Project Summary In the developing nervous system, proper axon outgrowth and pathfinding are regulated by multiple guidance molecules, their receptors and intracellular signaling pathways. 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), a pattern 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 gene expression by binding specifically to the 3’untranslated region (3’UTR) of target mRNAs, thus repressing translation and/or inducing mRNA degradation. Our preliminary studies indicate that the Robo1 3’UTR is required for regulation of protein expression in the developing spinal cord. Gga-miR-92, a highly conserved miRNA, is differentially expressed in the developing chicken spinal cord, and regulates Slit sensitivity via suppression of cRobo1 expression in commissural neurons, thereby controlling CA projection and midline crossing. The mature gga-miR-92 has the same sequence as mmu-miR-92a and mmu-miR-92b except the tenth base at the 5’ end of mmu-miR-92b. Mmu-miR-92b can also repress mRobo1 expression by targeting its 3’UTR. Therefore, we hypothesize that mmu-miR-92a and mmu-miR-92b, like gga-miR-92, specifically regulate mRobo1 expression in commissural neurons to control Slit/Robo1-mediated CA guidance in the developing mouse spinal cord. We will examine the role of mmu-miR-92a and mmu-miR- 92b in (1) suppressing mRobo1 expression in developing commissural neurons (Aim 1) and (2) Slit/mRobo1-mediated spinal CA guidance (Aim 2). Studies in this proposal will advance our understanding of molecular mechanisms underlying Slit/Robo-mediated axon guidance.