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Fig. 3 Nrf2a-depleted zebrafish exhibit reduced tissue repair responses after injury. (A-I) Tissue repair performance assessment. Caudal fin amputations were performed without injury to the notochord (dotted line) at 2 dpf, then the potential of tissue repair was evaluated at 2 (E-I) or 3 (B-D) days post-amputation (dpA). (B-C) Control MO and nrf2a MO were caudal fin amputated. Representative imaging of injured tail fin (scale bars, 100 μm) (B) and measurement of regenerated fin areas, lengths and widths (C) (n = 14–18 from 3 independent experiments, Two-tailed Student t-test). Error bars represent standard error of the mean (SEM). The regenerated fin areas were defined as the region between the amputation edge and the end of the extended fin. The regenerated fin lengths were measured from the notochord tip to the end of the extended fin. The regenerated fin widths were defined as the max width measured from the amputation edge to the end of the extended fin. (D) Inhibition of nrf2a was carried out in WT and CF animals by injecting the nrf2a morpholino. Measurement of regenerated tail fin areas, lengths and widths in the presence or absence of nrf2a (n = 16 from 3 independent experiments, Two-way ANOVA with Tukey's multiple comparisons test). (E) SHG and high magnification bright field imaging showing epithelial projection associated with collagen fibbers (scale bar, 10 μm). (F) Representative bright field microscopy of injured caudal fin showing extended collagen fibber-containing epithelial projections (arrows) pushing the healing plane forward (scale bars, 100 μm). Morpholino knockdown of nrf2 expression reduced the appearance of projections, suggesting that Nrf2a modulates the formation of collagen fibber in zebrafish. (G) Proportion of larvae forming visible projections (G; n = 14–16, Fisher test). ≈ 90 % of control larvae had visible projections, compared to only ≈ 50 % of nrf2a MO. (H) Mean ± SEM number of projections per larvae (H; n = 14–16, Two-way ANOVA, Tukey's multiple comparisons test). (I) Measurement of wound contour length in the presence or absence of nrf2a (n = 14–18 from 3 independent experiments, Two-way ANOVA with Tukey's multiple comparisons test). Nrf2a deficiency impairs tissue repair in zebrafish by affecting the formation of collagen and epithelial projections.