Example of a vigorously regenerating ccn2a +/+ heart and a poorly regenerating ccn2a −/− heart section. (E) Sagittal sections of 30 dpa ventricles stained with AFOG. Dot plots show highest area covered by collagenous scar on a tissue section from each heart. The statistical significance of differences was evaluated by a wound-recovery χ2 test. Data indicate the percentage of total hearts represented by each score. Color-coded bar chart indicates the degree of regeneration: green, complete blue, moderate red, very poor. (C,D) Semi-quantitative analysis of scarring in ccn2a +/+, ccn2a +/− and ccn2a −/− hearts (n=9 each) at 60 dpci (C), and ccn2a +/+, ccn2a +/− and ccn2a −/− hearts (n=7 each) at 150 dpci (D). Arrows and arrowheads indicate the collagenous scar and atrioventricular valves, respectively. (A,B) Representative bright-field images of 12 μm sagittal paraffin sections of 60 dpci (A) and 150 dpci (B) hearts stained with acid fuchsin-orange G (AFOG muscle, yellowish red fibrin, brick red collagen, blue).
dpci, days post cryoinjury dpa, days post amputation.Ĭcn2a mutant hearts exhibit increased scarring after injury. Arrowheads indicate EGFP-expressing cells. (G) BACccn2a:EGFP expression and tropomyosin (magenta marks CMs) immunostaining on a sagittal section of a 4 dpci heart. Arrowheads indicate EGFP expression overlapping with mCherry positive-cells in the injured tissue. (F) BACccn2a:EGFP and kdrl:HRAS-mCherry expression in a sagittal section of a 4 dpci heart. Arrowheads and arrows indicate ccn2a- and ccn2b-expressing cells in the injured tissue, respectively. (D,E) Representative images of ccn2a and ccn2b expression on sagittal sections of 7 dpci (D) and 7 dpa (E) adult zebrafish hearts. The statistical significance of differences was evaluated using a two-tailed Student's t-test (GraphPad Prism). Values are normalized to the mean of the sham control. (B,C) qPCR-based small-scale screen to identify dynamically expressed extracellular matrix genes in 4 dpci adult zebrafish hearts (B), and qPCR analysis of ccn2a expression during the early stages of heart regeneration (C) (n=3, each sample represents a pool of 6 hearts). (A) Schematic depiction of the known processes involved in heart regeneration. Ccn2a is expressed by endocardial cells in injured zebrafish hearts.
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