A Rare Nestin-Expressing Granule Cell Precursor Subpopulation Underlies SHH Medulloblastoma Formation
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**eLife Assessment
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In this valuable study, the authors identified a rare population of Nestin-expressing cells within the external granule layer of the early postnatal mouse cerebellum. They demonstrated that these cells are distinct from Sox2+ progenitors and can give rise to medulloblastoma. Collectively, the findings provide convincing evidence that this unique Nestin+ population is susceptible to oncogenic transformation and may underlie the preferential emergence of Sonic hedgehog-driven medulloblastomas.
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Abstract
Granule cell precursors (GCPs) drive the major postnatal expansion of the cerebellum and are the cells of origin of sonic hedgehog medulloblastoma (SHH MB). Although GCPs are often treated as a uniform population, increasing evidence suggests they are heterogeneous, and whether specific subpopulations show distinct tumorigenic competence remains unclear. Here, we identified a rare Nestin-expressing GCP subpopulation in the normal early postnatal cerebellum and showed that it is spatially restricted, molecularly distinct and highly competent to form tumors. These cells are enriched in the posterior external granule layer and co-express Atoh1 . Using different SHH MB mouse models, we showed that when this rare subpopulation of GCPs is targeted they can give rise to SHH MB with an efficiency comparable to targeting a larger number of Atoh1 -expressing GCPs and that tumors derived from Nestin-expressing GCPs arise in the posterior-lateral cerebellum. Single cell RNA sequencing revealed that Nestin-expressing GCP have a transcriptome indicating reduced neuronal differentiation and enrichment for stem cell genes compared to bulk GCPs and more closely align with SHH MB cells. Together, our findings reveal functionally important heterogeneity within the GCP lineage and suggest that SHH MB arises preferentially from a small subpopulation of GCPs that express Nestin.
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**eLife Assessment
**
In this valuable study, the authors identified a rare population of Nestin-expressing cells within the external granule layer of the early postnatal mouse cerebellum. They demonstrated that these cells are distinct from Sox2+ progenitors and can give rise to medulloblastoma. Collectively, the findings provide convincing evidence that this unique Nestin+ population is susceptible to oncogenic transformation and may underlie the preferential emergence of Sonic hedgehog-driven medulloblastomas. -
Reviewer #1 (Public review):
Summary:
GCPs, which drive postnatal cerebellar growth and can give rise to SHH-MB, are not uniform. The authors show that GCPs include a rare Nestin-expressing subpopulation with distinct molecular features. This subpopulation is spatially restricted, enriched for stem cell-like properties, and shows a high competency for tumor formation comparable to larger GCP pools, with tumors preferentially arising in the posterior-lateral cerebellum. Overall, the findings indicate that SHH-MB might originate preferentially from this small, tumor-competent Nestin-expressing GCP subset.
Strengths:
(1) The authors use a breadth of approaches from histology, mouse genetics, and single-cell RNA sequencing.
(2) Throughout, this paper uses very elegant genetic approaches, such as the double Nes-FlpoER; Atoh1-FSF-Cre; …
Reviewer #1 (Public review):
Summary:
GCPs, which drive postnatal cerebellar growth and can give rise to SHH-MB, are not uniform. The authors show that GCPs include a rare Nestin-expressing subpopulation with distinct molecular features. This subpopulation is spatially restricted, enriched for stem cell-like properties, and shows a high competency for tumor formation comparable to larger GCP pools, with tumors preferentially arising in the posterior-lateral cerebellum. Overall, the findings indicate that SHH-MB might originate preferentially from this small, tumor-competent Nestin-expressing GCP subset.
Strengths:
(1) The authors use a breadth of approaches from histology, mouse genetics, and single-cell RNA sequencing.
(2) Throughout, this paper uses very elegant genetic approaches, such as the double Nes-FlpoER; Atoh1-FSF-Cre; LSL-Smo-M2, to generate tumors only from Atoh1+; Nes+ double-positive cells. This intersectional genetic experiment makes for a very clear answer.
(3) The findings reported in this manuscript are valuable since they reveal a novel GCP subpopulation defined by spatial and molecular identity. Some of their experiments suggest that these cells could represent the main cell-of-origin of SHH MB. The experiments are carefully performed, and the evidence is convincing.
Weaknesses or elements that could be improved:
(1) A transgenic Nestin-CFP mouse is used in this study. However, it is not clear whether CFP accurately reflects the Nestin protein. Figure 1: After the promoter is turned off, these cells might remain positive for CFP for longer than they are positive for Nestin, due to CFP protein stability. Is the Nestin protein present in these cells? Nestin double immunofluorescence with CFP and Sox2 and Barhl1 could be performed to address this. Related to this comment, it is also important to note that this is a rat promoter transgene. So the transgene might not reflect exactly the endogenous Nestin expression.
(2) Could the posterior restriction of Nestin-CFP be due to the timing (P1) at which the authors looked? In other words, if they look earlier, would the authors see Nestin-CFP cells more anterior?
(3) Since only one medulloblastoma mouse model (Smo-M2) is used to conclude that "the Nes-expressing GCP population in the normal cerebellum is transcriptionally closer to SHH MB tumor cells than the remainder of the GCPs", the findings might not apply to other SHH-MB models. This should be mentioned.
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Reviewer #2 (Public review):
Summary:
In this manuscript, the authors studied transgenic reporter mice to profile Nestin expression in the postnatal mouse cerebellum. They discovered a small population of Nestin+; Atoh1+ granule neuron precursors (GNPs) in the external granule cell layer (EGL). Using immunostaining, qPCR, and RNA-sequencing, the authors showed that these Nestin+ cells are not identical to Sox2+ cells (e.g., the majority of Nestin+ cells are Sox2-). Using various mouse genetic strategies, including an elegant intersectional strategy that specifically targets Nestin+; Sox2+ cells, the authors showed that Nestin+ cells are capable of initiating Sonic hedgehog (SHH) medulloblastoma when they express the SmoM2 allele that drives constitutively active SHH signaling. Lastly, the authors profiled the transcriptomes of these …
Reviewer #2 (Public review):
Summary:
In this manuscript, the authors studied transgenic reporter mice to profile Nestin expression in the postnatal mouse cerebellum. They discovered a small population of Nestin+; Atoh1+ granule neuron precursors (GNPs) in the external granule cell layer (EGL). Using immunostaining, qPCR, and RNA-sequencing, the authors showed that these Nestin+ cells are not identical to Sox2+ cells (e.g., the majority of Nestin+ cells are Sox2-). Using various mouse genetic strategies, including an elegant intersectional strategy that specifically targets Nestin+; Sox2+ cells, the authors showed that Nestin+ cells are capable of initiating Sonic hedgehog (SHH) medulloblastoma when they express the SmoM2 allele that drives constitutively active SHH signaling. Lastly, the authors profiled the transcriptomes of these cells and showed that they display enriched stem cell genes and are closer to the transcriptomes of GNP-like cells in medulloblastoma compared to Nestin- GNPs in the developing cerebellum.
Strengths:
(1) The comprehensive mouse genetics experiments, in combination with immunostaining, lineage tracing, and RNA-seq studies, provided compelling evidence that rare Nestin+ cells are present in the EGL, predominantly at the posterior lateral cerebellum in early postnatal mice.
(2) The intersectional genetics experiment unequivocally show that Nestin+; Atoh1+ cells can be oncogenically transformed by SmoM2, leading to SHH medulloblastoma.
(3) The more stem cell-like transcriptomic features of the Nestin+ GNPs compared to Nestin- GNPs provide support for the heterogeneity of this transient progenitor cell population, with implications for development, congenital diseases, and tumors from the cerebellum.
Weaknesses:
Main comments:
My main concern relates to whether these Nestin+; Atoh1+ cells are restrictively localized in the EGL. Both the title "A Rare Nestin-Expressing Granule Cell Precursor Subpopulation Underlies SHH Medulloblastoma Formation" and what the authors described throughout the manuscript propose that Nestin+; Atoh1+ cells in the EGL are the cell-of-origin of SHH medulloblastoma. To definitively conclude this, the authors need to comprehensively analyze all regions of the developing cerebellum.
Most importantly, are Nestin+; Atoh1+ cells present in the rhombic lip? Are there any rhombic lip cells genetically labeled in their intersectional mouse mutants (e.g., the Atoh1Frt-Cre/+; Nes-FlpoER; R26LSL-SsmoM2/+ mice)?
If Nestin+; Atoh1+ cells are present at non-EGL regions in the developing cerebellum, the authors would have to reconsider many of their conclusions and also the title of this paper.
Additional comments:
(1) To investigate Nestin expression, the authors used Nes-CFP transgenic mice expressing CFP from promoter/enhancer sequences from the rat Nes gene (Encinas et al., 2006). Given that Nestin expression is of central importance for this study, it is important to validate that these reporter mice faithfully report Nestin protein expression (e.g., by co-labeling CFP with Nestin antibody and systemically comparing signals throughout the cerebellum, ideally in several developmental stages).
(2) The authors mostly presented immunostaining data of the cerebellum from P1 mice. It is important to systematically profile the appearance and disappearance of these Nestin+, Atoh1+ cells in mouse cerebellum across developmental stages (e.g., embryonic, early, and late postnatal stages).
(3) How different is the proliferative ability of the Nestin+ versus Nestin- GNPs at various developmental stages? Also, the difference between EdU+; Barhl1+; Nestin+ and EdU+; Barhl1+; Nestin- cells is quite small despite statistical difference (Figure 1N). Do the authors think this very small EdU incorporation difference can translate into a biological difference (in developmental and/or disease context)?
(4) Lines 145-147: "Compared to double-negative cells, Atoh1 and Nes were significantly higher in the double-positive fraction, supporting the identity of the cells as a previously unrecognized rare population of GCPs at P1 that expresses both the GCP marker Atoh1 and ventricular zone marker Nes." Nestin is not a ventricular zone marker. This should be rephrased.
(5) In Figure 3, the authors showed mouse survival data and concluded that Nes-driven and Atho1-driven SHH medulloblastoma models show similar tumor penetrance. This is not an entirely accurate description of their data. The Nes-SmoM2 mice displayed significantly longer survival compared to the Atoh1-SmoM2 mice (Figure 3B). This conclusion needs to be revised.
(6) In Figure 5, the authors showed that genes enriched in cluster 10 included Sox2, Nes, Wls, and Wnt1, while Neurod1 and Rbfox3 were preferentially expressed in the other GCP clusters. They conclude that cluster 10 represents a less differentiated, more stem-like GCP state, potentially positioned upstream in the lineage hierarchy. While these few markers are useful, it is more informative to formally support this conclusion by comparing the stem cell transcriptomic signature (using a larger gene list) between cluster 10 and other GCPs.
(7) In Figure 5, the authors performed gene ontology analysis and showed that cluster 10 is enriched for biological processes linked to WNT signaling and proposed that this molecular profile supports their identity as a transient, developmentally plastic population within the GCP lineage related to the rhombic lip. The authors are recommended to use an orthogonal approach (i.e., immunostaining to compare nuclear localization of beta-Catenin) to validate their transcriptome-based finding.
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