An investigation of guidance mechanisms regulating neuronal migration

描述（由申请人提供）：本项目主要研究神经元迁移的分子和细胞基础。许多类型的神经元通过响应接触依赖和接触无关的信号来迁移到适当的目的地；异常的神经元迁移是多种出生缺陷的基础。神经元迁移受细胞粘附分子的影响，细胞粘附分子发挥双重作用，使神经元参与影响迁移的细胞相互作用，并调节对神经投射的生长和指导重要的信号机制。初步实验表明，n -钙粘蛋白（Ncad）是神经系统中一种重要的细胞粘附分子，是面部branchiommotor neuron （FBMN）迁移所必需的。本提案中概述的实验旨在通过调节粘附依赖性相互作用和后脑环境中对分子引导信号的反应来验证Ncad是FBMN迁移所必需的这一总体假设。在Aim 1中，将使用实时成像技术详细分析Ncad缺陷胚胎的神经元迁移行为，以探索Ncad如何调节FBMN迁移和引导。此外，细胞移植将测试Ncad在fbmn中是自主还是非自主地调节其迁移。在初步实验中，我发现Ncad耗竭阻止了FBMN的迁移，一些神经元异常地迁移到中线，这表明有一个吸引物中线提示引导FBMN迁移。目的2将验证Ncad调节FBMN对分子引导提示SDF1a的反应的假设。这将通过在野生型和缺乏Ncad的胚胎中使用SDF1a的异位定位来测试SDF1a作为fbmn引诱线索的能力，并检查Ncad在调节fbmn对SDF1a的反应中的作用来实现。最后，另一种向神经元提供定向线索的方法是通过预先铺设的轴突束，这依赖于神经元和轴突束之间形成的细胞粘连。目的3将验证Ncad是FBMN和内侧纵束膜（MLF）之间粘附依赖性相互作用促进FBMN迁移所必需的假设。为了研究面部神经元和MLF之间的动态相互作用，我将在野生型和ncad缺乏的胚胎中使用免疫荧光和延时显微镜。我还将通过手术操作去除MLF来研究MLF在面部神经元引导中的重要性。这些实验将测试Ncad在调节FBMNs和MLF之间粘附依赖性相互作用中的重要性。总之，本提案的目的将确定Ncad在神经元迁移和指导中的作用，并增加我们对神经元迁移如何调节的理解。

DESCRIPTION (provided by applicant): This project is focused on understanding the molecular and cellular basis of neuronal migration. Many types of neurons undergo migration to reach their appropriate destination by responding to both contact-dependent and contact-independent signals; aberrant neuronal migration underlies a variety of birth defects. Neuronal migration is influenced by cell adhesion molecules, which play a dual role allowing neurons to engage in cellular interactions that influence migration as well as regulating signaling mechanisms important for the outgrowth and guidance of neural projections. Preliminary experiments have shown that N-cadherin (Ncad), a prominent cell adhesion molecule in the nervous system, is required for facial branchiomotor neuron (FBMN) migration. The experiments outlined in this proposal aim to test the overarching hypothesis that Ncad is required for FBMN migration by modulating both adhesion-dependent interactions and the response to molecular guidance cues within the hindbrain environment. In Aim 1 a detailed analysis of neuronal migratory behavior in Ncad-deficient embryos will be performed using live imaging to explore how Ncad regulates FBMN migration and guidance. Further, cell transplantation will test whether Ncad functions autonomously or non-autonomously in FBMNs to mediate their migration. In preliminary experiments I find Ncad- depletion arrests FBMN migration and some neurons aberrantly migrate into the midline, suggesting an attractant midline cue guides FBMN migration. Aim 2 will test the hypothesis that Ncad modulates FBMN response to the molecular guidance cue SDF1a. This will be accomplished by using ectopic localization of SDF1a in wild type and Ncad-deficient embryos to test the ability of SDF1a to act as an attractant cue to FBMNs and to examine the role of Ncad in modulating the response of FBMNs to SDF1a. Lastly, another means of providing directional cues to neurons is through following pre-laid axon tracts, which relies upon the formation of cellular adhesions between neurons and the axon tract. Aim 3 will test the hypothesis that Ncad is required for adhesion-dependent interactions between the FBMNs and the medial longitudinal fascicle (MLF) to facilitate FBMN migration. To examine the dynamic interaction between facial neurons and the MLF I will utilize immuno- fluorescence and time-lapse microscopy in wild type and Ncad-deficient embryos. I will also investigate the importance of the MLF in facial neuron guidance by removing the MLF using surgical manipulations. These experiments will test the importance of Ncad in regulating adhesion-dependent interactions between FBMNs and the MLF. Together the aims of this proposal will determine the role of Ncad in neuronal migration and guidance and add to our understanding of how neuronal migration is regulated. PUBLIC HEALTH RELEVANCE: Neuronal migration is critical for the ability of neurons to navigate their environment to reach their proper target. Understanding how neurons migrate is important because defects in neuronal migration result in human neurological diseases, most commonly encompassing mental retardation, epilepsy and autism. The proposed experiments will extend our current knowledge of the molecular and cellular basis of neuronal migration.