An investigation of guidance mechanisms regulating neuronal migration

描述（由申请人提供）：该项目的重点是了解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性和接触非依赖性信号进行迁移以到达其适当的目的地;异常的神经元迁移是各种出生缺陷的基础。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，允许神经元参与影响迁移的细胞相互作用以及调节对神经投射的生长和引导重要的信号传导机制。初步实验表明，N-钙粘蛋白（Ncad），一个突出的细胞粘附分子在神经系统中，是所需的面鳃神经元（FBMN）迁移。本提案中概述的实验旨在测试总体假设，即通过调节粘附依赖性相互作用和对后脑环境中分子指导线索的反应，Ncad是FBMN迁移所需的。在目标1中，将使用实时成像对Ncad缺陷胚胎中的神经元迁移行为进行详细分析，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中是否自主或非自主地发挥作用以介导它们的迁移。在初步的实验中，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，这表明吸引中线线索引导FBMN迁移。目的2将检验Ncad调节FBMN对分子导向因子SDF 1a的反应的假设。这将通过使用SDF 1a在野生型和Ncad缺陷型胚胎中的异位定位来完成，以测试SDF 1a作为FBMN的引诱剂线索的能力，并检查Ncad在调节FBMN对SDF 1a的反应中的作用。最后，向神经元提供方向线索的另一种方法是通过跟随预先铺设的轴突束，这依赖于神经元和轴突束之间细胞粘附的形成。目的3将检验以下假设：FBMN和内侧纵束（MLF）之间的粘附依赖性相互作用需要Ncad，以促进FBMN迁移。为了研究面神经元和MLF之间的动态相互作用，我将在野生型和Ncad缺陷型胚胎中利用免疫荧光和延时显微镜。我还将调查的重要性，MLF在面神经元的指导下，通过去除MLF使用手术操作。这些实验将测试Ncad在调节FBMN和MLF之间的粘附依赖性相互作用中的重要性。这项提案的目的将确定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.