Spatiotemporal lineage derepression by cell crowding-induced ETV4 inactivation

All extant amniotes employ a single-layered epithelium of epiblasts as a starting material for gastrulation, suggesting the necessity of the epithelial structure for three germ layer derivation. Using a human embryonic stem cell (hESC) epithelium as a model system, we found that local epithelial crowding derepresses the neuroectoderm fate by spatiotemporal inactivation of ETV4. ETV4 serves as a genetic toggle switch that links cell density to lineage fates. Mechanistically, cell crowding blocks FGF receptor endocytosis by reduced cell-extracellular matrix (ECM) interaction. Disrupted endocytosis decreases ETV4 protein stability by ERK inactivation. Mathematical modeling of epithelial crowding demonstrates that the cooperativity of integrin-ECM interaction transforms the gradient of crowdedness into bistable ETV4 transition, which ensures the switch-like function of ETV4 in lineage determination. Our results propose local cell crowding in a stem cell epithelium as a key cellular mechanism for spatiotemporal regulation of lineage fates. mRNA profiles of ETV4 +/- and low/high density H9 hESCs.