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Fig. 6
Transgene coupling-mediated fluorescence expression in the adult heart increases after injury. a–c Cardiac injury induces NATC reporter expression. 6 mpf Tg(ubb:NATC);Tg(myl7:creER) fish were injected with 4-OHT intraperitoneally 30 days prior to cryoinjury. a Whole-mount immunostaining revealed that NTR-mCherry+ cardiomyocytes (red) were localized mainly adjacent to the damaged area (dashed line), identified by disorganized cardiac cells (DAPI+, blue). b Cytoplasmic NTR-mCherry (red) and nuclear DAPI (blue) labeling shows sham- and injury-induced NATC-activated cardiomyocyte morphologies; arrowheads point to double-labeled cardiomyocytes. c Ratios of mean gray values of NATC and DAPI labeling were calculated from maximum intensity projections of 200 μm thick confocal images of individual hearts. d Many NTR-mCherry+ cardiomyocytes (red) detected mainly at the lesion border zone (disorganized cardiac cells are revealed by DAPI staining, blue) were phospho-histone H3 (pH3, green) positive. e, f The majority of mitotic cardiomyocytes at the lesion border zone show NATC reporter expression. e NTR-mCherry (red) and myosin heavy chain (MHC, green) expression were detected by immunostaining of 6 mpf sham-operated and cryoinjured Tg(ubb:NATC); Tg(myl7:creER) hearts, in which Cre had been activated by 4-OHT treatment from 24 to 40 hpf. NTR-mCherry+ cardiomyocytes undergoing mitosis (white circles) were detected with an 8 h EdU pulse (EdU+ nuclei are in white) and their numbers were quantified as percentages of total numbers of mitotic cardiomyocytes (f). Cardiac cells were visualized by DAPI (blue) staining. Representative Tg(ubb:NATC);Tg(myl7:creER) hearts from four sham and six injured hearts (a–d), and three sham and three injured hearts (e, f) are shown/analyzed. Images are maximum or average intensity projections (a, e) and 3D volume renderings (d, e) of 200–300 (a), 20 (b, e), and 300 (d) μm thick confocal stacks. Bars and error bars in c, f represent mean ± S.E.M. Each circle (n = 4 in c and 3 in f) and triangle (n = 6 in c and 3 in f) represents a heart. *p ≤ 0.05, **p ≤ 0.01 (two-tailed student’s t-test). Scale bars: 5 μm (b), 20 μm (e), 30 μm (d), 50 μm (a)