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.
This proposal seeks to study how the great diversity of cerebellar neuronal and glial cells arises during embryogenesis. Determining the developmental trajectory by which molecularly distinct cell types are produced and assembled into cerebellar circuits will be central to understanding developmental cerebellar disorders associated with autism, and other intellectual and motor deficits.
