SIGNALING PATHWAYS IN SKIN PATTERNING AND POLARITY PROJECT SUMMARY A major challenge in developmental biology is to understand how single cells are coordinated with one another to establish their orientations with respect to tissue and body axes. The coordination of neighboring cells in the plane of tissue is called tissue polarity or planar cell polarity (PCP). PCP is involved in various developmental processes, and defects in PCP cause many developmental disorders in humans. Increasing evidence also suggests that PCP is involved in certain cancers. The long-term goal of our study is to uncover fundamental mechanisms of mammalian PCP in normal development and disease. In this proposal, we address several unanswered central questions in the PCP field by elucidating mechanisms of Frizzled6 (Fz6), a key membrane PCP protein that controls the polarity of mouse skin. In preliminary studies, we discovered: (1) overexpression of Wnt5a, a member of the Wnt family of ligands for Fz receptors, disrupts hair follicle orientations; (2) knockout of Fz6 results in significant transcriptional changes in the skin; (3) Astrotactin-2 (Astn2), a recently identified genetic modifier of Fz6, binds to Fz3 and Fz6 in vitro. Based on available literature and our preliminary data, we hypothesize that Astn2 is a cofactor for Wnt5a-Fz6 signaling in skin PCP and Fz6 regulates a previously undefined transcriptional network to establish skin polarity. We will test our hypothesis with the following three aims: (1) to determine if Wnt ligands, especially Wnt5a, function as orienting cues in Fz6-mediated skin PCP; (2) to elucidate transcriptional mechanisms governing Fz6-mediated skin PCP; and (3) to elucidate mechanisms of Astn2 in modifying the skin polarity defect in Fz6 knockout mice. The proposed research is innovative. We will combine unique mouse models that we have generated and a novel in vitro PCP system that we have developed to elucidate the mechanisms of Fz6 in skin PCP. The proposed research is significant. The aims will unveil mechanistic insights into several new components in the Fz6-mediated PCP pathway, including upstream ligands, cofactors, and downstream effectors. Since PCP is a fundamental, conserved pathway that regulates a wide range of developmental processes, these data will also improve our understanding of the pathogenesis of PCP-related diseases, such as open neural tube, cleft palate, cystic kidney disease, hearing loss, ciliopathies, and heart defects.