MOLECULAR REGULATION OF LINEAGE SPECIFICATION OF THE MOUSE CEREBELLUM

摘要 除了感觉-运动处理外，小脑还参与高级认知 功能。因此，小脑病理和功能障碍与许多衰弱的发育有关。 自闭症、谱系障碍和其他智力缺陷等疾病。研究新技术的产生与发展 小脑中神经元和神经胶质细胞的适当数量和多样性将促进我们对 小脑环路的组装和小脑相关疾病的细胞基础。在.期间 胚胎发育过程中，各种GABA能小脑神经元，如浦肯野细胞、深部中间神经元 小脑核团和小脑皮质起源于明确的小脑室带 发展窗口。相比之下，小脑谷氨酸能神经元起源于小脑的菱形核 唇部，第二生发区，在第四脑室带和顶板之间的交界处 脑室。不同类型小脑细胞产生的分子机制是 不完全理解。特别是，菱形嘴唇是如何产生小脑核神经元的， 颗粒细胞和单极刷状细胞在时间上有限制的顺序，以及如何为不同的前体 构成小脑唯一输出的小脑核团在很大程度上是未知的。我们建议使用 单细胞RNA测序(ScRNAseq)研究细胞的发育程序 小鼠小脑神经原基的定型化与分化。小鼠胚胎小脑的初步研究 第(E)13.5天已经证明了scRNAseq在分类细胞类型或细胞方面的可行性和能力 各国之间的合作以及重建发展轨迹。申请的具体目的如下：1. 确定小鼠小脑的细胞组成和谱系关系。要测试 假设小脑细胞类型在细胞出生时获得一致性分子特征，我们将 进行大规模定量scRNAseq以鉴定细胞群体及其定义分子 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 由scRNAseq确定的不同细胞群的时空控制的出生和迁移。这个 组织学研究将通过以特定谱系为目标的小鼠突变的特征来加强。2. 确定小脑核团规范背后的分子机制。测试我们的 转录因子Meis2、Pax6和Orig2控制小脑发育的工作假说 细胞核，我们将在鸡和小鼠胚胎中使用电穿孔分析，并利用小鼠遗传学来确定 这些转录因子的功能增强或丧失如何影响小脑核团的规格。

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.