MOLECULAR MECHANISMS OF GRANULE CELL MIGRATION

这项研究的总体目标是了解分子 神经胶质细胞引导的神经元迁移机制及其遗传调控 在发育中的哺乳动物大脑中。分析其分子机制 关于迁徙，拟议的研究将使用体外模型系统， 我们实验室开发的，用于确定细胞黏附配体的作用 在小脑颗粒神经元沿神经胶质细胞的运动中 导游。我们将分析抗神经胶质细胞抗体的作用。 与Astroactin的配基结合，并将其与抗体的作用效果进行比较 神经元-神经元配体L1、N-CAM、N-钙粘蛋白和TAG-1，以及 纤维连接蛋白和层粘连蛋白的受体，整合素。迁移的细胞将是 视频增强差分干涉衬度显微镜成像 在微培养中检查频率、移动率或细胞学 迁移的颗粒神经元。在同伴研究中，我们将发展 染料标记颗粒迁移动力学的成像方法 小脑组织切片中的神经元，并评估其对 抗细胞黏附配体抗体的原位迁移。 提供有关预测的蛋白质结构的信息 Astroactin，包括它的整体结构，与 膜和与其他细胞黏附配体的同源性，亲和力- 纯化的抗Astroactin抗体将用于克隆 Astroactin活性的100KD蛋白质组分。我们将使用我们的 生物测试证明推测的astroactin克隆编码 Astroactin活性的功能表位。Astrotactin克隆 将在分子水平上进行分析，并对Astroactin的功能进行分析 PC12神经元中已识别克隆的表达将对cDNA进行XAME分析 和编织颗粒细胞。 为了分析神经元迁移的遗传调控，我们将 继续我们对神经性突变小鼠的研究，这些小鼠在 神经胶质引导的迁徙、编织和蜿蜒的尾巴。曲折的尾巴是一种 令人兴奋的新突变，细胞组织严重中断 仅限于小脑前叶。离体 重组实验将被用来确定细胞的位置 曲折尾巴基因的作用。颗粒细胞迁移的动力学研究 在曲折的尾巴小脑的组织切片中进行比较 来自Weaver的结果。分析细胞间的相互作用是否与 神经元或轴突为神经胶质引导的神经元提供“停止信号” 迁移，我们将检查小脑颗粒神经元是否终止 在体内和体外遇到神经元或轴突通道时的迁移 模型系统。

The overall goal of this research is to understand the molecular mechanism of glial-guided neuronal migration and its genetic regulation in the developing mammalian brain. To analyze the molecular mechanism of migration, the proposed research will use an in vitro model system, developed in our laboratory, to define the role of cell adhesion ligands in the locomotion of the cerebellar granule neuron along the glial guide. We will assay the effects of antibodies gainst the neuron-glia ligand astrotactin, and compare these with effects of antibodies against the neuron-neuron ligands L1, N-CAM, N-cadherin and TAG-1, and of the receptor for fibronectin and laminin, integrin. Migrating cells will be imaged with video-enhanced differential interference contrast microscopy in microcultures to examine the frequency, rate of movement or cytology of migrating granule neurons. In companion studies, we will develop methods to image the dynamics of migration of dye-labeled granule neurons in tissue slices of cerebellum, and assess the effects of antibodies against cell adhesion ligands on migration in situ. To provide information on the predicted protein structure of astrotactin, including its overall structure, interaction with the membrane and homology with other cell adhesion ligands, affinity- purified anti-astrotactin antibodies will be used to clone cDNAs for the 100KD protein component of the astrotactin activity. We will use our biological assays to demonstrate that putative astrotactin clones encode functional epitopes of the astrotactin activity. Astrotactin clones will be analyzed at the molecular level and the function of astrotactin cDNAs will be xamined by expression of identified clones in PC12 neurons and weaver granule cells. To analyze the genetic regulation of neuronal migration, we will continue our studies on neurological mutant mice with defects in glial-guided migration, weaver and meander tail. Meander tail is an exciting new mutation with a severe disruption of cellular organization restricted restricted to the anterior lobe of the cerebellum. In vitro recombination experiments will be used to define the cellular site of action of the meander tail gene. The dynamics of granule cell migration in tissue slices of meander tail cerebellum will then be compared with results from weaver. To analyze whether cell-cell interactions with neurons or axons provide the "stop signals" for glial-guided neuronal migration, we will examine whether cerebellar granule neurons terminate migration upon encountering lanes of neurons or axons in and in vitro model system.