Apical membrane modeling shapes organ lumina, defines membrane microdomains and leads cell movement during epithelial morphogenesis. The effector molecules and structural dynamics underlying these processes are still largely unknown in any species. We propose to analyze the molecular basis of apical membrane modeling in Caenorhabditis elegans tubulogenesis. The tubular organs of this transparent roundworm are derived from single-layered epithelia, providing a unique opportunity to study tube formation throughout development in a living organism. At the same time, its well characterized genetics allows for efficient molecular genetic analysis. We have shown that erm-1, the ancestral ortholog of the putative membrane-cytoskeleton linking ezrin, radixin, moesin (ERM) family, is required to shape the apical surfaces of the major internal organs of C. elegans, and to form intestinal microvilli and excretory canal canaliculi. We will attempt to dissect the mechanisms by which erm-1 exerts this morphogenetic function.
In Aim 1, we will conduct an erm-1 genetic suppressor screen, as a specific approach to identify erm-1-interacting molecules involved in this process. Numerous proteins have been identified as interacting with vertebrate ERMs in diverse structural and signaling processes. The in vivo relevance of these data has been difficult to assess in the complex vertebrate system, given the redundancy among ERMs and their presumed developmental role.
In Aim 2 we will conduct a candidate RNAi screen with orthologs of these proposed ERM-interacting molecules, to directly examine their effect on C. elegans morphogenesis. This screen utilizes C. elegans genome-wide databases, resources, and mutant collections.
Aim 3 will confirm and characterize the genes identified in both screens by genetic, biochemical, morphological and functional assays;and attempt to define their effect on apical membrane modeling. Ordering these genes by epistasis should generate a construction kit for apical membrane modeling in epithelial morphogenesis. Given the fundamental nature of morphogenesis, and the unusually high conservation of ERMs among species, we expect to gain insight into processes relevant to human organ morphogenesis, particularly to intestinal and renal tube morphogenesis, and the diseases affecting these processes.
