Supplementary Figure S1 Enlarged coronary artery branches in Edn1-knockout mice. a-d, Coronary angiography by ink injection in wild-type (a, b) and

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1 Supplementary Figure S1 Enlarged coronary artery branches in Edn1-knockout mice. a-d, Coronary angiography by ink injection in wild-type (a, b) and Edn1-knockout (Edn1-KO) (c, d) hearts. The boxed areas in a and c are magnified in b and d, respectively. Edn1-knockout mice show partial enlargement in the coronary septal branches (arrows). e-j, Co-immunostaining of wild-type (eg) and Edn1-knockout (h-j) septal branches for CD31 and αsma with merged images. Septal branch partially lacks SMCs (white arrowheads). All samples were harvested at E17.5. RV/LV, right/left ventricle; scale bars, 100 µm (a, c), 40 µm (b, d, e-j).

2 Supplementary Figure S2 Branching patterns of the septal branch. Coronary angiography by ink injection at E17.5. There are two types of branching patterns; right-sided pattern (a) and left-sided pattern (b). About 90% of wild-type ICR mice show the right-sided pattern. Scale bars, 100 µm.

3 Supplementary Figure S3 Serial observation of coronary artery formation in wild-type mice. a- e, Coronary angiography in mouse developing hearts. Each magnified image is depicted in the lower panel. All septal branches are first originated from the left coronary artery (LCA) just after the opening of coronary ostia at E13.5 (a). At the same time, small vessels sprout out from the right coronary artery (RCA) (arrowhead in a). These vessels grow towards the septal branch and reach it around E14.5 (b, arrowhead), forming a communicating artery between RCA and the septal branch around E15.5 (c, arrowhead). This communicating artery becomes predominant and the left-sided communication gradually regresses around E16.5 (d). Up to E17,5, the final branching pattern is established (e). f-h, Hematoxylin-eosin staining of sections from ink-injected hearts. The same samples shown in a, b and c are sectioned and stained. At E13.5, a sprout from RCA (yellow arrowhead) is growing into the roof of the right ventricle beneath the condensed mesenchyme (f). At 14.5, the sprout reaches the septal branch to make an end-to-side anastomosis (g, yellow arrowhead). At E15.5, the RCA-septal branch communication is established in the right side of the interventricular septum (h, yellow arrowhead). i, An ink-injected septal branch of an adult mouse. The communicating artery from RCA runs beneath the surface of the supraventricular crest and connects to the septal branch passing in the septomarginal trabeculation. Ao, aorta; CM: condensed mesenchyme, PM, papillary muscle; RCA/LCA, right/left coronary artery; RV/LV, right/left ventricle; RVOT, right ventricular outflow tract; SC, supraventricular crest; ST, septomarginal trabeculation; TV, tricuspid valve. Scale bars, 1mm (i), 100 µm (f, g, h).

4 Supplementary Figure S4 Dilatation of the proximal portion of coronary arteries in an EdnraEdnra knockout mouse. Coronary angiography (a) and hematoxylin-eosin hematoxylin eosin staining of the horizontal section (b, c) at the conal level (yellow line in a) reveals an enlarged proximal left coronary artery near the connection to the septal branch (arrows). The boxed area in b are magnified in c. LVOT, left ventricular outflow tract, MV, mitral valve; RV, right ventricle; SB, septal branch. Scale bars, 100 µm.

5 Supplementary Figure S5 Combinations of the septal branch dilatation and the ventricular septal defect in Ednra-knockout mice. At the E17.5, the septal branch has no dilatation and interventricular septation was completed in all wild-type mice and some Ednra-knockout (KO) mice (Normal) (From the left column). Among Ednra-KO mice, some show the septal branch dilatation (white arrowheads) but no ventricular septal defect (VSD) (type1). Others exhibit ventricular septal defect (red arrowheads) without septal branch dilatation (type2) or both anomalies simultaneously (type3). See also Supplementary Table S2. LVOT, left ventricular outflow tract; RV, right ventricle. Scale bars, 200 µm in middle panels; 100 µm in lower panels.

6 Supplementary Figure S6 Wnt1-Cre-labeled cells at E13.5-E14.5. a, b, Staining for β- galactosidase activity in the aortic root region at E13.5. The boxed area in a is magnified in b. Wnt1- Cre-labeled cells have already appeared around coronary arteries at E13.5. c-e, Co-immunostaining for β-galactosidase (c) and αsma (d) with merged images (e) in the proximal coronary arteries. Wnt1-Cre-labeled cells do not express αsma at this stage. f, g, Staining for β-galactosidase activity in the aortic root region at E14.5. The boxed area in f is magnified in g. h-j, Co-immunostaining for β- galactosidase (h) and αsma (i) with merged images (j) in the proximal coronary arteries. Wnt1-Crelabeled cells express αsma at this stage. k, l, Staining for β-galactosidase activity in the interventricular septal region at E14.5. The boxed area in k is magnified in l. m-o, Co-immunostaining for β-galactosidase (m) and CD31 (n) with merged images (o). p-r, Co-immunostaining for β- galactosidase (p) and αsma (q) with merged (r). Wnt1-Cre-labeled cells are also detected around endothelial cells, however they did not express αsma. CA, coronary artery; PV, pulmonary valve primordium; RV/LV, right/left ventricle; SB, septal branch. Scale bars, 100 µm in a, b, f, g, k, l; 40µm in c-e, h-j, m-r.

7 Supplementary Figure S7 Wnt1-Cre-labeled cells migrate into the heart but do not contribute to coronary artery SMCs in Ednra-knockout embryos. a, b, c, Staining for β-galactosidase activity in Ednra-heterozygous (Hetero) (a) and Ednra-homozygous (KO) (b, c) embryos at E13.5 (a, b) and E14.5 (c). d, Co-immunostaining for Ednra-driven EGFP (d), β-galactosidase (e) and with merged images (f) in a coronary artery in the conal region of an E14.5 Ednra-knockout embryo. Wnt1-Crelabeled cells are not found around the coronary artery. Red arrowheads indicate the condensed mesenchyme. Ao, aorta; CA, coronary artey; PA, pulmonary artery; PV, pulmonary valve primordium; RV/LV, right/left ventricle; VSD, ventricular septal defect. Scale bars, 200 µm in a-c; 40 µm in d-f.

8 Supplementary Figure S8 Wnt1-Cre-labeled cells around ascending aorta. Staining for β-galactosidase activity in the wild-type (a), Ednra-heterozygous (Ednra Hetero) (b) and Ednra-homozygous (Ednra KO) (c) embryos with the Wnt1-Cre/R26R allele. There are no significant differences in the distribution patterns of Wnt1-Cre-labeled cells within the tunica media of the ascending aorta between the groups. Ao, aorta; PA, pulmonary artery. Scale bars, 200 µm.

9 Supplementary Figure S9 Wnt1-Cre-labeled cells in the tunica media of the ascending aorta. Co-immunostaining of the aortic wall of Ednra heterozygous (Ednra Hetero) (a-c) and homozygous (Ednra KO) (d-f) embryos for β-galactosidase and αsma with merged images. Wnt1-Cre-labeled cells are detected similarly in the inner layer of the tunica media of the ascending aorta in both groups. Scale bars, 40 µm.

10 Supplementary Figure S10 Edn1 expression in developing interventricular septum. In situ hybridization reveals that Edn1 expression in the endocardium of atrioventricular cushions and interventricular septum at E12.5 (a) and E13.5 (b). EC, endocardial cushion; IVS, interventricular septum. Scale bars, 40 µm.

11 Supplementary Figure S11 Quail-chick chimera of the postotic NC. a, Scheme illustrating the position of the postotic NC grafted. The quail postotic NC between the r6 and r8 (somite 3) was orthotopically grafted to chick embryos. b, QCPN immunostaining for quail nuclei, indicating massive contribution of QCPN-positive cells to the SMCs in great vessels. c, 3D reconstruction of immunostained sections used 160 frontal sections of 4 µm each. QCPN-labeled cells distribute mainly to the pulmonary artery and the ascending aorta. QCPN-labeled cells are also seen in the semilunar valve and adventitial tissues, however they rarely get into the interventricular septum below the conotruncal region. Ao, aorta; IVS: interventricular septum; PA, pulmonary artery; RV/LV, right/left ventricle.

12 Supplementary Figure S12 Preotic NCCs represent a non-cardiomyocyte population. Immunostaining for the heart from a quail-chick chimera in which the chick midbrain and preotic hindbrain (r1-r5) neural folds were bilaterally excised and replaced by orthotopic quail grafts at 3-7 ss. QCPN-positive preotic NCCs migrated into the heart do not express desmin, a marker of cardiomyocytes. Scale bars, 40 µm.

13 Supplementary Figure S13 Proepicardium-derived cells contribute to the septal branch. Quail proepicardium was orthotopically grafted to the chick heart. Immunostaining for QCPN reveals quailderived proepicardial cells contributed not only to the free wall arteries (right coronary artery; RCA), but also to the septal branch. Ao, aorta; LV, left ventricle. Scale bars, 500 µm in a, 40 µm in b,c.

14 Supplementary Figure S14 Subdivided regions of preotic NC grafts. Scheme illustrating two regions of the preotic NC we grafted; between midbrain and r5 (left) and between midbrain to r2 (right).

15 Supplementary Figure S15 Rhombomere 4-derived NCCs labeled in R4::Cre;Z/AP mice contribute to heart development. a, At E11.5, AP-labeled NCCs are strongly expressed in the second PA (PA2) and the outflow tract. b, Frontal section of the E11.5 pharyngeal region reveals that AP-labeled cells specifically contribute to PA2 mesenchyme. c, Short axis section of the E11.5 outflow tract reveals AP-labeled cells in outflow cushion tissues. d, At E14.5, PA2-derived ear structures are stained for AP, indicating the specificity of R4-Cre labeling. OC, outflow cushion; PA1-6, the first to sixth PAs. Scale bars, 200 µm in b; 100 µm in c.

16 Supplementary Figure S16 Split coronary septal branch in preotic NC-ablated chick embryos. a, Ink injection reveals septal branch split into two lumens with jagged contours in an ablated embryo. b, c, 2D (b) and 3D (c) reconstruction images also visualize the same type malformation. One hundred and ninety horizontal sections of 10 µm each were used for the reconstruction.

17 Supplementary Figure S17 Septal branch enlargement in bosentan-treated embryos. Chick embryos treated with bosentan, an endothelin receptor antagonist, show characteristic lower beak defects (d) compared with control embryos (a). Immunostaining for αsma (b, e) and TOPRO (e, f) also reveals that bosentan-treated embryos hold enlarged coronary artery septal branches (white arrows), which are similar to preotic NC-ablated chick embryos and Edn1/Ednra-knockout mice. Scale bars, 40 µm.

18 Supplementary Figure S18 Novel contribution of preotic NCCs to heart development. Preotic NCCs migrate into the heart and differentiate into coronary artery SMCs, contributing to normal coronary artery formation. Endothelin signaling is involved in NC-dependent coronary formation.

19 Supplementary Table S1 Branching patterns of the septal branch at each embryonic stage. E 17.5 Right Left Both WT (n=21) 19 (90%) 2 (10%) 0 (0%) Ednra -/- (n=9) 0 (0%) 9 (100%) 0 (0%) Edn1 -/- (n=9) 0 (0%) 9 (100%) 0 (0%) E 15.5 Right Left Both WT (n=11) 3 (27%) 5 (45%) 3 (27%) Edn1 -/- (n=4) 0 (0%) 4 (100%) 0 (0%) E 14.5 Right Left Both WT (n=5) 0 (0%) 4 (80%) 1 (20%) Edn1 -/- (n=5) 0 (0%) 5 (100%) 0 (0%) WT, wild-type Supplementary Table S2 Incidence of the septal branch dilatation and ventricular septal defect in Ednra-KO mice at E17.5 WT n=17 Ednra-KO n=19 Normal (Dilatation(-)/VSD(-)) 17 4 Type1 (Dilatation(+)/VSD(-)) 0 5 Type2 (Dilatation(-)/VSD(+) 0 3 Type3 (Dilatation(+)/VSD(+)) 0 7 Supplementary Table S3 Incidence of coronary artery abnormalities* in preotic neural crest ablation. Somites 7 Control (n=28) Sham (n=12) Midbrain to r5 (n=24) r1/2/3 (n=5) r3/4/5 (n=10) Normal Abnormal Somites 8 Control (n=10) Sham (n=8) Midbrain to r5 (none) r1/2/3 (n=17) r3/4/5 (n=18) Normal Abnormal * Coronary artery abnormalities were defined when the septal branch was dilated more than twice in diameter compare to the proximal left coronary artery, when the septal branch was partially split into two or more lumens, and/or when the embryo had extra ostia.