MOLECULAR REGULATION OF LINEAGE SPECIFICATION OF THE MOUSE CEREBELLUM

小鼠小脑谱系规范的分子调控

基本信息

批准号：
10158523
负责人：
JAMES Y.H LI
金额：
$ 41.45万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10158523
关键词：
Adult Affect Alar Anatomy Biological Assay Birth Brush Cell Cell Lineage Cells Cerebellar Cortex Cerebellar Diseases Cerebellar Nuclei Cerebellum Chick Complex Data Development Disease Electroporation Embryo Embryonic Development Functional disorder Gene Expression Generations Genetic Genetic Transcription Glutamates Heterogeneity Histologic Human Individual Interneurons Knowledge Lateral Link Lip structure Medial Midbrain structure Molecular Molecular Profiling Motor Mus Mutation Neuraxis Neuroglia Neurons Nuclear Output Pathology Pattern Pilot Projects Population Purkinje Cells Regulation Regulator Genes Role Sensory Specific qualifier value Structure Testing Time Ventricular autism spectrum disorder cell motility cell type cognitive function data mining developmental disease gamma-Aminobutyric Acid granule cell histogenesis histological studies in vivo insight loss of function malformation migration motor deficit mouse genetics novel programs single-cell RNA sequencing spatiotemporal transcription factor transcriptome

项目摘要

SUMMARY In addition to its sensory-motor processing, the cerebellum is also involved in higher cognitive function. Accordingly, cerebellar pathology and dysfunction are linked to many debilitating developmental diseases like autism spectrum disorder and other intellectual deficits. Studying the generation of the proper number and diversity of neurons and glia in the cerebellum will advance our knowledge of the assembly of cerebellar circuits and the cellular basis of cerebellum-related disorders. During embryogenesis, various GABAergic cerebellar neurons, such as Purkinje cells, interneurons of deep cerebellar nuclei and the cerebellar cortex, arise from the cerebellar ventricular zone in defined developmental windows. By contrast, cerebellar glutamatergic neurons arise from the cerebellar rhombic lips, the second germinal zone, at the interface between the ventricular zone and roof plate of the forth ventricle. The molecular mechanisms underlying the generation of different cerebellar cell types are incompletely understood. In particular, how the rhombic lip gives rise to cerebellar nuclear neurons, granule cells and unipolar brush cells in temporally restricted orders, and how precursors for different cerebellar nuclei, which form the sole output of the cerebellum, are largely unknown. We propose to use single-cell RNA-sequencing (scRNAseq) to investigate the developmental programs underlying cell specification and differentiation in mouse cerebellar anlage. A pilot study of mouse cerebella at embryonic day (E) 13.5 has demonstrated the feasibility and power of scRNAseq in classifying cell types or cell states, and reconstructing developmental trajectories. The specific aims of the application as follows: 1. Define the cellular composition and lineage relationship in the mouse cerebellum. To test the working hypothesis that cerebellar cell types acquire coherence molecular signatures at cell birth, we will perform large-scale quantitative scRNAseq to identify cell populations and their defining molecular features in mouse cerebella between E11.5 and adult. The scRNAseq data will be used to infer the trajectory of cell lineages. Histological analyses will be performed to validate and define the spatiotemporally controlled birth and migration of various cell groups identified by scRNAseq. The histological studies will be augmented by characterizing mouse mutations that target defined lineages. 2. Determine the molecular mechanism underlying the specification of cerebellar nuclei. To test our working hypothesis that transcription factors Meis2, Pax6, and Olig2 control the development of cerebellar nuclei, we will use electroporation assays in chick and mouse embryos, and mouse genetics to determine how gain- or loss-of-function of these transcription factors affects the specification of cerebellar nuclei.

概括除了感觉运动处理之外，小脑还参与高级认知功能。因此，小脑病理学和功能障碍与许多发育衰弱有关。自闭症谱系障碍和其他智力缺陷等疾病。研究一代小脑中神经元和神经胶质细胞的适当数量和多样性将增进我们对小脑的认识小脑回路的组装和小脑相关疾病的细胞基础。期间胚胎发生、各种 GABA 能小脑神经元，例如浦肯野细胞、深部中间神经元小脑核和小脑皮质，起源于定义的小脑脑室区发展窗口。相比之下，小脑谷氨酸能神经元来自小脑菱形神经元嘴唇，第二生发区，位于心室区和第四生发区顶板之间的界面处心室。不同小脑细胞类型产生的分子机制是不完全理解。特别是菱形唇如何产生小脑核神经元，颗粒细胞和单极刷细胞按时间限制的顺序，以及不同的前体细胞如何小脑核是小脑的唯一输出，但人们对它知之甚少。我们建议使用单细胞 RNA 测序 (scRNAseq) 用于研究细胞的发育程序小鼠小脑原基的规范和分化。小鼠胚胎小脑的初步研究第（E）13.5天证明了scRNAseq在细胞类型或细胞分类方面的可行性和力量状态，并重建发展轨迹。申请的具体目的如下： 1. 定义小鼠小脑中的细胞组成和谱系关系。测试假设小脑细胞类型在细胞出生时获得了一致的分子特征，我们将进行大规模定量 scRNAseq 来识别细胞群及其定义分子 E11.5 和成年小鼠小脑的特征。 scRNAseq 数据将用于推断细胞谱系的轨迹。将进行组织学分析以验证和定义 scRNAseq 识别的各种细胞群的时空控制出生和迁移。这通过表征针对特定谱系的小鼠突变，将增强组织学研究。 2. 确定小脑核规格背后的分子机制。来测试我们的转录因子 Meis2、Pax6 和 Olig2 控制小脑发育的工作假设细胞核，我们将在鸡和小鼠胚胎中使用电穿孔测定，以及小鼠遗传学来确定这些转录因子功能的获得或丧失如何影响小脑核的规格。