Fig. 4

nkx2.5 is necessary to maintain ventricular identity in late-differentiating cardiomyocytes derived from the SHF. MF20/S46 immunofluorescence distinguishes ventricular myocardium (white) from atrial myocardium (green) in non-transgenic (A-F) and Tg(hsp70l:nkx2.5-EGFP) (G-L) embryos. Confocal projections of wild-type (A-C, G-I) and nkx2.5^-/- (D–F, J–L) hearts depict cardiomyocyte nuclei with Tg(-5.1myl7:nDsRed2) (red). Ventral views, arterial pole to the top, at 55 hpf. (B,C,E,F,H,I,K,L) White dots outline the MF20⁺ cardiomyocyte borders in the OFTs of hearts in (A,D,G,J), respectively. White arrows indicate ectopic S46⁺ cells; “A” denotes atrium. All embryos were heat-shocked at 11 somites. (A–C) In wild-type non-transgenic hearts, the late-differentiating cardiomyocyte population exhibits MF20, but not DsRed, fluorescence due to the delay in expression of Tg(-5.1myl7:nDsRed2) at the arterial pole. (D–F) Similarly, in non-transgenic nkx2.5^-/- hearts, cardiomyocytes expressing MF20, but not DsRed, are present at the arterial pole. A few ectopic S46⁺ cardiomyocytes are also visualized in this region (DsRed nuclei). (G–I) In transgenic wild-type hearts, MF20, but not DsRed, fluorescence at the arterial pole designates the delayed differentiation of these SHF-derived cardiomyocytes. (J–L) Despite the rescue of the cardiac chamber morphology and identity, excess S46⁺ cardiomyocytes are visualized at the arterial pole of transgenic nkx2.5^-/- hearts where the late-differentiating population accretes (DsRed nuclei).