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First published online August 25, 2008
doi: 10.1242/10.1242/dev.019919

Development 135, 3161-3171 (2008)
Published by The Company of Biologists 2008
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Hedgehog signaling to distinct cell types differentially regulates coronary artery and vein development

Kory J. Lavine1, Fanxin Long1,3, Kyunghee Choi2, Craig Smith1 and David M. Ornitz1,*

1 Department of Developmental Biology, Washington University School of Medicine, St Louis, MO 63110, USA.
2 Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO 63110, USA.
3 Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA.


Figure 1
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  		Fig. 1. Subepicardial and intramyocardial blood vessels represent venous and arterial lineages. (A-F) Immunofluorescent staining of cryosections for PECAM (A,D; red) and lacZ (B,E; green). Staining of E12.5 Efnb2-lacZ mice (A-C) reveals that intramyocardial (arrow), but not subepicardial (arrowhead) blood vessels express the arterial marker, ephrin B2. Similar analysis of E12.5 Ephb4-lacZ (D-F) mice demonstrates that subepicardial (arrowhead), but not intramyocardial, blood vessels (arrow) express the venous marker Ephb4. (G-L) Immunofluorescent staining of cryosections from Efnb2-lacZ (G-I) and Ephb4-lacZ (J-L) adult hearts shows that the spatial relationship between larger arteries and veins is conserved in the adult heart. Green, PECAM (G,J); red, lacZ (H,K). (C,F,I,L) Merges of fluorescent signals from A and B, D and E, G and H, and J and K, respectively.

 

Figure 2
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  		Fig. 2. Dermo1-Cre is active in the perivascular cell lineage. (A-C) lacZ staining of Dermo1-Cre/Rosa26-lacZ hearts showing that at E12.5 (A) Dermo1-Cre marks cells localized in patches of the epicardium that are undergoing EMT (bracket with asterisk, white arrowhead) and around intramyocardial blood vessels (arrow). (B) lacZ staining of postnatal day 10 (P10) hearts demonstrating that Dermo1-Cre-expressing cells give rise to perivascular interstitial cells (arrow), but not the vascular endothelium (arrowhead). (C, inset) Dermo1-Cre-positive cells also give rise to the interstitium of the valvular leaflets (asterisk). (D-F) Immunofluorescent staining of cryosections for lacZ (D, red) and PECAM (E, green) confirming that Dermo1-Cre marks cells positioned immediately adjacent to the intramyocardial blood vessels (arrow in D,E) and in patches of the epicardium (bracket with asterisk). Merge of D,E is shown in F. (G-L) Immunofluorescent staining for lacZ (G,J, green), cardiac actin (H, red) and smooth muscle actin (K, red) demonstrating that Dermo1-Cre marked cells (arrows) express smooth muscle actin and not cardiac actin. Bracket indicates position of the epicardium. (I,L) Merged images of G,H and J,K, respectively. (M-O) Immunofluorescent staining for VEGFA (M, green) and smooth muscle actin (N, red), revealing that smooth muscle cells located within the myocardial wall (arrows) express VEGFA. Asterisk denotes VEGFA-expressing myocardial cells. O, merged image of M,N.

 

Figure 3
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  		Fig. 3. HH signaling to the cardiomyoblast and pericyte is essential for coronary development. (A,B) Whole-mount PECAM immunohistochemistry demonstrating that, compared with control (A), Smomlc2v; dermo1 CKO heart (B) displays an arrest in coronary development. Bracket indicates atrial ventricular groove. (C) β-Galactosidase staining of an E12.5 Mlc2v-Cre/Rosa26R heart, demonstrating that Mlc2v-Cre does not efficiently target cardiomyoblasts located in the atrial ventricular grove (AVG, bracket). By contrast, Mlv2v-Cre efficiently targets the ventricular myocardium (area below bracket). (D,E) Cryosections of hearts stained with in situ probes for Ptch1 revealing that, compared with controls (D), Smomlc2v; dermo1 CKO hearts (E) lack both myocardial (asterisk) and perivascular (arrow) sources of Ptch1 expression. (F-K) Immunofluorescent staining of cryosections for PECAM (red) and VEGF ligands (green) reveal that, compared with controls (F,H,J), Smomlc2v; dermo1 CKO hearts (G,I,K) display diminished expression of VEGFA (F,G), VEGFB (H,I) and VEGFC (J,K). White arrowhead indicates (PECAM-positive) endocardial cells that appear unaffected in these conditional mutants. Red arrow in A,D denotes orientation of tissue sections in relation to whole-mount photographs (B, base; A, apex).

 

Figure 4
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  		Fig. 4. Myocardial HH signaling is required for coronary vein development. (A-D) Whole mount immunohistochemistry for PECAM, demonstrating that both control (A) and Smomlc2v CKO (B) hearts form a vascular plexus that encases the entire ventricle. High magnification of A,B demonstrates that the vascular plexus of Smomlc2v 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, Smomlc2v 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), Smomlc2v 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), Smomlc2v 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 Smomlc2v CKO hearts compared with control hearts.

 

Figure 5
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  		Fig. 5. Defective subepicardial mesenchyme development in Smomlc2v 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, Smomlc2v 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), Smomlc2v CKO hearts (I,J) contain only blood vessels growing within the myocardial wall (yellow arrow) and fail to develop subepicardial mesenchyme (white arrowheads, J).

 

Figure 6
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  		Fig. 6. Perivascular HH signaling is essential for coronary artery growth. (A-D) Whole-mount immunohistochemistry for PECAM showing that both control (A) and Smodermo1 CKO (B) hearts contain a vascular plexus that encases the entire ventricle. Higher magnification demonstrates that the vascular plexus of Smodermo1 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 Smodermo1 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 Smodermo1 CKO (H) hearts, demonstrating that Smodermo1 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 Smodermo1 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), Smodermo1 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 Smodermo1 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 Smodermo1 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 Smodermo1 CKO hearts (arrow).

 

Figure 7
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  		Fig. 7. Myocardial and perivascular HH signaling is required for tissue-specific VEGF ligand expression. (A,B) Cryosections of hearts stained with in situ probes for Ptch1. Although control hearts (A) contain expression of Ptch1 in both cardiomyoblasts (asterisk) and perivascular cells (arrow), Smomlc2v CKO hearts (B) express Ptch1 only in perivascular cells (arrow). (C-H) Immunofluorescent staining of cryosections for PECAM (red) and VEGF ligands (green). Compared with control hearts (C,E), which express VEGFA and VEGFB in both cardiomyoblasts (asterisk) and perivascular cells (arrow), Smomlc2v CKO hearts (D,F) express VEGFA and VEGFB in only perivascular cells (arrow). VEGFC is expressed in perivascular cells of both control (G) and Smomlc2v CKO (H) hearts (arrows). (I,J) Cryosections of hearts stained with an in situ probe for Ptch1. Although control hearts (I) express Ptch1 in both cardiomyoblasts (asterisk) and perivascular cells (arrow), Smodermo1 CKO hearts (J) express these transcripts in only the cardiomyoblast (asterisk). (K-P) Immunofluorescent staining of cryosections for PECAM (red) and VEGF ligands (green). Compared with control hearts (K,M), which express VEGFA and VEGFB protein in both cardiomyoblasts (asterisk) and perivascular cells (arrow), Smodermo1 CKO hearts (L,N) express VEGFA and VEGFB in only cardiomyoblasts (asterisk). VEGFC is expressed in perivascular cells of control (O), but not Smodermo1 CKO (P) hearts. (Q,R) Quantitative analysis of VEGF expression demonstrating statistically significant alterations in VEGF ligand expression in Smomlc2v CKO (Q) and Smodermo1 CKO (R) hearts compared with control hearts. Black bars represent myocardial expression and grey bars represent perivascular expression. Asterisk indicates a statistically significant difference compared with controls (P<0.01).

 

Figure 8
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  		Fig. 8. Coronary arteries and veins represent distinct vascular lineages. (A) H&E stained section of an E12.5 heart, demonstrating that coronary veins (black arrowhead) contain both red blood cells and rosettes of undefined cells (white arrow), whereas coronary arteries (black arrow) contain only red blood cells. (B) High magnification of the outlined area in A. (C-E) Immunofluorescent staining of E12.5 hearts demonstrating that although both coronary veins (C, arrowhead) and coronary arteries (C, arrow) express PECAM, only coronary veins express CD45 (D, arrowhead) and SCA1 (E, arrowhead). Cells located within coronary veins express CD45 (D, arrow) indicating that they are of hematopoietic origin. Blue, PECAM; green, CD45; Red, SCA1. (F-H) Immunofluorescent staining of SCL/TAL1-CD4 knock-in E12.5 hearts, demonstrating that SCL/TAL1 is expressed in coronary veins (arrowhead) but not in coronary arteries (arrow). Red, PECAM; green, CD4 (SCL/TAL1). H, merge of fluorescent signals in F,G. (C-G) Immunofluorescent signals are superimposed on DIC images. (A,C-H) Taken at 400x magnification. (I) The developing coronary vascular plexus is composed of two distinct subsets of blood vessels: coronary arteries and veins. (J) Model describing the origins of the coronary arterial and venous lineages. Coronary veins are derived from hemangioblasts, whereas coronary arteries are probably derived from endothelial cells. (K) Model describing the signaling events that coordinately control coronary artery and vein development. Perivascular HH signaling controls coronary artery growth by regulating perivascular expression of VEGFA, VEGFB and VEGFC. Myocardial HH signaling controls myocardial VEGFA and VEGFB expression, which is required for coronary vein growth, and in combination with perivascular VEGF expression, positively regulates coronary artery growth.

 

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