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: 10003759
• 负责人: Timothy Petros
• 金额: $ 96.02万
• 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/10003759
• 关键词: ATAC-seq; Adopted; Adult; Axon; Back; Brain; Brain Diseases; Brain region; Cell Transplantation; Cell Transplants; Cells; Chromatin; Classification; Code; Complex; Corpus striatum structure; DNA; DNA Methylation; DNA Modification Process; Development; Disease; Electrophysiology (science); Embryo; Embryonic Development; Environment; Enzymes; Epigenetic Process; Epilepsy; Etiology; Ganglia; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Code; Genetic Transcription; Genomic DNA; Goals; Harvest; Heterogeneity; Heterotopic Transplantation; Hippocampus (Brain); Human; In Vitro; Injections; Interneuron function; Interneurons; Label; Life; Link; Logic; Maps; Medial; Mediating; Mental disorders; Methylation; Minority; Morphology; Mus; Nervous system structure; Neurons; Neurosciences; Nuclear; Nucleotides; Parvalbumins; Pattern; Pharmaceutical Preparations; Play; Population; Population Heterogeneity; Process; Property; Protocols documentation; Publishing; Reporter; Reporting; Research Design; Resistance; Role; Schizophrenia; Signal Transduction; Site-Specific DNA-Methyltransferase (Adenine-Specific); Somatostatin; Source; Stem cells; Synapses; System; Techniques; Testing; Time; Transplantation; Untranslated RNA; Variant; Viral; autism spectrum disorder; base; cell type; design; environmental change; epigenetic regulation; epigenomics; experimental study; genome wide association study; histone modification; in vivo; in vivo evaluation; insight; nervous system disorder; neurochemistry; neurodevelopment; new technology; novel strategies; post-transplant; postnatal; progenitor; programs; protein structure; sequencing platform; single cell sequencing; tool; transcription factor; transcriptome

HOW THE ENVIRONMENT SCULPTS INTERNEURON DIVERSITY AND MATURATION The composition of interneuron subtypes varies significantly between different brain regions. Numerous experiments indicate that general interneuron classes (e.g., parvalbumin- (PV) or somatostatin-expressing (SST)) are determined as cells become postmitotic during embryogenesis, the role that the brain environment plays in interneuron fate determination and maturation remains unknown. To explore this issue, we harvested early postnatal interneuron precursors (P0-P2) from specific brain regions and transplanted them into wildtype hosts either homotopically (cortex-to-cortex) or heterotopically (cortex-to-hippocampus). This technique allows us to determine if transplanted interneurons adopt properties of the host environment (indicating a strong role for the environment in regulating interneuron diversity) or if they retain subtype features more consistent with the donor region. Our findings indicate that the environment largely determines the composition of interneuron subtypes in a brain region regardless of donor region. However, some interneuron subtypes seem to be more genetically predefined and resistant to environmental influences. These findings were published in Cell Reports in late 2017. We have continued these studies by harvesting interneurons from the striatum and transplanting them to distinct brain regions (as well as graft interneurons from other regions into the striatum), which has led to additional important insights into how the brain environment sculpts interneuron maturation. Additionally, we have established a single cell sequencing platform in the lab that we are currently adapting for transplanted interneurons. We are reharvesting grafted interneurons 3 weeks post-transplantation so that we can compare the transcriptome of endogenous, homotopic and heterotopically transplanted cells to characterize (in an unbiased manner) how environmental changes influence the transcriptome of transplanted interneurons. IDENTIFYING A ROLE FOR EPIGENETICS IN EARLY INTERNEURON FATE DECISIONS 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, DNAme and histone modification 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 will characterize the epigenomic landscape of progenitor cells in distinct embryonic brain regions and integrate these findings with transcriptome analysis using the recently developed single cell ATAC-seq protocols. In a more targeted approach, we will investigate the role of the histone modification enzyme Ezh2 in interneuron development. These combined approaches will generate a more complete picture of a cells state during initial fate decisions. 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. EXPLORING THE LOGIC OF SYNAPTIC CONNECTIVITY OF CHANDELIER CELLS Understanding synaptic connectivity is one of the most complex questions in neuroscience. Chandelier cells, a subset of PV+ interneurons, form unique synaptic contacts on the axon initial segments (AIS) of excitatory projection neurons, with a small handful of ChCs contacting each AIS. This specific connectivity pattern presents an intriguing situation to explore the logic of this synaptic connectivity. Do chandelier cells consistently synapse on AIS with other similar targeting chandelier cells, or instead are these connections randomly distributed between chandelier cells? We are currently using several strategies involving Brainbow reporters to explore the synaptic connectivity logic of chandelier cells in the mouse cortex.

环境如何塑造中间神经元的多样性和成熟度 不同大脑区域之间的中间神经元亚型的组成存在显着差异。大量实验表明，一般的中间神经元类别（例如，小清蛋白（PV）或表达生长抑素（SST））是在胚胎发生过程中细胞有丝分裂后确定的，但大脑环境在中间神经元命运决定和成熟中所起的作用仍然未知。为了探讨这个问题，我们从特定的大脑区域收获了早期出生后中间神经元前体（P0-P2），并将它们同伦（皮质到皮质）或异位（皮质到海马）移植到野生型宿主中。这项技术使我们能够确定移植的中间神经元是否采用宿主环境的特性（表明环境在调节中间神经元多样性方面发挥着重要作用），或者它们是否保留与供体区域更一致的亚型特征。我们的研究结果表明，无论供体区域如何，环境在很大程度上决定了大脑区域中神经元亚型的组成。然而，一些中间神经元亚型似乎更具遗传性，并且对环境影响具有抵抗力。这些发现于 2017 年底发表在《Cell Reports》上。我们继续进行这些研究，从纹状体中采集中间神经元并将其移植到不同的大脑区域（以及将其他区域的中间神经元移植到纹状体中），这为大脑环境如何塑造中间神经元成熟提供了更多重要见解。此外，我们在实验室建立了一个单细胞测序平台，目前正在适应移植的中间神经元。我们在移植后 3 周重新收获移植的中间神经元，以便我们可以比较内源性、同位和异位移植细胞的转录组，以（以公正的方式）表征环境变化如何影响移植的中间神经元的转录组。 确定表观遗传学在早期中间神经元命运决定中的作用 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，但表观遗传机制在此过程中的作用尚未得到研究。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，DNAme 和组蛋白修饰已被报道可调节许多干细胞和发育关键过程中的转录和染色质（核 DNA 和相关蛋白质）结构。这个想法特别重要，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) 映射到非编码区域，这意味着基因表达的表观遗传调控可能是某些疾病病因的基础。为此，我们将描述不同胚胎大脑区域中祖细胞的表观基因组景观，并使用最近开发的单细胞 ATAC-seq 协议将这些发现与转录组分析相结合。通过更有针对性的方法，我们将研究组蛋白修饰酶 Ezh2 在中间神经元发育中的作用。这些组合方法将在最初的命运决定过程中生成更完整的细胞状态图。 开发一种新方法来识别初始中间神经元命运决定背后的遗传级联 纵向追踪特定群体内基因表达的能力对于理解表达变化如何介导发育和可塑性至关重要。以前旨在识别 SST 或 PV 命运中间神经元特异基因和转录因子的筛选基本上不成功，因为几个问题严重阻碍了此类研究。首先，这些中间神经元起源于内侧神经节隆起（MGE），这是一个异质祖细胞群，产生中间神经元和各种 GABA 能投射神经元，使得很难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于研究 MGE 祖细胞没有帮助。在理想的情况下，我们希望识别正在经历命运决定的 MGE 祖细胞中活跃转录的基因，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可诱导形式的 DNA 腺嘌呤甲基化酶识别 (DamID)，这将使我们能够标记 MGE 祖细胞的转录组。当我们拥有区分特定中间神经元细胞类型的工具时，可以在成熟时收获标记的细胞。然后对甲基化的基因组 DNA 进行分析，使我们能够及时回顾以确定在特定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，以确定基因甲基化在小鼠中是否像在 mESC 中一样发挥作用。我们还在寻求一种替代病毒策略，该策略将使我们能够在注射到小鼠胚胎后暂时激活 Dam，然后在成体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。 探索枝形吊灯细胞突触连接的逻辑 了解突触连接是神经科学中最复杂的问题之一。吊灯细胞是 PV+ 中间神经元的一个子集，在兴奋性投射神经元的轴突初始段 (AIS) 上形成独特的突触接触，并有少量 ChC 与每个 AIS 接触。这种特定的连接模式为探索这种突触连接的逻辑提供了一个有趣的情况。枝形吊灯细胞是否与其他类似的目标枝形吊灯细胞在 AIS 上一致地突触，或者这些连接是否随机分布在枝形吊灯细胞之间？我们目前正在使用多种涉及 Brainbow 记者的策略来探索小鼠皮层中枝形吊灯细胞的突触连接逻辑。