ABSTRACT Neural crest cells constitute a multipotent cell population that gives rise to diverse cell lineages. The neural crest arising from the postotic hindbrain is known as the ‘cardiac’

neural crest, and contributes to the great vessels and outflow tract endocardial cushions, but the neural crest contribution to structures within the heart remains largely controversial.

Here we demonstrate that neural crest cells from the preotic region migrate into the heart and differentiate into coronary artery smooth muscle cells. Preotic neural crest cells

preferentially distribute to the conotruncal region and interventricular septum. Ablation of the preotic neural crest causes abnormalities in coronary septal branch and orifice formation.

institution. Download PDF SIMILAR CONTENT BEING VIEWED BY OTHERS PERINATAL ANGIOGENESIS FROM PRE-EXISTING CORONARY VESSELS VIA DLL4–NOTCH1 SIGNALLING Article 08 September 2021 COORDINATION

OF ENDOTHELIAL CELL POSITIONING AND FATE SPECIFICATION BY THE EPICARDIUM Article Open access 06 July 2021 _HAPLN1A_+ CELLS GUIDE CORONARY GROWTH DURING HEART MORPHOGENESIS AND REGENERATION

Article Open access 13 June 2023 INTRODUCTION The neural crest (NC), first identified by Wilhelm His in 1868 as ‘Zwischenstrang’, the intermediate cord1, is a multipotent stem cell

population that originates from the border between the developing neural plate and surface ectoderm. NC cells (NCCs) can differentiate into a wide variety of cell types, including neurons,

glia, pigment cells and craniofacial bones and cartilages in different developmental contexts2,3. In cardiovascular development, the cardiac NC arising from the postotic hindbrain

corresponding to rhombomeres (r) 6–8 contributes to the formation of the tunica media of pharyngeal arch (PA) artery-derived great vessels, the aorticopulmonary septum and the outflow tract

endocardial cushions4. Ablation of the cardiac NC in chick embryos results in aortic arch artery anomalies and persistent truncus arteriosus5,6,7. It has long been controversial whether and

how the NC contributes to the formation of coronary arteries that supply the entire heart. Coronary arteries are generally accepted to form by ingrowth of angiogenic precursors into the

aorta8,9,10, with the integration of migrating proepicardial mesenchyme as the main source of smooth muscle cells (SMCs)11,12,13. On the other hand, quail–chick chimera experiments have

shown that the cardiac NC does not differentiate into coronary SMCs, although ganglia of NC origin are associated with the proximal portion of coronary arteries10,14. However, ablation of

the cardiac NC results in disruption of the normal orifice formation and deployment of coronary arteries, indicating the role of NCCs in determining the spatial arrangement of the coronary

vasculature14. By contrast, studies in mice have indicated the possible direct involvement of NCCs as the source of SMCs in the proximal coronary stems by using an NC-specific _Wnt1_-Cre

recombinase system15. The apparent discrepancy may be due to differences in species, but no definite explanation has been given for it. NC development involves multiple cell-to-cell

interactions through a variety of signalling molecules. Endothelin-1 (Edn1), a 21-amino acid peptide known as the most potent vasoconstrictor16, is a key regulator of craniofacial and

cardiovascular development, acting mainly on NCCs expressing Edn receptor type-A (Ednra), a G-protein-coupled receptor17,18,19. Inactivation of the Edn1–Ednra signalling leads to

mandibular-to-maxillary homeotic-like transformation and cardiovascular anomalies, involving the outflow tract and aortic arch arteries20,21,22. In the present study, we newly identified

anomalous coronary artery formation in _Edn1_ and _Ednra_ knockout (KO) mice, which was associated with changes in the fate of NCCs. Furthermore, experiments in avian embryos first revealed

that early-migrating preotic NCCs, rather than cardiac (postotic) NCCs, differentiate into SMCs and contribute directly to coronary artery formation partially through Edn1/Ednra-dependent

mechanisms. Our findings may provide a solution to the controversy on the contribution of NCCs to coronary artery development. RESULTS CORONARY DEFECTS IN THE ABSENCE OF EDN SIGNALLING

During the analysis of cardiac phenotypes in _Ednra_ KO mice, we often encountered abnormal structures in the interventricular septum. In E17.5 wild-type (WT) embryos, septal branches of the

coronary artery were found along the right ventricular side of the interventricular septum (Fig. 1a). _Ednra_ KO embryos instead showed large cavities in the septum (Fig. 1d). These

cavities contained red blood cells and were lined by flat cells with endothelial morphology, indicating their vascular origin (Fig. 1e). In ventricular free walls, coronary arteries with

apparently normal size were identified in both WT and _Ednra_ KO mice (Fig. 1c). To characterize the nature of these abnormalities, we performed coronary angiography in E17.5 WT and KO

embryos using an ink injection technique. This method revealed the branching structures of coronary arteries with a smooth contour and tapering toward the periphery in WT mice (Fig. 1g). By

contrast, about 50% of _Ednra_ KO embryos showed focal dilatation in the septal branch with a jagged contour (Fig. 1i). This phenotype was also found in _Edn1_ KO embryos with a similar

penetrance (Supplementary Fig. S1). Next we performed immunohistochemical analysis with anti-CD31 and anti-α-smooth muscle actin (αSMA) antibodies as markers for endothelial/endocardial

cells and SMCs, respectively. In E17.5 WT mice, major coronary branches have a single and continuous smooth muscle layer surrounding an endothelial layer (Fig. 1k–m). In _Ednra_ KO mice,

however, enlarged septal branches partially lacked the SMC layer, although the endothelial layer appears intact (Fig. 1n–p), and a number of thin branches were sprouted from the enlarged

vessels (Fig. 1j). These observations suggest that a disturbance in the formation of smooth muscle layer may cause the dilatation of coronary arteries in _Ednra_ KO mice. Coronary

angiography revealed another difference in coronary artery morphology between WT and _Ednra_- or _Edn1_ KO mice. In 90% of E17.5 WT mice on an Institute of Cancer Research (ICR) background,

septal branches perfusing the interventricular septum originated from the right coronary artery (Supplementary Fig. S2 and Supplementary Table S1). Serial observation during coronary

formation indicated that this branching pattern was generated by remodelling, in which septal branches initially originating from the left coronary artery before E14.5 (Fig. 2a and

Supplementary Fig. S3) became connected to the right coronary artery through a communicating vessel around E15.5 (Fig. 2b and Supplementary Fig. S3). This resulted in disappearance of the

proximal part of the initial communication to the left coronary artery (Supplementary Fig. S3). The communicating artery runs beneath the surface of the supraventricular crest and connects

to the septal branch passing in the septomarginal trabeculation in adult mice (Supplementary Fig. S3). In _Edn1_ KO mice, septal branches originated from the left coronary artery at E14.5 as

in WT embryos (Fig. 2e). However, the communicating vessel to the right coronary artery did not form in _Edn1_ KO mice (Fig. 2f). Consequently, septal branches remained as branches from the

left coronary artery in all the _Edn1_ KO mice examined (Supplementary Table S1). Dilatation of the septal branch was also obvious at E15.5 (Fig. 2f). _Ednra_ KO mice also exhibited the

same phenotype (Supplementary Table S1). In addition, the whole contour of coronary arteries, especially in the conotruncal region, was also affected in _Edn1_- and _Ednra_ KO mice (Figs 1i

and 2f and Supplementary Fig. S1), with dilatation of the proximal portion of the left coronary artery in some cases (Supplementary Fig. S4). These results suggest that the Edn signalling

may be critical for coronary artery development in mice. As _Edn1_ KO mice show ventricular septal defect with about 50% penetrance20, it seemed possible that the coronary artery phenotype

of the _Edn1_- and _Ednra_ KO mice might be secondary to ventricular septal defect (VSD). To test this possibility, we examined possible correlation between the incidence of VSD and coronary

artery dilatation in serial sections of WT and _Ednra_ KO mice. Among 19 KO mice on the ICR background, VSD and coronary dilatation were observed in 10 and 12 mice, respectively

(Supplementary Table S2). None of the 17 WT mice demonstrated such abnormalities (Supplementary Table S2 and Supplementary Fig. S5). Notably, three mice showed VSD without coronary

dilatation and, conversely, five mice showed coronary dilatation without VSD (Supplementary Table S2 and Supplementary Fig. S5). This result suggests that VSD and coronary dilatation are

independent of each other in _Ednra_ KO mice. _WNT1-CRE_-LABELLED CELLS CONTRIBUTE TO CORONARY SMOOTH MUSCLE To test whether the coronary artery abnormalities are due to NC defects, we used

_Wnt1-Cre_/R26R reporter mice that express β-galactosidase from the ROSA26 locus upon Cre-mediated recombination. Although the contribution of _Wnt1-Cre_-labelled cells to the walls of

proximal coronary arteries has been previously reported15, their distribution patterns and ultimate cell types remain elusive. Therefore, we first analysed the temporal and spatial

distribution of _Wnt1-Cre_-labelled cells along the coronary vasculature. At E13.5, _Wnt1-Cre_-labelled cells were found in the conotruncal region to form the condensed mesenchyme as

previously reported for NCCs7,15, and also around the proximal coronary vessels, although they did not express contractile marker proteins (Supplementary Fig. S6). At E14.5, the expression

of αSMA was detected in labelled cells around the proximal coronary arteries (Supplementary Fig. S6). Labelled cells were also detected around more distal vessels in the interventricular

septum, but they did not express αSMA (Supplementary Fig. S6). At E17.5, labelled cells were found to converge on the proximal portion of coronary arteries (Fig. 3a–c). Immunohistochemical

analysis revealed that these labelled cells were incorporated into the αSMA-positive smooth muscle layer together with _Wnt1-Cre_-negative cells, whereas many _Wnt1-Cre_-labelled cells

outside the smooth muscle layer remained αSMA-negative (Fig. 3d–f). We could not find _Wnt1-Cre_-labelled cells in any coronary branches in the apical region. At intermediate locations,

however, labelled cells were observed in the septal branch (Fig. 3g–i) contributing directly to the αSMA-positive SMCs (Fig. 3j–l). Next we analysed the distribution of _Wnt1-Cre_-labelled

cells in _Ednra_ KO embryos. At E13.5–E14.5, _Wnt1-Cre_-labelled cells were abundantly found in the conotruncal region and formed the condensed mesenchyme in KO embryos as seen in WT and

heterozygous embryos (Supplementary Fig. S6, S7). However, they were not detected around the proximal coronary arteries in KO embryos (Supplementary Fig. S7), unlike in WT embryos

(Supplementary Fig. S6). Also at E17.5, no labelled cells were found around the enlarged septal branch in KO embryos, whereas a number of labelled cells were observed around the septal

branch in heterozygous embryos (Fig. 4a–d). Consequently, the abnormal septal branch in KO embryos lacked _Wnt1-Cre_-labelled SMCs, which were constantly observed in the septal branch of

heterozygous embryos (Fig. 4e–j). Even in the proximal region, _Wnt1-Cre_-labelled cells, although appearing in the vicinity of the vessel wall, were hardly detectable in the smooth muscle

layer in _Ednra_ KO embryos, whereas heterozygous littermates showed incorporation of labelled cells into the smooth muscle layer as observed in WT embryos (Fig. 4k-p). By contrast, labelled

cells were abundantly incorporated into the tunica media of the aorta and had differentiated into SMCs in _Ednra_ KO embryos, as well as in heterozygous or WT littermates (Supplementary

Figs S8 and S9), indicating that the contribution of _Wnt1-Cre_-labelled cells to the wall of the aorta and coronary arteries are differentially regulated in terms of their requirement for

Edn signalling. _EDN1_ AND _EDNRA_ EXPRESSION IN MOUSE DEVELOPING HEART To investigate the involvement of Edn signalling in coronary development, we examined _Edn1_ and _Ednra_ expression in

developing heart. At E12.5, when primary coronary vascular plexus starts to form23, _Edn1_ was expressed in the outflow and atrioventricular cushion endocardium and developing

interventricular septum (Fig. 5a and Supplementary Fig. S10). At E13.5–E14.5, _Edn1_ expression appeared in the endothelium of the proximal portions of coronary arteries in addition to the

cushion and septal endocardium (Fig. 5b and Supplementary Fig. S10). _Ednra_ expression, mimicked by knocked-in _EGFP_ expression18,24, was detected broadly in cardiomyocytes and aortic

SMCs, and also in _Wnt1-Cre_-labelled cells in the condensed mesenchyme and close to the endocardium at E12.5 (Fig. 5d–f). This region is likely to be the site where the communicating artery

between the right coronary artery and the septal branch is forming (Supplementary Fig. S3). At E13.5 and E14.5, _Ednra-EGFP_ was expressed in _Wnt1-Cre_-labelled cells in condensed

mesenchyme and around coronary arteries (Fig. 5g–l). These results indicate that Edn1 secreted from the cushion endocardium and later from the coronary endothelium may recruit

_Wnt1-Cre_-labelled cells by acting on Ednra, and promote their differentiation into coronary artery SMCs. PREOTIC NCCS CONTRIBUTE TO CORONARY ARTERY SMOOTH MUSCLE To assure that

_Wnt1-Cre_-labelled cells contributing to the coronary artery represented NC derivatives, we used the quail–chick chimera technique for accurate lineage tracing. However, chimeric

replacement of the postotic NC at the level of r6–r8 in chick embryos followed by staining with a quail-specific QCPN antibody showed no contribution of quail cells to the wall of coronary

arteries, although some were detected within the conotruncal and septal region (Supplementary Fig. S11 and Supplementary Movie S1). We then hypothesized that more anterior NCCs might

contribute to the coronary arteries and generated quail–chick chimeras in which the midbrain and preotic hindbrain (r1–r5) neural folds were bilaterally excised and replaced by orthotopic

quail grafts at 3–7 somite stages (ss; Fig. 6a). Surprisingly, replacement of the preotic NC resulted in a significant number of QCPN-positive quail cells distributing into the heart,

especially in the interventricular septum (Fig. 6b–d). Quite a few QCPN-positive cells were found to be incorporated into the smooth muscle layer of the coronary arteries in a pattern

similar to that of _Wnt1-Cre_-labelled cells in mouse embryos (Fig. 6b), whereas many other QCPN-positive cells were scattered within cardiac tissues, especially in papillary muscles in the

right ventricle (Fig. 6c). Co-immunostaining for desmin, a marker for cardiomyocytes, indicated that QCPN-positive cells within the cardiac tissue represented a non-cardiomyocyte population

(Supplementary Fig. S12). To obtain the whole pattern of their distribution, we reconstructed the sections staining and plotted QCPN-positive cells (Fig. 6d). They showed a unique pattern of

distribution, that is, quail cells were abundant in the upper part of the interventricular septum (Fig. 6d and Supplementary Movie S2). Immunohistochemical analysis revealed that these

quail cells contributed to SMCs and possibly non-muscle cells (Fig. 6e–g). SM22α, another smooth muscle marker, was expressed in QCPN-positive cells in the septal branch (Fig. 6h–m) In small

vessels around the conotruncal region, QCPN- and SM22α-positive SMCs were abundantly found (Fig. 6n–p). Orthotopic grafting of quail proepicardial tissues into chick embryos revealed that

QCPN-negative SMCs in the septal branch, as well as in arteries in the free wall, are predominantly of proepicardial origin (Supplementary Fig. S13), as reported previously11,12,13. Then we

subdivided the grafted region of the preotic NC (Supplementary Fig. S14) and found that all regions of the preotic NCCs contributed to papillary muscles. However, we found no contribution of

NCCs originating from the midbrain to r2 in the septal branch or upper part of the interventricular septum, showing that the cells that originated from r3 to r5 might mainly contribute to

coronary artery smooth muscle. As both r3 and r5 provide only a small population of migratory NCCs25, we examined whether r4 is responsible for NC contribution to coronary artery smooth

muscle by using _R4::Cre;Z/AP_ transgenic mouse embryos, in which r4-derived NCCs were specifically and permanently labelled by alkaline phosphatase (AP) expression. At E9.5, AP-positive

cells were detected in the outflow tract in addition to the second PA (PA2; Fig. 6q). At E11.5, AP-positive cells were found in PA2 mesenchyme and cardiac outflow cushion tissue, but not in

the third to sixth pharyngeal arteries contributed by postotic NCCs (Supplementary Fig. S15). At E14.5, AP-positive cells were detected in the conotruncal region and formed a layer

surrounding proximal coronary arteries in addition to their contribution to PA2-derived ear structures (Fig. 6r and Supplementary Fig. S15). These results indicate that the contribution of

preotic NCCs to cardiovascular development is distinct from that of postotic NCCs, and that NCCs from r4 mainly contribute to coronary artery SMCs. Finally, we examined spatiotemporal

relationship between preotic and postotic NCCs by labelling premigratory crest cells with fluorescent dyes. We injected CM-DiI (red) and CFDA/DiO (green) into the neural folds at the levels

of r3/4 (preotic) and somites 1/2 (postotic) in chick embryos at 8 ss and examined fluorescent signals after 3 days of incubation (Fig. 7a). Interestingly, migration of preotic crest cells

preceded that of postotic cells in the similar pathway within the cushion tissue (Fig. 7b–d), indicating that the anteroposterior order of NCCs corresponds to their proximodistal location

within the cardiac region through different timing of migration. PREOTIC NC ABLATION CAUSES CORONARY ARTERY MALFORMATIONS Next we ablated the preotic NC from the midbrain to r5 and analysed

the coronary morphology after 12 days of incubation to examine its role in coronary development. Unlike in postotic NC-ablated embryos, no great vessel anomalies were observed in preotic

NC-ablated embryos (Fig. 8a). Instead, coronary artery malformations were detected in about one-third of ablated embryos (Supplementary Table S3). Some preotic NC-ablated embryos showed

extra ostium formation even in the non-coronary cusp, where the extra ostium is never formed in normal embryos (Fig. 8c). Coronary angiography also revealed abnormally enlarged septal

branches like those of _Edn1-/Ednra_ KO mice (Fig. 8e). To avoid any artificial effect caused by intravascular injection, we also generated reconstructed serial sections for

three-dimensional analysis. Angiography, stained sections and reconstructed images all showed dilated septal branches (Fig. 8g), and reconstitution image recaptured the same finding of the

coronary angiography (Fig. 8i–l). Furthermore, we also found a fistula between the septal branch and the right ventricle or other types of anomalies, including septal branch split into two

lumens or more by a pillar-like structure in ablated embryos (Fig. 8k and Supplementary Fig. S16). The enlarged septal branch partially lacked the marker of SMCs (Fig. 8m). These deformities

might reflect abnormalities in the remodelling process. We also performed a region-specific preotic NC ablation before 7 ss, and most frequently found septal branch malformations when the

NC between r3 to r5 was ablated (Supplementary Table S3). No abnormalities appeared when the ablation was performed at later stages (Supplementary Table S3). Considering that cardiac

(postotic) NC ablations are routinely performed after 7 ss, our findings are consistent with the earlier migration of preotic NCCs than postotic cardiac NCCs. Finally, we examined whether

inactivation of Edn signalling can cause coronary artery abnormalities also in chick embryos using bosentan, an Edn receptor antagonist. Bosentan-treated embryos displayed enlarged septal

branches as in _Edn1_ and _Ednra_ KO mice and preotic NC-ablated chick embryos (Supplementary Fig. S17). This result suggests that Edn signalling is involved in the contribution of NCCs to

coronary artery formation in both mouse and chick embryos. DISCUSSION The regional destination and cell fate of NCCs correspond to their premigratory position within the neural tube along

the anteroposterior axis2. The contribution of NCCs to cardiac development has been attributed uniquely to those arising from the postotic hindbrain, referred to as the cardiac NC4. In this

study, we first identified the contribution of the preotic cranial NC, which primarily gives rise to the craniofacial skeleton3, to developing coronary arteries and adjacent cardiac tissues

(Supplementary Fig. S18). This study has also reconciled the long-lasting controversy on the contribution of NCCs to coronary artery development, and has shown that the discrepancy between

mouse and chick/quail experiments is not due to a species difference. The distribution pattern of preotic NCCs and their contribution to cardiovascular development are obviously different

from those of the postotic cardiac NC. Although cardiac NCCs arising from the postotic hindbrain migrate into the posterior (caudal) PAs and form the tunica media of PA artery-derived great

vessels, the aorticopulmonary septum and the outflow tract endocardial cushions, preotic NCCs migrate into the heart beyond the conotruncal region and give rise to coronary SMCs and

mesenchymal-like cells, especially in the interventricular septum and papillary muscles of the right ventricle. This fate is limited to the NCCs that migrate very early from the neural folds

of the midbrain and preotic (r1–r5) hindbrain, which may explain why this cell population has hitherto escaped detection. Together with dual labelling experiments, the spatiotemporal order

of NCCs along the anteroposterior axis appears to correspond to their postmigratory positions within the cardiovascular system along the inflow–outflow axis. Ablation of the postotic and

preotic NC in chick embryos leads to different types of coronary artery defects, indicating their roles in coronary artery development through different mechanisms. In the absence of

postotic NCCs, the coronary arteries show a variance in their site of origin and an asymmetry in the deployment of SMCs14. However, concomitant persistent truncus arteriosus with changes in

NC-related structures, such as the myocardial sheath, and no direct contribution of postotic NCCs to the coronary smooth muscle make the direct involvement of the postotic NC obscure. By

contrast, ablation of the preotic NC results in malformed septal branches, which correspond to the direct contribution of the crest cells, and redundant formation of the coronary orifices

without obvious great vessel anomalies. These results suggest that the preotic NC may be more directly involved in coronary artery formation by controlling the integrity of the vascular wall

and determining the site of orifices in a manner distinct from those for the postotic NC. Preotic NCCs migrating into the heart appear to differentiate into other cell types in addition to

SMCs. Many of these cells distribute mainly in the interventricular septum and papillary muscles in a scattered pattern. Recently, Fukuda and colleagues26,27 have reported the presence of a

NC-derived stem cell population in the heart, which can differentiate into various cell types, including cardiomyocytes. By contrast, our present study shows that preotic NCCs do not

differentiate into cardiomyocytes, at least in embryonic stages (Supplementary Fig. S12). The comparison between these NC derivatives deserves further study. It is also noteworthy that

r4-derived NCCs, which constitute PA2 ectomesenchyme, are the major population of preotic NCCs migrating into the heart and contributing coronary artery formation. PA2 mesoderm-derived cells

have been reported to migrate into the heart as the leading edge of the second heart field28,29. Possible interaction between migratory preotic NCCs and PA2-derived mesodermal cells may

also warrant further investigation. We also showed the role of Edn signalling in the remodelling of coronary arteries in mouse and chick embryos. The similarity of the _Edn1_-/_Ednra_-null

phenotype in mouse to the NC ablation in chick suggests that the preotic NC is the target of Edn signalling in coronary remodelling. We have previously reported that Edn1 activates the

G-protein-coupled receptor Ednra on preotic NCCs migrating into the first PA, and specifies mandibular identity through Gq/G11-dependent signalling18. This and the present findings indicate

that Edn1 activates different signalling and genetic programmes in preotic NCCs in different contexts. Interestingly, coronary artery dilation in the interventricular septum is observed in

NC-specific G12/G13-deficient mice rather than Gq/G11-deficient mice30. Ednra has been reported to couple with G12/G13 as well as Gq/G11 and activate Rho signalling in various cell types,

including vascular SMCs31,32. Rho signalling is a key regulator of various cell behaviours, such as migration, differentiation and remodelling, in vascular SMCs33,34. Taken together, we

speculate that Edn1/Ednra may activate Rho signalling in preotic NCCs through a G12/G13-dependent pathway to modulate coronary artery remodelling. In the present study, we detected _Edn1_

expression in cushion endocardium and proximal coronary endothelium, near which _Ednra_-expressing NCCs were distributed at the critical stage for coronary formation around E12.5–E14.5. This

expression pattern suggests the possibility that the Edn signalling may serve as a local cue for the recruitment of preotic NCCs and their differentiation into SMCs in the coronary

vasculature. Vascular SMCs originate from different sources depending on the region11. In the coronary arteries, SMCs have been proved to be mainly of proepicardial origin12,35,36,37. On the

other hand, the heterogeneity of coronary artery SMCs has also been suggested by a number of studies, and NCCs and second heart field-derived cells have been considered as candidate

sources15,38,39. However, lack of direct evidence has lead many investigators to question the NC contribution to coronary artery SMCs11. Our present findings provide direct evidence by

identifying the preotic NC as another source. Especially, preotic NCCs preferentially contribute to the septal and conotruncal regions, which appear less accessible to epicardial cells

compared with the ventricular free walls. The origin and patterning of the coronary arteries exhibit remarkable diversity among vertebrate species40. In fish, the coronary circulation

originates from postbranchial vessels corresponding to the dorsal aorta. After lungs replace gills during evolution, the origin gradually shifts from the dorsal aorta to the ventral aorta,

which is the closest supply of oxygenated blood to the heart. Such phylogenetic diversity may be associated with differences in migration and interaction of heterogeneous cell populations

involving NCCs. Indeed, ablation of the preotic NC affects the number and position of coronary ostia and vascular morphology. Furthermore, the interventricular septum, where preotic NCCs

mostly migrate, is a novel structure in vertebrate evolution and is phylogenetically diverse in its morphology and patterns of blood supply41,42. These findings lead us to speculate that

preotic NC may provide species-specific patterning information as observed in craniofacial development43,44. Heterogeneity of coronary SMCs gives another implication in the pathophysiology

of coronary artery disease, a major cause of death throughout the world. Previous studies have suggested that susceptibility to atherosclerosis is highly associated with cell lineage in

humans and experimental animals45,46,47. NC-derived SMCs and mesoderm-derived SMCs are shown to be different in growth and agonist-induced gene expression patterns48,49. A recent report

demonstrated that NC-derived SMCs were more prone to calcification than those of mesodermal origin50. Taken together with these previous findings, our results may provide a new basis for the

understanding of the pathophysiology of coronary artery disease. METHODS ANIMALS _Edn1_ KO (_Edn1__–/–_) mice21, _Ednra_ KO (_Ednra__EGFP/EGFP_, _EGFP_-knock-in) mice24, _Wnt1-Cre_/R26R

reporter mice15,51 and _R4::Cre;Z/AP_ mice52,53 have been described previously. Mutant mice were maintained on a mixed C57BL/6J × ICR background and were housed in an environmentally

controlled room at 23±2 °C, with a relative humidity of 50–60% and under a 12-h light:12-h dark cycle. Genotypes were determined by PCR on the tail-tip or amnion DNA using specific primers

provided in Supplementary Table S4. Embryonic ages were determined by timed mating with the day of the plug being E0.5. Fertilized Hojuran chicken (_Gallus gallus_) eggs and Japanese quail

(_Cortunix japonica_) eggs were obtained from Shiroyama Keien Farms (Tochigi, Japan) and Motoki Hatchery (Saitama, Japan), respectively, and were incubated in a humidified atmosphere at 37 

°C until the embryos reached appropriate stages. All animal experiments were approved by the University of Tokyo and Tokyo Women’s Medical University Animal Care and Use Committee, and were

performed in accordance with the institutional guidelines. INK INJECTION Hearts were collected from mouse and chick embryos at appropriate stages and dipped into heparinized PBS. Then, ink

(Kiwa-Guro, Sailor, Japan) was gently injected from the ascending aorta using a glass micropipette. Samples were fixed in 4% paraformaldehyde solution and dehydrated in sequential ethanol

solution. To visualize the septal branch, dehydrated samples were immersed in BABB (1:2 benzyl alcohol to benzyl benzoate). Some injected samples were re-dehydrated in ethanol solution and

embedded in paraffin. Consecutive section (10 μm) was stained with haematoxylin-eosin stain. Β-GALACTOSIDASE STAINING LacZ expression was detected by staining with X-Gal

(5-bromo-4-chloro-3-indolyl β-D-galactoside) for β-galactosidase activity. Section staining was performed as described previously54. For sections, samples were embedded in optical coherence

tomography compound, cryosectioned and subjected to X-gal staining. Some sections were counter stained with haematoxylin (Muto Pure Chemicals, Japan). Z/AP STAINING Z/AP staining has been

described previously55. E9.5, E11.5 and E14.5 embryos were fixed in 4% paraformaldehyde and rinsed in PBS. After a fixation, samples were rinsed in NTMT (NaCl 0.1 M, Tris–HCl 0.1 M(pH9.5),

MgCl2 0.05 M and Tween20 (0.1%). Nitroblue tetrazolium (3.5 μl; Roche 1383213) and 5-bromo-4-chloro-3-indolyl phosphate (3.5 μl; Roche 1383221) per ml of NTMT were used for the staining.

Hearts were dissected and stained as whole mount or cryostat sections. IMMUNOHISTOCHEMISTRY Embryo cryosections (12–16 μm) were immunostained using the following antibodies: rat monoclonal

anti-GFP (Nacalai Tesque, Kyoto, Japan; 1:200), rabbit anti-GFP (Medical and Biological Laboratories, Nagoya, Japan; 1:250), mouse monoclonal anti-quail cell (QCPN; Developmental Studies

Hybridoma Bank; 1:30), Rabbit polyclonal anti-β-galactosidase (Rockland; 1:1,000), mouse monoclonal anti-actin α-smooth muscle (1A4; Sigma; 1:1,000), rabbit anti-SM22α (Abcam; 1:200), rat

anti-mouse CD31 (BD Pharmingen; 1:100) and mouse anti-desmin (Developmental Studies Hybridoma Bank; 1:100). Signals were visualized with FITC (fluorescein isothiocyanate)- or

TRITC-conjugated secondary antibodies specific for the appropriate species. Some sections were treated with biotin-conjugated secondary antibodies and visualized using the VECTASTAIN ABC

System (Vector Laboratories), streptavidin-FITC (Dako; 1:200), streptavidin-TRITC (Beckman Coulter; 1:200), or streptavidin-APC (eBioscience; 1:200). Nuclei were visualized with TO-PRO-3

(Molecular Probes). Fluorescent signals were visualized with a computer-assisted confocal microscope (Nikon D-ECLIPSE C1 and EZ-C1 software (Nikon). _IN SITU_ HYBRIDIZATION _In situ_

hybridization was performed on cryosections (16 μm) using the _Edn1_ probe as previously described21,24. THREE-DIMENSIONAL RECONSTRUCTIONS Each paraffin-embedded section (4–10 μm) was used

for reconstruction. Digital images of the stained sections were loaded into Amira (Visage Imaging, Inc.) with a voxel size appropriate to section thickness. Images were aligned and concerned

regions (QCPN-positive signals or the septal branch) were labelled. The labels were resampled to iso-volumetric voxel dimensions, and these smoothed data sets were transformed into a

surface by triangulation. The number of triangles was reduced using the SmoothSurface module of Amira. QUAIL–CHICK CHIMERA AND ABLATION OF THE NC Quail–chick chimeras have been described

previously7,56. After an incubation of fertilized quail and chicken eggs in a humid chamber at 37 °C, window was cut through the shell and embryo were visualized with India ink (black ink,

Rotring, Germany) diluted 1:10 in saline solution, injected into the subgerminal cavity. Stages of host chicken and donor quail embryos were match as closely as possible. Ablation of the

neural folds, including premigratory NC, was performed by manual extirpation using electrolytically sharpened tungsten needles. For premigratory cranial NC (from the midbrain to the r5) of

chimera and ablation, the 4- to 7-ss embryos were used. Because of whole staining embryos using HNK1 antibody (data not shown), cranial NCCs of the 8-ss embryos were migrated. For

premigratory cardiac NC (from the r6–r8 and somite 4) of chimera and ablation, the 6- to 9-ss embryos were used. Sham-operated embryos were produced by tearing the vitelline membrane with no

further surgery. For control embryos the windows were opened and the somite with no treatment were counted. QUAIL–CHICK PROEPICARDIUM CHIMERA Quail–chick proepicardium (PEO) chimeras have

been described previously57. Stage 16–17 quail hearts were inspected to confirm that the PEO was not attached to the dorsal wall of the heart. Using an eggshell membrane, the quail PEO was

isolated together with the sinus venosus and transplanted behind the chick heart. After an appropriate incubation period, the embryos were killed, and their hearts were subjected to

immunostaining. _IN-OVO_ FLUORESCENT DYE INJECTION Method has been described previously29,58. Fluorescent dye injection was performed on 8-ss chick embryos. Micropipettes were filled with

CM-DiI (Molecular Probes) or CFDA/DiO mixture (1:1; Molecular Probes) in ethanol or N,N-dimethylformamide (Wako) was diluted 1:1, or 1:2 in tetraglycol (Sigma) to yield working solutions of

1.0, or 2.5 mg ml−1, respectively. PHARMACOLOGICAL INACTIVATION OF EDN SIGNALLING _IN OVO_ The method has been described previously59. Thirty microlitre olive oil drop was introduced onto

the shell membrane at 48 h of incubation. At day 12, embryos were collected. In the bosentan-treated group, bosentan (Actelion, Ltd) was suspended in the oil at the 5 mg ml−1, whereas only

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embryo: a model of mispatterning of the branchial arch derivatives. _Development_ 125, 4931–4941 (1998). CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank M.L. Kirby

for many worthwhile advices; A. McMahon and H. Kokubo for _Wnt1-Cre_ mice; P. Soriano and A. Aiba for R26R mice; S. Yokoyama for introduction of ink for coronary angiography; H. Nagao, Y.

Fujisawa and S. Kushiyama for technical assistance; and Actelion Pharmaceuticals Ltd for the bosetan usage. This work was supported by the Global COE Program (Integrative Life Science Based

on the Study of Biosignaling Mechanisms), the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and grants-in-aid for scientific research (KAKENHI 22-2950 to

Y.A., 20591310 to S.M.-T., and 09004092 and 24249047 to H.K.) from the Japan Society for the Promotion of Science (JSPS), Japan, and grants-in-aid for scientific research (09156294 to H.K.)

from the Ministry of Health, Labour and Welfare of Japan. Y.A. is a Research Fellow of the Japan Society for the Promotion of Science (DC1). AUTHOR INFORMATION Author notes * Yuichiro Arima

and Sachiko Miyagawa-Tomita: These authors contributed equally to this work AUTHORS AND AFFILIATIONS * Department of Physiological Chemistry and Metabolism, Graduate School of Medicine,

University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan Yuichiro Arima, Rieko Asai, Daiki Seya, Koichi Nishiyama, Ki-Sung Kim, Yasunobu Uchijima, Yukiko Kurihara & Hiroki

Kurihara * Division of Cardiovascular Development and Differentiation, Department of Pediatric Cardiology, Medical Research Institute, Tokyo Women’s Medical University, 8-1 Kawada-cho,

Shinjuku-ku, Tokyo, 162-8666, Japan Sachiko Miyagawa-Tomita & Kazuhiro Maeda * Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, Basel, CH-4058, Switzerland

Maryline Minoux & Filippo M. Rijli * Faculté de chirurgie dentaire, 1, place de l'hpital, Strasbourg, 67 000, France Maryline Minoux * University of Basel, Basel, CH-4056,

Switzerland Filippo M. Rijli * Department of Cardiovascular Medicine, Faculty of Life Sciences, Kumamoto University, 2-2-1 Honjo, Kumamoto, Kumamoto, 860-0811, Japan Hisao Ogawa Authors *

Yuichiro Arima View author publications You can also search for this author inPubMed Google Scholar * Sachiko Miyagawa-Tomita View author publications You can also search for this author

inPubMed Google Scholar * Kazuhiro Maeda View author publications You can also search for this author inPubMed Google Scholar * Rieko Asai View author publications You can also search for

this author inPubMed Google Scholar * Daiki Seya View author publications You can also search for this author inPubMed Google Scholar * Maryline Minoux View author publications You can also

search for this author inPubMed Google Scholar * Filippo M. Rijli View author publications You can also search for this author inPubMed Google Scholar * Koichi Nishiyama View author

publications You can also search for this author inPubMed Google Scholar * Ki-Sung Kim View author publications You can also search for this author inPubMed Google Scholar * Yasunobu

Uchijima View author publications You can also search for this author inPubMed Google Scholar * Hisao Ogawa View author publications You can also search for this author inPubMed Google

Scholar * Yukiko Kurihara View author publications You can also search for this author inPubMed Google Scholar * Hiroki Kurihara View author publications You can also search for this author

inPubMed Google Scholar CONTRIBUTIONS Y.A., S.M.-T. and H.K. conceived the study and designed experiments. Y.A. and S.M.-T. performed main parts of the mouse and chick–quail experiments,

respectively. K.M. performed histological analysis. R.A. performed _in situ_ hybridization. D.S. performed β-galactosidase staining. M.M. and F.M.R. provided _R4::Cre;Z/AP_ mouse samples.

K.N., K.-S. K. and Y.U. helped with mouse analysis. H.O. supported the initiation of this study. Y.K. contributed to KO mice and pharmacological experiments. Y.A., S.M.-T. and H.K.

coordinated the experimental work, analysed the data and wrote the manuscript with contributions from all authors. CORRESPONDING AUTHOR Correspondence to Hiroki Kurihara. ETHICS DECLARATIONS

COMPETING INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURES AND TABLES Supplementary Figures S1-S18 and Supplementary Tables

S1-S4 (PDF 3085 kb) SUPPLEMENTARY MOVIE S1 3D reconstruction of quail-chick chimera sections, representing the distribution of postotic NCCs. (MOV 2678 kb) SUPPLEMENTARY MOVIE S2 3D

reconstruction of quail-chick chimera sections, representing the distribution of preotic NCCs (MOV 2276 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS

ARTICLE Arima, Y., Miyagawa-Tomita, S., Maeda, K. _et al._ Preotic neural crest cells contribute to coronary artery smooth muscle involving endothelin signalling. _Nat Commun_ 3, 1267

(2012). https://doi.org/10.1038/ncomms2258 Download citation * Received: 16 May 2012 * Accepted: 05 November 2012 * Published: 11 December 2012 * DOI: https://doi.org/10.1038/ncomms2258

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