Cellular and Genetic Origins of Astrocytes

描述(申请人提供)：人脑由神经元和神经胶质细胞组成。胶质细胞约占脑细胞的90%，与多发性硬化症、癫痫发作和阿尔茨海默病等破坏性疾病有关。我们对最普遍的胶质细胞星形胶质细胞的发育知之甚少。这项提议将确定神经干细胞形成星形胶质细胞的基本机制。它还旨在产生有用的标记，以研究星形胶质细胞在发育和人类疾病中的作用。这项拟议的研究的目标是识别一组星形胶质细胞发育的遗传标记，并绘制它们在中枢神经系统中的细胞起源图。有待检验的基本假设是，星形胶质细胞从发育中的CMS中的不同位置发育而来。我们有三个特定的目标：特定的目标1是确定和表征转录因子的活性，这些转录因子唯一地标记并可能调节星形胶质细胞的发育。在前期工作中，我们询问了哺乳动物的转录组，以确定在发育中的脊髓中与星形胶质细胞共定位的转录因子。将进行表达分析和功能获得和功能丧失筛查，以优先产生标记并可能调节中枢神经系统星形胶质细胞谱系异质性的转录因子抗体。具体目标2是开发纤维星形胶质细胞和原浆星形胶质细胞的细胞类型和阶段特异性标记。我们已经开发了荧光激活细胞分选(FACS)方法，用于急性采集灰质星形胶质细胞并通过表达谱进行分析。通过从Gensat转基因小鼠中分离在(1)泛星形胶质细胞和(2)白质星形胶质细胞区段中表达绿色荧光蛋白(GFP)的细胞，我们将在不同的中枢神经系统区域识别纤维星形胶质细胞、原浆星形胶质细胞和星形胶质细胞前体的特异性标记。具体目标3是确定星形胶质细胞多样性是否与胚胎脊髓和大脑中起源的发育部位相关。我们将使用一组cre/lox转基因小鼠来定位星形胶质细胞的异种祖细胞结构域，以及它们在胚胎中枢神经系统中的最终细胞起源。基于在Aims1/2中获得的信息，我们将产生一个条件报告转基因小鼠品系，该品系将在星形胶质细胞中唯一表达FACS可选择标记GFP。这将使星形胶质细胞亚群的急性纯化成为可能，从而进一步区分星形胶质细胞的异质性。这些研究将阐明神经干细胞产生星形胶质细胞的机制，并为了解星形胶质细胞在人类发育中的不同作用提供有用的遗传学工具，对癫痫、脑癌和神经变性等人类疾病的诊断和治疗具有实际意义。

DESCRIPTION (provided by applicant): The human brain is made up of neurons and glia. Glia comprise about 90% of brain cells and are involved in devastating disorders such as multiple sclerosis, seizures and Alzheimer's disease. We know little about development of the most prevalent glial cells called astrocytes. This proposal will identify fundamental mechanisms by which neural stem cells give rise to astrocytes. It is also intended to generate useful markers to investigate roles for astrocytes during development and in human disease. The goal of the proposed research is to identify a set of genetic markers for development of astrocytes and to map their cellular origins in the central nervous system. The underlying hypothesis to be tested is that astrocytes develop from heterogeneous locations in the developing CMS. We have three specific aims: Specific Aim 1 is to identify and characterize activity of transcription factors that uniquely mark and may regulate astrocyte development. In preliminary work, we have interrogated the mammalian transcriptome to identify transcription factors that co-localize with astrocytes in the developing spinal cord. Expression analysis and gain- and Ioss-of-function screens will be conducted to prioritize generation of antibodies to transcription factors that mark and may regulate astrocyte lineage heterogeneity in the CNS. Specific Aim 2 is to develop cell type- and stage-specific markers for fibrous and protoplasmic astrocytes. We have developed fluorescent activated cell sorting (FACS) protocols for acute harvest of gray matter astrocytes and analysis by expression profiling. By sorting cells from Gensat transgenic mice that express green fluorescent protein (GFP) in (1) pan-astroglial and (2) white matter astrocyte compartments, we will identify markers specific for fibrous astrocytes, protoplasmic astrocytes and astrocyte precursors in various CNS regions. Specific Aim 3 is to determine whether astrocyte diversity correlates with developmental site of origin in the embryonic spinal cord and brain. We will use a cohort of cre/lox transgenic mice to fate map heterogeneous progenitor domains for astrocytes and their ultimate cellular origins in the embryonic CNS. Based on information obtained in Aims1/2, we will generate a floxed conditional reporter transgenic mouse Iine that will express the FACS-selectable marker GFP exclusively in astroglia. This will enable acute purification of astrocyte subsets for further discrimination of astrocyte heterogeneity. PUBLIC HEALTH RELEVANCE These studies will elucidate the mechanisms by which neural stem cells give rise to astrocytes and provide useful genetic tools for understanding the diverse roles of astrocytes in human development, with practical implications for diagnosis and treatment of human disorders such as epilepsy, brain cancer and neurodegeneration.