Fine-tuning ERK activity enables proliferation-differentiation balance during lineage specification of human embryonic stem cells

ERK is a key signaling mediator controlling both proliferation and lineage specification during embryo development. How ERK choreographs differentiation and proliferation to achieve balanced developmental outcomes in lineages with variable ERK activities remains unclear. To investigate this, we established multiplex quantitative live-cell imaging to track human pluripotent stem cell differentiation into mesendoderm (ME), a lineage specified by gastrulation morphogens and dependent on high ERK activity. We found that distinct morphogen combinations generate varying ERK activity levels, which correlate with heterogeneous ME fate choices despite relatively uniform cell cycle dynamics. To dissect how heterogenous ERK levels directly modulate and coordinate ME differentiation and proliferation, we engineered a synthetic spectrum of titrated ERK activities. Our results showed that ERK fine-tunes ME differentiation potential and cell division speed under nonoverlapping activity ranges, enabling quantitative control of ME fate specification without major effect on cell cycle progression. Mechanistically, this uncoupling stems from differential transcriptional and translational sensitivities of ME-specifying genes versus cell cycle genes to ERK input. Together, our findings reveal how a single signaling pathway quantitatively balances differentiation and proliferation during lineage commitment and embryogenesis.