Characterizing the role of migration, proliferation, and contact-mediated repulsion in oligodendrocyte progenitor cell tiling

项目摘要 中枢神经系统(CNS)中轴突的髓鞘形成是电活动在大脑中传播所必需的 神经元与轴突健康的维持。少突胶质细胞前体细胞(OPC)引起髓鞘形成 中枢神经系统的细胞，称为少突胶质细胞。虽然大多数OPC分化为少突胶质细胞，但 OPC的种群在整个生命周期中仍然没有分化，并且均匀地分布在中枢神经系统。OPC如何占用空间 在发育和保持他们的间隔到成年期是未知的。成年小鼠大脑皮质的活体成像 斑马鱼幼虫的脊髓表明，OPC经历了一个称为瓷砖的过程。我们给瓷砖下了定义 作为OPC在开发期间经历的动态过程，其中它们建立并保持均匀的间隔 以及在非病理条件下不重叠的不同领域。在瓷砖铺设期间，OPC还展示 接触介导的排斥(CMR)。CMR是一个迁移OPC将收回其流程并 在与其他OPC接触后改变他们的迁徙方向。然而，分子机制是 OPC用于促进平铺和CMR尚不清楚。此外，脱髓鞘疾病导致OPC增加 集群，这意味着本地平铺机制的中断。理解基本机制 对OPC-OPC相互作用的规范将提供对OPC瓷砖和OPC瓷砖发展过程的洞察 调节OPC向脱髓鞘病变迁移和募集的潜在靶点。这样做的目的是 提案旨在阐明促进OPC在开发过程中快速平铺的机制，方法是 研究贴片过程中的三个重要过程：(1)OPC增殖，(2)OPC迁移，(3) OPC CMR。为了研究开发过程中的OPC平铺，我将使用斑马鱼(Danio Rerio)作为脊椎动物模型 系统。这个系统的相对简单和斑马鱼胚胎和幼虫脊髓的清晰度允许 用于可视化OPC在大部分发展中的中枢神经系统中的迁移、增殖和CMR。我将使用 基于以往文献的候选基因方法，以确定潜在的增殖和CMR的介导物。 此外，我将调查从无偏见的小分子筛查中确定的候选对象，以确定小说 OPC在发育过程中迁移的中介物。然后我将使用CRISPR/Cas9基因的组合 编辑技术、药理抑制剂、活体成像和免疫组织化学来表征 OPC的增殖、迁移和CMR在发育性OPC瓦片中的作用。总体而言，这项工作将扩大我们的 了解OPC瓷砖和相互作用，并有可能为脱髓鞘提供翻译目标 疾病，如多发性硬化症。

Project Summary Myelination of axons in the central nervous system (CNS) is required for the propagation of electrical activity in neurons and maintenance of axonal health. Oligodendrocyte progenitor cells (OPCs) give rise to the myelinating cells of the CNS, known as oligodendrocytes. While the majority of OPCs differentiate into oligodendrocytes, a population of OPCs remains undifferentiated and evenly distributed in the CNS throughout life. How OPCs space during development and maintain their spacing into adulthood is unknown. In vivo imaging in adult mouse cortex and the spinal cord of zebrafish larvae demonstrate that OPCs undergo a process termed tiling. We define tiling as the dynamic process that OPCs undergo during development where they establish and maintain even spacing and distinct territories that, under non-pathological conditions, do not overlap. During tiling, OPCs also exhibit contact-mediated repulsion (CMR). CMR is a process whereby migrating OPCs will retract their processes and change their migratory direction following contact with other OPCs. However, the molecular mechanisms that OPCs use to facilitate tiling and CMR are unknown. Additionally, demyelinating diseases cause increased OPC clustering, which signifies a disruption in local tiling mechanisms. Understanding the fundamental mechanisms of that regulate OPC-OPC interactions will provide insight into the developmental processes of OPC tiling and potential targets for modulating OPC migration and recruitment to demyelinated lesions. The purpose of this proposal is to elucidate the mechanisms that facilitate the rapid tiling of OPCs during development by investigating three important processes during tiling: (1) OPC proliferation, (2) OPC migration, and (3) OPC CMR. To investigate OPC tiling during development, I will use zebrafish (Danio rerio) as a vertebrate model system. The relative simplicity of this system and clarity of the spinal cord of zebrafish embryos and larvae allow for visualization of OPC migration, proliferation, and CMR in a large portion of the developing CNS. I will use a candidate gene approach based on previous literature to identify potential mediators of proliferation and CMR. Additionally, I will investigate candidates identified from an unbiased small molecule screen to identify novel mediators of OPC migration during developmental tiling. I will then use a combination of CRISPR/Cas9 gene editing techniques, pharmacological inhibitors, in vivo imaging, and immunohistochemistry to characterize the role of OPC proliferation, migration, and CMR to developmental OPC tiling. Overall, this work will expand our understanding of OPC tiling and interactions and potentially provide translational targets for demyelinating diseases, such as multiple sclerosis.