Uncovering single-cell transcriptional dynamics in somitogenesis in live zebrafish embryos

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Project Summary/Abstract In vertebrate development, somites—morphological segments that prefigure the bones and muscles of the adult—are formed rhythmically and sequentially at the posterior end of the elongating body axis of the embryo. This rhythmic specification is dictated by a highly conserved biological clock that consists of an oscillatory gene regulatory network. In humans, failure of this network to robustly oscillate in individual cells or to synchronize tissue-wide oscillations across neighboring cells has been associated with developmental defects such as scoliosis. Despite years of research identifying the molecular components of this oscillatory network, we still do not understand the mechanisms affected by important disease-causing mutations in these components. Crucially, to date, the vast majority of information about this highly dynamic developmental process stems from inferences from fixed tissue or from fluorescent reporters whose maturation time is too slow to reveal the mechanistic basis of these mutations at the level of their transcriptional dynamics. To overcome this major obstacle, we established the MS2 system to measure the dynamics of transcriptional initiation of genes within this network in individual cells of living, developing zebrafish embryos, a widespread model of somitogenesis and scoliosis. This new ability empowers us to revisit the molecular processes underlying vertebrate segmentation from the standpoint of the dynamics of individual cells. Using this technology, we have discovered that the smooth protein oscillations that characterize somitogenesis are produced by bursts of transcriptional initiation. Here, we propose to characterize zebrafish somitogenesis in healthy conditions and to uncover (i) the molecular origins of transcriptional bursting in somitogenesis and (ii) how these bursts are coordinated between neighboring cells through signaling pathways in order to produce the coherent tissue-wide oscillations necessary for healthy development. We envision that our work will set the stage for precisely diagnosing the molecular underpinnings of disease phenotypes in vertebrate development. Further, the experimental and computational technologies developed here will empower the biology community to launch explorations into the single-cell nature of vertebrate development dynamics on par with what is currently attainable in invertebrate animals. Ultimately, this knowledge—combined with the innovated technologies and analyses proposed here—will make it possible to rationally modify these transcriptional dynamics at will for bioengineering or therapeutic purposes.