For over 40 years, the Bicoid-hunchback (Bcd-hb) system in the fruit fly embryo has been used as a model to study how positional information in morphogen concentration gradients is robustly translated into step-like responses. A body of quantitative comparisons between theory and experiment have since questioned the initial paradigm that the sharp hb transcription pattern emerges solely from diffusive biochemical interactions between the Bicoid transcription factor and the gene promoter region. Several alternative mechanisms have been proposed, such as additional sources of positional information, positive feedback from Hb proteins or out-of-equilibrium transcription activation. By using the MS2-MCP RNA-tagging system and analysing in real time, the transcription dynamics of synthetic reporters for Bicoid and/or its two partners Zelda and Hunchback, we show that all the early hb expression pattern features and temporal dynamics are compatible with an equilibrium model with a short decay length Bicoid activity gradient as a sole source of positional information. Meanwhile, Bicoid’s partners speed-up the process by different means: Zelda lowers the Bicoid concentration threshold required for transcriptional activation while Hunchback reduces burstiness and increases the polymerase firing rate.

During development, cell identity is established reproducibly among individuals through the expression of specific genes at the correct time and correct location in space. How genes extract and combine both positional and temporal information from different transcription factor (TF) profiles along polarity axes remain largely unexplored. Here, we showcase the classic hunchback gene in fruit fly embryos, with focus on 3 of its main TFs: Bicoid, Zelda and Hunchback proteins. We constructed a series of synthetic MS2 reporters, where the numbers and combination of binding sites for each TF are varied. Using live imaging of transcription dynamics by these synthetic reporters and modeling tools, we show that i) a Bicoid-only synthetic reporter needs 3 more Bicoid binding sites than found in the hunchback promoter to recapitulates almost all spatial features of early hb expression but takes more time to reach steady state; ii) Hunchback and Zelda binding sites combined with Bicoid sites both reduce the time to reach steady state and increase expression at a different step in the activation process: Zld sites lower the Bicoid threshold required for activation while Hb sites increase the polymerase firing rate and reduce bursting; iii) the shift of the Bicoid-only reporter induced by a reduction by half of Bicoid concentrations indicates that the decay length of the Bicoid activity gradient is lower than the decay length of the Bicoid protein gradient. Altogether, this work indicates that Bicoid is the main source of positional information for hunchback expression and places back the Bicoid system within the physical limits of an equilibrium model.

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