Non-canonical Wnt signaling and cell motility

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 DESCRIPTION (provided by applicant): The molecular mechanism(s) by which a cell becomes polarized for directional migration remains poorly understood. The non-canonical Wnt pathway has recently been shown to play important roles in cell polarization and migration, which are required for gastrulation and neural fold closure during vertebrate embryogenesis. Importantly defects in non-canonical Wnt signaling are implicated human birth defects disorders including in neural fold closure disorders such as spina bifida and in cancer metastasis. To date, how the non-canonical Wnt signaling pathway regulates changes to the actin cytoskeleton remains at best poorly defined. Our studies from the prior funding period have established that the Formin protein Dishevelled- associated activator of morphogenesis (Daam1) provides a crucial link between Dishevelled (Dvl) and the small GTPase Rho for gastrulation. Importantly, our studies demonstrate Daam1 is required for Wnt-dependent cytoskeletal changes, although the underlying biochemical details are not known. To further identify factors required downstream of Daam1 for cytoskeletal changes, we performed a yeast two-hybrid screen and isolated two new proteins in addition to others we have published. Co-immunoprecipitation and GST-pulldown assays confirm that these proteins are bona-fide Daam1- interacting factors and their interaction and subcellular co-localization with Daam1 in mammalian cells is Wnt-regulated. Furthermore, over-expression or depletion of these proteins, similar to Daam1, blocks gastrulation in the Xenopus embryo and results in an open neural tube phenotype characteristic of spina bifida. We have further cloned and begun characterization of the second vertebrate Daam family member Daam2. Our preliminary studies show that similar to Daam1, Daam2 plays a functional role in non-canonical Wnt signaling but Daam2 in contrast to Daam1 regulates vertebrate neural tube closure. In building a model for how non-canonical Wnt signaling through the Daam proteins regulate cytoskeletal changes, we hypothesize Daam1 and Daam2 are key modulators of the actin cytoskeleton for cellular motility during gastrulation. In this competitive renewal proposal, we will investigate how the two new identified proteins that bind to Daam1 functions to mediate cytoskeletal changes for cell motility during gastrulation using mammalian cells, Xenopus and zebrafish embryos. Second, we will characterize the role of Daam2 as a regulator of non-canonical Wnt signaling during vertebrate gastrulation. We will further delineate whether Daam2 and Daam1 have non-redundant functions during gastrulation and well as defining effector proteins common and specific to Daam1 and/or Daam2. These studies together will significantly advance our understanding of how non-canonical Wnt signaling through the Daam family of Formin proteins regulate cell polarity and cell motility required during vertebrate gastrulation and neural fold closure. These studies will further provide additional insights into how defects in Wnt signaling contribute to birth defects such as spina bifida and cancer metastasis.