Identifying the genetic basis for phenotypic variation is a central question in evolutionary biology and can be studied in detail using model organisms. Fecundity variation in different isolates of C. briggsae has been observed previously, but the genetic causes of this variation are unclear. Crosses between C. briggsae advanced-intercross recombinant inbred lines (AI-RILs) and parental strains yield near isogenic line (NIL) strains, which I created to provide a powerful genetic resource to fine-map the basis for fecundity and other trait differences. Phenotypic analysis of the NILs shows the complexities of possible epistatic interactions on phenotypic expression. These NIL strains contribute a valuable genetic resource toward the long-term goal of identifying the genes responsible for differences in fecundity in this species. The elucidation of the basis for this trait variation will also contribute further into the mechanisms for how genotype and phenotype and environment all interact.

Identifying the genetic basis for phenotypic variation is a central question in evolutionary biology and can be studied in detail using model organisms. Fecundity variation in different isolates of C. briggsae has been observed previously, but the genetic causes of this variation are unclear. Crosses between C. briggsae advanced-intercross recombinant inbred lines (AI-RILs) and parental strains yield near isogenic line (NIL) strains, which I created to provide a powerful genetic resource to fine-map the basis for fecundity and other trait differences. Phenotypic analysis of the NILs shows the complexities of possible epistatic interactions on phenotypic expression. These NIL strains contribute a valuable genetic resource toward the long-term goal of identifying the genes responsible for differences in fecundity in this species. The elucidation of the basis for this trait variation will also contribute further into the mechanisms for how genotype and phenotype and environment all interact.

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)

No description available.

Comparative study of homologous structures in closely related species allows the identification of changes in gene regulatory mechanisms and their impact on the evolution of developmental processes. Nematodes, the invertebrate roundworms, are well suited for such studies, especially the Caenorhabditis briggsae and its famous cousin C. elegans. These two worms diverged from a common ancestor roughly 30 million years ago, yet appear morphologically almost identical. My Ph.D. thesis has focused on a set of nuclear factors in C. briggsae that negatively regulate cell proliferation to generate the hermaphrodite-specific mating and egg-laying organ, i.e., vulva. To this end, I have taken a two-pronged approach: one, developing resources to facilitate genetic and genomic studies in this species, and two, characterizing the roles of a novel class of genes and known repressors of vulval development. My work has uncovered substantial differences in the underlying genetic networks that regulate vulva formation in C. briggsae and C. elegans. / Thesis / Candidate in Philosophy

Evolutionary variations during vulval development in C. elegans and its related nematode species are well analyzed. The formation of C. elegans vulva involves many complex cell-cell interactions that are mediated through well conserved EGF/EGFR/RAS, LIN12/Notch and WNT signaling pathways. These pathways specify distinct cell fates of the six epidermal vulval precursor cells (VPCs), P(3-8).p. pry-1/Axin in C. elegans is identified as a part of destruction box complex that mediates (beta)-catenin degradation and is known to negatively regulate canonical Wnt signaling pathway during its development. I focused on the genetic analysis of pry-1 I Axin function in C. briggsae, sister species to C. elegans, to study inter-species comparisons of vulva formation. Three alleles, lin(sy5353), lin(sy5411) and lin(sy5270) were genetically mapped to LG I using standard genetic and in del mapping techniques. Interestingly, a unique simultaneous Multi vulva and Vulvaless (Muv-Vul) phenotype was observed during vulva formation in Cbr-pry-1 alleles, resulting from the varying induction potentials of the VPCs along the A/P axis, compared to Cel-pry-1 animals. In order to analyze these phenotypic differences between Cel-pry-1 and Cbr-pry-1 in greater detail, I dissected vulval development in sy5353 animals. VPC competence analysis was done through cell lineages and ablations studies, while the C. briggsae vulval cell fate markers were used for cell fate specification analysis. Cell ablations revealed that P7.p and P8.p in Cbr-pry-1 animals exhibited non-competence towards anchor cell signaling. Additionally, gonad-independent inductions was observed in P(3-8).p cells and they adopted 2° cell fate specifications. Using RNAi approach, Cbr-pry-1 interactions with other vulval pathway genes were dissected and it was observed that Cbr-lin-12 is involved in VPC competence of P7.p and Cbr-pop-1 exhibited different regulatory levels during vulval development compared to C. elegans. Thus, it can be inferred that the mechanisms of vulva formation in C. briggsae has evolved through changes in the competence of VPCs. / Thesis / Master of Science (MS)

Abstract Collagen prolyl 4-hydroxylases (C-P4Hs) and related enzymes catalyze the hydroxylation of certain proline residues in animal collagens and plant hydroxyproline-rich proteins, respectively. Animal C-P4Hs and their isoenzymes have been characterized to date from humans, rodents, insects and nematodes. Most of the animal C-P4Hs are α2β2 tetramers in which protein disulphide isomerase (PDI) serves as the β subunit, but the nematode C-P4Hs characterized so far have unique molecular compositions. Two P4Hs have been cloned from the plant Arabidopsis thaliana and one from the Paramecium bursaria Chlorella virus-1, these being monomeric enzymes. This thesis reports on the identification of a large P4H family in the green alga Chlamydomonas reinhardtii and the cloning and characterization of one member, Cr-P4H-1. This is a soluble monomer that hydroxylates in vitro several peptides representing sequences found in C. reinhardtii cell wall proteins. Lack of its activity led to a defective cell wall structure, indicating that Cr-P4H-1 is essential for proper cell wall assembly and that the other P4Hs cannot compensate for the lack of its activity. Two C. elegans genes, Y43F8B.4 and C14E2.4, predicted to code for C-P4H α subunit-like polypeptides were analyzed. Three transcripts were generated from Y43F8B.4, one of them coding for a functional C-P4H α subunit named PHY-4.1. C14E2.4 turned out not to be a C-P4H α subunit gene, as a frame-shift led to the omission of codons for two catalytically critical residues. PHY-4.1 formed active tetramers and dimers with PDI-2 and had unique substrate requirements in that it hydroxylated certain other proline-rich sequences besides collagen-like peptides. Inactivation of the Y43F8B.4 gene led to no obvious morphological abnormalities. Spatial expression of the phy-4.1 transcript and PHY-4.1 polypeptide was localized to the pharynx and the excretory duct. Taken together, these data indicate that PHY-4.1 is not involved in the hydroxylation of cuticular collagens but is likely to have other substrates in vivo. Cloning and characterization of the PHY-1 and PHY-2 subunits from the closely related nematode Caenorhabditis briggsae revealed distinct differences in assembly properties between the C. elegans and C. briggsae PHY-2 subunits in spite of their high amino acid sequence identity. Genetic disruption of C. briggsae phy-1 resulted in a less severe phenotype than that observed in C. elegans, evidently on account of its more efficient assembly of the C. briggsae PHY-2 to an active C-P4H explaining the milder phenotype. Rescue of C. elegans and C. briggsae phy-1 mutants was achieved by injection of a wild-type phy-1 gene from either species.

The regulation and evolution of the inverted repeat transposable element Tel, found in the nematode Caenorhabditis elegans, was studied. The stability of Tel elements in the N2 strain genome was investigated by cloning seventeen N2 Tel elements. To examine their structural integrity, sixteen cloned N2 Tel elements were restriction mapped and, in the case of some variants, their DNA was partially sequenced. Two restriction site variants, Tcl(Eco).12 and Tcl(Hpa-).9, were found. Tel(1.5).10b had lost 89 bp from one end, while Tcl(1.7).28 contained a 55 bp insertion. Two additional elements, Tcl(0.9).2 and Tcl(0.9).14, had different internal deletions. Each element was about 900 bp in length. The majority of Tel elements cloned from the N2 strain were found to have identical restriction maps. Somatic excision of Tel elements in the N2 genome was demonstrated. Tel elements in N2 are apparently both structurally and functionally intact. Nevertheless, mobilization of Tel elements in the N2 germline is restricted. Two new transposable element families, Barney (also known as TCbl) and TCb2, were discovered in a closely related nematode, Caenorhabditis briggsae due to Tel identity. These two families, distinguished through differential inter-element hybridization, showed multiple banding differences between strains. The open reading frames (ORFs) of Tel and Barney share 71% DNA sequence and 74% amino acid sequence identity. The putative terminus of Barney exhibits 68% identity with the 54 bp terminal repeat of Tel. Partial sequencing of TCb2 revealed that its ORF is equally diverged from Barney and Tel. The basis of the sequence heterogeneity observed in the C. briggsae transposons and not in the C. elegans transposons could be due to either horizontal transfer or alternate paths of divergence. Significant sequence identity was found between Tel, Barney, and HB1 (a transposable element from Drosophila melanogaster) within their coding regions and terminal repeats. These sequence similarities define a subclass of inverted repeat transposable elements inhabiting two different phylla, Arthropoda and Nematoda. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

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)