VISUALIZING AND UNDERSTANDING THE ESTABLISHMENT OF PLANAR CELL POLARITY IN MAMMALIAN DEVELOPMENT

Abstract

Planar cell polarity (PCP) is a highly conserved, fundamental developmental pathway that regulates the coordination of cellular structures and behaviors along the plane of a tissue. This pathway is controlled by a set of core PCP membrane-associated complexes that undergo a transition from uniform to asymmetric distribution over developmental time. The core PCP complexes consist of three transmembrane proteins: Fz which localizes on one side of the cell, Vangl which opposes Fz localization, and Celsr1 which localizes to both sides of the cell and bridges the opposing Fz-Vangl at cell-cell junctions. Chapter 1 is a comprehensive literature review about the intracellular mechanisms regulating the trafficking of PCP proteins. Much of what we know of PCP is from studies in Drosophila, however, in vertebrates, multiple orthologs of PCP components exist and have expanded functions in key developmental processes, such as neural tube closure and convergent extension. Furthermore, mutations in PCP components have been linked to developmental defects, including in humans. What features of PCP are conserved and diverge between Drosophila and vertebrates? Which components regulate vertebrate PCP? How do PCP proteins function across vertebrate tissues? What are the dynamics of PCP proteins as cells rearrange and divide during vertebrate morphogenesis? New tools are necessary to investigate the cell biology and function of PCP components during vertebrate development. In this thesis, we present five novel mouse models to study core PCP protein localization, dynamics, and function in an in vivo context. Chapter 2 discusses and characterizes endogenously tagged Celsr1-3xGFP, Fz6-3xGFP, and tdTomato-Vangl2 mouse lines. The three fusion proteins show proper function, expression and localization in the embryonic skin and allow for analysis of expression and localization of these proteins across various tissues in the mouse. Live imaging of Fz6-3xGFP in epidermal basal cells reveals that PCP proteins are dynamic during tissue rearrangements and divisions, while global, average polarity is preserved. Furthermore, super-resolution STED microscopy of Fz6-3xGFP and tdTomato-Vangl2 resolves their localization between the two sides of a junction revealing a complex and clustered organization along junctions. In Chapter 3, using CRIPSR-Cas9-targeted Celsr1 and Celsr2 knockout mouse lines, we show that Celsr1 is the primary adhesion GPCR involved in epidermal PCP, where it stably enriches at junctional interfaces to recruit Fz6 and Vangl2. These novel mouse tools significantly advance the PCP field by providing a means to dissect an understudied and key vertebrate developmental pathway at unprecedented spatial and temporal resolution. Our findings further our understanding of PCP functions in embryonic development and provide insights into how it is established and maintained during tissue rearrangement and growth.