Identification of transcription factors that regulate astrocyte differentiation

调节星形胶质细胞分化的转录因子的鉴定

• 批准号: 8930212
• 负责人: Ye Zhang
• 金额: $ 8.8万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8930212
• 关键词: Address; Affect; Amyotrophic Lateral Sclerosis; Animals; Astrocytes; Autistic Disorder; Biological; Brain; Cell Culture Techniques; Cell Cycle; Cell Differentiation process; Cells; Cerebral cortex; Ciliary Neurotrophic Factor; Data; Data Set; Defect; Development; Disease; Electroporation; Event; Evolution; Gene Expression; Gene Expression Profile; Generations; Genes; Glioma; Health; Human; In Vitro; Intelligence; Knock-out; Knockout Mice; Knowledge; Lead; Light; Malignant Neoplasms; Mentors; Methods; Modeling; Molecular; Molecular Profiling; Mus; Mutant Strains Mice; Nervous System Physiology; Neuraxis; Neuroglia; Patients; Phase; Play; Property; RNA Sequences; Regulation; Research; Research Personnel; Rest; Retina; Role; Serum; Small Interfering RNA; Spinal Cord; Staging; Stem cells; Stroke; Techniques; Testing; Time; Training; Transcriptional Regulation; Traumatic Brain Injury; Viral; Virus Diseases; astrocyte progenitor; astrogliosis; brain size; cell type; cognitive ability; effective therapy; gain of function; improved; in utero; in vivo; innovation; knock-down; nervous system disorder; overexpression; prevent; progenitor; programs; stem cell differentiation; transcription factor; transcriptome sequencing; treatment strategy

 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.

 描述（由申请人提供）：星形胶质细胞是一种主要类型的神经胶质细胞，在神经系统的发育和功能中发挥关键作用。星形胶质细胞的功能障碍涉及神经系统疾病，包括神经胶质瘤、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被调节的仍然知之甚少。人类星形胶质细胞增殖的增加有助于人类进化中大脑尺寸的扩大，并且对人类智力具有潜在的重要性。然而，星形胶质细胞的不受控制的增殖可导致神经胶质瘤。人类和小鼠星形胶质细胞增殖和分化调节的机制差异尚不清楚。由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。虽然星形胶质细胞的转录调控已经在脊髓中进行了研究， 尽管在脊髓和视网膜中，调节脑中星形胶质细胞增殖和分化的转录因子仍然难以捉摸。在我的初步研究中，我使用了创新的方法从小鼠大脑中纯化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。我用这种无偏的方法鉴定了三种星形胶质细胞特异性转录因子。缺乏这些因素之一的小鼠 大大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急性纯化星形胶质细胞及其祖细胞的方法，并优化了防止这些星形胶质细胞变得反应性的培养条件，这是现有方法的主要局限性。基于这些结果，我建议测试的假设，TH三个星形胶质细胞特异性的转录因子是必要的和足够的星形胶质细胞分化，这些因素的差异调节的基础上增加的星形胶质细胞在人脑与小鼠大脑相比。在K99阶段，我将测试的必要性和充分性，这些转录因子在星形胶质细胞的增殖和分化，使用现有的敲除小鼠品系，并结合在体外和体内的分子操作技术，包括病毒感染和子宫内电穿孔。我将从指导实验室获得分子操作方面的专业知识。我还将研究这三个转录因子之间的相互作用，并确定是否由这三个因子形成的转录级联顺序调节星形胶质细胞的规格，增殖和成熟。最后，作为一个独立的研究者，我将利用K99阶段的分子操作培训，并检查三个转录因子在人类星形胶质细胞发育中的作用与新的纯化和培养方法，我开发。我还将研究人类星形胶质细胞增加的机制。这项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不知道其分化转录调控的主要细胞类型。此外，从该项目中获得的知识有可能促进胶质瘤的治疗。