Quantitative characterization of a vertebrate segmentation clock response to biomechanical signals during zebrafish somitogenesis

项目摘要 生物振荡器对于多种细胞、生理和发育过程是必不可少的，例如 细胞分裂心跳和体节发生受损的生物振荡器会导致失眠， 癌症，并对发育和分化有重大影响。分段时钟，一个生物 振荡器从斑马鱼到人类都很保守，在调节周期性体节的形成中起着关键作用 在脊椎动物胚胎体节发生过程中。尽管分裂时钟的核心分子参与者 长期以来，时钟被发现嵌入在一个大型的细胞内和细胞间网络中，以及它如何响应 复杂的机械和生物化学微环境在很大程度上仍然是未知的。这个目标 一个建议是开发一种体外测定，使复杂的过程的定量解剖 参与了斑马鱼的体节发生。前体中胚层（PSM）细胞，参与细胞分化的前体细胞， 体细胞发生，将从斑马鱼胚胎中分离出来，培养并在一系列的 具有可调机械信号（衬底刚度和机械拉伸）的微机械工具 生理范围。将进行实时成像以跟踪细胞行为，监测其振荡 行为、细胞内信号传导活动和细胞力学（包括细胞骨架收缩性和细胞力学）。 刚度）作为基底刚度和机械拉伸的函数。重要的是，我们的研究将在 对于单细胞以及在细胞间通讯被保留的细胞集落的情况下。 总之，我们提出的研究将导致新的知识，如何机械和生化 微环境共同调节自我组织成发育模式的PSM细胞。

Project Summary Biological oscillators are essential to a variety of cellular, physiological and developmental processes, such as cell divisions, heartbeats, and somitogenesis. Impaired biological oscillators cause diseases from insomnia to cancer and have a significant impact on development and differentiation. Segmentation clock, a biological oscillator well-conserved from zebrafish to humans, plays a key role in regulating the periodic somite formation during vertebrate embryo somitogenesis. Although the central molecular players of the segmentation clock have been long identified, the clock is embedded in a large intra- and inter-cellular network, and how it responds to the complicated mechanical and biochemical microenvironments remains largely unknown. The goal of this proposal is to develop an in vitro assay that enables the quantitative dissection of the complex processes involved in the zebrafish somitogenesis. Presomitic mesoderm (PSM) cells, the precursor cells involved in the somitogenesis, will be isolated from zebrafish embryos and cultured and examined under an array of micromechanical tools with tunable mechanical cues (both substrate rigidity and mechanical stretching) across a physiological range. Live imaging will be conducted to track cell behaviors, to monitor their oscillatory behaviors, intracellular signaling activities and cell mechanics (including both cytoskeletal contractility and cell stiffness) as a function of substrate rigidity and mechanical stretching. Importantly, our studies will be conducted for both single cells as well as in the context of cell colonies where cell-cell communications are preserved. Together, our proposed studies will lead to new knowledge about how the mechanical and biochemical microenvironments jointly regulate PSM cells that self-organize into developmental patterns.