Investigating netrin1-mediated commissural axon guidance in the developing spinal cord

项目总结/摘要 神经系统的功能取决于神经回路的正确形成， 发展当轴突顶端的生长锥使用细胞中的分子线索时， 环境来引导轴突延伸。Netrin 1是一种轴突引导线索，最初被认为是一种长距离的， 从底板（FP）散发的可扩散的化学引诱物。然而，巴特勒实验室最近的工作， 其他团体认为这种模式是不正确的。在小鼠脊髓中，netrin 1由FP 细胞和神经祖细胞（NPC）在心室区（VZ）。在没有netrin 1或其 受体Dcc，轴突支配VZ和连合轴突停止或解束。这些表型 只有当netrin 1从NPC中去除而不是FP细胞中去除时才能观察到，这表明NPC来源的netrin 1 负责引导轴突延伸。我们的研究表明，NPC衍生的netrin 1沉积在 脊髓的软脑膜表面（边缘），在那里它充当促进连合的粘合基质 轴突生长我的目标是确定netrin 1被传送到软膜的机制 脊髓的表面。在视觉系统中，netrin 1可以被切割成片段，同种型，具有独特的 空间和生物特性。这使我相信netrin 1的分裂有助于其运输到 软膜面我的初步数据表明netrin 1亚型存在于脊髓中，并且netrin 1是 差异切割以允许其定位于脊髓的不同区域。此外，我的数据 表明netrin 1转运是由马达蛋白Kif 1a介导的。在目标1中，我将跟踪netrin 1的路径 使用高分辨率显微镜从VZ到软膜表面，并表征netrin 1序列 异构体，使用质谱法。在目标2中，我将使用SiRNA敲低和功能获得研究， 研究KiF 1a是否是netrin 1运输所必需的。研究这些机制对1） 更好地了解神经发育障碍的基础; 2）修复和 修复受损电路。

Project Summary/Abstract The function of the nervous system is dependent on the correct formation of neural circuits during development. Circuits are generated when growth cones at the tips of axons use molecular cues in the environment to guide axon extension. Netrin1 is an axon guidance cue first thought to acts as a long-range, diffusible, chemoattractant that emanates from the floor plate (FP). However, recent work by the Butler lab and other groups, has suggested this model is incorrect. In the mouse spinal cord, netrin1 is expressed by both FP cells and neural progenitor cells (NPCs) in the ventricular zone (VZ). In the absence of either netrin1 or its receptor Dcc, axons innervate the VZ and commissural axons either stall or defasciculate. These phenotypes are only observed when netrin1 is removed from NPCs and not the FP cells, suggesting NPC-derived netrin1 is responsible for guiding axon extension. Our studies suggest that NPC-derived netrin1 is deposited on the pial surface (margin) of the spinal cord, where it acts as an adhesive substrate that promotes commissural axon outgrowth. My objective is to define the mechanisms that allow netrin1 to be transported to the pial surface of the spinal cord. In the visual system, netrin1 can be cleaved into fragments, isoforms, with unique spatial and biological properties. This led me to believe that cleavage of netrin1 facilitates its transport to the pial surface. My preliminary data suggests that netrin1 isoforms exist in the spinal cord, and that netrin1 is differentially cleaved to permit its localization to different regions of the spinal cord. Furthermore, my data suggests that netrin1 transport is mediated by the motor protein Kif1a. In Aim 1, I will track the path of netrin1 from the VZ onto the pial surface using high resolution microscopy and characterize the sequence of netrin1 isoforms, using mass spectrometry. In Aim 2, I will use SiRNA knockdown and gain-of-function studies to investigate if KiF1a is necessary for the transport of netrin1. Investigating these mechanisms is critical to 1) gaining a better understanding of the basis of neurodevelopmental disorders and 2) the repair and regeneration of damaged circuits.