Mechanical forces are critical but poorly understood inputs for organogenesis and wound healing. Calcium ions (Ca) are critical second messengers in cells for integrating environmental and mechanical cues, but the regulation of Ca signaling is poorly understood in developing epithelial tissues. Here we report a chip-based regulated environment for microorgans that enables systematic investigations of the crosstalk between an organ's mechanical stress environment and biochemical signaling under genetic and chemical perturbations. This method enabled us to define the essential conditions for generating organ-scale intercellular Ca waves in Drosophila wing discs that are also observed in vivo during organ development. We discovered that mechanically induced intercellular Ca waves require fly extract growth serum as a chemical stimulus. Using the chip-based regulated environment for microorgans, we demonstrate that not the initial application but instead the release of mechanical loading is sufficient, but not necessary, to initiate intercellular Ca waves. The Ca response depends on the prestress intercellular Ca activity and not on the magnitude or duration of the mechanical stimulation applied. Mechanically induced intercellular Ca waves rely on IPR-mediated Ca-induced Ca release and propagation through gap junctions. Thus, intercellular Ca waves in developing epithelia may be a consequence of stress dissipation during organ growth.
This protocols document describes the design considerations and software tools to design a microfluidic device, fabrication protocols for making master molds and the final polydimethylsiloxane (PDMS) device, and testing of the completed microfluidic device.
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