A Rare Nestin-Expressing Granule Cell Precursor Subpopulation Underlies SHH Medulloblastoma Formation

Salsabiel El Nagar author has email address
Yinwen Liang
Daniel N Stephen
Synphen H Wu
Alexandra L Joyner author has email address

Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States
MangroveNeuro, Biolabs@NYULangone, Long Island City, United States
Capital Medical University and Chinese Institutes for Medical Research, Beijing, China
Department of Neurology, Westchester Medical Center, Valhalla, United States
Biochemistry, Cell and Molecular Biology Program, Weill Cornell Graduate School of Medical Sciences, New York, United States

https://doi.org/10.7554/eLife.111770.1

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Figures and data

Nes-expressing granule cell precursors (GCPs) are enriched in the posterior cerebellum and display reduced proliferation.
(A-D) Immunofluorescence staining of sagittal cerebellar sections from Nes-CFP mice at P1 showing CFP (green), BARHL1 (red) and DAPI (blue). Sections represent four distinct levels on the lateral-medial axis. Panels (A’-D’) and (A’’-D’’) show high-magnification images of anterior and posterior regions indicated by white dashed boxes in A-D. White arrowheads indicate examples of CFP⁺ BARHL1⁺ double positive cells. Yellow dashed circle indicates a cochlear nucleus. N>3 mice (E) Quantification of the CFP⁺ BARHL1⁺ cells normalized to EGL length along the lateral-medial axis in the anterior cerebellum and posterior cerebellum. The posterior cerebellum corresponds to the last cerebellar lobule (fold) delineated by white brackets in panel A-D, whereas the anterior corresponds to the remaining EGL. N=5 mouse samples. (F) Total number of CFP⁺ BARHL1⁺ cells per cerebellum at P1 in the posterior, anterior and entire cerebellar compartments. Total number of CFP⁺ BARHL1⁺ in the posterior is 12258 ± 106 cells and in the anterior is 5931 ± 76 cells. N=4 mouse samples. (G-J) Immunofluorescence staining of sagittal cerebellar sections from Nes-CFP mice at P1 showing CFP (green), SOX2 (red) and BARHL1 (blue). Panels show high-magnification images of the posterior cerebellum at four distinct lateral-medial levels. White arrowheads indicate some CFP⁺ SOX2⁺ BARHL1⁺ triple positive cells. N>3 mice. (K) Quantification of the number of CFP⁺ BARHL1⁺, SOX2⁺ BARHL1⁺ and CFP⁺ SOX2⁺ BARHL1⁺ cells per section in the posterior EGL of Nes-CFP cerebella at P1. N=4 mouse samples (L) Percentage of CFP⁺ BARHL1⁺ (69.2±4.2%), SOX2⁺ BARHL1⁺ (10.9±4.5%) and CFP⁺ SOX2⁺ BARHL1⁺ (19.8±1.3%) cells per in the posterior EGL of Nes-CFP cerebella at P1. N=4 mouse samples. (M) Immunofluorescence staining of a midsagittal cerebellar section from Nes-CFP mice at P1 showing CFP (green), EdU (red) and BARHL1 (blue). (M’) High magnification image of the posterior EGL. White arrowheads indicate some CFP⁺ EdU⁺ BARHL1⁺ the triple positive cells. (N) Quantification of the proportion of EdU⁺ cells within BARHL1⁺ CFP^- and BARHL1⁺ CFP⁺ populations. N=4 mousse samples. Statistical significance was determined using an unpaired t-test. Scale bar: A-D, M: 500µm; A’-D’, A’’-D’’, G-J, M: 50µm. Data are represented as mean ±SEM.

Nes-expressing GCPs can be isolated by flow cytometry and co-express Atoh1.

(A) Representative flow cytometry analysis of transgene negative (wild-type), Atoh1-GFP and Nes-CFP mouse cerebella at P1 used as controls and of Nes-CFP; Atoh1-GFP mouse cerebella to isolate the 4 cell populations GFP^- CFP^- (double negative), GFP⁺ CFP^- (Atoh1⁺ only), GFP- CFP+ (Nes⁺ only) and GFP⁺ CFP⁺ (double positive cells) in the Atoh1-GFP; Nes-CFP mouse cerebella. (B) Quantification of each population as a percentage of total live cells from P1 Nes-CFP, Atoh1-GFP cerebella. N=4 mouse samples. Percentages of each population ± SEM are: GFP^- CFP^-: 20.25±7.87; GFP⁺ CFP^-:68.70±8.55; GFP^- CFP⁺: 9.93±0.84; GFP⁺ CFP⁺: 1.19±0.23. (C-D) RT-qPCR analysis of sorted cell populations showing relative gene expression (fold change relative to GFP^- CFP^- cells). Panel (C) shows the single positive populations (GFP⁺ CFP^- and GFP^-CFP⁺ cells) and (D) shows the double positive population GFP⁺ CFP⁺. N=3 and 4 biological replicates, normalized to Gapdh, respectively. See also Figure 2–Source data 1. Data are represented as mean ±SEM.

Nes-driven and Atho1-driven SHH medulloblastoma models show similar tumor penetrance and preferential growth of tumors laterally.

(A) Schematic representation of transgenes and gene loci in mosaic mutant tumor models Atoh1-FlpoER; R26^{FSF-GFPcre/SmoM2}; Trp53^fl/fl (Atoh1-SmoM2; Trp53^flfl) and Nes-FlpoER; R26^{FSF-GFPcre/SmoM2}; Trp53^fl/fl (Nes-SmoM2; Trp53^flfl) before and after tamoxifen injection at P0. (B) Kaplan-Meier curves of Atoh1- and Nes-driven mouse tumor cohorts with and without Trp53 ablation: Atoh1-SmoM2, Atoh1-SmoM2; Trp53^fl/fl, Nes-SmoM2 and Nes-SmoM2; Trp53^fl/fl. Statistical significances were calculated using the log-rank test and P values are shown. (C-J) Immunofluorescence staining of sagittal cerebellar sections from an Atoh1-SmoM2; Trp53^flfl (upper panel) and Nes-SmoM2; Trp53^flfl (lower panel) mouse at P35 showing GFP (tumor cells, green) and DAPI (blue). Sections are showed at four distinct lateral-medial levels. Tumor lesions are delineated by white dashed lines. N>3 mouse samples. (K-N) Immunofluorescence staining of sagittal cerebellar section from an Atoh1-SmoM2; Trp53^flfl (upper panel) and Nes-SmoM2; Trp53^flfl (lower panel) mouse at end stage showing GFP (tumor cells, green) and DAPI (blue). Sections are showed in the hemispheres (lateral) and vermis (medial). Tumors are delineated by white dashed lines. N>3 mouse samples. Scale bar: C to N: 1mm.

The Nes-driven SHH MB model targets approximately ten times fewer cells than the Atoh1-driven model.
(A-D) Immunofluorescence staining of sagittal cerebellar sections from Atoh1-FlpoER; tdTom mice collected at P2 following tamoxifen injection at P0, showing RFP (red) and DAPI (blue). Sections are shown at four distinct lateral-medial levels. (A’-D’) High-magnification images of boxed regions in panels A-D. White arrowheads indicate the tdTomato⁺ cells (RFP) in the EGL. N>3 mouse samples. (E-H) Immunofluorescence staining of sagittal cerebellar sections from Nes-FlpoER; tdTom mice collected at P2 following tamoxifen injection at P0, showing RFP (red) and DAPI (blue). Sections are shown at four distinct lateral-medial levels. (E’-H’) High-magnification images of boxed regions in panels (A-D). White arrows indicate the tdTomato⁺ cells (RFP) in the EGL. N>3 mouse samples. (I) Quantification of the number of tdTomato⁺ (RPF) cells in the EGL along the lateral-medial axis in Atoh1-FlpoER; tdTom and Nes-FlpoER; tdTom mouse cerebella at P2 following tamoxifen injection at P0. N=3 mice. (J) Quantification of the number total of tdTomato⁺ cells in the EGL per cerebellum in both genotype Atoh1-FlpoER; tdTom and Nes-FlpoER; tdTom. N=3 mice. Statistical significance was determined using an unpaired t-test. Scale bar: A-D, E-H: 500um and A’-D’, E’-H’: 50um. Data are represented as mean ±SEM.

Single cell RNA-seq analysis identifies a Nes-expressing GCP subpopulation with distinct transcriptional features.
(A) UMAP visualization of 5 samples of P1 cerebella cells showing 29 clusters. 4 samples were cells isolated from the posterior cerebellum and enriched from GCPs from individual mice and 1 sample (n=3 pooled) was GFP⁺ (Atoh1⁺) CFP⁺ (Nes⁺) cells isolated by FACS. Atoh1-expressing GCP clusters (0, 1, 2, 3, 6, 7, 8, 10, 13) and Nes-expressing progenitor (NEP) clusters (4, 12, 14, 17, 18, 19, 21, 22) are indicated. See also Figure 5-Source data 1. (B–C) Feature plots showing expression of Barhl1 (GCP marker) and Nes across all clusters. (D) UMAP of integrated GCP clusters clusters (0, 1, 2, 3, 6, 7, 8, 10, 13 in part A) used for focused analysis. See also Figure 5–Source data 2. (E–I) Expression of Nes, Atoh1, Mki67, Gli1 and Otx2 across the integrated GCP dataset. See also Figure 5-Source data 2. (J) Cell-cycle phase assignment showing G1, S, and G2/M populations in integrated GCP dataset. See also Figure 5-Source data 3. (H) UMAP highlighting in red cells in cluster 10 of the integrated GCP dataset that represent Nes+ GCPs and in blue cells in clusters 1, 3 and 6 expressing posterior cerebellum GCP markers. Grey indicates remainder of cells. (L) Volcano plot showing differentially expressed genes between cluster 10 and clusters 1, 3, and 6 in the integrated GCP dataset. Genes enriched in cluster 10 (right) include Sox2, Wnt1, and Wls genes enriched in clusters 1, 3, and 6 (left) include Gli1 and Neurod1. See also Figure 5-Source data 4, 5. (M-N) GO enrichment plots showing biological processes upregulated in cluster 10 compared with other GCP clusters (1, 3, 6) in the integrated GCP dataset, including pathways related to cell-cycle progression, DNA replication, and oxidative metabolism. See also Figure 5–Source data 6, 7.

Nes-expressing GCPs appear transcriptionally closer to SHH MB tumor cells than Nes- GCPs.

(A) UMAP visualization of an integrated dataset of the GCP clusters 10, 1, 3 and 6 at P1 (see Figure 5D) with the GCP-like tumor cells from a SmoM2 model dataset (Lao et al., 2026). See also Figure 6-Source data 1. (B) UMAP showing the same integrated dataset as in (A) colored by sample of origin, distinguishing P1 GCP cells and SmoM2 GCP-like tumor cells. (C, E, G) UMAP highlighting in blue the SmoM2 GCP-like tumor cells and in red all GCPs (C), Nes^- GCPs (E) or Nes⁺ GCPs (G). (D, F, H) Volcano plots showing differential gene expression of all GCPs (D), Nes^- GCPs (F) or Nes⁺ GCPs (H) compared to SmoM2 GCP-like tumor cells. See also Figure 6-Source data 2-4.

A dual recombinase strategy confirms SHH MB tumors can initiate from Nes-expressing GCPs (Atoh1⁺).
(A) Kaplan-Meier survival curve of Atoh1^Frt-Cre/+; Nes-FlpoER; R26^LSL-SsmoM2/+ mice following tamoxifen induction at P0. (B-E) Immunofluorescence staining of sagittal cerebellar sections from Atoh1^Frt-Cre/+; Nes-FlpoER; tdTom mice collected at P4 following tamoxifen injection at P0 showing RFP (red) and DAPI (blue). Sections are shown at four distinct lateral-medial levels. (B’-E’) High magnification images of boxed regions in Panels (B-E). White arrowheads indicate the tdTomato⁺ cells (RFP) in the EGL. N>3 mouse samples. Scale bars: B-E: 500um, B’-E’: 50um

The Nes-CreER line labels a much broader progenitor population of cells than the Nes-FlpoER line.
(A-D) Immunofluorescence staining of sagittal cerebellar sections from Nes-CreER; tdTom mice (Nes-CreER; R26^LSL-tdTom) collected at P2 following tamoxifen injection at P0, showing RFP (red) and DAPI (blue). Sections are shown at four distinct lateral-medial levels. (A’-D’) High magnification images of anterior cerebellum. (A’’-D’’) High magnification images of posterior cerebellum. N= 3 mice. Scale bars: A-D: 500um and A’-D’, A’’-D’’: um

ScRNA-seq workflow and additional characterization of the Nes-expressing GCP clusters.
(A) Number of cells from each sample used for downstream analyses after filtering. Samples CFP1 to CFP4 represent 1 animal each and CFP-GFP represent 3 animals pooled. (B) Violin plots showing the number of features, the RNA counts and the percent of mitochondrial RNA counts in each sample. (C) Dot plot graph showing expression levels of cell type marker genes across all clusters for all cells. See also Figure 5-Source data 1. (D) UMAPs of all cells shown by samples. CFP1 to CFP4 are the posterior GCP samples from Nes-CFP mice (one mouse per sample) at P1 and CFP-GFP corresponds to the sample from Atoh1-GFP; Nes-CFP mice where CFP⁺ GFP⁺ cells were isolated by FACS (n=3 mice combined). (E) Dot plot graph showing expression levels of GCP markers across the integrated GCP dataset. See also Figure 5-Source data 2. (F) Feature plots showing expression of posterior marker Tlx3 in GCP clusters in the integrated GCP dataset. See also Figure 5-Source data 2.

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