Mutations in Grl lead to enhanced CM proliferation and reduced fibrotic scar tissue following injury. (A) The sgRNA target sequence of the grl allele (blue) and the PAM (yellow) designed in the first exon of grl for mutation generation. (B) Targeted deletion mutations induced by CRISPR/Cas9 technique at the grl genes. The WT sequence is shown at the top. Deletions are shown as red dashes. The mutation deletion is indicated at the right of each sequence. (C) Experimental design for PCNA and Mef2 immunostaining and fibrotic scar (AFOG) analysis. (D-F) Representative AFOG staining images (blue for collagen, red for fibrin) of injured ventricles from WT sibling and grl5nt−/− fish at 30 dpa, scored as ‘class 1’ (complete regeneration) (D), ‘class 2’ (partial regeneration) (E) and ‘class 3’ (blockade in regeneration) (F). (G) Quantification of regenerative status of ventricles from WT sibling fish (n=6, sections=194) and grl5nt−/− fish (n=8, sections=288) at 30 dpa. Heart sections were scored according to the criteria described in Materials and Methods. Histograms show the percentage of heart regeneration represented by each score for each group. ***P<0.001, Chi-square test. (H-K) Section images of injured ventricles from WT (H) and mutant grl5nt−/− fish (I), as well as WT (J) and grl19nt−/− fish (K) at 7 dpa, stained with anti-PCNA (green) and anti-Mef2 (red) antibodies. Insets show higher-magnification images of the dashed boxes. Arrowheads indicate proliferating CMs. (L) Quantification of CM proliferation indices in 7 dpa ventricles of grl+/+ (n=6) and grl5nt−/− fish (n=6). ***P<0.001, Student's t-test (unpaired, two-tailed). (M) Quantification of CM proliferation indices in 7 dpa ventricles of grl+/+ (n=4) and grl19nt−/− fish (n=5). **P<0.01, Student's t-test (unpaired, two-tailed). Data presents as mean±s.e.m. Scale bars: 100 µm.
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