Abstract: During embryonic development, tightly choreographed patterns of gene expression—shallow gradients, sharp steps, narrow stripes—specify cell fates. The prediction of developmental outcomes from these transcription factor patterns and from regulatory DNA sequence remains an open challenge in physical biology that requires a quantitative understanding of the mechanisms that dictate the flow of information along the processes of the central dogma. We used stripe 2 of the even-skipped gene in Drosophila embryos as a case study in the dissection of the regulatory forces underpinning a key step along the developmental decision-making cascade: the generation of cytoplasmic mRNA patterns via the control of transcription in individual cells. Using live imaging, theoretical and computational approaches, we developed a comprehensive toolkit to watch the regulation of the entirety of the central dogma in real time. We found that the transcriptional burst frequency is modulated across the stripe to control the mean mRNA production rate. However, we discovered that bursting alone cannot quantitatively recapitulate the formation of the stripe, and that control of the window of time over which each nucleus transcribes even-skipped plays a critical role in stripe formation. Theoretical modeling led to the discovery that these two regulatory strategies—the analog control of the mean transcription rate by bursting and the binary control of the transcription time window—obey different kinds of regulatory logic, suggesting that the stripe is shaped by the interplay of two distinct molecular processes. Our work provides an example of how biological numeracy can be used as a driver for discovery as well as a stark reminder that reaching a predictive understanding of developmental decision-making will require a precise understanding of how gene expression is regulated not only across space, but also over time.
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