SE George et al. (1999) International C. elegans Meeting "Still Searching for components of the VAB-1 pathways"

AD Chisholm, SE George

[International C. elegans Meeting, 1999]

We are identifying the mechanisms by which the C. elegans Eph receptor tyrosine kinase, VAB-1, signals in neural and epithelial morphogenesis. VAB-1 has both kinase-dependent and kinase-independent functions during C. elegans development (George et al, Cell 92:633). The kinase-dependent (forward signaling) function of VAB-1 is mediated by its cytoplasmic kinase domain, possibly through tyrosine phosphorylation of the juxtamembrane domain. Proteins that interact with the cytoplasmic domain of Eph receptors have been identified in vertebrates using the yeast two-hybrid system. Candidate molecules include a low molecular weight protein tyrosine phosphatase and adaptor proteins such as Nck, Grb2, and Grb10. We are using a modified yeast two-hybrid screen to identify genes that function in VAB-1 forward signaling. Initial progress and methodology of the screen will be presented. The nature of the VAB-1 kinase-independent function is unknown but may involve the extracellular domain. Our genetic and biochemical studies indicate that VAB-2 (a C. elegans ephrin) is a ligand for VAB-1 and that it appears to mediate the kinase-independent function of VAB-1 (see Chin-Sang et al abstract). vab-2 mutations display synthetic lethality with vab-1 kinase mutations. We also observe synthetic lethality between vab-1 kinase mutations and a mutation in the receptor protein tyrosine phosphatase, PTP-1, suggesting that PTP-1 may be acting in a parallel pathway to the VAB-1 kinase activity (see Harrington et al abstract). To identify genes that are acting in the VAB-1 kinase-independent pathway or in parallel pathways we are performing screens to identify mutations that are synthetic lethal with vab-1 . Progress of the screen will be presented.

George SE et al. (1997) International C. elegans Meeting "EPH RECEPTOR TYROSINE KINASE SIGNALING IN C. ELEGANS MORPHOGENESIS"

Chisholm AD, George SE

[International C. elegans Meeting, 1997]

The C. elegans epidermis arises from a dorsal sheet of cells that spreads laterally to enclose the embryo on the ventral side. This ventral closure is essential for normal embryonic development. Mutations in vab-1 and vab-2 cause variable defects in ventral closure and epidermal morphogenesis. vab-1 encodes a member of the EPH family of receptor protein tyrosine kinases (WBG 14#5, p66). EPH RPTKs are the largest family of RPTKs and have been implicated in axonal guidance and morphogenesis in vertebrates. We have identified the molecular lesions in twelve vab-1 alleles. Null mutations bear deletions of parts of the extracellular domain. Missense mutations in the ligand-binding domain of VAB-1 cause partial loss of function and may define residues important for ligand binding. Mutations affecting only the kinase domain cause weak mutant phenotypes, suggesting that kinase activity may not be essential for all of VAB-1!s functions. A vab-1-GFP reporter construct is expressed in hypodermal cells during ventral closure. vab-2 encodes a member of the family of EPH RPTK ligands (see abstract by Lynch and Chisholm). The similar phenotypes of vab-1 and vab-2 mutants suggest that VAB-2 may be a ligand for VAB-1. The vab-1 null phenotype is more severe than those of vab-2 putative null alleles suggesting that VAB-1 could interact with other ligands in addition to VAB-2. We are taking genetic and molecular approaches to identify additional components of EPH RPTK signal transduction in C. elegans.

Georges Alves et al. (2001) International C. elegans Meeting "Molecular characterization of the seu-2 gene involved in axon guidance"

Georges Alves, Joe Culotti

[International C. elegans Meeting, 2001]

Our laboratory has identified seu-2 as a suppressor of axon guidance defects induced by ectopic UNC-5 expression (Colavita & Culotti (1998) Dev. Biol. 194: 72-85). The seu-2(ev523) mutation on its own does not cause axon guidance defects, but was probably picked up in this suppressor screen because the screen is sensitized to the dose of components of the UNC-5 axon guidance signaling pathway. Since the seu-2(ev523) mutant does not show any obvious phenotype, we had to use the rescue of the dorsalward axon guidance phenotype induced by ectopically expressed UNC-5 to identify a rescuing region. Using overlapping cosmids, we identified a rescuing fragment containing two predicted genes that encode a putative seven-pass transmembrane protein and a serine-threonine phosphatase PP2A. The two coding regions are very close and are likely to constitute an operon. To determine which one of these genes is actually seu-2 , we are attempting to rescue the mutant phenotype with constructs carrying a premature termination mutations in each gene. The sequence analysis of the transcripts from both genes has not revealed any amino-acid change suggesting that the seu-2(ev623) mutation may affect untranslated regions of the gene. We are investigating the expression pattern of the gene using a translational fusion to GFP. Preliminary results reveal expression in dorsal and ventral muscles, in motorneurons and in amphid and phasmid neurons throughout development. We will check expression in touch neurons by using a longer promoter region and observing the reporter transgene expression during embryonic development. To further characterize the seu-2 locus genetically and molecularly, we are currently doing a non-complementation screen to isolate new mutant alleles of the seu-2 gene. The molecular analysis of the new mutations should provide new insights into the function of SEU-2 and possibly identify different functional domains of the protein.

Wilhelm, Thomas et al. (2015) International Worm Meeting "A C.elegans RNAi screen identifies novel genes with antagonistic pleiotropic effects."

Medina, Rebeca, Wilhelm, Thomas, Richly, Holger, Byrne, Jonathan

[International Worm Meeting, 2015]

In 1957, George C. Williams published the antagonistic pleiotropy hypothesis of aging, which was later mathematically supported by Brian Charlesworth. This theory predicts that some genes mediate beneficial effects early in life when natural selection is strong, but are detrimental late in life when natural selection is weak. Natural selection favors these beneficial effects on fitness early in life at the expense of enriching harmful effects late in life; hence leading to the evolution of aging.We screened 800 chromatin regulators and transcription factors in order to identify novel genes that exhibit antagonistic pleiotropy. Genes were inactivated in the post-reproductive phase by the use of RNAi from day nine of adulthood onwards. The screen led to the identification of several genes that significantly extend lifespan upon late-life inactivation, while shortening lifespan when inactivated early in life.The forkhead box (FOX) A transcription factor pha-4 represents a top candidate of our screen. Late-life inactivation of pha-4 resulted in a strong lifespan extension of 33%. In contrast, inactivation of pha-4 from the first day of adulthood reduced C.elegans lifespan. This is of particular interest, as pha-4 has been shown to mediate diet-restriction and germline-less induced longevity. Our findings indicate that, depending on the age, pha-4 is either needed for normal lifespan and longevity or exhibits detrimental effects. We are currently aiming to identify and to characterize the biological processes underlying the observed negative effects of pha-4 late in life.Generally, genes that behave according to the antagonistic pleiotropy hypothesis may represent promising drug targets to fight age-associated diseases. Our findings emphasize that timing needs to be addressed to fully understand how genes and gene networks impinge on the aging process.

Tiller, George R. et al. (2013) International Worm Meeting "Potential Roles of Peroxidases in C. elegans Innate Immunity."

Garsin, Danielle A., Tiller, George R.

[International Worm Meeting, 2013]

ROS (reactive oxygen species) are produced at mucosal epithelial surfaces, as part of the innate immune response to combat invading pathogens. The proteins responsible for this ROS production belong to the NOX/DUOX (NADPH Oxidase and Dual Oxidase) family and include C. elegans Ce-Duox1/BLI-3. It was previously shown that in response to infection with Enterococcus faecalis, BLI-3 released ROS, and reduction of bli-3 expression caused the worms to be significantly more susceptible. Additionally, DUOXs are known to generate ROS for use by peroxidases, which then generate more potent oxidation products that aid in the host's innate immune response. An example is human lactoperoxidase (hLPO), which uses ROS from hDUOX2 to produce hypothiocyanite to facilitate clearance in the airway epithelia. Based on this information, we hypothesized that BLI-3-generated ROS may act as a substrate for peroxidases to generate antimicrobial agents in the C. elegans immune response. Using bli-3 as the query, WormBase.org was searched for homologous genes. The genes-of-interest were screened for a susceptibility-to-pathogen phenotype in C. elegans using RNAi (RNA interference) to reduce their expression. Genes necessary for full resistance against pathogen were further tested for general fitness defects using longevity assays. F49E12.1 RNAi worms incubated with E. faecalis at 25 deg C displayed an increased susceptibility phenotype (P-value = 0.0011; median survival: 189 and 165 hours, V.C. and F49E12.1, respectively). As determined by Amplex Red, when infected, F49E12.1 RNAi worms display increased H2O2 levels relative to vector control (P-value = 0.0068) which suggests F49E12.1 utilizes H2O2 for a, currently, unknown purpose. Interestingly, F49E12.1 possesses significant homology to peroxidases that are crucial for innate immunity in crustaceans as well as mammals. Therefore, the current focus is to elucidate how the putative peroxidase F49E12.1 contributes to the C. elegans innate immune response.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.

Karin Kiontke et al. (2008) Development & Evolution Meeting "New Phylogeny Of Caenorhabditis Including Recently Discovered Species"

David Fitch, Karin Kiontke, Marie-Anne FELIX

[Development & Evolution Meeting, 2008]

Recently, seven new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to 17, 10 of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10, 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Whereas none of them is likely to be the sister species of C. elegans, we now know of two close relatives of C. briggsae-C. sp. 5 and C. sp. 9. C. sp. 9 can hybridize with C. briggsae in the laboratory [see abstract by Woodruff et al.]. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. This species is easier to cultivate than C. japonica and may be a better candidate for comparative experimental work. Two of the new species branch off before C. japonica as sister species of C. sp. 3 and C. drosophilae+C. sp. 2, respectively. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and five from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last two years. There is no indication that we are even close to knowing all species in this genus.

Lynch AS et al. (1997) International C. elegans Meeting "VAB-2 ENCODES AN EPH RECEPTOR TYROSINE KINASE LIGAND THAT FUNCTIONS IN C. ELEGANS EPIDERMAL MORPHOGENESIS."

Chisholm AD, Lynch AS

[International C. elegans Meeting, 1997]

A key step in morphogenesis of the C. elegans epidermis is ventral closure. vab-1 and vab-2 mutants display similar defects in epidermal ventral closure and morphogenesis. vab-1 encodes a receptor tyrosine kinase (RTK) of the EPH family (see abstract by George and Chisholm). vab-2 is defined by six alleles which cause morphogenesis defects similar to those caused by hypomorphic vab-1 alleles. We have found that vab-2 encodes a member of the family of ligands for EPH RTKs, and that the molecular changes associated with different alleles of vab-2 are consistent with partial or complete loss of gene function. Vertebrate EPH ligands are either transmembrane proteins or membrane-anchored via a glycosylphosphatidylinositol (GPI) motif. At present, we are unsure whether VAB-2 is transmembrane or membrane-anchored; however the VAB-2 predicted receptor binding domain is 40% identical to those of transmembrane EPH ligands, and ~30% identical to GPI-anchored ligands. EPH RTKs and their ligands function in vertebrates to control the growth of axons, and are also implicated in morphogenetic pathways. Given the similarity of the vab-1 and vab-2 phenotypes, VAB-1 and VAB-2 might interact as receptor and ligand in a signalling pathway involved in the control of epidermal morphogenesis.

Schartner, Caitlin M. et al. (2015) International Worm Meeting "X-chromosome evolution: divergence of X-sequence motifs that drive dosage compensation across Caenorhabditis species."

Meyer, Barbara J., Lo, Te-Wen, Schartner, Caitlin M.

[International Worm Meeting, 2015]

Dosage compensation (DC) across Caenorhabditis species exemplifies an essential process that has undergone rapid co-evolution of protein-DNA interactions central to its mechanism. In C. elegans, recruitment elements on X (rex sites) recruit a condensin-like DC complex (DCC) to hermaphrodite X chromosomes to balance gene expression between the sexes. Recruitment assays in vivo showed that C. elegans rex sites do not recruit the DCC of C. briggsae, and vice versa. To understand how DC complexes and X chromosomes evolved to use different X targeting sequences, we compared DCC subunits and binding sites in C. elegans to those in three species of the C. briggsae clade (15-30 MYR diverged): C. briggsae, its close relative C. nigoni (C. sp. 9), and C. tropicalis (C. sp. 11). By raising antibodies and introducing endogenous tags with TALENs or CRISPR/Cas9, we showed that homologs of both SDC-2, the pivotal X targeting factor, and DPY-27, a DCC-specific condensin subunit, bind X chromosomes of XX animals. Although the DCC shares key components across these four species, the binding sites differ. First, ChIP-seq studies in C. briggsae and C. nigoni identified DCC binding sites that are homologous across these close relatives but differ from C. elegans sites in sequence and location. Second, C. elegans sites use motifs enriched on X (MEX and MEXII) to drive DCC binding, but these motifs are not in C. briggsae or C. nigoni DCC sites and are not X-enriched. Third, we found an X-enriched motif at DCC binding sites of C. briggsae and C. nigoni that is not X-enriched in C. elegans. An oligo with the C. briggsae motif recruits the DCC in C. briggsae, but a similar oligo lacking the motif fails to recruit, establishing the importance of the motif. Fourth, another motif was found in C. briggsae and C. nigoni that shares a few nucleotides with MEX, but its functional divergence was shown by C. elegans recruitment assays. Fifth, two endogenous C. briggsae X-chromosome regions with strong C. elegans MEX motifs fail to recruit the C. briggsae DCC, as assayed by ChIP-seq and recruitment assays. None of these DCC motifs is enriched on the C. tropicalis draft X sequence, supporting further binding site divergence within the C. briggsae clade. Ongoing ChIP-seq studies in C. tropicalis will help determine how C. elegans and C. briggsae clade motifs are evolutionarily related. Comparison of DCC targeting mechanisms across these four species allows us to characterize a rarely captured event: the recent co-evolution of a protein complex and its rapidly diverged target sequences across an entire X chromosome.