(A–C) The 3 basic stages of trabeculation (initiation, assembly, and extension) and their corresponding sections of Tie2^+/fl (D–F), Tie2-cko (G–I), and Tie2^–/– (K and L) embryos dual immunostained with troponin T (red) and endomucin (green) antibodies. Nuclei are counterstained with DAPI (blue). CJ, cardiac jelly; CM, cardiomyocytes; End, endocardium. At stage 1 (initiation, around E9.0), as the inner layer CMs delaminate into the lumen and form sheet-like protrusions (myocardial lamina, white arrowheads), the endocardium sends out sproutings to penetrate the thick cardiac jelly and make direct touchdown (arrows) with the outer layer of the myocardium in the control embryo. In Tie2-cko, however, the endocardial sproutings have not yet reached the outer layer of the myocardium (yellow arrowheads). At stage 2 (assembly, E9.25), as endocardial sproutings progress laterally beneath the myocardial lamina, they later assemble to isolated short trabecular clusters. At stage 3 (extension, E9.5), finger-like, long trabecular structures are formed by extension. More endocardial touchdown endpoints were detected in the control than in Tie2-cko and Tie2^–/– ventricles. Although the myocardial wall in Tie2^–/– ventricles (K and L) was able to thicken to become multicellular and form sheet-like protrusions (myocardial lamina, white arrowheads), assembly and extension of trabeculae were impaired. Scale bars: 100 μm. A representative of more than 8 images was chosen for each panel. (J) Quantification of endocardial touchdown endpoints in control, Tie2-cko, and Tie2^–/– embryos at E9.25 and E9.5 (n = 6 per group). **P < 0.01, 1-way ANOVA.