The coordination of miRNAs in axon guidance

适当的轴突生长和寻路对于建立精确的神经网络至关重要， 发展轴突导向缺陷与多种神经系统疾病有关。 引导受体表达的时空调节促进神经元对 以形成准确的神经元布线。在脊椎动物中， Robo 1在交叉前低而在交叉后高的连合轴突（CA）中，作为一个神经元， “分子开关”调节对Slit排斥的敏感性并引导CA中线交叉。但 Robo 1表达的微调时空调控机制在很大程度上仍然存在 未知microRNAs（miRNAs）通过特异性结合三个引物来调节靶基因的表达。 靶mRNA的非翻译区（3 'UTR），从而抑制翻译和/或诱导 mRNA降解。我们最近的研究表明，鸡Robo 1（cRobo 1）3 'UTR是必需的， 调节发育中脊髓的蛋白表达，miR-92抑制cRobo 1 表达以调节Slit敏感性，从而控制CA投射和中线穿越。 有趣的是，另一种高度保守的miRNA miR-219 a也抑制cRobo 1的表达。异位 miR-219 a在交叉后连合神经元中的表达导致底板中CA的停滞。 因此，我们认为miR-92和miR-219 a都是Robo 1的负调控因子， 通过在3 'UTR处靶向其mRNA以控制Slit/Robo 1介导的CA投射来在CA中表达 和中线交叉。为了检验这一假设，我们将首先确定是否内源性 miR-219 a特异性调控鸡胚连合神经元cRobo 1的表达 脊髓中线穿越（目标1）：我们将（1）确定时空表达 miR-219 a和cRobo 1在连合神经元中的模式，（2）检查内源性miR-219 a和cRobo 1的活性。 miR-219 a在鸡脊髓交叉前连合神经元中的表达，以及（3）解开 miR-219 a介导的cRobo 1抑制的潜在机制。第二，我们会专注于 研究miR-219 a在Slit/Robo 1介导的CA生长和转化中的功能重要性 体外和CA投射和体内寻路（Aim 2）。最后，我们将探讨合作的 miR-92和miR-219 a在Slit/Robo 1介导的脊髓CA引导中抑制cRobo 1表达 (Aim 3）：我们将研究miR-92和miR-219 a在（1）抑制cRobo 1 通过靶向其3 'UTR表达，（2）调节CA中cRobo 1局部蛋白质合成，（3）Slit/ cRobo 1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Robo 1表达，从而调节Slit敏感性， 胚胎脊髓发育过程中Slit/Robo 1介导的CA指导。

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.