Drewnik, E. et al. (2017) International Worm Meeting "Tissue-specific activity of pathway components regulating C. elegans embryonic epidermal contraction."

Mains, P., Drewnik, E., Smit, R.

[International Worm Meeting, 2017]

Epidermal morphogenesis plays a significant role in driving early elongation of the C. elegans embryo. Interactions between actin and myosin components result in contraction of the cytoskeleton, driving shape changes in epidermal cells that squeeze the worm from an ovoid embryo to a vermiform larva. The actin cytoskeleton differs between lateral and dorsal/ventral (DV) cells, with an actin meshwork in the lateral epidermis and parallel bundles in dorsal/ventral epidermis. Contraction is thought to be strongest in lateral epidermal cells, whereas DV cells are proposed to play a more passive role, though still generate tension. I am investigating tissue-specific activity of the genetic components involved in regulating contraction using transgenic rescue and RNAi techniques. Contraction/elongation is regulated by let-502/Rho-binding kinase and mel-11/myosin phosphatase working antagonistically in one branch of the elongation pathway, while pak-1/p-21 activated kinase and fem-2/PP2c phosphatase work in a parallel pathway. let-502 activity is regulated antagonistically by rhgf-2/Rho-GEF (Guanine exchange factor) and rga-2/Rho-GAP (GTPase activating protein). Both rhgf-2 and rga-2 are ubiquitously expressed in the epidermis; however, it has been shown by our lab that rhgf-2 activity is sufficient in lateral epidermal cells, and shown by the Labouesse lab that rga-2 is sufficient in DV cells (Diogon et al. Development, 2007). Evidence from our lab also suggests that fem-2 is sufficient in DV cells. Although let-502 and mel-11 are expressed ubiquitously throughout the embryonic epidermis, it is yet to be determined in which cells the genes are necessary or sufficient. I have designed constructs to test rescue of let-502, mel-11, and pak-1 mutants in a tissue-specific manner under the control of lateral (ceh-16) and DV (elt-3) promoters. Additionally, I have generated constructs restricting RNAi activity in an rde-1(null) background to lateral or DV cell populations to test phenocopy. These methods will determine whether let-502, mel-11, and pak-1 are sufficient and/or necessary in lateral, DV, or both cell types. Preliminary evidence for let-502 suggests tissue-specific rescue following our model, where lateral epidermal expression rescues and DV expression does not.

Ouellette, Marie-Helene et al. (2021) International Worm Meeting "Neurogenetics of modulatory cholinergic signaling in C. elegans interneurons"

Hendricks, Michael, Ouellette, Marie-Helene

[International Worm Meeting, 2021]

We previously identified a small circuit that integrates both sensory and motor input, involving a glutamatergic interneuron (RIA) that receives cholinergic feedback from head motor neurons (SMDs). RIA is functionally and spatially subdivided into compartments corresponding to sensory input and reciprocal motor domains. Motor input, which is mediated specifically through GAR-3 mAChRs, is received in phase with head movement during locomotion, leading to compartmentalized local calcium events and gait regulation. We found that a novel phenotype we named "head lifting" (movement of the worm's anterior section along the Z axis) appears to strongly correlate with disruption of RIA compartmentalization. Head lifting arises from bilateral (DV) activation of head muscles and occurs preferentially at particular points in the locomotion cycle. Here, we characterize this phenotype and use it to probe the genetics of compartmentalized signals in RIA.

Kouki Noguchi et al. (2005) International Worm Meeting "Studies on localization mechanisms of pos-1 mRNA in C.elegans embryos."

Kouki Noguchi, Yuji KOHARA

[International Worm Meeting, 2005]

Maternal pos-1 mRNA localizes to posterior half of embryo at the 1st cleavage and localized to germ lineage in early embryogenesis. We are trying to elucidate this localization mechanisms. For this purpose, first, we injected various reporter RNA into the gonad of adult hermaphrodite and examined the RNA distribution in embryos by in situ hybridization when the reporter RNA reached to the embryos (4 hours after the injection). When we used a RNA construct (Cap - pos-1 5'-UTR - lacZ - pos-1 CDS- pos-1 3'-UTR - polyA, using pJK370 vector kindly provided by T. Evans), although the localization of the RNA was not observed, the RNA signals disappeared after 1st cleavage, probably due to degradation. Interestingly, the dagaradation depended on the presence of pos-1 sequence. This suggest that a balance between degradation and protection might produce the localized distribution of pos-1 mRNA. However, this phenomenon may be caused by the nature of the reporter RNA that are supplied artificially into cytoplasm, therefore, secondly, we are trying to examine the distribution of reporter RNA that are transcribed from the genome in nuclei. For this purpose, by the genomic co-injection method, we obtained the transformant lines that expressed reporter RNAs (expression vector are constructed from pJH4.52 vector kindly provided by G. Seydoux) maternally. Although it took time to device to detect the low level expression of the reporter construct, currently we are analyzing these strains. We will present more data and discuss about the localizing mechanism at the meeting.

David B Guiliano et al. (2001) International C. elegans Meeting "Operon Resolution, Usage and Diversity of SL2-like spliced leaders in the phylum Nematoda"

Mark L Blaxter, David B Guiliano, Villius Pigaga

[International C. elegans Meeting, 2001]

Nematodes represent an extremely diverse phylum with five major clades containing both free living and parasitic representatives (Blaxter et. al. 1998). The genome of the clade V nematode Caenorhabditis elegans contains many gene clusters which are transcribed as polycistronic pre-messenger RNAs. Unfortunately there is currently no information on the genomic organization of any nematodes other than the free living rhabditids Oscheius brevesophaga, C. elegans and C.briggsae (Evans et. al. 1997). One particularly interesting question is whether these operon-like structures are conserved in other nematode species and if they are do they utilize SL2-like spliced leaders to resolve the polycistronic transcripts. We have employed a directed approach using ribosomal protein operons found in the C. elegans genome and the Nematode EST datasets (Parkinson et. al. 2001) to identify a potential operon in a selection of clade V, IV, and III nematodes. In the clade III filarial nematode Brugia malayi we have determined that the rpL27a and rpP1 are transcribed as a polycistronic unit. However, the second transcript in the unit (rpP1) exclusively receives SL1 when it is transpliced. Conversely in the clade V diplogasterid Pristionchus pacificus we have found that a diverse set of SL2-like sequences transpliced to rpP1. In a survey of the genome sequence of C. elegans we identified several additional SL2-like sequences that had not been previously described. This data indicates that while SL2 usage may have evolved in a clade V ancestor, there have been independent expansions of SL2-like sequences in the rhabditids and diplogasterid. We are currently examining the rpP1 of the clade IV nematode Strongyloides ratti to determine if it utilizes SL2-like spliced leaders.

Anthony S Rogers (2005) International Worm Meeting "Full Length Transcripts in WormBase"

Anthony S Rogers

[International Worm Meeting, 2005]

UnTranslated Regions (UTRs) of messenger RNAs have been shown to be important in the regulation of several C.elegans genes such as tra-2(1) and fem-3(2). To give an improved representation of UTR regions in WormBase predicted full length transcripts are now generated each release. These are built up automatically from the available transcript data and the current CDS structure. Non-genomic transcript sequence such as Trans-spliced leaders and polyA tails are masked before aligning the transcript to the genome using BLAT(3). Identifying TSL and polyA sequences prior to genome alignment helps to prevent misalignments and means individual features can be made to represent these sequences. UTR structures are built from the subset of transcripts which have been computationally shown to agree with the CDS prediction. This new method of representing full length transcripts allows us to build objects that more closely resemble their biological reality with trans-spliced leader sequence and polyadenylation information included where applicable. It is also possible to show where there is evidence of multiple UTRs for a single CDS.As of WS140 there are 25869 transcripts derived from 22410 CDSs representing the 19735 protein coding genes. 1) Translational regulation of tra-2 by its 3' untranslated region controls sexual identity in C.elegans Goodwin EB, Okkema PG, Evans TC and Kimble J. Cell, 1993 75:329-339 2) Control of the sperm-oocyte switch in Caenorhabditis elegans hermaphrodites by the fem-3 3' untranslated region. Ahringer JA, Kimble JE Nature, 1991 349:346-348 3) BLAT The BLAST-Like Alignment Tool W. James Kent. Genome Research (2002) Vol. 12, Issue 4, 656-664

Crittenden SL et al. (1995) International C. elegans Meeting "GENES CONTROLLING GLP-1 ASYMMETRY IN THE EARLY C. ELEGANS EMBRYO"

Evans TC, Crittenden SL, Rudel D, Schuster D, Binder J, Kimble JE

[International C. elegans Meeting, 1995]

We have begun to investigate the genetic control of embryonic polarity in the early C. elegans embryo. GLP-1, a membrane receptor required for inductive cell interactions, is normally restricted to AB and its descendants by translational repression of maternal glp-1 mRNA both prior to the 2-cell stage as well as in P1 and its descendants (Evans et al., 1994). Our approach has been to screen mutant embryos with a-GLP-1 antibodies for an altered pattern of GLP-1 expression. We first examined existing mutant embryos. The par genes influence many aspects of embryonic asymmetry, including P-granule segregation and asymmetric cleavage; we find that mutant embryos of four par genes (par-1, -2, -3, and -4) mislocalize GLP-1, but those of two others (par-5 and par-6) do not . Therefore, proper localization of GLP-1 is not necessarily coupled to other asymmetries, such as asymmetric cleavage. The skn-1, mex-1 and pie-1 genes influence blastomere identity, but do not affect GLP-1 localization. Finally, among 17 temperature sensitive emb and zyg maternal effect lethal (Mel) mutants examined to date, only one, emb-8, mislocalizes GLP-1. We have also screened for new mutants that disrupt GLP-1 expression. We have currently screened nearly 5000 genomes and have identified seven mutants with altered GLP-1. Two are new alleles of par-4; the other five are being characterized currently. Of these five, three have symmetrical and synchronous early divisions similar to the pars, while the other two appear to be more like wild type. Thus, we may have identified new loci that affect specific steps in the process of localizing GLP-1.

Gao DL et al. (1996) Midwest Worm Meeting "Analysis of the Distal Tip Cell (DTC)/Germline Interaction in C. elegans Gonad"

Kimble JE, Gao DL

[Midwest Worm Meeting, 1996]

Analysis of the individual components of a signal transduction pathway is greatly facilitated by the separation of the signal and receptor into well-defined cells or tissues. The cells participating in an inductive interaction are clearly defined as signaling or receiving members of the interaction. We have focused on the induction of the germ line by the somatic distal tip cell (DTC). The DTC resides at the distal end of the germ line and induces mitotic divisions in the germ line throughout postembryonic development. We have previously shown that lag-2 is expressed in the signaling DTC cell and that a LAG-2::b-Gal fusion protein is taken up into the receiving germline tissue (1). Others in the Kimble lab have shown that glp-1 is expressed in the receiving tissue the germ line (2). This is consistent with the idea that LAG-2 is a signaling ligand for the receptor GLP-1. Therefore, this GLP-1 mediated inductive interaction is both well-defined and accessible to manipulation. We have built a battery of lag-2::reporter constructs with myc, b-gal, and GFP epitope tags inserted either in the extracellular or intracellular domain or fused to the promoter of the lag-2 gene. By utilizing these reporter constructs and anti-GLP-1 antibodies, we have systematically investigated the distribution of LAG-2 at different developmental stages of the animal and in different mutant backgrounds. In addition, we have explored the ability of our tagged proteins to signal GLP-1 and induce germline mitosis. In the course of these experiments, we have discovered a novel dominant-negative LAG-2 created by insertion into the extracellular domain, suggesting that LAG-2 may normally function as oligomers or dimers. (1) Henderson, Gao, Lambie & Kimble(1994). Development 120, 2913-2924. (2) Crittenden,Troemel,Evans & Kimble(1994). Development 120, 2901-2911.

Evans TC et al. (1991) International C. elegans Meeting "DEVELOPING AN IN VIVO ASSAY FOR TRANSLATIONAL CONTROL OF GERMLINE RNAs."

Evans TC, Kimble JE

[International C. elegans Meeting, 1991]

Some genes, such as fem-3 and glp-l, act zygotically to control germline fates, and matemally to control embryonic fates. The maternal RNA (or protein) products of such genes must be carefully regulated. In particular,fem-3 directs spermatogenesis in L4l and its RNA accumulates during oogenesis for use during embryogenesis2. Thus, fem- 3 must be active in L4's to promote spermatogenesis and inactive in adults to allow oogenesis. Recent studies suggest that the 3'UTR offem- 3 is important for fem-3 inactivation: fem-3(gf) mutations may cause inappropriate translation offem-3 RNA due to removal of a negative regulatory site in the 3'UTR (Ahringer and Kimble abstract). In addition, transformation experiments suggest a possible role for the S'UTR in positive regulation offem-3 at the post-transcriptional level (T. Evans, WBG 11(4)). To analyze the translational regulation of fem- 3 and other maternal RNAs, we are developing a method to assay germline translation in vivo. RNAs are made in vitro from plasmids in which a reporter gene ( -galactosidase or ,B-glucuronidase) is flanked by fem-3 regulatory sequences. Capped and polyadenylated reporter RNAs are then injected into adult germlines or soma, and protein expression is assayed. Our preliminary results show weak germline expression from RNAs containing afem-3(8fl 3'UTR, but no expression from RNAs with a fem-3(+) 3'UTR. We do not know yet whether there is a significant difference between weak and no expression. Both RNAs provide strong expression when injected into somatic tissue (including intestine, hypodermis and probably somatic gonad). Thus, the germline appears to inhibit the activities of these RNAs. Attempts to optimize germline expression are currently underway. We hope to use this assay to explore the gerrnline and embryonic regulation of matemal RNAs, and to identify genes required for this regulation.

Lee, Song-Hua et al. (2009) International Worm Meeting "A genome scale gene expression analyses reveals that Burkholderia pseudomallei suppresses Caenorhabditis elegans immunity via targeting a GATA transcription factor."

Nathan, Sheila, Tan, Man-Wah, Lee, Song-Hua

[International Worm Meeting, 2009]

The gram-negative soil bacteria Burkholderia pseudomallei is the etiological agent of melioidosis in humans and animals, causing significant mortality and morbidity in South-east Asia and northern Australia, where it is endemic. The innate immune system is the first line of defense against this pathogen but the mechanisms of innate immunity to this pathogen remain to be fully elucidated. B. pseudomallei can infect the nematode Caenorhabditis elegans and upon ingestion complete mortality is observed by 48 hours. Immune responses of C. elegans to pathogens involved several conserved transcription factors, including the FOXO/DAF-16 and GATA/ELT-2 transcription factors. To investigate host immune responses to B. pseudomallei infections, we performed a genome-wide transcriptome analysis on age-matched adult worms infected for 2, 4, 8 and 12 hours. Our analyses revealed that approximately 6% of the C. elegans genes were transcriptionally regulated when compared to uninfected animals. Enriched among induced genes included those in the metabolism functional category, such as genes encoding cytochrome P450 and UDP-glucuronosyltransferase, as well as defence-related genes. In contrast, genes involved in the aging process, lipid metabolism and response to oxidative stress were robustly repressed upon B. pseudomallei infection. Interestingly, the proportion of genes that were suppressed increased with time of infection, suggesting that suppression of host defence genes may be an important virulence mechanism employed by B. pseudomallei. Another gram-negative human pathogen Pseudomonas aeruginosa is able to suppress C. elegans immunity by ejecting the transcription factor DAF-16 from the nucleus of intestinal cells (Evans et al. 2008). We showed that immune suppression by B. pseudomallei is distinct from that of P. aeruginosa. Instead, it involves the targeting of an intestinal GATA transcription factor ELT-2 and the down regulation of ELT-2 transcriptional targets. Evidence for this novel mechanism of host immune suppression will be reported.

Onami S et al. (1997) International C. elegans Meeting "CHANGES IN POLY(A) TAIL LENGTH OF MATERNAL MESSAGES."

Kohara Y, Onami S

[International C. elegans Meeting, 1997]

In Drosophila and Xenopus, lots of maternal mRNA have been reported whose translation is regulated through cytoplasmic polyadenylation; their poly(A) tails elongate when their translation is activated. Aiming to fish out the maternal mRNA of C. elegans whose translation is regulated through cytoplasmic polyadenylation, we have developed a method to assess the poly(A) tail length of given mRNA in embryos at a specific stage. First, we examined the poly(A) tail lengths of several known maternal mRNA whose translation is considered to be regulated. Maternal glp-1 mRNA exists in embryo before the first division and is detected in both blastomeres at 2cell stage, while GLP-1 protein first appears at 2cell stage and is restricted in AB blastomere(Evans et al., Cell 77:183). In order to examine the poly(A) tail length of glp-1 mRNA, total RNA was extracted from 15 embryos at the same stage. An RNA-oligonucleotide-tag was added to its 3'-end. This RNA was examined by RT-PCR using a glp-1-specific and a tag-specific primer. Results showed that glp-1 mRNA has poly(A) tail of -40 bases in matured oocyte while 2cell stage embryo contains glp-1 mRNA with longer poly(A) tail(>110bases). 2cell stage embryo also contains glp-1 mRNA with shorter poly(A) tail(-25bases). These results suggest that cytoplasmic polyadenylation is involved in the translational regulation of glp-1 mRNA. Fem-3 mRNA and skn-1 mRNA were examined and elongation of their poly(A) tails during embryogenesis was also observed. We are currently examining the exact time at which the poly(A) tails start to elongate. Also, we are examining their poly(A) tail lengths in AB and P1 blastomere.