The brain relies for its function on a precise and complex pattern of axonal connections. The broad long-term goal of this project is to understand how this pattern of axon connections is set up during development. When such connections fail to form properly, or are subsequently lost, this can lead to a broad range of neurodevelopmental, psychiatric and neurodegenerative disorders. This proposal focuses particularly on RNA-based regulatory mechanisms. A key advantage of regulating gene expression at the mRNA level is that protein expression can be directed to specific subcellular regions with temporal and spatial specificity – an important advantage in neurons, which have a high degree of spatial organization. Accordingly, RNA-based regulation plays key roles in axon guidance, neuron migration and synapse plasticity, although the specific mechanisms remain poorly understood. Here, RNA-based mechanisms will be studied in regulation of the microtubule cytoskeleton (Aim 1), and in axon pathway selection at a complex choice point (Aim 2). Aim 1 focuses on the microtubule cytoskeleton, which has crucial roles in neuron structure and motility. Our recent work has now identified a mechanism for RNA-based regulation of microtubules. Specifically, microtubule plus-end protein APC binds tubulin Tubb2b mRNA, at a site required for Tubb2b translation in axons, formation of dynamic microtubules in the growth cone, and neuron migration in vivo. This opens up a new field of investigation into RNA-based regulation of the microtubule cytoskeleton. One goal will be to investigate coordinated regulation of specific tubulin mRNAs which have APC binding sites in their 3'UTR and cause most human tubulinopathies. Another objective will use time-lapse imaging to understand specifically how RNA-based regulation controls microtubule dynamics, including fundamental new models for both microtubule initiation at the minus end, and assembly at the plus- end. In addition to axons, these mechanisms will be characterized in formation of synaptic spines. Aim 2 will continue studies of commissural axon guidance at the spinal cord midline, a well-characterized model of developmental axon pathfinding. RNA-based regulation is known to occur within commissural axons, including upregulated translation of mRNAs in distal axon segments that have crossed the midline intermediate target. However, little has been known of the mechanisms, including the RNA-binding proteins involved, their downstream mRNA targets, or upstream regulatory pathways. This Aim will characterize specific RNA-binding proteins that display highly selective expression on axon segments, strong and distinct phenotypes in midline guidance, and interactions with mRNAs regulated in axons at the midline; as well as upstream ligands and receptors that interact physically and functionally with these RNA based regulatory mechanisms. These studies will provide novel information on fundamental mechanisms of axon development, and...