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Fig. 5. Defective subepicardial mesenchyme development in Smo^mlc2v CKO hearts. (A-F) Immunofluorescent staining for cardiac actin (B,E; green) and PECAM (A,D; red), confirming that although control hearts (A-C) contain both subepicardial (white arrowhead) and intramyocardial (white arrow) blood vessels, Smo^mlc2v CKO hearts (D-F) contain only blood vessels growing within the myocardial wall (yellow arrow). (A,B,D,E) Fluorescent signals are superimposed on DIC images for orientation. (C,F) Merge of fluorescent signals in A,B and D,E, respectively. Red arrowheads in B,E denote position of subepicardial mesenchyme. (G-J) Immunofluorescent staining for PECAM (G,I; red), cardiac actin (G-J; green), WT1 (H,J; red) and DAPI (H,J; blue) demonstrating that compared with control hearts (G,H) that contain well-developed subepicardial mesenchyme (denoted by white arrowheads, H), Smo^mlc2v CKO hearts (I,J) contain only blood vessels growing within the myocardial wall (yellow arrow) and fail to develop subepicardial mesenchyme (white arrowheads, J).

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