Vulval development in C. elegans serves as powerful paradigm to understand the interplay of diverse signal transduction pathways during organogenesis. Previous studies have demostrated that the canonical Wnt signaling pathway plays a pivotal role in the development of the vulva in C. elegans and helps in establishing the 20-10-20 vulval induction pattern of the vulval precursor cells (VPCs). The main focus of my masters research project was to get an understanding of how this vulval induction pattern, established in response to Wnt signaling has evolved in other closely nematode species, particularly C. briggsae. We fmd that the Wnt signaling pathway has evolved to positively as well as negatively regulate the competence of VPCs in C. briggsae. We demonstrate that while mutations inpry-1/Axin in C. elegans result in Multivulva (Muv) phenotype, mutations in the C. briggsae pry-1 gene give rise to a novel MultivulvaVulvaless (Muv-Vul) phenotype. This phenotype is characterized by VPCs anterior to P6.p frequently adopting induced cell fates while those posterior to P6.p frequently adopt a non-induced fate. Furthermore, we also show that the functioning of the Wnt signaling pathway in C. briggsae is dependent upon the activity of key regulators of the Wnt pathway such as the TCFILEF-1 family member pop-1, the f3-catenin bar-] and the hox gene lin-39. Taken together, the fmdings from this study show that while a conserved canonical Wnt pathway confers competence on VPCs in both C. elegans and C. briggsae, the final outcome nonetheless seems to have diversified. / Thesis / Master of Science (MSc)

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A Thesis Submitted to the School of Graduate Studies in the Partial Fulfillment of the Requirements for the Degree Master of Science / Caenorhabditis elegans and one of its close relatives, Caenorhabditis briggsae, are animal models that are commonly used for comparative studies to understand the evolution of developmental mechanisms and gene function. Although the two species appear nearly identical morphologically, comparative genomic analyses have revealed interesting differences between the genomes. Whether such differ- ences contribute to changes in developmental mechanisms and signalling pathways is an active area of research. One of the most well studied phenotypes associated with C. elegans signalling pathways are those that affect the specification of vulval tissue. Within the system of vuval development, mutants that exhibit the Mul- tivulva (Muv) phenotype are important as they show inappropriate divisions of vulva cells, which model tumour formation. Comparing gene function in different species genetic backgrounds can lead to an understanding of how genetic differ- ences contribute to different responses in cancer development. Genetic screens, conducted in our laboratory, yielded several genes whose loss of function results in a Muv phenotype and identified a novel regulator of C. briggsae vulval devel- opment, Cbr-ivp-3. Using the nematode C. briggssae as experimental system, we have characterized the tumour suppressor gene, Cbr-ivp-3, which impacts cell sig- nalling and cell division. I have carried out molecular genetic analyses of ivp-3 in both C. briggsae and C. elegans and have begun to characterize the functional role of Cbr-ivp-3. The findings in this thesis suggest that Cbr-ivp-3 is functioning to negatively regulate EGF/Cbr-lin-3. / Thesis / Master of Science (MSc) / The nematodes Caenorhabditis elegans and Caenorhabditis briggsae, are commonly used for comparative studies to understand the evolution of developmental mechanisms and gene function. Although both species appear morphologically similar, comparative genomic analyses reveal differences between genomes. Comparing gene function in different genetic backgrounds can lead to an understanding of how genetic differences contribute to different responses in cancer development. Genetic screens have yielded several genes whose loss of function results in a Multivulva phenotype, showing inappropriate division of vulva cells, modeling tumor formation. We have carried out molecular genetic analyses of ivp-3, a novel regulator of C. briggsae vulval development, in both species and have found that Cbr-ivp-3 is regulating vulva development by negatively regulating EGF/Cbr-lin-3.

Caenorhabditis elegans and its congener, C. briggsae are excellent animal models for the comparative study of developmental mechanisms and gene function. Gupta lab is using the vulval tissue in these nematodes as a system to investigate conservation and divergence in signal transduction pathways. Genetic screens conducted earlier in our laboratory recovered several mutants that cause multivulva (Muv) phenotype. The Muv genes act as tumor suppressors and negatively regulate the proliferation of vulval precursors. Genetic and molecular work on these genes has revealed that C. briggsae vulva developmental utilizes novel genes representing a new phenotypic class termed ‘Inappropriate Vulva cell Proliferation (IVP)’ (Sharanya et al., 2015). This indicates that the signaling mechanism in C. briggsae specifies vulval cell fates differently from C. elegans. Interestingly, it has been found that Cbr-ivp mutants show higher levels of Cbr-lin-3 (EGF) transcript, indicating that these genes act genetically upstream of Cbr-lin-3, similar to SynMuv family members in C. elegans. Moreover, RNAi knockdown of the Cbr-lin-3 transcript resulted in the suppression of the multivulva phenotype in mutant animals. Similar suppression was also observed when a MAP kinase inhibitor was used in the previous study. In addition, the role of two other novel negative regulators of cell proliferation, Cbr-lin(bh1) and Cbr-lin(bh3) was also investigated. Preliminary findings on these regulators suggested that both Cbr-lin(bh1) and Cbr-lin(bh3) exhibiting a heritable Muv phenotype and are found to be located on Chromosome I and III respectively. Identification of novel genes and further characterization will help us understand the molecular function of genes and their involvement in the regulation of vulval cell differentiation. The findings of my research work will provide a background for future studies to understand the role of novel genes in reproductive system development. Overall, these results provide evidence that although the morphology of vulva is similar in the two nematode species, underlying mechanisms of development appear to have diverged. / Thesis / Master of Science (MSc)