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. (2000) West Coast Worm Meeting "Global patterns of expression patterns in muscle using mRNA-Tagging"

Roy PJ, Kim SK

[West Coast Worm Meeting, 2000]

Gene networks that control development must endow each unique cell with distinguishing properties. Microarray technology along with the completed C. elegans genome now makes it possible to know these gene networks in a comprehensive manner. Our microarrays contain PCR fragments representing nearly every predicted C. elegans gene. By hybridizing labeled cDNA probes from different samples to the microarray, one can simultaneously determine the expression differences between those two samples for every gene. A limitation of current microarray technology is that RNA is isolated from whole worms, in turn, making tissue specific expression patterns difficult to detect. We have developed a strategy to circumvent this problem of identifying the mRNA profiles in specific tissues or cells by using an approach we call "mRNA-tagging". To isolate cell-specific mRNA, we are using previously characterized promoters to drive the expression of epitope-tagged protein that binds mRNA in a non-biased fashion in a defined set of cells. By immunoprecipitating the tagged mRNA-binding protein from whole animal lysates, cell-specific mRNA is co-immunoprecipitated. The abundance of each mRNA species isolated from these distinct cells is then assayed using microarrays. To identify genes expressed in muscle, we are using the well-characterized promoter myo-3 (Okkema et al., Genetics 135, 385-404) to drive the expression of the epitope-tagged mRNA-binding protein in body wall muscle. To date, we have successfully and reproducibly co-immunoprecipitated muscle-specific RNA, as assayed through both northern and microarray analysis. Control germ line and neuronal RNA are not enriched. We are currently optimizing our mRNA-tagging protocol to identify gene expressed in muscle, and are also extending our studies to identify neuronal and hypodermal genes.

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.

Ginzburg VE et al. (2000) East Coast Worm Meeting "Semaphorin 1a and 1b mutants exhibit male tail morphological defects."

Ginzburg VE, Roy PJ, Culotti JG

[East Coast Worm Meeting, 2000]

The semaphorin protein family has been implicated in axon guidance and morphogenesis in vertebrates and invertebrates, as described by Kolodkin et al., Cell 75, 1389-1399, 1993. Analysis of the C.elegans genome has revealed three genes that encode semaphorins designated Sema-1a, Sema-1b and Sema-2a according to their predicted protein structure. Recently, P. Roy has isolated and characterized mutants of the secreted semaphorin Sema-2a and found it has mild axon guidance defects and epidermal cell migration defects (P. Roy et., al 2000). To understand what role transmembrane semaphorins play in the development of C.elegans we decided to utilize reverse genetics to isolate Sema-1a and Sema-1b mutants. With mutants of all three semaphorin genes, we can study their functions using classical genetics, and moreover we can look for downstream components by designing various enhancer and suppressor screens. First, we have constructed a deletion library of approximately 1.7 million haploid genome, using a standard mutagenesis protocol. We have designed several sets of nested PCR primers for Sema-1a and Sema-1b genes. Screening of the deletion library found several candidates for deletions in the genes and we isolated two alleles of Sema-1a (ev715, ev708) and one allele of Sema-1b (ev709). The alleles were sequenced and revealed that ev715 is a deletion of ~300bp that puts the next three exons out-of-frame, ev708 is a first intron deletion of ~800bp and ev709 is a deletion of two exons. We found that backcrossed versions of the mutants exhibit several abnormalities, which we are further characterizing. Ectopic male tail ray1 (R1) and swollen head are two common phenotypes for both genes. The penetrance of an ectopic R1 in ev715 is 41% (n=201), and in ev709 is 17% (n=170). The penetrance of this unique phenotype is enhanced in a Sema-1a, Sema-1b double mutant (ev715 ev709) to 91% (n=202). Currently, we are attempting to rescue these phenotypes with the cloned genes. We are also further characterizing single, double and triple semaphorin mutants. P>P>

Damian Marlee et al. (2005) International Worm Meeting "Expression Profiling of the Excretory Cell"

Steve Von Stetina, David M Miller, Kathie Watkins, Alfred L George, Clay Spencer, Damian Marlee

[International Worm Meeting, 2005]

The excretory cell in C. elegans is critical for osmoregulation. This H-shaped cell is made up of a large ectodermal cell body, located beneath the posterior pharyngeal bulb, and bilateral tubular canals that run the length of the animal directly beneath the hypodermis. We propose to define a gene expression fingerprint of the excretory cell as a first step toward identifying genes with key roles in excretory cell development and function. These studies will exploit available Affymetrix microarray gene 'chips' representing most genes in the nematode genome coupled with a molecular genetic strategy for isolating enriched populations of mRNAs from single cell populations.1 We have used microparticle bombardment to create transgenic animals for expression localization studies and for mRNA tagging. Transgenic lines expressing GFP driven by the clh-4 promoter of, a chloride channel previously shown to be expressed solely in the excretory cell2, were created and, as expected, GFP expression was localized to the excretory cell. Next, we used the clh-4 promoter to drive expression of an epitope tagged poly-A-binding protein (3XFLAG-PAB-1); anti-FLAG-staining detects excretory-specific expression in this transgenic line. Co-immunoprecipitation with anti-FLAG antibody enables capture of excretory cell-specific mRNAs cross-linked to FLAG-PAB-1. These mRNAs will be amplified, biotin labeled, and applied to the C. elegans Affymetrix array. A comparison to microarray profiles obtained from all cells should reveal excretory enriched transcripts. We intend to apply this strategy to identify excretory cell transcripts as the excretory cell develops in embryonic and larval stages as well as to define homeostatic excretory mRNAs in the adult. We expect that this information will identify homologs in vertebrate/mammalian osmoregulatory systems that can be tested in RNAi experiments to define their roles in the differentiation and function of nematode excretory cell. 1. Roy PJ, Stuart JM, Lund J, Kim SK. Nature 2002; 418:975-979. 2. Nehrke K, Begenisich T, Pilato J, Melvin JE. Am J Physiol Cell Physiol. 2000 279(6):C2052-66

Tanentzapf G et al. (1998) East Coast Worm Meeting "Analysis of the C. elegans Homologue of the Drosophila Gene crumbs - Progress Report"

Tepass U, Culotti JG, Roy PJ, Tanentzapf G

[East Coast Worm Meeting, 1998]

We present the ongoing characterization of the C. elegans homologue of the Drosophila gene crumbs. Previous work in Drosophila has demonstrated that crumbs is necessary for the establishment of the apical pole in ectodermally derived epithelia. The crumbs gene encodes for a transmembrane protein that contains both EGF like repeats and domains similar to the G-domain of laminin A. Through the C. elegans genome project we have found a homologue of the crumbs gene, called crb-1, and have been analyzing its function. The C. elegans crumbs gene shows 37% similarity in overall protein sequence and also shows very high structural similarity in the arrangement of the EGF like repeats, G-domain of laminin A like motifs, and the cytoplasmic domain. Using various promoter constructs we characterized the expression pattern of the crb-1 gene We have confirmed the pattern observed with these promoter constructs using a GFP-tagged crumbs protein. The crb-1 gene is expressed in a complex pattern embryonically in such tissues as the gut, the pharyngeal primordia, and various components of the sensory nervous system in the head region, this expression pattern is mostly the same in the adult. In collaboration with the lab of Dr. J. Culotti we have obtained a line with a TC1 insertion in the crb-1 gene. We have used this line to generate two deletion alleles both of which are predicted to generate frameshift null mutations. Both of these mutant lines are viable and are wild type in appearance, movement, and behaviour. By crossing our crb-1 promoter constructs into the mutant lines we have made lines which express a reporter gene in the cells that would be predicted to be affected in the mutant. Our results indicate that these cells are present and have no obvious defects in their shape and morphology in the mutant background. Finally since crb-1 is expressed in the socket and amphid sheath cells of the head sensilla we have looked at the integrity of these cells by DI staining, and in addition tested the chemosensation of our mutants. We found that both lines are wild type for both assays. We are currently extending our analysis to prove conclusively that the mutations we made are indeed null alleles. An explanation for the lack of phenotype might be that other mechanisms for the specification and maintenance of epithelial polarity exist in C. elegans which can compensate for the lack of crb-1. Therefore we are in the process of cloning full length cDNA of the crb-1 gene in order to carry out overexpression studies which we hope will reveal any function crb-1 has in controlling epithelial polarity. In addition, in order to determine whether crb-1 is a functional homologue of the Drosophila crumbs we will test if crb-1 is able to rescue the crumbs mutant phenotype in Drosophila.

Warren CE et al. (1997) International C. elegans Meeting "BETA-6-N-ACETYLGLUCOSAMINYLTRANSFERASES IN C. ELEGANS."

Roy PJ, Culotti JG, Warren CE, Dennis JW

[International C. elegans Meeting, 1997]

Systematic searches of the DNA sequence databases have revealed in C. elegans a series of predicted genes homologous to mammalian glycosyltransferases which, if enzymatically active, could confer glycosylation similar to mammals. We have detected GnT-I, beta-3-GalT, C4-GnT and GnT-V by enzyme assay of C. elegans lysates. In whole animals, lectin binding repertoires consistent with mammalian-like glycosylation patterns have been observed histochemically(1). Mutations in sqv-3, a beta-4-galactosyltransferase homolog cause developmental defects in vulval formation and sperm-egg interaction(2). These observations make the nematode an attractive model organism for the study of glycobiology. We have focused on 2 C. elegans genes homologous to the mammalian glycosyltransferases GnT-V (yk126h8) and C2-GnT (F44F4.6). We have obtained and sequenced these cDNAs. The predicted proteins are 59.9% similar, 36.7% identical for the GnT-V homolog yk126h8 and 52.3% similar, 29.4% identical for the C2-GnT homolog F44F4.6. We created a frozen Tc1-insertion mutant bank and isolated Tc1 insertion lines for both homologues from which we are currently generating deletion alleles. In addition, the transcript 5! ends are being mapped, the gene-products are being tested for enzyme activity and the expression pattern during C. elegans development is being assessed using GFP reporter protein fusions to the genomic promoter regions in transgenic animals. (1) G Borgonie et al Histochem 101:379 (2) T Herman & B Horvitz ECWM 1996 #6

Suzuki Y et al. (1997) International C. elegans Meeting "MUTATION OF A C. ELEGANS BMP HOMOLOGUE (dbl-1) APPEARS TO AFFECT BODY SIZE AND MALE TAIL PATTERNING"

Culotti JG, Wood WB, Morris GA, Yandell MD, Roy PJ, Suzuki Y

[International C. elegans Meeting, 1997]

Bone morphogenetic proteins (BMPs) of the transforming growth factor-beta (TGF-beta) superfamily play important roles in development. To further explore their importance in C. elegans, we undertook to find a mutation in the molecularly identified C. elegans BMP-related gene dbl-1 V (decapentaplegic, BMP2,4-like)(1). Screening of a Tc1 insertion library for a mutant allele identified a mutant line that has a Tc1 in exon 4, C-terminal to the first cysteine in the predicted mature domain of the processed protein. The mutation is recessive, and the mutant animals are small (Sma) and male abnormal (Mab). The same phenotypes result from mutations in the daf-4, and sma-2, 3, 4 genes, which encode components of a TGF-beta signal transduction pathway(2). This suggests that dbl-1 may encode the previously unidentified ligand for this pathway. We are currently testing genetic interactions between dbl-1 and these genes. (1) Yandell et al. (1993) WBG 12(5):82; (1994) WBG 13(4):65; (1996) WBG 14(2):41 (2) Savage et al. (1996) Proc. Natl. Acad. Sci. USA 93:790-794

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.

Calhoun, Adam et al. (2011) International Worm Meeting "Spatial memory in C. Elegans."

Chalasani, Sreekanth, Sharpee, Tatyana, Calhoun, Adam

[International Worm Meeting, 2011]

Worms are known to increase the frequency of turning and reversal events in response to removal of a food stimulus. This keeps the animal in a small area near the last observation of food. The increase in turning frequency is known to be regulated by dopamine and glutamate (Hills et al 2004). We have found that the search strategy is modified by the spatial pattern of food that is experienced. Worms adapt quickly to new food environments, learning spatial patterns on short timescales. The dopamine deficient cat-2 mutant is unable to learn different spatial patterns, suggesting that dopamine is required for spatial memory. References Hills T, Brockie PJ, Maricq AV (2004). Dopamine and glutamate control area-restricted search behavior in Caenorhabditis elegans. J Neurosci 24(5): 1217-1225.