Fine-tuning of ERK oscillation frequencies enables proliferation differentiation balance during mesendoderm fate specification

It is well documented that differentiation of stem cells is generally associated with and facilitated by an elongated cell cycle. However, for lineage specification requiring high ERK activity, which also promotes proliferation, it is unclear how stem cells simultaneously negotiate the “proliferation-differentiation” decision. Here we investigate this question using multiplex quantitative live-cell imaging to track embryonic stem cells (ESC) differentiation into mesendoderm (ME) lineage with high ERK activity. We show that ERK activity increase manifested by a heightened ERK oscillation frequency drove robust ME differentiation accompanied by a modest cell cycle acceleration. We profile the responses of ME differentiation and cell cycle length as a function of a synthetic ERK oscillatory activity gradient. Our results reveal that these two processes took place in distinct ranges of ERK oscillation frequency, with ME differentiation requiring higher level for ERK oscillation to secure efficient ME protein translation. Interactions between gastrulation morphogens recapitulated an ERK oscillation gradient, potentiating heterogeneous ME fates while maintaining a comparable level of proliferation. Our study thus provides mechanistic insights into how ERK fine-tunes proliferation and differentiation during cell fate specification Overall design: hESCs were treated with CHIR99021 in combination with different concentrations of the Ulix inhibitor. Polysome profiling was performed by sucrose gradient ultracentrifugation, and RNA was extracted from pooled gradient fractions for RNA-seq analysis. The collected fractions included CHIR-total-RNA and gradient pools CHIR-2–3, CHIR-4–5, CHIR-6–7, and CHIR-8–11, which represent RNA populations ranging from free/lowly translated mRNAs to heavy polysome-associated mRNAs. Sample names containing labels such as “CHIR-U0_15” indicate cells treated with CHIR99021 together with 0.15 µM Ulix inhibitor; additional samples include treatments with 1.25 µM and 5 µM Ulix.