Cellular Crowding Influences Extrusion and Proliferation to Facilitate Epithelial Tissue Repair

Epithelial wound healing requires a complex orchestration of cellular rearrangements and movements to restore tissue architecture and function after injury. While it is well-known that mechanical forces can affect tissue morphogenesis and patterning, how the biophysical cues generated after injury influence cellular behaviors during tissue repair is not well understood. Using time-lapse confocal imaging of epithelial tissues in living zebrafish larvae, we provide evidence that localized increases in cellular crowding during wound closure promote the extrusion of non-apoptotic cells via mechanically regulated stretch-activated ion channels (SACs). Directed cell migration toward the injury site promoted rapid changes in cell number and generated shifts in tension at cellular interfaces over long spatial distances. Perturbation of SAC activity resulted in failed extrusion and increased proliferation in crowded areas of the tissue. Together, we conclude that localized cell number plays a key role in dictating cellular behaviors that facilitate wound closure and tissue repair. Movie S1 Movie S1 Epithelial extrusion during homeostasis. Maximum intensity confocal projection images of 4 dpf (day post-fertilization) larvae acquired every 15 minutes for 1.5 hours of Et(Gal4-VP16)^zc1044a;Tg(UAS-E1b:Lifeact-EGFP) during homeostasis. Scale bar, 50 μm. Movie S2 Movie S2 Visualizing early cellular events after injury in a living epithelial tissue. Maximum intensity confocal projection images acquired every 15 minutes for 1.5 hours of Et(Gal4-VP16)^zc1044a;Tg(UAS-E1b:Lifeact-EGFP) after injury. Scale bar, 50 μm. Movie S3 Movie S3 Cellular crowding promotes extrusion at the wound site after injury. Maximum intensity confocal projection of time-lapse imaging of Tg(Ubi:H2A-EGFP-2A-mCherry-CAAX) after amputation. Images were acquired every 5 minutes and 51 seconds for 1.5 hours. Scale bar, 50 μm. Movie S4 Movie S4 Strain rate after amputation. Strain rate maps of untreated (left) and Gd3+ treated (right) tail fins over 30min post-amputation; time-frame = 1min. Color code gives the magnitude of simple strain rate in s^-1. Blue represents negative strain rate (contraction), red represents positive strain rate (extension) and green represents no strain.