Robin Schneider et al. (2006) European Worm Meeting "The Loss of the Single APC Homolog apr-1 Causes Hyperplasia in the C. elegans Intestine"

Olaf Bossinger, Gisela Helbig, Robin Schneider

[European Worm Meeting, 2006]

Robin Schneider, Gisela Helbig and Olaf Bossinger. In humans APC is a powerful tumour suppressor that is mutated in most cases of sporadic or congenital colorectal cancer. In these forms of cancer, cells lose their ability to control the cell cycle and cell migration along the crypt-villus axis is disturbed. The C. elegans intestine is a simple epithelial tube that consists of only 20 cells (E-cells). Depletion of the C. elegans APC homolog APR-1 (Hoier et al., 2000) by bacterial RNAi causes hyperplasia of E-cells. In contrast, apr-1(RNAi) embryos contain up to 40 E-cells, which in most cases achieve to arrange into a tube-like structure. The use of gut-specific RNAi against APR-1 and the gain-of-function phenotype of the cycline dependent kinase CDC-25.1 (Clucas et al., 2002) suggests that intestinal cell proliferation is under organ-specific control. Preliminary data indicate that C. elegans homologs of the vertebrate WNT pathway are also involved in the regulation of endodermal cell proliferation. A detailed analysis of gut-specific hyperplasia phenotypes will be presented and discussed with regard to the involved pathways.

Christopher W Brey et al. (2001) International C. elegans Meeting "ENHANCEMENT OF STEINERNEMA CARPOCAPSAE DESICCATION TOLERANCE BY GENETIC IMPROVEMENT"

Randy Gaugler, Christopher W Brey

[International C. elegans Meeting, 2001]

The entomopathogenic nematode (ENP) Steinernema carpocapsae , is an important biological control agent for a wide range of soil dwelling insect pests. However, the field efficacy of this ENP is limited by its sensitivity to high drought and salinity conditions. We report efforts to improve the desiccation tolerance of S. carpocapsae by transforming it with the trehalose-6-phosaphate synthase (tps1) and glycogen synthase(gsy1)genes. Trehalose-6-phosphate synthase and glycogen synthase are enzymes involved in the biosynthesis of trehalose, a disaccharide that accumulates to stabilize the lipid biomembranes in many organisms when in response to stress. To increase desiccation tolerance by genetic modification, we have cloned gene tps1 from yeast and C. elegans into expression vectors pJJ436 and pPD95.67, respectively. In addition, we also cloned gsy1 from Steinernema feltiae into expression vector pJJ436. Vector pJJ436 contained the Ce sq-1 promoter, whereas pPD95.67 contained the promoter of the tps1 Ce gene. All vectors contained the gfp transformation gene which was used as a selection marker. Vector constructions (yeast: pJYeTr.1; C. elegans : pP67CeTr.2; S. feltiae : pJSfTr.1) were microinjected independently into young S. carpocapsae females (48 h from infective juvenile stage). Injected females were mated with noninjected males for 2-4 days and progeny were screened for gfp expression. After selecting and retaining gfp expressing individuals for three generations, F3 progeny were tested for desiccation tolerance. We will present details of our methodology and results in our presentation.

Nathalie Velarde et al. (2005) International Worm Meeting "F-actin dynamics in early C. elegans development"

Nathalie Velarde, Fabio Piano

[International Worm Meeting, 2005]

Actin has been shown to play key roles during early development in the C. elegans embryo (Schneider and Bowerman, 2003). However, it has been difficult to faithfully reproduce F-actin dynamics in vivo during early embryogenesis. To visualize F-actin we have generated a transgenic line that uses the F-actin binding domain of Drosophila moesin to decorate endogenous actin filaments with GFP (GFP::Moe). The GFP::Moe fusion line appears to be specific for filamentous actin and allows the visualization of F-actin dynamics in C. elegans in embryos. Preliminary evidence shows that F-actin is very dynamic in many cellular processes from prior to fertilization through the first mitotic cellular division. During fertilization a posterior actin cap forms and then dissipates. Prior to the first cell division F-actin becomes enriched in the anterior, similar to the anterior PAR proteins. As seen in par-3 mutants (Kirby et. al., 1990), depleting the embryo of PAR-6 with RNAi prevents this enrichment, suggesting that PAR-6 and the other anterior PARs may be required for F-actin to accumulate in the anterior. We have also observed the presence of highly dynamic actin comets in the early embryo. Preliminary evidence suggests that these comets may be involved with endocytic processes. Using results from large-scale RNAi surveys, we are employing the GFP::Moe line to perform secondary screening of genes associated with pseudocleavage defects to further characterize this process. We will present our progress in using this GFP::Moe line to study genetic interactions involving actin dynamics in early development.

Mihail Sarov et al. (2008) European Worm Meeting "A first version of the C. elegans Transgeneome: genome scale set of tagged fosmid transgenes"

Vlarie Reinke, Stewart Kim, Anthony Hyman, Francis Stewart, John Murray, Robert Waterston, Michael Snyder, Mihail Sarov, Mei Zhong

[European Worm Meeting, 2008]

Protein tagging with fluorescent and/or affinity epitopes provides a. generic way to address gene function. Recently we have developed a 96 well. format recombineering pipeline that allows us to rapidly convert large. genomic DNA clones such as BACs or fosmids into tagged transgenes [1]. Due. to their large size, these transgenes usually contain most cis regulatory. elements and correctly recapitulate the endogenous patterns of gene. expression. Ballistic transformation of fosmid transgenes allows us to. routinely generate low copy integrated worm lines, with stable and. reproducible expression of the tagged protein in variety of tissues and. developmental stages including germline and early embryos.. The high efficiency and fidelity of this approach allows us to engineer. genome scale sets of fosmid transgenes with unprecedented throughput.. Generation of a comprehensive "Transgeneome" resource with a tagged. transgene for most C. elegans genes is now within reach. As a first step. towards this goal we started with several targeted projects that aim to. determine the physical interactions and the localization of more than 1000. proteins involved in transcription regulation, chromatin structure and. function and cell division. As a part of the ModENCODE consortium we have. already generated hundreds of transgenic constructs and tens of integrated. transgenic worm lines. That allowed us to test more than 20 different tag. combinations and we now have validated tags that perform very well for. protein localization, purification and chromatin immunoprecipitation. At. the meeting we will present results illustrating the power of the approach. as well as some of the lessons we learned from the large-scale application. of the method. We will discus how the community can access these resources. and we hope to initiate collaborations that will translate this research to. other functional gene sets.. 1. Sarov, M., S. Schneider, A. Pozniakovski, A. Roguev, S. Ernst, Y. Zhang,. A.A Hyman, and A.F Stewart. 2006. A recombineering pipeline for. functional genomics applied to Caenorhabditis elegans. Nature Methods 3:. 839-844.

Mihail Sarov et al. (2007) International Worm Meeting "Recombineering based high throughput protein tagging in C. elegans."

Mihail Sarov, Francis Stewart, Anthony Hyman

[International Worm Meeting, 2007]

The methods for protein tagging in C. elegans so far have been inefficient and largely dependent on artificial cDNA based constructs, which can lack important regulatory elements. The recent generation of a genomic fosmid library[1] opened the possibility for direct modification of any gene of interest in its native genomic environment by recombineering in E. coli. We recently published a method for precise in vivo engineering of large genomic clones into GFP tagged transgenes for ballistic transformation[2]. Using such transgenes, we reproduce known and document new expression patterns. We further demonstrate that the tagged protein can complement the function of its endogenous counterpart. Applying the latest developments of the recombineering field and introducing several innovations of our own we have now streamlined the protocol to allow rapid 96 well format liquid culture processing in a continuous recombineering pipeline that takes just four days. This unprecedented throughput and the achieved economy of scale allow us to rapidly produce thousands of tagged transgenes. Our method has become the basis for a large-scale project aimed at comprehensive description of the expression pattern (through fluorescent reporters) and the DNA binding sites (through affinity tag based chromatin immunoprecipitation) of more than 400 C. elegans transcription factor genes (as part of the NIH funded ModENCODE program). At the meeting we will discus the advantages of a distributed, community based collaboration model that will bring this technology to any worm lab and will make the genome wide protein tagging a feasible goal. 1. Perkins, J., K. Wong, R. Warren, J.E. Schein, J. Stott, R. Holt, S. Jones, M.A. Marra, and D. Moerman. 2005. A Caenorhabditis elegans fosmid library. In 15th International C. elegans Conference, University of California, Los Angeles. 2. Sarov, M., S. Schneider, A. Pozniakovski, A. Roguev, S. Ernst, Y. Zhang, A.A. Hyman, and A.F. Stewart. 2006. A recombineering pipeline for functional genomics applied to Caenorhabditis elegans. Nature Methods 3: 839-844.

Alison Slinskey Legg et al. (2007) International Worm Meeting "C. elegans research for high school teachers and students: The Gene Team."

Lew Jacobson, Alison Slinskey Legg

[International Worm Meeting, 2007]

The Gene Team is a 5-year program to explore and refine a novel mechanism for engaging high school teachers and high school students in biomedical research and inquiry-based learning, with a focus on the genetics of model organisms. The objectives are: (a) To improve the knowledge and process skills of high school biology teachers; (b) To introduce high school students to the practice of scientific research and develop their skills in scientific thinking; (c) To design and implement inquiry-based curricular modules using model organisms for high school biology classes. The core of the program is a 7-week summer session (40 hrs/week), during which participants in a single dedicated 1000 sq. ft. lab work in parallel on three distinct research projects involving bacteria, yeast and C. elegans. Nine participating research labs provide projects (3 each year), with the intention that the "products" (mutants, constructs, data) of the summer research are returned to the originating labs for further investigation. The novelty of the team concept is that the participants work as peers despite a wide diversity of age and experience: The 2006 Gene Team was composed of 3 high school teachers, 6 high-school students, 2 research-experienced undergraduates and 3 graduate students, under the daily supervision of a faculty member and a program staff member. Simultaneously, the teacher-participants work at the Curriculum Roundtable with a group of master teachers and University faculty to design and refine inquiry-based learning modules, targeted at state curricular standards for biology in grades 9-12. Modules may be classroom-based or lab-based, and we provide equipment, supplies and staff support for implementation during the school year. Modules are field-tested and iteratively refined through one academic year before wider dissemination. The Gene Team is supported by a Science Education Partnership Award (SEPA) from the National Center for Research Resources, NIH.

Li, Rachel C.K. et al. (2011) International Worm Meeting "Genetic and molecular investigation of PKD-2 and LOV-1 involved in male C. elegans perception of sex pheromone."

Li, Rachel C.K., Chow, King L.

[International Worm Meeting, 2011]

Transient receptor potential polycystin (TRPP) complex PKD1/PKD2 in human are important in kidney's function, and their mutations are associated with polycystic kidney disease. The C. elegans counterparts, PKD-2/LOV-1 localized to the ciliated endings of the male specific neuron CEMs, however, are indispensable for the male response to sex pheromone. In this study, we aim to identify additional PKD-2/LOV-1 polycystin signaling molecules required for sex pheromone perception. atp-2 and cwp genes are potential pkd-2 and lov-1 interacting partners based on their roles in male sensory behavior and their co-expression in CEMs. In our pheromone assays, all cwp mutant males respond normally while males with atp-2 knocked-down are defective, which suggest the distinct requirement of atp-2 in this sex-specific behavior. The interdependence between pkd-2, lov-1 and atp-2 will be ascertained by testing atp-2 RNAi males or its cell specific over-expression in pkd-2 and/or lov-1 mutants by pheromone assay. In addition, a pkd-2 promoter deletion analysis has been performed to identify upstream regulatory components of PKD-2/LOV-1 TRPP channel synthesis. As genes co-expressed in a specific cell type may share a common cis-regulatory motif, identifying the signature motif for CEM-specific expression may help uncover potential pkd-2 interacting partners by a genome-wide search for genes with a CEM-signature sequence. For the cellular function of PKD-2/LOV-1 TRPP complex in CEMs, we hypothesize that this complex is directly activated by sex pheromone and the signal is relayed intracellularly. This hypothesis will be evaluated by an in vitro system using Drosophila Schneider cells to investigate if physiological response can be triggered via PKD-2/LOV-1 TRPP complex by sex pheromone stimulation. Results from this experiment would be informative in defining the functional activity of these TRPP channels in the sex pheromone perception, shedding light on the biology and signaling of polycystin complex. (This study is supported by Research Grants Council, Hong Kong.).

H Hwang et al. (1999) International C. elegans Meeting "Nucleotide-sugar biosynthesis and glycosylation are involved in vulval morphogenesis"

H Hwang, HR Horvitz

[International C. elegans Meeting, 1999]

Eight sqv ( sq uashed v ulva) genes ( sqv-1 to 8 ) have a mutant vulval morphogenetic phenotype that is defined by reduced separation between the anterior and posterior halves of the vulva in the L4 (1). SQV-3 and SQV-8 are glycosyltransferase-like proteins, and SQV-7 is similar to a putative nucleotide-sugar transporter (2). We have previously reported the cloning of sqv-1 and sqv-4 (3). Both SQV-1 and SQV-4 are similar to enzymes involved in nucleotide-sugar metabolism. These molecular identities suggest that glycosylation is important in shaping the vulva during development. We have now determined biochemically that SQV-4 is a UDP-glucose dehydrogenase. Recombinant SQV-4 expressed in E. coli can reduce NAD in the presence of UDP-glucose, suggesting that UPD-glucose is being converted to UDP-glucuronate. Like known UDP-glucose dehydrogenases, SQV-4 appears to act specifically on UDP-glucose, as it fails to reduce NAD using any of the many other nucleotide-sugars tested. Mutant recombinant SQV-4 derived from either of the two genetically identified mutant alleles does not show detectable activity. Preliminary antibody staining using a rabbit polyclonal antibody directed against a GST::SQV-4 indicates that SQV-4 is localized to the vulval cells and several other tissues, including seam cells. This result agrees with the expression of SQV-4::GFP fusion protein and supports a model in which SQV-4 acts in the vulval cells during vulval development. We also are trying to determine the functions of the other four cloned sqv genes using biochemical assays and heterologous complementation. Lastly, we are mapping and cloning the three remaining sqv genes. 1. Herman, T. et al. (1999). PNAS 96 : 968-973. 2. Herman, T. and Horvitz, H.R. (1999). PNAS 96 : 974-979. 3. Hwang, H. and Horvitz, H.R. (1998) East Coast C. elegans meeting, p. 103.

Isaac RE et al. (1995) International C. elegans Meeting "THE METABOLISM AND INACTIVATION OF AF1 AND PF1 BY NEMATODE NEPRILYSIN."

Coates D, Priestly RM, Sajid M, Isaac RE

[International C. elegans Meeting, 1995]

AF1 (Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH2) and PF1 (Ser-Asp-Pro-Asn-Phe-Leu-Arg-Phe-NH2) have potent effects on the locomotory muscle of Ascaris suum. AF1 is inactivated by three neuropeptide-degrading enzymes found in crude plasma membranes of A.suum muscle. One of these enzymes is an endopeptidase which hydrolyses the Glu3-Phe4 bond of AF1 and which is inhibited by phosphoramidon (IC50, 0.7M), a potent inhibitor of mammalian neprilysin (neutral endopeptidase 24.11, enkephalinase). A phosphoramidon-sensitive endopeptidase is also present in membranes prepared from Caenorhabditis elegans and we are characterising the enzyme by biochemical and molecular approaches. The enzyme appears to be a membrane protein which can be solubilised with detergents but it does not partition into the detergent-rich layer formed during the temperature induced phase separation of Triton X-114 and therefore does not behave like a typical integral membrane protein. The enzyme attacks peptide bonds comprising the amino group of hydrophobic amino acids and hydrolyses the Asn4-Phe5, Phe5-Leu6 and the Arg7-Phe8 peptide bonds of the C.elegans peptide, PF1. Phe-X-Arg-Phe-NH2 is normally the core sequence for bioactivity and therefore cleavage of PF1 within this sequence will result in inactivation. The endopeptidase has a pH optimum of 7.8, and is inhibited by EDTA and 1,10 bis phenanthroline, indicating the involvement of a bivalent metal ion at the active site. Phosphoramidon, thiorphan, SCH 39370, SCH 32615 and SQ 28603 are inhibitors of mammalian neprilysin and all inhibit the C.elegans endopeptidase. The nematode enzyme is also inhibited by Co2+, Cu2+, Zn2+ and Fe2+ but not by Ca2+, Mg2+ and Mn2+. A putative neprilysin gene has been identified in the genome of C.elegans as part of the genome sequencing project and we are now screening for a Tc1 insertion induced knockout of this gene to determine the effect of neprilysin gene inactivation on nematode behaviour.

Sonnhammer ELL et al. (1998) European Worm Meeting "Bioinformatics driven functional genomics: a multifront approach to understand complex pathways with a "simple" little worm"

Vaz Gomes A, Sonnhammer ELL, Krause MW, Schrimpf S, Wahlestedt C

[European Worm Meeting, 1998]

C. elegans has been a useful model organism for developmental studies because of its ease of culture, simple anatomy, and available genetics. These attractive features are now be augmented by a wealth of molecular data from a multitude of nematode research labs as well as data provided by the completion of the full genome sequencing project. Already, the genomic information is allowing accurate detection of C.elegans orthologues to various genes relevant in human diseases or other conserved pathways (Mushegian et al., 1997). The time is right to take a bioinformatics driven approach to functional studies. The combination of the complete genomic sequence, bioinformatics, and an experimentally facile organism makes C. elegans an excellent system in which to dissect complex signaling pathways. A large body of signal-transduction pathways involving seven-transmembrane G-protein coupled receptors mediate responses to different types of chemicals like odorants, neurotransmitters, hormones, and also to more !physical! stimuli, like osmotic pressure, temperature, pressure, etc. Many members of these types of pathways have been studied in some detail in C. elegans while other potential candidates have so far only been identified !in silico! (Sonnhammer & Durbin, 1997). Clearly, the nematode has been, and will continue to be, helpful in identifying potential roles for these factors in regulating behaviour and responses to environmental cues, or in crucial developmental processes. The emergence of RNA-mediated interference (RNAi) (Fire et al., 1998) provides a powerful technique that will facilitate the identification of phenotypes associated with the elimination of single or combinations of multiple signalling factors. To improve biochemical analysis in the worm, we aim to apply the new generation of protein 2D-gels analysis systems. We expect this to become a powerful tool (e.g. Bini et al., 1997), for example revealing mutation effects and more accurate proteomics. This is especially important for genes encoding transcription factors, where changes of the expression pattern might be detected most easily at the protein level. For such studies the main requirement is non-lethality, health and fertility of the mutation, so that enough material can be obtained for the analysis. REFERENCES Bini, L., Heid, H., Liberatori, S., Geier, G., Pallini, V. & Zwilling, R. (1997) Electrophoresis18, 557-562 Fire, A., Xu, SQ., Montgomery, M.K., Kostas, S., Driver, S.E. & Mello, C. (1998) Nature 391, 806-811 Mushegian, A.R., Bassett, D.E.Jr, Boguski, M.S., Bork, P. & Koonin, E.V. (1997) PNAS 94, 5831-5836 Sonnhammer, E. & Durbin, R. (1997) Genomics 46, 200-216