An investigation of guidance mechanisms regulating neuronal migration

描述(由申请者提供)：本项目侧重于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对接触依赖和接触非接触信号的反应进行迁移以到达合适的目的地；异常的神经元迁移是各种出生缺陷的基础。神经元的迁移受到细胞黏附分子的影响，黏附分子起着双重作用，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至关重要的信号机制。初步实验表明，N-钙粘附素(Ncad)是神经系统中一种重要的细胞黏附分子，它是面部分支运动神经元(FBMN)迁移所必需的。这项建议中概述的实验旨在通过调节后脑环境中的黏附依赖相互作用和对分子指导线索的反应来检验总体假设，即Ncad是FBMN迁移所必需的。在目标1中，将使用实时成像对Ncad缺陷胚胎的神经元迁移行为进行详细的分析，以探索Ncad如何调节FBMN的迁移和引导。此外，细胞移植将测试Ncad在FBMN中是自主还是非自主地发挥作用，以调节它们的迁移。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，这表明吸引人的中线线索引导FBMN迁移。目的2验证Ncad调节FBMN对分子引导信号SDF1a的反应的假设。这将通过利用SDF1a在野生型和Ncad缺乏的胚胎中的异位定位来测试SDF1a作为吸引信号对FBMN的能力，并研究Ncad在调节FBMN对SDF1a的反应中的作用。最后，另一种向神经元提供定向线索的方法是跟踪预先铺设的轴突束，这依赖于神经元和轴突束之间细胞粘连的形成。目的3将验证Ncad在FBMN和内侧纵束(MLF)之间的黏附依赖性相互作用以促进FBMN迁移的假说。为了检测面部神经元和MLF之间的动态相互作用，我将在野生型和Ncad缺陷的胚胎中使用免疫荧光和时间推移显微镜。我还将通过外科手术移除MLF来研究MLF在面神经元指导中的重要性。这些实验将测试Ncad在调节FBMN和MLF之间依赖黏附的相互作用方面的重要性。总之，这项提议的目的将决定Ncad在神经元迁移和引导中的作用，并增加我们对神经元迁移是如何调控的理解。

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.