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Mechanisms regulating interneuron diversity and maturation

调节中间神经元多样性和成熟的机制

基本信息

批准号：
10908179
负责人：
Timothy Petros
金额：
$ 193.57万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/10908179
关键词：
ATAC-seq Ataxia Telangiectasia Axon Biological Assay Brain Brain Diseases Brain region CRISPR/Cas technology Candidate Disease Gene Cell Cycle Cell Transplantation Cells Chromatin Communication Complex Development Disease model EZH2 gene Electrophysiology (science)Embryo Embryonic Development Enhancers Environment Epigenetic Process Epilepsy Equilibrium Gene Expression Gene Expression Regulation Genes Genetic Genetic Transcription Goals Guide RNA Homologous Gene Human In Vitro Interneuron function Interneurons Knockout Mice Label Libraries Link Logic Lysine Manuscripts Medial Methyltransferase Modification Molecular Morphology Mus Mutation Nature Nervous System Neurons Parvalbumins Play Polycomb Population Predisposition Primates Production Property Prosencephalon Publications Publishing Reporting Repression Role Schizophrenia Seizures Series Signal Transduction Somatostatin Source Specificity Techniques Testing Transgenic Mice Weaver Syndrome autism spectrum disorder candidate identification embryonic stem cell epigenetic regulation experimental study follow-up gene repression genomic locus histone methylation in vivo insight interest loss of function mouse model multiple omics neurochemistry neurodevelopment neurogenesis postmitotic precursor cell progenitor single-cell RNA sequencing tool transcriptome transcriptome sequencing

项目摘要

LOSS OF EZH2 IN MGE PROGENITORS ALTERS INTERNEURON FATE Enhancer of zeste homolog 2 (Ezh2) is the methyltransferase component of the Polycomb Repressive Complex 2 (PRC2), which is critical for trimethylation of histone 3 at lysine 27 (H3K27me3) and results in gene repression. Mutations in EZH2 and dysregulation of H3K27me3 can lead to neurodevelopmental abnormalities such as Weaver Syndrome and ataxia-telangiectasia. During cortical neurogenesis, H3K27me3 is a critical epigenetic modification that regulates cellular maturation rate, and in turn, determines when precursor cells exit the cell cycle. Loss of function studies reveal that Ezh2 plays a critical role in epigenetic regulation of neuronal fate and maturation in some brain regions, but a role for Ezh2 in forebrain GABAergic interneurons has not been explored. Here, we removed Ezh2 from the medial ganglionic eminence (MGE) to study its role in interneuron development. We find that loss of Ezh2 shifts interneuron fate, with an increase in somatostatin-expressing (SST+) and a decrease in parvalbumin-expressing (PV+) interneurons in multiple brain regions. Intrinsic electrophysiological properties are normal, but PV+ interneurons display increased axonal complexity. We also observe fewer MGE-derived interneurons at P5, indicating reduced interneuron production during development. Last, we utilized single cell Multiome and CUT&Tag assays to characterize transcriptional and H3K27me3 differences in the MGE between WT and KO mice. Our results reveal a critical role for Ezh2 in interneuron fate and maturation. Single cell multiome (RNA-seq and ATAC-seq) analysis revealed genetic and epigenetic changes in the embryonic MGE that are consistent with these cell fate changes. Lastly, CUT&Tag analysis revealed differential changes in H3K27me3 levels at specific genomic loci, with some genes displaying a relative increase in H3K27me3 levels. Thus, loss of Ezh2 in the MGE causes significant changes in interneuron fate, morphology, and gene expression and regulation. A manuscript describing these results will be submitted soon and posted on bioRxiv. A ROLE FOR EPIGENETIC REGULATION OF MGE-DERIVED INTERNEURONS Our recent publications established a ground truth of transcriptional and epigenetic states in four distinct embryonic brain regions that give rise to distinct subtypes of forebrain interneurons (RhodesPetros 2022; LeePetros 2022). We are now expanding on these findings to characterize how various mutations alter the transcriptome, epigenetic environment and ultimately cell fate. In one study, we are exploring how disruption of a candidate Nkx2.1 enhancer alters the fate of MGE-derived interneurons. In another study, we are studying how disruption of histone methylation at a specific residue effects interneuron fate and leads to mice with enhanced seizure susceptibility. These studies are ongoing. MECHANISMS REGULATING FATE DETERMINATION OF CGE-DERIVED INTERNEURONS While significant progress has been made characterizing mechanisms regulating initial fate decisions of MGE-derived interneurons, our understanding of CGE-derived interneurons lags significantly behind. This is in part because we lack genetic tools to specifically target and manipulate CGE-derived cells. There is an expansion of CGE-derived interneuron subtypes in humans and primates compared with mice, so a better understanding of the developmental trajectory of these cells is warranted. To this end, we are currently performing several experiments to better understand the developmental logic of CGE-derived cells. First, we previously performed cell transplant assays of postmitotic MGE-derived interneuron precursors to better understand how the brain environment influences cell fate (QuattrocoloPetros, Cell Reports 2017; QuattrocoloPetros JoVE 2018). We are currently performing similar cell transplantation experiments to determine whether specific CGE-derived interneuron subtypes are derived from distinct regions of the CGE. By combining this spatial logic with the scRNA-Seq and scATAC-seq studies we published last year (RhodesPetros Nature Communications 2022; LeePetros eLife 2022), we hope to link early transcription and chromatin accessibility profiles in CGE progenitors with mature interneuron fates. Second, we have developed a Perturb-Seq approach to identify candidate genes that promote CGE-derived interneurons. We are using CRISPR-Cas9 mouse embryonic stem cell (mESC) to activate or repress an array of genes involved in neurodevelopment, using a lentiviral library consisting of numerous gRNAs targeted to genes of interest. We will follow up any intriguing, fate-determining gene candidate hits from this Perturb-Seq screen to explore their possible role in CGE-derived interneurons. Third, based on insights from our scRNA-seq experiments (LeePetros eLife 2022), we have generated new transgenic mouse models with the goal of specifically targeting CGE-derived interneurons during development. We are currently testing the specificity and efficacy of this mouse line to label CGE-derived interneurons (or specific subsets) during neurodevelopment. In combination, this series of experiments should significantly increase our understanding of mechanisms regulating CGE-derived interneurons.

MGE祖细胞中EZH 2的缺失改变了中间神经元的命运 zeste增强子同源物2（Ezh 2）是多梳抑制复合物2（PRC 2）的甲基转移酶组分，其对于组蛋白3在赖氨酸27（H3 K27 me 3）处的三甲基化至关重要并导致基因抑制。EZH 2的突变和H3 K27 me 3的失调可导致神经发育异常，如韦弗综合征和共济失调-毛细血管扩张症。在皮质神经发生过程中，H3 K27 me 3是一种重要的表观遗传修饰，调节细胞成熟速率，进而决定前体细胞何时退出细胞周期。功能丧失研究表明，Ezh 2在某些脑区神经元命运和成熟的表观遗传调控中起关键作用，但Ezh 2在前脑GABA能中间神经元中的作用尚未探索。在这里，我们将Ezh 2从内侧神经节隆起（MGE）中取出，以研究其在中间神经元发育中的作用。我们发现Ezh 2的丢失改变了中间神经元的命运，在多个脑区中生长抑素表达（SST+）增加，小白蛋白表达（PV+）中间神经元减少。内在电生理特性正常，但PV+中间神经元显示轴突复杂性增加。我们还观察到P5时较少的MGE衍生的中间神经元，表明发育过程中中间神经元的产生减少。最后，我们利用单细胞Multiome和CUT&Tag测定来表征WT和KO小鼠之间MGE中的转录和H3 K27 me 3差异。我们的研究结果揭示了Ezh 2在中间神经元的命运和成熟中的关键作用。单细胞多基因组（RNA-seq和ATAC-seq）分析揭示了胚胎MGE中的遗传和表观遗传变化，这些变化与这些细胞命运变化一致。最后，CUT&Tag分析揭示了特定基因组位点处H3 K27 me 3水平的差异变化，其中一些基因显示H3 K27 me 3水平的相对增加。因此，MGE中Ezh 2的缺失会导致中间神经元命运、形态以及基因表达和调节发生显着变化。描述这些结果的手稿将很快提交并发布在bioRxiv上。 MGE源性中间神经元的表生调节作用我们最近的出版物在四个不同的胚胎脑区域中建立了转录和表观遗传状态的基础事实，这些区域产生了不同的前脑中间神经元亚型（RhodesPetros 2022; LeePetros 2022）。我们现在正在扩展这些发现，以描述各种突变如何改变转录组，表观遗传环境和最终细胞命运。在一项研究中，我们正在探索候选Nkx2.1增强子的破坏如何改变MGE衍生的中间神经元的命运。在另一项研究中，我们正在研究组蛋白甲基化在特定残基上的破坏如何影响中间神经元的命运，并导致小鼠具有增强的癫痫易感性。这些研究正在进行中。 CGE诱导的中间神经元命运决定的调控机制虽然已经取得了显着的进展，MGE衍生的interneurons的初始命运决定的调节机制，我们的理解CGE衍生的interneurons明显落后。这部分是因为我们缺乏专门针对和操纵CGE衍生细胞的遗传工具。与小鼠相比，在人类和灵长类动物中存在CGE衍生的中间神经元亚型的扩展，因此有必要更好地了解这些细胞的发育轨迹。为此，我们目前正在进行几项实验，以更好地了解CGE衍生细胞的发育逻辑。首先，我们之前对有丝分裂后MGE衍生的中间神经元前体进行了细胞移植试验，以更好地了解大脑环境如何影响细胞命运（QuattrocolPetros，Cell Reports 2017; QuattrocolPetros JoVE 2018）。我们目前正在进行类似的细胞移植实验，以确定特定的CGE衍生的中间神经元亚型是否来自不同区域的CGE。通过将这种空间逻辑与我们去年发表的scRNA-Seq和scATAC-seq研究（RhodesPetros Nature Communications 2022; LeePetros eLife 2022）相结合，我们希望将CGE祖细胞中的早期转录和染色质可及性特征与成熟的中间神经元命运联系起来。第二，我们已经开发了一种Perturb-Seq方法来识别促进CGE衍生的中间神经元的候选基因。我们正在使用CRISPR-Cas9小鼠胚胎干细胞（mESC）来激活或抑制参与神经发育的一系列基因，使用由靶向感兴趣基因的众多gRNA组成的慢病毒文库。我们将跟踪任何有趣的，决定命运的基因候选命中从这个扰动序列筛选，以探索他们在CGE衍生的中间神经元的可能作用。第三，基于我们scRNA-seq实验（LeePetros eLife 2022）的见解，我们已经生成了新的转基因小鼠模型，目标是在发育过程中特异性靶向CGE衍生的中间神经元。我们目前正在测试这种小鼠系在神经发育过程中标记CGE衍生的中间神经元（或特定子集）的特异性和有效性。结合起来，这一系列的实验应该显着增加我们的了解机制调节CGE衍生的中间神经元。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Neocortical Projection Neurons Instruct Inhibitory Interneuron Circuit Development in a Lineage-Dependent Manner.

DOI：
10.1016/j.neuron.2019.03.036
发表时间：
2019-06-05
期刊：
Neuron
影响因子：
16.2
作者：
Wester JC;Mahadevan V;Rhodes CT;Calvigioni D;Venkatesh S;Maric D;Hunt S;Yuan X;Zhang Y;Petros TJ;McBain CJ
通讯作者：
McBain CJ

Transcriptional heterogeneity of ventricular zone cells in the ganglionic eminences of the mouse forebrain.

小鼠前脑神经节隆起的心室区细胞的转录异质性。

DOI：
10.7554/elife.71864
发表时间：
2022-02-17
期刊：
eLife
影响因子：
7.7
作者：
Lee DR;Rhodes C;Mitra A;Zhang Y;Maric D;Dale RK;Petros TJ
通讯作者：
Petros TJ

Temporal Dynamics and Neuronal Specificity of Grin3a Expression in the Mouse Forebrain.

DOI：
10.1093/cercor/bhaa330
发表时间：
2021-03-05
期刊：
Cerebral cortex (New York, N.Y. : 1991)
影响因子：
0
作者：
Murillo A;Navarro AI;Puelles E;Zhang Y;Petros TJ;Pérez-Otaño I
通讯作者：
Pérez-Otaño I

NMDARs Drive the Expression of Neuropsychiatric Disorder Risk Genes Within GABAergic Interneuron Subtypes in the Juvenile Brain.

DOI：
10.3389/fnmol.2021.712609
发表时间：
2021
期刊：
Frontiers in molecular neuroscience
影响因子：
4.8
作者：
Mahadevan V;Mitra A;Zhang Y;Yuan X;Peltekian A;Chittajallu R;Esnault C;Maric D;Rhodes C;Pelkey KA;Dale R;Petros TJ;McBain CJ
通讯作者：
McBain CJ

An epigenome atlas of neural progenitors within the embryonic mouse forebrain.