Cerebellar development relies on the coordinated proliferation and differentiation of progenitors from the ventricular zone (VZ) and rhombic lip (RL). To systematically map their spatiotemporal dynamics, we performed EdU pulse labeling by injecting pregnant mice with EdU and collecting embryonic cerebella at daily intervals over five consecutive days as well as an acute half-an-hour post EdU injection. EdU labeling identifies actively dividing progenitor cells at the time of injection. As development progresses, EdU+ cells can be tracked to study their differentiation and migration, revealing the temporal dynamics of VZ and RL progenitor-derived neurons in the cerebellum. Using multiplex immunohistochemistry with VZ- and RL-derived cell-type specific markers, we tracked the spatial distribution and differentiation of EdU-labeled cells, distinguishing VZ- and RL-derived progenitor lineages. Additionally, we outline a strategy to isolate EdU+ cells for single-cell RNA sequencing (scRNA-seq) and ATAC sequencing (ATAC-seq), enabling a comprehensive molecular characterization of progenitor fate transitions. This approach provides a high-resolution developmental trajectory of cerebellar progenitors, offering new insights into the regulatory mechanisms driving cerebellar neurogenesis and their disruptions in neurodevelopmental disorders.

The cerebellar ventricular zone (VZ) is the primary source of progenitor cells that give rise to all cerebellar GABAergic neurons, including Purkinje cells (PCs) and interneurons (INs). While the VZ has been well studied in mice, much less is known about its role in human brain development. In this study, we investigated how progenitors and neurons form in the human cerebellar VZ, using in situ hybridization, immunohistochemistry, and single-cell RNAseq analysis. Our findings reveal several key differences from the mouse model. We found that Purkinje cells are generated during a brief two-week period, even before the cerebral cortex begins to develop. Interneurons, on the other hand, start differentiating a few weeks later and mature on a timescale of months to years. A unique feature of human cerebellar development is the presence of specialized inner and outer subventricular zones (SVZ), which are absent in mice. Most differentiation occurs in these regions, with the first wave taking place in the outer SVZ. Additionally, we observed variations in Purkinje cell arrangement and number, including a subset of Purkinje cells that continue expressing cell cycle genes, suggesting a more complex and prolonged developmental profile compared to mice. By characterizing these developmental processes, our study provides new insights into human cerebellar development, highlighting important structural and temporal differences from animal models. These findings may have implications for understanding neurodevelopmental disorders.