Directing hierarchical cell fate decisions through sequential pulses of minimal signaling alphabets

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Abstract Inductive signals can direct cell fate decisions, yet the diversity of cell identities far exceeds the available signaling repertoire. While developing systems resolve this paradox by applying temporal sequences of minimalistic signals to hierarchically organized gene regulatory networks (GRNs), the rules governing robust sequential fate navigation remain largely unknown. To address this, we modeled how sequential signals steer trajectories toward arbitrary fates within a Waddington-like manifold, enumerating parameters and combinatorial logics of cross-inhibition and self-activation (CIS) modules, and various ways of applying the sequential signals. We uncover a fundamental conflict between commitment stability and inductive plasticity that severely limits sequential fate navigation. Crucially, introducing non-inductive gap intervals systematically resolves this bottleneck by dissipating kinetic leakage without sacrificing upstream stability. Finally, we demonstrate that polarization-division cycle can autonomously generate signals meeting these temporal requirements. Together, our findings provide a minimal model demonstrating the feasibility and design principles for driving hierarchical cell fate decisions through sequential pulses of a restricted signaling repertoire.