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.