Subepicardial endothelial cells invade the embryonic ventricle wall to form coronary arteries

Abstract

Coronary arteries bring blood flow to the heart muscle. Understanding the developmental program of the coronary arteries provides insights into the treatment of coronary artery diseases. Multiple sources have been described as contributing to coronary arteries including the proepicardium, sinus venosus (SV), and endocardium. However, the developmental origins of coronary vessels are still under intense study. We have produced a new genetic tool for studying coronary development, an AplnCreER mouse line, which expresses an inducible Cre recombinase specifically in developing coronary vessels. Quantitative analysis of coronary development and timed induction of AplnCreER fate tracing showed that the progenies of subepicardial endothelial cells (ECs) both invade the compact myocardium to form coronary arteries and remain on the surface to produce veins. We found that these subepicardial ECs are the major sources of intramyocardial coronary vessels in the developing heart. In vitro explant assays indicate that the majority of these subepicardial ECs arise from endocardium of the SV and atrium, but not from ventricular endocardium. Clonal analysis of Apln-positive cells indicates that a single subepicardial EC contributes equally to both coronary arteries and veins. Collectively, these data suggested that subepicardial ECs are the major source of intramyocardial coronary arteries in the ventricle wall, and that coronary arteries and veins have a common origin in the developing heart.

Figure 1

Subepicardial and intramyocardial vessels in developing heart. (A) Coronary vessel coverage over the surface of the heart from E11.5 to E14.5 (n = 9-13). Graph calculates area encircled by dotted lines. (B) PECAM staining of E10.5 to E15.5 hearts showing that subepicardial coronary vessels (arrows) develop before intramyocardial vessels (arrowheads). Right panels are boxed regions and results are representative of 5-10 hearts per time point. (C) Quantification of the number of PECAM⁺ subepicardial and intramyocardial coronary ECs in the left ventricle from E10.5 to E15.5. (D) Continuity analysis by quantifying the number of PECAM⁺ cells within 20 μm of endocardial (Endo.) or subepicardial (Subepi.) ECs at E11.5 to E14.5. *P < 0.05; n = 6-10. White bar = 0.5 mm; black bar = 200 μm; yellow bar = 50 μm.

Figure 2

Apln and AplnCreERT2 expression map in early developing heart. (A) Whole mount in situ hybridization shows no Apln expression in PEO at E9.5 and Apln expression in a coronary vessel-specific pattern at E11.5 and E12.5. (B) qRT-PCR analysis of heart ventricles shows that pan expression of endothelial markers Pecam and Vegfr2 is similar in different stages, whereas Apln expression begins at E11.5 and subsequently increases. *P < 0.05. n = 4-10. (C) ECs were purified by FACS of dissociated Tie2-GFP hearts. GFP+ and GFP− populations were processed for qRT-PCR. Compared with the pan EC markers Vegfr2 and Pecam, expression of Apln was significantly increased in ECs from E11.5 to E14.5. n = 4-9. (D-E) E12.5 hearts were dissected into either the entire ventricle (V) or apex only (A) for qRT-PCR analysis. At E12.5, Apln expression level was significantly lower in the apex compared with the ventricle, suggesting that most Apln⁺ sprouting coronary ECs have not reached the apex. NS, not significant; *P < 0.05; n = 8-10. (F-J) CreERT2 (ESR, yellow arrowheads) is not expressed in TBX18⁺, WT1⁺, or RALDH2⁺ (pro)epicardial cells (white arrowheads) or within heart (h) from E9.5 to E12.5. White asterisks indicate atrioventricular groove. (K) Apln-LacZ expression is restricted to the surface of ventricle wall at E12.5 (white arrowheads). Black line outlines the epicardium. PEO, proepicardium; SV, sinus venosus; V, ventricle; A, atrium; LV, left ventricle; RV, right ventricle; Li, liver. Black bar = 0.5 mm; white bar = 100 μm.

Figure 3

Apln expression map at E9.5 to E12.5. (A-D) Apln is expressed in PECAM⁺ vascular ECs, but not in endocardial ECs. Asterisks indicate AV groove; red arrow indicates Apln⁺PECAM⁺ vascular ECs. PA, pharyngeal arch; Li, liver; h, heart; PEO, proepicardial organ. Representative of three hearts for each time point. White bar = 100 μm.

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.

Figure 5

Subepicardial ECs are the major sources of intramyocardial coronary vessels in the ventricle wall. Differential labeling of coronary vessels in the ventricular septum (VS) when dosed at E10.5 and E12.5. Labeled cells in the septum only with the E12.5 tamoxifen injection show that the E10.5 dose is inactive by this time. *P < 0.05; NS, not significant; n = 3-4. White bar = 100 μm. LV, left ventricle; RV, right ventricle; VS, ventricular septum.

Figure 6

BI formation and coronary vessels in ventricle septum. (A-C) Penetration distance from endocardium (Endo) and avascular distance from BI. n = 10. (D) Consecutive slides of Sema3d^LacZ/+ heart showing BI on epicardium (X-gal+) arises from endocardium and connects with chamber (double red arrow). (E) Majority of Endo-derived BIs appear on ventral side of ventricle septum (black lined area). *P < 0.05; n = 42. (F) Expansion of endocardium-derived ECs of BIs in Fog2 mutants. White dotted line indicates impaired SV-derived coronary plexus; white arrows indicate PECAM⁺ BIs. (G) In remote area of VS, endocardium sinusoids become narrow strand of PECAM⁺Lectin⁻ cells (white arrowheads), with loss of connection with chamber circulation. White dotted lines outline ventricle septum. Representative of three embryos. Black bar = 0.5 mm; white bar = 100 μm.

Figure 7

Single subepicardial EC contributes to both coronary arteries and veins. (A, B) E10.5-labeled subepicardial ECs give rise to arterial cells (NOTCH1⁺, red arrowheads) and venous cells (COUP TFII⁺, green arrowheads) at E15.5. White arrowheads indicate COUP TFII⁺ endocardial cells. (C) Schematic representation of clones recovered from Apln^CreERT2/+;Rosa26^Rainbow/+ embryos with low-efficiency recombination. (D, E) Representative coronary vessel clone in an E15.5 Apln^CreERT2/+;Rosa26^Rainbow/+ heart shown in whole mount (D) and section (E). (F) Coronary ECs in one clone were positive for both arterial (NOTCH1) and venous (COUP TFII) markers. Yellow arrowheads indicate subepicardial venous EC (RFP⁺;COUP TFII⁺); white arrowheads point to intramyocardial arterial ECs (RFP⁺;NOTCH1⁺). (G) Percentage of intramyocardial and subepicardial coronary ECs within single Apln^CreERT2/+;Rosa26^Rainbow/+ clone compared with the distribution of total vessels. *P < 0.05; n = 5 for all vessels; n = 28 for clonal vessels. (H) VE-Cad-CreERT2-induced coronary clones (white) containing sister cells within the AV junction are distributed throughout the thickness of the heart (double yellow arrow). White dotted line indicates epicardium; red dotted line indicates border between trabecular and compact myocardium. (I) Quantification of the percentage of subepicardial (Subepi., blue) and intramyocardial (Intramyo., red) ECs within each clone at E13.5. A similar graph demonstrating the distribution of total vessels in E13.5 left ventricle (10×) is shown for comparison. n = 3-6. LV, left ventricle; RV, right ventricle; VS, ventricular septum. Yellow bar = 100 μm; white bar = 0.5 mm.

Figure 8

Second heart field (Isl1, Tbx1, and Mef2c)-traced endocardial cells do not adopt a coronary EC fate. (A) Schematic illustrating the experimental design for testing the contribution of endocardial cells to coronary vessels in the embryonic ventricle wall. Endocardial cells and their progenies are labeled in red. (B) Isl1-Cre labeled NFATc1⁺ endocardial cells at E10.5 (yellow arrowheads). Dotted white line outlines the epicardium (left panel). Whereas Isl1-Cre labeled endocardial cells at E10.5, intramyocardial coronary ECs were not labeled at E15.5 (white arrowheads, right panel). Inset shows percentage of Isl1-Cre-traced cells (GFP⁺PECAM⁺) in both compartments. CA, coronary artery; Endo., endocardium. *P< 0.05; n = 6. (C, D) Neither Tbx1-Cre nor Mef2c-Cre lineage-traced endocardial cells contribute to intramyocardial ECs in the compact myocardium at E15.5 (white arrowheads). (E) Right lateral whole-mount view of Mef2c-Cre;Rosa26^RFP/+ heart shows an unlabeled mature right CA (white arrowheads). (F) Mef2c-Cre labeled endocardial cells and coronary ECs in the VS at E15.5, but very few coronary ECs in the ventricle wall (RV). *P < 0.05; n = 4. White bar = 100 μm; red bar = 1 mm. LV, left ventricle; RV, right ventricle; VS, ventricular septum; Epi, epicardium; Endo., endocardium; Comp., compact layer; Trab., trabecular layer.

Figure 9

Schematic showing subepicardial ECs as a major source for intramyocardial coronary vessels. During embryogenesis, subepicardial ECs (blue) located beneath the epicardium (brown) migrate along the surface of the heart between E11.5-E12.5 (blue arrow). Subepicardial ECs then migrate into the compact myocardium to become intramyocardial coronary arteries and capillaries at E13.0-E15.5 (red arrows).