Roy PJ et al. (1995) International C. elegans Meeting "Characterization of Semaphorin I and II."

Roy PJ, Zheng H, Culotti JG

[International C. elegans Meeting, 1995]

Semaphorins are a family of proteins implicated in nerve guidance, found in a number of invertebrates and vertebrates, initially described by Kolodkin et al., Cell 75, 1389-1399. Through degenerate PCR, we have identified a C.elegans semaphorin I (Ce-SemaI) cDNA fragment. By splicing a clone we isolated from a cDNA library together with one obtained from the Coulson cDNA sequencing project, we constructed a full length cDNA (2.7kB) which corresponds to the indicated size on Northern blots. Y. Kohara then isolated Ce-SemaI independently and mapped it to YACS, y53C6 and y54E5. We probed overlapping cosmids defined by the two YACS with Ce-SemaI and identified the cosmids (F14B11 and K09A10) which contain the gene encoding Ce-SemaI. The Ce-SemaI gene physically maps within 80 kB to the left of unc-54 on the right arm of chromosome I. Restriction mapping and sequencing of the gene enabled us to construct two minigenes from the Ce-SemaI gene and cDNA, and several reporter constructs utilizing 3.8 kB of predicted promoter sequence, lac-Z and GFP. We are currently using a subclone which contains only the Ce-SemaI gene, and the two minigenes to try to rescue prospective mutants which lie just to the left of unc-54; i.e., let-49, unc-122, let-207 and let-206. Also, Y. Kohara's cDNA sequencing project recently yielded a clone which encodes a second C. elegans semaphorin which differs from Ce-SemaI. In combination with Kohara's clones, we have generated and used a 5'RACE product to construct a full length cDNA (2.4kB), which matches the predicted size indicated by Northern analysis.

Roy PJ et al. (1997) International C. elegans Meeting "C.elegans Semaphorin II (els-2) Nulls are let, unc, dpy and Deformed."

Culotti JG, Warren CE, Zheng H, Roy PJ

[International C. elegans Meeting, 1997]

Many members of the semaphorin/collapsin family are required for proper neural development and survival; Drosophila semaphorin II is required for viability and can selectively inhibit proper neural synaptic arborization (Matthes et al., 1995, Cell. 81:631-639) . To further investigate the role of semaphorin during development we have cloned C. elegans semaphorin II (els-2) using a cDNA fragment isolated in Y. Kohara's lab. Reporter genes driven by els-2 promoter and enhancer fragments exhibit a complex embryonic expression pattern including alternating stripes of dorsal hypodermal cells. Neuronal expression is seen in many motorneurons, sensory neurons, and various ganglia. Muscle expression is also observed. An els-2 Tc1 insertion allele (ev573) was isolated from our transposon library. Two els-2 deficiency alleles (ev574 and ev575) were generated from ev573. Both ev574 and ev575 delete the first exon, the 5'UTR and possibly promoter and enhancer elements. Brood size for strains homozygous for ev574 and ev575 are very low. Survivors are uncoordinated, dumpy, and severely deformed, especially near the posterior. Phenotypic and genetic analysis is ongoing.

Roy RD et al. (1997) International C. elegans Meeting "Food-dependent regulation of cell division and postembryonic development in C. elegans."

Ambros VR, Roy RD

[International C. elegans Meeting, 1997]

Postembryonic development in C. elegans requires precise coordination between environmental factors, developmental effectors and the cell cycle apparatus. When L1 larvae emerge in the absence of food they remain viable but in an immature, undeveloped state. Upon feeding, postembryonic cell divisions are induced and the animal begins to develop. We are interested in understanding the basis of this food-dependent induction of postembryonic development. To perform a genetic analysis of this developmental switch we designed a S-phase-specific reporter strategy to isolate mutants that enter cell divison cycles in a food-independent manner. Conversely, in another screen using hydroxyurea, an S-phase inhibitor, we select for conditional mutants that do not develop in the presence of food. Candidate mutants for each category are currently being characterised. We are also examining the roles of known cell cycle regulators in this pathway.

Peter J Roy et al. (2001) International C. elegans Meeting "Identifying muscle genes that change transcription levels in response to varying cholinergic signals using full-genome microarrays."

Peter J Roy, Stuart K Kim

[International C. elegans Meeting, 2001]

The excitatory neurotransmitter acetylcholine (ACh) not only controls muscle cell contraction, but can also lead to a change in muscle cell physiology. For example, in mammals, chronic exposure to the ACh agonist nicotine can desensitize cells, leading to addiction. To better understand how a post-synaptic cell responds to ACh, we are examining the genome-wide transcriptional changes in C. elegans striated muscle cells in response to varying cholinergic signal. To do this, we first needed to develop a method to measure relative gene expression levels in single tissues in C. elegans. Previous DNA microarray experiments have compared gene expression levels from entire worms rather than from specific tissues. A major limitation to this approach is that gene expression changes in one tissue, such as muscle, could be obscured by expression in other tissues. To isolate muscle mRNAs, we use the myo-3 promoter (a gift from A. Fire) to drive expression of an epitope-tagged poly(A)-mRNA-binding protein in striated muscle. The mRNA/tagged-protein complex is co-immunoprecipitated from whole-animal lysates using antibodies against the epitope tag. mRNAs significantly enriched in muscle are identified by comparing the immunoprecipitated mRNA to the mRNA present in the initial worm extract using DNA microarrays. We call this technique "mRNA-tagging". L1-muscle mRNA-tagging (6 repeats) revealed 1453 genes that were significantly enriched, including most characterized muscle-specific genes (positive controls) and excluding most non-muscle genes (negative controls). We repeated the entire experiment using L2 larvae (6 repeats) and found that there were few significant differences between L1 and L2 muscle. Together, these results indicate that mRNA-tagging successfully identifies genes expressed in striated muscle. To profile transcriptional changes in striated muscle in response to ACh, we either excited muscle cells using the nicotinic ACh agonist levamisole or prevented normal muscle contraction using an ACh receptor mutant. We then used mRNA tagging to identify genes expressed in excited and mutant muscle, and compared those genes to each other and to those expressed in "normal" muscle. 304 genes show increased expression and 851 genes show decreased expression in levamisole-excited muscle, relative to normal or mutant muscle. The gene expression data indicates that ACh excitation decreases the relative expression of ACh receptors and other ligand-gated ion channels, which may represent new ACh receptor candidates. Specifically, the expression levels of seven ligand-gated receptors, two 2nd messenger-gated receptors, and two characterized ACh receptor genes (lev-1 and unc-29), were significantly lower in the levamisole-treated or normal worms than they were in the mutant. The overall effect may be to desensitize cells to acetylcholine following strong activation, and to sensitize cells in ACh receptor mutants.

RD Roy et al. (1999) International C. elegans Meeting "Extra distal tip cells in cki-1(RNAi) animals do not result from duplication of existing distal tip cells."

V Ambros, E Lambie, RD Roy

[International C. elegans Meeting, 1999]

Cyclin-dependent kinase inhibitors (CKIs) play a key role in arresting cell cycle in response to various physiological, environmental, and developmental cues. RNA-mediated interference of cki-1 activity (cki-1(RNAi) results in pleiotropic phenotypes. Misregulation of cell division control following cki-1(RNAi) permits progenitor cells to divide before being appropriately determined. This is likely the basis of some of the observed vulval patterning defects (extra VPCs and anchor cells), the germline defect (extra distal tip cells (DTCs)) and the inability to form differentiated dauer alae (hypodermal cell divisions). To address whether extra DTCs arise from duplications of existing cells or whether they are produced from an ectopic source in cki-1(RNAi), the production of extra DTCs was examined in lag-2::GFP; cki-1(RNAi) animals. The cki-1(RNAi)-induced ectopic DTCs do not appear before the late L3 stage as per lag-2::GFP expression. Therefore laser ablation of distal tip cells in the early L3 (from picking L2 lethargus animals) should eliminate the production of DTCs in the adult if the extra DTCs result from duplication of endogenous DTCs. DTCs are observed in nearly all ablated animals indicating that DTCs arise earlier and simply are not determined until later, or they stem off from another lineage later in development. To test if these cells are born earlier, similar ablations were performed in the somatic gonad progenitors in L1 animals. Removal of Z1.a and Z4.p resulted in the production of DTCs in the adult despite removal of these progenitor cells. Removal of Z1.p and Z4.a lead to only 2 DTCs per animal suggesting that the extra DTCs arise from these cells and not from duplications of the DTCs later. Despite the appearance of a cki-1(RNAi)-induced cell fate transformation, a more consistent interpretation would lie in the ability of cki-1 to block cell divisions until cells are appropriately determined. Premature divisions that occur in cki-1(RNAi) may permit daughter cells to retain mother cell fates and hence give rise to lineage modifications. This possibility will be confirmed in chromosomal mutants of cki-1.

MOLIN, L. et al. (2019) International Worm Meeting "Screen for new actors of muscle aging."

Bessereau, JL., ROY, C., SOLARI, F., MOLIN, L.

[International Worm Meeting, 2019]

We previously demonstrated that muscle aging in C. elegans involves a stereotyped sequence of events characterized by a dramatic decrease in the expression of genes encoding proteins required for muscle contraction, followed by a change in mitochondria morphology, and an increase in autophagosome number (Mergoud et al., 2018). To further characterize the mechanisms underlying muscle aging we used CRISPR/CAS9 to generate a transcriptional reporter that mirrors the down-regulation of tnt-2 (troponine T) expression with age. We used CRISPR-CAS9 to generate a knock-in strain, in which tagRFP-T fluorescence in body wall muscles (BWM) and protein level decrease with age. Using this reporter strain we performed a semi-clonal screen after EMS mutagenesis in order to identify genes whose mutation maintains tagRFP-T fluorescence with age. Details of the screening strategy and some preliminary results will be presented. Mergoud dit Lamarche A., Molin L., Pierson L., Mariol MC., Bessereau JL., Gieseler K., Solari F. (2018). UNC-120/SRF independently controls muscle aging and lifespan in Caenorhabditis elegans. Aging Cell. https://doi.org/10.1111/acel.12713

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.

Dana Byrd et al. (2006) Development & Evolution Meeting "Understanding the Molecular Properties of a Stem Cell Niche"

Judith Kimble, Dana Byrd, Daniel Hesselson

[Development & Evolution Meeting, 2006]

Distal tip cells (DTCs) serve at least two critical roles during development of the C. elegans hermaphrodite gonad. They function as migratory leader cells to regulate morphogenesis of the U-shaped gonad arms and as niche cells to regulate germline proliferation. We have taken a microarray approach to determine the gene expression profiles of the DTC during development to further explore the molecular basis of its leader and niche cell functions. We are enriching for DTC transcripts by immunoprecipitation of Flag-tagged PAB-1 (poly-A binding protein) with bound RNAs (Roy et al. 2002). DTC expression will be validated with promoter-GFP constructs, and function will be assessed by cell-specific RNAi. Our progress will be described.

Sinchita Roy Chowdhuri et al. (2004) Mid-west Worm Meeting "The C. elegans T-box gene tbx-2 is required for pharyngeal development"

Sinchita Roy Chowdhuri, Peter G Okkema, Yen Huynh

[Mid-west Worm Meeting, 2004]

T-domain transcription factors are crucial developmental regulators in all animals. In the vertebrate heart, T-domain factors interact genetically and physically with NK-2 homeodomain factors to promote cardiomyocyte differentiation. C. elegans contains 20 T-box genes, many of which have not been functionally characterized. We have focused on tbx-2 , a member of the Tbx2 subfamily that includes factors involved in vertebrate heart development. Loss of C. elegans tbx-2 function, either by RNAi or in deletion mutants, results in feeding defects, L1 arrest and abnormal pharyngeal morphology, although most markers of pharyngeal differentiation are expressed. These phenotypes are similar to those of animals defective in the pharyngeal muscle specific NK-2 homeobox gene ceh-22 , and we hypothesize tbx-2 functions with ceh-22 in pharyngeal muscle development. A ceh-22::gfp reporter is expressed in tbx-2 mutants, although the number of ceh-22::gfp expressing cells is reduced. We are currently examining the tbx-2 expression pattern to understand its role in pharyngeal development and to determine if it is co-expressed with ceh-22 . In addition we are currently examining possible genetic and physical interactions between tbx-2 and ceh-22 .

Mariam Alexander et al. (2005) International Worm Meeting "A Screen For Genes Required for Muscle Arm Development."

Mariam Alexander, Peter J Roy

[International Worm Meeting, 2005]

In C. elegans, body wall muscles (BWMs) extend membrane projections called muscle arms to the nerve cord to form the neuromuscular junction. Our lab and others have provided evidence that there is likely two distinct phases of muscle arm development; embryonic muscle arms arise by a passive mechanism (CR Norris, IA Bazykina, DH Hall, EM Hedgecock, Worm Breeder's Gazette 14(5): 37), while larval arms actively seek out motor axons (Dixon and Roy, submitted). Although the mechanism by which larval muscle arms reach motor axons is not clear, one model is that a chemotropic cue secreted by motor axons guide muscle arm migration to the nerve cord. To better understand muscle arm development, I performed a forward genetic screen to isolate mutants with muscle arm extension defects. I have screened 36,000 haploid genomes and isolated five mutants with highly penetrant and expressive muscle arm defects. Complementation tests show that tr50 is allelic to unc-60, a gene encoding the C. elegans ADF/cofilin orthologue, while tr51 is allelic to unc-22, which encodes twitchin. The remaining mutants do not map near other genes known to be required for larval muscle arm extension, including lev-11 and unc-54 (Dixon and Roy, submitted), suggesting that these genes may provide novel insight into the development of muscle arms. Surprisingly, one of mutants that has a dramatic reduction in the number of muscle arms (tr64) is not uncoordinated. This suggests that proper muscle arm development is not necessary for wild type movement. I am currently refining the map positions of the genes corresponding to the remaining mutations.