Mapping the developmental profile of ventricular zone-derived neurons in the human cerebellum

Abstract

The cerebellar ventricular zone (VZ) is the primary source of progenitors that generate cerebellar GABAergic neurons, including Purkinje cells (PCs) and interneurons (INs). This study provides detailed characterization of human cerebellar GABAergic neurogenesis using transcriptomic and histopathological analyses and reveals conserved and unique features compared to rodents. We show that the sequential progression of neurogenesis is conserved and occurs before 8 postconception weeks. Notably, PC differentiation occurs in the outer subventricular zone (SVZ), a region absent in the mouse cerebellum. Human PCs are generated during a compact two-week period before the onset of cerebral cortex histogenesis. A subset of human PCs retain proliferative marker expression weeks after leaving the VZ, another feature not observed in rodents. Human PC maturation is protracted with an extensive migration and reorganization throughout development with dendritic arborization developing in late gestation. We define a continuous transcriptional cascade of PC development from neuroepithelial cells to mature PCs. In contrast, while human interneuronal progenitors are born beginning in early fetal development, they exhibit an even more protracted differentiation across late gestation and into postnatal ages. These findings show dynamic developmental process for human cerebellar GABAergic neurons and underscore the importance of the embryonic environment, with early disruptions having potentially significant impacts.

Keywords: Purkinje; cerebellum; development; human; ventricular zone.

Fig. 1.

Single nucleus RNAseq of the human cerebellum at CS21-23 reveals two distinct developmental trajectories. (A) Illustration of a midsagittal section of the human embryo with the dorsal (d, red arrowhead) and ventral ends (v, black arrowhead) of the VZ/SVZ marked. The regions in gray indicate the RL and RL-trajectories, while those in yellow indicate the VZ and SVZ. (B) KI67 expression at CS14 (32 postconception days), CS19 (47.5pcd), CS21 (52pcd), and CS23 (56.5pcd). Yellow, light and dark green vertical lines indicate VZ, inner and outer SVZ respectively. Sections have been counterstained with DAPI. [Scale bar, 100 μm (red).] (C) UMAP embedding of 20,749 nuclei from the developing human cerebellum between CS21–CS23. (Left) Transcriptionally distinct clusters are colored by cell type annotation, Carnegie stage, cell cycle phase, and inferred pseudotime using scFates (8), where darker colors indicate less differentiated cells. The SOX14+ GABA CN cluster is present throughout the subpanels of Fig. 1C, except for the scFates pseudotime subpanel, where it was removed to facilitate pseudotime reconstruction. (Right) Cells colored by broad cell type annotation and quantified proportionally by Carnegie stage. (D) Expression of key genes used to annotate cellular clusters in (A). (E) Volcano plot for DEGs in the NE cluster of (C) and RNAscope ISH expression of DEGs in the NE cluster—LRP2, PTPRZ1, WNT7B. [Scale bar, 100 μm (white).] (F) Expression plots for GABAergic PC lineage marker genes. Expression of cluster-specific marker genes across pseudotime as determined in Fig. 1C as cells differentiate into PCs from the VZ. (G) UMAP embedding as in Fig. 1C colored by expression of GABAergic lineage marker genes.

Fig. 2.

Compartmentalization of the human cerebellar VZ. (A–G) RNAscope in situ hybridization indicating RNA expression of PTF1A, MKI67 (Inset), OLIG2, GSX1, LHX1, and SKOR2 in the human cerebellum at ages CS19 (A and F), CS20 (B), CS21 (G), CS23 (C), 8PCW (D), and 10PCW (E). [Scale bar, 100 μm (red); 200 μm (black).] Black and red arrow heads (A–E) in the PTF1A images mark the ventral and dorsal limit of VZ/SVZ expression, while the purple arrowhead in the GSX1 images tracks the extent of dorsal GSX1 expansion. Yellow arrowheads in C and G indicate OLIG2 expression in the nuclear transitory zone (NTZ). Arrowheads in F and G indicate the dorsal and ventral limit of PTF1A (purple), OLIG2 (blue), and GSX1 (orange) expression. Higher magnification images show that while the GSX1+ domain partially overlaps with the PTF1A domain, it does not fully coincide. Additionally, OLIG2 and GSX1 are not coexpressed, confirming that these two domains are distinct.

Fig. 3.

Spatial transcriptomics of compartments of the human cerebellar VZ and SVZ. (A) Representative immunofluorescent section from medial cerebellum at CS20 immunostained with KI67 (Green) and DAPI (Blue) used as input for GeoMx spatial transcriptional profiling. Regions of RNA capture are indicated. (Scale bar, 250 μm.) (B) Transcriptional heatmap from CS19/20 GeoMx samples (n = 3) reveals a differentiation cascade from VZ to oSVZ. (C) Spatial differential expression analysis from the indicated comparisons identifies VZ and oSVZ-specific gene expression profiles.

Fig. 4.

Distribution of PIPs and INs across human cerebellar development. (A–M) Expression of PAX2, PVALB, and STT in the developing human cerebellum. [Scale bar, 100 μm (black); 200 μm (red); 500 μm (blue).] (N) Timeline of PIPs development in humans.

Fig. 5.

Arrangement of PCs in the developing human cerebellum. (A–J) Expression of Calbindin in the developing human cerebellum. White and red asterisks in (A) and (B) point to the upper and lower PC plate. Arrows in (C) and (D) indicate the likely migrating PCs and PC axons respectively. [Scale bar, 50 μm (red), 100 μm (white), 200 μm (yellow), 500 μm (blue), 1000 μm (green).] (K) Timeline of PC development in humans documenting changes in PC morphology. Drawings of PCs have been traced from actual images in SI Appendix, Fig. S4.

Fig. 6.

Single cell/nucleus developmental trajectory of human cerebellar inhibitory neurons across gestation and adulthood. (A) UMAP embeddings of 6 published datasets integrated with CS21-23 (6-8PCW) snRNAseq samples from this study colored by cell sample age, cell type, and cell cycle phase. (B) Marker gene expression plots for progenitors (SOX2, MKI67, PTF1A, PRDM13), GABAergic neurons (LHX1), PCs (SKOR2, PCP4, ESRRB, CALB1, FOXP1, FOXP2, EBF1, EBF2), and INs (PAX2, GRIK3, KCNA2, SORCS3, NXPH1) indicate discrete cell states along branching lineages toward PC and IN terminal fates across ages CS21 (6PCW)—52 y. (C) UMAP embeddings of the subset of interneuronal trajectory from the integrated human cerebellar GABAergic neuron atlas in Fig. 6A colored by cell sample age, cell type, and cell cycle phase. (D) Expression plots for GABAergic lineage and Inhibitory interneuron marker genes. (E) UMAP embeddings of the subset of PC-lineage cells. (F) Expression plots for GABAergic lineage and PC marker genes. (G) Marker gene expression over PC-lineage pseudotime using Slingshot reveals a transcriptional trajectory of differentiation from neuroepithelial VZ cells to CALB1+ Purkinje cells (PCs).

Fig. 7.

A subset of human PCs express cell cycle genes. (A) RNAscope in situ hybridization indicating RNA expression of PRDM13 at CS19, CS21. (B) Comparison of relative cell fractions positive for MKI67, PRDM13 (P = 0.9775), and SKOR2 (P = 2.932e−13) in human and mouse cerebellar scRNAseq datasets. Statistical significance was assessed using a two-sample proportion test using the R package {stats} function prop.test() (C) MKI67 PRDM13 and MKI67 SKOR2 expression at CS22. (D) Immunofluorescence sections confirming the presence of PCs (SKOR2+) positive for proliferative marker PCNA. [Scale bar, 50 μm (white); 100 μm (red).]