Pelletier, Katharine et al. (2015) International Worm Meeting "Evolution of hermaphroditism in Caenorhabiditis species."

Pelletier, Katharine, Pilgrim, Dave, Reidy, Keith

[International Worm Meeting, 2015]

Hermaphroditism (sperm production in the XX ovotestis) is one of the traits that has made C. elegans such a powerful genetic model, but most species in the genus have a male/female mating system. The genetic pathway used to determine somatic and germline sex in C. elegans is well understood; however the genes in the pathway are poorly conserved within Caenorhabditis. We have been interested in how sex determination, a rapidly evolving process in Caenorhabditis, can provide insights into how biological pathways can be modified from a common set of ancestral genes to provide novel traits, such as the XX hermaphrodite. Previous work from our lab and the Haag lab has shown that the control of sperm production in a female gonad is different in C. briggsae as compared to C. elegans. fem mutants in C. elegans result in XX and XO females, while in C. briggsae both XX and XO fem mutants are hermaphrodites.We have now extended that work, by isolating and characterizing suppressors of Cb-tra-2 ts mutations, in hope of identifying missense mutations which will help with the analysis of Fem protein function. Over 70 tra-2 suppressors were identified in the Pilgrim and Haag labs by looking for the restoration of self-fertility to tra(ts) animals at the restrictive temperature. As expected, most of these lie in the Cb-fem genes, however some represent novel phenotypes/alleles not previously observed in either system. I will present the current understanding of the C. briggsae sex determination pathway and how this differs from C. elegans. Additionally, I will discuss how comparative genomics within the Caenorhabditis species may provide us with clues to the relationship between molecular changes and the evolution of novel traits. .

Pilgrim, Dave et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "THE SEX DETERMINING PATHWAY IN CAENORHABDITIS; TRA SUPPRESSORS AND FEM GENES"

Reidy, Keith, Pilgrim, Dave, Egydio de Carvalho, Carlos, Dewar, Jill

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

Sex determination in Caenorhabditis is a rapidly evolving process that can provide insights into how biological pathways can be modified from a common set of ancestral genes. C. elegans and C. briggsae are two of three androdiocious species within the Elegans group. These two species are both morphologically and developmentally similar but their genetic control of hermaphroditism is different. XX animals in these species are somatically female but are capable of producing and storing sperm before switching to oocyte production. Previous work from our lab and Eric Haag's showed that the function of the Tra genes was similar between the two species, while mutations in C. briggsae fem-2 or fem-3 genes resulted in somatic but not germline feminization, suggesting that the sperm/oocyte switch must be controlled downstream of the Fem genes in C. briggsae XX animals. We have now extended that work, by isolating and characterizing suppressors of Cb-tra-2 and Cb-tra-3 ts mutations, in hope of identifying missense mutations which will help with the analysis of Fem protein function.Over 70 tra-2 suppressors were identified in the Pilgrim and Haag labs and several tra-3 suppressors in our lab by forward genetics (looking for the restoration of self-fertility to tra(ts) animals at the restrictive temperature). The results of these screens and the ongoing characterization will be presented. The majority of the tra-2 suppressors are alleles of the three fem genes, as expected, including a large set of mutations in Cb-fem-1, which had not been previously isolated by reverse genetics. A small set of interesting mutations complemented the fem genes and are phenotypically distinct from any other mutants described in C. elegans or C. briggsae. WGS has identified candidates for these suppressors and we are currently in the process of mapping the mutations to confirm that the lesion is responsible for their unique phenotypes, and the alleles are being separated from the tra-2 background to allow characterization of their phenotype.

Stefan Taubert et al. (2004) West Coast Worm Meeting "Biochemical Characterization of the C. elegans Nuclear Hormone Receptor NHR-49"

Keith R Yamamoto, Stefan Taubert, Marc Van Gilst

[West Coast Worm Meeting, 2004]

Nuclear Hormone receptors (NHRs) are a family of ligand-regulated transcription factors involved in diverse metazoan functions, such as sexual differentiation, organ development, and regulation of metabolism. The C. elegans genome contains over 270 NHR genes, approximately five times as many as the human genome. For most of these no function has been described yet. We focus on characterizing NHR-49, which regulates fat metabolism, and lifespan (Marc van Gilst and Keith R. Yamamoto, unpublished). Interestingly, this mirrors the functions of human Peroxisome-proliferator-activated-receptor a (PPARalpha), while the sequence of NHR-49 suggests similarity to another human receptor, Hepatocye Nuclear Receptor 4alpha (HNF4alpha). Since HNF4alpha dimerizes via its ligand-binding-domain (LBD), we utilized yeast two-hybrid based technology to assess whether the LBDs of NHR-49 and 12 related HNF4alpha-like NHRs, can also homodimerize. While several of these LBDs readily dimerized (including NHR-49), some others did not (such as DAF-12). Since the homodimerization of NHR-49's LBD is predicted to occur via two conserved salt-bridges (E5/K55 and E50/R93), we mutated the E50 and K55 residues to alanines. We will present the effect of these mutations on LBD homodimerization and activation of a transcriptional reporter in yeast. In an attempt to identify ligands of NHR-49 we treated worms with clofibrate, a synthetic PPARa ligand used to treat hyperlipidemia. This treatment caused a strong reduction of worm body fat levels, and was accompanied by lethality at higher concentrations. However, similar effects were observed in a strain harboring an NHR-49 deletion, suggesting that the effects of clofibrate are independent of NHR-49. We are currently characterizing changes in the transcriptional program in response to clofibrate treatment.

S Taubert et al. (2004) European Worm Meeting "CHARACTERIZATION OF THE C. ELEGANS NUCLEAR HORMONE RECEPTOR NHR-49"

S Taubert, K Yamamoto, M Van Gilst

[European Worm Meeting, 2004]

Nuclear Hormone receptors (NHRs) are a family of ligand-regulated transcription factors involved in diverse metazoan functions, such as sexual differentiation, organ development, and regulation of metabolism. The C. elegans genome contains over 270 NHR genes, approximately five times as many as the human genome. For most of these no function has been described yet. We focus on characterizing NHR-49, which regulates fat metabolism, and lifespan (Marc van Gilst and Keith R. Yamamoto, unpublished). Interestingly, this mirrors the functions of human Peroxisome-proliferator-activated-receptor a (PPARa), while the sequence of NHR-49 suggests similarity to another human receptor, Hepatocye Nuclear Receptor 4a (HNF4a). Since HNF4a dimerizes via its ligand-binding-domain (LBD), we utilized yeast two-hybrid based technology to assess whether the LBDs of NHR-49 and 12 related HNF4a-like NHRs, can also homodimerize. While several of these LBDs readily dimerized (including NHR-49), some others did not (such as DAF-12). Since the homodimerization of NHR-49's LBD is predicted to occur via two conserved salt-bridges (E5/K55 and E50/R93), we mutated the E50 and K55 residues to alanines. We will present the effect of these mutations on LBD homodimerization and activation of a transcriptional reporter in yeast. In an attempt to identify ligands of NHR-49 we treated worms with clofibrate, a synthetic PPARa ligand used to treat hyperlipidemia. This treatment caused a strong reduction of worm body fat levels, and was accompanied by lethality at higher concentrations. However, similar effects were observed in a strain harboring an NHR-49 deletion, suggesting that the effects of clofibrate are independent of NHR-49. We are currently characterizing changes in the transcriptional program in response to clofibrate treatment.

Nathalie Pujol et al. (2003) International Worm Meeting "Morphogenesis and sensory behaviour associated with a single Toll homologue"

Nathalie Pujol, Jean-Jacques Remy, Jonathan Ewbank

[International Worm Meeting, 2003]

Toll is a conserved cell-surface receptor that plays two major roles in Drosophila: in dorso-ventral patterning in the early embryo and in anti-microbial innate immunity (1). Many of the vertebrate homologues of Toll, the Toll-Like Receptors or TLRs, are known to be involved in innate immunity. In contrast to both insects and vertebrates, the nematode C. elegans possesses just one TLR, TOL-1, essential for early development (2). Through 4D Nomarski microscropy analyses carried out in A. Chisholms lab (UCSD), we have found that in tol-1 null mutants both the closure of the gastrulation cleft and epidermal ventral closure are abnormal. In vitro, Toll has been shown to function as a cell-adhesion molecule (3). Our results suggest a role for TOL-1 in the adhesion or migration of epidermal cells in vivo. We are undertaking genetic screens for suppressors of the tol-1 phenotype in the hope of identifying novel genetics partner for TOL-1, as well as directly testing its role in cell adhesion. Unlike Toll, tol-1 appears not to play a direct role in nematode defence against infection. On the other hand, adult worms have a tendency to avoid the bacterial pathogen Serratia marcescens and TOL-1 is necessary for this behaviour (2). We have found that this behaviour varies as a function of the worms age and population density, suggesting that it includes contributions both from the bacteria and the worms themselves. We are currently testing worm with defects either in sensory behaviours (e.g. glr-1, nmr-1) or in secretion (e.g. daf-22) to see whether we can identify mutants that phenocopy the tol-1 repulsion defect. Preliminary tests that oppose attraction to diacetyl and avoidance of copper (4), also suggest that tol-1 might play a more general role in sensory integration. 1. M. P. Belvin, K. V. Anderson, Annu Rev Cell Dev Biol 12, 393-416 (1996). 2. N. Pujol et al., Curr Biol 11, 809-21 (2001). 3. F. J. Keith, N. J. Gay, Embo J 9, 4299-306. (1990). 4. T. Ishihara et al., Cell 109, 639-49 (2002).