Confocal imaging performed on ( A–D) klf2a:H2B-GFP ; vmhc:mCherry-NTR or ( J–M) Tp1:d2GFP; vmhc:mCherry-NTR ventricle-ablated hearts at 24 hpt (6 dpf) reveals that ( D, M) gata2a-/- mutant hearts as well as wild-type hearts treated with ( B, K) blebbistatin (Blebb) or ( C, L) tricaine (Tric) exhibit reduced klf2a:H2B-GFP or Tp1:d2GFP expression when compared to ( A, J) ethanol (EtOH) sham-treated hearts. Quantitation of the relative average fluorescence intensity in control (black bars) and ventricle-ablated (gray bars) hearts confirms reduced ( E) klf2a:H2B-GFP or ( N) Tp1:d2GFP expression in gata2a-/- mutant hearts as well as wild-type hearts treated with blebbistatin or tricaine ( klf2a:H2B-GFP n = 12 control EtOh; 5 control Blebb; 7 control Tric; 9 control gata2a-/-; 11 MTZ EtOH; 6 MTZ Blebb; 6 MTZ Tric; 9 MTZ gata2a-/-. Tp1:d2GFP n = 12 control EtOh; 6 control Blebb; 7 control Tric; 9 control gata2a-/-; 11 MTZ EtOH; 6 MTZ Blebb; 6 MTZ Tric; 9 MTZ gata2a-/-). Whole-mount in situ hybridizations at 24 hpt (6 dpf) show that ( F–I) klf2a and ( O–R) notch1b expression is decreased in vmhc:mCherry-NTR ventricle-ablated hearts treated with ( G, P) blebbistatin (n = 0/8 klf2a; n = 2/12 notch1b) or ( H, Q) tricaine (n = 0/7 klf2a; n = 0/10 notch1b) as well as in ( I, R) gata2a-/- ventricle-ablated hearts (n = 3/9 klf2a; n = 5/12 notch1b) when compared to ( F, O) hearts sham-treated with ethanol (n = 7/7 klf2a; n = 10/10 notch1b). All confocal images shown are maximum intensity projections. V, ventricle; A, atrium; dpf, days post-fertilization; hpt, hours post-MTZ treatment. Dashed lines outline the heart. Bars: 50 μm. ( E, N) Mean + s.e.m. ANOVA, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001. The following figure supplements are available for Figure 3.
