Functional Interrogation of Chromatin Remodeling Gene in Brain Development

背景|神经细胞的发育和哺乳动物大脑的形成正是由基因表达来组织的。基因转录受转录因子和许多表观遗传因子的调控，包括染色质重塑蛋白。虽然关于转录因子如何调控哺乳动物大脑中特定细胞类型的各种发育过程和功能结果已经有了丰富的知识，但对于染色质重塑蛋白在这些过程中的作用却知之甚少。目标和方法|我们在NSERC DG中的目标是研究其中一种蛋白质CHD8是如何控制神经元亚型的转录组的，这对这些亚型的形态发育有什么影响，以及这如何反过来影响生理功能。在本项目中，我们将携带Chd8fl/fl的小鼠与ROSA 26-TD-番茄和特定细胞类型的Cre系杂交。我们将从神经元和神经胶质亚型特异的Cre系开始，包括NEX-Cre，VIAAT-Cre和GFAP-Cre。我们的具体目标是：-目标1：在特定类型的脑细胞中去除CHD8后，识别和比较全基因组的转录特征。CHD8的缺失和TD-番茄的同时标记使我们能够分析来自脑组织的同质细胞群，并使用RNA-Seq进行全基因组转录分析。-目的2：确定CHD8完全缺失在特定类型神经元中引起的形态变化。我们将进行检测，以检测CDH8去除后的存活、迁移、树突状突起生长和突触密度。-目的3：确定在亚型细胞中完全敲除CHD8导致的功能结果。我们将使用切片电生理学来表征兴奋性和抑制性神经元的动作电位、兴奋性和抑制性输入。结果和意义|NSERC DG将培养高素质的人才，其中包括一名博士后研究员和两名博士生，他们将带着丰富的神经科学研究专业知识离开我的实验室。这些实验将提供直接的体内证据，证明染色质调节因子(CHD8基因)如何影响亚型特异性转录组，并赋予神经元和神经胶质细胞不同、复杂的特性，这将使我们能够全面了解哺乳动物大脑发育的基因调控。我们的研究结果、见解和方法将为解决特定染色质重塑蛋白在未来导致独特的神经元和神经胶质亚型规范的规则提供一种易于处理的策略，这是神经科学中的一个基本问题，也是我的实验室的长期目标。

Background | Development of nerve cells and the formation of the mammalian brain is precisely organized by gene expression. Gene transcription is regulated by groups transcriptional factors (TFs) and many epigenetic factors, including chromatin remodeling proteins. While a wealth of knowledge exists about how TFs regulate various developmental processes and functional outcomes of specific cell types the mammalian brain, little is known about the contribution of chromatin remodeling proteins in such processes. Objectives and Approaches | Our aims in this NSERC DG are to examine how one of these proteins, Chd8, controls the transcriptome of neuronal subtypes and what impact this has on the morphological development of these sub-types, and how this, in turn, affects physiological function. We will cross mouse harboring Chd8fl/fl together with Rosa 26-td-tomato and cell-type-specific Cre lines in this project. We will start with neuronal, and glial subtype-specific Cre lines, including NEX-Cre, VIAAT-Cre, and GFAP-Cre. Our specific aims are: - Aim 1: To identify and compare the genome-wide, transcriptional signature, after removing of Chd8 in specific types of brain cells. The deletion of Chd8 and simultaneously labeling with td-tomato allows us to analyze a homogeneous population of cells from brain tissues, and to carry out genome-wide transcriptional analysis using RNA-Seq. - Aim 2: To determine the morphological alterations caused by completely Chd8 deletion in specific types of neurons. We will carry out assays to examine the survival, migration, dendritic outgrowth and synaptic density resulting from removal of Cdh8. - Aim 3: To determine the functional outcomes caused by completely knocking out of Chd8 in subtypes of cells. We will characterize both excitatory and inhibitory neurons on their firing of action potential, excitatory and inhibitory inputs using slice electrophysiology. Outcomes and Significance | This NSERC DG will lead to the training of Highly Qualified Personnel, including one postdoc fellow and two Ph.D. students who will leave my lab armed with substantial expertise in neuroscience research. These experiments will provide a direct, in vivo evidence regarding how a chromatin regulator (Chd8 gene) contribute to subtype-specific transcriptome, and endow neurons and glial cells with diverse, complex properties, which will enable a comprehensive picture for the genetic regulation of brain development in mammals. The results, insights, and methods that will come out of our studies will provide a tractable strategy for addressing the rules by which specific chromatin-remodeling proteins can lead to unique neuronal and glial subtype specification in the future, which is a fundamental question in neuroscience, and a long-term goal of my lab.