Characterization of let-765/nsh-1 and its role in RAS signalling in Caenorhabditis elegans

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(Thesis) Ph.D.
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The broad diversity of cell types generated during development of a multicellular organism result from cell-fate decisions. Many fate decisions are directed by the actions of signalling pathways such as the RAS/map kinase (MAPK) pathway via the transcriptional regulation of target genes. The differential expression of target genes is, in turn, responsible for the determination of cell fate. In C. elegans, a RAS/MAPK pathway acts downstream of the epidermal growth factor (EGF) receptor to regulate cell fates in several tissues. In particular, development of the vulva has provided an opportunity to dissect the function of RAS pathway components as well as positive and negative regulators of the pathway. I have identified the essential gene, let-765, as notch signalling pathway homolog-1 (nsh-1), a DExD/H box protein. To investigate the role of let-765 with respect to C. elegans development, I performed a genetic and molecular analysis of the gene. let-765 was found to be broadly expressed throughout development and an assessment of reduction-of-function phenotypes suggests that it is likely required for RAS pathway-directed processes, including larval viability, vulval induction, and development of the male tail and posterior ectoderm. By investigating genetic interactions between the EGFR/RAS/MAPK pathway and let-765, I have demonstrated that it promotes RAS pathway activity and is required for vulval induction. These studies indicate that let-765 acts upstream of the ligand and antagonizes the repressive activity of the synthetic multivulva (synMuv) genes. Specifically, LET-765 appears to positively regulate the transcription of lin-3 EGF, which is required to specify vulval cell fates. The variety of phenotypes generated by a loss or reduction of let-765 function is consistent with a role for LET-765 regulating multiple processes through interactions with factors in addition to the EGFR/RAS pathway. A novel role for DExD/H box proteins in transcriptional regulation has been proposed recently and, together with the data demonstrating a role for LET-765 in the regulation of lin-3 transcription, I propose that LET-765 may interact with transcriptional regulatory complexes to promote gene expression. In summary, this study has provided novel insight into the control of LIN-3/EGF expression and the EGFR/RAS pathway during C. elegans development.
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