Transient fibrosis resolves via fibroblast inactivation in the regenerating zebrafish heart

Fig. 1.. Intracardiac fibroblasts contribute to transient fibrosis during zebrafish heart regeneration. (A and B) Transcriptome analysis of wt1a:GFP+ cells isolated from adult zebrafish hearts. (A) Ingenuity pathways enriched in wt1a:GFP+ ventricular cells compared with all GFP− cells. A number of differentially expressed genes are shown; the x axis shows −log10[Benjamini–Hochberg adjusted (B.H.) P values]. (B) Heatmap indicating up-regulation of fibrotic marker genes (underlined) in the wt1a:GFP+ cell fraction. (C–F) postnb mRNA in situ hybridization followed by anti-GFP immunohistochemistry on sections of wt1a:GFP ventricles without injury (C and D) and at 7 d postinjury (E and F). Arrowheads indicate wt1a:GFP+ cells. (G–P) Immunofluorescence staining of sections of wt1a:GFP;col1a2:mCherry-NTR double-transgenic hearts without injury (G–K) or at 7 dpi (L–P). Red arrowheads mark col1a2:mCherry+ cells; green arrowheads mark wt1a:GFP+ cells; and yellow arrowheads mark double-positive cells. Asterisk in L indicates injured area. (Q and R) FACS-sorted cells from the wt1a:GFP;col1a2:mCherry ventricular apex without injury (Q) or at 7 dpi (R). Representative examples from a total of four hearts…

Fig. 2.. Endocardium-derived cells contribute to transient fibrosis during zebrafish heart regeneration. (A) Analysis of the transcriptome of kdrl:mCherry+ cells FACS-sorted from the ventricular apex of hearts with no injury or at 7 dpi was performed. Volcano plot. Light blue, false discovery rate (FDR) >0.05, abs[log fold change (LFC)] <1; orange, FDR >0.05, abs(LFC) >1; green, FDR <0.05, abs(LFC) >1. (B) Ingenuity pathway analysis including the number of differentially expressed genes. (C–F) Immunofluorescence with anti-GFP and anti-mCherry of a heart section from an adult fli1a:GFP;col1a2:mCherry-NTR zebrafish. Yellow arrowheads mark double-positive cells at the injury area (IA; asterisk). Green arrowheads mark fli1a:GFP+ cells negative for mCherry. (G and H) FACS-sorted cells from fli1a:GFP;col1a2:mCherry hearts without injury or at 7 dpi. Shown are two representative examples from a total of four hearts per condition analyzed. (I) The percentage of fli1a:GFP+ cells that express more than 100 a.u. of mCherry, with the threshold corresponding to the maximum value of mCherry detected in endocardial cells from uninjured hearts. (J) The percentage of col1a2:mCherry+;fli1a:GFP+ cells…

Fig. 3.. periostin b expression marks an activated cardiac fibroblast population upon ventricular cryoinjury. (A–E) Whole-heart immunofluorescence in the postnb:citrine; fli1a:dsRedEx double-transgenic line. Whole-heart (A) and zoomed-in views (B and C) of the ventricular apex of an uninjured heart and a heart at 7 dpi (D and E). Perivascular cells can be observed in C. postnb:citrine, green; fli1a:dsRedEx, red. (F and G) Immunofluorescence staining of a sagittal heart section of a postnb:citrine;kdrl:mCherry zebrafish (F). Asterisk in F marks the injury area. (G) Zoomed-in view of the injured ventricular apex. postnb:citrine, green; kdrl:mCherry, red. (H) Quantification of proliferating postnb+ cells postinjury (mean ± SD; ****P < 0.0001, **P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparisons test). (I) FACS-sorted cells from kdrl:mCherry;postnb:citrine hearts. No double-positive cells were detected. (J and K) Transcriptome analysis of postnb:citrine+ cells isolated from the ventricular apex. (J) Ingenuity pathway analysis. The x axis shows −log10(B.H. P values). (K) Volcano plot. Light blue, FDR >0.05, abs(LFC) <1; orange, FDR <0.05, abs(LFC) <1; green, FDR <0.05, abs(LFC)…

Fig. 4.. Persistence of postnb-derived cells in the regenerated myocardium. (A) 4-Hydroxytamoxifen was added to postnb:CreERT2;ubb:Switch fish at 3 and 4 dpi, and hearts were dissected at different dpi. (B–I) Immunofluorescence staining of heart sections at 7 (B–E) or 90 dpi (F–I). (J) Percentage of postnb-derived cells at the injury area. Symbols show individual measurements, and boxes and whiskers show mean ± SD; **P = 0.0064 by two-tailed unpaired t test. (K–M) postnb-derived mCherry+ cells were sorted from the ventricular apex at 7 and 60 dpi, and transcriptome analysis was performed on isolated mCherry+ cells. (K and L) Ingenuity pathway analysis. Bars represent pathways enriched in postnb-derived cells compared with the remainder of cells in the injury area at 7 and 60 dpi. Numbers of differentially expressed genes are indicated. The x axis shows −log10(B.H. P values). (M) Volcano plot. Light blue, FDR >0.05, abs(LFC) <1; orange, FDR >0.05, abs(LFC) >1; green, FDR <0.05, abs(LFC) >1. (N) Heatmap of the top 200 differentially expressed genes between 7 and 60 dpi in postnb-traced cells and their expression in wt1a:GFP+ cells from uninjured hearts. [Scale bars, 25 µm (C–E and G–I) and…

Fig. 5.. Genetic ablation of collagen 1a2-expressing cells impairs cardiomyocyte proliferation in the cryoinjured heart. (A) col1a2:mCherry-NTR adult animals were cryoinjured and treated with metronidazole (Mtz) from 4 to 6 dpi. BrdU injection was performed 1 d before fixation. (B–G) Immunofluorescence using anti-mef2 and anti-MHC to mark cardiomyocytes, and anti-BrdU in col1a2:loxP-tagBFP-loxP-mCherry-NTR (control) and col1a2:mCherry-NTR fish treated with Mtz and BrdU as described in A. Arrowheads mark BrdU+ cardiomyocytes. (H) Quantification of BrdU+ cardiomyocytes in col1a2:mCherry-NTR and control hearts. Shown are individual measurements and median ± interquartile range; ***P = 0.0004 by Mann–Whitney test, n = 23 fish per condition, from two different experiments (blue and red dots). Three whole-heart ventricle sections were quantified per point. [Scale bars, 10 µm (C and E–G) and 100 µm (B and D).]