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

项目摘要/摘要 神经系统的功能依赖于神经回路的正确形成 发展。当轴突顶端的生长锥体使用分子线索时，就会产生回路 引导轴突延伸的环境。Netrin1是一种轴突引导信号，最初被认为是起远程作用的， 从底板(FP)发出的可扩散的化学吸引剂。然而，巴特勒实验室最近的工作和 其他团体表示，这种模式是不正确的。在小鼠脊髓中，Netrin1由FP和Fp共同表达 脑室带(VZ)内的细胞和神经前体细胞(NPC)。在缺少netrin1或其 DCC受体，轴突支配VZ，连合轴突停滞或脱束。这些表型 只有当从NPC而不是FP细胞中去除netrin1时才能观察到，这表明鼻咽癌来源的netrin1 负责引导轴突延伸。我们的研究表明，鼻咽癌来源的netrin1沉积在 脊髓的硬膜表面(边缘)，在那里它起促进连合的粘附性基质的作用 轴突生长。我的目标是定义允许netrin1传输到pial的机制。 脊髓的表面。在视觉系统中，netrin1可以被切割成片段、异构体、具有唯一性 空间和生物特性。这让我相信，netrin1的裂解有助于将其运输到 Pial表面。我的初步数据表明，netrin1亚型存在于脊髓中，而netrin1是 不同的裂解以允许其定位于脊髓的不同区域。此外，我的数据 提示netrin1的转运是由运动蛋白Kif1a介导的。在目标1中，我将跟踪netrin1的路径 使用高分辨率显微镜从VZ到软膜表面，并对netrin1的序列进行分析 异构体，使用质谱学。在目标2中，我将使用siRNA敲除和功能获得研究来 调查是否需要KiF1a来运输netrin1。研究这些机制对1)至关重要 更好地了解神经发育障碍的基础和2)修复和 损坏电路的再生。

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.