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

项目总结/摘要 在脊椎动物的发育过程中，体节- 成体-在胚胎的伸长体轴的后端有节奏地和顺序地形成。 这种节奏的规范是由一个高度保守的生物钟，包括一个振荡基因 监管网络。在人类中，这种网络在单个细胞中无法稳健振荡或同步 跨越相邻细胞的组织范围的振荡与发育缺陷有关， 脊柱侧凸 尽管多年的研究确定了这种振荡网络的分子组成部分，我们仍然没有 了解这些成分中重要致病突变的影响机制。最关键的是， 到目前为止，关于这种高度动态的发展过程的绝大多数信息都来自于 来自固定组织或来自成熟时间太慢而不能揭示细胞的荧光报告子的推断。 这些突变在转录动力学水平上的机制基础。 为了克服这一主要障碍，我们建立了MS 2系统来测量转录动力学， 在活的、发育中的斑马鱼胚胎的单个细胞中，这个网络中的基因启动， 躯体发育和脊柱侧凸的模型。这种新的能力使我们能够重新审视分子过程 从单个细胞的动力学的角度来看，潜在的脊椎动物分割。使用此 技术，我们已经发现，平滑的蛋白质振荡的特点，体节发生是 由转录起始的爆发产生。在这里，我们建议以斑马鱼的体节发生为特征， 健康的条件下，并揭示（i）在体细胞发生转录爆发的分子起源和（ii） 这些突发如何通过信号通路在相邻细胞之间协调， 健康发育所必需的连贯的组织范围的振荡。我们设想，我们的工作将为 在脊椎动物发育中精确诊断疾病表型的分子基础的阶段。 此外，这里开发的实验和计算技术将使生物界能够 对脊椎动物发育动力学的单细胞性质进行探索， 目前在无脊椎动物中可获得的。最终，这种知识-结合创新 技术和分析提出这里-将有可能合理地修改这些转录 生物工程或治疗目的的任意动力学。

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.