SMARCA4 Loss and Mutated β-Catenin Induce Proliferative Lesions in the Murine Embryonic Cerebellum

Almost all medulloblastomas (MB) of the Wingless/Int-1 (WNT) type are characterized by hotspot mutations inCTNNB1, and mouse models have convincingly demonstrated the tumor-initiating role of these mutations. Additional alterations inSMARCA4are detected in ∼20% of WNT MB, but their functional role is mostly unknown. We, therefore, amended previously described brain lipid binding protein (Blbp)-cre::Ctnnb1(ex3)fl/wtmice by the introduction of floxedSmarca4alleles. Unexpectedly, mutated and thereby stabilized β-catenin on its own induced severe developmental phenotypes in male and femaleBlbp-cre::Ctnnb1(ex3)fl/wtmice in our hands, including a thinned cerebral cortex, hydrocephalus, missing cerebellar layering, and cell accumulations in the brainstem and cerebellum. An additional loss of SMARCA4 even resulted in prenatal death for most mice. RespectiveBlbp-cre::Ctnnb1(ex3)fl/wt::Smarca4fl/recmutants (male and female) 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 bothCtnnb1andSmarca4were negatively selected and did not show tumorigenic potential after transplantation in adult female 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 toward the unraveling of tumorigenic mechanisms induced by aberrant WNT signaling and SMARCA4 deficiency.