Identification of novel C. elegans Wnt signaling targets functioning in the specification and differentiation of larval seam cells and vulval precursor cells

The Wnt signaling pathway is one of the key extracellular pathways involved in many developmental processes in vertebrates and invertebrates. In the nematode C. elegans, the two Wnt pathways named the Wnt/BAR-1 canonical (WBC) pathway and the Wnt/β-catenin asymmetry (WBA) pathway function in cell fate specification, polarity and migration. We and others have shown that the WBC pathway functions in the specification of the vulval precursor cells (VPCs) that form the hermaphrodite vulva, and the WBA pathway functions in the specification of epithelial seam cells during their larval asymmetric stem cell-like divisions. My research project has focused on the identification of Wnt signaling targets functioning in the VPCs and seam cells using microarray analysis.;The Wnt pathway was over-activated in worms using a heat shock inducible stable variant of BAR-1/β-catenin, and under-activated using a dominant negative variant of POP-1/TCF. The transcript pools from the VPCs and seam cells were enriched by the 'mRNA tagging' method and I used microarray analysis to identify 240 putative Wnt pathway targets showing an average fold change of ≥ 1.5 in the Wnt pathway over-activation samples compared to the under-activation samples. Of these, expression analysis and phenotypic characterization was conducted for 50 genes. I found 29 genes are expressed in the seam cells, hypodermis and/or vulval cells during larval development. RNA interference and mutant analysis revealed five genes with defects in terminal seam cell number. This work has also uncovered a previously unexplored role of Wnt signaling and of eight putative Wnt targets in influencing the lateral fusion of differentiated seam cells, and formation of the cuticular structure called alae. Additionally, 12 putative Wnt targets showed defects in vulval induction, differentiation and morphogenesis.;I have also identified the GATA transcription factor encoding genes egl-18 and elt-6, which are known embryonic regulators of seam cell specification, as putative Wnt targets in larval seam cells. Genetic and epistasis analysis suggests that these factors act reiteratively downstream of the Wnt pathway in specifying seam cell fate during larval asymmetric seam cell divisions, unraveling a novel connection between two important regulators of seam cell fate specification. Finally, I have defined for the first time, the L2/L3 larval transcriptome of the seam cells and VPCs. This work has filled a significant gap in our understanding of C. elegans Wnt targets functioning in seam cell and vulval development.