Fig. 4. Myocardial HH signaling is required for coronary vein development. (A-D) Whole mount immunohistochemistry for PECAM, demonstrating that both control (A) and Smo^mlc2v CKO (B) hearts form a vascular plexus that encases the entire ventricle. High magnification of A,B demonstrates that the vascular plexus of Smo^mlc2v CKO (D) hearts is less dense than controls (C). (E,F) 3D reconstructions of cryosections stained with antibodies to PECAM, showing that although E13.5 control hearts (E) display both subepicardial (white arrowhead) and intramyocardial (white arrow) blood vessels, Smo^mlc2v CKO hearts (F) contain only a single layer of vasculature (yellow arrow). (G,H) Histological sections of PECAM stained hearts reveal that, compared with E13.5 controls (G), Smo^mlc2v CKO hearts (H) do not contain blood vessels growing within the subepicardial space (red arrowheads) and only possess blood vessels growing with the myocardial wall (yellow arrowhead). Black arrow and arrowheads indicate, respectively, intramyocardial and subepicardial blood vessels in control heart (G). (I-T) Immunofluorescent staining for Efnb2-lacZ (I-N) and Ephb4-lacZ (O-T) E13.5 hearts with antibodies against PECAM (I,L,O,R; red) and lacZ (J,M,P,S; green). In contrast to control hearts (I-K,O-Q), which contain ephrin B2-expressing intramyocardial vessels (white arrows) and Ephb4 -expressing subepicardial vessels (arrowheads), Smo^mlc2v CKO hearts (L-N,R-T) possess only a single set of vasculature expressing ephrin B2 (yellow arrows). (U) Model depicting the changes in the coronary vasculature seen in Smo^mlc2v CKO hearts compared with control hearts.