Multimodal Validation of the Existence of Transitional Cerebellar Progenitors in the Human Fetal Cerebellum

The developing human cerebellum comprises a series of transient progenitor states that are essential for generating diverse neural subtypes, yet the identity and validation of intermediate cell populations bridging stem-like and lineage-committed neuronal precursors remain limited. In our previous single-cell transcriptomic study, we identified a distinct transitional cerebellar progenitor (TCP) population enriched in specific progenitor zones such as the rhombic lip during human fetal cerebellar development. To address the concerns raised in a Matters Arising regarding the existence of the TCP cells, we provide additional multimodal validations of this population. Rigorous reanalysis of our single-cell transcriptomic data, applying stringent quality control measures, validated the quality of TCP cells and their classification as a transcriptionally distinct population. Multiple orthogonal validations of TCP signature genes (SOX11 and HNRNPH1) using RNAscope in situ hybridization, Xenium-based spatial transcriptomics, and immunohistochemistry on additional fetal cerebellar samples across different stages demonstrated the consistent presence of TCPs in the rhombic lip, transitioning from PRTG⁺ stem-like zones in the ventricular zone at early developmental stages to the subventricular zone overlapping with EOMES⁺ unipolar brush cell precursors at later stages. TCP-like populations were also independently identified in two fetal cerebellar single-nucleus transcriptomic atlases, and their gene signature was enriched in a cell population associated with aggressive medulloblastomas. Collectively, these multimodal validations confirm the existence of a transitional progenitor population in the human fetal cerebellum, with implications for cerebellar lineage progression and medulloblastoma origin.