Figure 1.

Epicardial Duox-generated H₂O₂ associated with myocardial regeneration. (A-C) After imaging of Tg(tcf21:nucEGFP), we performed RNA in situ hybridization with duox probes on 10-μm cryosections of Tg(tcf21:nucEGFP) hearts at sham, 7 and 14 dpa. Note the induced expression of duox and its colocalization with epicardial reporter Tg(tcf21:nucEGFP) in injured areas (arrowheads) at 7 and 14 dpa (B, C) while little staining at sham (A). Scale bar for A-C, 50 μm. (D) Cardiac-specific expression of Hyper in a zebrafish embryo. Hyper was restricted in the Tg(myl7:Hyper) transgenic heart at 48 hpf. (E-H) Ex vivo Hyper heart images from sham control (E), 7 dpa (F), 14 dpa (G) and 30 dpa (H). Spatially resolved H₂O₂ image, indexed by the ratio between the F₄₈₈ and F₄₀₅ images of Hyper (left panels), is presented in pseudocolor. (I) Ratiometric Hyper signals (F₄₈₈/F₄₀₅) averaged over the regenerative zone of injured heart during the first month of regeneration after resection. n = 3. (J) Representative transmural spatial profiles of the Hyper signal at 14 dpa (red line) and sham (green line) hearts. (K) Ex vivo calibration of Hyper F₄₈₈/F₄₀₅ ratio as a function of ambient H₂O₂ concentration. n = 3. Arrow denotes the average F₄₈₈/F₄₀₅ ratio seen in regenerative zones at 14 dpa. (L-O) Redox signal showing greater epicardial and myocardial oxidization during regeneration. A 14 dpa Tg(myl7:hyper) heart was labeled with Redox sensor cc-1. Images show Hyper signals at 488 (L) and 405 nm (M), and Redox sensor cc-1 signals at 555 nm excitation (N), and the merging of L and N (O). Note that the Redox sensor cc-1 signal (arrowheads), indicative of intracellular oxidization, was most conspicuous in epicardial cells lacking Hyper expression, and also overlapped with the Hyper signal (arrows) in adjacent myocardium. Scale bars, 100 μm.