Robert Barstead et al. (2001) International C. elegans Meeting "The C elegans Knockout"

Bin Shen, Shohei MItani, Don Moerman, Malini Viswanathan, Robert Barstead, Keiko Gengyo-Ando, Jamie Osborn, Miwa Tamura, Gary Moulder, Jeff Holmes, Yuji KOHARA, Sheldon McKay, Steven Jones, Etsuko Machiyama, Erin Gilchrist, Sachiko Noguchi, Mark Edgley, Anil Dsouza, Martin Lansdale

[International C. elegans Meeting, 2001]

Roberts TM (1983) International C. elegans Meeting "crawling C elegans spermatozoa contain 2-nm filaments."

Roberts TM

[International C. elegans Meeting, 1983]

Andrew Roberts et al. (2001) International C. elegans Meeting "TGF-beta Signaling and Gene Expression"

Rick Padgett, Shi-lan Wu, Andrew Roberts, Cole Zimmerman

[International C. elegans Meeting, 2001]

TGF-beta like signaling in C. elegans occurs in two pathways, the dauer and the SMA/MAB pathways. While they are distinct in function, both pathways share a common Type II receptor, Daf-4. Although the major components of both pathways are fairly well studied, downstream effectors and target genes have yet to be characterized. To address this question we have chosen to do global expression studies using cDNA microarrays. The first experiment involves comparing mRNA generated in daf-4 mutant animals to N2 mRNA from a comparable developmental stage. We are in the process of analyzing this data. We are particularly interested in expression changes that affect cell cycle regulation and in identifying novel targets of TGF-beta signaling. In a complimentary experiment, screens are underway to characterize the link between TGF-beta and the cell cycle using genetic screens built around an rnr::GFP reporter. Mutants identified by this screen should represent downstream effectors of TGF-beta that influence cell cycle control. Together these experiments should provide a wealth of information for our continued study of TGF-b signaling and the cell cycle. In addition, because our lab works mainly with the SMA/MAB pathway, we are interested in downstream effector molecules that are influenced solely by this pathway. Previous and ongoing experiments in the lab include a screen for small body size, as well as lon-2 and lon-1 suppressor screens, which have already uncovered many of the essential components of the pathway (see abstracts from Gumienny and Zimmerman). Experiments are underwayto analyze mRNA from animals expressing a dbl-1 overexpression construct (Long body phenotype) and compare that to mRNA isolated from sma-6 mutant animals. Because dbl-1 is the SMA/MAB specific ligand, and sma-6 is the SMA/MAB specific Type I receptor, this should give us great insight into the downstream targets of the pathway. When coupled with the information already available from our genetic screens, these experiments will shed much needed light on the targets of TGF-beta signaling in C. elegans.

Robert T Fazzio et al. (2001) International C. elegans Meeting "A Role for VAV-1 in Pharyngeal Contraction"

Robert T Fazzio, Jerry E Mellem, Mary C Beckerle, Andres V Maricq

[International C. elegans Meeting, 2001]

VAV, VAV-2 and VAV-3 comprise a family of vertebrate guanine nucleotide exchange factor proteins that function in the activation of Rho signaling pathways. This activation occurs in the form of a VAV-mediated exchange of GDP for GTP on Rho in response to cellular stimulation. Once activated, Rho pathways promote an organized remodeling of the actin cytoskeleton, which results in processes such as cellular motility, proliferation and growth. Misregulation of these signaling pathways through specific mutations of the vav gene results in the constituative activation of Rho family members and oncogenesis. To better understand the role of VAV in the regulation of Rho signaling, we have undertaken a genetic and molecular analysis of VAV in C. elegans . We have previously reported the cloning and expression pattern of the C. elegans vav homolog ( vav-1 ). Briefly, vav-1 encodes a 1000 amino acid protein that shares 34% identity with vertebrate family members and contains the domains predicted to be important for nucleotide exchange factor activity. VAV-1 is localized to pharyngeal muscle, g1 gland cells, body wall muscle, and is sometimes found in vulval tissues and somatic gonad. We have also reported that the deletion of vav-1 results in larval lethality, presumably caused by the disruption of synchronous pharyngeal muscle contraction. Electrophysiological analysis of vav-1 null animals suggests that the pharyngeal defect may result from inadequate conduction between individual muscle cells. This hypothesis is based on results obtained from Electropharyngeograms (EPG) performed on the vav-1 mutant: EPG analyses of vav-1 animals produce traces in which the depolarization wave is extended over an abnormally long period of time relative to wild type EPGs. This might indicate that each pharyngeal muscle cell is depolarizing at its own pace, uncoupled from other cells. That vav-1 mutant pharyngeal muscle tends to fibrillate, rather than pump, supports the uncoupled hypothesis. We are now performing experiments designed to further study the vav-1 null phenotype. Recently, we identified that a limited expression of vav-1 in the pharynx was enough to rescue this lethality. Further tissue-specific expression studies are now attempting to localize the vav-1 requirement to pharyngeal muscle cells or g1 gland cells. We are also performing a directed mutational analysis to identify the potential for VAV-1 to function in nucleotide exchange.

Robert M Steven et al. (2001) International C. elegans Meeting "THE IDENTIFICATION OF PROTEINS THAT INTERACT WITH UNC-73/TRIO IN C. ELEGANS"

Terry Kubiseski, Joseph Culotti, Tony Pawson, LiJia Zhang, Robert M Steven

[International C. elegans Meeting, 2001]

C. elegans unc-73 , also known as trio in mammals and Drosophila , was the first gene of this family to be identified with a role in axon pathfinding. The axons of many neurons fail to reach their target tissues and often travel along abnormal pathways in unc-73 mutant animals. Recently it was also discovered that trio is required for the correct formation of photoreceptor, CNS and motor axon pathways in Drosophila . Experiments in both C. elegans and Drosophila reveal that UNC-73/Trio function involves signaling through the Rac GTPase and its downstream effectors to regulate the cytoskeletal rearrangements necessary for growth cone migrations. unc-73 is a complex gene predicted to encode seven overlapping transcripts. Together the intracellular proteins encoded by these transcripts contain, from N terminus to C terminus, eight spectrin-like repeats, a tandem Dbl homology (DH) and pleckstrin homology (PH) domain combination, an SH3 domain, a second DH/PH domain combination, an immunoglobulin domain and a fibronectin type III domain. DH domains are known to have RhoGEF activity, which is the ability to activate members of the Rho family of GTPases. The first UNC-73 DH/PH domain specifically activates the Rac GTPase while the second domain is specific to Rho. Interestingly, animals with a mutation that eliminates the activity of the first DH domain are uncoordinated (Unc), presumably as a result of axon guidance defects, while a deletion of the exons encoding the second DH domain results in early larval lethality. Two approaches are being used to identify proteins that interact with UNC-73. First, a genetic screen is being set up based on the observation that overexpression of a construct encoding a truncated form of UNC-73, containing just the DH-1/PH-1 and SH3 domains, in wild-type animals results in a severe Unc phenotype, similar in character to that of the moderate to severe alleles of unc-73 . An integrated line containing this construct is fully penetrant for the Unc phenotype and will be used in a screen for nonUnc suppressors. Mutations in genes that might be expected to suppress this dominant phenotype include those that act downstream of the Rac GTPase pathway. Mutations in genes upstream of unc-73 that activate the UNC-73 DH1 domain may also suppress. Second, a yeast two hybrid screen was used to identify several proteins that potentially interact with UNC-73. One of the interacting proteins, also confirmed by pull-down experiments with bacterially expressed proteins, is an actin-binding protein. This is consistent with our hypothesis that UNC-73 is involved in the regulation of the actin cytoskeleton.

AB Roberts et al. (1999) International C. elegans Meeting "Genetic analysis of genes involved in hypodermal function in Caenorhabditis elegans"

C Clucas, AB Roberts, IL Johnstone

[International C. elegans Meeting, 1999]

The C. elegans cuticle is a complex multilayer stucture consisting predominately of a group of small collagen-like proteins known as cuticular collagens. The collagen genes show differential regulation during the nematode life cycle with different genes being expressed at specific stages e.g. dpy-7 is an early collagen gene and is expressed about 4 hours earlier than the col-12 collagen gene. The cuticle plays a central role in the maintenance of post-embryonic body shape and is synthesised and secreted by the hypodermal cells. Previously, a forward genetic screen using strains carrying either col-12/GFP or dpy-7/GFP fusion transgenes had generated several embryonic lethal mutants with phenotypes suggesting hypodermal abnormalities. Genetic and molecular characterisation of 2 of these mutant alleles - ij013 and ij017 is currently underway. ij013 does not express col-12/GFP . It elongates and then collapses, has functional body wall muscle and apparently normal gut development. ij017 shows an abnormal pattern of col-12/GFP expression. It elongates and then collapses back with herniations appearing on the hypodermis. Both mutants have normal dpy-7/GFP expression patterns which, with the evidence of elongation, suggests the presence of a dysfunctional cuticle. Mapping of the mutant genes has been performed using STS mapping and classical genetic crosses. ij013 has been assigned to Chromosome V between unc-51 and pha-4 . Complementation tests show that ij013 is distinct from pha-4 . ij017 has been placed between lin-31 and stP101 on Chromosome II. Phenotypic rescue using cosmids covering these regions is currently being performed.

Robert D Slone et al. (2001) International C. elegans Meeting "Dissecting mechanisms of X-adrenoleukodystrophy neurotoxicity using C. elegans"

Monica Driscoll, Heather Thieringer, Robert D Slone

[International C. elegans Meeting, 2001]

X-linked adrenoleukodystrophy (X-ALD) is a progressive degenerative disorder that affects the nervous system and adrenal glands in affected males. X-ALD is a peroxisomal disease in which peroxisome structures are present in the cell, but very long chain fatty acids (VLCFA) are not transported into the peroxisome to undergo degradation. Consequently, excess VLCFAs accumulate especially in the adrenal gland and the brain. The protein that is missing or defective is the ALD protein (ALDP), which is a member of the ABC transporter protein family. In a related finding, a gene that works directly upstream of the ALDP in the degredation of VLCFAs, the VLCFA CoenzymeA synthetase, is implicated in neurodegeneration in the Drosophila . This enzyme normally activates the VLCFAs for subsequent degradation in the peroxisome. In the fly, absence of this gene (called bubblegum ), causes late-onset neurodegeneration. These two findings imply that excess very long chain fatty acids in the cell may have a role in triggering neurodegeneration. The mechanism through which this occurs is unknown but is of clear clinical interest. We have identified homologs for both the ALDP and the VLCFA synthetase genes in the C. elegans genome. While there are numerous fatty acid synthtases in the worm, there is just one protein (Y65B4Bl.5) that contains high homology to the two signature motifs that are specific for the growing "bubblegum" family of VLCFA synthetases. Members of this "bubblegum" family of synthetases are expressed in the nervous system in human, mouse, and fly. We are examining the expression pattern of the C. elegans homolog Y65B4BL.5 with GFP analysis. Preliminary experiments using RNAi suggest that knockdown of this gene may result in embryonic lethality. Due to the possibility of the neural expression of this gene, and the associated difficulty of targeting RNAi to the nervous system, we have constructed a neuron-specific foldback RNA construct under control of the mec-7 promoter. Identification of neurodegeneration as a result of knockdown of this gene in the worm would enable us to genetically dissect the cellular and molecular pathogenesis of X-linked adrenoleukodystrophy. We will report our results on developing this model for VLCFA toxicity.

Robert Barstead et al. (2005) International Worm Meeting "The C. elegans Gene Knockout Consortium"

Angela Fisher, Candice Navaroli, Allison Hay, Robert Barstead, Anna Rankin, Lucy Liu, Joanne Lau, Nadereh Rezania, Mark Edgley, Owen Dadivas

[International Worm Meeting, 2005]

The C. elegans Gene Knockout Consortium (http://www.celeganskoconsortium.omrf.org/) pursues systematic targeted gene disruption in the nematode, both upon request for the research community and in the larger context of the whole set of genes for which human orthologs exist (about 7,000 genes). This work is currently based on a reverse-genetics PCR method, generating single-gene deletions. All deletions are stabilized as homozygous or balanced heterozygous strains, the deletion breakpoints are sequenced, and the strains and data are placed immediately into the public domain through our collaborators at the Caenorhabditis Genetics Center (CGC: http://biosci.umn.edu/CGC/CGChomepage.htm) and WormBase (http://www.wormbase.org/). To date the Consortium has generated deletions in more than 1800 genes, and more than 1300 of these have been stabilized and archived at the CGC. Distribution of Consortium strains accounts for an increasing proportion of distributions from the CGC every year, and stood at 20% for the calendar year 2004.The reverse genetics PCR method using UV/TMP as the mutagen has evolved in our hands into a robust system for efficient generation of deletion mutations. However, some target genes have been refractory to this method, and we therefore continue to evaluate other isolation/detection methods (for example, see abstracts on array CGH and resequencing at this meeting). We will report on our latest progress using all methods.Additional authors for whom inadequate space was allowed by abstract submission system: Peter Huang (UBC and BC Genome Sciences Centre), Jason Maydan (UBC), Sheldon McKay (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Roberts TM et al. (1981) International C. elegans Meeting "directed membrane flow on the pseudopods of C elegans spermatozoa."

Ward S, Roberts TM

[International C. elegans Meeting, 1981]

Roberts J et al. (1985) International C. elegans Meeting "C elegans DNA that directs segregation in S Cerevisiae."

Roberts J, Stinchcomb DT, Mello CC, Hirsh DI

[International C. elegans Meeting, 1985]