Fig. 3

Early expression of nkx2.5 actively maintains ventricular identity during chamber emergence. (A-R) Ventral views, anterior to the top, at 52 hpf in non-transgenic (A-F) and Tg(hsp70l:nkx2.5-EGFP) (G-R) embryos. MF20/S46 immunofluorescence (A,D,G,J,M,P) distinguishes ventricular myocardium (red) from atrial myocardium (green) and in situ hybridization depicts expression of vmhc (B,E,H,K,N,Q) and amhc (C,F,I,L,O,R) in genotypically wild-type (A-C,G-I,J-L) and nkx2.5^-/- (D-F,M-O,P-R) embryos. In comparison to non-transgenic nkx2.5^-/- embryos (D-F), transgenic nkx2.5^-/- embryos retain appropriate chamber-specific identity following heat shock at 11 somites (M-O) and 21 somites (P-R). However, ectopic S46⁺ cardiomyocytes near the OFT in the non-transgenic nkx2.5^-/- embryo (D) are also present in the transgenic nkx2.5^-/- embryo following heat shock at 11 somites (M), whereas complete rescue of ventricular chamber identity is achieved following heat shock at 21 somites (P). White arrows indicate ectopic S46⁺ cells (D,M). Black arrows highlight the corresponding ectopic amhc⁺ population (F,O). (S) Bar graph indicates numbers of atrial, ventricular, and total cardiomyocyte nuclei; the transgene Tg(-5.1myl7:nDsRed2) in both cardiac chambers facilitates cell counting at 48 hpf. Mean and standard error of each data set are shown without detection of statistically significant differences between non-transgenic wild-type and transgenic nkx2.5^-/- embryos following heat shock at 11 somites and 21 somites (p>0.01), indicating rescue of chamber-specific cardiomyocyte cell numbers in transgenic nkx2.5^-/- siblings. All embryos were genotyped following cardiomyocyte cell counting.