Harrington, Sean et al. (2017) International Worm Meeting "The discovery of neuroactive small molecule tools using a high-throughput egg laying screening pipeline."

Yip, Chris, Harrington, Sean, Roy, Peter, Giuliani, Maximiliano, Au, Aaron

[International Worm Meeting, 2017]

The neuromuscular circuit that governs C. elegans egg-laying is well understood and the majority of regulatory elements within it are highly conserved. The egg-laying system has been exploited previously to probe the polypharmacology of established neuromodulators such as clozapine (Karmacharya et al. (2011), Brain Res., 1393(1), 91-99) and imipramine (Weinshenker et al. (1999), J of Neurosci., 19(22), 9831-9840). Findings from these studies translated to a mouse model demonstrating the conserved nature of components of the egg-laying system. Here, we aim to identify novel small-molecule modulators of neuromuscular function by screening for those that modulate egg-laying behaviour in the worm. To do so, we have developed a high-throughput screening pipeline that takes advantage of the amenability of the egg-laying phenotype to computational counting. Using this screening pipeline, we have screened through 4642 molecules and identified 83 molecules that either stimulate or suppress egg laying behaviour at a concentration of 60 M or less. Many of these molecules have no reported relevant bioactivities in the literature. Several of our hits are bioactive in a vertebrate model (Danio rerio) suggesting the possibility of a conserved mechanism of action (Burns et al. (2015), Nature Comm., 6(1), doi:10.1038/ncomms8485). We are currently characterizing the mechanism of action of these molecules using four parallel approaches: 1) testing their interaction with a panel of human neuronal targets (NIH Psychoactive Drug Screening Project - Besnard et al. (2012), Nature, 492(7428), 215-220); 2) thermal proteome profiling using quantitative mass spectrometry to identify candidate targets in worms (Franken et al. (2015), Nature Protoc., 10(10), 1567-1593); 3) forward genetics screens to identify candidate targets (Kwok et al. (2006), Nature, 441(7089), 91-95); & 4) understanding their interaction with a panel of C. elegans egg laying mutants. We hope that our hits will become useful tools for future biological investigations, and that a subset might have therapeutic potential to modulate neuromuscular activity in more complex systems.

Dernburg AF et al. (1997) International C. elegans Meeting "Cytological investigation of homologous pairing during meiotic prophase"

Dernburg AF, Villeneuve AM

[International C. elegans Meeting, 1997]

Pairing and recombination between homologous chromosomes are essential for their faithful segregation during the first meiotic division. The mechanisms leading to homologous chromosome pairing and synapsis are still only poorly understood in any experimental organism. C. elegans provides a highly advantageous model system in which genetic and cytological approaches can be combined to study this process. We are developing tools and methods to enable us to examine chromosome pairing in the light microscope in order to address several specific questions: Do special sites on each chromosome (particularly the genetically-defined !pairing centers!) become associated earlier than others? Do chromosome rearrangements that act as crossover suppressors disrupt homologous pairing as dramatically as genetic evidence seems to suggest? Are gene products that are required for meiotic recombination associated with particular regions of the chromosomes? To answer these and other questions, we are using several approaches, including fluorescence in situ hybridization (FISH) methods, and labeling of chromosomes in living worms using the GFP-Lac repressor binding method developed by Aaron Straight, Andrew Murray, and Andy Belmont for other experimental systems. By applying high-resolution 3-dimensional microscopy, we hope to obtain a better understanding of rules governing the organization and interaction of chromosomes in the meiotic prophase nucleus.

Konno, Hiroyuki et al. (2015) International Worm Meeting "Mechanism and significance of the maternal mex-3 mRNA localization in C. elegans."

Kohara, Yuji, Noguchi, Koki, Konno, Hiroyuki

[International Worm Meeting, 2015]

In C. elegans, several maternal mRNA localizations are observed, however, the mechanism and significance are not clear. Here, we studied anterior mRNA localization of maternal mex-3 mRNA. mex-3 mRNA localizes to anterior half of embryo from the late 1-cell stage to the 4-cell stage. Localization pattern of MEX-3 protein is similar to that of mex-3 mRNA, raising the hypothesis that mex-3 mRNA localization contributes to MEX-3 protein localization. We identified a CCCH-type zinc finger protein MEX-5 as a trans-acting element. Transgene assay revealed that 54-nucleotide sequence on mex-3 3'-untranslated region is the cis-acting element for the mRNA localization. This sequence is AU-rich and fulfills the feature of the MEX-5 binding sites. We made mex-3 mutant which lacks the cis-acting element by genome editing technology. Interestingly, the localization of not only mex-3 mRNA but also MEX-3 protein were not observed in this mutant. We are going to discuss the significance of mex-3 mRNA localization.

Gogonea CB et al. (1997) International C. elegans Meeting "EXPRESSION OF THE REPORTER GENE MEC-12::LACZ IN LOCOMOTORY AND CHEMOSENSORY MUTANTS OF C. ELEGANS"

Siddiqui ZK, Siddiqui SS, Gogonea CB

[International C. elegans Meeting, 1997]

Mutations in close to 100 genes affect locomotion. These mutants show uncoordinated locomotion, ranging from slow movement, poor backing, colier, kinkier, and paralyzed phenes (J. Hodgkin, 1997: Appendix in C. elegans II). Previously it was shown that mutants in at least 25 genes harbor defects in the axonal growth and process placement of the six touch receptor neurons (Siddiqui, 1990). A reporter gene mec-12::lacZ has been shown to express in the set of six touch receptor neurons (Fukushige et al., 1997). So far, a variety of cellular defects in the process placement, axonal guidance and axonal growth of the touch cells and the PVR in 15 genes (unc-4, unc-5,unc-30, unc-33, unc-44, unc-51, unc-53, unc-55, unc-61, unc-71, unc-76, unc-83, unc-86, and unc-119) have been observed. Defects include ectopic branching of axons, fore- shortened processes, misplaced cell body positions, extra commissures, and a lack of staining. In addition, we have examined the reporter gene expression in the background of various mechano- sensory mutations (Chalfie and Au, 1989). These results will be summarized. We thank the Caenorhabdtis Genetics Center, and Martin Chalfie for various mutant strains.

Roy, Peter et al. (2021) International Worm Meeting "A C. elegans Motor-Centric Screening Pipeline Yields Novel and Selective Nematicidal Scaffolds"

Stagljar, Igor, Pyche, Jacob, Yip, Christopher, Gilleard, John, Snider, Jamie, Zasada, Inga, Harrington, Sean, Lautens, Mark, Guiliani, Maximillano, Dowling, James, Jiang, Yan, Wong, Vicotria, Choo, Ken-Loon, Palmeira, Bruna, Cutler, Sean, Volpatti, Jonathan, Redman, Elizabeth, Au, Aaron, Haeberli, Cecile, Knox, Jessica, Kitner, Megan, Roy, Peter, Keiser, Jennifer, Vaidya, Aditya, Kim, Yong-Hyun, Burns, Andrew

[International Worm Meeting, 2021]

Nematode parasites of humans, livestock and crops pose a significant burden to human health and welfare. Alarmingly, our arsenal of effective nematocidal compounds is being depleted. Parasitic nematodes of animals have rapidly evolved resistance to anthelmintic drugs, and traditional nematicides used for crop protection have been restricted or banned because of poor phylum-selectivity. Here, we present our C. elegans-based discovery pipeline focused on lethal molecules that also induce motor defects. This pipeline yielded multiple new and selective nematicidal small molecule scaffolds (i.e., structurally-related families of molecules). We show that one of these scaffolds, tentatively called the APPs, stimulates neurotransmitter release and immobilizes larvae of multiple nematode parasites of plants and mammals in vitro. At similar concentrations, APP-1 does little to model flies, fish, plants or human cells. Forward genetic screens of 100,000s of mutant genomes failed to yield resistant animals that resist the lethal effects of APP-1, suggesting that resistance to the APPs in the field may not be easily generated. Hence, the APPs represent a novel, selective and potentially useful addition to our nematocidal armament.

Yang, Zhe et al. (2015) International Worm Meeting "A C. elegans model for Human Antigen R (HuR)."

Yang, Zhe, Buechner, Matthew

[International Worm Meeting, 2015]

Human antigen (Hu) proteins HuB, HuC, HuD, and HuR are a family of RNA-binding protein that stabilize mRNA by binding to AU-rich elements (ARE) in the 3'UTR. HuR is universally expressed in human cells, and stabilizes many tumor-related genes such as bcl-2 and XIAP. HuR is upregulated in many cancer cell types involved in breast and colon cancer. EXC-7 is the sole homologue of the Hu proteins in C. elegans. EXC-7 maintains the single-cell tubular canals of the excretory cell by stabilizing sma-1 mRNA, encoding betaH-spectrin. Mutants in exc-7 exhibit small fluid-filled cysts throughout shortened excretory canals, as well as a misshapen tailspike. Both EXC-7 and HuR contain 3 RNA-recognition motifs (RRMs), with a region between the 2nd and 3rd RRM that appears to be needed for intracellular transport. We are investigating the functional equivalence of these proteins, by determining whether human HuR can functionally replace EXC-7 in C. elegans. If so, then C. elegans could be developed as a model for studying HuR in vivo, especially for screens for chemicals that interfere with HuR function, using excretory canal phenotype as a measure of HuR activity.We have injected the hur coding region driven by various promoters to express HuR within the excretory canals. HuR appears to affect both the length and diameter of the excretory canal lumen. We plan to attach GFP to HuR to visualize the position and movement of this protein within the long excretory canals, and to make worm/human chimeric proteins to investigate domain activity of these proteins.

Lundquist EA et al. (1995) International C. elegans Meeting "mec-8 ENCODES A PUTATIVE RNA-BINDING PROTEIN AND AFFECTS SPLICING OF unc-52 TRANSCRIPTS"

Rogalski TM, Shaw JE, Moerman DG, Herman RK, Lundquist EA

[International C. elegans Meeting, 1995]

mec-8 mutants are defective in mechanosensation (Chalfie and Au, 1989), dye-filling of amphid and phasmid neurons (Perkins et al., 1986) and attachment of body wall muscle to hypodermis (Lundquist and Herman, 1994). The DNA sequence of the mec-8 gene indicates that it can encode a protein containing two copies of an 80-amino acid RNA recognition motif. We propose that MEC-8 is a trans-acting pre-mRNA processing factor. mec-8 mutations enhance viable unc-52 mutations: the mec-8; unc-52(viable) double mutant resembles the unc-52 null mutant, which is paralyzed and arrested at the twofold stage of elongation, with severe body wall muscle defects (Williams et al. 1994). unc-52 produces many alternatively spliced transcripts that encode proteoglycans found in the basement membrane adjacent to muscle cells (Rogalski et al., 1993). We have used RT-PCR to assay the patterns of alternative splicing of unc-52 transcripts. The concentration of a subset of splice products is clearly reduced in mec-8 animals. We propose that the muscle defects of mec-8 animals and the synthetic lethality of mec-8; unc-52(viable) animals are the result of defects in the generation of certain unc-52 alternatively-spliced transcripts. We have obtained evidence that mec-8 also regulates the processing of its own pre-mRNAs. We propose that MEC-8 controls the splicing of transcripts of other loci that are involved in mechanosensory and chemosensory neuron function. Finally, we suggest that two loci identified by mutations that suppress mec-8 defects, smu-1 and smu-2, may also be involved in pre-mRNA processing.

Kuersten S et al. (1995) International C. elegans Meeting "IN VITRO PROCESSING OF MONO- AND POLYCISTRONIC TRANSCRIPTS IN A C. ELEGANS WHOLE CELL EXTRACT."

Kuersten S, Blumenthal TE

[International C. elegans Meeting, 1995]

Pre-mRNA splicing in C. elegans occurs in three distinct, but related, forms: Cis-splicing (intron removal) and SL1 or SL2 trans-splicing. In trans-splicing, 22 nt leaders are spliced onto the 5' ends of mRNAs. SL1 trans-splicing is specified by an AU-rich intron-like sequence (an "outron") followed by a functional 3' splice site, present at the 5' end of the pre-mRNA. In contrast, SL2 trans-splicing occurs at 3' splice sites present between genes in polycistronic transcription units. In order to determine the signals that mediate trans-splicing specificity, we have developed a whole cell embryo extract capable of at least partially reproducing these processing events in vitro. This extract is capable of performing at least four distinct pre-mRNA processing events: Cis-splicing, SL1 trans-splicing, SL2 trans-splicing and cleavage/polyadenylation. However, the extract is relatively inefficient; RT-PCR is required to detect spliced products. When the extract is incubated with a transcript derived from the rol-6 gene, which is normally trans-spliced to SL1, a trans-spliced product is observed. The presence of this product is ATP-dependent and the transcript is spliced to SL1. In addition, an intron is also removed but more ATP appears to be required for cis-splicing. Both capped and uncapped rol-6 RNAs are spliced to SL1 in the extract, indicating that, at least in vitro, the presence of a 5' cap on the pre-mRNA isn't required for SL1 trans-splicing. Processing of a polycistronic transcript also occursi n vitro, including both cleavage/polyadenylation and trans-splicing at the correct internal site between genes. The downstream mRNA is spliced to both SL2 and SL1, in about equal proportions, even though this mRNA receives exclusively SL2 in vivo. Thus, the in vitro system only partially recapitulates the in vivo specificity. This in vitro system should allow us to dissect the signals that specify SL2 trans-splicing.

Lawry, S.Terese et al. (2019) International Worm Meeting "Using the Million Mutation Project strains to obtain a range of touch insensitive mutants."

Chalfie, Martin, Walker, Matt, Lawry, S.Terese

[International Worm Meeting, 2019]

The Million Mutation Project (MMP; Thomson et al., Genome Res. 23: 1749-1762, 2013) has become an outstanding resource for mutations in many C. elegans genes. More recently Timbers et al. (PLoS Genet. 12: e1006235, 2016) used a subset of MMP strains to identify genes that could be mutated to give a dye-filling (Dyf) phenotype. Because most of the mutations in the MMP strains are homozygous, we were intrigued about the possibility that the MMP strains could be used to identify mutations having subtle phenotypic effects. We also wanted to assess how complete a screen utilizing the MMP strains would be. To this end we have undertaken a screen of the MMP strains to identify those with extreme to slight defects in the gentle touch response. Classical forward genetic screens (Chalfie and Sulston, 1981; Chalfie and Au, 1989) identified 18 genes necessary for gentle touch sensitivity, although others have also been found (e.g., among genes whose null phenotype is lethality). We have currently screened about 1700 of the 2007 MMP strains. 111 of these strains have an abnormal gentle touch response. 73 of these strains have mutations affecting the protein sequence, UTRs, or splice sites of genes previously known to mutate to touch insensitivity. In particular, we identified strains with mutations in all but one gene (mec-17) found in our initial forward mutageneses. mec-17 is extremely difficult to mutagenize to touch insensitivity; all four mutations in MMP strains failed to make the animals touch insensitive. Thus, the MMP collection, at least for viable phenotypes, appears to be essentially complete. Using MMP defects in the known touch genes that did and did not produce touch sensitivity, we were able to look for important domains within the encoded proteins. We are now identifying causative mutations in the remaining 38 strains, starting with the strains that exhibit a more extreme phenotypic defect. Because the MMP strains are sequenced, identifying the causative mutation should be relatively quick using genetic mapping. We are conducting several other screens using this resource and strongly recommend it to our colleagues.

Fujita M et al. (2000) Midwest Worm Meeting "EXC Proteins Regulate Epithelial Cytoskeletal Morphology"

Fujita M, Buechner M, Suzuki N, Sakamoto H, Nishiwaki K, Hall DH, Matsumoto K

[Midwest Worm Meeting, 2000]

exc mutations alter the shape of the apical lumen of the excretory canal from a smooth narrow tube to wide cysts of varying shape, depending on the gene affected. We previously cloned the exc-7 gene, which encodes a homologue of the neural mRNA processing protein ELAV. sma-1 and exc-3 are likely target mRNAs regulated by ELAV; the sma-1 gene contains in its 3' UTR the AU-Rich Element normally bound by ELAV proteins. We now report the cloning of the exc-5 gene; it encodes a guanine nucleotide exchange factor specific to rho-type GTPases. Mutant animals missing this protein produce apical cytoskeleton, but do not properly maintain this material at the apical surface. The basolateral surface appears to be unaffected save for a mild guidance defect. In contrast, the canal cell of animals that overexpress the exc-5 gene show no canal extension; instead, long lumenal tube of normal width forms folded up inside the canal cell body. This phenotype is also seen in mosaic animals deficient in basement membrane formation, such as lam-1 and epi-1. The EXC-5 protein appears to regulate maintenance of the ratio of apical to basolateral surface produced in epithelial cells; too little EXC-5 protein perturbs placement of apical cytoskeleton, while too much perturbs placement of basolateral cytoskeleton. While these effects may be present in other epithelia, the extremely low diameter:length ratio, as well as the extreme separation of apical and baso-lateral surfaces of the excretory canals, magnify the effects of exc-5 mutations in this cell. Attempts to construct animals doubly homozygous mutant for exc-5 (rh232) and exc-7 (rh252) were unsuccessful; this combination appears to be lethal. EXC-7 appears to determine lateral placement of cytoskeletal elements along the length of the canal, while EXC-5 regulates placement at the apical vs. basolateral surface. In mutants missing either one of these elements, we believe that enough material is properly placed for the cells to function, although maintenance of cell shape is affected, while animals missing both of these complementary elements of cell morphology cannot survive.