Molecular genetic study of reproductive system development in C. elegans and related nematode species

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My NSERC-funded research program focuses on understanding genetic networks that control organ formation in eukaryotes by dissecting molecular mechanisms of similarities and differences in gene function in nematodes. To this end, we are conducting a comparative analysis of reproductive system development, specifically the hermaphrodite vulva, in the nematode (worm) Caenorhabditis elegans and its sister species C. briggsae. A large collection of   mutants were isolated earlier in our lab through forward genetic screens. The mutants belong to three distinct phenotypic classes: vulvaless (Vul), multivulva (Muv), and protruding vulva (Pvl). To date, 17 Vul, Muv and Pvl genes have been studied in some detail. Ten of these are cloned and function investigated in some detail (Seetharaman et al., Dev Biol 2010; Sharanya et al., G3 2012; Sharanya et al., Evol Chamberlin et al., manuscript under review). These genes function at different steps of the vulval induction process and participate in Wnt-beta-Catenin and EGFR-Ras signalling pathways. Interestingly, we have also uncovered evolutionary changes in some of the genes, leading us to propose that developmental mechanisms have diverged between the two nematode species. NSERC funding will enable us to further investigate the mechanism of evolutionary changes in gene function and pathways. Towards this, we will be focusing on the Muv genes that show differences in cell induction pattern compared to the C. elegans orthologs. In collaboration with Dr. Chamberlin's lab (Ohio State), we have molecularly characterized four of the Muv mutants that define a new phenotypic class termed 'inappropriate vulva proliferation (ivp).' The genes are Cbr-htz-1 (H2A.z), Cbr-spr-4 (REST/NRSF), Cbr-gon-14 (hAT transposon family), and Cbr-ivp-3 (Ribonuclease H-like superfamily). Although all four genes are conserved in C. elegans, there are major functional differences. While mutations in any of the C. elegans orthologs, except gon-14, cause normal vulva phenotype, C. briggsae mutants are nearly fully penetrant Muv. To understand the genetic pathway of ivp genes, RNA-seq experiment was carried out. The results revealed that C. briggsae Muv mutants exhibit misregulation of germline genes, leading us to propose that the Muv genes may act to promote differentiation of somatic cells. Future experiments will focus on understanding the mechanism of action of ivp genes in C. briggsae and their functional differences from C. elegans. To this end, we plan to characterize the interacting genes and downstream targets through a combination of forward and reverse genetic (such as CRISPR and RNAi) approaches. These experiments should reveal whether C. briggsae Muv genes define new pathways by altered regulation of known C. elegans orthologs or whether existing developmental pathways are used differently. Ultimately, the findings will uncover the conservation and divergence in developmental mechanisms in metazoans.