Opposing actomyosin pools establish epithelial polarity during naive pluripotency exit

Epithelial cell polarity is fundamental for embryonic development and organ function. During organogenesis, epithelia often develop from unpolarized precursor cells. How mammalian epithelial cells establish polarity de novo is unclear, in part due to challenges of observing this process in real time in nontransformed cells. Here, we leverage 3D spheroid culture of mouse embryonic stem cells, fluorescent protein knock-in, and live imaging to study epithelial polarity establishment. We show that apical myosin activity, regulated by myosin light chain kinase (MLCK), is crucial for proper epithelial polarity in this system. MLCK-dependent actin flows carry ZO-1, a tight junction component, to the apical junction. A second pool of myosin, regulated by Rho kinase, localizes basally and modulates ZO-1 recruitment to the membrane. MLCK inhibition also disrupts localization of atypical protein kinase C and podocalyxin, supporting a broader role of MLCK-dependent myosin in establishing the apical membrane. Our results support a model in which distinct apical and basal actomyosin pools coordinate epithelial polarity establishment.