Myocardial progenitors in the pharyngeal regions migrate to distinct conotruncal regions

Authors


Abstract

Background: The cardiac progenitor cells for the outflow tract (OFT) reside in the visceral mesoderm and mesodermal core of the pharyngeal region, which are defined as the secondary and anterior heart fields (SHF and AHF), respectively. Results: Using chick embryos, we injected fluorescent-dye into the SHF or AHF at stage 14, and the destinations of the labeled cells were examined at stage 31. Labeled cells from the right SHF were found in the myocardium on the left dorsal side of the OFT, and cells from the left SHF were detected on the right ventral side of the OFT. Labeled cells from the right and left AHF migrated to regions of the ventral wall of the OFT close to the aortic and pulmonary valves, respectively. Conclusion: These observations indicate that myocardial progenitors from the SHF and AHF contribute to distinct conotruncal regions and that cells from the SHF migrate rotationally while cells from the AHF migrate in a non-rotational manner. Developmental Dynamics 241:284–293, 2012. © 2011 Wiley Periodicals, Inc.

INTRODUCTION

Congenital heart diseases (CHD) are diagnosed in 19–75 per 1,000 live births, and the incidence of moderate to severe forms of CHD is approximately 6/1,000 live births (Hoffman and Kaplan, 2002). Many of these life-threatening forms of CHD are caused by abnormal development of the heart outflow tract (OFT, conotruncal region), including conotruncal heart defects, such as persistent truncus arteriosus (PTA), double outlet right ventricle (DORV), tetralogy of Fallot (TOF), and transposition of the great arteries (TGA). In chick cardiogenesis, the OFT originates from the second lineage of heart-forming regions, which include the secondary and anterior heart fields (SHF and AHF), which are found in the visceral mesoderm beneath the floor of the pharynx caudal to the OFT and in the mesodermal core of the first and second pharyngeal arches, respectively (Waldo et al., 2001; Mjaatvedt et al., 2001). During mouse cardiogenesis, the OFT and right ventricle both originate from the pharyngeal mesoderm designated as a second heart field (Kelly et al., 2001; Buckingham et al., 2005). Accordingly, it is suggested that the abnormal development of the second lineage of heart-forming regions could cause conotruncal heart defects. In the chick embryo, laser ablation of the right side of the SHF leads to conotruncal defects including TOF and pulmonary atresia (Ward et al., 2005). In addition, morphological observations have suggested that conotruncal heart defects, including TOF, DORV, and TGA, are caused by the arrest of normal rotation in the nascent OFT and great arteries (Lomonico et al., 1988; Bostrom and Hutchins, 1988). 22q11.2 deletion syndrome, which is the most common interstitial chromosome deletion found in humans, is frequently associated with conotruncal heart defects. Genetic studies in humans and mice have clarified that TBX1/tbx1, which is expressed in the second heart field at the onset of/during the elongation of the OFT, is the gene responsible for this syndrome (for reviews, see Lindsay 2001; Baldini 2005; Nakajima 2010). A shortened and straight OFT associated with hypoplastic OFT cushion ridges is observed in mouse models of TGA (Yasui et al., 1995; Nakajima et al., 1996; Kitamura et al., 1999; Ai et al., 2006). However, the genetic and morphogenetic alterations that lead to TGA morphology are largely unknown (Bajolle et al., 2006). According to these observations, it is strongly suggested that arrested or altered development of the second lineage of heart-forming regions causes conotruncal heart defects. However, the etiological link between spatiotemporal alterations in the second lineage of heart-forming regions and the spectrum of particular conotruncal heart defect is not well understood.

At the gastrula stage, the prospective heart cells reside in the left and right anterior lateral plate mesoderm. Experiments using isl1-null mutant mice have detected two sets of cardiogenic precursors. The most lateral cells in the paired heart-forming mesoderm move to the ventral midline, fuse with each other, and form a single primitive heart tube, from which the atria and left ventricle will later develop. Medially located cells in the heart-forming mesoderm are incorporated into the second heart field and later give rise to the cardiac OFT and right ventricle (Cai et al., 2003; Moreno-Rodriguez et al., 2006; Abu-Issa and Kirby 2008; Nakajima et al., 2009). A fluorescent dye–labeling experiment involving chicks showed that the right side of the SHF caudal to the OFT migrates spirally to the left side of the OFT and contributes to the formation of the myocardium on the left side of the conus region. However, the fate of the myocardial progenitors on the left sides of the SHF and AHF in the mesodermal core of the pharyngeal arches has not been examined because the chick embryo lies with its right side facing upwards; thus, only the right side of the SHF is accessible (Ward et al., 2005). Another dye-marking experiment showed that the cranial paraxial mesoderm at the axial level of rhombomeres 1–4 migrates into the developing OFT through the anterior pharyngeal arches and contributes to the myocardium/endocardium of the developing OFT (Tirosh-Finkel et al., 2006). In the present study, we used a fluorescent dye–labeling experiment involving chick embryos to investigate the destinations of myocardial progenitors in the second lineage of heart fields in the pharyngeal regions. We found that myocardial progenitor cells originating from the SHF and AHF contribute to the formation of distinct conotruncal regions and that progenitors from the SHF migrate rotationally while cells from the AHF move in a non-rotational manner.

RESULTS

The Right and Left SHF Migrate Rotationally to Distinct OFT Regions

First, we examined the fates of the right and left SHF. To do this, DiI was pressure injected into these regions at stage 14, and then the embryos were re-incubated and sacrificed at stage 31. In the mature hearts into which DiI had been injected into the right SHF at stage 14 (n=7), DiI was detected on the left side of the OFT at stage 31 (arrow in Fig. 1A2). The ventricles were cut transversely beneath the atrioventricular canal, and the intracardiac distribution of the DiI-positive cells was examined using a fluorescence stereoscopic microscope in the apex to base direction. DiI-positive cells were distributed on the left side of the OFT (arrowheads in Fig. 1B2) as well as in the proximal endocardial cushion tissues, mainly in the left proximal cushion (* in Fig. 1B2). Serial sections were then cut and stained with anti-sarcomeric α-actinin antibody to identify the myocardium (Fig. 1C1–4, 1D1–4). The sections were then observed under a fluorescent microscope, and the distribution of DiI-positive cells was plotted on a scheme of the heart (R3 in Supp. Fig. S2, which is available online). As a result, we found that DiI was distributed in the left lateral to dorsal myocardial wall of the OFT (Fig. 1C2, D2, blue dots in Supp. Fig. S2-R3) as well as in the proximal left endocardial cushion tissue (Fig. 1C2, red dots in Supp. Fig. S2-R3). In some hearts (3/7), DiI was detectable in the dorsal myocardial wall of the left ventricular OFT (LVOT) (Fig. 1D2–4, Supp. Fig. S2-R3). Immunostaining for sarcomeric α-actinin showed that DiI was distributed in both α-actinin-positive and -negative regions, indicating that DiI-positive cells were distributed not only in the myocardium but also in the endocardial cushion mesenchyme. Our observations indicated that the progenitors in the right SHF migrate to the left side of the OFT, as described previously (Ward et al., 2005).

Figure 1.

The DiI injected into the right SHF was distributed on the left side of the OFT. DiI was injected into the right SHF at stage 14, and then the embryos were reincubated and sacrificed at stage 31. A, B: DiI-positive cells were observed on the left side of the OFT (arrow in A2). Intracardiac observations viewed from the apex showed that DiI was distributed on the left side of the OFT (arrowheads in B2) and in the left proximal endocardial cushion tissue (asterisk in B2). C, D: Serial cross-sections were cut parallel to the atrioventricular canal and stained with anti-sarcomeric α-actinin antibody (cardiac muscle) and DAPI (nuclei). DiI-positive cells were seen in the myocardium as well as the endocardial cushion tissue on the left side of the OFT (C2–4). The dorsal myocardium of the left ventricular OFT also contained DiI-positive cells (D2-4). C4 and D4, merged images; LA, left atrium; LV, left ventricle; LVOT, left ventricular outflow tract; RA, right atrium; RV, right ventricle; RVOT, right ventricular outflow tract. Scale bar = 250 μm (C1, D1), 100 μm C2–4), or 25 μm (D2–4).

In stage-31 hearts into which DiI had been injected into the left SHF at stage 14 (n=8), DiI was observed in the ventral region of the OFT as a sharp band beneath the pulmonary valve (conus region, arrow in Fig. 2A2). Intracardiac observations from the apical viewpoint showed that DiI was distributed in the right ventral region of the right ventricular OFT (RVOT) (arrowheads in Fig. 2B2) as well as in proximal left and right endocardial cushions (asterisk in Fig. 2B2). Serial sections that had been stained with anti-sarcomeric α-actinin antibody showed that DiI-positive cells were located in the ventral myocardial wall of the RVOT (conus region, Fig. 2C2, Supp. Fig. S2-L3) as well as in myocardium beneath the pulmonary valve (Fig. 2D2, Supp. Fig. S2-L3). In all hearts (8/8), DiI was distributed in the ventral wall of the RVOT close to the pulmonary valve, and in two of eight hearts DiI was observed in the myocardium ventral to the LVOT (Supp. Fig. S2-L3).

Figure 2.

The DiI injected into the left SHF was distributed in the ventral wall of the OFT. DiI was injected into the left SHF at stage 14, and then the embryos were reincubated and sacrificed at stage 31. A, B: DiI-positive cells were observed as a sharp band in the ventral wall of the OFT (arrow in A2). Intracardiac observations from the apex showed that DiI was distributed in the ventral wall of the OFT (arrowheads in B2) and in the left proximal endocardial cushion tissue (asterisk in B2). C, D: Serial cross-sections were cut parallel to the atrioventricular canal and stained with anti-sarcomeric α-actinin antibody (cardiac muscle) and DAPI (nuclei). The ventral myocardium of the RVOT (right ventricular outflow tract) contained DiI-positive cells (C, D). Note that the ventral wall of the LVOT (left ventricular outflow tract) also contained DiI-positive cardiomyocytes (Supp. Fig. S2). LA, left atrium; LV, left ventricle; LVOT, left ventricular outflow tract; RA, right atrium; RV, right ventricle; RVOT, right ventricular outflow tract. Scale bar = 250 μm (C1, D1) or 100 μm (C2–4, D2–4).

To further confirm the results of the above experiments, we performed a double (DiI and DiO) labeling experiment, in which DiO was injected into the right SHF and DiI was injected into the left SHF, and the hearts of the embryos were examined at stage 31. As a result, we found that the right SHF-derived DiO-positive cells were distributed on the left side of the OFT (arrows in Fig. 3) and the left SHF-derived DiI-positive cells were located in the right ventral wall of the OFT (arrowheads in Fig. 3).

Figure 3.

The right and left SHF migrate spirally to the left and right sides of the OFT, respectively. DiO was injected into the right SHF, and DiI was injected into the left SHF at stage 14, and then the embryos were reincubated and sacrificed at stage 31. The right SHF-derived DiO-positive cells were distributed on the left side of the OFT (arrows), and the left SHF-derived DiI-positive cells were located on the right ventral side of the OFT (arrowheads). A1–4: Ventral view of the stage-31 heart; B1–4: Cross-section indicated by line B1 in A1; A4 and B4, merged images. LA, left atrium; LV, left ventricle; LVOT, left ventricular outflow tract; RA, right atrium; RV, right ventricle; RVOT, right ventricular outflow tract.

The above experiments suggested that the myocardial progenitors from the right SHF contribute to the formation of the left dorsal wall of the OFT in the mature heart and that progenitor cells from the left SHF help to form the right ventral region of the OFT. Our results also suggested that progenitors from both SHF are capable of migrating not only to the myocardial wall but also to the proximal endocardial cushions, from which the OFT septum as well as the supraventricular crista later develop.

The Right and Left AHF in the Second Pharyngeal Arches Contribute to the Ventral Wall of the OFT

In addition to mouse cardiogenesis (Kelly et al., 2001), it has been reported that mesodermal tissue from the first and second pharyngeal arches contributes to the OFT in chicks (Mjaatvedt et al., 2001; Tirosh-Finkel et al., 2006). Immunostaining patterns showed that mesenchymal cells that were contiguous with the OFT myocardium in the first and second pharyngeal arches expressed Nkx2.5 and Islet1 (Supp. Fig. S3). Therefore, we next examined the fate of mesenchymal tissue in the right and left second pharyngeal arches. In stage-31 hearts into which DiI had been injected into the right second pharyngeal arch at stage 14 (n=12), DiI was observed in the right lateral wall of the conus region (arrow in Fig. 4A2). Intracardiac observations from the apical viewpoint showed that DiI-positive cells were located in the ventral wall of the RVOT close to the LVOT (arrowhead in Fig. 4B2). Serial sections showed that DiI was distributed in the right ventral myocardium of the RVOT adjacent to the LVOT as well as in the proximal right endocardial cushion (Fig. 4C2–4, Supp. Fig. S2-R2).

Figure 4.

Heart progenitor cells from the right and left pharyngeal arches migrated to the ventral wall of the right ventricular OFT close to the aortic and pulmonary valves, respectively. DiI was injected into the right or left side of the second pharyngeal arch, and then the embryos were reincubated and sacrificed at stage 31. A–C: DiI-labeled cells from the right second pharyngeal arch were distributed on the right ventral side of the OFT (arrow in A2). Intracardiac observations viewed from the apex showed that DiI-positive cells derived from the right second pharyngeal arch were distributed in the ventral wall of the right ventricular OFT close to the tricuspid and aortic valves (arrowhead in B2). Serial cross-sections stained with anti-sarcomeric α-actinin antibody showed that DiI-positive cells were located in the ventral right ventricular myocardium close to the left ventricular OFT (C2–4). D–F: DiI-labeled cells derived from the left pharyngeal arch were distributed as a sharp band in the ventral wall of the right ventricular OFT (arrow in D2). Intracardiac observations showed that DiI was present in the ventral wall of the right ventricular OFT close to the pulmonary valve (arrowheads in E2). Serial sections stained with anti-sarcomeric α-actinin antibody showed that DiI-positive cells were distributed in the myocardium ventral to the right ventricular OFT close to the pulmonary valve (F2–4). C4 and F4, merged images; LA, left atrium; LV, left ventricle; LVOT, left ventricular outflow tract; RA, right atrium; RV, right ventricle; RVOT, right ventricular outflow tract. Scale bar = 250 μm (C1, F1) or 100 μm (C2–4, F2–4).

DiI-labeled cells from the left second pharyngeal arch were observed as a sharp boundary band in the ventral wall of the RVOT at stage 31 (arrow in Fig. 4D2, n=15). Intracardiac observations showed that DiI was distributed in the right ventral wall of the OFT close to the pulmonary valve as well as in the proximal right endocardial cushion (arrowheads in Fig. 4E2, Supp. Fig. S2-L2). Serial sections showed that DiI was mainly distributed in the right ventral myocardial wall of the RVOT (Fig. 4F2–4, Supp. Fig. S2-L2).

The above observations suggested that cardiogenic cells in the right and left second pharyngeal arches (AHF) migrate to the ventral wall of the RVOT and that cells originating from the right second pharyngeal arch migrate to lateral regions that are further to the right (close to the right atrioventricular canal and LVOT) than the areas to which cells derived from the left second pharyngeal arch migrate. To confirm this, we performed double injection experiments, in which DiI was injected into the right second pharyngeal arch and DiO was injected into the left second pharyngeal arch, and examined the embryos at stage 27 (Fig. 5). As shown in Figure 5, the right pharyngeal arch–derived DiI-marked cells were mainly distributed on the right side of the OFT (large arrows in Fig. 5), and the left pharyngeal arch-derived DiO-marked cells were located in the ventral wall of the OFT (large arrowheads in Fig. 5). Cross-sections of the OFT region showed that DiI-positive cells were present in the right ventrolateral myocardial wall of the OFT (large arrow in Fig. 5B2), and some DiI-positive cells were located in the cushion mesenchyme (small arrows in Fig. 5B2). In addition, the DiO-positive cells were distributed in the ventral myocardial wall of the OFT (large arrowhead in Fig. 5B3) as well as in the cushion tissue (small arrowheads in Fig. 5B3).

Figure 5.

DiI-labeled cells from the right second pharyngeal arch migrated to the right lateral wall of the OFT, and DiO-labeled cells from the left second pharyngeal arch migrated to the ventral wall of the OFT. DiI was injected into the right second pharyngeal arch (r-ph2), and DiO was injected into the left second pharyngeal arch (l-ph2), and then the embryos were examined at stage 27. The right pharyngeal arch-derived DiI-marked cells were distributed in the right ventrolateral wall of the OFT (large arrows), and the left pharyngeal arch-derived DiO-marked cells were located in the ventral wall (large arrowheads). Cross-sections of the OFT showed that DiI-positive cells were located in the right ventrolateral myocardial wall of the OFT (large arrow in B2), and some DiI-positive cells were detected in the cushion mesenchymal tissue (small arrows in B2). DiO-marked cells were seen in the ventral myocardial wall of the OFT (large arrowhead in B3) as well as in cushion tissue (small arrowheads in B3). A1–4: Ventral view of the stage-27 heart. B1–4: Cross-section indicated by line B1 in A1; A4 and B4, merged images.

The distribution of the dye-marked cells derived from the left second pharyngeal arch was similar to that of cells originating from the left SHF. However, serial sections revealed that the cells from the left pharyngeal arch were distributed in more proximal regions (close to the tricuspid valve) than those from the left SHF (Supp. Fig. S2-L2, L3). To confirm this, we performed double injection experiments, in which DiI was injected into the left SHF via the pericardial coelom and DiO was injected into the left second pharyngeal arch via the pharyngeal ectoderm, and then the embryos were examined at stage 31. As shown in Figure 6, left SHF-derived DiI-positive cells were distributed on the ventral side of the conus region (arrows in Fig. 6) close to the pulmonary valve, and DiO-positive cells from the left second pharyngeal arch were present on the ventral side of the conus region close to the aortic valve or inflow tract (arrowheads in Fig. 6).

Figure 6.

DiI-labeled cells from the left SHF migrated to the ventral wall of the OFT close to the pulmonary valve, and DiO-labeled cells from the left second pharyngeal arch migrated to the ventral wall of the OFT close to the aortic valve. DiI was injected into the left SHF, and DiO was injected into the left second pharyngeal arch (l-ph2), and the embryos were examined at stage 31. The left SHF-derived DiI-marked cells were distributed in the ventral wall of the OFT close to the pulmonary valve (arrow in A2), and the left second pharyngeal arch-derived DiO-marked cells were located in the ventral wall of the OFT close to the inflow tract (arrowhead in A3). Cross-sections showed that DiI-positive cells were located in the right ventral myocardial wall (arrow in B2), and DiO-marked cells were seen in the ventral wall of the OFT close to the LVOT (arrowhead in B3). A1–4: Ventral view of the stage-31 heart. B1–4: Cross-section indicated by line B1 in A1; A4 and B4, merged images. LA, left atrium; LV, left ventricle; LVOT, left ventricular outflow tract; RA, right atrium; RV, right ventricle; RVOT, right ventricular outflow tract.

DiI Injection Into the First Pharyngeal Arches

We finally examined the contributions of the right and left first pharyngeal arches to the mature heart. We found that the cells originating from the first and second pharyngeal arches on the right (Supp. Fig. S2-R1 and R2) and left (Supp. Fig. S2-L1 and L2) sides displayed similar distributions in the heart at stage 31. Furthermore, when the first pharyngeal arches were labeled with DiI/DiO at stage 12 and observed at stage 31, the distribution pattern of the dye-marked cells was similar to that observed in hearts labeled at stage 14 (Supp. Fig. S4).

DISCUSSION

In the present study, we showed that dye-marked cells in the right SHF migrated to the left dorsal side of the OFT in the mature heart and that marked cells in the left SHF moved to the right ventral side of the OFT adjacent to the semilunar valves. In addition, the progenitor cells in the left pharyngeal arches (AHF) migrated to the ventral region of the OFT close to the pulmonary valve, and cells from right pharyngeal arches (AHF) migrated to the right ventral side of the OFT close to the aortic valve (Fig. 7). These observations suggest that during OFT development (1) cells from the SHF migrate rotationally in a counterclockwise direction towards the OFT (viewed from the apex), (2) myocardial progenitor cells from the AHF migrate to the ventral wall of the OFT in a non-rotational manner, and (3) heart progenitors in the SHF and AHF contribute to the formation of distinct conotruncal regions. A previous developmental anatomy study involving human embryos showed that the junction of the OFT and the great arteries undergoes rapid rotation between Carnegie stages 15 and 19 (Lomonico et al., 1986). Ward et al. (2005) showed that the dye-labeled right side of the SHF migrates spirally to the left side of the OFT in chicks. Transgenic mice carrying the y96-Myf5 nlacZ-16 gene, which is a marker of a subpopulation of the RVOT myocardium, display counterclockwise rotation of the OFT (Bajolle et al., 2006). Taking these observations together, it is evident that the left and right SHF migrate rotationally to add to the developing OFT, while the left and right AHF migrate to the ventral wall of the RVOT in a non-rotational fashion. These results also indicate that heart progenitors in the SHF and AHF contribute to distinct conotruncal regions, suggesting that spatiotemporal alterations in either of these second heart-forming regions causes the spectrum of particular conotruncal heart defects.

Figure 7.

Schematic representations showing the fates of the SHF and AHF. A: Ventral views of the secondary heart field (SHF) and anterior heart field (AHF) in the pharyngeal regions of a stage-14 chick embryo. The AHF is located in the core mesoderm of the first and second pharyngeal arches (Ph1 and Ph2), and the SHF is located in the visceral mesoderm of the pharynx (at the sites of the future third pharyngeal arches) caudal to the distal end of the outflow tract (OFT). B, C: Schematic showing the myocardium derived from the SHF and AHF shown in A. B: Ventral view of the mature heart. C: Apical view (the heart was cut along the broken line shown in B and viewed internally from the apex). AHF, anterior heart field; Ao, ascending aorta; LV, left ventricle; LVOT, left ventricular outflow tract; OFT, outflow tract; PA, pulmonary artery; Ph, pharyngeal arch; RV, right ventricle; RVOT, right ventricular outflow tract; SHF, secondary heart field.

Retrospective clonal analysis of mice showed that a single progenitor cell in the anterior pharyngeal arches gives rise to both heart and skeletal muscles; i.e., cells from the first pharyngeal arches contribute to the masseter muscles and right ventricle, and cells from the second pharyngeal arches give rise to muscles involved in facial expression and the conus region and that there is a clonal relationship between the right (or left) facial expression muscles and the respective myocardium at the base of the aorta (or pulmonary trunk) (Lescroart et al., 2010). These findings suggest that in mice the heart progenitors in the right second pharyngeal arch give rise to the subaortic myocardium and the cells in the left second pharyngeal arch go on to produce the subpulmonic myocardium (fig. 6 in Lescroart et al., 2010). Our results are basically consistent with their results, i.e., that the DiI-marked cells in the right second pharyngeal arch contribute to the myocardium ventral to the aortic valve and cells from the left second pharyngeal arch contribute to the ventral wall of the RVOT close to the pulmonary valve. However, our results showed that the dorsal myocardial wall of both semilunar valves originate from the right SHF (Fig. 1D, Supp. Fig. 2-R3). The difference between the results of Lescroart et al. (2010) and our results can be attributed to methodological differences because it is not feasible to mark all progenitor cells in the pharyngeal region. Another possibility is that species differences affected the results, i.e., the myocardium consisting of the dorsal region of the OFT beneath the semilunar valves is derived from the right side of the SHF in chicks. It was also reported that progenitor cells in the first pharyngeal arches give rise to the myocardium in the right ventricular free wall in mice, whereas our dye-marking experiments showed that dye-marked cells from the first pharyngeal arches migrated to the conus region. Carbon particle and dye-marking experiments involving chicks have shown that the straight (primitive) heart tube gives rise to the right ventricle (De la Cruz et al., 1977; Mjaatvedt et al., 2001), and Tbx5, a marker gene for the left ventricle, is expressed in the posterior region of the straight heart tube in chicks (Yamada et al., 2000); therefore, it is possible that the fusion of the right and left cardiogenic anterior lateral plate mesoderms occurs at the site of the right ventricle during chick cardiogenesis while it occurs in the left ventricle in mice. The fate maps of the second lineage of heart-forming regions in the pharyngeal regions are basically the same in chick and mouse; however, there might be small differences between the species.

In the present report, we showed that heart progenitors in the SHA/AHF migrate to form the conus myocardium as well as conotruncal endocardial cushions. Our observations suggest that in addition to the SHF, the mesenchymal populations in the first and second pharyngeal arches are capable of migrating into the nascent OFT to generate not only the myocardium but also endothelial/mesenchymal tissue, as shown in mouse cardiogenesis (Kelly et al., 2001; Cai et al., 2003). In chick cardiogenesis, some of the cranial paraxial mesoderm migrates into the OFT through the first and second pharyngeal arches and contributes to the OFT myocardial and endocardial populations (Tirosh-Finkel et al., 2006). Experimental manipulations have suggested that the cephalic mesoderm anterior to the heart tube gives rise to the myocardium of the OFT in chicks (Mjaatvedt et al., 2001). In addition to the mesodermal components, it is well established that cardiac neural crest cells contribute to the aortico-pulmonary septum and the tunica media of the arch arteries through the third, fourth, and sixth pharyngeal arches (Nishibatake et al., 1987). Waldo et al. (2005) reported that dye-marked cells in the SHF at stage 18 give rise to the proximal wall of the aorta and pulmonary trunk. Taking these observations together, it is suggested that progenitor cells in the SHF/AHF migrate to the developing OFT and give rise to the myocardium/endocardium prior to stage 18. Thereafter, cells from the SHF form the tunica media of the proximal regions of the great arteries, and cardiac neural crest cells from the posterior three pairs of pharyngeal arches establish the aortico-pulmonary septum as well as the distal walls of the great arteries.

Mice possessing mutations affecting the development of the second heart field show common OFT morphological features, such as a short OFT and hypoplastic conotruncal cushion ridges (Washington Smoak et al., 2005; Théveniau-Ruissy et al., 2008; High et al., 2009; Watanabe et al., 2010). Elongation/rotation of the OFT and the formation of conotruncal ridges play crucial roles in the establishment of ventriculoarterial alignment as well as septation (Yasui et al., 1995; Nakajima et al., 1996; Qayyum et al., 2001; Okamoto et al., 2010); therefore, altered development of the heart progenitors in the pharyngeal region can induce conotruncal malformation. Further experiments are necessary to clarify the etiologic link between spatiotemporal alterations in the second heart-forming region and specific conotruncal heart defects.

EXPERIMENTAL PROCEDURES

Chick Embryos

Fertilized eggs, which were purchased from Shiroyama Farm (Kanagawa, Japan), were incubated at 37°C and 60–70% humidity. After 42–48-hr incubation, 3 ml of egg albumin were removed using a syringe pump, and a fenestration (1.5 × 2 cm) was made on the shell surface. To facilitate visualization of the embryo, 10% India ink/Tyroide's solution was injected into the yolk sac beneath the embryo, which was then staged according to Hamburger and Hamilton (1951), and stage-14 embryos were used for the subsequent experiments.

Pharyngeal Mesenchyme and Visceral Mesoderm Cell Tracing In Ovo

To trace pharyngeal or visceral mesoderm cells, the embryos were pressure injected with DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate, Molecular Probes, Eugene, OR) or DiO (3,3′-dioctadecyloxacarbocyanine perchlorate, Molecular Probes), as described previously (Yanagawa et al., 2011). A stock solution of DiI was prepared in dimethylsulfoxide (DMSO) (2.5 mg/mL) or absolute ethanol containing 10% glycerol (1 mg/mL), and freshly prepared working solution (1:100 to 500 in PBS [phosphate-buffered saline]) was injected into the embryos. A stock solution of DiO in dimethylformamide (DMF) (2.5 mg/mL) was freshly diluted (1:200) in PBS and used. After removing the perivitelline membrane covering the pharyngeal region, the embryo was carefully exteriorized from the nascent amniotic membrane, making it possible to rotate the embryo in order to view its left and right sides. DiI or DiO was pressure injected into the visceral mesoderm of the pericardial coelom (SHF, Supp. Fig. S1A) or the pharyngeal core region (AHF, Supp. Fig. S1B and S5) via the external surface of the pharyngeal arch using a pulled glass capillary needle (20 μm in external diameter) equipped with a pressure injector (40 psi, 20–40 msec; Narishige, Tokyo, Japan). After confirming the location of the injection site under a fluorescent stereoscopic microscope (Leica, Tokyo, Japan), the fenestration in the eggshell was covered with cellophane tape, and the embryo was incubated until stage 31. At stage 31, each embryo had its heart extirpated, fixed with 4% paraformaldehyde in PBS for 30 min, observed under a stereoscopic fluorescent microscope, and photographed. After the observation period, the hearts were additionally fixed for 12 hr, embedded in OCT compound (Sakura, Tokyo, Japan), and frozen in liquid nitrogen.

Fluorescence Microscopy

Serial frozen sections (8 μm) were cut on a cryostat, mounted on slides, and air-dried. After being rinsed in PBS, the sections were coverslipped using mounting medium and observed under a conventional fluorescent microscope (Olympus, Tokyo, Japan). DiI-positive regions were then plotted on a scheme of the heart (Supp. Fig. S2). Sections containing DiI-positive cells were subjected to immunohistochemistry for sarcomeric α-actinin to identify the myocardium (Sakata et al., 2007). Slides containing DiI-positive sections were selected and incubated in PBS for 12 hr at 4°C to remove the coverslip. The samples were then rinsed in PBS, blocked for 1 hr with 1% bovine serum albumin/PBS, incubated with anti-sarcomeric α-actinin antibody (diluted 500× in blocking solution, clone EA53; Sigma, St. Louis, MO) for 2 hr at room temperature, rinsed with PBS, and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Cappel, Cochranville, PA) for 1 hr at room temperature. Nuclei were stained with DAPI (4′6′-diamino-2-phenylindole dihydrochloride) for 20 min, rinsed with PBS, and mounted.

Acknowledgements

The authors thank S. Uoya and H. Yoshimoto for their technical assistance.

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