scRNA-seq experiments reveal distinct cardiomyocyte clusters in adult zebrafish hearts.

(A) Uniform manifold approximation and projection (UMAP) clustering of cmlc2+ single-cells from adult hearts. (B-D) Feature plot of ckmt2a (B), phlda2 (C), actn1 (D), expression in cardiomyocyte clusters of adult hearts. (E) Identification of cardiomyocyte clusters based on gene ontology analysis.

phlda2:EGFP+ cells specifically label primordial cardiomyocytes in adult zebrafish hearts.

(A) phlda2+ cells visualized in whole cardiac sections and whole-mount heart from adult phlda2:EGFP animals. n=8. Scale bar, 100 µm. (B) Confocal slices indicating phlda2+ cells in uninjured phlda2:EGFP;cmlc2:mCherry ventricles. White arrows represent phlda2+/cmlc2+ cells. n=8. Scale bar, 10 µm. (C) Confocal slices indicating phlda2+ cells in adult phlda2:mCherry;deltaC:EGFP ventricles. n=12. Scale bars, 20 µm. (D) Confocal slices indicating phlda2+ cells in adult phlda2:EGFP;tcf21:mCherry ventricles. n=10. Scale bars, 10 µm. All data are representative of two independent experiments.

Primordial cardiomyocytes are required for morphogenesis of myocardium and coronary vascularization.

(A) Compact and trabecular muscle in ventricular sections from Mtz-treated cmlc2:EGFP and cmlc2:EGFP;phlda2:mCherry-NTR animals. Animals were treated with Mtz during the juvenile stage and analyzed 30 dpt. Boxed area is enlarged with high magnification. Arrows represent the compact muscle and arrowheads represent trabecular muscle. N = 6-8. Scale bar, 20 µm. (B) Quantification of cmlc2⁺ cell area relative to heart area within 100 µm of the heart wall from experiments in (A). Mann-Whitney rank-sum test. (C) Visualization of gata4+ cardiomyocytes in whole-mounted juvenile gata4:EGFP and gata4:EGFP;phlda2:mCherry-NTR hearts after Mtz treatment. Boxed area is enlarged with high magnification. Arrowheads represent EGFP signals in phlda2 cell–depleted hearts. n=6-7. Scale bars, 20 µm. (D-E) Quantification of the percentage of EGFP+ pixels (D) and EGFP intensity (E) on the ventricular surface from experiments in (D) and (E). Mann-Whitney rank-sum test. (F) Coronary vessels in whole-mounted deltaC:EGFP and deltaC:EGFP;phlda2:mCherry-NTR hearts. Animals were treated with Mtz during the juvenile stage and analyzed at 30 dpt. Arrows represent the broken vessel fragments. n=5. Scale bars, 20 µm. (G-H) Quantification of vessel length/heart area (G) and vessel fragments (H) on the ventricular surface from experiments in (F). All data are representative of three independent experiments.

Primordial cardiomyocytes are not essential for myocardial regeneration and coronary revascularization.

(A) Section images of Mtz-treated gata4:EGFP or gata4:EGFP;phlda2:mCherry-NTR ventricles at 7 dpa assessed for gata4:EGFP+ CMs in the injury site. Adult animals were treated with Mtz before amputation. Dashed line indicates amputation plane. N = 8. Scale bars, 20 µm. (B) Quantification of EGFP+ pixels from experiments in (A). Scale bars, 20 µm. Mann-Whitney rank-sum test. (C) Section images of Mtz-treated cmlc2:EGFP or cmlc2:EGFP;phlda2:mCherry-NTR ventricles at 30 dpa assessed for muscle recovery. Adult animals were treated with Mtz before amputation. Dashed line indicates amputation plane. N = 9 - 10. Scale bar, 20 µm. (D) Quantification of regeneration indices from experiments in (C). Myocardial regeneration is categorized as follows: 1 = complete regeneration of a new myocardial wall; 2 = partial regeneration; and 3 = a strong block in regeneration. (E) Section images of Mtz-treated deltaC:EGFP or deltaC:EGFP;phlda2:mCherry-NTR ventricles at 30 dpa assessed for coronary vessels in the injury site. Adult animals were treated with Mtz before amputation. Dashed line indicates amputation plane. N = 5 - 6. Scale bars, 20 µm. (F) Quantification of EGFP+ pixels from experiments in (E). Scale bars, 20 µm. Mann-Whitney rank-sum test. (G) Section images of Mtz-treated wt siblings or phlda2:mCherry-NTR ventricles at 30 dpa assessed for AFOG staining collagen/fibrin deposition in the injury site. Adult animals were treated with Mtz before amputation. Dashed line indicates amputation plane. N = 9. Scale bars, 20 µm. (H) Quantification of scar score from experiments in (G). The scar score is categorized as follows: 1 = hearts without persisting collagenous scar; 2 = hearts with scar remnants that were covered by a new myocardium; 3 = the absence of a new myocardium around the wound. The Chi-squared test was performed. All data are representative of three independent experiments.

Primordial cardiomyocytes have limited regenerative capacity.

(A) Section images of phlda2:mCherry-NTR ventricles without injury and during regeneration at 7, and 60 dpa. n =11-13. Dashed line indicates amputation plane. Scale bar, 20 μm. (B) Section images of phlda2:mCherry-NTR ventricles without Mtz treatment and after 7 or 30 days of Mtz treatment. n = 12-15. Scale bar, 20 μm. (C) Section images of ventricles of vehicle- or Mtz-treated phlda2a:mCherry-NTR at 30 dpa. Animals were treated with Mtz during the juvenile stage and amputated as adults. Dashed line indicates amputation plane. n = 12. Scale bars, 20 µm. (D) Section images of phlda2:mCherry-NTR;gata4:EGFP ventricles at 7 dpa assessed for phlda2+ cells and gata4:EGFP+ CMs in the injury site. Box area is shown in higher magnification. Arrows represent phlda2+ cells; arrowheads represent gata4+ cells. Dashed line indicates amputation plane. n = 12. Scale bar, 20 µm. All data are representative of two independent experiments.