A complete model of the molecular mechanisms governing cell polarization and the migration events during gastrulation and neural tube closure remains incomplete. While it is known that the non-canonical Wnt signaling pathway plays a critical role in the cytoskeletal rearrangements and cellular responses required for these events to occur, vital elements of this pathway remain missing. Of principal concern, is that aberrant Wnt signaling has been linked to birth defects such as spina bifida and implicated in cancer metastasis. Therefore, a more thorough investigation of the molecules involved in this pathway remains an essential goal. Our past work has shown that one essential component of Wnt-mediated cytoskeletal changes during gastrulation is Dishevelled-associated activator of morphogenesis protein (Daam1). Daam1 bridges the gap between Dishevelled (Dvl) and the small GTPase Rho and is required for Wnt-dependent Rho activation, but the biochemical details of this process are still undefined. To identify additional factors that might be involved n this process, we performed a yeast two-hybrid screen using a C-terminal region of Daam1. During this screen we identified SRGAP2, a RhoGAP protein. RhoGAP proteins catalyze the transition of GTP-bound Rho to GDP-bound Rho, inactivating it. Thus SRGAP2 may play a key role in modulating Daam1 mediated Rho activation. Confirmation of SRGAP2 as a genuine Daam1- binding partner was determined by co-immunoprecipitation and GST-pulldown assays. The two proteins also show subcellular co-localization just below the cellular plasma membrane and also along the actin stress fibers. Additionally, in a manner similar to Daam1, overexpression or depletion of SRGAP2, in Xenopus laevis embryos, produces gastrulation defects and neural tube disclosures typical of spina bifida. The continued delineation of the non-canonical Wnt signaling pathway, requires a better understanding of the mechanisms involved in Daam1 mediated Rho activation. We hypothesize that Daam1 and SRGAP2 are key modulators of Wnt-dependent Rho activation and the cytoskeletal rearrangements necessary for vertebrate gastrulation and neural tube closure. This grant application is designed to investigate the role of SRGAP2 in non-canonical Wnt signaling and how it functions to mediate the cytoskeletal rearrangements and cellular responses required for vertebrate gastrulation and neural tube closure. We will approach our investigation using three specific aims. First, we will examine the functional domains of SRGAP2 and Daam1 required for their interaction, and examine if this interaction is Wnt-dependent. Second, we will use Xenopus laevis embryos to explore the role of SRGAP2 during gastrulation and neural tube closure. In the third aim, we will investigate the subcellular localization patterns of SRGAP2 and Daam1, how this pattern is altered by Wnt treatment and how changes in this pattern may affect the actin cytoskeletal. These experiments together will provide a more thorough understanding of how SRGAP2 functions to regulate Wnt-induced Rho activity levels and changes to the cytoskeleton and how this regulation affects the cellular motility events of gastrulation and neural tube closure. Importantly, they will provide additional insights into how defects in this process are manifested in birth defects disorders such as spina bifida and also in cancer metastasis.
Understanding the development of human cancers and birth defect disorders remains dependent on identifying key signaling molecules and their signal transduction pathways that contribute to this pathology. One key-signaling molecule that has been demonstrated to play causative roles in human cancer and birth defect disorders is the Wnt protein. Our studies are focused on understanding the role of a new protein called SRGAP2 that transduces one aspect of Wnt signaling termed non-canonical Wnt signaling. We propose that these studies can provide important new insights into the mechanisms regulating embryogenesis as well as gaining insights into how deregulated Wnt signaling contributes to cancer formation and birth defect disorders including spina bifida.
