SMARCA4 loss and mutated ß-catenin induce proliferative lesions in the murine embryonic cerebellum [scRNA-seq]
Almost all medulloblastomas (MB) of the Wingless/Int-1 (WNT) type are characterized by hotspot mutations in CTNNB1, and mouse models have convincingly demonstrated the tumor-initiating role of these mutations. Additional alterations in SMARCA4 are detected in around 20% of WNT MB, but their functional role is mostly unknown. We therefore amended previously described Blbp cre::Ctnnb1(ex3)fl/wt mice by the introduction of a floxed Smarca4 allele. Unexpectedly, mutated ß-catenin on its own induced severe developmental phenotypes in Blbp cre::Ctnnb1(ex3)fl/wt mice in our hands, including a thinned cerebral cortex, hydrocephalus, missing cerebellar layering, and non-proliferative cell accumulations in the brain stem and cerebellum. An additional loss of SMARCA4 even resulted in prenatal death for most mice. Respective Blbp cre::Ctnnb1(ex3)fl/wt::Smarca4fl/fl mutants developed large proliferative lesions in the cerebellum evolving from E13.5 to E16.5. Histological and molecular analysis of these lesions by DNA methylation profiling and single-cell RNA sequencing suggested an origin in early undifferentiated SOX2-positive cerebellar progenitors. Furthermore, upregulated WNT signaling, altered actin/cytoskeleton organization, and reduced neuronal differentiation were evident in mutant cells. In vitro, cells harboring alterations in both Ctnnb1 and Smarca4 were negatively selected and did not show tumorigenic potential after transplantation in adult recipient mice. However, in cerebellar explant cultures, mutant cells displayed significantly increased proliferation, suggesting an important role of the embryonic microenvironment in the development of lesions. Altogether, these results represent an important first step towards the unravelling of tumorigenic mechanisms induced by aberrant WNT signaling and SMARCA4 deficiency. Overall design: Whole cerebella of E16.5 embryos (cre-negative controls and Blbp-cre::Ctnnb1(ex3)fl/wt::Smarca4fl/fl mutants with cerebellar lesions) were analyzed by single-cell RNA sequencing