Project Summary
Proper axon outgrowth and pathfinding is essential for establishing precise neural networks of the
nervous system 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
3’untranslated region (3’UTR) of target mRNAs, thus repressing translation and/or inducing mRNA
degradation. Our preliminary studies indicate that chicken Robo1 (cRobo1) 3’UTR is required for regulation
of protein expression in developing chicken spinal cords. miR-92, a highly conserved miRNA, suppresses
cRobo1 expression in a miR-92 miRNA response element (MRE)-dependent manner. miR-92 and cRobo1
are differentially expressed in the developing chicken spinal cord with a mutually exclusive expression
pattern. Ectopic expression of miR-92 in postcrossing commissural neurons results in CAs stalling in the
floor plate. Therefore, we propose that miR-92 is a negative regulator of Robo1 expression in CAs by
targeting its mRNA at the 3’UTR, thereby regulating Slit sensitivity to control CA projection and midline
crossing. To test this hypothesis, we will first determine whether endogenous miR-92 specifically regulates
cRobo1 expression in commissural neurons of embryonic chicken spinal cords during midline crossing
(Aim 1): we will (1) examine the activity of endogenous miR-92 in precrossing commissural neurons of
chicken spinal cords, (2) determine the subcellular expression patterns of miR-92 and cRobo1 in
commissural neurons, and (3) untangle the mechanisms underlying miR-92-mediated repression of
cRobo1. Secondly, we will focus on studying the functional importance of miR-92 in Slit/Robo1-mediated
CA outgrowth and turning in vitro and CA projection and pathfinding in vivo (Aim 2). Finally, we will identify
novel miRNAs targeting to cRobo1 in commissural neurons and examine their roles in Slit/Robo1-mediated
CA guidance during midline crossing (Aim 3). These proposed experiments will support a model that
specific miRNAs suppress Robo1 expression, thereby modulating Slit sensitivity to control Slit/Robo1-
mediated CA guidance during embryonic spinal cord development.