Figure 6. Endocardial attenuation of Tie2 results in enhanced CM proliferation.

(A and B) BrdU pulse labeling and coimmunostaining for BrdU (red) and troponin T (green) showing more BrdU-positive CMs (arrows) but fewer BrdU-positive ECs (arrowheads) in the trabecular zone of Tie2-cko embryos at E11.5 than those in the control group. (C) Quantification of BrdU-positive nuclei as a percentage of total nuclei in myocardium indicated that Tie2-cko displayed higher proliferation rates of CMs in the trabecular zone at E11.5 and E12.5 (n = 5 per group). **P < 0.01, 2-way ANOVA. (D and E) Compared with the control littermates (D), Bmp10 expression (red, in situ hybridization) in the trabecular CMs of Tie2-cko embryos (E) at E10.5 was enhanced. Endocardium was labeled with endomucin antibody (green). (F and G) Dual immunostaining of control and Tie2-cko heart sections for the cell cycle inhibitor p57 (red) and endomucin (green) at E11.5 showing significantly reduced p57 expression in the mutant endocardium. (H and I) Dual immunostaining of control and Tie2-cko heart sections for p-Erk1/2 (red) and CD31 (green) at E11.5 showing Erk1/2 hyperphosphorylation in the mutant trabecular myocardium. (J) Western blot analysis of E12.5 control and Tie2-cko hearts confirming significantly enhanced phosphorylation of Erk1/2 in the mutants. α-Tubulin was used as a loading control. Scale bars: 50 μm. For the studies in A–I, more than 5 embryos per genotype collected from at least 3 independent litters were analyzed. A representative of more than 10 images was chosen for each panel. For the studies in J, 58 embryonic hearts per genotype harvested from 23 independent litters were analyzed, and data are expressed as a representative of 3 independent experiments.