SUMMARY Epithelial cells have essential functions in tissue integrity and selective molecular transport. which depend upon the differential targeting of proteins to plasma membranes during polarity establishment and maintenance. Loss of epithelial polarity is associated with serious, life threatening diseases including the progression of cancers, polycystic kidney disease, and cystic fibrosis. Despite the importance of polarity, surprisingly little is known about the mechanisms that direct apical and basolateral proteins to the correct surfaces, and almost nothing is known about how cells break symmetry to establish polarity during in vivo organogenesis. Recent advances in genomic editing, tissue specific protein depletion, and live imaging make these questions approachable at a previously unattainable level. In the simple epithelium of the embryonic C. elegans intestine, PAR-3 (and other apical polarity proteins) move from puncta on lateral membranes to the future apical surface in a microtubule-dependent manner. Depletion of microtubules delays but does not abolish polarity establishment, suggesting other parallel pathways are involved in polarization, which may include actomyosin and/or membrane trafficking based on in vitro studies and parallel roles for these pathways in other contexts. Consistent with this hypothesis, my preliminary data show that basolateral polarity proteins initially move to the future apical surface by a different route than apical proteins, before being retargeted to basolateral membranes. Additionally, my preliminary data implicate membrane trafficking in polarity establishment as RNAi depletion of secretory proteins (SEC-23 or SNAP-29) abrogates apical polarity. Thus, I will test the hypothesis that parallel pathways involving microtubules, actin, and/or membrane trafficking are required to break symmetry and establish apical-basolateral polarity in the intestinal epithelium. In Specific Aim 1, I will determine the mechanisms by which proteins move to apical versus basolateral plasma membranes, using live imaging of fluorescently tagged endogenous proteins to define their relative movements during polarization. I will test the roles of the cytoskeleton in polarity establishment by live imaging embryos after chemically or genetically perturbing the cytoskeleton. A forward genetic screen in a sensitized background lacking microtubules will identify additional parallel pathways involved in polarity establishment. In Specific Aim 2, I will determine the requirement for membrane trafficking in polarity establishment by depleting SEC-23 or SNAP-29 specifically within the intestine. Chemical and RNAi screens will identify additional membrane trafficking proteins involved in polarity establishment, which will be validated with tissue specific depletion. Site directed mutagenesis will determine the sequences required for the targeting of basolateral proteins. In vivo and ex vivo manipulation of intestinal geometry will determ...