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How differentiating progenitor cells can attain a distinct differentiated cell-identity is a challenging problem given the promiscuous nature of critical transcription factors and the typically fluctuating signaling environment in which cells exist. Here we test the hypothesis that a unique and robust differentiated cell identity can result from a core component of the differentiated state doubling up such that it has key functions in the differentiated state and also works at low levels as a positive-feedback signaling mediator that drives progenitor cells to differentiate. Using live single-cell fluorescence imaging analysis in the adipocyte differentiation system, we show that FABP4, one of the most abundant core components that buffers lipids in differentiated adipocytes, regulates at low levels expression of PPARG, the master regulator of adipocyte differentiation. Adipocyte progenitor cells transition from a priming phase of differentiation to a second phase during which low levels of FABP4 and PPARG mutually reinforce each other’s expression. In this way both proteins reach a threshold where cells irreversibly commit to the differentiated state. Before and after reaching this threshold, PPARG transcriptionally increases FABP4 expression while fatty-acid loaded FABP4 binds to and increases PPARG activity. Together, our study suggests a control principle for robust cell identity, whereby a core component of the differentiated state also promotes differentiation from its own progenitor state. HIGHLIGHTS Only a small fraction (~10%) of the maximally reached FABP4 levels in cells is needed to commit cells to the differentiated state. Mature adipocytes express levels of FABP4 ten times the minimal level needed to commit cells to the differentiated state, thus providing an explanation for why the mature adipocyte state is so stable. Fatty-acid loaded FABP4 binds to and activates PPARG, thereby turning on PPARG positive feedback loops that increase PPARG expression. FABP4 critically controls the second phase of adipogenesis between activation of the feedback loops and reaching the threshold to differentiate.

SUMMARY Terminal cell differentiation is essential for developing and maintaining tissues in all multi-cellular organisms. However, when and how the opposing processes of proliferation and terminal cell differentiation regulate permanent exit from the cell cycle and the number of differentiated cells is not well understood. Here we use adipogenesis as a model system to measure in live single cells the moment of terminal cell differentiation during cell-cycle progression. We show that terminal cell differentiation occurs through a competition during a lengthening G1 phase whereby cells either commit to terminally differentiate and close the window of proliferation, or commit to the next cell cycle and extend the window of proliferation until a later G1 phase. By regulating when cells terminally differentiate, different strengths of mitogen and differentiation stimuli control the number of terminally differentiated cells produced per progenitor cell. Together, our study suggests that tissue development, maintenance and regeneration is regulated by a competition mechanism that speeds up or delays the time when cells commit to terminally differentiate. HIGHLIGHTS Progenitor cells decide to terminally differentiate by using a competition process that can last over multiple cell cycles and engages during each lengthening G1 phase The cyclin-CDK inhibitor p21 is induced by PPARG and extends G1 duration, and also triggers permanent cell-cycle exit once PPARG reaches the threshold for terminal differentiation The system architecture regulates the number of differentiated cells by speeding up or delaying the time when cells commit to terminally differentiate and close the window of proliferation