CycD2 potentiates cardiogenic activity of GATA4. (A) CycD2 stimulates cardiogenic activity of the rat GATA4. Animal cap explants injected with 100 pg of CycD2 mRNA, 300 pg of Gata4 mRNA (G4), or the two combined were analyzed for expression of indicated markers at stage 34. Myl7 and myh6 are exclusively expressed in cardiac myocytes, endodermin (a2m) is an endodermal marker, smooth muscle actin (acta2) marks smooth muscle, and globin (hba1) is a marker of blood. Note that stimulation of GATA4 activity by CycD2 is restricted to induction of cardiac but not other cell fates. (B) S160 is important for cardiogenic activity of GATA4; 400 pg of indicated GATA4 constructs were injected. Animal explants were cultured as above, and expression of indicated markers were determined by RT-PCR. (C) S160 is required for stimulation of GATA4 activity by CycD2; 100 pg of CycD2 mRNA and 300 pg of the indicated GATA4 constructs were injected per embryo. Expression of myl7, myh6, and odc1 were determined by RT-PCR. The dotted lines between lanes in A and C indicate that the image shown was assembled either from two gels run in parallel or derives from the same gel after splicing out unnecessary lanes. (D) Immunofluorescence assay to determine the nuclear localization of the indicated GATA4 constructs in stage 6–7 embryos. Anti-GATA4 antibody was used. (Scale bar: 0.1 mm.)