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Three-dimensional and molecular analysis of the developing human heart
Sizarov, A.
Publication date
2011
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Citation for published version (APA):
Sizarov, A. (2011). Three-dimensional and molecular analysis of the developing human heart.
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chapter 5
Three-Dimensional and Molecular Analysis
of the Arterial Pole of the
Developing Human Heart
Aleksander Sizarov
Wouter H Lamers,
Timothy J Mohun,
Nigel A. Brown,
Robert H Anderson,
Antoon FM Moorman
Abstract
Background – Labeling experiments in chicken and mouse embryos revealed important roles for different
cell lineages in the development of the arterial pole of the heart. These data can only fully be exploited when integrated into the continuously changing morphological context, and compared with the patterns of gene expression in the human embryo. As yet, studies on the formation of separate ventricular outlets and arterial trunks in human embryos are exclusively based on histologically stained sections.
Methods and Results – We performed immunohistochemical analyses of serially sectioned human embryos,
along with three-dimensional reconstructions. Development of the arterial pole of the heart involves several parallel and independent processes of the formation and fusion of endocardial ridges, remodeling of the aortic sac, and closure of an initial aorto-pulmonary foramen through formation of the aortico-pulmonary septum. Expression patterns of the transcription factors ISL1, SOX9, and AP2α show that, in addition to fusion of the SOX9-positive endocardial ridges, intra-pericardial protrusion of the mesenchyme derived from the neural crest contributes to the separation of the developing ascending aorta from the pulmonary trunk. The non-adjacent walls of the intra-pericardial arterial trunks are formed through addition of ISL1-positive cells to the distal outflow tract, while the facing parts of the walls form from the protruding mesenchyme.
Conclusions – The morphogenetic steps, along with the gene expression patterns reported in this study are
comparable to those reported for the mouse, and confirm the involvement of mesenchymal tissues derived from endocardium, mesoderm and migrating neural crest cells in the septation process of the outflow tract.
Introduction
Malformations of the arterial pole of the heart, encompassing the ventricular outlets and arterial trunks, constitute almost one third of all cardiac malformations and are oftenly incompatible with life.1,2 The malformations are markedly diverse, combining abnormalities in ventriculo-arterial connections and septation, along with valvar and vascular defects.3 This strongly suggests an intimate association of separate morphogenetic and molecular pathways in the formation of the outflow tract. Existing descriptions of the developing outflow tract, however, are ambiguous,4 making the existing literature quite inaccessible, even for the morphological expert.
Studies of the shaping and septation of the outflow tract in several experimental animals have revealed an important role of the mesenchymal tissues derived from neural crest5 and second heart field,6,7 albeit thus far with limited integration of the molecular and lineage analyses into the complex three-dimensional (3D) context. Studies of the human outflow tract, however, have been based only on histologically stained sections, which have resulted in conflicting interpretations. Thus, the role of the so-called aortico-pulmonary septum in the septation of the distal outflow tract and the development of the intra-pericardial arterial trunks has remained controversial.8,9 So as to gain insight into the intricate development of the distal component of the human outflow tract, therefore, we performed an integrated molecular and morphological study on the tissues involved in the separation of the systemic and pulmonary arterial channels. The morphogenetic steps, along with the gene expression patterns reported in this study are comparable to those reported for mouse, lending support to extrapolations of the mechanisms governing development of the arterial pole derived from experimental studies, to the human situation.
Material and Methods
Human Embryos
Collection of human embryonic material and preparation for histological studies were described previously.10 In total, we used seven Carnegie stage 12-13 embryos, five stage 14-15 embryos, five stage 16 embryos, four stage 18 embryos, and 3 stage 21-23 embryos. We included only embryos considered normal. Since blood was not removed from the embryos, immunohistochemical stainings with some fluorochromes resulted in strong erythrocyte autofluorescence. Collection and use of the human embryonic material for research were approved by the Medical Ethical Committees of the Universities of Tartu, Estonia, and
Amsterdam, the Netherlands. In addition, the high resolution episcopic micropscopy (HREM) technique11 was applied to 13 embryos at stages 13-17. These embryos, which were judged as unsuitable for gene expression studies, were kindly provided by the MRC–Wellcome Trust Human Developmental Biology Resource, managed by the Institute for Human Genetics of Newcastle University, United Kingdom.
Immunohistochemistry and Three-Dimensional Reconstruction
Immunofluorescent staining and 3D reconstructions, as well as the preparation of the EFIC models, were performed as previously described.10,11 Detailed description of the protocols and antibodies is given in the Data Supplement.
Limitations of Our Study
Assessment of cellular lineage should ideally be performed by means of genetic or physical labeling of cells, clearly impossible in human embryos. The second best alternative, albeit with limitations, is to study patterns of gene expression. Particularly, neural crest-derived cells expressing the DNA-binding activator protein AP2α12,13 could only be detected during their migration. The non-standardized fixation, and limited numbers of available embryos, did not permit complete optimization of the staining protocol for some antibodies, and prevented assessment of biological variation between specimens at similar developmental stages. Despite these potential problems, immunohistochemical stainings proved to be reproducible. While the immunohistochemically stained serial sections allow 3D molecular analysis of the structures, reconstructions have to be corrected for surface distortions due to uneven stretching and occasional damage of the sections. By comparing our reconstructions with images from the HREM datasets, nonetheless, we confirmed that no spurious structures had been introduced, nor important details lost, during the corrections. The use of HREM technology allows exquisite assessment of the internal morphology in almost any desired section plane, but, in the human embryo, does not readily permit the distinction between tissue types other than by apparent morphological differences.
Results
We describe the outflow tract as the outlet portion of the developing heart within the coelomic, or pericardial, cavity. This outlet portion is a highly dynamic structure, with cells continuously added distally from the pharyngeal mesoderm,7,14 and with its proximal part concomitantly differentiating into right ventricular myocardium.15-17 From the stance of lineage, in none of the stages studied does the outflow component consists of the same cells. To facilitate the understanding of the complex morphogenetic changes encountered, we encourage the reader to examine the results along with the interactive 3D-PDF (accessible at http://3d.hfrc.nl).
Stage 12-13 – the Tubular Outflow Tract and Aortic Sac
In the looped and chamber-forming heart of the stage 12-13 embryo, which corresponds to 26-30 days of development, we distinguish a venous sinus, left and right atrial pouches, an atrioventricular canal, left and right ventricles and a tubular outflow tract (Figure 1). At this stage, the outflow tract is entirely myocardial and forms a serpentine-like connection between an inconspicuous dorso-caudally located right ventricle and the aortic sac, a manifold embedded in the pharyngeal mesenchyme and giving rise to three pairs of symmetric aortic arch arteries (Figure 1B,C,E). The myocardial wall of the outflow tract is contiguous with the epithelial lining of the coelomic cavity (Figure 1C,E). The lumen of the outflow tract is bordered by endocardium expressing connexin40 (Figure 1F,G). Between the endocardium and myocardium, the cardiac jelly contains sparse mesenchymal cells expressing the transcription factor SOX9 (Figure 1I), indicating their endocardial origin through endothelial-mesenchymal transformation.18 There is no indication of septation (Figure 1E,F,G), with the lumen of the intra-pericardial outflow tract continuing extra-pericardially as the aortic sac and aortic arches (Figure 1B,C,E). From the proximal part of the bilateral sixth aortic arches, the lumina of the pulmonary arteries originate and run caudally (Figure 1C). ISL1, marking the pharyngeal mesoderm,14 is expressed in the myocardial wall of the outflow tract, and in mesenchymal cells invading the cardiac jelly (Figure 1H). This mesenchymal tissue is the first indication of the formation of non-myocardial columns, which become more clearly distinguishable in the next stages. The expression of AP2α, a transcription factor expressed in migrating cardiac neural crest cells,12,13 indicates already the presence of these cells within the outflow tract mesenchyme (Figure 1J).
Stage 14-15 – Septation of the Proximal Outflow Tract and Remodeling of the Aortic Sac
At stage 14-15, which corresponds to 31-36 days of development, the outflow tract has become relatively shorter, concomitant with the ventricularization of its proximal part15-17 (Figure 2A). The cells making up the myocardial wall of the outflow tract, nonetheless, continue to express ISL1 (Figure 2H). The mesenchyme within the proximal outflow tract has now become more densely populated, forming a pair of endocardial ridges (asterisks in Figure 2F). These endocardial ridges approximate each other distally, dividing the lumen into prospective aortic and pulmonary channels, which connect the right ventricular cavity and the primary, or interventricular, foramen with the distal outflow tract in a slightly spiral fashion (dotted lines in Figure 2E). The distal part of the outflow tract remains undivided at this stage, and resembles the tubular outflow tract in the stage 13 heart (Figure 2E,G). Its distal myocardial wall is still contiguous with the epithelial lining of the coelomic cavity. The myocardial component shows virtual lack of proliferation (Figure 3A*), as previously reported for the rat.19 The lumen of the distal outflow tract now continues extra-pericardially as a longitudinal arterial channel (Figure 2C,E), which is about 4 times narrower than the original aortic sac. Similar to previous reports,20,21 we consider this
Figure 1. 3D and molecular analysis of the cardiac outflow tract at stage 13. The outflow tract (OFT) is entirely
myocardial (panels A,E) and connects the right ventricle (RV) with the aortic sac (as) (panel B). Panels E,F and G show the tubular nature of the outflow tract at this stage. Note that the lumen of the outflow tract is lined by endocardium expressing connexin40 (Cx40). Panel D shows the contour of the stage 13 embryo, the heart of which was reconstructed. The contour of this heart (red and blue) is superimposed to the figure. It clearly illustrates the ambiguity of terms dorsal/ventral and cranial/caudal in describing the morphogenesis of the pharyngeal region. Note, that the third (3rd), fourth (4th) and sixth (6th) aortic arches originate directly from the aortic sac. The dotted line in panel C refers to the distance, by which the origins of the tiny right and left pulmonary arteries are separated from each other at this stage. Panels G through J show sections through the distal outflow tract, which were incubated with antibodies as indicated. The asterisk in G and H points to the appearance of the mesenchymal columns within the distal outflow tract (see text). Scale bars (panels F-J), 200 m. Abbreviations: AVC, atrioventricular canal; LA/RA, left/right atrium; LSCV, left superior cardinal vein; LV, left ventricle.
Figure 2. 3D and molecular analysis of the cardiac outflow tract at stage 14-15. The asterisks in panels A and B point
to the intra-pericardial mesenchymal columns disrupting the myocardial wall of the distal outflow tract ventrally and dorsally. Panel C shows the appearance of the longitudinal arterial channel, the future ascending aorta (AAo) giving rise to the third (3rd) and fourth (4th) aortic arches. Note the separate origin of the sixth aortic arches (6th) from the unseptated distal outflow tract (asterisk in panel E) as the result of the remodeling of the aortic sac, which is no longer recognizable as a distinct entity at this stage. Panel D shows the contour of stage 15 embryo, the heart of which was reconstructed. The arrow in panels A and E point to the characteristic bend of the myocardial outflow tract dividing it into proximal and distal parts. Dotted lines in panel E refer to the spirally oriented channels in the proximal outflow tract connecting unseptated distal outflow tract (asterisk) with the right ventricle (white line) and primary foramen (green line). The dotted lines correspond to the channels referred by white and green arrows in panel F. Note, that at this stage endocardial ridges are recognizable only in the proximal part of the outflow tract (yellow asterisks in panel F), while the distal part resembles the tubular outflow tract of the previous stage (panel G). The dotted line in panel C points to the decreasing distance between the origins of the left and right pulmonary artery branches with the proximal parts of 6th aortic arches being still interposed between them. Panels G through J show sections of two different embryos, which were incubated with antibodies as indicated. The star in panels E and in G through J points to the mesenchyme located between the 4th and 6th and between the right and left aortic arches, while asterisk points to the ventral ISL1-positive mesenchymal column (see text). Scale bars (panels F-J), 200 μm. Abbreviations: pv, pulmonary vein; tr, trachea; for other abbreviations see Figure 1.
arterial channel as the future extra-pericardial ascending aorta. The sixth aortic arches, giving rise to the right and left branches of the pulmonary arteries, originate from the caudal aspect of the junction between distal outflow tract and aortic sac, while the fourth and third arches arise from the developing ascending aorta more cranially (Figure 2C,E). At this stage, however, both the forming distal aortic and pulmonary channels originate from the unseptated distal part of the outflow tract (asterisk in Figure 2E), which can therefore be considered as an aortopulmonary foramen.
The mesenchyme of the pharyngeal region bordering the distal outflow tract is molecularly diverse. Columns of ISL1-positive and SOX9-negative mesenchymal tissue are recognizable at the ventral and dorsal aspects of the myocardial outflow tract (Figure 2A,B,G-I). These columns are in direct continuity with the ISL1-positive pharyngeal mesenchyme and interdigitate with the endocardium-derived mesenchyme expressing SOX9 (Figure 2I). The columns are devoid of AP2α, do not express α-smooth muscle actin (αSMA), and show virtually no proliferation, while the endocardially-derived mesenchyme is αSMA- and Ki67-positive (Figures 2J and 3A,A*). More proximally, the ends of these ISL1-positive mesenchymal columns
Figure 3. Expression of αSMA and proliferation marker Ki67 in the developing distal outflow tract. Sister sections are
incubated with antibodies as indicated. Note that the myocardial outflow tract remains devoid of proliferation. The asterisks in panels A and A* point to the Ki67- and αSMA-negative columns of pharyngeal mesenchyme invading the distal outflow tract. Note, that at stage 16 these columns are no longer recognizable, while other moderately proliferating non-myocardial tissue (# in panels B and B*) protrudes between the developing ascending aorta (AAo) and pulmonary trunk (PT) and expresses strongly αSMA. The endocardial ridges are also moderately αSMA-positive (asterisks in B and B*). At stage 18 (panels C and C*) two perpendicularly oriented arterial trunks are discernable with separate wall expressing αSMA and Ki67. The pulmonary trunk continues as a broad arterial duct (duct). Mesenchymal tissue is present in between the walls of the arterial trunks, which does not express αSMA (# in panel C). Scale bars, 200 μm. Abbreviations: lb, lung bud; for other abbreviations see previous Figures.
form the future intercalated ridges (not shown). Dorsally, the unseptated distal outflow tract is bordered by the ISL1-positive pharyngeal mesenchyme interposed between the origins of the symmetric sixth and fourth pairs of aortic arches (star in Figures 2E,G and 4A). We consider this tissue, which on sagittal cuts slightly protrudes into the distal outflow tract, to be the first sign of aortopulmonary septation. The AP2 -positive cells are seen along the sixth aortic arches
Figure 4. 3D morphology of the distal outflow tract as assessed by the high-resolution episcopic microscopy. Panels
A through A** show different views of the unseptated distal outflow tract at stage 14-15 with the dorsal wall between fourth (4th) and sixth (6th) aortic arches slightly protruding intrapericardially (star), leading to appearance of the future extrapericardial ascending aorta (AAo). Panels B through B** show different views through the septated distal outflow tract at stage 16. Note, that progressive protrusion of the dorsal wall of the outflow tract intrapericardially leads to formation of the so-called aortico-pulmonary septum (#) separating devloping ascending aorta from the future pulmonary trunk (PT). Yellow asterisks point to the endocardial ridges. See text for further description.
and ventral to the ISL1-positive protruding mesenchyme (Figure 2J), suggesting the migration route of the neural crest-derived cells.5 As AP2α is no longer expressed in the non-migrating neural crest-derived cells, these cells are not seen in the SOX9-positive endocardial ridges of the proximal outflow tract (not shown).
Stage 16 – Septation of the Distal Outflow Tract
At stage 16, thus 4-6 days later, the myocardial part of the outflow tract no longer expresses ISL1 (Figure 5H), and has shortened further, whereas the mass of the right ventricle has become visible ventrally (Figure 5A). The columns of ISL1-positive mesenchyme, clearly discernable ventrally and dorsally at earlier stages, are no longer identifiable. Instead, the right and left aspects of the distal outflow tract wall are no longer myocardial, and express ISL1 and αSMA (Figures 3B and 5G,H). The endocardial ridges, expressing SOX9 and being negative for AP2α, are now clearly seen also in the distal part of the outflow tract (Figure 5G-J). The approximation of the ridges to each other divides the lumen of the distal outflow tract into the future intra-pericardial ascending aorta and pulmonary trunk, the spiral orientation of which (dotted lines in Figure 5E) already reflects the topographic relationships of the intra-pericardial arterial trunks as seen in the formed heart.9 The aortopulmonary foramen is closed through progressive intrapericardial protrusion of the pharyngeal mesenchyme, which in previous stages bordered dorsally the ldistal outflow tract (Figure 4B,B*). This protruding mesenchyme, which forms an aortico-pulmonary septum,20-22 is ISL1- and SOX9-negative, and expresses AP2α (Figure 5H-J), suggesting that migration of neural crest-derived cells ventral to the ISL1-positive pharyngeal tissues constitutes the mechanism driving the progressive protrusion. Intrapericardially, the septum acquires an oblique plane, which accommodates the almost perpendicular relationship between the aortic and pulmonary channels. As result, the future pulmonary trunk locates to the left of the midline and the developing ascending aorta to the right. The pulmonary trunk continues extrapericardially as the pair of the sixth aortic arches, from which the left and right pulmonary arteries arise (Figure 5B,C,E). The third and fourth aortic arches originate symmetrically from the extrapericardial portion of the ascending aorta. In the middle and distal portion of the outflow tract, small intercalated ridges, mesenchyme of which is now ISL1-negative and expresses SOX9, together with other two outflow tract ridges, indicate the formation of the arterial valvar primordia.23 The non-myocardial wall of the distal outflow tract bordering laterally the developing intra-pericardial arterial trunks are ISL1- and αSMA-positive, but do not express AP2α (Figures 3B* and 5H,J). AP2α, however, is expressed in the mesenchyme surrounding the aortic arch arteries, supporting the notion of a distinct developmental origin of the non-facing walls of intra-pericardial arterial trunks and the arterial channels arising from them.24
Figure 5. 3D and molecular analysis of the cardiac outflow tract at stage 16. Note the shortening of the muscular outflow
tract and the mass of the right ventricle (RV) recognizable ventrally (Panel A). Panels B and C show the extra-pericardial portions of the perpendicularly oriented ascending aorta (AAo) and pulmonary trunk (PT), which are separated from each other by the mesenchymal tissue of the aortico-pulmonary septum (#), shown in cross-sections in panels G-J. Panel E shows the relations between developing ascending aorta and pulmonary trunk (white and green dotted lines, respectively resembling already the situation seen in the formed heart, albeit yet largely locating within the myocardial outflow tract. The dotted lines in panel E correspond to the aortic (white arrow) and pulmonary channels (green arrow), shown in panels F and G. The dotted line in panel C points to the further decreasing distance between the origin of the pulmonary artery branches. Panel D shows the contour of the stage 16 embryo, the heart of which was reconstructed. Panels G through J show sections through the distal outflow tract of two different embryos, which were incubated with antibodies as indicated. Note the appearance of the molecularly distinct aortopulmonary septum (#) interposed between ascending aorta and pulmonary trunk. The contact between this septum with the outflow tract ridges (yellow asterisks) is marked by dashed line. The yellow arrows in panel J point to the sharp border of the AP2α expression domain. Scale bars (panels F-J), 200 μm. For abbreviations see previous Figures.
Stages 18-23 – Formation of the Intrapericardial Arterial Trunks
Another 5 to 7 days later, at stage 18, the cardiac outflow tract has acquired the configuration as seen in the formed heart (Figure 6A), with complete separation of the blood streams at the level of the outflow tract (Figures 4C,F and 6C-E), albeit with a still patent interventricular foramen. The intrapericardial outflow tract can now be divided into arterial and myocardial components. The distal myocardial part surrounds the developing arterial valves, while the proximal part forms the non-proliferating myocardial infundibula of both ventricles (Figures 3C* and 6A,E). The mesenchymal tissue between the proximal right and left ventricular outflow tracts (asterisk in Figure 6C,D) begins to myocardialize.25 The most distal intrapericardial portion of the outflow tract has now become non-myocardial and constitutes the arterial trunks, which spiral round one another, and have separate walls strongly expressing αSMA (Figure 3C). The mesenchymal wall of the pulmonary trunk, but not of the aorta, weakly expresses the cardiac transcription factor NKX2-5 (Figure 6F). In contrast to the ISL1-negative myocardial component of the outflow tract, the smooth muscular walls of the arterial trunks express ISL1, reflecting the ongoing addition of new cells to the arterial pole of the heart from the pharyngeal mesoderm, but now as smooth muscle cells.26 The loose mesenchyme between the intra-pericardial arterial trunks is contiguous with the mediastinal mesenchyme surrounding the developing trachea and esophagus. It is negative for ISL1, SOX9 and αSMA (Figures 6G,H,3C), but expresses AP2α, similar to the mesenchymal protrusion seen at stage 16 (Figures 5J,6J). The walls of the arterial trunks are largely negative for AP2α, although the facing walls of the intra-pericardial aorta and pulmonary trunk express weakly this factor (Figure 6J), suggesting their origin from the neural crest-derived mesenchyme of the protrusion. The developing ascending aorta only connects to the fourth pair of aortic arch arteries (Figure 6A-C). The pulmonary trunk connects to the sixth aortic arch arteries forming now the bifurcation of the pulmonary trunk. The right-sided arch artery distal to the origin of the right pulmonary artery has almost regressed, while the left-sided arch has enlarged, and is recognizable as the arterial duct (Figure 6B,C,E).
Discussion
Studies on the development of the arterial pole in the human heart20,21,23,27-31 have thus far remained necessarily descriptive, since no gene expression data has been available. Experimental studies have revealed some morphogenetic mechanisms, demonstrating the distinct cell populations that play an important role in the development of the outflow tract, but have often failed to place their findings into a proper morphological context. Moreover, the curved form of the pharyngeal region has rendered unambiguous descriptions of the developing cardiac outflow tract very difficult, often making the previous literature inaccessible even for
Figure 6. 3D and molecular analysis of the cardiac outflow tract at stage 18. Panel A shows the further shortened
myocardial outflow tract along with formation of the right ventricular infundibulum. The intra-pericardial ascending aorta (AAo) and pulmonary trunk (PT) are now surrounded by their own non-myocardial walls (panels A and C), which resembles the relations between the great arteries in the formed heart. The asterisks in panels C and D refers to the myocardiolizing mesenchymal septum between the left and right outflow tracts (LVOT/RVOT). Panels E through J show sections through the right ventricular outflow tract, developing pulmonary valve (PuV), pulmonary trunk and ascending aorta. Sections were incubated with antibodies as indicated. Note that the tissue (#) between great arteries is ISL1-negative and expresses AP2α, which is also expressed in the facing walls of the arterial trunks. Interestingly, we observed weak expression of the “cardiac” homeobox factor NKX2-5 in the wall of the pulmonary trunk, but not in the wall of the ascending aorta (panel F), reflecting the cardiogenic potential of these mesenchymal cells of the pulmonary truncal wall. The proximal parts of the sixth arches form now the bifurcation of the main pulmonary artery (dotted line in panel B). Note, that the left-sided sixth arch (6th) persists and becomes the arterial duct (duct), while the right-sided sixth arch distal to the origin of the right pulmonary artery regresses. In this embryo, both fourth aortic arches (4th) are of roughly equal size, while the third arches are no longer recognizable. Scale bars (panels D-I), 200 μm. Abbreviations: AoV, aortic valve; AVJ, atrioventricular junction; for other abbreviations see previous Figures.
experts, and has underscored many controversies.4 In this study, we present a 3D and molecular analysis of the dynamic changes that occur during the development of the outflow tract in the human heart. We show that, during stages 14 through 16, separation of the aortic and pulmonary channels occurs through fusion of the endocardial ridges with each other and additionally with a protrusion of the neural crest-derived pharyngeal mesenchyme bordering the distal outflow tract dorsally, after which the separate walls of the intra-pericardial arterial trunks are formed through addition of mesenchymal cells to the distal outflow tract. Our 3D analysis also reveals that, at stage 14-15, the aortic sac remodels due to formation of a dorsal protrusion bordering the distal outflow tract and leads to the formation of the future extra-pericardial ascending aorta. In the next few days, this so-called aortico-pulmonary septum occupies a more oblique plane reflected in the right-sided position by the ascending aorta and a left-sided dorsally oriented position by the pulmonary trunk. The contribution of three sources of mesenchyme, endocardium, pharyngeal mesoderm and neural crest, to the septation of the outflow tract, correlates well with the great variety of malformations involving the cardiac outflow tract.
Septation of the Cardiac Outflow Tract and Establishment of the Correct Ventriculo-Arterial Connections
Subsequent to the initiation of chamber formation, differentiating cells from the ISL1-positive cardiac progenitor pool continue to be added to the arterial and venous poles of the heart.7,14 At the arterial pole, this contribution results in the formation of a relatively long, serpentine-like outflow tract, which initially is a tubular structure connecting the developing right ventricle with the aortic sac feeding the pharyngeal arch arteries. Over a short period, encompassing about 3 weeks of human development, the tubular outflow tract becomes converted into two separate, spirally aligned, channels with myocardial and arterial components. At the same time, the bulk of the mesenchymal ridges within the outflow tract is transformed into the myocardial free-standing subpulmonary infundibulum and the paired arterial valves.8,9 One of the most controversial issues regarding the development of the outflow tract remains the mechanism of division of the single lumen of the distal outflow tract into the left and right ventricular outlets. On the basis of distinct clustering of mesenchymal cells, and their relation to the lumen and myocardial walls of the outflow tract, a leading role in septation was assigned either to a so-called aortopulmonary septal complex, exclusively to the endocardial ridges, or to a combination of these structures.9 Our 3D analysis, facilitated by molecular stainings, reveals several morphogenetic processes occurring at consecutive developmental stages, albeit partly overlapping, which have received relatively little attention in these previous reports.
The formation and fusion of the of spirally oriented endocardial ridges28 separates the lumen of the outflow tract into the aortic and pulmonary channels. As early as stage 16, the
orientation of the lumens of the developing left and right ventricular outlets resembles already the definitive arrangement of the separate, and correctly aligned, left and right outflow tracts. Our immuno-histochemical analyses reveal substantial molecular diversity in the mesenchymal tissues participating in the division of the developing outflow tract of the human heart. The endocardial ridges express transcription factor SOX9, which is crucial for formation of intracardiac mesenchyme.18 The mesenchyme of the endocardial ridges, which in the mouse and chicken become densely populated with neural crest-derived cells,32-34 is negative for ISL1, and moderately expresses αSMA, along with the proliferation marker Ki67. At stage 13, we observed expression of AP2α within the loose mesenchyme of the most distal part of the outflow tract and in the mesenchyme surrounding the pharyngeal pouches, which reflects the route of migration of the cardiac neural crest cells toward the cardiac outflow tract.33,35 The endocardial ridges proximally to the myocardial border of the outflow tract, however, do not express AP2α, supporting the notion that the neural crest-derived cells lose their AP2α expression as they lose their migratory phenotype.13 The strict complementarity of the expression domains of AP2α and ISL1 in the pharyngeal mesenchyme shows that the migrating neural crest-derived cells do not mix with the mesoderm of the second heart field (Figures 2H,J and 5H,J). At stage 14-15, when the distal outflow tract is still unseptated, two intra-pericardial columns of pharyngeal mesenchyme are clearly recognizable, invading the dorsal and ventral aspects of the distal outflow tract. These columns have a distinct molecular phenotype, expressing ISL1, but being devoid of SOX9, AP2α, αSMA and Ki67. In the next stage, when the distal outflow tract is septated, no remnants of these mesenchymal columns are recognizable, since most probably they have been transformed into intercalated ridges of the developing arterial valve primordia, and into the non-facing walls of the intra-pericardial ascending aorta and pulmonary trunk.
The migration of cells derived from the neural crest cranial to the sixth aortic arches forms a progressively protruding oblique plane, the so-called aortico-pulmonary septum,20-22 causing a rightward shift of the ascending aorta and a leftward shift of the pulmonary trunk and sixth arches (Figure 7).The intra-pericardial protrusion of this neural crest-derived septum and its fusion with the endocardium-derived distal outflow tract ridges closes the aortopulmonary foramen, which is the communication initially present in the distal outflow tract (Figures 4A,B and 5E,G). Such an interpretation is comparable to previous labeling studies concluding that initial septation of the distal outflow tract involves migration of peribranchial mesenchyme along the sixth aortic arches intrapericardially to form the aortico-pulmonary septum.36 A recent study of the Ripply3-deficient mice underscores the importance and sufficiency of such migration. These mice display complete absence of the third and fourth aortic arches, but have normally formed sixth arches, and separated intra-pericardial arterial trunks.37 The formation of the
valves of the arterial trunks and the fate of the myocardializing mesenchymal septum initially present between the left and right outflow tracts are not discussed here, as we have concentrated on the analysis of the separation of the distal outflow tract into the intra-pericardial ascending aorta and pulmonary trunk.
Formation of the Intrapericardial Arterial Trunks and Remodeling of the Aortic Sac
When initially formed, the outflow tract of the developing human heart possesses a single lumen, and has exclusively myocardial wall. The intra-pericardial outflow tracts of the formed heart, however, possess in addition to the myocardial ventricular outlets a smooth-muscular part, namely the intra-pericardial arterial trunks.9 As we have discussed, the process of septation in the distal outflow tract at stage 16 already results in the appearance of two channels oriented relative to each other in a fashion resembling the relationships between the intra-pericardial arterial trunks of the formed heart. At this stage, the intrapericardial outlets are surrounded by the developing arterial valves, and are located within the non-proliferating myocardial outflow tract, while the lateral aspects of the distal outflow tract wall bordering the developing ascending aorta and pulmonary trunk, possess already a non-myocardial phenotype. Two stages later the walls of the intrapericardial arterial trunks distal to the arterial valves, acquire a smooth-muscle phenotype. In the formed heart, only the subpulmonary outflow tract remains myocardial, while normally the subaortic outlet displays fibrous aorto-mitral continuity.9 Since the possibility of
Figure 7. Schematic representation of the septation of the distal outflow tract. Note that this is a highly simplified
two-dimensional view of the morphologically complex 3D structure. At stage 14-15 (panel A) the aortopulmonary foramen within the distal outflow tract, which give rise to the extra-pericardial sixth aortic arches and the future ascending aorta, is dorsally bordered by ISL1-positive mesenchyme. At both sides of this ISL1-positive mesenchyme neural crest-derived cells are migrating ventrally along and cranial to the sixth aortic arches. At stage 16 (panel B), spirally oriented endocardial ridges have been formed and fused also in the distal outflow tract dividing the ascending aorta and pulmonary trunk with progressive protrusion of the neural crest-derived mesenchyme resulting in the obliquely oriented aortico-pulmonary septum (#), which closes the aortopulmonary foramen. At stage 18 (panel C), the continuous addition of ISL1-positive cells at the arterial pole results in the formation of the intra-pericardial arterial trunks, the facing walls of which are formed by neural crest-derived mesenchyme. The star in panels A-C points to the ISL1-positive mesenchyme at the level of the sixth aortic arches, which remains interposed between them. Abbreviations: L/R, left and right.
trans-differentiation of existing cardiomyocytes into smooth muscular cells was experimentally excluded,34 and no evidence for removal by apoptosis could be found either,19 the fate of these myocardial cells remains to be assessed. The myocardium distal to the arterial valves is removed by apoptosis at later stages, as demonstrated in chicken.17 The continuous addition of ISL1-positive cells from the pharyngeal mesenchyme, now dubbed second heart filed, to the distal end of the septated outflow tract, which differentiate into smooth muscle cells, produces the walls of the intra-pericardial arterial trunks.26 In agreement, we observed ISL1 expression in the walls of the intra-pericardial arterial trunks in the stage 18 embryonic human heart, suggesting conservation of the mechanism of formation of the arterial walls through continuous addition of differentiating cells from the ISL1-positive pharyngeal mesenchyme. The facing walls of the arterial trunks, however, were also positive for AP2α, indicating a neural crest origin.35,38
The unseptated tubular outflow tract of the stage 12-13 embryonic heart continues within the pharyngeal mesenchyme as the aortic sac. Unlike a previous report,39 we were unable to find such a structure in human embryos older than stage 14. During stage 14-15 the initial sac is remodeled through growth of the surrounding pharyngeal mesenchyme, and is no longer recognizable as a morphological entity. This remodeling is the first sign of aortopulmonary septation promoting correct route of migration of the neural crest-derived intrapericardial mesenchyme cranial to the sixth aortic arches to close the aortopulmonary foramen, thus achieving separation of the intra-pericardial ascending aorta from the pulmonary trunk (Figure 7).
Acknowledgments
We are indebted to the personnel of the Gynaecology Department of the Tartu University Hospital, to Dr. M. Aunapuu and Prof. A. Arend from the Anatomy Institute of the University of Tartu, Estonia, for their help with the collection of the human embryos. We thank J.Hagoort for his continuous support and invaluable help with the preparation of the interactive 3D-PDF file. We thank drs. M.B. van den Hoff, D.J. Henderson and B. Chaudhry for stimulating scientific discussions and support.
Sources of Funding
This work was supported by the European Community’s Framework Programmes’ contracts LSHM-CT-2005-018630 ("HeartRepair") and Health-F2-2008-223040 (“CHeartED”). Collection of human embryonic material was supported by grant 7301 from the Estonian Science Foundation.
Disclosures
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Supplemental Methods
Immunofluorescence and Immunohistochemical Staining
Paraffin-embedded embryos were sectioned at 7 to 10 μm. Sections were mounted onto silane-coated slides, deparaffinized in xylene, rehydrated in graded ethanol series and washed in phosphate-buffered saline (PBS, pH 7.4).
Triple immunofluorescent staining was performed as described previously.1 Here we report the results of
staining with the following primary antibodies: goat polyclonal for connexin40, rabbit polyclonal for NKX2-5 (both from Santa Cruz Biotechnology; diluted 1:250), goat polyclonal for Islet-1 (1:250; Neuromics), rabbit polyclonal for SOX9 (1:250; Chemicon), rabbit polyclonal for Ki67 (1:500; Monosan), mouse monoclonal for α-smooth muscle actin (1:500; Sigma-Aldrich), and mouse monoclonal for AP2α (1:25; Developmental Studies Hybridoma Bank). Some combinations of the primary antibodies contained a mouse monoclonal antibody for troponin I (1:250; Chemicon) as myocardial marker. After washing in TNT (100 mM Tris, pH 7.4; 150 mM NaCl; 0.05% Tween-20, Sigma-Aldrich) sections were incubated at room temperature for 1.5-2 hr in the dark with a mix of fluorochrome-coupled secondary antibodies containing donkey-anti-goat Alexa568, chicken-anti-rabbit Alexa488 and donkey-anti-mouse Alexa680 (all diluted 1:250; Molecular Probes, Invitrogen).
Three-Dimensional Reconstructions
Three-dimensional reconstructions from the fluorescently stained serial sections were performed using Amira software (www.amiravis.com), essentially as described previously.1,2 Myocardium was labeled on basis of the
positivity for troponin I. The extent of the labels for other structures, which have not been specifically stained on the sections, was defined according to general embryology knowledge.3,4 Some structures (e.g, pharyngeal
mesenchyme, celomic wall, veins and arteries) were not labelled in their entirety for the sake of clarity.The 3D data from the Amira viewer were exported into Adobe Acrobat 9 Pro Extended (Adobe Systems Inc., www.adobe.com) to generate the file in interactive 3D portable document format.5
Preparation of the Models using High Resolution Episcopic Micropscopy
Human embryos obtained from the Human Developmental Biology Resource (www.hdbr.org) were dehydrated through graded methanol and infiltrated from 24 to 48 hours with JB-4 methacrylate embedding solution (Polysciences) supplemented with eosin (0.27g/100ml) and acridine orange (0.06g/100ml) essentially as described.6 The same mix was used for polymerisation using the manufacturer’s protocol, except that 50% more catalyst was employed. After 24-48 hours, blocks were baked for a minimum of 48 hours at 90ºC before storage at 4ºC. Blocks were sectioned at 2 to 3 μm thickness using a Leica SM2500 microtome and successive block face images captured using a Hamamatsu Orca HR CCD camera attached to Olympus MVX optics, with GFP excitation and emission filters. Sectioning and image capture were automated via Image-Pro 6 software (Media Cybernetics). Grey levels of raw data sets were adjusted to provide optimal tissue contrast using Photoshop CS3 (Adobe Systems Inc.). For 3D volume rendering, the heart region of each embryo was sub-sampled to a final dataset size of 250-300 Mb, inverted and rendered using OsiriX 3.8 software (www.osirix-viewer.com).
Supplemental References
1. Sizarov A, Anderson RH, Christoffels VM, Moorman AFM. Three-dimensional and molecular analysys of the venous pole of the developing human heart. Circulation. 2010;122:798-807.
2. Sizarov A, Ya J, de Boer BA, Christoffels VM, Lamers WH, Moorman AF. Development of the building plan of the human heart: morphogenesis, growth and differentiation. Circulation. 2011;123:1125-1135.
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5. de Boer BA, Soufan AT, Hagoort J, Mohun TJ, van den Hoff MJB, Hasman A, Voorbraak FJM, Moorman AFM, Ruijter JM. The interactive presentation of 3D information obtained from reconstructed datasets and 3D placement of single histological sections with the 3D portable document format. Development. 2011;138:159-167.
6. Weninger WJ, Mohun TJ. Three-dimensional analysis of molecular signals with episcopic imaging techniques. Methods Mol
