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

项目摘要 生物振荡器对各种细胞、生理和发育过程都是必不可少的，例如 细胞分裂、心跳和体细胞发生。生物振荡器受损会导致从失眠到 癌症并对发育和分化有重大影响。分段时钟，一种生物时钟 从斑马鱼到人类都很保守的振荡器，在调节周期性体节的形成中起着关键作用。 在脊椎动物胚胎的体细胞发育过程中。尽管分段时钟的中心分子参与者 时钟已经被发现很久了，它被嵌入到一个巨大的细胞内和细胞间网络中，以及它如何响应 复杂的机械和生化微环境在很大程度上仍不清楚。这样做的目的是 建议开发一种体外试验，使复杂过程的定量分析成为可能 参与斑马鱼的体细胞发育。分裂前中胚层(PSM)细胞，前体细胞参与 体细胞发生，将从斑马鱼胚胎中分离出来，并在一系列 具有可调机械线索(基板硬度和机械拉伸)的微机械工具 生理范围。将进行实时成像来跟踪细胞行为，以监测它们的振荡 行为、细胞内信号活动和细胞力学(包括细胞骨架收缩和细胞 硬度)作为衬底硬度和机械拉伸的函数。重要的是，我们的研究将进行 对于单个细胞以及在细胞-细胞通信被保存的细胞克隆的上下文中。 总之，我们提出的研究将带来关于机械和生化的新知识 微环境共同调节PSM细胞，使其自组织成发育模式。

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.