Mechanisms regulating interneuron diversity and maturation

调节中间神经元多样性和成熟的机制

• 批准号: 10468555
• 负责人: Timothy Petros
• 金额: $ 116.61万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10468555
• 关键词: Adult; Affect; Axon; Back; Brain; Brain Diseases; Brain region; Candidate Disease Gene; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Code; DNA; DNA Methylation; DNA Modification Process; Data; Databases; Development; Disease; Disease model; Electrophysiology (science); Embryo; Embryonic Development; Enhancers; Epigenetic Process; Epilepsy; Equilibrium; Etiology; Future; Ganglia; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Code; Genetic Transcription; Genetic Variation; Genomic DNA; Goals; Harvest; Heterogeneity; Human; In Vitro; Injections; Interneuron function; Interneurons; Label; Link; Maps; Medial; Mediating; Mental disorders; Methylation; Molecular; Morphology; Mus; Nervous system structure; Neuroglia; Neurologic; Neurons; Nuclear; Pharmaceutical Preparations; Play; Population; Population Heterogeneity; Process; Property; Prosencephalon; Radial; Reporting; Research Design; Role; Schizophrenia; Signal Transduction; Single Nucleotide Polymorphism; Site-Specific DNA-Methyltransferase (Adenine-Specific); Source; System; Techniques; Testing; Time; Tissue-Specific Gene Expression; Transgenic Mice; Untranslated RNA; Ventricular; Viral; XCL1 gene; autism spectrum disorder; base; cell type; design; epigenetic regulation; epigenomics; experimental study; genome wide association study; histone methylation; histone modification; in vivo; in vivo evaluation; insight; nerve stem cell; nervous system disorder; neurochemistry; neurodevelopment; neurogenesis; novel strategies; postnatal; progenitor; promoter; protein structure; single-cell RNA sequencing; stem cells; subventricular zone; tool; transcription factor; transcriptome

CHARACTERIZING THE EPIGENETIC LANDSCAPE DURING EMBRYONIC NEUROGENESIS While most studies have focused on genes that regulate initial interneuron fate decisions during embryogenesis, a role for epigenetic mechanisms in this process has not been investigated. There is ample evidence that the epigenetic code plays critical roles during neurodevelopment, notably at cell state changes. In particular, DNA and histone modifications often follow specific rules termed the epigenetic code, similar to the genetic code. Collectively, DNA methylation and histone modifications have been reported to regulate transcription and chromatin (nuclear DNA and associated proteins) structure in many stem cell and developmentally critical processes. This idea is particularly relevant since epigenetic changes are observed in many neurological and psychiatric diseases and most single-nucleotide variants (SNVs) identified in diseases-specific GWAS studies map to non-coding regions, implying epigenetic regulation of gene expression may underlie some disease etiologies. To this end, we have characterized the epigenomic landscape of progenitor cells in distinct embryonic brain regions using single cell ATAC-sequencing. By integrating this data with our own single cell transcriptome analysis, we have established a ground state of chromatin accessibility and gene expression at the single cell level throughout the embryonic forebrain. We have uncovered numerous 'high confidence' promoter-enhancer interactions that may play important roles in fate determination of specific neuronal subtypes from distinct embryonic brain regions. We have begun to integrate these findings with neurological and psychiatric databases of human SNPs to determine if there are specific embryonic brain regions and/or cell types that display preferential accessibility at disease-associated SNPs. Following up on these initial observations, we are currently performing more targeted approaches to understand how perturbation of several genes critical for histone methylation affect chromatin accessibility, gene expression and ultimately cell fate. We hope to perform similar sets of experiments on additional disease-related genes in the future. DEFINING THE TRANSCRIPTIONAL HETEROGENEITY OF VENTRICULAR ZONE RADIAL GLIA CELLS The ventricular zone (VZ) of the nervous system contains radial glia cells that were originally considered relatively homogenous in their gene expression. However, a detailed characterization of transcriptional diversity in these VZ cells has not been reported. Here, we performed single-cell RNA sequencing to characterize transcriptional heterogeneity of neural progenitors within the VZ and subventricular zone (SVZ) of the mouse embryonic cortex and ganglionic eminences (GEs). By using a transgenic mouse line to enrich for VZ cells, we detect significant transcriptional heterogeneity within VZ and SVZ progenitors, both between forebrain regions and within spatial subdomains of specific GEs. Additionally, we observe differential gene expression between E12.5 and E14.5 VZ cells, which could provide insights into temporal changes in cell fate. Together, our results reveal a previously unknown spatial and temporal genetic diversity of telencephalic VZ cells that will aid our understanding of initial fate decisions in the forebrain. We are currently establishing CRISPR-based strategies in mouse ESCs to manipulate candidate genes and determine their role in interneuron fate determination and maturation. DEVELOPING A NOVEL APPROACH TO IDENTIFY GENETIC CASCADES UNDERLYING INITIAL INTERNEURON FATE DECISIONS The ability to longitudinally track gene expression within defined populations is essential for understanding how changes in expression mediate both development and plasticity. Previous screens that were designed to identify genes and transcription factors specific to SST- or PV-fated interneurons were largely unsuccessful because several issues significantly hinder these types of studies. First, these interneurons originate from the medial ganglionic eminence (MGE), which is a heterogeneous population of progenitors that gives rise to both interneurons and a variety of GABAergic projection neurons, making it difficult to segregate interneuron progenitors from other cell types. Additionally, many markers that define mature interneuron subtypes are not expressed embryonically, and thus these class-defining markers are not helpful for studying MGE progenitors. In an ideal scenario, we would like to identify actively transcribed genes in MGE progenitors undergoing fate decisions while retaining the capacity to identify whether these cells become PV- or Sst-expressing interneurons in the postnatal brain. To this end, we developed a spatially and temporally inducible form of DNA adenine methylase identification (DamID) that will allow us to label the transcriptome of MGE progenitors. Labeled cells can be harvested at maturity when we have the tools to distinguish specific interneuron cell types. Then the methylated genomic DNA will be analyzed, allowing us to retrospectively look back in time to identify candidate fate determining genes expressed in specific interneuron populations. Our initial tests in mESCs were promising, as we observed drug-inducible genomic DNA methylation in the appropriate expected experimental conditions. Based on these promising results, we have since generated mouse lines from these mESCs. We are currently testing the in vivo function of the Dam methylation system to determine if the genetic methylation is functioning in the mouse as it did in the mESCs. We are also pursuing an alternate viral strategy that will allow us to temporally activate Dam after injection into the mouse embryo, then harvest specific interneuron cell types in the adult to retroactively look at actively transcribed genes throughout development.

胚胎神经发生过程中表观遗传景观的表征 虽然大多数研究都集中在胚胎发生过程中调节初始中间神经元命运决定的基因上，但表观遗传机制在这一过程中的作用尚未研究。有充分的证据表明，表观遗传密码在神经发育过程中起着关键作用，特别是在细胞状态变化中。特别是，DNA和组蛋白修饰通常遵循称为表观遗传密码的特定规则，类似于遗传密码。总的来说，DNA甲基化和组蛋白修饰已被报道在许多干细胞和发育关键过程中调节转录和染色质（核DNA和相关蛋白）结构。这个想法是特别相关的，因为表观遗传变化在许多神经和精神疾病中观察到，并且在疾病特异性GWAS研究中鉴定的大多数单核苷酸变体（SNV）映射到非编码区，这意味着基因表达的表观遗传调节可能是某些疾病病因的基础。为此，我们已经使用单细胞ATAC测序表征了不同胚胎脑区域中祖细胞的表观基因组景观。通过将这些数据与我们自己的单细胞转录组分析相结合，我们已经在整个胚胎前脑的单细胞水平上建立了染色质可及性和基因表达的基态。我们已经发现了许多“高置信度”的启动子-增强子相互作用，可能在不同胚胎脑区域的特定神经元亚型的命运决定中发挥重要作用。我们已经开始将这些发现与人类SNPs的神经学和精神病学数据库相结合，以确定是否有特定的胚胎脑区域和/或细胞类型在疾病相关的SNPs上显示出优先的可及性。在这些初步观察的基础上，我们目前正在进行更有针对性的方法，以了解对组蛋白甲基化至关重要的几个基因的扰动如何影响染色质可及性、基因表达和最终细胞命运。我们希望在未来对其他疾病相关基因进行类似的实验。 室状带放射状胶质细胞跨膜异质性的定义 神经系统的脑室区（VZ）含有放射状胶质细胞，这些细胞最初被认为在其基因表达方面相对同质。然而，在这些VZ细胞中的转录多样性的详细表征还没有报道。在这里，我们进行了单细胞RNA测序，以表征小鼠胚胎皮层和神经节隆起（GE）的VZ和脑室下区（SVZ）内的神经祖细胞的转录异质性。通过使用转基因小鼠系富集VZ细胞，我们检测到VZ和SVZ祖细胞内显著的转录异质性，前脑区域之间和特定GE的空间子域内。此外，我们观察到E12.5和E14.5 VZ细胞之间的差异基因表达，这可以为细胞命运的时间变化提供见解。总之，我们的研究结果揭示了一个以前未知的空间和时间的遗传多样性的端脑VZ细胞，这将有助于我们了解最初的命运决定在前脑。我们目前正在小鼠胚胎干细胞中建立基于CRISPR的策略，以操纵候选基因并确定它们在中间神经元命运决定和成熟中的作用。 一种识别初始中间神经元命运决策遗传级联的新方法 在特定人群中纵向追踪基因表达的能力对于理解表达的变化如何介导发育和可塑性至关重要。先前设计用于鉴定SST或PV命运的中间神经元特异性的基因和转录因子的筛选在很大程度上是不成功的，因为几个问题显著阻碍了这些类型的研究。首先，这些中间神经元起源于内侧神经节隆起（MGE），其是产生中间神经元和各种GABA能投射神经元的祖细胞的异质群体，使得难以将中间神经元祖细胞与其他细胞类型分离。此外，许多定义成熟中间神经元亚型的标记物在胚胎中不表达，因此这些类别定义标记物对研究MGE祖细胞没有帮助。在理想的情况下，我们希望在经历命运决定的MGE祖细胞中识别活跃转录的基因，同时保留识别这些细胞是否成为出生后大脑中表达PV或Sst的中间神经元的能力。为此，我们开发了一种空间和时间诱导形式的DNA腺嘌呤甲基化酶鉴定（DamID），这将使我们能够标记MGE祖细胞的转录组。当我们有工具区分特定的中间神经元细胞类型时，标记的细胞可以在成熟时收获。然后将分析甲基化的基因组DNA，使我们能够及时回顾，以确定在特定的中间神经元群体中表达的候选命运决定基因。我们在mESC中的初步测试是有希望的，因为我们在适当的预期实验条件下观察到药物诱导的基因组DNA甲基化。基于这些有希望的结果，我们已经从这些mESC中产生了小鼠品系。我们目前正在测试Dam甲基化系统的体内功能，以确定基因甲基化是否在小鼠中发挥作用，就像在mESC中一样。我们还在寻求一种替代的病毒策略，这将使我们能够在注射到小鼠胚胎中后暂时激活Dam，然后在成人中收获特定的中间神经元细胞类型，以追溯性地观察整个发育过程中活跃转录的基因。