advanced review organogenesis of the vertebrate...

Advanced Review

Organogenesis of the vertebrateheartLucile Miquerol1,2 and Robert G. Kelly1,2∗

Organogenesis of the vertebrate heart involves a complex sequence of eventsinitiating with specification and differentiation of myocardial and endocardial cellsin anterior lateral mesoderm shortly after gastrulation, followed by formationand rightward looping of the early heart tube. During looping, the heart tubeelongates by addition of second heart field progenitor cells from adjacentpharyngeal mesoderm at the arterial and venous poles. Progressive differentiationis controlled by intercellular signaling events between pharyngeal mesoderm,foregut endoderm, and neural crest-derived mesenchyme. Regulated patternsof myocardial gene expression and proliferation within the embryonic heartdrive morphogenesis of atrial and ventricular chambers, while cardiac cushions,precursors of the definitive valves, form in the atrioventricular and outflowregions. In amniotes, separate systemic and pulmonary circulatory systems ariseby septation and remodeling events that divide the atria and ventricles into leftand right chambers. Cardiac neural crest cells play a key role in dividing thearterial pole of the heart into the ascending aorta and pulmonary trunk. Duringthe remodeling phase the definitive cardiac conduction system, that coordinatesthe heartbeat, is established. In addition, the epicardium, critical for regulatedventricular growth and development of the coronary vasculature, spreads over thesurface of the heart as an epithelium from which cells invade the myocardiumto give rise to diverse cell types including fibroblasts and smooth muscle cells.Cardiogenesis thus involves highly coordinated development of multiple cell typesand insight into the different lineage contributions and molecular regulation ofeach of these steps is expanding rapidly. © 2012 Wiley Periodicals, Inc.

How to cite this article:WIREs Dev Biol 2013, 2:17–29. doi: 10.1002/wdev.68

INTRODUCTION

The heart is the first organ to form and functionin the developing vertebrate embryo. The basic

steps of heart organogenesis have been documentedin the literature by anatomists and embryologists,with experimental embryology in amphibian andavian models providing insights into inductive eventsand dynamic processes during heart development. Inaddition, molecular studies, including transgenesis,Cre-mediated genetic labeling, and forward andreverse genetics in mouse and zebrafish models,have accelerated our understanding of how different

∗Correspondence to: [email protected] Marseille Universite, Developmental Biology Institute ofMarseille Luminy, Marseille, France2CNRS UMR7288, Marseille, France

cell lineages contribute to the heart and providedinsight into the mechanisms regulating cardiogenesis.The requirement for cardiac function from earlystages of development highlights the importanceof interactions between this genetic blueprint andfunctional parameters, including hemodynamics andelectrical activity. In addition to providing ahighly tractable model for organogenesis the clinicalimportance of heart development is reflected in thehigh incidence of congenital heart defects in man.The sequence of events that accompany early heartmorphogenesis is common to all vertebrate speciesincluding heart tube formation, rightward looping,heart tube elongation by addition of progenitor cellsfrom adjacent pharyngeal mesoderm, followed bycardiac chamber, cushion, and valve morphogenesis.In amniotes, septation and remodeling events during

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the early fetal period generate a four-chambered organwith separate systemic and pulmonary circulatorysystems. In this review these different stages arediscussed sequentially, with emphasis on mouse heartdevelopment, and recent findings and controversiesare highlighted. While heart development in zebrafish,amphibians and avians will also be considered,the reader is directed to recent reviews for adetailed discussion of the important contributionof these models to our understanding of heartorganogenesis.1–3

Early Heart Tube DevelopmentThe heart is a mesodermal derivative and cardiac pre-cursor cells gastrulate through the anterior regionsof the primitive streak and move laterally to giverise to anterior lateral mesoderm4 (Figure 1(a)). Asthe embryonic coelom forms, cardiac specificationand differentiation take place in anterior lateralsplanchnic mesoderm through a combination of pos-itive and negative intercellular signaling events.2,5

These include prodifferentiation bone morphogeneticprotein (BMP) and fibroblast growth factor (FGF)signals from underlying pharyngeal or foregut endo-derm and negative signals, including β-catenin/WNTsignaling, from midline structures, the activity ofwhich is blocked by procardiogenic WNT inhibitors.6

Collectively these signals define the precise regionwhere cardiac genes are first activated, includingthose encoding transcription factors such as thehomeodomain transcription factor NKX2-5, the T-box factor TBX5, the Zinc-finger factor GATA4,the MADS-box factor MEF2C, and the cardiac spe-cific chromatin remodeling subunit SMARCD3 (for-merly known as BAF60C)7 (Figure 1(b)–(d)). Tran-scriptional regulation plays an important role inorchestrating heart development, highlighted by theincidence of congenital heart disease causing muta-tions in cardiac transcription factors.7 Combinatorialtranscriptional activation drives the cardiomyogenicprogram in the cardiac crescent and three of these fac-tors, GATA4, TBX5, and SMARCD3, are sufficient

(a) (b) (c) (d)

(e) (f) (g) (h)

FIGURE 1 | Early heart tube (HT) development. (a) Future cardiac cells gastrulate through the anterior region of the primitive streak (PS) andmigrate (arrows) to form lateral anterior mesoderm (AM). (b) The cardiac crescent (CC) forms in anterior splanchnic mesoderm underlying the headfolds (HF). Late differentiating second heart field (SHF) cells are positioned medially. (c) Transverse section at the level of the dotted line in(b) showing positive signals from underlying endoderm [bone morphogenetic protein (BMP) and fibroblast growth factor (FGF)] and negative signalsfrom the midline (β-catenin/WNT). (d) Transcription factors cooperatively activating the cardiomyogenic genetic program. (e) The linear HT ischaracterized by an anterior arterial pole (AP) and posterior venous pole (VP). (f) Transverse section at the level of the dotted line in (e) showing theventral HT attached to the dorsal mesocardium (DM) and comprised of an outer myocardial layer (MC) and inner endocardial tube (EC) separated bycardiac jelly (CJ). SHF cells are situated in medial splanchnic mesoderm in the dorsal pericardial wall (DPC wall) underlying the pharynx (Ph). (g) Aslooping initiates the AP of the HT is attached to the first (PAA1) and second (PAA2) pharyngeal arch arteries. (h) Sagittal section after breakdown ofthe DM showing the transverse pericardial sinus (TPS), and location of SHF cells in the DPC wall. A, anterior; C, coelom; D, dorsal; End, endoderm; L,lateral; M, medial; N, node; NT, neural tube; P, posterior; PM, paraxial mesoderm; SoM, somatic mesoderm; SpM, splanchnic mesoderm; V, ventral.Color code: pink, early differentiating myocytes and derivatives; green, anterior SHF and derivatives; blue, posterior SHF; yellow, endoderm.

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WIREs Developmental Biology Organogenesis of the vertebrate heart

to activate cardiomyogenesis in non-cardiac regionsof the embryo.8 Furthermore, GATA4 and TBX5,together with MEF2C, can reprogram cardiac fibrob-lasts directly to a cardiomyogenic fate.9 Epigeneticmechanisms, such as Polycomb mediated gene silenc-ing, are also important in preventing inappropriategene expression in cardiac progenitor cells.10,11

Morphogenetic movements associated withforegut closure transform the cardiac crescent intoa linear heart tube in the ventral midline of theembryo (Figure 1(e) and (f)). The heart tube comprisesan outer myocardial layer and inner endocardialtube and is characterized by an anteriorly positionedoutflow, or arterial pole, and a posterior inflow,or venous pole (Figure 1(e) and (f)). Cardiac jelly,secreted by the myocardium, separates the myocardialand endocardial layers and plays a role in earlycardiac function. The stage at which myocardial andendocardial lineages diverge is uncertain12; recentevidence suggests that the endocardium is at leastin part derived from distinct vascular endothelialprogenitor cells.13 The linear heart is initially opendorsally to ventral pharyngeal endoderm, forminga trough-like structure suspended in the pericardialcavity by the dorsal mesocardium (Figure 1(f)). Thedorsal mesocardium rapidly breaks down behind thecentral part of the heart tube, creating the transversepericardial sinus and isolating the tubular heart in theventral region of the embryo (Figure 1(g) and (h)).

Heart Tube ExtensionThe heart tube subsequently loops to the right, inthe first morphological manifestation of embryoniclaterality (Figure 1(g)). The mechanisms drivingrightward looping, downstream of asymmetric Nodalexpression in left lateral plate mesoderm, areunknown. Concomitant with looping, the heart tubeundergoes a dramatic increase in length. Heart tubeelongation is largely driven not by proliferation ofcells within the linear heart tube, which has beenshown to have extremely low cell-cycle times,14 butby addition of cardiac progenitor cells from outsidethe early heart to the arterial and venous poles. Thesecells originate in splanchnic mesoderm contiguous andmedial to the cardiac crescent and have been termedthe second heart field.15 Second heart field cells inthe dorsal pericardial wall become separated fromthe heart tube when the dorsal mesocardium breaksdown, maintaining continuity with the heart only atthe poles (Figure 1(b)–(h)).

Second heart field cells are characterized by con-tinued proliferation and differentiation delay relativeto cells giving rise to the linear heart tube and aproliferative center has been identified in the dorsal

pericardial wall in avians.14 Retrospective clonal anal-ysis in the mouse has demonstrated that the secondheart field corresponds to a second cardiac lineagethat diverges from cells giving rise to the linear hearttube at or prior to gastrulation (Figure 2(a)).16 DiIlabeling, cell lineage analysis, and Cre tracing exper-iments have demonstrated that cells from the secondheart field contribute progressively to the arterial poleof the heart tube to give rise to right ventricular andoutflow tract myocardium and smooth muscle at thebase of the great arteries.16–20 In contrast, the linearheart tube predominantly gives rise to the left ven-tricle. At the venous pole of the heart, second heartfield cells give rise to atrial, atrial septal, and inflowtract myocardium18,21–23 (Figure 2(b)–(d)). Myocar-dial investment of the caval and pulmonary veinscontinues significantly after second heart field addi-tion at the arterial pole of the heart is complete.24,25

Progenitor cells contributing to the arterial and venouspoles have been termed the anterior and posterior sec-ond heart field, respectively, although the boundariesbetween these populations are as yet poorly defined.Recent genetic lineage tracing suggests that a subpop-ulation of arterial pole progenitor cells may originatein a HOX gene expressing region of the posteriorsecond heart field.26 An important insight from thesestudies is that different regions of the definitive heartare prefigured in distinct cardiac progenitor cell popu-lations. Certain of these subpopulations, in particularthose giving rise to the distal outflow tract and atrialseptum, are highly relevant clinically, as perturbationof their development results in common congenitalheart defects.

Second heart field cells express genes encodingthe transcription factors ISL1 and TBX1, and thefibroblast growth factors FGF8 and FGF10.15 ISL1regulates signaling pathways required to coordinatesecond heart field deployment and is essential forheart tube elongation.18 ISL1 positive cells have beenidentified in the fetal and early postnatal heart andappear to be resident cardiac stem cells that poten-tially contributing to later growth of the heart.27,28

TBX1 regulates proliferation and differentiation delayin the second heart field, including FGF ligand geneexpression, and is required for development of thedistal outflow tract.29–31 Both Isl1 and Tbx1 areexpressed in multipotent cardiovascular progenitorcells in differentiating embryonic stem cells, givingrise to myocardium, smooth muscle, and endothelialcells.28,29 How such multipotency is temporally andspatially encoded in the second heart field in vivo iscurrently unclear.

Arterial pole development is coordinated bya dialog between the second heart field, neural








(a)

(b) (c) (d)

(e) (f) (g)

FIGURE 2 | The second heart field (SHF). (a) The lineage relationship between progenitor cell populations giving rise to different regions of theheart. The outflow tract is exclusively derived from the SHF and left ventricle from the linear heart tube. The lineage relationship with craniofacialskeletal muscles derived from pharyngeal mesoderm is indicated. (b, c) Left lateral view (b) and sagittal section (c) showing anterior (green) andposterior (blue) SHF contributions to the heart at E9.5. Core pharyngeal arch mesoderm of arches 1 (PA1CM) and 2 (PA2CM) is visible in (b).(d) Embryonic heart at midgestation (E10.5) showing parts of the heart derived from the anterior SHF (green), linear heart tube (pink), and posteriorSHF (blue). The anterior SHF also gives rise to a cuff of smooth muscle at the arterial pole (SmM). At this stage, the arterial pole of the heart isattached to pharyngeal arch arteries (PAA) 3, 4, and 6. (e) Transverse section at the level of the dotted line in (b) showing juxtaposition betweenanterior SHF cells (green) and neural crest-derived mesenchyme (CNC, orange) lateral to and underlying the pharynx (Ph). (f) Enlargement of theboxed area in (e) showing intermingled CNC and mesodermal (Mes) mesenchymal cells and the balance between fibroblast growth factor (FGF) andbone morphogenetic protein (BMP) signals regulating proliferation and differentiation during outflow tract elongation. (g) Left lateral view of anE14.5 head showing craniofacial skeletal muscles derived from the first pharyngeal arch (muscles of mastication, light green) and second pharyngealarch (muscles of facial expression, dark green) pharyngeal arches. A-SHF, anterior second heart field; AVC, atrioventricular canal; D, dorsal; d-OFT,distal outflow tract; DPC wall, dorsal pericardial wall; End, endoderm; LA, left atrium; LV, left ventricle; OFT, outflow tract; p-OFT, proximal outflowtract; P-SHF, posterior second heart field; RA, right atrium; SoM, somatic mesoderm; V, ventral. Color code as for Figure 1.

crest-derived cells, and pharyngeal endoderm(Figure 2(e) and (f)). A balance between propro-liferative β-catenin/WNT and FGF signaling path-ways and prodifferentiation BMP and non-canonicalWNT pathways coordinates progressive heart tubeextension.15,32,33 This balance is modulated by neuralcrest influx into the pharyngeal region that brakesthe process of heart tube elongation by reducingFGF signal reception in second heart field cells.34

Pharyngeal mesoderm expressing Tbx1 and Isl1also gives rise to a subset of craniofacial skeletalmuscles35,36 (Figure 2(b) and (g)). Clonal analysishas revealed a lineage relationship between thesecraniofacial muscles and second heart field-derivedmyocardium, indicating the existence of bipotential

myogenic progenitor cells in pharyngeal mesoderm.37

Furthermore, first arch-derived muscles share a lin-eage relationship with right ventricular myocardiumand second arch-derived muscles with outflow tractmyocardium, reflecting the progressive caudal dis-placement of the outflow tract during pharyngealmorphogenesis37 (Figure 2(g)). While a second heartfield has been identified in all vertebrate speciesanalyzed, including Xenopus and zebrafish,38,39 Isletexpressing cells adjacent to precardiac cells have anexclusively skeletal myogenic fate in the protochordateCiona intestinalis.40 A subpopulation of skeletal myo-genic progenitor cells in pharyngeal mesoderm maythus have been diverted to the heart during vertebrateevolution.



(a) (b) (c) (d)

FIGURE 3 | Chamber morphogenesis. (a) Left lateral view showing ballooning of the ventricular and atrial chambers (brown) and the poorlyproliferative atrioventricular (AVC) and outflow tract (OFT) myocardium (blue). (b) Chamber and non-chamber genetic programs are established bybone morphogenetic protein (BMP) signaling and T-box transcription factor activity. (c) During ballooning morphogenesis cells from the OFTcontribute to the right ventricular free wall while cells from the AVC canal contribute to the left ventricular free wall (white arrows). (d) Cushiondevelopment in the OFT and AVC regions of the midgestation heart. Distal outflow tract cushions (d-OFT) are colonized by neural crest-derivedmesenchyme (orange); proximal outflow tract (p-OFT) and AVC cushion mesenchyme is derived by epithelial to mesenchymal transition of theunderlying endocardium (gray). IC, inner curvature; LA, left atrium; LV, left ventricle; OC, outer curvature; RA, right atrium; RV, right ventricle.

Cardiac Chamber and CushionMorphogenesisHeart tube extension is complete by midgestationin the mouse at which stage the heart growsby proliferation of cells within the heart. Highlyregulated patterns of gene expression and proliferationresult in formation of right and left ventricular andatrial working chamber myocardium on the outercurvature of the looped heart tube by a process ofballooning morphogenesis41 (Figure 3(a)–(c)). Lowrates of proliferation are maintained at the innercurvature of the heart tube, including the outflow tractand atrioventricular canal region. These proliferativedifferences are regulated by complex overlappingexpression patterns of T-box containing transcriptionfactors.42 TBX20 and TBX5 promote chambermyocardial development while TBX2 and TBX3repress the chamber genetic program and promotecushion development in the atrioventricular canaland outflow tract (Figure 3(b)). An underlyingmechanism has been identified involving competitionbetween the transcriptional activator TBX5 andthe transcriptional repressors TBX2 and TBX3 forinteraction with NKX2-5 at target promoters.43 BMPsignaling plays a patterning role upstream of Tbx2and Tbx3 expression in the atrioventricular canalregion; BMP signaling intermediates are antagonizedthrough sequestration by TBX20 at sites of chambermyocardium formation.44 Cellular, as well asproliferative, mechanisms underlie cardiac ballooningas cell lineage and vital dye labeling experimentshave shown that Tbx2 expressing cells in theatrioventricular canal contribute to the left ventricularfree wall,45 and cells from the outflow tract give rise toa significant part of the right ventricle46 (Figure 3(c)).

Cardiac cushions in the atrioventricular and out-flow tract regions function in preventing regurgitation

and ensuring directional blood flow in the embry-onic heart. Myocardial-derived BMP signals overlyingsites of cushion formation trigger an epithelial tomesenchymal transition in endocardial cells throughactivation of endothelial Notch and Transform-ing growth factor-β signaling.47 Endocardial-derivedcushion mesenchyme will subsequently give rise to thedefinitive cardiac valves. At this stage, cardiac neu-ral crest cells enter the arterial pole of the heart andcontribute to mesenchyme in the distal outflow tractcushions (Figure 3(d)).

Cardiac Septation and Valve DevelopmentEarly events of heart tube organogenesis, includingheart tube formation and extension, as well as cham-ber and cushion morphogenesis, are shared by allvertebrate species. However, adaptation to a terres-trial existence during vertebrate evolution necessitatedpartial (amphibians, reptiles) or total (amniotes) sepa-ration of systemic and pulmonary circulatory systems.This is achieved by extensive morphological remod-eling during early fetal development resulting in theconvergence of ventricular, outflow tract, atrioventric-ular and atrial septa. The ventricular septum forms atthe interface between linear heart tube and secondheart field-derived myocardium, defined by a steepgradient of Tbx5 expression48 (Figure 4(a)). This sep-tum has a muscular component at the cardiac apexand a membranous component underlying the out-let septum, where outflow tract and atrioventricularcushions converge to complete ventricular septation49

(Figure 4(b)). Neural crest cell migration into theoutflow tract cushions plays a critical, though incom-pletely understood, role in cushion formation and divi-sion of the outflow tract into the base of the ascendingaorta, outlet of the left ventricle, and pulmonary trunk,






(a) (b) (c)

FIGURE 4 | Cardiac septation. (a) Ventricular and outflow tract septation are underway at E12.5. The aorticopulmonary septum (APS) originatingbetween the fourth (PAA4) and sixth (PAA6) pharyngeal arch arteries separates the ascending aorta and pulmonary trunk (PT) and converges withfusing outflow tract cushions. Counterclockwise outflow tract rotation (arrow) positions the ascending aorta above the left ventricle. (b) Septation iscomplete by E14.5 in the mouse; the aorta (Ao) is connected to the left ventricle and the PT to the right ventricle. Three semilunar valve leaflets formthe outlet valves at the base of the ascending aorta (AoV) and pulmonary trunk (PV). The PT is connected to the descending aorta by the arterial duct(DA). Subpulmonary myocardium (blue, SPMc) surrounds the base of the PT. The interventricular septum has muscular (muIVS) and membranous(meIVS) components. (c) Atrial and atrioventricular septation showing the primary (PAS) and secondary (SAS) atrial septa and the dorsalmesenchymal protrusion (DMP). The mitral (MV) and tricuspid (TV) valves connect the atria to the left and right ventricles. IVS, interventricularseptum; LA, left atrium; LCCA, left common carotid artery; LSCA, left subclavian artery; LV, left ventricle; RA, right atrium; RCCA, right commoncarotid artery; RSCA, right subclavian artery; RV, right ventricle; VV, venous valve.

outlet of the right ventricle.50 Cardiac neural crest cellinflux is regulated by semaphorin, endothelin, andBMP signaling pathways, among others.50 Separa-tion of the ascending aorta and pulmonary trunk ismediated by the aorticopulmonary septum, originat-ing in the dorsal wall of the aortic sac between thefourth and sixth arch arteries, and convergence ofthis septum with the distal outflow tract cushions.51

A muscular outflow tract septum is generated bydirected myocardial invasion, or myocardialization, ofthe fused outflow tract cushions, a process regulatedby the planar cell polarity signaling pathway.52,53

Anticlockwise rotation of the arterial pole of the heartpositions the base of the pulmonary trunk over theright ventricle and the ascending aorta over the leftventricle (Figure 4(a) and (b)).54,55 Correct alignmentof the left ventricle with the ascending aorta is a pivotalstep in isolating systemic and pulmonary blood flows.Defects in the septation process itself, or in the prioraddition of second heart field cells required for the nec-essary elongation and alignment of the outflow tract,lead to a spectrum of conotruncal congenital heartdefects including common arterial trunk, overridingaorta, tetralogy of Fallot, and double outlet rightventricle.

Atrial septation initiates with formation ofthe dorsal mesenchymal protrusion, a posteriorsecond heart field derivative giving rise to theprimary atrial septum and part of the atrioventricularseptal complex (Figure 4(c)). A cell death inducedhole in the primary atrial septum bypasses thepulmonary circulation during fetal development.56

At birth, this communication is closed by thesecondary atrial septum, an infolding of the atrialroof, concomitant with closure of the arterial ductconnecting the pulmonary trunk and descendingaorta, thus definitively isolating the systemic andpulmonary circulatory systems. Interestingly, sonichedgehog signaling in specific second heart fieldprogenitor cell populations is required both for atrialseptation and development of myocardium at the baseof the pulmonary trunk (Figure 4(b) and (c)).22,23 Thesource of SHH ligand is future pulmonary endoderm,suggesting that during vertebrate evolution lungs mayhave coevolved with those parts of the heart requiredfor development of an independent pulmonarycirculation. In amphibians, ventricular and outflowtract septation is incomplete. It has recently beenshown that the outlet septum in Xenopus is derivedfrom the second heart field and that, in contrast to thesituation in amniotes, neural crest cells do not invadethe arterial pole of the amphibian heart.57

Concomitant with cardiac septation, endocar-dial cushions in the atrioventricular canal and outflowtract become remodeled to form valve leaflets. Cush-ion compaction is associated with the appearance ofhighly organized extracellular matrices and connectivetissue architecture.47,58 The mitral and tricuspid valvesregulate atrial blood flow into the left and right ven-tricles, respectively (Figure 4(c)). In the outflow tract,two intercalated cushions form in the provalve region.After division of the outflow tract, the ascendingaorta and pulmonary trunk each have three semilunarvalve leaflets that form close to the interface between



myocardium and smooth muscle cells at the base ofthe great arteries (Figure 4(b)).

Conduction System DevelopmentThe heart beats from the early heart tube stage andcardiac rhythm and electrical propagation are reg-ulated by the cardiac conduction system. Defectsin conduction system development underlie cardiacarrhythmias. In the early tubular heart, pacemakeractivity initiates in the posterior left region of thevenous pole and as the heart tube elongates becomesprogressively restricted to the forming sinoatrialnode59 (Figure 5(a) and (b)). The molecular mecha-nisms underlying localized differentiation of pace-maker cells in newly forming myocardium involvethe intersection of different transcription factor activi-ties (Figure 5(c)). These include negative regulation byPITX2, expressed in the left atrium and sinus horn,restricting activity to the right side of the heart, andSHOX2 repression of NKX2-5, together with positiveregulation of sinoatrial node differentiation by TBX18and TBX3.24,60–62 Subsequent fast conduction veloc-ity in atrial myocardium is conferred by expression ofthe gap junction subunits GJA1 and GJA5.

Electrical activity is slowed in the atrioventric-ular canal region by the specific gene expressionprofile of this region of the heart regulated by thetranscription factors TBX2 and TBX3, repressors

of Gja1 and Gja5 expression.42 Conduction delayin the atrioventricular region ensures that ventricu-lar contraction occurs after that of the atria. In theearly heart ventricular contraction initiates adjacentto the atrioventricular canal. As cardiac septationproceeds, atrioventricular myocardium contributes tothe atrial and ventricular walls and is replaced by aring of fibrous tissue that isolates ventricular fromatrial myocardium; electrical continuity is maintainedonly through the atrioventricular or central conduc-tion system.63 This includes the atrioventricular node,positive for the gap junction subunit GJA1, that delayselectrical activity between atria and ventricles, anda GJA5-positive fast ventricular conducting compo-nent comprised of the atrioventricular or His Bundleat the top of the muscular interventricular septumand right and left bundle branches on the suben-docadial surface of the ventricular septal walls. Theperipheral ventricular conduction system consists ofa network of fast conducting Purkinje fibers on thesubendocardial ventricular surface that ensure syn-chronous myocardial contraction following an apexto base pattern.64 All components of the conductionsystem are specialized cardiomyocytes. Clonal analy-sis in avian and mouse embryos has demonstratedthat the ventricular conduction system originatesfrom common progenitors with adjacent workingmyocytes.65,66 Conductive cells are less proliferative

(a) (b) (c)

(d)

FIGURE 5 | Conduction system morphogenesis. (a) Cartoon showing the origin of different components of the conduction system in themidgestation mouse heart including the sinoatrial node (SAN) at the boundary between the right sinus horn (RSH) and right atrium (RA), slowconducting atrioventricular canal myocardium (AVC, blue), and nascent ventricular trabeculae (green). Fast and slow conducting regions areillustrated by full and broken white arrows, respectively. (b) Configuration of the conduction system in the fetal heart after completion of septation,showing the central conduction system composed of the atrioventricular node (blue, AVN) and atrioventricular (or His) bundle (AVB), connected tothe right (RBB) and left (LBB) bundle branches (green). The peripheral ventricular conduction system consists of a network of subendocardial Purkinjefibers in the right (RPF) and left (LPF) ventricles (green). Ventricular myocardium is characterized by subendocardial trabeculae (T) and a compactmyocardial layer (CM) adjacent to the epicardium (EPC). An example of clonally related myocytes in different layers of a transmural polyclonal cone(PCC) is illustrated, showing a contribution to compact (dark brown), trabecular (light brown) and conductive (green) myocytes. (c) Summary of thecombinatorial positive and negative acting transcription factors regulating gene expression in the SAN. (d) Biphasic development of the ventricularconduction system during which a limited proliferation step follows differentiation of conductive myocytes from a common progenitor with workingchamber myocytes. ENC, endocardium; LA, left atrium; LSH, left sinus horn; LV, left ventricle; OFT, outflow tract; RV, right ventricle; VV, venous valve.







than working myocytes and differentiation is fol-lowed by limited outgrowth (Figure 5(d)). Neuralcrest-derived cells play a role in insulating componentsof the central conduction system.67 The transcriptionfactors TBX3, TBX5, NKX2-5, ID2 and IRX3 havebeen shown to be required for ventricular conductionsystem development. While TBX3 plays a key rolein controlling development of the central conductionsystem,68 TBX5, NKX2-5, and their common targetgene Id2, regulate morphogenesis of the ventricularconduction system. Hypoplasia of the central andperipheral conduction system on haploinsufficiency ofTbx5 or Nkx2-5 reveals that precise levels of thesefactors are required to coordinate conduction systemdevelopment.69–71 IRX3 regulates levels of Gja1 andGja5 expression in the ventricular conduction systemand is thus required for acquisition of the conductivephenotype.72

The ventricular conduction system formson the subendocardial surface of the ventricularmyocardium. This region of the heart is charac-terized by transient myocardial projections calledtrabeculae that form in response to signals from theoverlying endocardium. Endothelin, neuregulin, andNotch signaling pathways operate across the closeconjunction of endocardium and myocardium to ini-tiate trabeculation and promote conduction systemdifferentiation.73–75 Clonal analysis has revealed thatthe ventricular wall is composed of polyclonal conesresulting from low levels of proliferation in trabecu-lar myocardium and elevated proliferation in compactmyocardium adjacent to the epicardium.76,77 Spongytrabecular myocardium is thought to maximize oxy-genation and ventricular function prior to estab-lishment of the coronary circulation.78 Progressive

compaction of trabeculae during fetal developmentaccompanies Purkinje fiber differentiation. Failure ofventricular compaction results in hypertrabeculationand cardiomyopathy in the adult. Transcriptional dif-ferences between subepicardial and subendocardiallayers of ventricular myocardium also play importantroles in cardiac function. Subepicardially enrichedexpression of the potassium channel KCND2, forexample, is required for the ventricular repolarizationgradient and is restricted to this region of the ven-tricular wall by subendocardial expression of thetranscriptional repressor IRX5.79

Epicardial and Coronary VascularDevelopmentIn addition to the myocardium and endocardium,a third layer of cells, the epicardium, forms a thinepithelium on the outer surface of the heart atmidgestation in the mouse. The epicardium is derivedfrom the proepicardial organ, situated adjacent tothe venous pole of the heart. Cells from the proepi-cardial organ attach to the ventricular surface andprogressively spread over the surface of the heart80

(Figure 6(a)). These cells express genes encoding thetranscription factors WT1, TBX18, and SCX, as wellas FGF ligands, IGF2, PDGFA, SEMA3D, and theretinoic acid synthesizing enzyme ALDH1A2 (for-merly known as RALDH2).81–83 The epicardium thusacts as a signaling center, stimulating ventricularmyocardial growth in the subepicardial layer leadingto a thickened compact myocardial layer.82,83 In themouse, cardiomyocyte proliferation becomes severelyrestricted after birth,84 however a recent study hasdemonstrated that the neonatal mouse heart has the

(a) (b) (c)

FIGURE 6 | Epicardial development. (a) Right lateral view of a midgestation heart showing attachment of cells derived from the proepicardialorgan (PEO) and spreading of epicardial cells (blue) over the external surface of the heart (black arrows). Endothelial progenitor cells originating fromthe sinus venosus (red) also spread over the surface of the heart (white arrows). (b) Cartoon showing epicardial-derived (black arrows) cardiacfibroblasts (CF) and smooth muscle cells (SmM) in the ventricular wall associated with coronary endothelial cells (red, orange). See text for discussionof the contribution of epicardially derived cells to coronary endothelium and myocardium. (c) Scheme showing epicardial contributions to myocardialproliferation and coronary vasculature. Proliferative signals are indicated by gray arrows. CM, compact ventricular myocardium; CV, coronary vascularendothelial cells; ENC, endocardium; EPC, epicardium; LV, left ventricle; OFT, outflow tract; RA, right atrium; RV, right ventricle; T, trabeculae.













capacity to extend myocyte proliferation, conferring atransient potential for cardiac regeneration.85

In response to myocardial signals, a subsetof cells in the epicardial layer undergo β-catenindependent oriented cell division perpendicular tothe epicardial basement membrane, followed by anepithelial to mesenchymal transition and invasion ofthe subepicardial space and underlying myocardium86

(Figure 6(b)). Epicardially derived cells contributeto interstitial cardiac fibroblasts, which play fur-ther roles in stimulating myocardial growth87 andin constituting the fibrous matrix of the heart, andto smooth muscle cells associated with the coronaryvasculature (Figure 6(b) and (c)). The range of celltypes to which epicardially derived cells contributeis currently being reevaluated using genetic labelingapproaches. A contribution of epicardially derivedcells to coronary endothelium proposed in the avianheart80,88 has been observed in the mouse usingScx- and Sema3d- but not Tbx18-Cre genetic tracingexperiments.81,89 Together these results reveal molec-ular compartmentalization in the proepicardial organand suggest that the epicardium may contain diverseprogenitor cell populations.81 Similarly, a contribu-tion of epicardially derived cells to myocardium hasbeen debated. While certain Cre studies support such acontribution,81,90,91 others have suggested that epicar-dially derived cells may only give rise to myocardiumat a low level after epicardial activation in response todamage in the adult heart.92–94 Epicardially derivedcells also contribute to a population of mesenchymalstem cell-like cells resident in the adult heart that maybe involved in local tissue repair.95

The coronary vasculature develops at the time ofcardiac septation and provides an oxygenated bloodsupply to the growing ventricular wall. The originof coronary endothelial cells is currently debated andtransgene and lineage studies have recently identifieda population of cells derived from the sinus veno-sus that migrate over the heart from the venous poleand subsequently differentiate into arterial and venousendothelial cells96 (Figure 6(a)). Lineage experimentshave also implicated endocardial cells as a secondsource of coronary endothelial cells during growthof the compact myocardium.96 In addition, Scx- and

Sema3d- expressing proepicardial cells have recentlybeen suggested to contribute to coronary endothelialcells.81 FGF, VEGF, and hedgehog signaling pathwaysplay important roles in development of the coronaryvasculature.82 At the level of the great arteries, thecoronary plexus selectively invades the base of theaorta, through as yet unknown mechanisms. The rightand left coronary ostia are positioned centrally abovethe right and left pulmonary trunk facing aortic valveleaflets. Proximal coronary artery patterning defectsin embryos lacking myocardium at the base of thepulmonary trunk suggests the potential for negativeas well as positive signals in directing sites of ostiaformation.31 While coronary smooth muscle is pre-dominantly epicardially derived, Cre-tracing analysisand ablation experiments in avian embryos have sug-gested that the second heart field may contribute tosmooth muscle cells associated with coronary arteriesproximal to the coronary ostia.19,20

CONCLUSION

Recent molecular and cell lineage studies have dra-matically accelerated the pace of research into heartdevelopment. However, many fundamental questionsremain, in particular concerning the timing and mech-anism of divergence between different cell lineages,such as myocardial and endocardial progenitor cellsor conducting and working myocytes. Key currentresearch questions pertain to prepatterning in earlycardiac progenitor cells, dissecting the complex itera-tive roles of signaling pathways and regulatory genesin different steps of heart development, refining ourunderstanding of the relative contribution of myocar-dial proliferation and stem cell populations to cardiacgrowth and addressing the dynamic nature of manyof the steps documented in this review. Additionalefforts in different vertebrate model systems aim tobetter understand the interaction between genes andnon-genetic parameters, including hemodynamics andelectrical activity. Advances in our understanding ofheart development will provide mechanistic insightinto congenital heart defects in human patients aswell as driving efforts to promote cardiac repair in thedamaged heart.

ACKNOWLEDGMENTS

Supported by the European Community’s FP7 contract CardioGeNet (Health-2007-B-223463) and theFondation pour la Recherche Medicale.



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FURTHER READINGHeart Development. Current Topics in Developmental Biology. Bruneau BG, ed. 2012, 100. Available at: http://www.sciencedirect.com/science/bookseries/00702153.

Kirby ML. Cardiac Development. New York, NY: Oxford University Press; 2007. ISBN: 0-19-517819-X

Rosenthal N, Harvey RP, eds. Heart Development and Regeneration. San Diego, CA: Academic Press, Elsevier; 2010. ISBN:978-0-12-381332-9

De la Cruz MV, Markwald RR, eds. Living Morphogenesis of the Heart. Birkhauser, Basel: Bikhauser; 1998. ISBN:0-8176-4037-1

Schoenwolf GC, Bleyl SB, Brauer PR, Francis-West PH, eds. Larsen’s Human Embryology. 4th ed. Philadelphia, Pennsylvania:Churchill Livingstone, Elsevier; 2009. ISBN: 978-0-443-06811-9

Spotlight issue on Cardiac Development of Cardiovascular Research. In: Franco D, Kelly RG, eds. 2011, 91. Available at:http://cardiovascres.oxfordjournals.org/content/91/2.toc.


http://www.sciencedirect.com/science/bookseries/00702153.

http://www.sciencedirect.com/science/bookseries/00702153.

http://cardiovascres.oxfordjournals.org/content/91/2.toc

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