Morphogenesis of outflow tract rotation during cardiac development: The pulmonary push concept

Authors

  • Roderick W.C. Scherptong,

    1. Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands
    2. Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
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  • Monique R.M. Jongbloed,

    Corresponding author
    1. Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands
    2. Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
    • Department of Anatomy and Embryology, Leiden University Medical Center, Postal zone: S-1-P, P.O. Box 9600, 2300 RC Leiden, The Netherlands
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  • Lambertus J. Wisse,

    1. Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
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  • Rebecca Vicente-Steijn,

    1. Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
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  • Margot M. Bartelings,

    1. Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
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  • Robert E. Poelmann,

    1. Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
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  • Martin J. Schalij,

    1. Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands
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  • Adriana C. Gittenberger-De Groot

    1. Department of Cardiology, Leiden University Medical Center, Leiden, The Netherlands
    2. Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands
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Abstract

Background: Understanding of cardiac outflow tract (OFT) remodeling is essential to explain repositioning of the aorta and pulmonary orifice. In wild type embryos (E9.5–14.5), second heart field contribution (SHF) to the OFT was studied using expression patterns of Islet 1, Nkx2.5, MLC-2a, WT-1, and 3D-reconstructions. Abnormal remodeling was studied in VEGF120/120 embryos. Results: In wild type, Islet 1 and Nkx2.5 positive myocardial precursors formed an asymmetric elongated column almost exclusively at the pulmonary side of the OFT up to the pulmonary orifice. In VEGF120/120 embryos, the Nkx2.5-positive mesenchymal population was disorganized with a short extension along the pulmonary OFT. Conclusions: We postulate that normally the pulmonary trunk and orifice are pushed in a higher and more frontal position relative to the aortic orifice by asymmetric addition of SHF-myocardium. Deficient or disorganized right ventricular OFT expansion might explain cardiac malformations with abnormal position of the great arteries, such as double outlet right ventricle. Developmental Dynamics 241:1413–1422, 2012. © 2012 Wiley Periodicals, Inc.

INTRODUCTION

Morphogenesis of the outflow tract (OFT) is a complex and delicately orchestrated process (Gittenberger-de Groot et al., 2005; Yutzey and Kirby, 2002; Waldo et al., 2001). During proper development of the OFT, the great arteries achieve their definitive morphologic relationship, with the aorta situated in a central position right posterior of the pulmonary trunk. Maldevelopment of the OFT results in an abnormal position of the aorta and pulmonary trunk, observed in some forms of congenital heart disease, such as tetralogy of Fallot, transposition of the great arteries, and double outlet right ventricle, often characterized by a side-to-side arrangement of the great vessels. The malpositioning of the great arteries and their respective orifices in these instances has major consequences, with early mortality without timely intervention. Knowledge of normal OFT development is mandatory as a first step in comprehension of the background of these malformations, as well as to aid in early (prenatal) diagnosis.

During embryonic development, the myocardium of the OFT is derived from the second heart field, also referred to as the anterior or secondary heart field (Kelly et al., 2001; Mjaatvedt et al., 2001; Waldo et al., 2001). In this report, we refer to this population as the anterior heart field, which includes the coelomic wall covering the pericardial cavity in the OFT region of the heart (Waldo et al., 2001). This population of cells expresses amongst others Isl1, Tbx1 and Tbx3, GATA4, Nkx2.5, and WT-1 (Brown et al., 2004; Cai et al., 2003; Kelly and Papaioannou 2007; Mesbah et al., 2008, 2012; Waldo et al., 2001). Expression of some of these genes, including Tbx3 and Isl1, is also observed in neural crest–derived cells, and a recent study indicates a dual origin (myocardial and neural crest cells) of Isl1 derivatives in the heart (Engleka et al., 2012; Mesbah et al., 2008). Anterior heart field cells contribute to the vascular wall of the great arteries and differentiate into a myocardial phenotype upon migration to the myocardial OFT. While at the beginning of development the myocardial OFT is short, orchestrated interaction between products of the aforementioned genes results in continuing addition of cells from the anterior heart field to the OFT, which lengthens in response. Although the exact time span of cellular addition from the anterior heart field to the myocardial OFT is unclear, addition until embryonic day (E) 11.5 in mouse was previously described (Xu et al., 2004). Using immunofluorescent stainings and deposits of Indian ink in chick, an increase of the myocardial part of the OFT up to stage HH24 has been demonstrated (Rana et al., 2007), corresponding to E12.5 in mouse.

The future aorta and pulmonary trunk and their respective orifices have a side-by-side position early in cardiac development. It is suggested that, besides lengthening, a rotational motion of the OFT is required for normal positioning of the aorta and pulmonary trunk and their respective orifices (Bajolle et al., 2006; Thompson and Fitzharris 1979; Thompson et al., 1987). Multiple developmental signaling factors such as Pitx2c and Fgf8/10, expressed in the anterior heart field in an asymmetrical fashion, have been reported as potential regulators of this rotational motion (Ai et al., 2006). Several mutant mouse models with a phenotype that includes OFT malformations are currently available, including models with alterations in expression in vascular endothelial growth factor (VEGF) signaling. We have previously studied VEGF 120/120 mutant mouse embryos that solely express the VEGF120 isoform, and found a high susceptibility of these embryos for OFT malformations, including tetralogy of Fallot and double outlet right ventricle (Stalmans et al., 2003; Van Den Akker et al., 2007). It was postulated that the anterior heart field–derived subpulmonary myocardium is highly sensitive for signaling for VEGF and Notch, which may underlie the observed malpositioning of the OFT vessels in the VEGF 120/120 mutants. However, a direct link with a contribution of the anterior heart field mesenchyme to the OFT has not been studied thus far. In general, studies in which the dynamics of OFT rotation are related to the expression of markers more typical for myocardial precursors in the anterior heart field during consecutive developmental stages, are currently lacking. Hence, it is unknown how and if the anterior heart field contribution to the myocardial OFT could be involved in OFT rotation and whether we are dealing with an already described shortening of the subaortic OFT (Watanabe et al., 1998, 2001) or with a marked, relative lengthening of the subpulmonary OFT, or both. The aim of the current study was to assess how the specific architecture of the anterior heart field could result in an asymmetric addition of OFT myocardium during cardiac development. We demonstrate asymmetry in contribution of myocardial precursors within the anterior heart field and postulate a basis for OFT rotation and for subsequent normal positioning of the aortic and pulmonary trunk orifices. Additionally, we studied this phenomenon in VEGF120/120 embryos, which have been described with a double outlet right ventricle.

RESULTS

Early Myocardial Outflow Tract Development

At embryonic day (E) 9.5 and E10.5, the OFT was not septated yet and was positioned entirely above the primitive right ventricle. At E9.5, the OFT consisted of a myocardial part lined on the inside by cardiac jelly (in subsequent stages developing to endocardial cushion tissue), and of an aortic sac, connecting to the pharyngeal arch arteries (Fig. 1). At this stage, the pro-epicardial organ at the venous pole of the heart could be distinguished and expressed WT-1. No epicardial covering of the primitive heart tube was present yet at this stage, with the exception of a few cells (Fig. 1a), while the coelomic wall covering the OFT was also still negative for WT-1. Nkx2.5 expression was observed in the ventral endoderm of the foregut, in mesenchymal cells at the level of the OFT as well as in the coelomic wall being more pronounced at the left side of the OFT (Fig. 1b). At stage E10.5, faint expression of MLC-2a was observed in the mesenchyme surrounding the aortic sac at the left side, where lining of the coelomic cavity joins the OFT myocardium (arrow in Fig. 1c). Here MLC-2a expression intensified along the myocardial OFT towards the body of the right ventricle, indicating myocardial differentiation (Fig. 1c). Expression of Isl1 was marked within the cardiac mesoderm and faded towards the differentiating myocardium of the OFT (Fig. 1d). Nkx2.5 was strongly expressed in the anterior heart field, including the coelomic wall, and in the OFT myocardium at E10.5 (Fig. 1f). The expression of both Isl1 and Nkx2.5 within the anterior heart field mesenchyme was observed as a central cluster of cells ventral to the foregut, extending preferentially on the left side of the aortic sac and surrounding the left 6th pharyngeal arch artery (Fig. 1d, f), forming the region surrounding the future pulmonary trunk and the ductus arteriosus. On the right side in the cardiac mesoderm, in the region bordering the aortic wall, expression of Isl1 and Nkx2.5 was absent at this stage. Moreover, the faint MLC-2a staining was missing. A WT-1-positive coelomic wall now covered the Nkx2.5-negative mesoderm on the right (putative aortic) side. Lacking at this stage was a WT-1-positive coelomic wall covering the Nkx2.5-positive population on the left (putative pulmonary) side (Fig. 1e). WT-1-positive epicardial cells, derived from the pro-epicardial organ, were found on the atrial and ventricular myocardium.

Figure 1.

Expression patterns on MLC-2a, Nkx2.5 Isl1, and WT-1 in myocardial and mesenchymal regions of the developing outflow tract. Sections through the outflow tract of a wildtype mouse heart embryonic day (E) 9.5 (a,b) and E10.5 (c–f). a: Expression pattern of WT-1. Besides a few WT-1-expressing epicardial cells (arrowhead), epicardial covering of the heart is largely absent at this stage. Expression of WT-1 in the coelomic wall in the region of the outflow tract (OFT) is also still largely absent. b: Nkx2.5 expression can be observed in the endoderm (End), as well as in the coelomic wall bordering the OFT (arrowhead), more pronounced on the left side as compared to the right. c: The myocardial marker MLC-2a stains the myocardium of the OFT. The myocardium is lined on the inside by endocardial cushion tissue (EC). A more faint MLC-2a staining is seen in the region of the second heart field mesoderm (arrow) being more prominent on the left side of the aortic sac (AoS). d: Expression of Isl1 is prominent in the endoderm (End) and the anterior second heart field mesoderm (asterisk). It extends along the left lateral side of the AoS up to the myocardial OFT. Isl1 expression is also found in some of the myocardial cells (M) and the coelomic wall lining (Coe, arrowhead in d), being also more prominent on the left side as compared to the right side. The faint staining in the right-sided mesenchyme most likely represents vascular smooth muscle cells of the developing arterial wall. e: Expression pattern of WT-1. Note the expression in the region of the coelomic wall at the right side (arrowhead), as compared to the lack of expression in the coelomic epithelium at the left side (open arrowhead). f: Nkx2.5 expression is markedly asymmetric (asterisk) and found more prominently of the left side of the OFT (future pulmonary trunk) and only sparsely on the right side (future aorta). Note the pronounced Nkx2.5 expression in the cells of the coelomic wall on the left side (arrowhead), as compared to the largely absent expression in the coelomic wall on the right side (open arrowhead). CJ, cardiac jelly; DAo, dorsal aorta; G, foregut; LA, left atrium; LV, left ventricle; RV, right ventricle; Bar = 100 μm.

Expression of Myocardial Progenitor Markers During Septation and Before Outflow Tract Rotation

At E11.5, septation of the aorta and pulmonary trunk and orifice level had initiated by condensed mesenchyme (Fig. 2a). A comparable expression pattern of Isl1, Nkx2.5, MLC-2a, and WT-1 expression with stage E10.5 was observed. A mesenchymal column of Nkx2.5-positive cells extended from the cardiac mesoderm in the anterior heart field to the left side of the OFT beneath the left 6th pharyngeal arch artery, where it connected to the subpulmonary myocardium (Fig. 2a–c, f,g). 3D-reconstruction of the OFT myocardium, based on the expression pattern of MLC-2a, demonstrated an anterior and posterior cranial extension of myocardium resulting in a saddle-shaped distal myocardial OFT border (Fig. 2d,e,h,i, dotted lines). The condensed mesenchyme of neural crest origin connects to the endocardial cushions at the top of the myocardial extensions (Gittenberger-de Groot et al., 2005). Mesenchymal expression of Nkx2.5 (indicated in yellow in the 3D-reconstructions) and Isl1 could be observed as a central cluster of cells located in the pre-pharyngeal mesoderm dorsal to the level of the OFT (Fig. 2i), extending in the indentation between the myocardial extensions on the left side where the pulmonary trunk connects to the subpulmonary myocardium (Fig. 2d). This area of the coelomic wall lacked WT-1 (Fig. 2j; 3D-reconstructions of epicardial covering are shown in Fig. 2k,l). Nkx2.5 expression was absent in the indentation between the myocardial extensions on the right side in the aortic orifice region (Fig. 2e) where the coelomic wall is positive for WT-1 (Fig. 2j,l). Similar to E10.5, Isl1 expression in the cardiac precursor cells showed an asymmetric distribution favouring the pulmonary side (not shown). In the OFT myocardium, the expression was lost whereas Nkx2.5 as well as MLC-2a was present in both the subaortic and subpulmonary myocardium. An interactive pdf file of a 3D-reconstruction of stage E11.5 is provided in Supp. Fig. S2a, which is available online).

Figure 2.

Asymmetric distribution of myocardial progenitors at the level of the outflow tract. Sections and 3D-reconstructions of an E11.5 heart. a: MLC-2a is expressed in the myocardium. The aortic sac is now septated indicated by the presence of condensed mesenchyme (CM) in between the ascending aorta (AAo) and pulmonary trunk (Pu), which are still positioned side by side with the aorta somewhat more to the right as compared to the pulmonary trunk. b: Histological section demonstrating colour coding of 3D-reconstructions in d, e, and h, i. Yellow: Nkx2.5-positive myocardial and vascular wall precursors. Light blue: endocardial cushion tissue merging with the CM of the aortopulmonary septum. Dark blue: Lumen of 6th pharyngeal arch artery (future ductus arteriosus) and Pu. Light red: Lumen of AAo, dorsal aorta (DAo), and outflow tract. Dark red: vascular wall on the aortic side. Light brown: myocardium. (Also see the colour coding bar at the inferior part of the figure.) c: Nkx2.5 expression is marked in the mesenchyme lining the pulmonary wall (arrow) and merging with the subpulmonary myocardium. This phenomenon is lacking on the aortic side. The faint staining in the right-sided mesenchyme most likely represents vascular smooth muscle cells of the developing arterial wall. d, e, and h, i. show 3D-reconstructions. Black lines in d indicate the sections of levels depicted in a,c and f–g. d: Left lateral view, showing the asymmetric expression of Nkx2.5 in the cardiac mesoderm (yellow) around the left branch of the 6th pharyngeal arch artery (6th l, blue) and extending in the pulmonary depression of the saddle-shaped orifice level (indicated by the dotted lines in d, e, h, and i). This is lacking on the aortic side of the orifice (e, right lateral view). f: Nkx2.5-stained section at the level of the outflow tract. Note that a column of Nkx2.5-positive myocardial precursor cells is present exclusively on the left side, where the Pu and right ventricular outflow tract are developing. g: Histological section (stained by MCL-2a) demonstrating colour coding of 3D reconstructions in d, e, h, and i, as related to the histological section shown in f. Yellow: Nkx2.5-positive myocardial and vascular wall precursors. Light blue: endocardial cushion tissue. Blue: left 6th pharyngeal arch artery (6th l, future ductus arteriosus). Red: vascular wall on the aortic side. (Also see the colour coding bar at the inferior part of the figure.) h: 3D-reconstruction, anterior view. i: 3D-reconstruction, posterior view. For explanations and colour coding, see above. j: WT-1 staining. Note the WT-1-expressing cells in the coelomic wall at the right (putative aortic) side (arrowhead), as compared to the low WT-1 expression on the left (putative pulmonary) side (open arrowhead). k: 3D reconstruction, left lateral view and (l) right later view, demonstrating the epicardial covering of the heart, indicated in brown. The remainder of the colour coding is as described above. Note the “bare” area at the left part of the outflow tract that is uncovered by epicardium at this stage (open arrowhead). In contrast, the right side of the OFT is covered by epicardium (arrowhead). AVC, atrioventricular canal; LA, left atrium; LV, left ventricle; OFT, outflow tract; RA, right atrium; RV, right ventricle; 6th r, right 6th pharyngeal arch artery. Bar = 200 μm.

Positioning of the Pulmonary Trunk and Aorta: The Pulmonary Push Concept

At E12.5, a distinct column of Nkx2.5-expressing mesenchymal cells was found exclusively within the indentation at the orifice level at the pulmonary side but not in the aortic region (Fig. 3a–c, Supp. Fig. S1). From stage E12.5 onwards, the entire heart was covered by WT-1-expressing epicardial cells. In contrast to previous stages, the coelomic epithelium at the left side of the outflow tract now also expressed WT-1 and, similar to previous stages, WT-1 was expressed in the coelomic epithelium at the right side. From E12.5 to E14.5, the orifice of the future pulmonary trunk became progressively positioned in an anterior and rightward direction (Fig. 3a, d, black dot and arrow). An interactive pdf file of a 3D-reconstruction of stage E12.5 is provided in Supp. Fig. S2b).

Figure 3.

Positional changes of the aorta and pulmonary artery during outflow tract development: The pulmonary push concept. a–c: 3D-reconstructions of stage E12.5. Colour coding: Dark blue: ductus arteriosus (DA) and short pulmonary trunk (Pu). Red: ascending aorta (AAo). (Also see colour coding bar at the inferior part of the figure.) a: Left lateral view. b: Cranial view. c: Right lateral view. The Nkx2.5-expressing mesenchymal cells (yellow) can be seen inserted in the pulmonary side of the indentation of the saddle-shaped orifice (a, b) whereas this is lacking on the aortic side (b, c) where the endocardial cushions and condensed mesenchyme are visible (indicated in light blue). d–f: 3D-reconstructions of stage E14.5. Dark blue: pulmonary trunk (Pu) and ductus arteriosus (DA), connecting distally to the descending aorta (DAo). d: Left lateral view. e: Cranial view. f: Right lateral view. Black dot and arrow (a, d) denote the cranio-anterior positional change of the pulmonary artery orifice from E12.5 to E14.5. The green dot (b, c, and e, f) denotes the pronounced expansion of the atrioventricular canal (AVC) in a rightward direction parallel to the OFT towards the aortic orifice. LV, left ventricle; Pa, pulmonary artery; RV, right ventricle. Bar =100 μm.

At E14.5, the pulmonary trunk and orifice reached their definitive position, which is anterior to the aortic orifice (Fig. 3d–f). The mesenchymal column of Nkx2.5-expressing cells was no longer present at E13.5 and E14.5, indicating that myocardial precursors are not incorporated anymore from the anterior heart field into the OFT. Concurrent with the repositioning of the pulmonary orifice, the atrioventricular canal became positioned below the aortic orifice as it expanded rightward during development (compare green dot and arrow in Fig. 3b and e [superior views] and c and f [right lateral views]). An interactive pdf file of a 3D-reconstruction of stage E14.5 is provided in Supp. Fig. S2c).

Abnormal Development: Expression of Myocardial Progenitor Markers in the VEGF120/120 Model

To test the hypothesis of the pulmonary push concept, development of the OFT was studied in VEGF120/120 mouse embryos of stage E10.5. For the current study, focus was directed specifically at the Nkx2.5- and Isl1-positive myocardial precursors at the OFT of the heart, as compared to results obtained in wild type embryos. In wild type embryos at stage E10.5, a well-organized Nkx2.5-positive, Isl1-positive cluster of cells was observed in the cardiac mesenchyme (Fig. 4a–c, asterisk in Fig. 4b), and an elongated column of Nkx2.5-positive cells was observed extending along the left side of the OFT (dotted area in Fig. 4b). In the stage-E10.5 VEGF120/120 embryos, a large cluster of cells was also observed in the cardiac mesenchyme (asterisk in Fig. 4e). However, in contrast to wild type, we observed an abnormal organisation of this Nkx2.5-positive anterior heart field mesoderm in front of the pharynx. The extension of this population along the pulmonary side of the OFT up to the pulmonary myocardium was very short with only a small area facing the coelomic cavity (Fig. 4d–f, compare the length of the dotted lines in Fig. 4b and e).

Figure 4.

Expression of myocardial progenitor markers in the VEGF120/120 model. Results in E10.5 in wildtype (a–c) and VEGF 120/120 (d–f) embryos. a–c: Three subsequent sections, stained for MLC-2a (a), Nkx2.5 (b), and Isl1 (c), are shown. In wild type embryos, a group of Isl1/Nkx2.5-positive cells are located behind in the heart in the pre-pharyngeal mesoderm (asterisk in b). An elongated column of Nkx2.5- (b) and Isl1- (c) positive cells can be observed extending along the left side of the outflow tract (OFT) (indicated by the dotted line in b). Upon differentiation, cells increasingly express the myocardial marker MLC-2a (a). d–f: Three subsequent sections in VEGF120/120 embryos, stained for MLC-2a (d), Nkx2.5 (e), and Isl1 (f) are shown. In VEGF 120/120 embryos, the cluster of Isl1- (f) and Nkx2.5- (e) positive precursors (asterisk in e) shows an abnormal organisation. The column of cells extending on the left side of the OFT is only very short (compared length of the dotted lines in b and e). AoS, aortic sac; End, endoderm; LA, left atrium; G, foregut; 6th, left sixth pharyngeal arch artery. Bar = 100 μm.

DISCUSSION

OFT remodeling is an essential part of heart development. Normal separation and positioning of the great arteries of the OFT is necessary for the heart to sustain its function as a central pump coordinating the blood flow through the systemic and pulmonary circulations (Bartman and Hove 2005). Congenital OFT malformations may severely compromise normal physiology, resulting in hemodynamic overload, cyanosis, and early death (Bashore, 2007). Knowledge of the developmental processes resulting in proper OFT formation and remodeling, as well as the pivotal cells or cell groups involved, is essential to comprehend when and why malformations, resulting in congenital heart disease, occur. In recent years, the relevance of the contribution of cells derived from the second heart field has been attested (Cai et al., 2003; Snarr et al., 2008; Vincent and Buckingham 2010), although the exact mechanism of OFT remodeling, including the final positioning of the aorta and the pulmonary trunk and their orifices, is unclear.

It has been shown that the anterior part of the second heart field plays a crucial role in normal OFT development together with neural crest and epicardium (Kirby et al., 1985; Gittenberger-de Groot et al., 2000; Waldo et al., 2005). During cardiovascular development, cells from the three above-described populations are recruited to the linear heart tube via both the arterial and the venous pole from E9.5 onwards (Kelly and Buckingham, 2002; Perez-Pomares et al., 2003; Poelmann and Gittenberger-de Groot, 1999). It has been demonstrated previously that the myocardial part of the OFT, comprising both the subaortic and subpulmonary myocardium, is derived from the second heart field (Cai et al., 2003). This myocardium has been shown to have a common lineage relationship to the right- and left-sided head muscles, respectively (Lescroart et al., 2010).

We have, based on results in the VEGF120/120 model, previously postulated that the second heart field–derived subpulmonary myocardium may be highly sensitive for signaling of factors like VEGF and Notch, which may underlie the observed malpositioning of the OFT vessels in the VEGF120/120 mutants, but did not show a direct link with a contribution of the second heart field mesenchyme to the OFT in this study (Van Den Akker et al., 2007).

The pulmonary trunk originates from the posterior left side of the aortic sac, whereas the putative aorta is originally positioned on the right side (Gittenberger-de Groot et al., 2005; Waldo et al., 2005). After OFT development is completed, however, the pulmonary trunk has obtained a right anterior position. A dynamic process is required during cardiac development, resulting in a rotational movement of the pulmonary trunk and orifice around the aorta.

A mechanism of cell death, predominantly present in the subaortic myocardium resulting in a shortening of the subaortic OFT, has been proposed as the mechanism for proper formation of the ventriculo-arterial connections (Watanabe et al., 1998, 2001; Schaefer et al., 2004). Cell death is a well-known mechanism in heart development and has been described to occur at the level of the OFT (Poelmann and Gittenberger-de Groot, 2005; Vuillemin and Pexieder, 1989). However, a mechanism of asymmetric active shortening of the OFT by cell death alone is unlikely to explain the complex positioning of the OFT and does not take into account a concomitant asymmetrical gaining of length of the subpulmonary myocardium, while the subaortic myocardium remains relatively short from the onset (Bartelings and Gittenberger-de Groot, 1991).

It was previously reported that asymmetrical expression of Fgf8/10, Pitx2c, and possibly Tbx1 within the anterior heart field is involved in the induction of asymmetrical OFT growth (Nowotschin et al., 2006; Brown et al., 2004). Experimental knock-out of these genes resulted in the association with a large variety of fundamental defects in OFT development (Liu et al., 2002). Especially relevant seems to be the co-expression of Tbx1 with Pitx2, an important gene in right-left signaling, in the pharyngeal mesoderm of the OFT. Crossing of heterozygous Tbx1 and Pitx2 mice results in cardiac anomalies including double outlet right ventricle (Nowotschin et al., 2006). Experiments using right- and left-sided second heart field labeling experiments in the chick OFT indicate contributions of second heart field precursors to distinct lateralized regions of the OFT (Takahashi et al., 2012). It was unclear whether other known markers of myocardial precursors within the anterior heart field demonstrate a similar asymmetric expression pattern that could explain the above-described dynamics of OFT remodeling. We have now shown that it is also applicable to Nkx2.5 and Isl1 expression. It is remarkable that the Nkx2.5- and Isl1-positive area of the anterior heart field was the last part of the heart to be covered by a WT-1-expressing coelomic epithelium, i.e., the epicardium. On the aortic side, which is supposed to originate from the right-sided pre-pharyngeal mesoderm (current study; Bajolle et al., 2008; Takahashi et al., 2012), the disappearance of the Nkx2.5-positive population at day 9.5 was followed by the start of expression of WT-1 in the coelomic wall. A possible interaction between Nkx2.5 and WT1 needs further study.

There is support from our studies that lengthening of the subpulmonary myocardium of the OFT is active until day 12.5, which is at least one day longer than posed in the literature (Xu et al., 2004; Viragh and Challice 1973) but in line with studies in chick that have indicated contributions to the OFT up to HH24 (correlating with mouse day12.5) (Rana et al., 2007).

The aim of the current study was to assess whether the anterior heart field architecture could provide clues relevant for normal positioning of the aorta orifice and pulmonary trunk during mammalian heart development. The main finding of the present study, based on 3D-reconstructions of consecutive developmental stages, was that myocardial precursor cells were asymmetrically positioned in the OFT during development, as they were distinctively observed at the pulmonary side, whereas they were almost absent at the aortic side. The current study shows for the first time that continued addition from the Nkx2.5-expressing mesenchymal myocardial precursors below the left 6th pharyngeal arch artery may push the future pulmonary artery orifice in an anterior and rightward direction.

In addition, we have shown that this addition was disorganized and the extension towards the left side of the OFT shorter than in wildtype in the VEGF120/120 model, which has a phenotype in which OFT malformations are predominant (Van Den Akker et al., 2007). A distinct developmental origin of second heart field–derived cells contributing to the myocardium at the base of the pulmonary trunk versus the myocardium at the base of the aorta was recently indicated (Bajolle et al., 2008). Clonal studies showed a larger number of cell clones in the subpulmonary region, indicative for a higher proliferative rate, whereas in the subaortic region clones were very small (Bajolle et al., 2008). The subpulmonary myocardium remained in a posterior position in the Pitx2c knock-out mouse that presents with OFT malformations such as transposition of the great arteries and double outlet right ventricle (Bajolle et al., 2006). We postulate that the dominant left-sided expression of Pitx2c induces the expression of Nkx2.5 within the anterior heart field, which might be disturbed in Pitx2c knock-out mouse embryos. In our study of the VEGF120/120 embryo, we could discern the malpositioning of the Nkx2.5-positive mesenchymal cells at the pulmonary side of the OFT in line with results of Bajolle et al. (2006) that indicate a malpositioning of OFT myocardium in Pitx2c mutant hearts with double outlet right ventricle. The disorganization of the Nkx2.5-positive SHF mesenchyme in front of the pharynx is indicative of an abnormal differentiation of the SHF. The small area of Nkx2.5-positive myocardial precursors covering the pulmonary side of the OFT could underlie the observed tetralogy of Fallot and double outlet right ventricles in the VEGF120/120 hearts (Van Den Akker et al., 2007) in which subpulmonary myocardium is insufficiently added.

In transposition of the great arteries, the arterial orifices are positioned in almost one plane (Bartelings and Gittenberger-de Groot, 1991), supporting the fact that addition of myocardium to the right ventricular OFT has either not taken place or was deficient. Similar observations have been made for mouse hearts with double outlet right ventricle with a marked shortening of the right ventricular OFT (Bartram et al., 2001; Molin et al., 2004). At this time, specific patterns in the second heart field for Nkx2.5 and Isl1 have not been reported. Results in the VEGF120/120 model in the current study support the postulated mechanism of a deficient and disorganized addition of myocardial precursors to the pulmonary side of the OFT. An asymmetric contribution of anterior heart field–derived myocardium around and specifically below the left 6th pharyngeal arch artery during crucial stages of OFT development provides an explanation for the dynamic process that results in positioning of the pulmonary artery orifice in its normal left anterior location. It must be questioned whether a true rotational motion, based on spiraling outflow tract ridges, occurs. The latter explanation has dominated the positional remodeling for several decades, and an abnormal torsion was considered the underlying mechanism for OFT tract abnormalities as observed in transposition of the great arteries (Chuaqui and Bersch 1973; Lomonico et al., 1988; Thompson and Fitzharris 1979; Thompson et al., 1987). This explanation for rotation of the outlet, however, has also been the subject of controversy over the past decades, and several authors have indicated that no such rotation occurs during normal development (Steding and Seidl, 1980, 1981; De la Cruz and Da Rocha 1956; Gittenberger-de Groot et al., 2005). Given the second heart field contributions to OFT remodeling, new concepts for related abnormalities occur. Obviously, proper positioning of the great arteries needs to occur in order to achieve the normal morphological relation of the aorta and pulmonary trunk. Results of our study support the occurrence of rotation. We propose that, rather than a mechanism based on spiraling of the endocardial OFT cushions, an asymmetrical lengthening of the right ventricular OFT causes the pulmonary trunk to be pushed towards its definitive position in front of the aorta, which is observed as a rotation.

Conclusions

We conclude that the addition of Nkx2.5-positive myocardial precursors from the anterior heart field occurs during normal development predominantly below the left branch of the 6th pharyngeal arch artery. We postulate that this results in a movement without spiralization of the OFT in which, due to the continued addition of right-sided myocardium, the pulmonary trunk orifice is pushed in a rightward and anterior direction, which presents a new explanation for the rotation of the pulmonary orifice and trunk. This mechanism is referred to as pulmonary push, which results in a rotation of the pulmonary orifice to an anterior position. As the term rotation has been intrinsically used in many ways and for many OFT structures, we prefer the use of “pulmonary push” to describe the mechanism of OFT positioning. Deficient or disorganized positioning of the Nkx2.5-positive precursors, as was observed in the VEGF120/120 mutant, might explain cardiac malformations with side-by-side position of the great arteries.

EXPERIMENTAL PROCEDURES

Embryonic Material and Immunohistochemical Procedures

The handling of all animals and embryos was according to the Guide for Care and Use of Laboratory Animals, as published by the NIH. Wildtype mouse embryos were obtained from the CLB-Swiss strain. In addition to studies of wildtype embryos and to test the hypothesis of the pulmonary push concept, VEGF+/120 mice were crossed to obtain VEGF120/120 embryos and VEGF+/ + wild type littermates. The day the vaginal plug was detected was designated embryonic day (E) 0.5. Pregnant female mice were sacrificed on consecutive days from E9.5 onward up until E14.5 for wildtype mice and, per day, three embryos were harvested for the study. For VEGF120/120 mice, stage E10.5 was studied. For further immunohistochemical staining, all embryos were embedded in paraffin after fixation in 4% paraformaldehyde in phosphate buffered saline (0.1 M, pH 7.2) and subsequent dehydration. Embryos were sectioned transversely to the body axis (5 μm). Due to the position of the heart in the thorax, the level of the outflow tract was therefore positioned in a more frontal plane. Sections were serially mounted on glass slides. Immunohistochemical staining was performed with antibodies against the myocardial marker MLC-2a (1/6,000, kindly provided by S.W. Kubalak, Charleston, SC); Nkx 2.5 (1/4,000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, SC-8697), expressed in atrial and ventricular and outflow tract myocardium, as well as in the mesenchyme of the second heart field; the second heart field marker Islet 1 (Isl1) (1/400, mouse monoclonal antibody, clone 39.4D5, Developmental Studies Hybridoma Bank, Iowa City, IA) and Wilms Tumor 1 (WT-1) (1/1000, Santa Cruz Biotechnology, Inc., sc-192), to show the coelomic wall covering and the pro-epicardial organ-derived epicardium. The slides were first incubated for 45 min using ABC-reagent (Vector Laboratories, Burlingame, CA, PK 6100), and then with 400 μg/ml 3-3′di-aminobenzidin tetrahydrochloride (DAB, Sigma-Aldrich, St. Louis, MO, D5637) dissolved in trismaleate buffer pH 7.6 to which 20 μl H2O2 was added. The latter incubation was done 5 min for MLC-2a and 10 min for Nkx 2.5, Isl1, and WT-1. Furthermore, counterstaining was done using 0.1% hematoxylin (Merck, Darmstadt, Germany) for 5 sec, and the slides were subsequently rinsed with tap water for 10 min. Finally, slides were dehydrated and mounted with Entellan (Merck).

Three-Dimensional Reconstruction

To describe the dynamics of OFT development within a spatial context, three-dimensional reconstructions were made from E9.5 to ED14.5 embryos. Micrographs of serial sections were processed using the AMIRA software package (Template Graphics Software, San Diego, CA) as described previously (Jongbloed et al., 2004). First, the myocardium was reconstructed using the expression pattern of MLC-2a, after which the expression of Isl1, WT-1, and Nkx2.5 was superimposed to depict myocardial progenitors within the anterior heart field. Isl-1 expression was used to demarcate the anterior heart field, Nkx2.5 was used to identify precursors of OFT myocardium (Waldo et al., 2001; Xu et al., 2004), and WT-1 to show which parts of the outflow tract were covered with epicardium.

Acknowledgements

We thank Ron Slagter for designing the animation of outflow tract remodelling. Roderick W.C. Scherptong was supported by an unrestricted educational grant of Actelion Pharmaceuticals Nederland BV (Woerden, The Netherlands).

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