Summary
Accurate knowledge of normal cardiac development is essential for properly understanding the morphogenesis of congenital cardiac malformations that represent the most common congenital anomaly in newborns. The heart is the first organ to function during embryonic development and is fully formed at 8 weeks of gestation. Recent studies stemming from molecular genetics have allowed specification of the role of cellular precursors in the field of heart development. In this article we review the different steps of heart development, focusing on the processes of alignment and septation. We also show, as often as possible, the links between abnormalities of cardiac development and the main congenital heart defects. The development of animal models has permitted the unraveling of many mechanisms that potentially lead to cardiac malformations. A next step towards a better knowledge of cardiac development could be multiscale cardiac modelling.
Résumé
La connaissance du développement normal du cœur est essentielle pour la compréhension de la genèse des malformations cardiaques congénitales, lesquelles représentent l’anomalie congénitale la plus fréquente chez le nouveau-né. Le cœur est le premier organe à se former durant le développement de l’embryon et sa formation se termine vers la huitième semaine de grossesse. Les études récentes provenant de la génétique moléculaire ont permis de spécifier le rôle des précurseurs cellulaires dans le champ du développement cardiaque. Dans cet article, nous décrivons les différentes étapes du développement cardiaque en insistant sur les processus d’alignement et de septation. Nous montrons aussi souvent que possible les liens entre les anomalies du développement cardiaque et les principales malformations cardiaques congénitales. Le développement des modèles animaux a permis de révéler de nombreux mécanismes à l’origine des malformations cardiaques. La prochaine étape pour une meilleure compréhension du développement cardiaque pourrait être la modélisation cardiaque multi-niveaux.
Background
The first functioning organ in the embryo is the heart. It begins to beat from 2 weeks of gestation (WG) onwards (4 weeks of amenorrhea) and is fully formed at 8 WG. The development of the heart is highly conserved through evolution and follows the same general pattern in all vertebrates. Fusion of the primary heart tubes is followed by a rightward looping of the newly formed linear heart tube, differentiation of the chambers and valves, and development of the conduction system and coronary circulation.
Congenital heart defects represent the most common congenital anomaly in newborns, with a prevalence of 8–10 per 1000 births . Delineating the normal sequence of heart development is essential for understanding the morphogenesis of congenital cardiac malformations. However, studying cardiac embryology is no easy task because it involves intricate structures and functions that evolve in space and time, and are closely interrelated. Moreover, understanding the developing heart requires a three-dimensional conceptualization that remains very complex for a human mind. In this article, the major processes involved in all stages of normal heart development are reviewed. Particular focus is given to those processes essential to the correct alignment and septation of cardiac structures. This provides a narrative through which congenital heart defects may be investigated, as sequential disruption of normal development.
The beginnings: formation of the primitive heart tube (days 15–21)
The heart starts to form at the beginning of the third WG. By the end of the second WG (day 15), the embryo is a flat disc made of two cell layers: the epiblast and the hypoblast. The primitive streak, which establishes the longitudinal axis of the embryo, appears at the median and caudal parts of the embryonic disc. At day 16, the epiblastic cells migrate towards the primitive streak and invaginate (gastrulation), leading to the differentiation of the embryo into three layers: ectoderm, mesoderm and endoderm.
The heart derives from the anterior mesoderm. At this stage, the mesodermal cells are still precardiac cells. However, the different axes of the embryo are already predetermined, particularly the left-right axis. Mesodermal cells differentiate into cardiac cells in response to induction signals from the endoderm, such as bone morphogenetic protein . In the mesoderm, there are five transcription factors that are considered to be the primordial genes involved in cardiac development and these are highly conserved through the evolution of animal species: NKX2.5, Mef2, GATA, Tbx and Hand . This ancestral genetic network controls the fate of the cardiac cells, the expression of protein-coding genes and cardiac morphogenesis. These genes regulate themselves and control their expression . Precardiac cells are multipotent and differentiate into myocardial, endothelial and smooth muscle cells by a phenomenon called progressive lineage restriction . Myocardial cells thus differentiate into chamber-specific myocytes (atrial and ventricular) and conduction cells .
Mesodermal precardiac cells migrate towards the cephalic pole of the embryo to form the cardiogenic crescent or first heart field (FHF). With cephalic then lateral inflexion of the embryo, the crescent migrates anteriorly and its two parts fuse on the midline to form the primitive linear heart tube ( Fig. 1 ). This tube consists of an inner endothelial layer and an external myocardial layer, separated by cardiac jelly.
Tissue origins: the cardiac fields
The heart does not develop solely from cells of the primary linear heart tube. Very early in cardiac development, a second population of cardiac cells is present at the medial and ventral parts of the FHF . This group of cells, called the second heart field (SHF), migrates medially and into the pharyngeal regions when the primary heart tube forms. SHF cells express the transcription factor islet-1 and differentiate into cardiac myocytes, smooth muscle cells and endothelial cells .
After the loop, the SHF is located within the pharyngeal mesoderm, at the inner curvature, between the outflow and inflow tracts. The role of the SHF is of major importance for the development of the four-chamber heart: cells from the anterior part of the SHF (anterior heart field) contribute myocardial cells to the right ventricle and to the outflow tract (OT), and smooth muscle cells to the base of the aorta and the pulmonary artery ; cells from the posterior part of the SHF (dorsal mesocardium) contribute myocardial cells to the walls of the atria and to the atrial septum, and smooth muscle cells to the walls of the systemic and pulmonary veins . The FHF then serves as a scaffold for building most of the heart from the cells of the SHF and gives rise only to the left ventricle and to the most primitive part of the atria, including the two appendages.
Two extracardiac cellular populations also contribute cells to the heart and vessels: the cardiac neural crest provides cells for the OT and the great arteries through migration to these areas; and the epicardium arises from the proepicardial organ, which is located at the posterior part of the heart, near the venous pole – proepicardial cells give rise to the epicardium, which covers the surface of the heart, and invade the myocardium to form fibroblasts and smooth muscle cells for the coronary arteries.
Looping, convergence and wedging
There are three steps that are fundamental to a proper alignment of cardiac structures, which is itself mandatory for normal cardiac septation. These three steps are looping, convergence and wedging . The intricate link between these processes and the development of the internal structures of the heart is illustrated on Fig. 2 .
Cardiac looping
Cardiac looping is the first manifestation of right-left lateralization in the embryo . The primitive straight heart tube loops to the right at 23–24 days of intrauterine life (D-loop), folding to the right into a S-shape, after an initial displacement to the left of the caudal part of the heart, termed ‘jogging’ ( Fig. 3 ). This step is crucial for the further morphology of the heart because it brings the future cardiac chambers into their relative spatial positions. The current theory about how the cardiac looping occurs is that the cilia within the primary node (or Hensen’s node) rotate, creating an extracellular flow current that determines the rightward bend of the tube . The anomalies of cardiac looping affect the laterality of the heart. If there is complete reversal of the loop, the heart is in situs inversus totalis or a complete mirror-image. The reversal can be incomplete and random, leading to all types of unusual segmental arrangements, often associated with heterotaxy syndromes.
Convergence
The loop creates two limbs in parallel, an inflow (proximal) limb and an outflow (distal) limb, separated by the inner curvature . The process of convergence brings the two limbs together craniocaudally, permitting alignment of the OT with the ventricular, atrioventricular (AV) and atrial septa . Immediately after cardiac looping (early looping stage), the inlet segment (atria and AV canal) is located entirely above the future left ventricle and the outlet segment (the conotruncus or OT) is located entirely above the future right ventricle, leading to both double-inlet left ventricle and double-outlet right ventricle types of AV and ventriculoarterial connection. From this stage on, the heart continues to grow by addition of myocardial cells from the SHF, both at its arterial pole (anterior heart field) and at its venous pole (posterior heart field or dorsal mesocardium).
The atria and the ventricles develop and differentiate along the anteroposterior and right-left axis.
Four transitional zones can be described in the developing heart . The endocardial cushions of the AV canal and the OT constitute two transitional zones, delimiting, respectively, the inlet segment and the outlet segment of the heart. The zone of junction between these two segments is the inner curvature, which is the pivot around which the remodelling of the AV and ventriculoarterial junctions will take place, including convergence and wedging ( Fig. 4 ). The cushions contribute to septation and to the formation of the cardiac valves.
The sinus venosus contributes to atrial septation and to the atrial conduction pathways.
The primary fold joins together the inner and the outer curvature ( Fig. 4 ), at the site of the future primitive ventricular septum; it contributes to ventricular septation and to the formation of the AV node and ventricular conduction pathways. The primary fold is also the starting point of the establishment of the right AV connection, which is initially absent.
The right ventricle and the ventricular OT grow rapidly by addition of myocardial cells from the SHF. At the same time, the right AV connection develops, along with the muscular bands of the right ventricle. This series of ‘morphogenetic shifts’ leads to alignment in the same sagittal plane of the AV canal, the future atrial and ventricular septa, and the developing OT . This alignment, or convergence, is absolutely necessary to further normal septation. During this process, the inner curvature (further ventriculoinfundibular fold) deepens and the endocardial cushions in the AV region grow and fuse to form the AV septum.
Cardiac malformations resulting from a defect occurring at the convergence stage are often severe, as they concern both the ventricles and the AV valves. They can be caused either by a lack of ventricular growth or by an absent or anomalous development of the right AV junction. These anomalies result in a malalignment between the atrial and ventricular septa, resulting itself in various congenital heart defects, including double-inlet ventricle, tricuspid atresia and ventricular hypoplasia.
Wedging
The anterior heart field, part of the SHF, facilitates the elongation of the OT by addition of myocardial cells, in response to the migration of cardiac neural crest cells towards the OT . Elongation of the OT is necessary for proper alignment (convergence) and wedging.
During wedging, the myocardial wall of the OT undergoes a counterclockwise rotation, viewed from the ventricular side, so that the aortic valve rotates behind the pulmonary trunk, going down and to the left to settle between the two AV valves, establishing mitral-aortic continuity ( Fig. 2 ). At the same time, the conal septum develops by fusion and muscularization of the endocardial cushions of the OT and is taken along leftwards by the rotation of the developing aortic valve, to join the upper primitive ventricular septum at the level of the upper division (the ‘Y’) of the septal band or septomarginal trabeculation, itself derived from the primary fold . The left part of the ventriculoinfundibular fold (‘subaortic conus’) then disappears, corresponding to the so-called ‘absorption of the subaortic conus’, establishing the mitroaortic fibrous continuity.
A malalignment between the OT and the ventricles inevitably results in a failure of fusion of the outlet septum with the primitive ventricular septum and in a ventricular septal defect (VSD) located between the two limbs of the Y of the septal band. This type of VSD is common to all so-called ‘conotruncal’ (or neural crest) defects. Tetralogy of Fallot can then be considered as a failure within the last step of cardiac looping – wedging – leading to a malalignment between the OT and the ventricles . In other words, tetralogy of Fallot may result from an arrest of rotation of the OT at the base of the great arteries .
Failure of myocardialization, leading to incomplete or abnormal convergence and wedging, is a major cause of many congenital heart defects, especially double-outlet right ventricle . Anomalies of both convergence and wedging produce malalignment of both inlet and outlet segments, while anomalies of wedging produce malalignment of the OT only.
The venous pole: atrial septation and development of the pulmonary veins
The venous pole consists of two parts: the sinus venosus and the primitive atrium, separated by the sinoatrial fold. The sinus venosus connects to the right atrium because of the asymmetric growth of the right part of the primitive atrium . Ultimately, the right atrium has two parts: the trabeculated part (right atrial appendage); and the sinus venosus, with its two valves (right [Eustachian and Thebesian valves] and left [atrial septum]). The sinus venosus receives the caval veins and the coronary sinus.
Development of the pulmonary veins
The common pulmonary vein takes its origin within the dorsal mesocardium (itself part of the posterior SHF), in the form of a mediopharyngeal cellular strand. In the beginning, the common pulmonary vein is connected to the sinus venosus, itself separated from the primitive atrium (the atrial appendages) by the sinoatrial fold . At this stage, connections between the pulmonary venous plexus and the vitelline and cardinal veins persist. Progressively, the common pulmonary vein incorporates within the left part of the primitive atrium, being pushed to the left by the growth of the vestibular spine, a structure also derived from the posterior part of the SHF. The incorporation of the pulmonary vein into the left atrium contributes to its identity: the left atrial wall consists of an inner vascular part, derived from pulmonary venous tissue, and an outer myocardial part . If there is a defect of incorporation of the common pulmonary vein into the left atrium, the primitive connections persist, leading to the various types of abnormal pulmonary venous return: either with the derivatives of the cardinal veins (right [innominate vein, superior vena cava, azygos vein]; left [coronary sinus]); or with the derivatives of the umbilicovitelline veins (portal vein, ductus venosus; inferior vena cava).
Direct drainage of all or part of the pulmonary veins within the morphologically right atrium is observed in heterotaxy syndromes; its mechanism is still unclear – possibly malposition of the septum primum or defect of the vestibular spine?
Atrial septation
At the beginning of the fifth week of intrauterine life, the septum primum (or primitive atrial septum) develops from the roof of the common atrium. Its inferior part is crescent-shaped with two extremities, anterior and posterior. At the posterior part of the common atrium, immediately underneath the septum primum and above the AV endocardial cushions, appears the vestibular spine (dorsal mesenchymatous protrusion, dorsal mesocardium), which derives from the posterior SHF and expresses Isl1 ( Fig. 5 A) . The inferior free edge of the septum primum is covered by a mesenchymal cap, which is considered as the anteroposterior extension of the vestibular spine . At its anterior extremity, the mesenchymal cap is continuous with the anterosuperior AV endocardial cushion. The space between these three mesenchymatous structures constitutes the primitive interatrial foramen or ostium primum.
