Transcriptional regulation of neural progenitor divisions and cell fate in the developing cortex

发育中皮层神经祖细胞分裂和细胞命运的转录调控

基本信息

批准号：
10659677
负责人：
Louis-Jan Pilaz
金额：
$ 41.5万
依托单位：
SANFORD RESEARCH/USD
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-15 至 2028-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10659677
关键词：
Affect Architecture Binding Biological Assay Birth Cell Cycle Cell Nucleus Cell Proliferation Cells Cerebral cortex Co-Immunoprecipitations Complex Core Protein Data Deacetylase Defect Development Disease Electroporation Embryo Embryonic Development Enhancers Epigenetic Process Erythroid Cells Etiology Gene Expression Genes Hematopoietic stem cells Human Hybrids Image Intellectual functioning disability Knockout Mice Lead Link Luciferases Macrocephaly Measures Missense Mutation Modeling Molecular Mus Mutation Neurodevelopmental Disorder Neurons Nucleosomes Pathway interactions Patients Phenocopy Phenotype Production Proliferating Proteins Publishing Regulation Regulon Reporting Repression Repressor Proteins Role Signal Transduction Testing Thick Tissues Transcriptional Regulation Yeasts autism spectrum disorder brain size cell type conditional knockout critical period experimental study histogenesis in utero in vivo insight knock-down mouse model nerve stem cell neuroregulation notch protein novel overexpression postnatal premature progenitor promoter protein complex response single-cell RNA sequencing transcription factor transcriptome

项目摘要

Summary The early expansion of the neural progenitor (NP) pool is a critical period, setting the stage for the development of the cerebral cortex. Alterations in NP proliferation during that period can have devastating consequences on neuron numbers and circuitry that could eventually lead to a variety of neurodevelopmental diseases such as autism, micro- and macrocephaly. Therefore, understanding the mechanisms that govern early NP expansion is central to understanding these diseases. Missense mutations or deletion of ZBTB7A lead to macrocephaly and intellectual disability but the mechanisms underlying these phenotypes are completely unknown. We discovered an enrichment for ZBTB7A in the nuclei of embryonic mouse NPs, and our Zbtb7a conditional KO (cKO) mouse model show increased cortical thickness at birth and an early expansion of the progenitor pool in embryonic cortices. Overexpression of ZBTB7A leads to opposite phenotypes, with premature differentiation of NPs. Next, we used CUT&RUN to identify ZBTB7A target genes during early cortical development. This analysis revealed that ZBTB7A binds to the promoters and enhancers of a regulon composed of transcription factors and cell-cycle regulators. We confirmed that ZBTB7A binds to the promoter of Hes5, and we observed that ZBTB7A can block Hes5 promoter activity in response to activated Notch. Finally, we used a novel in vivo BioID approach in E15 NPs to discover ZBTB7A interactors. This analysis suggests that ZBTB7A interacts with GATAD2A/B proteins, two components of the Nucleosome Remodeling and Deacetylase complex (NuRD) repressor complex. In this study, we will use a conditional knockout mouse model to further characterize how Zbtb7a impacts NP proliferation and the establishment of cortical architecture. In a second step, we will examine how ZBTB7A modulates Notch Signaling to control NP proliferation. We will use luciferase assays in primary NPs and in utero electroporation to test the relevance of ZBTB7A sub domains and how ZBTB7A patient mutations affect the expression of Notch targets. In a third step we will evaluate how ZBTB7A cooperates with the NuRD complex to regulate NP proliferation. For this we will use in vivo BioID as well as co-immunoprecipitation experiments to discover the composition of NuRD complexes interacting with ZBTB7A. We will repeat BioID experiments earlier in development and we will use co-immunoprecipitation to define which NuRD complex proteins associate with ZBTB7A in early NPs. The role of Gatad2b in NPs is completely unknown, therefore we will use IUE to test if knockdown of Gatad2b mimics NP proliferation phenotypes linked with Zbtb7a cKO, and to determine the requirement of Gatad2b in Zbtb7a overexpression phenotypes, including the repression of Notch targets. Altogether these studies will describe novel core principles that drive corticogenesis and will deepen our understanding of the etiology of neurodevelopmental disorders.

概括神经祖细胞（NP）池的早期扩张是一个关键时期，为神经祖细胞（NP）池的早期扩张奠定了基础。大脑皮层的发育。在此期间 NP 增殖的变化可能会产生毁灭性的影响对神经元数量和电路的影响最终可能导致各种神经发育障碍疾病，如自闭症、小头畸形和大头畸形。因此，了解控制机制早期 NP 扩展对于了解这些疾病至关重要。 ZBTB7A 错义突变或缺失导致巨头畸形和智力障碍，但这些表型背后的机制是完全未知。我们在胚胎小鼠 NP 的细胞核中发现了 ZBTB7A 的富集，并且我们的 Zbtb7a 条件性 KO (cKO) 小鼠模型显示出生时皮质厚度增加，并且早期胚胎皮质中祖细胞库的扩张。 ZBTB7A 的过度表达会导致相反的结果表型，具有 NP 的过早分化。接下来，我们使用CUT&RUN来鉴定ZBTB7A靶基因在早期皮质发育期间。该分析表明 ZBTB7A 与启动子和增强子结合由转录因子和细胞周期调节因子组成的调节子。我们确认 ZBTB7A 结合 Hes5 的启动子，我们观察到 ZBTB7A 可以阻断 Hes5 启动子活性，以响应激活缺口。最后，我们在 E15 NP 中使用了一种新颖的体内 BioID 方法来发现 ZBTB7A 相互作用子。该分析表明 ZBTB7A 与 GATAD2A/B 蛋白（核小体的两个组成部分）相互作用重塑和脱乙酰酶复合物 (NuRD) 阻遏复合物。在本研究中，我们将使用条件敲除小鼠模型进一步表征 Zbtb7a 如何影响 NP 增殖和建立皮质结构。第二步，我们将研究 ZBTB7A 如何调节 Notch 信号来控制 NP 增殖。我们将在原代纳米粒子和子宫内电穿孔中使用荧光素酶测定来测试以下内容的相关性： ZBTB7A 子结构域以及 ZBTB7A 患者突变如何影响 Notch 靶标的表达。在第三个步骤我们将评估 ZBTB7A 如何与 NuRD 复合物合作调节 NP 增殖。为此我们将使用体内 BioID 以及免疫共沉淀实验来发现 NuRD 的成分与 ZBTB7A 相互作用的复合物。我们将在开发早期重复 BioID 实验，并且我们将使用免疫共沉淀来确定哪些 NuRD 复合蛋白与早期 NP 中的 ZBTB7A 相关。角色 Gatad2b 在 NP 中的表达是完全未知的，因此我们将使用 IUE 来测试 Gatad2b 的敲低是否模拟 NP增殖表型与Zbtb7a cKO相关，并确定Zbtb7a中Gatad2b的需求过度表达表型，包括Notch靶标的抑制。总的来说，这些研究将描述驱动皮质生成并将加深我们对病因学的理解的新颖核心原理神经发育障碍。