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 ~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/wt mice by the introduction of floxed Smarca4 alleles. Unexpectedly, mutated and thereby stabilized β-catenin on its own induced severe developmental phenotypes in male and female Blbp-cre::Ctnnb1(ex3)^fl/wt mice 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. Respective Blbp-cre::Ctnnb1(ex3)^fl/wt::Smarca4^fl/rec mutants (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 both Ctnnb1 and Smarca4 were 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.

几乎所有 Wingless/Int-1 (WNT) 型髓母细胞瘤 (MB) 均以CTNNB1热点突变为特征，小鼠模型已令人信服地证明了这些突变的肿瘤启动作用。在约 20% 的 WNT MB 中检测到SMARCA4的其他改变，但它们的功能作用大多未知。因此，我们通过引入 floxed Smarca4等位基因修改了先前描述的脑脂质结合蛋白 ( Blbp ) -cre::Ctnnb1(ex3) ^fl/wt小鼠。出乎意料的是，β-连环蛋白自身的突变和稳定导致了我们手中的雄性和雌性Blbp-cre::Ctnnb1(ex3) ^fl/wt小鼠的严重发育表型，包括大脑皮层变薄、脑积水、小脑分层缺失和脑干和小脑中的细胞聚集。 SMARCA4 的额外缺失甚至导致大多数小鼠产前死亡。相应的Blbp-cre::Ctnnb1(ex3) ^fl/wt ::Smarca4 ^fl/rec突变体（雄性和雌性）在小脑中从 E13.5 演变成 E16.5 形成大的增殖性病变。通过 DNA 甲基化谱和单细胞 RNA 测序对这些病变进行组织学和分子分析，表明这些病变起源于早期未分化的 SOX2 阳性小脑祖细胞。此外，突变细胞中 WNT 信号传导上调、肌动蛋白/细胞骨架组织改变以及神经元分化减少都很明显。在体外，含有Ctnnb1和Smarca4改变的细胞被阴性选择，并且在移植到成年雌性受体小鼠中后没有表现出致瘤潜力。然而，在小脑外植体培养中，突变细胞表现出显着增加的增殖，表明胚胎微环境在病变发展中的重要作用。总而言之，这些结果代表了阐明异常 WNT 信号传导和 SMARCA4 缺陷诱导的致瘤机制的重要第一步。