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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 per condition were analyzed. (S) The percentage of wt1a:GFP+ cells that express more than 1,000 a.u. of mCherry, with the threshold corresponding to the maximum value of mCherry detected in nonactivated fibroblasts. (T) The percentage of col1a2:mCherry+;wt1a:GFP+ cells according to the thresholds shown in R. Graphs show individual measurements and means ± SD. ****P < 0.0001 by two-tailed t test. at, atrium; ba, bulbus arteriosus; v, ventricle. [Scale bars, 25 μm (H, I, M, and N), 50 μm (C–F), and 100 μm (G and L).]