The ER-resident proteins VMP1 and TMEM41B share a conserved DedA domain, which confers lipid scramblase activity. Loss of either gene results in embryonic lethality in mice and defects in autophagy and lipid droplet metabolism. To investigate their role in pluripotency and lineage specification, we generated Vmp1 and Tmem41b mutations in mouse embryonic stem cells (ESCs). We observed that ESCs carrying mutations in Vmp1 and Tmem41b show robust self-renewal and an unperturbed pluripotent expression profile but accumulate LC3-positive autophagosomes and lipid droplets consistent with defects in autophagy and lipid metabolism. ESCs carrying combined mutations in Vmp1 and Tmem41b can differentiate into a wide range of embryonic cell types. However, differentiation into primitive endoderm-like cells in culture is impaired, and the establishment of extra-embryonic endoderm stem (XEN) cells is delayed. Mechanistically, we show the deregulation of genes that are associated with WNT signaling. This is further confirmed by cell surface proteome profiling, which identified a significant reduction of the WNT-receptor FZD2 at the plasma membrane in Vmp1 and Tmem41b double mutant ESCs. Importantly, we show that transgenic expression of Fzd2 rescues XEN differentiation. Our findings identify the role of the lipid scramblases VMP1 and TMEM41B in WNT signaling during extra-embryonic endoderm development and characterize their distinct and overlapping functions.

内质网驻留蛋白 VMP1 和 TMEM41B 共享一个保守的 DedA 结构域，该结构域赋予脂质扰乱酶活性。任何一个基因的缺失都会导致小鼠的胚胎致死以及自噬和脂滴代谢缺陷。为了研究它们在多能性和谱系规范中的作用，我们在小鼠胚胎干细胞 （ESC） 中生成了 Vmp1 和 Tmem41b 突变。我们观察到携带 Vmp1 和 Tmem41b 突变的 ESC 表现出强大的自我更新和不受干扰的多能表达谱，但积累了 LC3 阳性自噬体和脂滴，这与自噬和脂质代谢缺陷一致。携带 Vmp1 和 Tmem41b 联合突变的 ESC 可以分化成多种胚胎细胞类型。然而，在培养物中分化为原始内胚层样细胞的分化受损，胚外内胚层干细胞 （XEN） 细胞的建立延迟。从机制上讲，我们显示了与 WNT 信号传导相关的基因的失调。细胞表面蛋白质组分析进一步证实了这一点，该分析确定了 Vmp1 和 Tmem41b 双突变 ESC 中质膜处 WNT 受体 FZD2 的显着减少。重要的是，我们表明 Fzd2 的转基因表达挽救了 XEN 分化。我们的研究结果确定了脂质扰乱酶 VMP1 和 TMEM41B 在胚胎外内胚层发育过程中 WNT 信号传导中的作用，并表征了它们不同和重叠的功能。