OLIGODENDROCYTE ONTOGENY AND DIFFERENTIATION

在中枢神经系统中，髓鞘由少突胶质细胞产生。 髓鞘形成的程度最终取决于分配， 少突胶质细胞祖细胞的增殖和形态学上 以及成熟少突胶质细胞的生化分化。 我们建议 在新生小鼠脑原代培养中研究这两种现象， 分析小鼠的几种神经突变对其的影响- JP、MSD、QK、SHI、MLD -其特征在于髓鞘形成不足。 的 将使用以下方法分析少突胶质细胞个体发育的群体动力学： 祖细胞的有限稀释分析， 增殖细胞和成熟细胞的免疫荧光计数。 的 分析少突胶质细胞分化的生化参数 通过确定各种髓鞘积累的发育概况 相关的脂质和蛋白质成分。 这将在整个 在克隆分辨微孔和单个少突胶质细胞中培养 使用各种免疫学技术来鉴定和定量 件. 我们还将分析髓鞘碱性蛋白的调控 在正常和突变培养物中的基因表达，使用北方杂交和RT-PCR， 与重组cDNA探针杂交和体外翻译， 免疫沉淀以定量MBP特异性mRNA，并使用两者 用多克隆和单克隆进行免疫印迹和免疫荧光 定量MBP相关多肽的抗体。 本分析的结果将详细描述 少突胶质细胞的个体发生和分化在细胞和分子水平 水平，并将揭示这一过程是如何调节， 遗传和表观遗传因素。 这将提供重要的洞察力， 大脑发育的过程以及出生缺陷对大脑发育的影响， 也将有助于阐明脱髓鞘疾病的病因 如多发性硬化症和控制髓鞘再生的因素， 中枢神经系统

In the central nervous system myelin is produced by the oligodendrocytes. The extent of myelination ultimately depends on the allocation and proliferation of oligodendrocyte progenitor cells and on the morphological and biochemical differentiation of mature oligodendrocytes. We propose to study these two phenomena in primary cultureof newborn mouse brain and to analyze the effect thereon of several neurological mutations of the mouse - jp, msd, qk, shi, mld - that are characterized by hypomyelination. The population dynamics of oligodendrocyte ontogeny will be analyzed using: limiting dilution analysis of progenitor cells, 3H-thymidine labeling of proliferative cells and immunofluorescent counting of mature cells. The biochemical parameters of oligodendrocytes differentiation will be analyzed by determining the developmental profile of accumulation of various myelin related lipid and protein components. This will be performed in whole culture, in clone-resolved microwells and in individual oligodendrocytes using a variety of immunological techniques to identify and quantitate components. We will also analyze the regulation of myelin basic protein gene expression in normal and mutant cultures using both Northern hybridization with a recombinant cDNA probe and in vitro translation and immunoprecipitation to quantitate MBP-specific mRNA, and using both immunoblotting and immunofluorescence with polyclonal and monoclonal antibodies to quantitate MBP-related polypeptides. The results of this analysis will provide a detailed description of oligodendrocyte ontogeny and differentiation at the cellular and molecular levels in primary culture and will reveal how this process is regulated by genetic and epigenetic factors. This will provide important insight into the process of brain development and the effect thereon of birth defects, and will also help to elucidate the pathoetiology of demyelinating diseases such as multiple sclerosis and the factors controlling remyelination in the central nervous system.