Multiscale coordination of planar cell polarity

PROJECT SUMMARY Planar cell polarity (PCP) is an ancient and near universal property of epithelial tissues in which subcellular protrusions and membrane-associated proteins asymmetrically localize and align within a tissue plane. PCP enables polarized cell behaviors such as directed cilia beating and collective cell motility, and mutations in PCP pathway components cause severe developmental malformations in mammals and structural birth defects in humans. Despite its ubiquity across species and its importance in embryonic development, basic principles of PCP establishment remain unknown, including how symmetry is initially broken, whether cell contact is required for asymmetry, and what is the minimal number of cells required to establish PCP. Our lab has developed the mouse epidermis as a powerful model system with which to decipher fundamental and conserved principles of PCP as well as mammalian innovations in the pathway. We have developed methods to perform long-term live imaging, FRAP, and super-resolution microscopy using endogenously-tagged PCP reporters. Together with strategies to generate mosaics and chimeras, the mouse skin is now on par with Drosophila in terms of genetic tools and techniques to study PCP, but in a mammalian system. Using these tools, our lab has directly addressed the question of how many cells are required to establish PCP. Inherent in this question is whether PCP proteins can partition cell autonomously, or if intercellular binding of PCP components between cells was necessary to initiate the process. Using chimeric mouse embryos in which dual-PCP reporter cells have been genetically uncoupled from their neighbors, we found that in the absence of intercellular interactions, PCP proteins do not polarize cell autonomously. Further, we demonstrated that intercellular binding of Celsr1 between just two cells is both necessary and sufficient to initiate the sorting of PCP complexes into asymmetric bridges, and formation of two Celsr1-enriched edges is sufficient to generate cellular-level asymmetry. Thus, intercellular binding of PCP complexes is the critical step that initiates sorting of opposing PCP complexes to generate asymmetry. Building on this discovery, this proposal seeks to define the molecular events downstream of Celsr1 homotypic binding that initiate the sorting of PCP complexes into asymmetric localizations (Aim 1), define the roles of cytosolic components Dvl and Pk and their phase separating behaviors in the process (Aim 2), and elucidate the molecular basis of Celsr1 asymmetric bridges (Aim 3). Combining state-of-the art genome engineering approaches, advanced live and super-resolution imaging combined with classic embryological approaches to generate embryos consisting of genetically distinct cells, successful completion of these aims with define the molecular mechanisms that break symmetry and amplify polarity during PCP establishment.