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mmp14benh1 is a bona fide mmp14b enhancer and is required for efficient heart regeneration. (A) Strategy for deletion of the 304-bp mmp14b-enh1 sequence. (B) PCR genotyping of a mmp14b+/+, mmp14b+/Δenh1, and mmp14bΔenh1/Δenh1 zebrafish. (C) RNAscope in situ hybridization for endogenous mmp14b on frontal sections of injured mmp14b+/+ and mmp14bΔenh1/Δenh1 adult zebrafish hearts at 7 dpa. Boxed regions are enlarged in (1) and (2). Arrows highlight mmp14b expression (magenta). (D) Relative expression in AU of mmp14b in control (sham) and injured (Amp) mmp14b+/+ and mmp14bΔehn/1Δenh1 zebrafish hearts 7 dpa. (E) Sections of hearts from adult mmp14b+/+ and mmp14bΔenh1/Δenh1, subjected to ventricular amputation and immunostained for cardiomyocyte nuclei (⍺-Mef2; magenta) and cycling cells (⍺-PCNA; green). Boxed regions are enlarged in (1 to 4). Arrows highlight PCNA/Mef2 double-positive cardiomyocytes. (F) Cardiomyocyte proliferation indices at 7 dpa in mmp14b+/+ and mmp14bΔenh1/Δenh1 hearts. Proliferation data were collected for 10 sections per heart and averaged to generate each data point. (G) Frontal sections of adult mmp14b+/+ and mmp14bΔenh1/Δenh1 hearts 30 dpa stained with AFOG to detect muscle (brown), fibrin (red), and collagen (blue). The asterisk highlights collagen-rich scar tissue. (H) Residual scar as a percentage of total ventricular area at 30 dpa in mmp14b+/+ and mmp14bΔenh1/Δenh1 hearts; data were collected for four to six sections per heart and averaged to generate each data point. The orange dashed lines approximate the amputation area. Scale bars, 100 μm [(C) and (G)] and 50 μm (E). Statistical significance in (D) was determined using one-way ANOVA with Tukey’s multiple comparisons test. Statistical significance in (F) and (G) was determined using Student’s t test. **P < 0.01; ***P < 0.001.
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