Figure 4

Subepicardial ECs contribute substantially to intramyocardial coronary vessels. (A) Tissue recombination assays using Apln^CreERT2/+;Rosa26^RFP/+ transgenic sinus venosus and atria (SV/A) portions, and WT ventricle with epicardium (V/Epi). In the presence of tamoxifen, marked coronary vessels (arrowheads) could be detected. No tamoxifen was used as control. No marked coronary vessels were detected when transgenic V/Epi was paired with WT SV/A. (B) Tie2-Cre;Rosa26^LacZ/+ transgenic SV/A was paired with WT V/Epi. Marked coronary vessels (arrowheads) form on the ventricle. Thus, the SV/A is the source of coronary vessels in this assay. (C) Genetic labeling strategy for lineage tracing of subepicardial ECs. (D) Whole-mount view and tissue section of Apln^CreERT2/+;Rosa26^LacZ/+ hearts stained with X-gal. Embryos were dosed with tamoxifen at E10.5, which restricts cell labeling to the subsequent 24 h. In section views, asterisks indicate endocardial cushion; black arrowheads point to sprouting subepicardial ECs (X-gal+); red lines with double arrows denote the thickness of ventricle wall. Black dotted lines outline the epicardium; red dotted lines indicate the border between the trabecular and compact myocardial layer. (E) Whole-mount view and tissue section of X-gal-stained E15.5 Apln^CreERT2/+;Rosa26^LacZ/+ hearts without tamoxifen injection. (F) Whole mount and sectional fluorescence of Apln^CreERT2/+;Rosa26^RFP/+ heart. (G) Example of sections from E15.5 Apln^CreERT2/+;Rosa26^mTmG/+ hearts used to quantify the percentage of coronary vessels derived from subepicardial ECs in the ventricle wall. Staining of RFP (tdTomato) and GFP (mG) represents lineage marker as Apln-derived cells. PECAM were used as pan endothelial markers. Tamoxifen was injected at E10.5 for F and G. White arrowheads indicate labeled intramyocardial ECs. Representative of 3. White bar = 0.5 μm, yellow bar = 50 μm. LV, left ventricle; RV, right ventricle; VS, ventricular septum.