MicroRNA regulation of guidance receptors in axonal pathfinding

项目摘要 正确的轴突生长和寻径对于建立精确的神经网络是必不可少的。 神经系统在发育过程中。轴突引导缺陷与多种神经疾病有关 精神错乱。引导受体表达的时空调节促进不同的反应 为了形成准确的神经元连接，神经元将引导线索。在脊椎动物中，引导的水平 受体Robo1在交叉前低，交叉后连合轴突(CA)高，作为一种 “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)通过与靶基因特异性结合来调节靶基因的表达 靶mRNA的3‘非翻译区(3’UTR)，从而抑制翻译和/或诱导mRNA 退化。我们的初步研究表明，鸡肉Robo1(CRobo1)3‘非编码区是调控所必需的 在发育中的鸡脊髓中的蛋白质表达。MIR-92，一种高度保守的miRNA，抑制 CRobo1以miR-92 miRNA反应元件(MRE)依赖的方式表达。MIR-92和cRobo1 在发育中的鸡脊髓中差异表达，并具有互斥表达 图案。MiR-92在交叉连合后神经元中的异位表达导致CAs停滞 地板。因此，我们认为miR-92是一种负性调控Robo1在CA中的表达 将其mRNA靶向3‘非编码区，从而调节狭缝敏感性以控制CA投射和中线 过马路了。为了验证这一假说，我们将首先确定内源性miR-92是否特异性地调节 鸡胚胎脊髓中线交叉时cRobo1在连合神经元中的表达 (目标1)：我们将(1)检测大鼠前交叉连合神经元内源性miR-92的活性。 鸡脊髓，(2)检测miR-92和cRobo1在鸡脊髓中的亚细胞表达 以及(3)解开miR-92介导的抑制MIR-92的机制。 CRobo1。其次，我们将重点研究miR-92在Sit/Robo1介导的功能中的重要性 CA的体外生长和翻转以及体内CA的投射和寻路(目标2)。最后，我们将确定 针对连合神经元中cRobo1的新型miRNAs及其在Sit/Robo1介导中的作用 中线穿越期间的CA指导(目标3)。这些拟议的实验将支持一个模型 特定的miRNAs抑制Robo1的表达，从而调节狭缝敏感性来控制狭缝/Robo1- 胚胎脊髓发育过程中介导的CA引导。

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.