Project Summary/Abstract The correct implementation of developmental programs depends on information encoded in an organism's DNA. Despite decades of work in dissecting the spatial control of gene expression in embryonic development, we know relatively little about the temporal control of these programs—largely due to reliance on dead, fixed tissues. Recently, our lab established new technologies for real-time measurements of input transcription factor concentration dynamics and output transcriptional activity in single cells of the early embryo of the fruit fly Drosophila melanogaster. Our measurements have revealed that the timing of transcription in development is under precise control. Here, we propose a dialogue between theory and experiment to dissect this largely unexplored layer of transcriptional control using the regulation of the hunchback gene by the activator Bicoid and the pioneer-like transcription factor Zelda as a case study. Recent work from our lab has suggested that hunchback transcription ensues as a result of the transition of its promoter through multiple transcriptionally silent states. In this model, Bicoid and Zelda catalyze the transitions between silent states, presumably by triggering the acetylation of nearby histones. In order to systematically test this model of transcriptional onset and reveal the molecular identities of the pathways involved in the dictating transcriptional onset, we will (i) exploit optogenetics to determine whether the timing Bicoid- and Zelda-driven transcriptional onset is independent of the control of mRNA production rates once transcription has already ensued, (ii) harness measurements of the distribution of transcriptional onset times in single cells to uncover the structure of the biochemical cascade leading to transcriptional onset and how this cascade is modulated by Bicoid and Zelda binding, and (iii) identify the histone acetylases and deacetylases invoked by Bicoid and Zelda to dictate transcriptional onset, a process that takes place at time scales that are not accessible by commonplace genome-wide approaches to dissect the chromatin landscape. Overall, our proposed work will establish a clear workflow for the dissection of this new layer of regulatory control given by the timing of transcription in development and for uncovering the underlying molecular pathways. This approach will be amenable to be implemented in other relevant genes in the fruit fly as well as in other workhorses of developmental biology. Further, we envision that our quantitative and predictive approach to dissecting developmental programs will empower future synthetic applications as well as reengineering of multicellular organisms, for example to fix developmental defects or to halt states of unchecked cellular proliferation.