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）中轴突的髓鞘化是神经元电活动传播所必需的。 神经元和维护轴突健康。少突胶质细胞祖细胞（OPCs）引起髓鞘形成， 中枢神经系统的少突胶质细胞。虽然大多数OPCs分化为少突胶质细胞， OPC群体在整个生命过程中保持未分化并且均匀地分布在CNS中。OPCs空间 在发育过程中，并保持他们的间隔到成年是未知的。成年小鼠大脑皮层的体内成像 和斑马鱼幼鱼的脊髓表明，OPCs经历了一个称为平铺的过程。我们定义平铺 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距 以及在非病理条件下不重叠的不同区域。在平铺过程中，OPC还表现出 接触介导排斥（CMR）。CMR是一个过程，通过该过程，迁移OPC将收回其过程， 在与其他OPC接触后改变迁移方向。然而，分子机制， OPC用于促进平铺和CMR是未知的。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 的调控OPC-OPC相互作用将提供深入了解OPC平铺的发展过程， 调节OPC迁移和募集至脱髓鞘病变的潜在靶点。这样做的目的 一个建议是阐明在开发过程中促进OPC快速平铺的机制， 研究平铺过程中的三个重要过程：（1）OPC增殖，（2）OPC迁移，和（3） OPC CMR。为了在开发过程中研究OPC平铺，我将使用斑马鱼（Daniorerio）作为脊椎动物模型 系统这个系统相对简单，斑马鱼胚胎和幼体的脊髓清晰， 用于在大部分发育中的CNS中观察OPC迁移、增殖和CMR。我会用 候选基因的方法，根据以前的文献，以确定潜在的介质的增殖和CMR。 此外，我将研究从无偏见的小分子筛选中鉴定的候选物，以鉴定新的 在发育平铺过程中OPC迁移的介质。然后我将使用CRISPR/Cas9基因 编辑技术、药理学抑制剂、体内成像和免疫组织化学来表征 OPC增殖，迁移和CMR对发育OPC平铺的作用。总的来说，这项工作将扩大我们的 了解OPC平铺和相互作用，并可能为脱髓鞘提供翻译靶点 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.