a–f Cryosections of Tg(Δ113p53:GFP) hearts at sham (a, a′), 4 (b, b′), 7(c, c′), 14 (d, d′), 21 (e, e′) and 30dpa (f, f′) were immunostained by anti-GFP (in green) and anti-MHC (MF20) (in red) antibodies. The nucleus were stained with DAPI (in blue). Scale bar, 50μm. g Average size of GFP⁺ cardiomyocytes on heart sections of Tg(Δ113p53:GFP) at sham, 4, 7, 14, 21 and 30dpa, was presented as the percentage of the total ventricular area. Each dot presented an individual heart. Data are means of 3 sections/heart from 3 hearts/time point. h, i RNA in situ hybridisation was performed with the DIG-labelled probe to detect both p53 and Δ113p53 on cryosections of WT hearts at sham (h) and 14dpa (i). The representative picture was taken from three hearts in each group. Scale bar, 50μm. j Relative mRNA expression of p53, Δ113p53 and p21 in the WT injury hearts at sham and 7dpa. The total RNA was extracted from a pool of at least 10 hearts in each group. k, l Cryosections of Tg(Δ113p53:GFP) hearts of p53^+/+ sibling (k) and p53^M214K mutant (l) at 14dpa were immunostained by anti-GFP antibody. The representative picture was taken from three hearts in each group. The white arrow heads indicate wounding site. Scale bar, 50μm. The experiments were repeated independently for at least three times with similar results. Statistical analysis was performed on relevant data using Student’s two-tailed t test in GraphPad Prism 5. The p values were represented by n.s. and asterisks. n.s., p>0.05. *p<0.05. **p<0.01. ***p<0.001.

a–e Cryosections of Edu-labelled Tg(Δ113p53:GFP) hearts at sham (a) and 7 dpa (b, c) were immunostained by anti-GFP (in green) and anti-MF20 (in red) antibodies. The nucleus were stained with DAPI (in blue). Framed area in b was magnified in c. The representative picture was taken from 3 to 7 hearts. Scale bar, 50 μm. Yellow arrows: Edu⁺/GFP⁻/MF20⁻ cells; white arrows: Edu⁺/GFP^-/MF20⁺ cells; white arrow head: Edu⁺/GFP⁺/MF20⁺ cells. The number of Edu⁺/MF20⁺ cells on heart sections of Tg(Δ113p53:GFP) at sham and 7 dpa, was presented as the percentage of the total MF20⁺ cells at the wound site (d). The number of Edu⁺/GFP⁺/MF20⁺ cells on heart sections of Tg(Δ113p53:GFP) at sham and 7 dpa, was presented as the percentage of the total GFP⁺/MF20⁺ cells at the wound site (e). Data are means of 4–6 sections/heart with the largest wound area from 3 to 7 hearts in different treatments. Scale bar, 50 μm. Each dot represents an individual heart. f A schematic diagram representing the 4HT-based Cre-LoxP system driven by Δ113p53 promoter. Δ113p53-P (blue arrow): the 3.6-kb DNA fragment from the upstream of Δ113p53 transcription start site; β-act2-P (blue arrow): the promoter of β-actin2; CreER (Brown bar): the coding region of tamoxifen-inducible Cre recombinase–oestrogen receptor fusion protein; LoxP (blue bar): the site of LoxP; DsRed (red bar): the coding region of DsRed; Stop (black bar): the translation stop codon; EGFP (light green bar): the coding region of EGFP gene; 4HT: the treatment of 4-hydroxytamoxifen. g Schematics of the cell lineage tracing experiment. Either sham or surgical Tg(Δ113p53:CreER; β-act2:RSG) zebrafish were treated with 4HT at 7 and 10 dpa as indicated. The treated surgical zebrafish were sampled at 14 and 30 dpa, while all of the treated sham zebrafish were sampled at 30 dpa. h–k Red and green fluorescence on the cryosections of Tg(Δ113p53:CreErt2;β-actin:RSG) hearts at sham (i), 14 (j) and 30 dpa (k) were from the en vivo DsRed and EGFP protein respectively. The nuclei were stained with DAPI (blue). Scale bar, 50 μm. The number of EGFP⁺ cells on heart sections of Tg(Δ113p53:CreER; β-act2:RSG) at 14 and 30 dpa, was presented as the percentage of the total DsRed⁺ cells at the resection site (h). Data are from the biggest section with most EGFP⁺ cells of every heart. Each dot represents an individual heart. l–n Fibrin clot stained with Masson’s trichrome on the crysections of Δ113p53^+/+ (l) and Δ113p53^M/M mutant hearts (m) at 30 dpa. Yellow dotted lines indicate the approximate injury area. Scale bar, 50 μm. Average injury area with fibrin clots on sections of Δ113p53^+/+ and Δ113p53^M/M mutant hearts at 30 dpa was presented as the percentage of the total ventricular area (n). Data are means of three sections/heart. Each dot represents the average injury area of an individual heart. The experiments were repeated independently for at least three times with similar results. Statistical analysis was performed on relevant data using Student’s two-tailed t test in GraphPad Prism 5. The p values were represented by n.s. and asterisks. n.s., p > 0.05. *p < 0.05. **p < 0.01. ***p < 0.001.

a Co-localisation of red (Δ113p53⁺ cells) (a′) and green fluorescence (gata4⁺ cells) (a′′) on the cryosections of Tg(Δ113p53:mCherry; gata4:EGFP) hearts at 14 dpa. The white arrow heads indicate co-labelling. The nucleus were stained with DAPI (in blue). The representative picture was taken from three hearts. Scale bar, 50 μm. b–e Cryosections of Tg(gata4:EGFP); Δ113p53^+/+ (b, b′, d, d′) and Tg(gata4:EGFP); Δ113p53^M/M hearts (c, c′, e, e′) at 7 and 14 dpa, were immunostained by anti-GFP (in green) and anti-MF20 (in red) antibodies. The nucleus were stained with DAPI (in blue). Scale bar, 50 μm. f Average size of EGFP⁺ cardiomyocytes on the edge of wound site in Tg(gata4:EGFP); Δ113p53^+/+ and Tg(gata4:EGFP); Δ113p53^M/M mutant zebrafish at 7 and 14 dpa, was presented as the percentage of the ventricular area at the resection site. Data are means of three sections/heart from six hearts/time point. Scale bar, 50 μm. Each dot represents an individual heart. The experiments were repeated independently for at least three times with similar results. Statistical analysis was performed on relevant data using Student’s two-tailed t test in GraphPad Prism 5. The p values were represented by n.s. and asterisks. n.s., p > 0.05. *p < 0.05. **p < 0.01. ***p < 0.001.

a, b The DsRed⁺ nucleus of cardiomyocytes (in red) at the resection site in Tg(myl7:nDsRed); Δ113p53^+/+(a) and Tg(myl7:nDsRed); Δ113p53^M/M (b) hearts were labelled by EdU (in green) at 14 dpa. Framed areas were magnified in a′ and b′. nDsRed: nuclear DsRed. The white arrow heads indicate co-labelling. Scale bar, 50 μm. c, d Cryosections of Tg(myl7:nDsRed); Δ113p53^+/+ (c) and Tg(myl7:nDsRed); Δ113p53^M/M (d) hearts at 7 dpa were co-stained by anti-DsRed and anti-PCNA antibodies. The nucleus were stained with DAPI (in blue). Framed areas were magnified in c′ and c″, or d′ and d″. The white arrow heads indicate co-labelling. Scale bar, 50 μm. e, f The number of co-labelled nDsRed⁺ nucleus of cardiomyocytes with either EdU (e) or PCNA (f) in Tg(myl7:nDsRed); Δ113p53^+/+ and Tg(myl7:nDsRed); Δ113p53^M/M hearts at 14 or 7 dpa, was presented as the percentage of the total nDsRed⁺ nucleus at the resection site. Data are means of three sections/heart. Each dot represents the average number of co-labelled nDsRed⁺ nucleus of cardiomyocytes with either EdU or PCNA in an individual heart. The experiments were repeated independently for at least three times with similar results. Statistical analysis was performed on relevant data using Student’s two-tailed t test in GraphPad Prism 5. The p values were represented by n.s. and asterisks. n.s., p > 0.05. *p < 0.05. **p < 0.01. ***p < 0.001.

(a–d) After surgery, Tg(Δ113p53:GFP) animals were treated with either DMSO (a, a′, c, c′) or DPI (b, b′, d, d′) daily at 3–7 or 7–14 dpa. The treated animals were sampled at 7 and 14 dpa and subjected to cryosection. Cryosections of hearts were immunostained by anti-GFP (in green) and anti-MHC (MF20) (in red) antibodies. The nuclei were stained with DAPI (in blue). Scale bar, 50 μm. e Average size of GFP⁺ cardiomyocytes on heart sections of Tg(Δ113p53:GFP) treated with DMSO or DPI at 7 and 14 dpa, was presented as the percentage of the ventricular area at the resection site. Data are means of three sections/heart. Each dot represents the average size of GFP⁺ cardiomyocytes in an individual heart. Scale bar, 50 μm. f–l Ex vivo HyPer heart images of either Δ113p53^+/+ (f, h, j) or Δ113p53^M/M mutant hearts (g, i, k) at sham, 10.5 and 21 dpa. Spatially resolved H₂O₂ image, indexed by the ratio between the F₄₈₈ and F₄₀₅ images of HyPer (below), is presented in pseudocolor. Ratiometric HyPer signals (F488/F405) averaged over the regenerative zone of injured heart at 3.5, 7, 10.5, 14, 17.5 and 21 dpa were presented as the difference to the average F₄₈₈/F₄₀₅ ratio at the apex of respective sham hearts (l). Each dot represents the ratiometric HyPer signal in an individual heart. Statistical analyses were performed on data from Δ113p53^+/+ and Δ113p53^M/M mutant hearts at the same time point. The experiments were repeated independently for at least three times with similar results. Statistical analysis was performed on relevant data using Student’s two-tailed t test in GraphPad Prism 5. The p values were represented by n.s. and asterisks. n.s., p > 0.05. *p < 0.05. **p < 0.01. ***p < 0.001.

Δ113p53:GFP was not detectable in the surgery hearts of p53^M214K mutant fish at 14 dpa. (A-B) Cryosections of Tg(Δ113p53:GFP) hearts of p53^+/+ (A) and p53^M214K mutant (B) at 14 dpa were immunostained by anti-GFP (in green) and anti-MHC (MF20) (in red) antibodies. The nucleus were stained with DAPI (in blue). The representative picture was taken from 3 hearts in each group. The white arrow heads indicate wounding site. Scale bar, 50 μm.

Validation of Tg(Δ113p53:CreER; β-act2:RSG) zebrafish. (A) Western blot with a monoclonal antibody against zebrafish Δ113p53 was performed to analyze the induction of Δ113p53 upon the treatment of a DNA damage drug, camptothecin (Campt). The Tg(Δ113p53:CreER; β-act2:RSG) zebrafish embryos at 1 day post fertilization (dpf) were treated with Campt for 24 hours. Afterwards, a part of untreated and Campt-treated embryos were divided and treated with 4HT for 2 hours. β-actin was used as the protein loading control. (B-E) Life images of red (DsRed) (B, C, D, E), green (EGFP) (B’, C’, D’, E’) and bright field (B”, C”, D”, E”) in Tg(Δ113p53:CreER; β-act2:RSG) zebrafish embryos treated with either Campt (C, C’, C”), or 4HT (D, D’, D”), or both (E, E’, E”) as described above. Scale bar, 500 μm.

Δ113p53^M/M mutant and WT uninjured hearts are comparative. (A-B) Trichrome Masson’s staining on the crysections of uninjured Δ113p53^+/+ (A) and Δ113p53^M/M mutant hearts (B) at 9 months of age. The representative picture was taken from 5 hearts in each group. Scale bar, 50 μm.

style='font-size:15px;line-height: 29.333335876464844px;font-family: "Times New Roman";'>Validation of Tg(Δ113p53:mCherry) zebrafish. (A) Diagram showing the Tg(Δ113p53:mCherry) reporter driven by Δ113p53promoter. Δ113p53-P (blue arrow): the 3.6 kb DNA fragment from the upstream of Δ113p53 transcription start site; mCherry (red bar): the coding region of mCherry. (B-C) Live images of red (mCherry) fluorescence in Tg(Δ113p53:mCherry) zebrafish embryos treated with Campt (C) or without Campt (B) for 24 hours. Scale bar, 500 μm.

Depletion of Δ113p53 has little effects on DNA damage response and apoptotic activity during heart regeneration. (A-B) Cryosections of Tg(myl7:nDsRed); Δ113p53^+/+ (A) and Tg(myl7:nDsRed); Δ113p53^M/M (B) hearts at 14 dpa were co-stained by anti-DsRed (in red) and anti-γ-H2AX (in green) antibodies. Framed areas were magnified in A’ and B’. (C-D) TUNEL assay (in green) and co-immunostaining with anti-DsRed antibody (in red) were performed on cryosections of Tg(myl7:nDsRed);Δ113p53^+/+ (C) and Tg(myl7:nDsRed); Δ113p53^M/M (D) hearts at 14 dpa. Framed areas were magnified in C’ and D’. The representative picture was taken from 6 hearts in each group. Scale bar, 50 μm.