Mechanisms of Astrocyte Development

描述（由申请人提供）：中枢神经系统包含一组特殊的胶质细胞，称为星形胶质细胞。成熟的视神经和视网膜星形胶质细胞对视网膜神经节细胞起代谢支持作用，并维持血视网膜屏障。这些细胞也可以转化为神经系统的破坏性成分，被称为反应性星形胶质细胞，它对轴突再生形成永久性障碍。发育中的视神经星形胶质细胞（星形胶质细胞前体）在眼睛中发挥着许多独特的作用。首先，星形胶质细胞前体作为神经节细胞轴突的引诱物，当它们离开眼睛向中枢神经系统其他部位的靶细胞生长时。其次，星形胶质前体细胞亚群迁移到眼睛内，在那里它们召唤来自眼外的内皮细胞进入，并作为内皮细胞形成初级视网膜血管的基质。我的实验室的长期目标是了解某些生长和模式因子，如骨形态发生蛋白（BMPs）和sonic hedgehog (SHH)，在视杯和神经的正常和反应性星形胶质细胞中直接分化和基因表达的机制。目前的研究目标是确定在星形胶质细胞分化中起关键作用的Pax2基因在发育过程中可能受BMP7和SHH调控的分子机制。该应用将验证SHH和BMP7通过调节抑制蛋白TLX的活性共同调控视网膜星形胶质细胞中Pax2的假设。本申请中提出的研究涉及多学科方法，包括1)体外和体内测定BMP和SHH通路成员与其他调节dna结合蛋白（如共免疫沉淀、染色质免疫沉淀和荧光素酶测定）的相互作用，2)体外和体内BMP通路的操作，3)体外和体内利用免疫组织化学表征视神经星形胶质细胞的表型特性，Western blots，原位杂交，定量聚合酶链反应和图像分析。来自这些研究的信息将进一步加深我们对视网膜和视神经星形胶质细胞发育的必要机制的理解，也可能增加我们对视网膜损伤或疾病后反应性星形胶质细胞如何发育的理解。

DESCRIPTION (provided by applicant): The central nervous system contains a specialized group of glial cells, known as astrocytes. Mature optic nerve and retinal astrocytes play a metabolically supportive role for retinal ganglion cells, as well as maintain the blood-retinal barrier. These cells can also transform into a destructive component of the nervous system, known as reactive astrocytes, which form permanent barriers to axonal regeneration. Developing optic nerve astrocytes (astrocyte precursors) play a number of roles that are unique to the eye. First, astrocyte precursors act as an attractant for ganglion cell axons as they leave the eye to grow towards other target cells in the rest of the central nervous system. Second, a subpopulation of the astrocyte precursor cells migrates into the eye where they beckon to endothelial cells from outside the eye to enter and act as the substrate upon which the endothelial cells form the primary retinal vasculature. The long term goal of my laboratory is to understand the mechanisms by which certain growth and patterning factors, such as bone morphogenetic proteins (BMPs) and sonic hedgehog (SHH), direct differentiation and gene expression in normal and reactive astrocytes in the optic cup and nerve. The goal of the current proposal is to determine the molecular mechanisms by which Pax2, a gene critical in astrocyte differentiation, may be regulated by BMP7 and SHH during development. This application will test the hypothesis that SHH and BMP7 cooperate in the regulation of Pax2 in primary retinal astrocytes by moderating the activity of the suppressor protein TLX. The studies proposed in this application involve a multi-disciplinary approach, including 1) in vitro and in vivo assays to determine interactions of BMP and SHH pathway members with other regulatory DNA-binding proteins such as co-immunoprecipitation, chromatin immunoprecipitation, and luciferase assays, 2) in vitro and in vivo manipulations of BMP pathways, 3) characterization of the phenotypic properties of optic nerve astrocytes in vitro and in vivo using immunohistochemistry, Western blots, in situ hybridization, quantitative polymerase chain reaction and image analysis. Information derived from these studies will further our understanding of the mechanisms necessary for the development of retinal and optic nerve astrocytes and may also increase our understanding of how reactive astrocytes develop following injury or disease to the retina. PUBLIC HEALTH RELEVANCE: Developing astrocytes in the optic stalk are critical for the path finding of retina ganglion cells as they leave the eye and the development of retinal vasculature. One transcription factor, Pax2, is necessary and sufficient for the differentiation of astrocytes. Our lab has proposed a novel mechanism whereby the intracellular signaling pathways of two factors found in the developing optic stalk, bone morphogenetic protein 7 and sonic hedgehog, cooperate in the modulation of repressor protein TLX at the Pax2 promoter. Funding of this application would result in 1) increased knowledge of the mechanisms involved in the development of retinal astrocytes and 2) will enhance our understanding of how these factors may work in the rest of the eye, and 3) augment our understanding of how reactive astrocytes may form in disease and following injury of the nervous system and perhaps provide therapeutic targets to treat reactive astrocytes.