The Coordination of miRNAs in Axon Guidance - Proper axon outgrowth and pathfinding is essential for establishing precise neural networks during development. Defects in axon guidance are implicated in a variety of neurological disorders. Spatiotemporal regulation of guidance receptor expression facilitates differential responses of neurons to guidance cues in order to form accurate neuronal wiring. In vertebrates, levels of the guidance receptor Robo1 are low in precrossing and high in postcrossing commissural axons (CAs), functioning as a ‘molecular switch’ to regulate sensitivity to Slit repulsion and guide CA midline crossing. However, the mechanism underlying the fine-tuned spatiotemporal regulation of Robo1 expression remains largely unknown. MicroRNAs (miRNAs) regulate target gene expression by binding specifically to the three prime untranslated region (3’UTR) of target mRNAs, thus repressing translation and/or inducing mRNA degradation. Our recent studies indicate that the chicken Robo1 (cRobo1) 3’UTR is required for regulation of protein expression in the developing spinal cord, and miR-92 represses cRobo1 expression to regulate Slit sensitivity thereby controlling CA projection and midline crossing. Interestingly, miR-219a, another highly conserved miRNA, also suppresses cRobo1 expression. Ectopic expression of miR-219a in postcrossing commissural neurons results in stalling of CAs in the floor plate. Therefore, we propose that both miR-92 and miR-219a function as negative regulators of Robo1 expression in CAs by targeting its mRNA at the 3’UTR to control Slit/Robo1-mediated CA projection and midline crossing. To test this hypothesis, we will first determine whether endogenous miR-219a specifically regulates cRobo1 expression in commissural neurons of embryonic chicken spinal cords during midline crossing (Aim 1): we will (1) determine the temporospatial expression patterns of miR-219a and cRobo1 in commissural neurons, (2) examine the activity of endogenous miR-219a in precrossing commissural neurons of chicken spinal cords, and (3) untangle the mechanisms underlying miR-219a-mediated repression of cRobo1. Secondly, we will focus on studying the functional importance of miR-219a in Slit/Robo1-mediated CA outgrowth and turning in vitro and CA projection and pathfinding in vivo (Aim 2). Finally, we will investigate the collaboration of miR-92 and miR-219a in repression of cRobo1 expression in Slit/Robo1-mediated spinal CA guidance (Aim 3): we will examine the collaboration of miR-92 and miR-219a in (1) suppression of cRobo1 expression by targeting its 3’UTR, (2) regulation of cRobo1 local protein synthesis in CAs, (3) Slit/ cRobo1-mediated CA guidance. These proposed experiments will provide a novel model that specific miRNAs collaboratively suppress Robo1 expression, thereby modulating Slit sensitivity to control Slit/Robo1-mediated CA guidance during embryonic spinal cord development.
