The main goal of this proposal is to understand how epithelia are formed in the roundworm C. elegans. Epithelial structures perform many necessary roles in the body, such as acting as barriers (e.g.: skin) or forming transport tubules (e.g. gut, blood vessels, etc.). To execute these diverse functions, epithelial cells must be highly polarized with distinct membrane domains. The regulation of epithelial polarity is of high importance, as dysregulation can result in human disease. For example, Wilms' Tumor, a childhood cancer, can be caused by a mutation in the Wilms' Tumor 1 (WT1) gene. WT1 mutant mice fail to form a complete kidney due to a defect in epithelium formation. In addition, the loss of epithelial polarity may contribute to cancer metastasis. Thus, it is crucial to determine the genes that regulate epithelial polarization. The current view is that the adhesion protein E-cadherin is necessary to initiate and maintain epithelial polarity. However, a cadherin-independent pathway that initiates epithelial polarization has been uncovered in many systems, including vertebrates. For example, the serine/threonine kinase LKB1 is sufficient to polarize single human intestine cells in the absence of cadherin-mediated signaling. In addition, C. elegans mutants lacking a functional cadherin still have polarized epithelia. To elucidate the mechanism of this cadherin-independent pathway, the Mango lab performed a genetic screen to discover mutants that fail to generate a foregut epithelium. Surprisingly, this screen identified regulators of cytokinesis, the central-spindlin components ZEN-4/kinesin and CYK-4/GAP. The failure to form a foregut epithelium resulted from a defect in polarity and not in cytokinesis. Further work from the Mango lab showed that CYK-4 regulates RhoA activity and actomyosin contractility to initiate polarization of the C. elegans zygote. This proposal aims to 1) Test the intriguing hypothesis that formation of a foregut epithelium requires the same genes that act to polarize the one-cell embryo and 2) Perform a genome-wide RNA interference (RNAi) screen to identify new genes regulating epithelium formation. In the first aim I will use fluorescently-tagged proteins and microscopy to document carefully the events during polarization of the foregut epithelium. I will also determine at which step ZEN-4 and CYK-4 act, and perform mutant analysis to generate a genetic pathway for epithelium formation. In the second aim, the unbiased, genome-wide RNAi screen for novel regulators of epithelial polarization will involve feeding dsRNA-expressing bacteria to knock-down gene expression. Positive candidates will be analyzed in detail to discover their role in establishing polarity. These results will add to our knowledge of this basic cell biological process and may identify therapeutic targets for human disease. ? ? ?
|Von Stetina, Stephen E; Liang, Jennifer; Marnellos, Georgios et al. (2017) Temporal regulation of epithelium formation mediated by FoxA, MKLP1, MgcRacGAP, and PAR-6. Mol Biol Cell 28:2042-2065|