Regulating the timing of enhancer transitions is key to defining sharp boundaries of Fushi tarazu expression in the Drosophila embryo
Coordinating the action of different enhancers is crucial to correctly specify cell fate decisions during development. Yet it remains poorly understood how the activity of multiple enhancers is choregraphed in time. To shed light on this question we used new live imaging approaches to quantify transcription and protein expression in single cells of Drosophila melanogaster embryos. We employed these tools to dissect the regulation of Fushi tarazu (Ftz), a transcription factor expressed in a series of stripes by two distinct enhancers: autoregulatory and zebra. The anterior edges of the Ftz stripes are sharply defined and specify essential signaling centers. Here, we determined the time at which each boundary cell commits to either a high-Ftz or low-Ftz fate using dynamic features of time-resolved Ftz protein traces. By following the activity of each enhancer individually, we showed that the autoregulatory enhancer does not establish this fate choice. Instead, it perpetuates the decision defined by zebra. This is contrary to the prevailing view that autoregulation drives the fate decision by causing bi-stable Ftz expression. Furthermore, we showed that the autoregulatory enhancer is not activated based on a Ftz concentration threshold, but through a timing-based mechanism. We hypothesize that this is regulated by a set of pioneer-like transcription factors, which have recently been shown to act as timers in the embryo. Our work provides new insight into the genetic mechanisms that directly regulate the dynamics of gene regulatory networks, and supports the emerging view that this regulation is vital for reliable cell fate specification.