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: 10691098
• 负责人: Timothy Petros
• 金额: $ 100.43万
• 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/10691098
• 关键词: Affect; Atlases; Axon; Brain; Brain Diseases; Brain region; Candidate Disease Gene; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Communication; DNA; DNA Methylation; DNA Modification Process; Data; Development; Disease; Disease model; Electrophysiology (science); Embryo; Embryonic Development; Enhancers; Epigenetic Process; Epilepsy; Equilibrium; Etiology; Future; Ganglia; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Code; Genetic Transcription; Genetic Variation; Goals; Heterogeneity; Hi-C; Higher Order Chromatin Structure; Human; In Vitro; Interneuron function; Interneurons; Label; Link; Maps; Mental disorders; Molecular; Morphology; Mus; Nature; Nervous System; Neuroglia; Neurons; Nuclear; Play; Population; Process; Property; Prosencephalon; Radial; Reporter; Reporting; Research; Research Personnel; Role; Schizophrenia; Series; Signal Transduction; Single Nucleotide Polymorphism; Source; Techniques; Tissue-Specific Gene Expression; Transgenic Mice; Untranslated RNA; Ventricular; autism spectrum disorder; base; epigenetic regulation; epigenome; experimental study; genome browser; genome wide association study; histone methylation; histone modification; in vivo; insight; nerve stem cell; nervous system disorder; neurochemistry; neurodevelopment; neurogenesis; progenitor; promoter; protein structure; single-cell RNA sequencing; stem cells; subventricular zone; 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 performed a series of experiments to characterize the transcriptome (scRNA-seq), chromatin accessibility (snATAC-seq), histone modifications (CUT&Tag) and higher order chromatin structure (Hi-C and Capture-C) in distinct neurogenic regions (LGE, MGE, CGE and cortex) of the embryonic mouse brain. This comprehensive analysis generated an 'Epigenome Atlas' of the embryonic brain at the initial stages of neurogenesis and revealed striking differences in the chromatin landscape between adjacent brain regions (Rhodes...Petros, Nature Communications 2022). Additionally, 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 made all of this data available in a searchable and modifiable format on the UCSC Genome Browser platform (https://www.nichd.nih.gov/research/atNICHD/Investigators/petros/epigenome-atlas). Following up on this initial study, 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 utilized a transgenic mouse line that specifically labels VZ cells with a fluorescent reporter and 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 (LGE, MGE and CGE). We detected significant transcriptional heterogeneity within VZ and SVZ progenitors, both between forebrain regions and within spatial subdomains of specific GEs (Lee...Petros, eLife 2022). 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 (1) manipulate candidate genes and determine their role in interneuron fate determination and maturation, and (2) identify new genes that are critical for generation of CGE-derived interneurons.

胚胎神经发生过程中表观遗传特征的研究 虽然大多数研究都集中在胚胎发育过程中调节初始神经元间命运决定的基因上，但表观遗传机制在这一过程中的作用尚未被调查。有充分的证据表明，表观遗传密码在神经发育过程中发挥着关键作用，特别是在细胞状态变化方面。特别是，DNA和组蛋白的修饰通常遵循被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。这一观点特别相关，因为在许多神经和精神疾病中观察到表观遗传变化，并且在疾病特异性GWAs研究中发现的大多数单核苷酸变异(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一些疾病病因的基础。为此，我们进行了一系列实验，以表征小鼠胚胎大脑不同神经源性区域(LGE、MGE、CGE和皮质)的转录组(scRNA-seq)、染色质可及性(snATAC-seq)、组蛋白修饰(Cut和Tag)和高阶染色质结构(Hi-C和Capture-C)。这一全面的分析生成了胚胎大脑在神经发生的初始阶段的“表观基因组图谱”，并揭示了相邻大脑区域之间染色质景观的显著差异(Rhodes...Petros，自然通讯2022)。此外，我们还发现了许多“高置信度”的启动子-增强子相互作用，这些相互作用可能在决定不同胚胎脑区特定神经元亚型的命运方面发挥重要作用。我们已经在加州大学基因组浏览器平台(https://www.nichd.nih.gov/research/atNICHD/Investigators/petros/epigenome-atlas).上以可搜索和可修改的格式提供了所有这些数据在这项初步研究的基础上，我们目前正在进行更有针对性的方法，以了解几个组蛋白甲基化关键基因的扰动如何影响染色质的可及性、基因表达和最终细胞命运。我们希望在未来对更多与疾病相关的基因进行类似的实验。 确定脑室带径向神经胶质细胞的转录异质性 神经系统的脑室带(VZ)含有放射状胶质细胞，最初认为它们的基因表达相对同质。然而，这些VZ细胞的转录多样性的详细特征还没有报道。在这里，我们利用转基因小鼠品系，用荧光报告特异性标记VZ细胞，并进行单细胞RNA测序，以表征小鼠胚胎皮质和神经节隆起(LGE、MGE和CGE)中VZ和室下区(SVZ)内神经前体细胞的转录异质性。我们在VZ和SVZ前体细胞中检测到显著的转录异质性，无论是在前脑区域之间，还是在特定GE的空间亚域内(Lee...Petros，eLife 2022)。此外，我们观察到E12.5和E14.5 VZ细胞之间的差异基因表达，这可能为细胞命运的时间变化提供洞察力。总之，我们的结果揭示了端脑VZ细胞以前未知的空间和时间遗传多样性，这将有助于我们理解前脑中最初的命运决定。我们目前正在小鼠胚胎干细胞中建立基于CRISPR的策略，以(1)操纵候选基因并确定它们在神经元间命运决定和成熟中的作用，以及(2)识别对CGE来源的中间神经元的产生至关重要的新基因。