NGN3 oscillatory expression controls the timing of human pancreatic endocrine differentiation

Understanding protein expression dynamics is crucial for the mechanistic understanding of cell differentiation. We investigate the dynamics and decoding of NEUROGENIN3 (NGN3), a transcription factor critical for pancreatic endocrine development. A knockin endogenous reporter shows that NGN3 protein oscillates with a 13-h periodicity in human induced pluripotent stem cell (hiPSC)-derived endocrine progenitors and is switched off as cells differentiate to β-like and pre-α cells. Increasing NGN3 protein stability results in one broad peak of expression instead of oscillations, with a larger peak-to-trough fold-change. This leads to precocious endocrine differentiation and earlier expression of key NGN3 target genes. Single-cell analysis of dynamics, mathematical modeling, and experimental validation suggest that NGN3 oscillations are decoded by fold-change detection (FCD) rather than the level of expression via an incoherent feedforward loop (IFFL) motif that explains both normal and precocious differentiation. Our findings suggest that oscillatory NGN3 dynamics control the timing of differentiation but not fate specification.