In vivo mechanisms of Dorsal-mediated transcriptional control in development - 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. These measurements have revealed that the transcriptional activity of individual genes is not constant in time; rather, bursts of gene expression arise when promoters transition between ON and OFF states. Transcriptional bursting in eukaryotes, particularly in developmental genes, is ubiquitous; most transcription factors control mRNA levels by modulating bursting frequency, duration or amplitude, or some combination thereof. Critically, while we have uncovered which bursting parameters transcription factors modulate to dictate transcriptional dynamics, we remain ignorant about how this control is implemented at the molecular level. Here, we propose to make progress toward uncovering the molecular mechanisms by which the Dorsal activator controls transcriptional bursting in the early embryo of the fruit fly Drosophila melanogaster. We will combine our cutting-edge imaging and computational technologies for simultaneously measuring Dorsal concentration dynamics and the instantaneous state of its target promoters (ON or OFF) in individual nuclei of living embryos. Specifically, we will (1) use our novel compound-state Hidden Markov model to determine whether Dorsal controls burst size, frequency, amplitude, or some combination thereof, in order to generate hypotheses about the mechanisms of action of this activator, (2) determine whether stable clusters of high Dorsal concentration that we recently discovered play an active role in regulating transcriptional dynamics, and (3) use cutting-edge lattice light-sheet and adaptive optics microscopy to detect functional Dorsal binding events at snail and determine how this binding triggers the cascade of biochemical events that leads to the initiation and maintenance of transcription. Overall, our proposed work will establish a clear workflow for the in vivo dissection of the molecular mechanisms of transcriptional control in development. This approach is amenable to implementation in other genes in the fruit fly as well as in other workhorses of developmental biology. We envision that our mechanistic insights will make it possible to derive theoretical models of developmental decision-making that 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 in cancer.
