Oscillatory co-expression of HES1 and HES5 enables a hybrid state in a cross-repressive transcription factor regulatory motif

Many cell fate decisions in the developing neural tube are directed by cross-repressive transcription factor (TF) motifs that generate bistability, such that cells express one TF but not both. Hybrid states in which cells express both cross-repressing fate determinants have been observed, but how these arise or persist remains unclear. Here, we focus on HES1 and HES5, which are auto-repressive oscillatory TFs that regulate neural progenitor maintenance and are expressed in adjacent dorsoventral progenitor domains in the developing spinal cord. Knockdown experiments demonstrate that HES1 and HES5 are cross-repressing in mouse spinal cord neural progenitors, and live-cell imaging in vitro shows that they can be co-expressed, defining a hybrid state. In this state, HES proteins co-oscillate in-phase within single cells. Computational modelling indicates that modulation of cross-repression strength or relative TF abundance destabilises this state, driving resolution towards a single oscillatory HES TF. This is consistent with in vivo analysis showing transient HES1 and HES5 co-expression, followed by progressive restriction to a single TF oscillator. Our findings suggest that oscillatory expression enables the co-existence of cross-repressing TFs, allowing hybrid states within a developmental bistable motif.