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

• 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
• 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

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.

总结 神经前体细胞库的早期扩增是一个关键时期，为神经前体细胞库的形成奠定了基础。 大脑皮层的发育。在此期间，NP增殖的变化可能具有破坏性 对神经元数量和电路的影响，最终可能导致各种神经发育 自闭症、小头症和大头症等疾病。因此，了解这些机制 早期NP扩增是理解这些疾病的关键。ZBTB 7A的错义突变或缺失 导致大头畸形和智力残疾，但这些表型背后的机制是 完全未知我们发现ZBTB 7A在胚胎小鼠NP的细胞核中富集， 我们的Zbtb 7a条件性KO（cKO）小鼠模型显示出生时皮质厚度增加， 胚胎皮质中祖细胞库的扩大。ZBTB 7A的过表达导致相反的结果。 表型，具有NP的过早分化。接下来，我们使用CUT&RUN来鉴定ZBTB 7A靶基因， 在早期皮质发育过程中。该分析显示ZBTB 7A结合至启动子和增强子， 由转录因子和细胞周期调节因子组成的调节子。我们证实了ZBTB 7A与 我们观察到ZBTB 7A可以阻断Hes 5启动子活性， 激活Notch最后，我们在E15 NPs中使用了一种新的体内BioID方法来发现ZBTB 7A相互作用物。 该分析表明ZBTB 7 A与核小体的两种组分GATAD 2 A/B蛋白相互作用 重塑和脱乙酰酶复合物（NuRD）阻遏物复合物。在本研究中，我们将使用条件 基因敲除小鼠模型，以进一步表征Zbtb 7a如何影响NP增殖和 皮质结构在第二步中，我们将研究ZBTB 7A如何调节Notch信号传导以控制NP 增殖我们将在原代NP和子宫内电穿孔中使用荧光素酶测定来测试以下的相关性： ZBTB 7A亚结构域以及ZBTB 7A患者突变如何影响Notch靶标的表达。在第三 第一步，我们将评估ZBTB 7A如何与NuRD复合物合作来调节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的组成 与ZBTB 7A相互作用。我们将在开发的早期重复BioID实验， 免疫共沉淀以确定早期NP中哪些NuRD复合物蛋白与ZBTB 7A缔合。的作用 Gatad 2b在NP中的表达是完全未知的，因此我们将使用IUE来测试Gatad 2b的敲除是否模拟 与Zbtb 7a cKO相关的NP增殖表型，并确定Zbtb 7a中Gatad 2b的需求 过表达表型，包括Notch靶的抑制。这些研究将描述 新的核心原则，驱动皮质生成，并将加深我们对病因的理解， 神经发育障碍