Genetic Studies of Cortex Structure and Development

皮层结构和发育的遗传学研究

• 批准号: 9976603
• 负责人: JOHN L. R. RUBENSTEIN
• 金额: $ 60.55万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-30 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9976603
• 关键词: Automobile Driving; Binding; Binding Proteins; Biological Assay; CRISPR/Cas technology; Cell Separation; Cell physiology; Cells; Cerebral Palsy; Cerebral cortex; Code; DNA sequencing; Development; Embryo; Enhancers; Epilepsy; Fibroblast Growth Factor; Foundations; Gene Expression Regulation; Gene Targeting; Generations; Genes; Genetic; Genetic Enhancer Element; Genetic Transcription; Genetic study; Histones; Human; Human Genetics; Mental Retardation; Mental deficiency; Mitotic; Molecular; Mutation; Nature; Neurons; Pathway interactions; Pattern; Phenotype; Regulator Genes; Regulatory Element; Research; Role; Schizophrenia; Signal Transduction; Structure; Testing; Transcriptional Regulation; Transgenic Mice; Translating; Ventricular; Work; autism spectrum disorder; chromatin immunoprecipitation; developmental disease; emx2 protein; epigenomics; experimental study; genetic information; genetic variant; in vivo; insight; loss of function; loss of function mutation; mutant; neuropsychiatric disorder; progenitor; promoter; public health relevance; response; subventricular zone; transcription factor; transcriptome sequencing

PROJECT SUMMARY Dysregulation of the cerebral cortex is central to human developmental disorders such as epilepsy, mental deficiency, autism and schizophrenia. During development, cortical progenitors generate the projection neurons of the different cortical subdivisions. Understanding the genetic circuitry controlling the development and function of these neurons provides an essential foundation for interpreting human allele variants that are enriched in people who have neuropsychiatric disorders. To elucidate this genetic circuitry, we must define the transcription factors (TF), and regulatory elements and of the coding regions that they control. The proposed research, which concentrates on cortical regionalization, involves the systematic identification of TFs, and the regulatory elements and genes downstream of TFs. Currently, the regional-specification functions of a few TFs in embryonic cortical progenitors are known, and little is known about their direct transcription targets, the nature of the regulatory elements that these TFs control, and the transcriptional circuitry that integrates development and function of these cells. Here we propose to make inroads into each of these components of the TF hierarchy regulating cortical development. Furthermore, we aim to elucidate transcriptional mechanisms through which patterning of cortical progenitors is transmitted to, and maintained in, cortical neurons. We hypothesize that enhancers active in the ventricular zone, subventricular zone and the cortical plate are differentially bound by TFs that drive expression of region/layer-specific genes in post-mitotic cortical neurons. The enhancers serve as protein-binding modules that translate rostrocaudal gradients of TFs in cortical progenitors into region-specific expression in cortical neurons. Herein we focus on the transcriptional mechanisms controlling the generation of different regions of the cerebral cortex (cortical regionalization). The Five Specific Aims extend upon our earlier work on regionalization of cortical progenitors by FGF-signaling, TFs and enhancer elements. Here we investigate transcriptional regulation of cortical patterning by defining the TFs, and other genes, that are regulated by COUPTF1, EMX2, and PAX6 (Aim 1). We then use chromatin immunoprecipitation-DNA sequencing (ChIP-Seq) to define the regulatory element (RE) and gene targets of COUPTF1, EMX2, and PAX6 (Aim 2). Next, we use fluorescent activated cell sorting (FACS) to purify cells from the VZ, SVZ, CP and layers 5&6 to elucidate the epigenomic states of REs (and genes) using Histone ChIP-Seq. This will help us understand the molecular mechanisms that transmit regional patterning information from cortical progenitors to neurons (Aim 3). Finally, we define the function of REs related to cortical patterning using transgenic mice to assess RE activity (Aims 4) and REs deletions to define their role in gene regulation (Aim 5). Once integrated with human genetic information, this will enable us to gain powerful insights into how abnormalities in specific gene networks cause human neuropsychiatric disorders.

项目摘要 大脑皮层的调节异常是人类发育障碍如癫痫、精神发育障碍、神经发育障碍和脑发育障碍的核心。 自闭症和精神分裂症。在发育过程中，皮质祖细胞产生投射， 不同皮层分区的神经元。了解控制发育的遗传电路 这些神经元的功能为解释人类等位基因变异提供了必要的基础， 在患有神经精神疾病的人身上得到了丰富的发现。为了阐明这种遗传电路，我们必须定义 转录因子（TF）和调控元件以及它们控制的编码区。拟议 集中于皮层区域化的研究涉及TF的系统识别，以及 转录因子下游的调控元件和基因。目前，一些TF的区域规格功能 在胚胎皮质祖细胞中的转录是已知的，而关于它们的直接转录靶点知之甚少， 这些转录因子控制的调控元件的性质，以及整合这些转录因子的转录电路， 这些细胞的发育和功能。在这里，我们建议对这些组成部分中的每一个进行深入研究， 调节皮层发育的TF层级。此外，我们的目标是阐明转录机制， 皮层祖细胞的图案通过该通道被传递到皮层神经元并在皮层神经元中维持。我们 假设在脑室区、脑室下区和皮质板中有活性的增强子 在有丝分裂后的皮质神经元中，它们被驱动区域/层特异性基因表达的TF差异结合。 增强子作为蛋白质结合模块，翻译皮质中TF的吻尾梯度 在皮质神经元中的区域特异性表达。在这里，我们专注于转录 控制大脑皮层不同区域生成的机制（皮层区域化）。的 五个特定目的扩展了我们早期通过FGF信号传导对皮质祖细胞进行区域化的工作， TF和增强子元件。在这里，我们研究转录调控皮层图案的定义， 转录因子和其他基因受COUPTF 1、EMX 2和PAX 6（Aim 1）调控。然后我们用染色质 免疫沉淀-DNA测序（ChIP-Seq），以确定调控元件（RE）和基因靶点， COUPTF 1、EMX 2和PAX 6（目标2）。接下来，我们使用荧光激活细胞分选（FACS）来纯化细胞 从VZ、SVZ、CP和第5和6层，使用组蛋白阐明RE（和基因）的表观基因组状态 ChIP测序这将帮助我们了解传递区域模式信息的分子机制 从皮质祖细胞到神经元（Aim 3）。最后，我们定义了与皮质图案相关的RE的功能 使用转基因小鼠评估RE活性（目的4）和RE缺失以确定其在基因调控中的作用 (Aim 5）。一旦与人类遗传信息相结合，这将使我们能够获得强有力的见解， 特定基因网络的异常导致人类神经精神障碍。