Identification of transcription factors that regulate astrocyte differentiation

 描述(申请人提供)：星形胶质细胞是一种主要类型的胶质细胞，在神经系统的发育和功能中发挥关键作用。星形胶质细胞功能障碍涉及神经疾病，包括胶质瘤、自闭症、肌萎缩侧索硬化症、创伤性脑损伤和中风。星形胶质细胞的增殖和分化是如何调控的，目前还知之甚少。人类星形胶质细胞的增殖增加有助于人类进化过程中大脑大小的扩大，并对人类智力具有潜在的重要意义。然而，星形胶质细胞的无节制增殖可能会导致胶质瘤。人类和小鼠星形胶质细胞增殖和分化调控的机制差异尚不清楚。由一种细胞类型特异性表达的转录因子是细胞分化的关键调节因子。虽然已经在脊髓中研究了星形胶质细胞的转录调控 在脊髓和视网膜中，调控脑内星形胶质细胞增殖和分化的转录因子(S)仍然难以捉摸。在我的初步研究中，我使用创新的方法从小鼠大脑中纯化了所有主要的细胞类型，并通过RNA测序获得了每种细胞类型的敏感和准确的转录组数据集。我用这种无偏见的方法确定了三个星形胶质细胞特有的转录因子。缺乏这些因素之一的小鼠 显著降低了星形胶质细胞基因的表达。此外，我开发了第一种从人脑中准确纯化星形胶质细胞及其祖细胞的方法，并优化了一种培养条件，以防止这些星形胶质细胞变得具有反应性，这是现有方法的主要限制。在这些结果的基础上，我建议检验这三个星形胶质细胞特异性转录因子是星形胶质细胞分化所必需和充分的假设，以及这些因素的不同调控是人脑中星形胶质细胞比小鼠脑中星形胶质细胞增加的基础。在K99阶段，我将使用现有的基因敲除小鼠系，以及包括病毒感染和宫内电穿孔在内的体外和体内分子操作技术的组合，测试这些转录因子在星形胶质细胞增殖和分化中的必要性和充分性。我将从指导实验室获得分子操纵方面的专业知识。我还将研究这三个转录因子之间的调控相互作用，并确定这三个因子形成的转录级联是否依次调控星形胶质细胞的规格、增殖和成熟。最后，作为一名独立的研究人员，我将利用K99阶段的分子操作训练，并用我开发的新的纯化和培养方法来检测这三种转录因子在人类星形胶质细胞发育中的作用。我还将研究人类星形胶质细胞增加的机制。这项拟议的研究有望填补大脑发育中的一个主要知识空白，因为星形胶质细胞是大脑中最后一种主要的细胞类型，对其分化的转录调控一无所知。此外，从这个项目中获得的知识有可能促进胶质瘤的治疗。

 DESCRIPTION (provided by applicant): Astrocytes are a major type of glia that play critical roles in the development and function of the nervous system. Malfunction of astrocytes are involved in neurological disorders including glioma, autism, amyotrophic lateral sclerosis, traumatic brain injury and stroke. How astrocyte proliferation and differentiation are regulated remains poorly understood. Increased astrocyte proliferation in humans contributes to the expansion in brain size in human evolution, and is potentially important for human intelligence. Unchecked proliferation of astrocytes, however, can lead to glioma. The mechanistic differences in the regulation of astrocyte proliferation and differentiation in humans and mice are unknown. Transcription factors specifically expressed by a cell type are key regulators of cell differentiation. Although transcriptional regulations of astrocytes have been studied in the spinal cord and the retina, the transcription factor(s) that regulate astrocyte proliferation and differentiation in the brain remains elusive. In my preliminary studies, I used innovative methods to purify all of the major cell types from mouse brains and obtained sensitive and accurate transcriptome datasets of each of the cell types by RNA-sequencing. I identified three astrocyte-specific transcription factors with this unbiased approach. Mice deficient for one of these factors have substantially reduced expression of astrocyte genes. In addition, I developed the first method to acutely purify astrocytes and their progenitors from human brains and I optimized a culturing condition that prevents these astrocytes from becoming reactive, which is a major limitation of existing methods. Building on these results, I propose to test the hypothesis that th three astrocyte-specific transcription factors are necessary and sufficient for astrocytes differentiation and that the differential regulation of these factors underlies the increase of astrocytes in human brains compared with mouse brains. In the K99 phase, I will test the necessity and sufficiency of these transcription factors in astrocyte proliferation and differentiation using existing knockout mouse lines, and a combination of in vitro and in vivo molecular manipulation techniques including viral infection and in utero electroporation. I will acquire expertise in molecular manipulations from the mentoring labs. I will also examine the regulatory interactions between these three transcription factors and determine whether a transcriptional cascade formed by the three factors sequentially regulate astrocyte specification, proliferation, and maturation. Finally, as an independent investigator, I will utilize K99 phase training in molecular manipulations and examine the role of the three transcription factors in human astrocyte development with the new purification and culturing method I developed. I will also investigate the mechanisms underlying the increase of astrocytes in humans. The proposed research is expected to close a major knowledge gap in brain development, as astrocytes are the last major cell type of the brain without knowledge of the transcriptional regulation of their differentiation. Moreover, knowledge obtained from this project has the potential to advance the treatment of glioma.