The coordination of miRNAs in axon guidance

适当的轴突生长和路径寻找对于建立精确的神经网络是必不可少的 发展。轴突引导缺陷与多种神经疾病有关。 引导受体表达的时空调节促进神经元对 引导线索，以形成准确的神经元连接。在脊椎动物中，引导受体的水平 Robo1在交叉前低，交叉后连合轴突(CA)高，起着 “分子开关”，调节对缝隙排斥的敏感性，引导CA中线交叉。然而， Robo1表达的微调时空调节机制在很大程度上仍然存在 未知。MicroRNAs(MiRNAs)通过与三个启动子特异结合来调节靶基因的表达 靶mRNAs的3‘非翻译区，从而抑制翻译和/或诱导 M RNA降解。我们最近的研究表明，鸡Robo1(CRobo1)3‘端非编码区是必需的 用于调节发育中的脊髓的蛋白质表达，而miR-92抑制cRobo1 表达来调节缝隙敏感性，从而控制CA投射和中线交叉。 有趣的是，另一种高度保守的miRNA miR-219a也抑制cRobo1的表达。异位 MiR-219a在交叉连合后神经元中的表达导致底板CA的停滞。 因此，我们认为miR-92和miR-219a都是Robo1的负调控因子 靶向3‘端非编码区调控Sit/Robo1介导的CA投射在CA中的表达 和中线交叉。为了检验这一假设，我们将首先确定内生性 MIR-219a特异性调节鸡胚胎连合神经元cRobo1的表达 中线交叉时的脊髓(目标1)：我们将(1)确定时间空间表达 MiR-219a和cRobo1在连合神经元中的表达模式，(2)内源性 MIR-219a在鸡脊髓预交叉连合神经元中的表达，以及(3)解开 MiR-219a介导的cRobo1抑制机制。第二，我们将重点关注 研究miR-219a在Sit/Robo1介导的CA生长和转入中的功能重要性 体外和CA投射及体内寻路(目标2)。最后，我们将调查 MIR-92和miR-219a在Sit/Robo1介导的脊髓CA引导中抑制cRobo1表达的作用 (目标3)：我们将研究miR-92和miR-219a在(1)抑制cRobo1方面的合作 通过靶向其3‘非编码区表达，(2)调节cRobo1在CAS中的局部蛋白质合成，(3)Sit/ CRobo1介导的CA引导。这些拟议的实验将提供一种新的模型， MiRNAs协同抑制Robo1的表达，从而调节狭缝对 胚胎脊髓发育过程中Control Sit/Robo1介导的CA引导。

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.