23 

Eiteneuer, Annika............................ 22 

Elewa, Ahmed............................... 106 

Ellefson, Marina L ........................... 58 

Ellis, E. Ann .................................. 218 

Engebrecht, JoAnne ..................... 165 

Ermolaeva, Maria ........................... 81 

Escobar Restrepo, Juan M ..... 56, 110 

Escobar, Juan MI .......................... 196 

Espiritu, Eugenel B ....................... 222 

Esquela-Kerscher, Aurora............. 126 

Estrada, Rodrigo........................... 152 

Ezcurra, Begona ........................... 108 

Ezzio, Catherine P ........................ 116 

F 

Fall, Gabe T .................................. 164 

Farhadifar, Reza ............................. 19 

Farooqui, Sarfarazhussain ........... 196 

Feddersen, Charlotte ...................... 12 

Feldman, Jessica L ........................... 2 

Fenker, Kristin............................... 153 

Fernandez, Anita G......................... 53 

Fields, Brandon .............................. 57 

Fischer, Greg .................................. 67 

Fitch, David................................... 197 

Fleming, John ................................... 5 

Fletcher, Evan............................... 116 

Flynn, Jonathan R .......................... 58 

Fodor, Andras ............................... 215 

Ford, Jason R ................................. 20 

Formstecher, Etienne ..................... 77 

Foster, Olivia................................... 51 

Fotopoulos, Nellie ......................... 193 

Francis, Joshua W .......................... 38 

Frand, Alison R ................................. 6 

Fraser, Andrew G.......................... 158 

Friday, Andrew J ........................... 160 

Frohli, Erika .................................... 56 

Frommolt, Peter .............................. 81 

Fujita, Masashi ............................. 223 

Furuta, Tokiko ................................. 20 

G 

Gabrhel, Casey............................... 67 

Gaidatzis, Dimos ............................ 29 

Gally, Christelle ............................. 194 

Garcia, L. Rene ............................ 188 

Garcia, Rebecca E. ...................... 214 

Gaudet, Jeb ............................ 78, 191 

Gautier, Megan K............................ 82 

Gavin, Amanda ............................. 116 

George, Carolyn ............................111 

Germani, Francesca ......................... 9 

Ghai, Vikas ................................... 107 

Ghosh, Srimoyee ............................ 59 

Gilbert, Jennifer M ........................ 154 

Gill, Hasreet .................................... 72 

Gleason, Elizabeth J..................... 156 

Gleason, Ryan J ........................... 127 

Glotzer, Michael .............................. 88 

Gnazzo, Megan M .......................... 89 

Gobel, Verena................................... 5 

Golden, Andy ............................ 32, 90 

Goldstein, Bob ................................ 88 

Gomes, Jose-Eduardo.................... 77 

Gomez, Raymarie......................... 128 

Gomez-Orte, Eva.......................... 108 

Gonczy, Pierre .............................. 221 

Gorjanacz, Matyas.......................... 25 

Gorman, Kevin.............................. 182 

Gotta, Monica ........... 22, 76, 109, 227 

Govindan, J. Amaranath ............... 162 

Grant, Barth .................................. 127 

Grants, Jennifer M ........................ 129 

Greenberg, M. Banks ..................... 82 

Greenstein, David ........... 13, 162, 179 

237

Greenstein, David ......................... 180 

Greiss, Sebastian ........................... 81 

Grimm, Julie ................................... 60 

Grussendorf, Kelly A ..................... 195 

Guang, Shouhong .......................... 39 

Guerrero, Francisco...................... 152 

Gumienny, Tina L .............. 61, 80, 218 

Gutierrez, Peter .............................. 56 

Guven-Ozkan, Tugba ................... 118 

H 

Ha, Dae Gon................................... 12 

Haag, Andrea.......................... 56, 110 

Hajnal, Alex........11, 56, 110, 196, 202 

Hale, Jared J .................................111 

Hall, David ................................ 5, 180 

Hall, Jenny .................................... 116 

Hamiche, Karim ............................ 198 

Hamill, Danielle R ......................... 224 

Han, Sung Min .............................. 149 

Hanna-Rose, Wendy ...................... 93 

Hansen, Angela ............................ 153 

Hansen, Jody M............................ 155 

Hardin, Jeff ................................... 206 

Harel, Sharon ............................... 198 

Harris, Todd W ................................ 45 

Haruta, Nami .................................. 21 

Haynes, Kelly.................................. 91 

Hegermann, Jan ............................. 23 

Heiman, Maxwell G ...................... 209 

Henderson, Melissa A ........... 156, 160 

Hengartner, Micheal O.................... 98 

Hermann, Greg ............................... 51 

Herrera, R Antonio ........................ 197 

Herrmann, Alyssa ......................... 205 

Herrmann, Christina ....................... 56 

Hersh, Brad .................................... 96 

Hirose, Takashi ............................... 97 

Hobert, Oliver ............................... 138 

Hoffman, Corey ............................ 189 

Hollis, Sarah E ...................... 113, 201 

238 

Holtackers, Rene ............................ 22 

Honda, Yu ....................................... 21 

Hong, Ray L .................................. 130 

Hoppe, Pamela E ............... 55, 62, 65 

Horvitz, Bob .......... 10, 95, 96, 97, 124 

Hubbard, E. Jane Albert ... 30, 43, 168 

Huelgas Morales, Gabriela ........... 157 

Hughes, Samantha ....................... 112 

Hunter, Jerrod ................................. 68 

Hurwitz, Michael ..................... 10, 101 

I 

Ikegami, Kohta................................ 34 

Imlay, Leah ..................................... 12 

Immerman, Lois .............................. 50 

Ishidate, Takao ............................. 106 

J 

Jacobs, Rene L ............................. 142 

Jacobson, Lewis ............................. 57 

Jaramillo-Lambert, Aimee ............... 90 

Jenes, Barnabas........................... 215 

Jenna, Sarah ................................ 198 

Ji, Jiaojiao ....................................... 39 

Ji, Ni .............................................. 131 

Jindra, Marek ................................ 186 

Jo, Jeanyoung .............................. 126 

Johnson, Casonya M ... 128, 137, 143, 

213 

Johnston, Wendy L ....................... 158 

Jones, Steven ............................... 177 

Jow, Margaret ............................... 152 

Jud, Molly ..................................... 153 

K 

Kadekar, Pratik ............................. 159 

Kang, Alan SR .............................. 190 

Kang, Lijun...................................... 15 

Kant, Sashi ................................... 136 

Kassim, Maher................................ 45 

Katic, Iskra .................................... 216

Katz, David J .................................. 38 

Keikhaee, Reza ............................ 225 

Keiper, Brett D ........................ 99, 160 

Keller, Martin................................... 98 

Kelly, Bill ....................................... 161 

Kemp, Benedict J ........................... 50 

Kemper, Kevin ...................... 104, 117 

Kemphues, Ken ................................ 1 

Kerr, Shana C ................................. 38 

Kershner, Aaron ...................... 28, 175 

Khan, Liakot...................................... 5 

Killeen, Marie T. ............................ 199 

Kim, Ahlee ........................................ 5 

Kim, Kyung Won ................... 232, 233 

Kim, Seongseop ........................... 162 

Kimble, Judith 28, 113, 174, 175, 232, 

233, 234 

Kintzele, Jason ............................... 62 

Kiontke, Karin ............................... 197 

Kirkconnell, Killeen S .................... 163 

Kivlehan, Emily ............................. 205 

Klompstra, Diana .......................... 226 

Kniss, Sarah ................................... 12 

Korswagen, Hendrik ..................... 131 

Korta, Dorota Z ............................... 30 

Kovacevic, Ismar ...................... 26, 70 

Kradolfer, David .............................. 56 

Kramer, Brendan .......................... 167 

Krause, Michael ............................ 219 

Kress, Elsa ..................................... 22 

Krizus, Aldis .................................. 158 

Kroetz, Mary B .............................. 200 

Kroft, Tim L ................................... 164 

Kruesi, William S .......................... 134 

Kubba, Saad ................................. 114 

Kugler, Hillel.................................... 43 

Kuhn, Jonathan A ........................... 69 

Kurhanewicz, Nicole ............... 48, 134 

Kurshan, Peri ................................ 217 

L 

Labbe, Jean-Claude ....................... 88 

Labella, Sara ................................ 160 

Labouesse, Michel........................ 194 

Laboy, Jocelyn T ............................. 63 

Lai, Allison ...................................... 53 

Lam, Karmen ................................ 199 

Lamelza, Piero.............................. 147 

Lancaster, Brett ............................ 132 

Landes, Ethan ................................ 64 

Lane, Latrisha S ............................. 65 

Langouet, Maeva ............................ 56 

Lascarez-Lagunas, Laura I ........... 102 

Law, Fiona ...................................... 66 

Lawrence, Katherine S ................. 165 

Lee, Myon-Hee ............. 113, 201, 232 

Lee, Zuo Yen ................................ 227 

Leger, Thibaud ................................ 76 

Leister, Lauren W ......................... 224 

L’Hernault, Steven W .................... 156 

Li, Ying .......................................... 127 

Lieb, Jason ..................................... 34 

Lilly, Michael ................................. 122 

Lin, Rueyling ..................... 40, 46, 118 

Lis, John T .................................... 134 

Liszewski, Walter ............................ 12 

Liu, Dennis.................................... 114 

Liu, Jun ..........................107, 111, 114 

Liu, Oliver ..................................... 192 

Llamosas, Estelle ......................... 136 

Lo, Te-Wen ..................................... 35 

Long, Ying..................................... 156 

Longhini, Katrina M......................... 88 

Low, Lloyd..................................... 146 

Lowry, Josh ............................. 54, 166 

Lu, Hang ....................................... 217 

Lun, Aaron .................................... 136 

Lyman Gingerich, Jamie ................. 67 

M 

Madric, Kenya............................... 126 

239

Maduro, Morris ....................... 41, 115 

Magistrado, Leila ............................ 41 

Magnuson, Lindsey ...................... 164 

Maier, Wolfgang ............................ 216 

Maine, Eleanor ............................. 178 

Mains, Paul E ................... 77, 84, 187 

Mandt, Rebecca ............................. 12 

Manjarrez, Jacob ............................ 68 

Mano, Itzhak ................................... 94 

Mao, Hui ......................................... 39 

Martin, Emmanuel ........................ 198 

Mason, D. Adam ........................... 205 

Massouh, Alan R .......................... 224 

Mathe-Fodor, Andrea .................... 215 

Mathews, Ellie ........................ 68, 133 

Mattaj, Iain W.................................. 25 

Mattingly, Brendan C .................... 195 

Maxwell, Colin ........................ 48, 134 

Mayers, Jonathan ........................... 23 

McClung, George ......................... 116 

McGhee, James ................... 132, 144 

McNally, Francis J .................... 58, 87 

Medina, Jessica .............................. 46 

Meli, Vijaykumar S ............................ 6 

Mello, Craig ............................ 36, 106 

Meraldi, Patrick ............................... 22 

Merlet, Jorge............................. 75, 76 

Messina, Kari .................................. 14 

Mets, Sarah .................................... 91 

Meyer, Barbara J .................... 35, 134 

Meyer, Hemmo ............................... 22 

Michael, Matthew.......................... 167 

Michaelson, David ........................ 168 

Michaux, Gregoire ........................ 228 

Middelkoop, Teije .......................... 131 

Mikl, Martin ................................... 135 

Miller, Kristine ................................. 52 

Miller, Michael ......................... 12, 149 

Mills, Erica S ................................... 38 

Mis, Emily K .................................... 53 

Moens, Luc ....................................... 9 

240 

Moerkamp, Asja .............................. 75 

Mohler, William A .............................. 3 

Mohnen, Megan.............................. 50 

Monahan, Kimberly B ..................... 49 

Moore, Julia L ................................. 44 

Morf, Matthias K ............................. 11 

Morf, Matthias ....................... 196, 202 

Morgan, Clinton T ......................... 232 

Morgan, Dyan E............................ 232 

Morrison, J. Kaitlin .......................... 99 

Moss, Eric G ......................... 104, 117 

Mueller, Louisa ............................. 202 

Mullen, Greg ........................... 68, 133 

Muller, Michael................................ 81 

Murphy, Shaun P ............................ 69 

Murray, John I ................................. 72 

Mushi, Juliet.................................... 12 

N 

Naar, Anders M ............................. 142 

Nabhan, Ahmad ............................ 169 

Naef, Felix .................................... 221 

Nance, Jeremy ..................... 203, 226 

Narbonne, Patrick ......................... 159 

Narlikar, Geeta.............................. 169 

Nash, Bruce .................................. 225 

Navarro Gonzalez, Rosa E ........... 157 

Navarro, Rosa E ........................... 102 

Navidzadeh, Nathan ..................... 159 

Neault, Mathieu ............................ 198 

Needleman, Daniel ......................... 19 

Nehrke, Keith .................................. 50 

Nelson, Christian R....................... 170 

Nesmith, Jessica ............................ 32 

Ngo, Minh ....................................... 69 

Nguyen, Jillian .................................. 3 

Nicholas, Hannah R...................... 136 

Niebergall, Lorissa J ..................... 142 

Nkengfac, Bernard........................ 198 

Noatynska, Anna ............................ 76 

Noble, Daniel ................................ 233

Norman, Kenneth ..................... 63, 79 

Nykamp, Keith .............................. 201 

O 

O’Connell, Kevin F........................ 181 

O’Flaherty, Brendan ........................ 64 

Oldenbroek, Marieke .................... 118 

Onami, Shuichi ....................... 16, 223 

Orozco, Jose M .............................. 70 

Ortiz Sanchez, Marco ................... 233 

Osterberg, Valerie ........................... 54 

Otsuka, Anthony J .......................... 71 

P 

Pacquelet, Anne ........................... 228 

Padgett, Richard W ...................... 127 

Paix, Alexandre............................. 171 

Palladino, Francesca .................... 139 

Panbianco, Costanza ..................... 76 

Parry, Jean M ................................. 72 

Paschal, Cate R............................ 170 

Patel, Anvi..................................... 122 

Patel, Falshruti B ............................ 73 

Patel, Tulsi .................................... 138 

Pattabiraman, Divya ....................... 74 

Patterson, Joseph R ............. 146, 182 

Pecreaux, Jacques ....................... 229 

Perlman, Benjamin ....................... 122 

Peters, Maureen A .......................... 50 

Petrella, Lisa N ............................. 172 

Phillips, Bryan T .................... 105, 121 

Piano, Fabio ................................... 53 

Piasecki, Brian ................................ 64 

Pickle, Catherine S ......................... 35 

Piekny, Alisa J ....................... 193, 208 

Pintard, Lionel..................... 75, 76, 77 

Pioppo, Lauren ............................. 116 

Plaga, Alexis R ............................. 224 

Podbilewicz, Benjamin.................... 60 

Pohl, Christian ................................ 44 

Poole, Daniel S ............................... 86 

Portman, Douglas ......................... 205 

Praitis, Vida .................................... 12 

Presler, Marc ............................ 14, 74 

Priess, James R ............................... 2 

Prodon, Francois ............................ 22 

Prouteau, Manoel ......................... 109 

Q 

Qadota, Hiroshi............................... 55 

Quach, Thanh K ........................... 137 

Quinn, Christopher C .................... 210 

R 

Rahe, Dylan P .............................. 138 

Rahimi, Sina ................................... 15 

Rakotomalala, Cedric ................... 139 

Ramani, Arun K ............................ 158 

Rand, Jim ............................... 68, 133 

Ranjan, Sinthu .............................. 114 

Rapoport, Veronika ....................... 130 

Reddien, Peter................................ 96 

Reedy, April R ................................. 55 

Refai, Osama M.............................. 78 

Rehain, Kathryn ............................ 173 

Reid, Anna .................................... 136 

Reiner, David J ............................... 49 

Rhoads, Robert E ......................... 160 

Rhos, Patrcia .................................. 78 

Richaudeau, Benedicte ...... 75, 76, 77 

Riche, Soizic ........................... 19, 229 

Rimann, Ivo .................................... 11 

Robert, Valerie J ........................... 139 

Robertson, Scott M ....................... 118 

Robertson, Scott ....................... 40, 46 

Rocheleau, Christian .............. 66, 120 

Rocheleau, Simon K ..................... 187 

Rohrschneider, Monica R ............. 203 

Rollins, Evvi .................................... 78 

Rose, Lesilee S ...................... 33, 222 

Rosu, Simona ................................. 17 

Rottiers, Veerle ............................. 142 

241

Roy, Debasmita .............................. 30 

Roy, Peter ....................................... 11 

Roy, Richard ......................... 148, 159 

Roy, Sarah H .................................. 92 

S 

Saenz-Narciso, Beatriz ................. 108 

Saito, Mako..................................... 92 

Salcini, Lisa .................................. 141 

Salem, Alex................................... 195 

Sanegre, Sabina ........................... 230 

San-Miguel, Adriana ..................... 217 

Santarella-Mellwig, Rachel ............. 25 

Santella, Anthony............................ 44 

Sarasija, Shaarika .......................... 79 

Sarkeshik, Ali ............................ 23, 86 

Satish, Shruthi ........................ 41, 115 

Sawin, Emma ................................. 10 

Schacht, Angela.............................. 12 

Schaeffer, Arielle ........................... 114 

Schartner, Caitlin M ........................ 35 

Scheckel, Claudia ........................... 29 

Schisa, Jennifer A ................. 146, 182 

Schneider, Jennifer ......................... 81 

Schuh, Amber ................................. 23 

Schultz, Robbie D ................... 80, 218 

Schumacher Tucker, Jennifer A ...... 47 

Schumacher, Bjoern ..................... 145 

Schumacher, Bjorn ......................... 81 

Schumacher, Jill M ......................... 20 

Schvarzstein, Mara ......................... 31 

Schwager, Francoise ...................... 22 

Schwarze, Katrin ............................ 23 

Schwendeman, Andrew R ............ 100 

Seidel, Hannah S.......................... 174 

Seiler, Jonas ................................... 22 

Sengupta, Madhu ......................... 146 

Session, Dane .............................. 163 

Seydoux, Geraldine 25, 171, 181, 231 

Shaham, Shai ........................... 7, 100 

Shakes, Diane C........................... 173 

242 

Shakes, Diane ................................ 14 

Shen, Kang ........................... 192, 217 

Shi, Herong................................... 114 

Shi, Xiaoqi ...................................... 45 

Shin, Heaji .............................. 28, 175 

Shin, Tae-Ho ................................. 106 

Shirayama, Masaki ....................... 106 

Shivas, Jessica ................................. 4 

Shivendra, Kishore ......................... 98 

Shorrock, Meghann ...................... 152 

Sifuentes, Margarita H. ................. 176 

Silva-Garcia, Carlos G .......... 102, 157 

Simionato, Elena .......................... 101 

Simske, Jeff ............................ 12, 204 

Singaravelu, Gunasekaran ............. 15 

Singh, Nirupama ........................... 125 

Singson, Andrew............................. 15 

Skop, Ahna ........................... 4, 86, 89 

Skorobogata, Olga........................ 120 

Smith, Harold E .................... 181, 219 

Smith, Jarrett ................................ 231 

Smith, Michele .............................. 205 

Smolikove, Sarit.................... 177, 183 

Snyder, Matthew P ....................... 178 

Song, Anren .................................. 160 

Song, Mi Hye ............................ 85, 91 

Sorokin, Elena P ........................... 234 

Soto, Martha C ..................... 3, 73, 83 

Spengler, Justin W.......................... 82 

Spike, Caroline ............................. 179 

Stanfield, Gillian.................... 153, 155 

Starich, Todd ................................. 180 

Starr, Daniel A ................................. 69 

Stein, Kathryn K.............................. 90 

Stein, Lincoln D .............................. 45 

Sternberg, Paul W .......................... 59 

Stetak, Attila.................................... 56 

Stock, Tyson ................................... 12 

Strome, Susan .............................. 172 

Subash, Jacob J ........................... 160 

Subasic, Deni ................................. 98

Sugimoto, Asako....................... 21, 22 

Sullivan-Wilson, Alexander ............. 12 

Sumiyoshi, Eisuke .......................... 21 

Sundaram, Meera V ....................... 72 

Swoboda, Peter .............................. 64 

Szewczyk, Nate .............................. 57 

Szymczak, Lindsey ....................... 114 

T 

Takayama, Jun ............................... 16 

Tanner, Kimberly D ....................... 214 

Tannoury, Hiba .............................. 151 

Taubert, Stefan ............................. 129 

Tavernier, Nicolas ..................... 75, 76 

Terasawa, Masahiro ....................... 21 

Tian, Chenxi ......................... 107, 114 

Tilleman, Lesley ................................ 9 

Tobin, David V ................................ 92 

Toulabi, Leila..................................111 

Towarnicky, Leah ............................ 14 

Toya, Mika ...................................... 21 

Tse, Yu Chung ................................ 88 

Tu, Zheng Jin ................................ 162 

Tuck, Simon .................................... 30 

U 

Udin, Gilles ................................... 109 

V 

Vadla, Bhaskar ..................... 104, 117 

Valbuena, Valeria S. M ................... 82 

Valfort, Aurore-Cecile ...................... 19 

Vallier, Laura G ............................. 140 

van Oudenaarden, Alexander ....... 131 

Vandamme, Julien ........................ 141 

Vanfleteren, Jacques R .................... 9 

VanGompel, Michael White ............ 33 

VanHoven, Miri ............................... 52 

Vargas, Chris .................................. 52 

Verbrugghe, Koen JC ..................... 24 

Vergara, Sandra ........................... 106 

Vertin, Eric ...................................... 91 

Via, Zachary ..................................111 

Vibbert, Jack ................................. 149 

Villanueva-Chimal, Angel E .......... 102 

Villeneuve, Anne ......... 17, 31, 74, 184 

Vine, Annalise ................................. 51 

Vlaeminck, Caroline.......................... 9 

Vora, Setu ..................................... 121 

Voronina, Ekaterina ...................... 171 

W 

Walck-Shannon, Elise M .............. 206 

Walker, Amy K .............................. 142 

Walker, Rachel ............................. 189 

Wallace, Andre ........................... 3, 83 

Waller, Bridget .............................. 207 

Walsh, Taylor A ............................. 156 

Walston, Timothy .......................... 207 

Walstrom, Katherine M ........... 82, 220 

Wang, Emily ................................. 156 

Wang, Haibin .................................... 8 

Wang, Peng .................................. 125 

Wang, Xiaochen ............................... 8 

Wang, Yuemeng ........................... 181 

Waters, Colin T ............................. 134 

Watts, Jenny ................................. 142 

Weber, Katherine P ...................... 122 

Wendland, Emily ........................... 159 

Werner, Michael .............................. 88 

Wernike, Denise ........................... 208 

Whipple, Lauren ........................... 205 

White, Ana .................................... 143 

Whitehurst, Rebecca E ................... 49 

Wiesenfahrt, Tobias ...................... 144 

Wightman, Bruce .................. 116, 122 

Williams, Ash ................................ 167 

Williams, Claire R ......................... 209 

Wilson, Luke D ............................. 164 

Winter, Ethan ................................ 173 

Wisidagama, Dona Roonalika ...... 130 

243

Witte, Kristen .................................. 23 

Wolters, Stefanie .......................... 145 

Wong, Chiyen ................................. 65 

Wood, Megan P ............................ 182 

Woollard, Alison .................... 112, 212 

Wright, Jane E ................................ 29 

Wu, Jui-Ching ............................... 222 

X 

Xiang, Shang .................................. 66 

Xu, Fei ............................................ 39 

Xu, Shawn ...................................... 15 

Xu, Tao ........................................... 86 

Xu, Xia .......................................... 178 

Xu, Yan ......................................... 210 

Y 

Yang, Qiutan ................................. 196 

Yang, Xiao-Dong ............................ 40 

Yates III, John ........................... 23, 86 

Yee, Callista .................................. 199 

Yilma, Zelealem .............................. 12 

Yin, Yizhi ............................... 177, 183 

Yochem, John ................. 54, 166, 225 

Yohannes, Lensa ............................ 12 

Yokoo, Rayka ............................... 184 

Yoo, Bum Ho ................................ 201 

Yucel, Duygu ................................ 136 

Yun, Sijung ................................... 219 

Z 

Zacharias, Amanda L ...................... 72 

Zaidel Bar, Ronen ......................... 211 

Zand, Tanya P ................................ 49 

Zanin, Esther .................................. 22 

Zarkower, David...................... 34, 200 

Zavolan, Michaela .......................... 98 

Zawadzki, Karl A ........................... 184 

Zetka, Monique ..................... 139, 160 

Zhang, Dongyan ............................. 91 

Zhang, Hongjie ................................. 5 

244 

Zhang, Yan ....................................... 8 

Zhou, Shan ..................................... 10 

Zhou, Xufei ..................................... 39 

Zouak, Melissa ............................. 229 

Zuckerman, Jennifer A .................. 103

VE. 

DR. 

NIVERSITY 

ELM 

D R. 

UNIVERSITY BUILDINGS, ACCOMMODATIONS, PARKING and POINTS OF INTEREST 

Conference Site Location Accommodation Location Parking Lot Location Point of Interest Location 

1. Memorial Union C1 A. Lowell Hall F2 Lot 6 C1 University Bookstore D2 

2 Union South & Hotel B2 B. Chadbourne Hall C2 Lot 29 C3 Walgreens D2 

C. University Inn D2 Lot 46 D2 Walgreens Pharmacy D2 

D. Doubletree Hotel E3 Lot 83 D2 Monona Terrace G2 

E Dahlmann Campus Inn D2 Overture Center F2 

1 

2 

3 

2012 

CAMPUS 

AVE. 

BREESE 

TER. 

DR. 

C. elegans Development, Cell Biology, & Gene Expression Meeting 

Camp Randall 

Stadium 


A B C D E 

F 

ENGINEERING 

Union South 

and Hotel 

2 

Chadbourne 

Hall 

B 

Memorial Union 

P6 

1 

Fountain 

Walgreens 

Pharmacy 

P29 

University 

Bookstore 

Lowell Hall 

Walgreens 

Lake St. 

Parking Ramp 

P83 

P46 

A 

E 

Dahlmann 

Campus Inn 

C 

University 

Inn 

D 

Doubletree 

Hotel 

A B C D E 

F 

Overture 

Center 

State 

Capitol 

G 

Walkway 

Monona 

Terrace 

LAKE 

MONONA 

G 

1 

2 

3

N 

W 

Studio B 

Studio A 

Workshops 

w Union Theater (1st floor) 

Slide Preview Room 

{ Rosewood (3rd Floor West) 

Meal/Buffet Rooms 

x Inn Wisconsin (2nd floor East) 

Phone 

Opening • Reception 

y Tripp Commons (2nd floor) 

Banquet/Dance 

Union South-See Campus Map 

� 

Entrance 

� 

� 

Play Circle 

Theater 

Wisconsin Union 

Theater 

Park Street 

Entrance 

Stairs 

Stairs to studiosW 

Rosewood 

Room 

CE Development Locations 

Registration 

{ Annex Room (2nd floor) 

Oral Sessions 

w Union Theater (1st floor) 

Poster Sessions 

u Great Hall (4th floor) 

v Reception Room (4th floor) 

z Main Lounge (2nd floor) 

E 

S 

Stairs to 

2nd floor 

Great Hall 

Phone 

• 

M 

Langdon 

Room 

3rd Floor West 

NOTE: Studios are not handicapped 

accessible. Accessible from West stairs only. 

W 

Stairs 

To Union Theater 

Box 

Office 

Stairs 

Hotel Rooms 

� 

Class of '24 

Recept. Room 

Elevator 

Browsing 

Library 

Stairs 

Stairs 

Stairs 

To Park Street 

4th Floor 

Elevator 

Capitol View 

� 

Main Lounge 

Outside Terrace Area 

Stairs 

Rathskeller 

MEMORIAL UNION 

Board 

Room 

Beefeaters 

Room 

To Terrace 

W/M 

Stairs 

Stairs 

Tripp Deck 

Annex Room 

Conference Headquarters rs 

To MU Games Room (downstairs) 

Paul Bunyan Room 

Art Gallery 

Elevator 

Main Entrance 

W 

Round 

Table 

Room 

Elevator 

Old Madison Room 

M 

� 

Tripp Commons 

Elevator 

Stairs 

M W 

Stairs 

Phone• 

Stairs 

Lakefront on Langdon 

Elevator 

� 

Central Reservations 

and Conference Services 

MU Floor Legend 

Floor Room in Use 

Fourth 

Third 

Second 

First 

3rd Floor East 

NOTE: Accessible from East elevator/ 

East end of building only. 

2nd Floor 

Profile Room 

Inn Wisconsin Room 

M 

W 

Inn WI Deck 

1st Floor 

Stairs Elevator Information Desk 

Tyme 

Phones 

Stairs 

Daily Scoop 

Deli 

Front 

Entrance

C. elegans Development, Cell Biology, & Gene Expression Meeting ...

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