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，为大脑皮层的发展。在此期间，NP增殖的改变可能会有毁灭性的神经元数和电路的后果最终可能导致各种神经发育诸如自闭症，小头和大脑的疾病。因此，了解管理的机制早期NP扩展对于理解这些疾病至关重要。 ZBTB7A的错义突变或删除导致脑畸形和智力残疾，但这些表型的机制是完全未知。我们在胚胎小鼠NP的核中发现了ZBTB7A的富集，以及我们的ZBTB7A有条件KO（CKO）小鼠模型显示出出生时皮质厚度增加，早期胚胎皮质中祖细胞池的扩展。 ZBTB7A的过表达导致相反表型，NP的过早差异。接下来，我们使用剪切和运行来识别ZBTB7A目标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剂组成的调节子。我们确认ZBTB7A与 HES5的启动子，我们观察到ZBTB7A可以响应于Hes5启动子活动激活的缺口。最后，我们在E15 NP中使用了一种新型的体内生物形式方法来发现ZBTB7A相互作用者。该分析表明ZBTB7A与Gatad2a/B蛋白（核小体的两个成分）相互作用重塑和脱乙酰基酶复合物（NURD）抑制剂复合物。在这项研究中，我们将使用有条件的敲除鼠标模型，以进一步表征ZBTB7A如何影响NP增殖和建立皮质建筑。在第二步中，我们将检查ZBTB7A如何调节Notch信号来控制NP 增殖。我们将在主要NP和子宫电穿孔中使用荧光素酶测定来测试 ZBTB7A子域以及ZBTB7A患者突变如何影响Notch靶标的表达。三分之一步骤我们将评估ZBTB7A如何与NURD复合物合作以调节NP的增殖。为此我们将使用体内生物治疗以及共免疫沉淀实验来发现NURD的组成复合物与ZBTB7A相互作用。我们将在开发中较早地重复生化实验，我们将使用共免疫沉淀以定义哪些复杂蛋白与早期NPS中的ZBTB7A相关。角色 NPS中的Gatad2b的Gatad2b是完全未知的，因此我们将使用IUE来测试Gatad2b模拟的敲低与ZBTB7A CKO相关的NP增殖表型，并确定Gatad2b在ZBTB7A中的需求过表达表型，包括抑制缺口靶标。这些研究总共描述新的核心原理，这些原则驱动皮质生成并将加深我们对病因的理解神经发育障碍。