A) Diagram of the VMZ explant assay. Isolated explants were cultured until stage 28 for analysis of Nkx2.5 and Tbx5 or stage 34 for αMHC expression by quantitative RT-PCR (B–D) or stage 40 for incidence of cTn-T+ explants by immunostaining (E,F).
B) N1 alone did not induce Nkx2.5, Tbx5 or αMHC through a dose range of 38–600 pg of injected mRNA, whereas the native heart field (HF) in DMZ explants expressed each marker as expected.
C,D) The effect of N1 plus a constant amount of C1 (C) or Crescent (D) on αMHC induction. The experiments are representative examples of at least 3 trials.
E) Incidence of VMZ explants with ectopic cTn-T+ myocardial tissue. p-values were calculated by the Chi-square test [**, <0.001; *, <0.05].
F) Whole mount view of stage 40 VMZ explants immunostained with anti-cTn-T showing that although N1 increased the incidence of cTn-T+ explants, they retained the small, dispersed foci characteristic of Crescent-induced myocardial tissue. Fluorescent image (red) is superimposed on brightfield image of explant.