Medulloblastoma encompasses a collection of clinically and molecularly diverse tumor subtypes that together comprise the most common malignant childhood brain tumor1–4. These tumors are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) following aberrant activation of the Sonic Hedgehog pathway (hereafter, SHH-subtype)3–8. The pathological processes that drive heterogeneity among the other medulloblastoma subtypes are not known, hindering the development of much needed new therapies. Here, we provide evidence that a discrete subtype of medulloblastoma that contains activating mutations in the WNT pathway effector CTNNB1 (hereafter, WNT-subtype)1,3,4, arises outside the cerebellum from cells of the dorsal brainstem. We found that genes marking human WNT-subtype medulloblastomas are more frequently expressed in the lower rhombic lip (LRL) and embryonic dorsal brainstem than in the upper rhombic lip (URL) and developing cerebellum. Magnetic resonance imaging (MRI) and intra-operative reports showed that human WNT-subtype tumors infiltrate the dorsal brainstem, while SHH-subtype tumors are located within the cerebellar hemispheres. Activating mutations in Ctnnb1 had little impact on progenitor cell populations in the cerebellum, but caused the abnormal accumulation of cells on the embryonic dorsal brainstem that included aberrantly proliferating Zic1+ precursor cells. These lesions persisted in all mutant adult mice and in 15% of cases in which Tp53 was concurrently deleted, progressed to form medulloblastomas that recapitulated the anatomy and gene expression profiles of human WNT-subtype medulloblastoma. We provide the first evidence that subtypes of medulloblastoma have distinct cellular origins. Our data provide an explanation for the marked molecular and clinical differences between SHH and WNT-subtype medulloblastomas and have profound implications for future research and treatment of this important childhood cancer.

Cancer stem cells (CSC) are remarkably similar to normal stem cells: both self-renew, are multipotent and express common surface markers, e.g., PROMININ-1 (PROM1, CD133)1. What remains unclear is whether CSC are the direct progeny of mutated stem cells, or more mature cells that reacquire stem cell properties during tumor formation. Answering this important question will require knowledge of whether normal stem cells are susceptible to cancer causing mutations; however, this has proved difficult to test since the identity of most adult tissue stem cells is not known. Here, using an inducible-Cre-nuclear(n)LacZ reporter allele knocked into the Prom1 locus (Prom1C-L), we show that Prom1 is expressed in a variety of developing and adult tissues. Lineage-tracing studies of adult Prom1+/C-L mice containing the Rosa26YFP reporter allele showed that Prom1+ cells are located at the base of crypts in the small intestine, co-express Lgr52, generate the entire intestinal epithelium, and are therefore likely to be the small intestinal stem cell. Prom1 was reported recently to mark CSC of human intestinal tumors that arise frequently as a consequence of aberrant Wingless (WNT) signaling3-5. Activation of endogenous Wnt signaling in Prom1+/C-L mice containing a Cre-dependent mutant allele of Beta-catenin (Ctnnb1lox(ex3)) resulted first in a gross disruption of crypt architecture and a disproportionate expansion of Prom1+ cells at the crypt base. Lineage-tracing demonstrated that the progeny of these cells replaced the mucosa of the entire small intestine with neoplastic tissue that was characterized by focal high-grade intraepithelial neoplasia and crypt adenoma formation. Although all neoplastic cells arose from Prom1+ cells in these mice, only 7% of tumor cells retained Prom1 expression. Our data indicate that Prom1 marks stem cells in the adult small intestine, which are susceptible to transformation into tumors retaining a fraction of mutant-Prom1+ tumor cells.

Medulloblastoma is a malignant childhood cerebellar tumour comprised of distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. We used single-cell transcriptomics to investigate intra-and inter-tumoural heterogeneity in twenty-five medulloblastomas spanning all molecular subgroups. WNT, SHH, and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronallike malignant populations, whereas Group 4 tumours were exclusively comprised of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, whose relative proportions distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide novel insights into the cellular and developmental states underlying subtypespecific medulloblastoma biology. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Summary Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Using H3K27ac and BRD4 ChIP-Seq, coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-Seq, that are responsible for subgroup divergence and implicate candidate cells-of-origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins.

The cerebellum develops from a restricted number of cell types that precisely organize to form the circuitry that controls sensory-motor coordination and some higher-order cognitive processes. To acquire an enhanced understanding of the molecular processes that mediate cerebellar development, we performed single-cell RNA-sequencing of 39,245 murine cerebellar cells at twelve critical developmental time points. Using recognized lineage markers, we confirmed that the single-cell data accurately recapitulate cerebellar development. We then followed distinct populations from emergence through migration and differentiation, and determined the associated transcriptional cascades. After identifying key lineage commitment decisions, focused analyses uncovered waves of transcription factor expression at those branching points. Finally, we created Cell Seek, a flexible online interface that facilitates exploration of the dataset. Our study provides a transcriptional summarization of cerebellar development at single-cell resolution that will serve as a valuable resource for future investigations of cerebellar development, neurobiology, and disease.