Genetic dissection of the role of macrophages in axonal myelination in zebrafish

描述（由申请人提供）：巨噬细胞和小胶质细胞是先天免疫系统的吞噬细胞，在神经系统中具有重要作用。髓鞘形成的神经胶质细胞用髓鞘包裹轴突，从而允许快速轴突传导。巨噬细胞和小胶质细胞的功能异常与许多神经退行性疾病和髓鞘相关疾病有关，如多发性硬化和周围神经病变。有趣的是，巨噬细胞的异常激活和髓鞘吞噬作用在脱髓鞘疾病中是突出的，这表明巨噬细胞和髓鞘生成神经胶质之间的相互作用。然而，对于巨噬细胞、小胶质细胞和有髓轴突之间相互作用的分子和细胞机制知之甚少。拟议研究的目标是开始在细胞和遗传水平上剖析这些关系，使用斑马鱼作为模型系统来解决有关巨噬细胞和小胶质细胞在有髓鞘轴突发育过程中的作用的问题。大胶质细胞和小胶质细胞从早期阶段就存在于脊椎动物胚胎中并发挥功能，但这些细胞在正常发育中的作用尚未得到很好的表征。本项目将研究这些免疫细胞在有髓轴突发育中发挥重要作用的假设。鉴于巨噬细胞在损伤后清除轴突和髓鞘碎片中的已知作用，第一个目的是通过分析缺乏巨噬细胞的斑马鱼突变体中有髓鞘轴突的组织和超微结构来测试这种相同的功能是否发生在胚胎中，甚至在未受损的神经中。本实验将提供关于巨噬细胞和小胶质细胞在有髓鞘轴突正常发育过程中可能发挥的作用的信息。为了鉴定参与巨噬细胞功能和轴突髓鞘形成的基因，将使用斑马鱼模型系统进行遗传筛选。除了使用已知标记物的髓鞘形成之外，还将针对巨噬细胞分布、活化和数量的缺陷筛选突变体。最后，将深入研究一些突变基因。表型研究，包括标记物研究、细胞移植、超微结构分析、巨噬细胞活化分析和延时成像（如果影响涉及异常细胞迁移），将在细胞水平上确定突变基因的功能。遗传作图和定位克隆将确定基因，并帮助在生物化学水平上确定其功能。这些实验将为巨噬细胞和小胶质细胞功能的机制及其与髓鞘形成的关系提供新的见解。该项目将揭示由异常免疫功能引起的各种神经系统疾病的潜在原因，并可能导致新的治疗方法。

DESCRIPTION (provided by applicant): Macrophages and microglia are phagocytic cells ofthe innate immune system that have important roles in the nervous system. Myelinating glial cells wrap axons with the myelin sheath and thereby allow for fast axonal conduction. Abnormal function of macrophages and microglia have been implicated in many neurodegenerative and myelin-related diseases, such as multiple sclerosis and peripheral neuropathies. Interestingly, aberrant activation of macrophages and myelin phagocytosis are prominent in demyelinating diseases, suggesting interactions between macrophages and myelinating glia. However, very little is known about the molecular and cellular mechanisms underlying the interactions between macrophages, microglia, and myelinated axons. The goal of the proposed research is to begin to dissect these relationships on a cellular and genetic level, using the zebrafish as the model system to address questions about the role of macrophages and microglia during development of the myelinated axons. Macrophages and microglia are present and functional in the vertebrate embryo from an early stage, but the roles of these cells in normal development have not been well characterized. This project will investigate the hypothesis that these immune cells play an essential role in development of myelinated axons. In light ofthe known role of macrophages in removing axonal and myelin debris after injury, the first aim is to test whether this same function occurs in the embryo even in undamaged nerves by analyzing the organization and ultrastructure of myelinated axons in zebrafish mutants lacking macrophages. This experiment will provide information on what role(s) macrophage and microglia may have during normal development of the myelinated axons. To identify genes involved in macrophage function and axonal myelination, a genetic screen will be conducted using the zebrafish model system. Mutants will be screened for defects in macrophage distribution, activation, and number in addition to myelination using known markers. Finally, a few mutated genes will be studied in depth. Phenotypic studies, including marker studies, cell transplantation, uitrastructural analysis, macrophage activation analyses, and time-lapse imaging if the effect involves abnormal cell migration, will define the function of the mutated genes at the cellular level. Genetic mapping and positional cloning will identify the genes and help define their functions at the biochemical level. These experiments will provide new insights into the mechanisms that dictate macrophage and microglia function and their relationship with myelination. This project will cast light on the potential causes of a wide array of neurological disorders derived from aberrant immune function, and may lead to new therapeutic approaches.