A transepithelial osmotic pressure gradient is required for rapid wound closure and barrier reconstitution of zebrafish tail fin wounds. (a) Simplified scheme of larval zebrafish tail fin epithelium ∼3 dpf. Putative cell–cell contacts are indicated. (b, left) Representative time-lapse montage of zebrafish larvae immersed in hypotonic (Hypo) or isotonic (Iso_NaCl, Iso_Sucrose) solutions at the indicated times after UV laser puncture injury. The actin cytoskeleton is labeled with GFP-Utr-CH. Bars, 50 µm. (b, right) Quantification of wound area as a function of time after injury. (c, left) Time-lapse montage of suprabasal AKT-PH-GFP (plasma membrane PIP3) expressing zebrafish larvae wounded and incubated in isotonic mounting agar (0′–40’), and overlaid with hypotonic fish bathing solution (40’–90’). Bars, 50 µm. (c, right) Quantification of wound area as a function of time after injury. Red indices, isotonic conditions. (d, left) Representative time-lapse montage of Tg(actb2:HyPer) larvae subjected to 1 mM H₂O₂ in hypotonic or isotonic bathing medium 1 h after tail fin tip amputation in isotonic bathing medium. The HyPer probe is reversibly oxidized by H₂O₂ that enters the fish through the wound, increasing the HyPer (E500/E420) emission ratio and probing for wound permeability as a function of time after injury. HyPer emission ratios are color-coded. Bars, 100 µm. (d, right) Quantification of oxidized tissue area as defined by high HyPer ratios (>0.64). Error bars indicate SEM of indicated (n) number of larvae. See Videos 1–3.