PROJECT SUMMARY: The formation of the brain’s intricate neural network begins in embryogenesis. Axon guidance is a critical developmental stage in which precise wiring of the central nervous system is achieved when axonal fibers connect with specific target cells. Errors in axon guidance can result in wiring defects that are associated with neurological disorders including epilepsy. The tips of axons have highly motile structures called growth cones that can sense and respond to extracellular guidance cues to direct axon migration. Growth cones depend on actin-based structures called filopodia to sense their surrounding environment and detect external guidance cues. The formation of filopodia is dependent on the bundling of parallel actin filaments by actin cross-linking proteins including Fascin1. During the guidance response, growth cone filopodia undergo remodeling, stabilizing in the direction of attractive cues and collapsing in response to repulsive cues. I hypothesize that the loss of Fascin1 in developing hippocampal neurons will result in erroneous axon guidance due to an inability to regulate filopodia remodeling. The studies proposed here will utilize molecular, cellular biology, and imaging techniques to study the role and regulation of Fascin1 in axon growth cones using cultured rat hippocampal neurons and an in vivo Drosophila model. In Aim 1.1, the effects of Fascin1 depletion via CRISPR-Cas9 editing on filopodia extension, persistence, and retraction will be studied. Three different axon guidance assays will be utilized to determine if Fascin1 depletion alters the ability of growth cones to undergo the guidance response. Aim 1.2 will study the spatiotemporal dynamics of Fascin1 in growth cones of cultured rat hippocampal neurons undergoing guided migration and the regulation of neuronal Fascin1 by protein kinase C (PKC). Previous work established that phosphorylation of Ser-39 of Fascin1 by PKC abrogates Fascin1’s filament bundling ability and I hypothesize that PKC regulates growth cone filopodia stability during axon guidance via this role. Aim 2 uses a Drosophila- based approach to study the role of Fascin1 in axon guidance in vivo. Drosophila express an single ortholog of mammalian Fascin1 called Singed and my preliminary studies indicate that the presence of Singed is required for proper formation of the mushroom body, a neuronal structure that is dependent on axon guidance to form. The studies proposed here will further the knowledge of cytoskeletal regulation during axon guidance which is of importance due to the association of errors in axon guidance with neurological disorders, including epilepsy.