Fig. 6. Perivascular HH signaling is essential for coronary artery growth. (A-D) Whole-mount immunohistochemistry for PECAM showing that both control (A) and Smo^dermo1 CKO (B) hearts contain a vascular plexus that encases the entire ventricle. Higher magnification demonstrates that the vascular plexus of Smo^dermo1 CKO hearts (D) is less dense compared with that of controls (C). (E,F) 3D reconstructions of cryosections stained with antibodies to PECAM showing that Smo^dermo1 CKO hearts (F) contain a normal compliment of subepicardial blood vessels (white arrowhead), but fewer intramyocardial blood vessels (green arrowhead) compared with control hearts (E). (G,H) Histological sections of PECAM-stained control (G) and Smo^dermo1 CKO (H) hearts, demonstrating that Smo^dermo1 CKO hearts contain similar numbers of subepicardial vessels (black arrowhead) but fewer intramyocardial vessels (green arrowhead) compared with controls. (I) Quantitation of the number of subepicardial and intramyocardial vessels per 20x field in control and Smo^dermo1 CKO hearts. Asterisk indicates a statistically significant difference compared with controls (P<0.001). (J-U) Immunofluorescent staining of Efnb2-lacZ (J-O) and Ephb4-lacZ (P-U) E13.5 hearts with antibodies against PECAM (J,M,P,S; red) and lacZ (K,N,Q,T; green). Compared with control hearts (J-L,P-R), Smo^dermo1 CKO hearts (M-O,S-U) contained fewer ephrin B2-expressing intramyocardial blood vessels (white arrows), but similar numbers of Ephb4-expressing subepicardial blood vessels (white arrowheads). (V) Model depicting the changes in the coronary vasculature seen in Smo^dermo1 CKO hearts compared with control hearts. (W,X) β-Galactosidase staining for Rosa26-lacZ, demonstrating that Dermo1-Cre-expressing cells are present in both control (W) and Smo^dermo1 CKO (X) hearts. In control hearts, Dermo1-Cre expressing cells are present in a perivascular distribution (black arrowhead), whereas these cells are scattered throughout the heart in Smo^dermo1 CKO hearts (arrow).