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.