Development of the human heart

Abstract In 2014, an extensive review discussing the major steps of cardiac development focusing on growth, formation of primary and chamber myocardium and the development of the cardiac electrical system, was published. Molecular genetic lineage analyses have since furthered our insight in the developmental origin of the various component parts of the heart, which currently can be unambiguously identified by their unique molecular phenotype. Moreover, genetic, molecular and cell biological analyses have driven insights into the mechanisms underlying the development of the different cardiac components. Here, we build on our previous review and provide an insight into the molecular mechanistic revelations that have forwarded the field of cardiac development. Despite the enormous advances in our knowledge over the last decade, the development of congenital cardiac malformations remains poorly understood. The challenge for the next decade will be to evaluate the different developmental processes using newly developed molecular genetic techniques to further unveil the gene regulatory networks operational during normal and abnormal cardiac development.

ing on growth, formation of primary and chamber myocardium and the development of the cardiac electrical system. Here, we will build on our previous review and on the latest molecular and cell biological studies that have channeled our insights. In addition, we will address post-natal cardiogenesis, since it has become evident that cardiac development is not complete at birth and that, contrary to traditional views, the adult heart can no longer be considered a post-mitotic organ.
In the healthy normal post-natal heart, oxygen-rich blood enters the left atrium, is propagated to the left ventricle and then pumped via the aorta into the systemic circulation. The oxygen-deprived blood, returning from the body, enters the right atrium and is propelled by the right ventricle via the pulmonary trunk toward the lungs. The cardiac conduction system orchestrates the efficient contraction-relaxation cycle of the atria and ventricles. The electrical impulse resulting in cardiac contraction is triggered in the sinus node, which is located at the entrance of the superior caval vein into the right atrium. The electrical impulse spreads through both atria, but cannot directly activate the ventricles due to the electrical isolation of the atria from the ventricles by the annulus fibrosus (also called insulating plane or fibrous continuity). The electrical impulse is delayed in the atrioventricular (AV) node, and then quickly propagated through the His-bundle (AV-bundle), which penetrates the insulating annulus fibrosis plane, via the bundle branches and the peripheral conduction system (the Purkinje fibers) to the cardiomyocytes. The Among the first cardiac-specific genes expressed are the transcription factors Islet1 and Nkx2.5. The area expressing these transcription factors is referred to as the heart-forming region (also cardiac crescent) and has a horseshoe-like shape. BMP inhibitors secreted by the neural tube regulate the medial expansion and FGF growth factors expressed by the endoderm, determine the posterior border of the of the heart forming region (Harvey, 2002;Moorman, Christoffels, Anderson, & van den Hoff, 2007;Sizarov et al., 2011;van den Hoff, Kruithof, & Moorman, 2004). At this stage differentiation proceeds quickly and the primitive cardiomyocytes start to spontaneously contract as a result of expression of (a) sarcomeric genes forming sarcomers, and (b) ion pumps and channels within the cell membrane allowing spontaneous depolarization. The contraction becomes polarized due to the electrical coupling of neighboring cells via gap junctions Moorman & Christoffels, 2003b;Tyser et al., 2016).
While the precardiac mesodermal cells are formed and migrate, a subset of cells undergoes epithelial to mesenchymal transition forming endocardial cells in between the precardiac mesoderm and the endoderm ( Figure 2). The endocardial cells form a network of small channels which coalesce into larger ones with ongoing development (Harris & Black, 2010). Although the myocardial and endocardial progenitors develop concomitantly, it has been demonstrated that individual cells differentiate either into endocardial cells or cardiomyocytes (Cohen-Gould & Mikawa, 1996). Despite the fact that endocardial and endothelial cells seem to be very similar at first glance, their transcriptomes differ (Harris & Black, 2010) and mouse and zebrafish mutants exist that can form endothelial cells but no endocardial cells (Lee, Stainier, Weinstein, & Fishman, 1994).
With ongoing development, the flat embryo acquires it threedimensional (3D)-shape as a consequence of folding, due to the fast growth of neural tissue at the end of the third week (CS9).
Misregulation of this process can result in ectopia cordis (For review see [Gabriel et al., 2014]). During folding, the heart acquires the shape of an inverted Y with two caudolateral inlets (also venous pole) and one craniomedial outlet (also arterial pole or outflow tract). The heart tube is organized in an outer layer of two to three layers of cardiomyocytes and an inner layer of endocardial cells. The myocardial and endocardial layers are separated by an extracellular matrix (cardiac jelly F I G U R E 1 From heart forming region to primary heart tube. Panels (a)-(c) provide a schematic representation of the formation of the linear heart tube from the heart forming region (HFR), as seen from the ventral side. Within the HFR the first heart field is indicated in light gray, the second heart field in dark gray, and the third heart field in black. AP refers to the arterial pole and VP to the venous pole of the primary heart tube mesocardium. At this stage the heart tube starts to show slow peristaltic-like contractions that are initiated at the venous pole.

| THE GROWTH AND LOOPING OF THE HEART TUBE
At the beginning of the fourth week of development (CS10) the straight heart tube undergoes looping. Looping is an elusive process during which the dorsal mesocardium ruptures along its midline and the heart tube bends to the right, acquiring a C shape. With ongoing development, the bending of the heart tube becomes more complex, acquiring an S-shape (Bayraktar & Manner, 2014;Manner, 2009).
While looping, the heart tube increases five-fold in length due to the continuous addition of newly differentiated cardiomyocytes, rather than by proliferation of the cardiomyocytes of the heart tube (de Boer, van den Berg, de Boer, Moorman, & Ruijter, 2012a;Soufan et al., 2006). The newly differentiated cardiomyocytes are derived   (de Boer et al., 2012b;Sizarov et al., 2011). These cardiac progenitor cells are often referred to as second heart field cells, as opposed to first heart field cells, from which the initial heart tube is formed (Buckingham, Meilhac, & Zaffran, 2005).
The distinction between these different populations is contentious, because different markers point to different borders . These second heart field progenitors express the transcription factor Islet1, while being added to the heart tube. Upon differentiation into cardiomyocytes, the cells cease proliferation, coinciding with the down-regulation of Islet1 and up-regulation of Nkx2.5 (Buckingham et al., 2005;Cai et al., 2003). The transcription factor Tbx1 was found to be a regulator of the segregation of the second heart field cells to the inflow and outflow pole of the heart (De Bono et al., 2018;Rana et al., 2014).

| FROM LINEAR TO FOUR CHAMBERED HEART
Upon rightwards looping, ventricular formation becomes evident at the outer curvature of the heart tube. At this location the cardiac jelly between the endocardial and myocardial layers disappears and myocardial protrusions, referred to as trabeculations, become evident at the luminal (endocardial) side. The cardiomyocytes of the forming ventricle start to express genes like atrial natriuretic factor (Anf/Nppa) and the gap-junction protein Connexin 40 (Gja5) (Houweling, Somi, van den Hoff, Moorman, & Christoffels, 2002). The T-box transcription factors Tbx5 and Tbx20 are important activators of the gene expression program in ventricular cardiomyocytes, whereas Tbx2 and Tbx3 are important repressors of the ventricular myocardium gene program in the developing atrioventricular region (Greulich, Rudat, & Kispert, 2011;Habets et al., 2002).
Prior to the appearance of the first trabeculations, mitotic spindles become oriented. Cardiomyocytes in which the spindles are parallel with the lumen of the heart tube contribute to the lengthening of the heart tube, while cardiomyocytes with mitotic spindles in the direction of the lumen will contribute to the trabeculation (Le Garrec et al., 2013;Meilhac, Esner, Kerszberg, Moss, & Buckingham, 2004).
When mitotic spindle orientation is disrupted, as is observed upon deletion of atypical protein kinase C (Prkc1), trabeculation formation is affected (Passer, van de Vrugt, Atmanli, & Domian, 2016). of Bmp10 in hypertrabeculation (Pashmforoush et al., 2004). The expression of the cell cycle inhibitor (Cdkn1c) in the cardiomyocytes F I G U R E 2 Schematic representation of the formation of the adult heart. With the onset of embryonic folding the left and right heart forming region (HFR). The yellow structures represent the endocardial cells (endo) that form the inner lining of the heart and in gray the formed primary myocardium. VP refers to the venous pole were the blood will enter the heart and AP to the arterial pole where the blood will leave the heart. For easy comparison of panels (a) through (d), a red line indicates the lateral border of the HFR and a blue line the medial border. With ongoing folding the HFR becomes positioned ventrally of the foregut (see also Figure 1). At the position where the lateral borders of the HFR meet, the linear heart tube is connected to the body wall via the dorsal mesocardium (dm) (panel d). The DM breaks due to which the heart is only attached to the body wall at the AP and VP (panel h-k). Whereas panels (a)-(d) show a dorsal view of the forming heart, panels (e)-(h) show a ventral view, illustrating the transition of linear heart tube to four-chambered heart. Only at the ventral site of the linear heart tube the differentiation of the embryonic ventricle (V) is locally initiated. The forming working myocardium of the chambers is indicated in blue (panels f-k). Note in panel (g) that at the dorsal side of the heart tube, primary myocardium is retained, which is referred to as the inner curvature (IC). Flanking the forming ventricle, primary myocardium is retained which is referred to as the inflow tract (IFT) and outflow tract (OFT). With ongoing development, the linear heart tube loops to the right and chamber formation becomes evident (panel h). At this stage the right ventricle (RV) starts to form when primary myocardium of the OFT differentiates into chamber myocardium. Moreover, upstream of the left ventricle (LV), the primary myocardium of the IFT locally differentiates into the left atrium (LA) and right atrium (RA). In the meantime, newly differentiated cardiomyocytes are added to the lengthening heart forming the sinus venous (SV) myocardium. The heart is connected to the blood circulation at the VP via the left and right cardinal vein (cv) and at the AP via the pharyngeal arch arteries (paa). Panel (j) shows a representation of the 5 week old human heart showing the expanding (ballooning) atria and ventricles, as well as the remnants of the primary myocardium of the IFT, AVC (atrioventricular canal), IC and OFT. The forming primary atrial septum (pAS) and ventricular septum (VS) are identified. Within the LA the attachment to the body wall is identified as the mediastinal mesenchyme (mm) through which the cardinal vein (cv) and the future pulmonary vein (pv) drain into the heart. In the formed heart (panel k) the primary myocardium of the IFT and AVC has differentiated into the central conduction system, comprising the sinoatrial node (sn), the atrioventricular node (avn), the His bundle (His) and the bundle branches (bb). Within the right atrium the superior and inferior caval veins (scv and icv) drain in the RA and the pulmonary veins (pv) in the LA. Flanking the chambers, valves are formed of which only the mitral valve (mv) and the tricuspid valve (tv) are shown. The semilunar valves cannot be shown in this representation of the trabeculations suggests active inhibition of proliferation of these cells (Kochilas, Li, Jin, Buck, & Epstein, 1999). Within the next six days of mouse development the ventricle increases 100-fold in volume, due to active cardiomyocyte division of the ventricular wall, resulting in outer ventricular wall thickening and increase in trabecular length. The lengthening of the trabeculations is the result of addition of cardiomyocytes to their base, rather than proliferation at their tips (de Boer, van den Berg, de Boer, et al., 2012a).
Although the initially formed trabeculations are long and slender compared to the compact outer myocardial layer, they become relatively short and thick with ongoing development. It should be noted that the absolute length of these adult thick trabeculations is much longer than the embryonic trabeculations. This change in appearance of the trabeculations is referred to as compaction. This process is complete by embryonic day (E)14.5 in mice and at about eight weeks of development (CS22) in human (Sedmera, Pexieder, Vuillemin, Thompson, & Anderson, 2000). How these local differences in proliferation are molecularly regulated is largely unknown. A number of cell cycle regulators were shown to result in hypoplastic ventricle upon functional impairment in mice (Berthet et al., 2006;Koera et al., 1997;Kozar et al., 2004;Moens, Stanton, Parada, & Rossant, 1993). Interestingly, in mice in which N-myc is deleted, the compact layer remains thin (Charron et al., 1992;Moens et al., 1993). In human, mutations in these genes might underlie nonventricular compaction.
During ventriclular development and heart tube lengthening, the atria begin to differentiate at the inflow region (Yutzey, Rhee, & Bader, 1994). The atria are formed symmetrically, but have a left-right identity from the outset. The left-sided identity is imposed by the transcription factor Pitx2c. As a consequence, the absence of Pitx2c expression results solely in right atrial identity and thus two morphologically identical atria, also referred to as right atrial isomerism. Vice versa, ectopic expression of Pitx2c at the right side, results in two morphologically left atria, that is, left atrial isomerism (Mommersteeg and others, 2007b). Like the forming ventricular myocardium the developing atrial myocardium is also marked by ANF and Cx40 expression. The initially formed atria are retained in the heart as the trabeculated, atrial appendages (Christoffels et al., 2000). This newly formed myocardium along the right common cardinal vein will become the dorsal aspect of the right atrium, in between the entrance of the superior and inferior caval vein, and is referred to as the sinus venarum in the adult heart.  (Aghajanian et al., 2014;Degenhardt et al., 2013]. APVC refers to a spectrum of abnormalities in which the pulmonary vein is not connected to the left atrium, but to the right atrium directly, or indirectly through the coronary sinus or the superior or inferior caval veins. This was underscored in sequencing SEMA3D in patients with APVC, identifying a mutation in SEMA3D that affects the function of SEMA3D (Degenhardt et al., 2013). Furthermore, a Scottish family with APVC was identified with a genomic alteration located in 4p12 (Bleyl et al., 2006), pointing perhaps to regulation of this developmental process by other genes. Although guided growth of the pulmonary vein toward the atrium seems to play an important role, it should be noted that, anatomically, the initial pulmonary venous return is a midline structure. Since the primary atrial septum develops at the right side of the pulmonary orifice, the pulmonary vein becomes incorporated into the left atrium.
Subsequent to the connection of the pulmonary vein to the atrium, a myocardial mantle forms around the pulmonary vein and its bifurcations. Mesenchymal cells flanking the pulmonary venous endothelium in the dorsal mesocardium differentiate into cardiomyocytes.
The population is, in contrast to the myocardium formed around the caval veins, derived from a Tbx18-negative, and Nkx2.5-and Isl1-positive progenitor population. These newly formed cardiomyocytes initiate rapid proliferation and migrate along the pulmonary veins forming a myocardial sleeve. Interestingly, in the absence of functional Pitx2c results in under population of the walls of the pulmonary veins (Mommersteeg et al., 2007a). In mice, the myocardial sleeve is found to extend deep into the lungs, up to the fifth bifurcation. In human, however, this sleeve only develops to the extent of the second bifurcation, and while this sleeve is being formed, it is also being taken up into the dorsal wall of the left atrium.
As a consequence, four pulmonary orifices are found in the left atrium and an extensive amount of smooth-walled myocardium in between these orifices.

| DEVELOPMENT OF THE CONDUCTION SYSTEM
The timing and coordination of muscular contraction in the adult heart is coordinated by the sinus node, AV node, AV bundle, bundle branches and the peripheral conduction system (Purkinje fibers). The car- The importance of Bmp-signaling is crucial in this process as AVCspecific deletion of the Bone morphogenetic protein receptor 1A (Bmpr1a) leads to impaired atrioventricular node and annulus fibrosis development (Gaussin et al., 2005;Stroud et al., 2007). AVC development is further confined by Notch-signaling (Rentschler et al., 2011) and transcriptional regulation through Tbx20 (Singh et al., 2009).
A cluster of cells on top of the forming ventricular septum will not follow the chamber myocardial gene expression program, but retain their primary myocardial phenotype. This group of cells will differentiate into the atrioventricular (His) bundle. Although this was already concluded by Keith andFlack in 1906 (Keith, Aberb, Eng, Flack, &Oxon, 1906), it took till the end of the century to visualize and follow these cells immunohistochemically using the GlN2 antibody Wessels et al., 1992) and later by expression studies of Tbx3 (Bakker et al., 2008;Hoogaars et al., 2004). The

| VENTRICULAR SEPTATION
The septum dividing the left and right ventricle in the formed heart is composed of the myocardial ventricular septum and the membranous septum.

When the right and left ventricles form the cells in between will
form the myocardial ventricular septum. The ventricular septum lengthens by a process called apposition, meaning that cells are added to the septum at its base (Harh & Paul, 1975). In human this process starts in the fourth week of development (CS12) (Sizarov et al., 2011).
It is thought that when apposition is aberrant that a hole or multiple holes will be formed in the ventricular septum. These left-right connections are referred to as muscular ventricular septal defects (VSDs).
The lumen in between the top of the interventricular septum and the inner curvature is referred to as the ventricular foramen. Because the bloodstream is laminar, particles introduced into the blood follow a specific path without lateral mixing (Hogers, deRuiter, Baasten, Gittenberger-de Groot, & Poelmann, 1995). These analyses showed that the blood flow is already separated into a left-and right-sided circulation prior to septation, and that blood from the right atrium passes through the ventricular foramen into the right ventricle during diastole and blood from the left ventricle passes through the ventricular foramen into the future aortic stream during systole. From this analysis it is obvious that the ventricular foramen never closes but becomes

| SEPTATION OF THE OUTFLOW TRACT
The outflow tract is a myocardial tube that runs from the developing ventricles to the aortic sac, which is embedded in the pharyngeal arches and is connected to three paired symmetrical pharyngeal arch arteries. The pharyngeal arch arteries remodel in to the arterial pole of the heart which already resembles the postnatal configuration at the end of the eight week of development (CS23) (Rana, Sizarov, Christoffels, & Moorman, 2013).
Initially, the myocardial OFT increases in length by the addition of newly differentiated cardiomyocytes to its distal border (Rana et al., 2007;Webb, Qayyum, Anderson, Lamers, & Richardson, 2003). These cardiomyocytes are derived from the second heart field (Kelly & Buckingham, 2002;Sizarov et al., 2012). From the fifth week of development (CS14) onward the myocardial OFT becomes shorter, due to the incorporation of the proximal OFT myocardium as part of the right ventricle. In the adult the distal myocardial border is found halfway at the level of the semilunar valves and below the coronary orifices. As a consequence, the OFT has a nonmyocardial portion in between its distal of myocardial border and the border of the pericardial cavity.
This nonmyocardial portion of the OFT will become the intrapericardial part of the aorta and pulmonary trunk (Rana et al., 2007;Sizarov et al., 2012).The cells of the nonmyocardial portion originate from both the second-heart field and the cardiac neural crest (Cai et al., 2003;Jiang, Rowitch, Soriano, McMahon, & Sucov, 2000;Zhou et al., 2017).
Septation of the OFT starts at its distal border in the sixth week of development (CS16) and proceeds in a proximal direction, to its completion a week later (CS18) (Sizarov et al., 2012). The OFT cushions lay in a spiraling fashion, reflecting the course of the aortic and pulmonary streams in the adult (Sizarov et al., 2012;Ya et al., 1998).
At the start of fusion of the OFT cushions, the proximal portion of the cushions is invaded by endocardial derived cells and by cardiac neural crest cells at its distal border. The migration of the neural crest cells into the heart is a complex process that is regulated by Wnt, Bmp, Fgf, and Semaphorin signaling (For review see [Stoller & Epstein, 2005]. Invasion of neural crest cells becomes evident with the formation of a protrusion of pharyngeal mesenchyme, termed the aorticopulmonary septum, that grows into the aortic sac and connects distally to the fused OFT cushions. The aorticopulmonary septum ensures that the aortic blood stream is guided into the left fourth pharyngeal arch artery and the pulmonary blood stream into the left and right sixth pharyngeal arch arteries (Anderson et al., 2009;Anderson et al., 2012;Rana et al., 2013). In the distal portion of the OFT, the cardiac neural crest cells form condensed pillars in the cushions and in the proximal portion of the OFT cushions, where they become dispersed (Waldo, Miyagawa-Tomita, Kumiski, & Kirby, 1998). In the distal portion of the OFT the neural crest cells will predominantly form the facing part of the walls of the aorta and pulmonary trunk. In the proximal portion of the OFT the cushions form upon fusion of the outlet septum, in which a large part of the mesenchyme disappears by apoptosis and is replaced by cardiomyocytes. A process referred to as myocardialization (van den Hoff et al., 1999;Ya et al., 1998). The myocardial outlet septum changes during the commitment of the aorta to the left ventricle largely into the freestanding subpulmonary infundibulum. In the adult right ventricle, remnants of the OFT myocardium and myocardial outlet septum are recognized as smoothwalled myocardium distal of the trabecular component (Anderson et al., 2009;Anderson et al., 2012). Even in the adult, this smoothwalled myocardium still has characteristics of the primary myocardium due to which it can serve as a source for arrhythmias in cardiac disease (Boukens et al., 2013).
When the OFT cushions do not fuse over their entire length, an anomaly known as a persistent truncus arteriosus or common arterial trunk arises. When the fusion defect is limited to the proximal OFT cushions, this results in a subarterial or outlet VSD Okamoto, Akimoto, Hidaka, Shoji, & Sumida, 2010). A transposition of the great arteries is found when the cushions are laid down in a parallel fashion rather than in a spiraling course. In a transposition of the great arteries the aorta and pulmonary trunk are situated next to each other in a frontal plane and at birth two separate blood circulatory systems are formed which is not compatible with life (Costell et al., 2002;Kirby, 2002). Because cardiac neural crest provide cells to the OFT and these cells secrete growth factors essential for normal development of the surrounding tissue, no or a limited number of cells invade the OFT a spectrum of abnormalities ranging from a common trunk, to pulmonary atresia and double outlet right ventricle arises (Hutson & Kirby, 2003;Stoller & Epstein, 2005). Moreover, when fusion of the aorticopulmonary septum and the proximal OFT cushions is impaired, this is recognized as an aorticopulmonary window Sizarov et al., 2012;Rana et al., 2013.

| THE CARDIAC CONNECTIVE TISSUES
Cardiomyocytes make up 90% of the volume of the adult heart muscle, but only comprise 50% in total cell number (Banerjee, 2007;Pinto et al., 2016). Of the nonmyocytes, more than half of the cells are endocardial/endothelial cells, approximately one third are fibroblasts and less than 10% are hematopoietic-derived cells (Pinto et al., 2016).
Already one month after birth the maximum number of cardiomyocytes in the human heart has been reached, being approximately 3.2 × 10 9 ± 0.75 × 10 9 cardiomyocytes (Bergmann et al., 2015). From first month after birth onward, the increase in size of the heart is almost exclusively due to a volume increase of the cardiomyocytes (hypertrophy), being almost 10-fold up to an age of 20 years (Bergmann et al., 2015). Moreover, only approximately 50% of the cardiomyocytes undergo a single round of cell division during an adult lifespan (Bergmann et al., 2009;Bergmann et al., 2015) and hardly any cardiomyocytes are supplement from a stem cell population (Jesty et al., 2012;van Berlo et al., 2014). A recent review extensively discusses this phase of heart development (Gunthel, Barnett, & Christoffels, 2018).
Cardiac fibroblasts have long been neglected functionally, but in the last decades their importance under normal physiological and pathological condition has started to become evident. Analysis of the cardiac fibroblast population in the heart also showed that they are not a single homogeneous group. Since the introduction of genetic lineage marking systems, it has become evident that fibroblasts are not only derived from the epicardial cells but also from endocardial, circulating and hematopoietic cells. In depth reviews on this topic have been recently published Swonger, Liu, Ivey, & Tallquist, 2016;Tallquist & Molkentin, 2017).

| EPICARDIUM AND ITS DERIVATIVES
The epicardium is derived from the proepicardium, a villous structure found immediately upstream of the heart tube at the beginning of the fourth developmental week (CS10). Within days (CS11) the villi contact the dorsal outer surface of the AVC and from that point cells spread and start to cover the entire heart with epicardium, a process complete at the end of the sixth week of development (CS16) (Hirakow, 1992). A subset of epicardial cells undergoes epithelial to mesenchymal transition giving rise to a mesenchymal layer between the epicardium and myocardium. Although not fully analyzed it is general contention that the epithelial to mesenchymal transition observed in the epicardium is largely comparable to epithelial to mesenchymal transition in the cushions. This formed sub-epicardial mesenchyme either remains or contributes to (1) the coronaries, (2) cardiac fibroblasts, (3) annulus fibrosus and (4) valve leaflets (Perez-Pomares & de la Pompa, 2011;Wessels et al., 2012).
Initially, it was thought that the entire coronary vessel tree would be formed from epicardial-derived cells by vasculogenesis and subsequently angiogenesis. However, studies using genetic lineage tracing experiments revealed that this appeared to be more complex.
Epicardial-derived cells contribute the coronary smooth muscle cells and adventitial fibroblasts. The coronary endothelium was found to be formed from endocardial-derived cells, endothelial cells of the sinus venosus, and a limited contribution of epicardial-derived cells. Indepth reviews on this topic were recently published (Perez-Pomares & de la Pompa, 2011;Sharma, Chang, & Red-Horse, 2017). When development of the coronary tree reaches completion, the two coronary arteries form by angiogenesis which tap into the lumen of the aorta (Bogers, Gittenberger-de Groot, Poelmann, Péault, & Huysmans, 1989 (Ieda et al., 2009).
Genetic labeling of the epicardial cells also revealed that the annulus fibrosis is derived from this cell population and a part of the cells of the atrioventricular valve leaflets (Aanhaanen et al., 2010;Wessels et al., 2012;Zhou and others, 2010). Their contribution to the valves will be discussed below.

| DEVELOPMENT OF THE VALVES
The valves are formed from a part of the four major cushions, as  Wessels et al., 2012). Panel (g) shows a schematic summary of the origin of the cellular contributions to the mitral, tricuspid and semilunar valves. In the pulmonary trunk The left cusp in both the pulmonary trunk and aorta is derived from the septal OFT cushion (blue). The right cusp in both the pulmonary trunk (PT) and aorta (Ao) is derived from the parietal OFT cushion (orange). The anterior cusp in the pulmonary trunk is derived from the right intercalated ridge and in the aorta the posterior cusp is derived from the left intercalated ridge (gray). The aortic or anterior leaflet of the mitral valve (MV) and the septal leaflet of the tricuspid valve (TV) are derived from the inferior (blue) and posterior (yellow) atrioventricular cushions (iAVC and sAVC, respectively). The posterior leaflet of the MV is derived from the left lateral atrioventricular cushion (llAVC, green) and the septal leaflet of the TV from the right lateral atrioventricular cushion (rlAVC, light blue). From the data shown in panels (a)-(f), one can infer that the posterior MV leaflet and the parietal leaflet of the TV comprise predominantly of epicardially-derived cells and both the aortic leaflet of the MV and the septal leaflet of the TV of endocardially-derived cells. (Modified from Lamers & Moorman, 2002) the intercalated OFT ridges (de Lange et al., 2004;de Vlaming et al., 2012;Lamers & Moorman, 2002;Snarr et al., 2008;Wessels et al., 2012). Although still to be experimentally evaluated, the development of the minor cushions and intercalated ridges is similar to that of the major cushions. As summarized in Figure 4, genetic marking of the epicardial or endocardial cell contribution to the AV cushions has revealed that the leaflet of the mitral valve that is attached to the left ventricular free wall is derived from the left lateral AV cushion; the leaflet that is attached to the ventricular septum is derived from the major cushions; the leaflet of the tricuspid valve that is attached to the right ventricular free wall is derived from the right lateral AV cushion; the leaflet that is attached to the ventricular septum is derived from the major cushions

| FUTURE PERSPECTIVE
Over the last two decades the field of heart development has taken large, and sometimes bold, new steps in understanding. In spite of this new knowledge, only in a discouraging low percentage of patients with cardiac malformations, a genetic or environmental cause can be found.
The hypothesis that several (genetic) events have to take place in one patient before a cardiac malformation can develop is becoming more and more likely. Therefore new insights are needed in order to under-