Insufficient proliferation in the myocardium to account for the increase of cardiomyocyte number in the zebrafish heart. (A,C,E) To quantify cardiomyocyte numbers at various stages of development, Tg(cmlc2:dsred2-nuc) embryos of 24, 36 and 48 hpf were fixed, stained using an α-DsRed antibody and imaged by confocal microscopy. Maximum projections of the DsRed signal is shown in these panels. The dotted line outlines the heart tube; the arterial pole (outflow) is to the top and the venous pole (inflow) to the bottom. Schematics of embryos at the various developmental stages are shown in the left bottom of panels A, C and E; white outlines the embryo and red represents the forming heart tube at each stage. The 24 hpf embryo is shown in dorsal view, the 36 and 48 hpf embryos in anterior view. V, ventricle; A, atrium. (B,D,F) Brightfield images of the embryos shown in A, C and E; dotted lines outline the eyes. (G) Phospho-His staining (brown) on transverse sections through the heart at 36 hpf. The dotted line indicates the myocardium; white arrowhead indicates a phospho-His-positive cell in the neural tube. (H) The total number of phospho-His-positive myocardial cells/embryo at 30 (n=3), 36 (n=3) and 48 hpf (n=6). Bars represent mean±s.e.m. (I,J) BrdU labeling from 24-48 hpf. White arrowheads indicate BrdU-positive (brown) endocardial cells; red arrowhead indicates a BrdU-positive blood cell; black arrow indicates BrdU-positive myocardial cells. (J) Enlargement of the black box in I indicating BrdU-positive cells in the arterial pole of the 48 hpf zebrafish heart; dotted lines outline the myocardium. Scale bars: 50 μm in A,C,E; 25 μm in G; 10 μm in I.
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