Abstract Identifying the design principles of complex regulatory networks driving cellular decision-making remains essential to decode embryonic development as well as enhance cellular reprogramming. A well-studied network motif involved in cellular decision-making is a toggle switch – a set of two opposing transcription factors A and B, each of which is a master regulator of a specific cell-fate and can inhibit the activity of the other. A toggle switch can lead to two possible states – (high A, low B) and (low A, high B), and drives the ‘either-or’ choice between these two cell-fates for a common progenitor cell. However, the principles of coupled toggle switches remains unclear. Here, we investigate the dynamics of three master regulators A, B and C inhibiting each other, thus forming three coupled toggle switches to form a toggle triad. Our simulations show that this toggle triad can lead to co-existence of cells into three differentiated ‘single positive’ phenotypes – (high A, low B, low C), (low A, high B, low C), and (low A, low B, high C). Moreover, the hybrid or ‘double positive’ phenotypes – (high A, high B, low C), (low A, high B, high C) and (high A, low B, high C) – can co-exist together with ‘single positive’ phenotypes. Including self-activation loops on A, B and C can increase the frequency of ‘double positive’ states. Finally, we apply our results to understand the cellular decision-making in terms of differentiation of naïve CD4+ T cells into Th1, Th2 and Th17 states, where hybrid Th1/Th2 and hybrid Th1/Th17 cells have been reported in addition to the Th1, Th2 and Th17 ones. Our results offer novel insights into the design principles of a multistable network topology and provides a framework for synthetic biology to design tristable systems.

Multistability in cellular differentiation enabled by a network of three mutually repressing master regulators