Organogenesis: Patterning the vertebrate heart

© 2002 Nature Publishing Group544 | JULY 2002 | VOLUME 3 www.nature.com/reviews/genetics

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Heart morphogenesis: an overviewHeart formation in vertebrates occurs through severalwell-established transitions (BOX 2). In mammals, heartprogenitor cells can be recognized morphologically as adistinct epithelial cell population in the cranio-lateralmesoderm of the late GASTRULATION embryo. These cellsadopt the shape of a crescent, extending laterally almostto the junction between the embryonic and extra-embryonic regions1. In this form, they are often referredto collectively as the cardiac crescent (FIG. 1a; BOX 2a). Thelinear heart tube that is subsequently formed (BOX 2c,d) isa transient structure that is composed of an innerendothelial tube shrouded by a myocardial layer. Thevarious cavities and constrictions that are present in thelinear heart tube betray the patterning events that havealready impinged on its structure 2. Even before heart-tube formation is completed caudally, the elongatingheart begins to adopt a rightward spiral form, in aprocess called cardiac looping (BOX 2e,f ). During looping,the future ventricles become distinct and balloon out-wards, and the atrial region and systemic venous tribu-taries are forced dorsally and cranially (BOX 2e,f ); thisbrings the primitive chambers into an alignment that isnecessary for their future integration (BOX 2g,h). The pre-cursors of the tricuspid and mitral valves are laid downduring looping. These valves form locally at the level of the atrioventricular canal (BOX 2) from cells of the

“The pattern which connects is a metapattern, a

pattern of patterns.”

Gregory Bateson

The developing heart is a highly modified muscular ves-sel. Separate but anatomically fused pumping units thatserve the systemic and pulmonary vascular circuits of thebody are integral parts of its form. Its structure and func-tion rely on the integrated development of numerousmodules, including chambers (atria and the ventricles),main vessels, valves, coronary circulation and a special-ized conduction system (BOX 1). In humans, the molecularand morphological events of heart formation seem to besensitive to genetic perturbation, and congenital heartdefects have been detected in ~1% of live births. In thisarticle, I briefly review the key events in early heart forma-tion and highlight recent advances in our understandingof heart patterning and morphogenesis. The goal of thisreview is to build a framework through which the rela-tionships between heart morphogenesis and molecularmechanisms can be appreciated, and from which congen-ital heart diseases can be understood. The emphasis willbe on mammalian heart development; however, othermodel systems bring an essential depth and perspective tothis field and data will be cited when they are likely toreflect generalities or when they provide a provocativecounterpoint to current knowledge.

PATTERNING THE VERTEBRATE HEARTRichard P. Harvey

The mammalian heart is crafted from a few progenitor cells that are subject to rapidly changingsets of instructions from their environment and from within. These instructions cause them tomigrate, expand and diversify in lineage, and acquire form and function. Molecular informationfrom various model systems, combined with increasingly detailed morphogenetic data, hasprovided insights into some of these key events. Many congenital heart abnormalities mightarise from defects in the early stages of heart development, therefore it is important tounderstand the molecular pathways that underlie the lineage specification and patterningprocesses that shape this organ.

Victor Chang Institute of Medical Research,384 Victoria Street,Darlinghurst 2010,New South Wales, Australia.e-mail: [email protected]:10.1038/nrg843

GASTRULATION

The highly integrated process ofcell movements, involving thewhole embryo, that leads toformation of the definitive tissue(germ) layers: endoderm,ectoderm and mesoderm.

NEURAL CREST CELLS

A migratory cell population thatarises at the lateral extremities ofthe embryonic neural plate, andwhich differentiates into variouscell types, depending on location.These include endothelial cells,smooth and skeletal muscle cells, bone, adrenal medulla,and cells of the sensory andautonomic nervous systems.

O R G A N O G E N E S I S

© 2002 Nature Publishing GroupNATURE REVIEWS | GENETICS VOLUME 3 | JULY 2002 | 545

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heart progenitors move through the NODE AND ORGANIZER

region and PRIMITIVE STREAK4,5, and take a lateral migratorypath towards the cranial and cranio-lateral parts of theembryo to form the cardiac crescent, which begins toexpress cardiac transcription factors3 (FIG. 1a,b; BOX 2a,b).The actual process of gastrulation might be relativelyunimportant for commitment to a cardiac cell fate. Forexample, mouse EPIBLAST cells will differentiate into car-diomyocytes if grafted directly into the heart-formingregion of post-gastrulation embryos, and cells from thecardiac crescent, when introduced back into the epi-blast, will re-gastrulate and contribute to different lin-eages, including heart3. However, such experimentsaddress more the plasticity of early mesodermal cellswhen placed in a new environment than they do thenature of commitment itself. Commitment to the heartlineages is now known to be progressive, spatially com-plex, and highly dependent on the evolving patterningprocesses and dynamic cell movements of gastrulation.Gastrulation allocates heart mesodermal cells to differ-ent embryonic niches from which they can be deployedat different times and in different ways to contribute toheart structure. So, the way that the heart field is per-ceived is shifting and aspects of its complexity will beaddressed in this and subsequent sections.

In most vertebrates, the heart progenitor cells lieadjacent to the progenitors of the head. In frogs andmammals, the tissue layers that are responsible forinducing the heart and head are topologically distinct,but are likely to be functionally analogous6–8. Headdevelopment requires that signalling by bone morpho-genetic proteins (BMPs), Wnt and Nodal ligands isblocked, and this is achieved in the extracellular milieuby specific inhibitors that are secreted from inducing tis-sues9,10. By contrast, heart lineages are induced at the lat-eral margins of the head precursor zone, where Wnt sig-nalling is still inhibited but BMP signalling ismaintained11,12 (FIG. 2). The endoderm that is in directcontact with cardiac mesoderm has received muchattention as a heart-inducing tissue in certain species13

and, indeed, members of the BMP family are expressedin endoderm, as well as in adjacent ectoderm and extra-embryonic tissues14–16.Various genetic and biochemicalperturbations in many organisms have shown a key rolefor BMP signalling in specifying and/or maintaining themyocardial lineage14,17–23. Furthermore, BMP-receptor-regulated transcription factors of the Smad family seemto directly activate genes that encode cardiac transcrip-tion factors24–27. In the multipotent P19 cell line, which isderived from a mouse embryonic carcinoma, dimethyl-sulphoxide-induced cardiogenesis is mediated by BMPand BMP-regulated Smads, and by the basic-leucine-zipper (bZIP) Smad-interacting protein, activatingtranscription factor 2 (Atf2), which can be phosphory-lated and activated by the mitogen-activated proteinkinase kinase kinase, Tak1 (REFS 22,28). The Tak1/Atf2pathway is one potential avenue for integrating BMPwith other signalling inputs. One such input might befrom the fibroblast growth factor Fgf8, which isexpressed in endoderm and seems to be induced byBMPs29. Fgf8 and BMPs are both required to induce

endocardial layer of the heart, which migrate and prolif-erate to form opposing tissue masses called endocardialcushions (BOX 2f ). Endocardial cushions also form in theoutflow region of the heart (BOX 2f ), giving rise to theaortic and pulmonary valves. NEURAL CREST CELLS that orig-inate in the hindbrain migrate into the outflow cushionsand participate in its septation. The outer layer of theheart (epicardium), the coronary circulation (BOX 1a)

and the interstitial fibroblasts of the heart are all derivedfrom a mesenchymal population located at the base ofthe developing heart called the pro-epicardial organ.

In the sections below, the molecular pathways guid-ing some of the transitions that take place during thedevelopment of the heart and that are outlined in BOX 2

will be described. Their order of presentation approxi-mately reflects the natural chronology of heart develop-ment, although it is essential to appreciate that theseevents overlap and interconnect.

Shaping the heart precursor zoneThe spatial coordinates of heart progenitor cells in thepre-gastrulation embryo have been mapped in severalvertebrate models3. During gastrulation in amniotes,

NODE AND ORGANIZER

Analogous structures inmouse/chick and frog embryos,respectively, that represent themain signalling centres in theearly period of body plandevelopment, and from whichthe axial lineages, such as theprechordal plate, notochord andgut endoderm, are derived.

PRIMITIVE STREAK

A morphogenetic furrowformed in embryos and throughwhich cells ingress atgastrulation.

EPIBLAST

Columnar epithelium that linesthe amniotic sac floor. This layergenerates endoderm andmesoderm by migration of cellsthrough the primitive streak.The remaining cells formectoderm.

Box 1 | The adult heart

The structural features of the adult human heart, whole (panel a) and in section (panel b),are shown. The structure of the mature heart reflects its embryonic development as amuscular tube (BOX 2) and the fact that its two pumping units work in series. The rightatrium (RA) receives venous blood from the body and passes it through the tricuspidvalve to the right ventricle (RV), which pumps it through the pulmonary artery to thelungs. Oxygenated blood from the lungs is returned to the left atrium (LA) of the heartthrough the pulmonary veins, and is passed to the left ventricle (LV) through the mitralvalve. Note that the tricuspid and mitral valves are supported by chords known aschordae tendineae (CT). From the left ventricle, blood is pumped through the aorta tothe arterial vascular circuit of the body. The coronary circulation branches off from theproximal portion of the aorta. The aorta and pulmonary artery are each guarded by avalve that prevents regurgitation. The heart beat is controlled by cells specialized forelectrical conduction that are organized into clusters (nodes) or tracts. The beat isinitiated at the sinuatrial node located at the junction between the right atrium and thesuperior caval vein. The electrical impulse is propagated throughout the atria and to theatrioventricular node (AVN), which is the conduit for the impulse to pass, after a delay, tothe ventricles. Rapid conduction occurs along the bundle of His and its bundle branchesto the ventricular apex, then throughout the ventricles by the Purkinje fibres. In the figurebelow, the compass indicates the orientation of the heart in the body. Ca, caudal(inferior); Cr, cranial (superior); L, left; R, right.

Superiorcaval vein Aorta

Pulmonaryartery

Left atrial appendage

SinuatrialnodeAVN

RV

Pulmonary valve

LA

LV

CTBundle of His

Mitral valve

Right atrial appendage

Inferiorcaval vein

Pulmonary veins

Purkinjefibres

Bundlebranches

RA

Coronaryarteries

Tricuspidvalve

Cr

Ca

R L

a b

Inter-ventricular septum

Aortic valve


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AMNIOTE

A reptile, bird or mammal, inwhich a membrane, called theamnion, separates the conceptusfrom its environment.

Box 2 | Heart development

This box depicts the main transitions that occur in early heart development in AMNIOTES (on the basis of the events in mousedevelopment; see main text for more details). The whole embryo or isolated heart is shown on the left, whereas on the right,a representative section (transverse in panels b and d; longitudinal in panels f and h) illustrates the main internal features.All views are ventral. Staging in days of embryonic development (E) is based on mouse development. The myocardium andits progenitors are indicated in red. The cardiac progenitors are first recognizable as a crescent-shaped epithelium (thecardiac crescent) at the cranial and cranio-lateral parts of the embryo (panels a and b). The progenitor population extendscranially and laterally almost to the junction between the embryonic and extra-embryonic regions of the embryo (red arrowin panel b). Next, heart progenitors move ventrally to form the linear heart tube, which is composed of an endothelial liningthat is shrouded by a myocardial epithelium (panels c and d). Note that the inflow region of the linear heart tube is locatedcaudally, and its outflow region is located cranially. The myocardium remains attached to the ventral foregut through thedorsal mesocardium in continuity with the dorsal pericardial mesoderm. The linear heart tube undergoes a complexprogression termed cardiac looping, in which the tubular heart adopts a spiral shape with its outer surface sweepingrightwards (panels e and f ). During looping, the inflow portion of the heart, including the common atrium, is forceddorsally and cranially so that it is now above the developing ventricles. The internal relief of the heart at this stage has becomecomplex (panel f ). Endocardial cushions (EC), the precursors of the tricuspid and mitral valves (BOX 1), are forming in theatrioventricular (AV) canal. Endocardial cushions also form in the outflow tract and these are the precursors of theaorticopulmonary septum,which divides the outflowtract into the aorta andpulmonary artery. Thesecushions also give rise to theaortic and pulmonaryvalves. Other features of thisdevelopmental stage are theformation of trabeculae (T),the spongiform layer ofmyocytes along the innersurface of the ventricles and the inter-ventricularseptum. During theremodelling phase of heartdevelopment (panels g andh), division of the heartchambers by septation iscompleted, and distinct leftand right ventricles (LV andRV, respectively) and leftand right atria (LA and RA,respectively) are evident.This is achieved by furtherspiralling of the heart tubesuch that the outflow regionbecomes wedged betweenthe developing ventricles onthe ventral side (panel g),and the inflow region spansthe ventricles dorsally(panel h). The chambersand vessels are now alignedas in the adult heart andbecome fully integrated.The muscular inter-atrialand inter-ventricular septaefuse with the non-muscularatrioventricular septum,which is derived from theendocardial cushions of theatrioventricular canal,therefore completing theseparation of the chambers.Ca, caudal (inferior); Cr,cranial (superior); L, left; R, right.

a b

c d

e f

g h

Cr

Ca

R L

Cr

Ca

R L

Cr

Ca

R L

T

RA LA

RV

EC

LV

T

Primitive foregutendoderm

Dorsalmesocardium

Foregut

EC

Inter-ventricularseptum

Cr

Ca

Inter-atrialseptum

AV septum

Inter-ventricular septum

Outflow tract

Common atrium

Neural epitheliumHead mesoderm

Intra-embryoniccoelom

Heart progenitors

EndocardiumMyocardium

Dorsal pericardialmesoderm

Forming right ventricleForming leftventricle

AV canal

Cardiac cresent E7.75

Linear heart tube E8.25

Looping heart E10.5

Remodelling heart E12.5


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induction of Nkx2-5 expression20. Conversely, in themouse, an early but not a late phase of Nkx2-5 expres-sion is inhibited in embryos that are mutant for theSmoothened gene (Smo), which encodes a membrane-bound signalling protein for Hedgehog-relatedligands42. This latter finding indicates that Hedgehogsignalling might be involved in the initiation of the car-diogenic programme in mammals. One Hedgehog lig-and, Indian hedgehog, is expressed during gastrulationin the endodermal layers that will make contact with thecardiac mesoderm43. Indian hedgehog can also induceexpression of the Bmp4 gene43, and this is consistentwith an upstream role for the gene in the cardiogenicpathway.

Early lineage decisionsOnce the cardiac progenitor zone has been established,the intra-embryonic coelom — a cavity that separatesprogenitors into heart mesoderm ventrally and pericar-dial mesoderm dorsally — is formed (FIG. 1c). At aroundthis time, a further lineage split occurs in the heart meso-dermal layer that separates myocardium — the muscularlayer of the heart — from endocardium, its endotheliallining44,45. Endocardial cells first appear between themyocardial and endodermal layers (FIG. 1b,c) and, in thechick, they express both myocardial and endocardialmarkers46,47. The underlying endoderm is required fortheir formation46. These findings indicate that cells of thecardiac crescent might be the common precursors formyocardial, pericardial and endocardial cells of theheart. Indeed, a recent analysis of the lineage fate of cellsin the mouse cardiac crescent using the Cre/Lox systemhas shown that all three of the above cell types have, atsome point, activated the myogenic transcription-factorgene Nkx2-5 (FIG. 1c), which is initially expressed in thecardiac crescent before intra-embryonic coelom forma-tion48,49. By contrast, however, experiments using retro-virus-mediated lineage tagging of single cells in chickembryos failed to reveal a common myocardial and

cardiogenesis29. Wnt inhibitors of the Frizzled-like andDickkopf families are also expressed in the endodermthat is juxtaposed to heart mesoderm and innodal/organizer tissues11,12 (FIG. 2). Although Wntinhibitors are necessary for heart induction, it is not yetclear whether they act directly on heart mesoderm, orwhether they act indirectly to confer a cranial characterto the endoderm11,12. Remarkably, however, the car-diomyogenic programme can be activated in posteriornon-cardiac mesoderm of chick and frog embryos inthe presence of both BMP and a Wnt inhibitor11,12,30;and, in frogs, such conditions can lead to formation ofectopic-beating myogenic heart tubes that are lined withendothelial cells12.

The cardiac crescent in amniotes is shaped not onlyby the positive influences that are provided by BMPs,Fgf8 and Wnt antagonists, but also through negativeinfluences from Wnts that are expressed in the neuraltube and from BMP signalling inhibitors that areexpressed in axial tissues30,31 (FIG. 2). BMPs might also actin a concentration-dependent manner to induce orrepress cardiogenesis29. Likewise, the balance of Wntsand Wnt antagonists might be crucial for determing cellfate12. In response to inductive signals, the cardiac cres-cent activates several transcriptional regulators of thecardiac programme — including Gata4/Gata5/Gata6,Nkx2-5 (NK2 transcription-factor related, locus 5),myocyte enhancer factor (Mef2b/Mef2c), Hand1/Hand2 (heart and neural crest derivatives expressedtranscript 1 or 2) and T-box 5/20 (Tbx5/Tbx20) (REFS 32–37) — and a positive cardiac cross-regulatorynetwork is established38. Several studies now indicatethat signalling inputs in addition to BMPs are requiredfor proper expression of these transcription-factorgenes14,19,20,39–42. For example, the expression of Nkx2-5seems to require several, temporally distinct signals. InXenopus, blocking BMP-mediated cardiogenesis by overexpressing dominant-negative forms ofBMP receptors inhibits only the maintenance not the

a

YS

b c

CC

CCFP

NP

PE

EP

SMCM

IEC

Figure 1 | The cardiac crescent. a | The cardiac crescent (CC), corresponding to the position of the heart progenitors, is shownhere in an embryonic stage (E) 7.5–8.0 mouse embryo (ventral view), and is highlighted by LacZ staining. The embryo was producedby crossing a mouse expressing Cre recombinase under the control of the cardiac-crescent-specific Nkx2-5 gene with a mousethat carries a Cre-dependent LacZ transgenic reporter48. The yolk sac (YS), which is part of the extra-embryonic region, is indicated. b | Transverse section through the caudal aspects of the cardiac crescent of the embryo shown in panel a. The box indicates theregion shown in panel c. c | Higher power view of the lateral wings of the cardiac crescent showing Nkx2-5–Cre expression inendocardial cell precursors that are located between cardiac mesoderm (CM) and subjacent endoderm. Adapted with permissionfrom REF. 48. EP, endothelial cell precursors; FP, foregut pocket; IEC, intra-embryonic coelom; NP, neural plate; PE, pharyngealendoderm; SM, somatic mesoderm.


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the extracellular matrix at the mesodermal–endodermalinterface. Blocking the interaction between fibronectinand integrin in avian embryos — for example, with anti-bodies58 — or in mice, by mutation of the fibronectin(Fn1) gene59, leads to complete or partial failure of thecardiac primordia to fuse, a condition termed cardiabifida. A more severe manifestation is seen in some Fn1mouse mutants on a genetic background from the 129Svinbred strain, in which cardiac cells reach the cranialregion, form a crescent and express myosin genes nor-mally, but never migrate ventrally to form a heart tube59.How the fibronectin gradient is established is unknown,although it might be formed downstream of morphogenand/or transcription-factor gradients that are establishedin the forming heart tube (see below).

Correct differentiation of embryonic endoderm iscrucially required for this second phase of migration,and several mutations affecting endoderm in zebrafishand mouse embryos partially disrupt the process, lead-ing to various degrees of severity of cardia bifida60,61.The mouse Gata4 and zebrafish Gata5 genes, for exam-ple, which encode transcription factors of the zinc-finger family, are required in endoderm for migration ofheart precursor cells to the midline, as well as for tran-scription of a host of cardiac genes62,63. The endodermeffect might be linked to the general failure of ventralmorphogenesis and foregut-pocket formation, and/orto the maturation of endoderm, which, as discussedabove, is a crucial source of cardiac-inducing andmigratory factors. The role of Gata4 in endoderm mightdiffer substantially from that in mesoderm, because theinteraction between Gata4 and a cofactor, Fog2, isrequired only in mesoderm64. The zebrafish mutationmiles apart, which also causes cardia bifida, has beenmapped to a gene encoding a G-protein-coupled recep-tor for sphingosine-1-phosphate, a bioactive lipid65.Members of this receptor family are known to mediatechemotaxis of human endothelial and aortic smooth-muscle cells, as well as many other biological processes66.Miles apart is required in cells other than cardiac precur-sors, indicating that its gene product probably indirectlycapacitates heart cells for migration65.

The primary heart fieldDuring the migrations discussed above, heart progeni-tors exist in a morphoregulatory field. Recent advancesin our understanding of signalling in the heart field haveuncovered new and important aspects of heart develop-ment. A morphoregulatory field has been described67 as“a dynamic region of developmental potency” for theformation of an organ or structure. A field is usuallydefined experimentally by assessing the potential ofsmall tissue explants taken from in and around theprogenitor region to differentiate into a particular struc-ture in vitro. A key finding is that fields are often largerthan the area that is fated to a specific tissue and are reg-ulative, in the sense that cells in a field that lie outside ofthe true progenitor zone can compensate for completeremoval or damage of those progenitors. In Xenopus,a regulative heart field occupies a region of cranio-ventral and lateral mesoderm that closely matches the

endocardial precursor cell45.Whenever endothelial prog-enitors were labelled, they gave rise only to endothelialprogeny, indicating that endocardium might already bespecified in the heart field.A possible explanation for thisapparent dichotomy is that the endothelium has severalorigins, and studies on avian embryos indicate that a dis-tinct population of endothelial precursor cells migratesinto the forming heart tube44,50.

Migration of heart precursorsTwo waves of cell migration bring heart progenitors tothe ventral midline where the heart tube is formed. Thefirst wave, a component of gastrulation, mobilizes heartand head mesendoderm from the node/organizer andprimitive streak towards the cranial regions of theembryo to form the cardiac crescent. This event requiresFgf8, as well as the basic helix–loop–helix (bHLH) tran-scription factors Mesp1 and Mesp2 (mesoderm posterior1 and 2) (REFS 51,52). Fgf4 is downregulated in mice thatare mutant for the Mesp1 and Mesp2 genes51,52, indicat-ing that Fgf4 might be their common target.Furthermore, it has been shown that the FGF receptor,Fgfr1, has a key role in cell migration duringgastrulation. FGF signalling through this receptor acti-vates the transcription-factor gene Snail, which theninduces an epithelial–mesenchymal transition in epiblastcells by repressing the expression of the gene encod-ing the calcium-dependent cell-adhesion molecule E-cadherin53. This might be a conserved mechanism forcell migration, because the FGF receptor Heartless isrequired for migration of heart mesoderm in flies54,55,and FGFs are broadly implicated in cell migration inother developing systems56. A second wave of migrationbrings established cardiac progenitor cells ventrally toform the heart tube57. This movement depends on the graded distribution of fibronectin along the cranio-caudal axis, and this fibronectin gradient is deposited in

Ectoderm

Neural plate

Inhibitory signalsfrom neuralplate (Wnts)

Headmesoderm

Anterior ectodermalsignal (BMP)

Anteriorendodermalsignal (BMP, Fgf8 and Wnt inhibitors)

Graded distribution of BMP activity

Cardiac mesoderm

Axialmesendodermalsignals (BMPinhibitors)

Figure 2 | Positive and negative signals that shape the cardiac progenitor zone.A transverse section of an E7.5–8.0 mouse embryo is used to illustrate the structure of thecardiac progenitor zone. Cardiac mesoderm is highlighted by LacZ staining (see also FIG. 1).Positive and negative interactions between tissue layers are denoted by arrows and bars,respectively. See main text for details. BMP, bone morphogenetic protein; Fgf8, fibroblast growthfactor 8; Wnt, wingless related.


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mesocardium and pericardium — as well as BRANCHIAL-

ARCH mesenchyme, migrate into the heart72–74; they areused to build the outflow tract, and possibly, in mouse,the right ventricle74 (FIG. 3). Explant assays show that theproximity of dorsal cells to right ventricular tissueunmasks their latent myogenic potency72. The cells ofthe secondary heart field express Fgf8 and Fgf10, whichmight confer on them migratory properties and/ormaintain myogenic potential29,73,74. These studies revealthe origin of a complete segment of the heart and oneway in which cells in a field can be held over for use inlater development. Defects in this process might under-lie the obvious outflow-tract malformations in mouseembryos that lack key cardiac transcription factors,such as Nkx2-5, Hand1 and Mef2c (REFS 76–78) and,indeed, those in human populations in which outflow-tract abnormalities account for about one-third of con-genital heart defects79. These data also account for thedelayed time of formation of the outflow tract75, andmight relate, in part, to the apparent differences in themode of transcriptional regulation of several individualgenes in different heart compartments24,80,81. The natureof interactions between the secondary heart-field cellsand migrating neural crest cells, which populate theoutflow tract and contribute crucially to its divisioninto the aortic and pulmonary arteries, will now be anactive area of research. Dorsal mesenchyme also con-tributes to formation of the atria82, indicating that sec-ondary heart-field cells might supplement the primarymyocardium at both poles of the developing heart.

Heart patterning in the cardiac crescentAs discussed above, the cardiac crescent gives rise toseveral progenitor populations, each with distinct tissuefates and cellular behaviours. These divisions mark thebeginning of heart-tube patterning and have a pro-found impact on heart form and function. Regionalgene-expression patterns seen in the cardiac crescenthighlight this point. In mouse, the Fgf8 and Fgf10genes, and an Fgf10-linked LacZ reporter transgene, areexpressed in a distinct domain that corresponds to themyocardial progenitor cells that are located closest tothe midline (FIG. 3, leftmost panel). This domain corre-sponds to the precursors of dorsal mesocardium andpericardial mesoderm — cells that constitute part ofthe secondary heart field74. So, it seems probable thatsecondary heart-field cells are already allocated andhave distinct cell behaviours at the cardiac crescentstage. Another mouse transgene — one that is con-trolled by a specific enhancer of the chick GATA6 gene— is expressed in cells of the cardiac crescent that are furthermost from the midline81 (FIG. 4a,b). In later development, expression of this transgene predomi-nantly marks cells that differentiate into the cardiac-conduction system, which is responsible for appropri-ately distributing the electrical impulse during theheart beat (FIG. 4c). These studies and others cited belowshow that unique regulatory domains are already estab-lished in the cardiac crescent, and strongly reinforce theview that it is during this period that the initial ele-ments of cardiac pattern are laid down.

expression domain of Nkx2-5 (REF. 68). Explants fromthis area have heart-forming potency, although potencygradually becomes restricted to an area correspondingprecisely to those precursors that will form the heart innormal development, and the restriction occurs at apoint that is downstream of Nkx2-5 expression. Cells ofthe heart field that lie outside of the definitive precursorpopulation are positioned most laterally, and normallycontribute to the dorsal mesocardium and dorsal peri-cardial mesoderm (BOX 2d). These are the epithelial layersthat are initially continuous with the myocardial layer ofthe heart tube on its dorsal side where it connects to theventral foregut68. The timing of myogenic differentia-tion and restriction of myogenic potential in lateral(regulatory) cells of the field seems to be mediated byNotch signalling69, which acts in cell-fate decisions inmany organisms70. Interestingly, expression of Serrate(which encodes one of the Notch ligands) is repressed inmyogenic cells of the heart as they differentiate, througha negative feedback loop that is activated in response toSerrate/Notch signalling. So, a tissue-specific interpreta-tion of Notch signalling is central to the resolution ofmyogenic and non-myogenic domains69.

The secondary heart fieldRegulative heart fields in amniotes have not beendefined. In chick embryos, for example, removing partor the entire heart field causes corresponding deficien-cies in the formed heart71,72. Even head mesoderm,which can autonomously differentiate into heart mus-cle in vitro when removed from inhibitory tissues30, isincapable of regulation in vivo 71. However, the dorsalmesocardium and pericardial mesoderm — tissues are regulatory for the frog heart — express the cardiactranscription-factor genes Nkx2-5 and Gata4(REFS 48,73), indicating that they could constitute a per-sisting (or secondary) heart field. Indeed, cells from thisregion of the embryo are now known to contribute toformation of the outflow tract72–74, a defined cardiacsegment that is added to the heart some time after theventricular and inflow regions have been formed75.Studies using vital dyes, as well as retroviral and trans-genic markers, have shown that cells that are dorsal tothe heart — which could potentially include dorsal

BRANCHIAL ARCHES

A series of paired segmentalstructures composed ofectoderm, mesoderm and neuralcrest cells that are positioned oneither side of the developingpharynx. In mammals, thebranchial arches contribute topharyngeal organs and to theconnective, skeletal, neural andvascular tissues of the head andneck.

Cr

Ca

R L

E7.75 E8.0 E8.25 E9.5

Primary heartfield

Secondary heart field

Formingheart tube

Right ventricle

Formingoutflow tract

Left ventricle

Atrioventricularcanal

Caudal heartprogenitors

Figure 3 | Primary and secondary heart fields. Drawings depict the relative position andmovement of secondary heart field cells (blue) relative to the primary heart field (red), from cardiac crescent through the looping stages of heart development in the mouse. Approximatestages in embryonic days of development (E) are shown. The compass indicates the body axes.Ca, caudal; Cr, cranial; L, left; R, right. Adapted with permission from REF. 74.


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later contribute to the caval veins91. Mouse embryos thatare treated with a RA synthesis inhibitor or a pan-RARantagonist also lack sinuatrial tissue89,92. The specificity ofthese treatments has now been confirmed genetically,with mouse embryos that have mutations in theRALDH2 gene (Aldh1a7) showing unlooped hearts thatlack a distinct sinuatrial region93 (FIG. 5c,d).

Correct sinuatrial development also requires Tbx5(REF. 94), the T-box family transcription-factor gene thatis mutated in Holt–Oram syndrome in humans. Tbx5 isexpressed in a graded fashion along the cranio-caudalaxis of the forming heart, with highest levels beingexpressed towards the caudal region33,95. Because Tbx5 isRA inducible93,96 and its graded distribution in the heartis flattened in Aldh1a7-mutant embryos93, RA signallingmight have a role in establishing its pattern. The heartand other tissues are known to be extremely sensitive tochanges in the dosage of T-box genes94, and gradedexpression patterns, as seen for Tbx5, might be crucial topatterning. Indeed, in homozygous Tbx5-knockoutmice, sinuatrial development is severely curtailed94 andtransgenic overexpression of Tbx5 in the ventricles leadsto abnormal ventricle morphology and downregulationof ventricle-specific genes96. These latter effects might bea mild example of the more striking phenotype that isseen in mouse and chick embryos that were treated withexcess RA at cardiac-crescent stages, in which heartprogenitors are improperly fused and most of the hearttissue acquires atrial characteristics89,96 (FIG. 5e,f).

The above findings further highlight the importanceof regional signalling events in the cardiac crescent toacquire pattern in the forming heart tube. Patterningevents are likely to be highly dynamic and linked in bothspace and time to the migratory events of gastrulation88.Interestingly, abnormalities in the timing of develop-mental events are often ignored as a possible cause ofcongenital heart defects. In the sections below, theprocesses that stabilize early patterning events and leadto chamber formation are discussed.

Establishing boundaries on the cranio-caudal axisThe heart is often regarded and depicted as a segmentedstructure97, although there is little evidence for aMETAMERIC construction. Should we then think of theheart tube as a continuum of regional specializations orare there developmental boundaries and, if so, how arethey formed and how do they relate to retinoid sig-nalling? The mammalian Hey genes encode members ofthe bHLH superfamily of transcription factors that arehomologous to the hairy/enhancer of split genes ofDrosophila, which are known to act in the Delta/Notchpathway98. Notably, Hey1 and Hey2 are also amongthose genes that are regionally expressed in heart prog-enitors at or before heart-tube fusion. At later stages,expression of Hey1 is restricted to the atria and outflowtract, and that of Hey2 to the ventricles98 — a comple-mentarity that is also evident in other tissues99. Hey pro-teins, like their Drosophila relatives that are active in theNotch pathway, seem to be transcriptional repres-sors100,101. Furthermore, Hey1 and Hey2 have beenshown to act downstream of Delta/Notch signalling

Heart-tube patterning: the cranio-caudal axisRegionalization in the cardiac crescent can also be seenin its cranio-caudal axis. Regional differences can beshown in chick embryos as a gradient in the rate atwhich clusters of myocyte contract, after explantationfrom different positions along the heart field83. Thisfunctional behaviour might relate to the opposingexpression gradients of Atp2a2 (ATPase, Ca2+ transport-ing, cardiac muscle, slow twitch 2) and Pln (phospho-lamban) — genes that encode proteins that regulateintracellular calcium flux and the timing of excitationand contraction84. Furthermore, only myocytes that areexplanted from the caudal region of the heart field acti-vate a myosin gene that is normally expressed only incaudal (sinuatrial) myocytes of the heart tube85.

It has long been known that excess retinoids cause arange of birth defects in vertebrates86. Furthermore, avianand rat models of vitamin A deficiency, as well as singleand multiple mouse knockouts for nuclear retinoic acidreceptor (RAR) and retinoid-X receptor (RXR) genes,have revealed that retinoids function in many aspects ofcardiac development86–88. The inherent cranio-caudalpositional identity in the cardiac crescent discussed aboveis sensitive to perturbation by retinoic acid (RA)85 and, inthe mouse, the TERATOGENIC effects of excess RA on theheart are effective only during cardiac-crescent stages89.Recent studies confirm a key role for RA signalling at theearliest stages of heart patterning. The distribution of reti-naldehyde dehydrogenase type 2 (RALDH2), which isresponsible for virtually all embryonic RA synthesis, andthe expression pattern of a retinoid-responsive transgene,show that both RA synthesis and activity are restricted tothe sinuatrial region of the heart field and forming hearttube89,90 (FIG. 5a,b). Furthermore, hearts from vitamin-A-deficient quail embryos are closed caudally and seem tolack sinuatrial tissue, including its venous tributaries that

TERATOGENIC

Able to cause birth defects.

METAMERIC

Composed of similar segments(metameres), as in the body planof segmented animals such asarthropods, and in embryonicstructures such as somites andrhombomeres of the hindbrain.

a

b

c

RAVR

EER

CC

AVB

Figure 4 | Regional expression of the GATA6–LacZ transgene in lateral aspects of thecardiac field. a | Embryonic day (E)7.5–8.0 mouse embryo showing expression of the transgene inthe cardiac crescent (CC). Oblique ventral view. b | Transverse section showing LacZ staining(arrowhead) only in the lateral aspect of the heart field (square bracket). c | Horizontal section of anE14.0 embryo showing transgene expression in right atrioventricular conduction ring (RAVR) andatrioventricular bundle (AVB). EER, extra-embryonic region. Reproduced with permission from REF.

81 © (2001) Elsevier Science.


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during SOMITOGENESIS and in vitro101–103. Hey proteins canheterodimerize with each other103, as well as with distinctbHLH proteins, including Hand1 and Hand2 (REF. 104),which might also act as transcriptional repressors104,105.Genetic studies in mice and zebrafish have shown thatHand genes are essential for specification and/or survivalof cardiomyocytes that are associated with specificchambers78,106–109. Indeed, in mouse embryos that aredoubly mutant for Nkx2-5 and Hand2, in which Hand1(and Hey2) are also downregulated, both left and rightventricles undergo apoptosis once they are specified106.The Notch signalling system can amplify and stabilizedifferences between cells — even small differencesbetween otherwise identical cells70 — resulting inboundary formation, cell-fate divergence and effects onthe cell cycle. Hey genes could work in this manner in theheart, for example, by creating stable differences betweenmyocardium of different chambers, or creating bound-aries between them. The congenital heart defects seen inhuman patients that carry mutations in Jagged1, whichencodes one of the Notch ligands, hint at direct involve-ment of upstream as well as downstream elements of theNotch pathway in the heart110.

Chamber formationIn amniotes, anatomical, electrophysiological and geneexpression data indicate that a specialized form ofmyocardium (the working myocardium of the cardiacchambers) is initially specified in defined zones at theouter curvature of the looping heart tube84,95. These dataindicate that working myocardium might form inresponse to the integration of cranio-caudal and dor-sal–ventral (D/V) patterning information in the form-ing heart.

One of the principal morphological manifestationsof ventricular specification at the outer curvature is theappearance of trabecular myocardium (trabeculae; seeBOX 2f,h), the spongiform inner muscular layer that is lessproliferative and more differentiated than the outerlayer. During the developmental period, it is likely thattrabeculae generate much of the contractile force of theheart and also serve to rapidly distribute the electricalimpulse for contraction throughout the ventricles. Inthe chick, the orientation of trabeculae is highlyordered111 and this intriguing feature of heart architec-ture is yet to be explored. Several genes are expressed atthe outer curvature in domains that overlap, at least inpart, the trabecular zone. This includes, in the mouse,genes that encode transcriptional regulators such asHand1, Cited1 and Irx1/Irx3/Irx5 (Iroquois-relatedhomeobox), the vasoactive hormone atrial natriureticfactor (Anf), conduction proteins of the connexin fam-ily and the muscle-specific cytoskeletal proteinChisel38,95,112,113. We can now define the outer curvature,and subdomains in it, as distinct transcriptional com-partments in the heart. Furthermore, it is evident thatchamber myocardium forms as a specialization of amore primitive form of muscle that is present in the pri-mary heart tube95, and activates conduction proteinsand cytoskeletal elements that are appropriate for thedemands of its specialized function.

SOMITOGENESIS

The process of progressiveformation, duringembryogenesis, of metamericmesodermal units (somites) thatrepresent the precursorstructures of dermis, skeletalmuscles and the axial skeleton.

a b

c d

e f

Figure 5 | Role of retinoic acid in heart development. a,b | Chick embryos (stage 9) showing expression of the atrial-specific myosin gene AMHC (blue staining; arrows), either alone(a) or together with immunohistochemical detection of RALDH2(retinaldehyde dehydrogenase type 2; orange; b), the enzymethat is responsible for retinoic acid (RA) synthesis in the embryo.Note the substantial overlap. Reproduced with permission fromREF. 147 © (2000) Elsevier Science. c,d | Scanning electronmicrographs of embryonic day (E)8.5 mouse embryos that arewild type (c) or homozygous mutant (d) for the RALDH2 gene.Note that the inflow systemic tributaries (indicated by arrows inc) are missing in the mutant heart, which is closed caudally.Reproduced with permission from REF. 93 © (2001) Company of Biologists Ltd. e,f | Results at E9.5 of treating mouseembryos at cardiac crescent stages with excess RA. Controluntreated embryos at this stage have a well-looped heart (notshown; see BOX 2, panel e). e | The zone of human alkalinephosphatase staining (blue), which indicates the expression of a sinuatrial-specific myosin transgene, is expanded in theincompletely fused and dysmorphogenic RA-treated heart. f | Immunostaining for the ventricle marker MLC2V (red),indicating severe diminishment of ventricular tissue in the RA-treated heart. Reproduced with permission from REF. 89 ©(1999) Company of Biologists Ltd. All views are ventral.


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neuregulin1 signalling pathway maintains expressionof Nkx2-5 and Tbx5, as well as numerous other car-diac transcription factor and downstream genes,in the developing left and possibly right ventricles (D. Li, M. Zhou and R.P.H., unpublished observa-tions). A rudimentary heart phenotype is also foundin mouse embryos that lack forkhead box H1a(Foxh1a), a gene that encodes Fast2, a winged helixDNA-binding and Smad-interacting factor that actsdownstream in the transforming growth factor-β(Tgf-β)(activin–Nodal) signalling pathway127,128.Fast2 has a key role in mesoderm formation andcranio-caudal patterning during gastrulation, andmany mutants do not form a heart at all. However,those that do fail to express markers of workingmyocardium, and the same is true of cardiomyocytesthat are derived by in vitro differentiation of homozy-gous-mutant embryonic stem cells (I. von Both and J. Wrana, personal communication).

An important repressive role for the T-box factorTbx2 in establishing the expression domains of Nppa inthe working myocardium has recently been shown129.Tbx2 is expressed in the myocardium of the formingheart in an evolving pattern that is always reciprocal tothat of Nppa. Furthermore, Tbx2 can form a repressivecomplex with Nkx2-5 on the proximal Nppa promoter,which competes with a Tbx5–Nkx2-5 complex thatpositively regulates Nppa expression. So, integration ofthe positive and negative modalities of the Nkx2-5 andTbx pathways helps to define the working myocardiumin the early heart. How signalling through Fast2 inte-grates with these pathways is yet to be explored.

The left–right frontierOne of the most vexing aspects of heart biology con-cerns the contribution of the embryonic left–right(L/R) axial system to heart patterning and morpho-genesis. This system specifies the lateral asymmetriesof the body and its organs. Its precise contributions toheart morphogenesis are poorly characterized; how-ever, on the basis of outcomes of disturbed laterality inhumans and animal models, we can clearly say that thepathway determines L/R differences in the morphol-ogy of the atria and their venous tributaries, and, morespeculatively, the direction and quality of ventricularbending. A spectrum of bizarre abnormalities canoccur if laterality is defective. These include mirror-image reversals of body asymmetry (situs inversus)and isomerisms, in which both the left and right partsof organs such as the heart and lungs develop withentirely left or right characteristics130,131. In the heart,only the atrial appendages (BOX 1) can be truly isomer-ized, as they derive from left and right cardiac primor-dia, respectively132. By contrast, the left (systemic) andright (pulmonary) ventricles arise in development in acranio-caudal arrangement, and receive contributionsfrom both left and right cardiac progenitor popula-tions132. The act of looping itself brings the ventriclesinto a L/R configuration. These fundamental differ-ences in L/R development of the atria and ventriclesare explored further below.

How are these patterns established? The neureg-ulin/Erbb signalling system is essential for the forma-tion of trabeculae114–116. Neuregulin1 is expressed inthe endocardium and signals to its membrane-boundtyrosine kinase co-receptors Erbb2 and Erbb4, whichare expressed in myocardium. Endocardium is sepa-rated from myocardium by a complex proteoglycanand glycosamino-glycan-rich matrix called car-diac jelly, and endocardium makes contact withmyocardium in numerous places through an invasiveprocess111. Neuregulin1 signalling might occur at thispoint of contact and/or across the cardiac-jelly matrixthrough its diffusible isoforms. Why trabeculae formonly on the outer curvature of the heart is probablyrelevant to the issue of how working myocardium isspecified. This spatial specificity seems not to be gov-erned by localized expression of the neuregulin co-receptor Erbb2, because the global expression of theErbb2 gene in myocardium through targeted integra-tion into the Nkx2-5 locus does not seem to expandthe zone of working myocardium117. The associationand functional coupling of Erbb2 with CD44, a mem-brane signalling protein that uses cardiac-jelly compo-nents such as hyaluronan as ligands118, hints at matrixelements being an important aspect of specification ofthe outer curvature. Indeed, trabeculae are completelylost in mouse embryos that have mutations in thehyaluronan synthetase-2 gene (Has2)119. Interestingly,the serotonin 2B receptor is also required for propergrowth and trabeculation of the ventricles, and fornormal levels of Erbb2 expression120. This receptor actsthrough the heterotrimeric GTPases Gα

qand Gα

11,

and embryos that are doubly mutant for their encod-ing genes also show a ventricular defect121. Studies onthe epidermal growth factor (EGF) receptor, anothermember of the Erbb family, indicate that the intracel-lular domain of Erbb receptors can act as an integra-tive platform for signalling through variousligand/receptor types, including G-protein-coupledreceptors, which activate the mitogen-activated protein kinase pathway122. Understanding these interactions might be necessary to fully appreciatechamber formation.

Several transcription factors are now implicated inthe formation of the outer-curvature zones that willbecome the working myocardium. The Holt–Oram T-box factor, Tbx5, interacts directly with the home-odomain factor Nkx2-5 and, in vitro, these factorscooperatively activate expression of the Anf (Nppa)123

and connexin 40 (Gja5) genes — markers of workingmyocardium in the embryo94,95. Both Tbx5 and Nkx2-5have also been implicated genetically in the formationof working myocardium. In homozygous-null Nkx2-5mutants, all tested markers of the outer curvature,including Nppa, Cited1, Chisel and Hand1, are abol-ished or severely downregulated38,77,112,124–126. Mutanthearts are unlooped and retain a molecular signaturethat is similar to that of the primary heart tube. Tbx5-mutant hearts also show poorly looped and hypoplas-tic hearts, and expression of the Nppa gene is abol-ished94. We have recently shown in the mouse that the


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Laterality and the Pitx2 gene. A key target of Nodal sig-nalling in left LPM and heart is the homeobox genepaired-like homeodomain transcription factor 2 (Pitx2)(FIG. 6a), known previously as the gene that is mutated inthe human autosomal dominant Rieger syndrome134. Thefunction of Pitx2 is not known, although overexpressionof Pitx2 in the right LPM of chick embryos induces for-mation of symmetrical unlooped hearts137, indicating apossible key role for this gene in heart looping. Curiously,ventricles bend normally to the right in Pitx2-knockoutmice, although mutant hearts develop septal and valvulardefects accompanied by right pulmonary and atrial iso-merism that is indicative of loss of left-sided identity138.Over-proliferation in trunk and body-wall mesoderm inPitx2 mutants indicates a possible role for this gene in regulating the cell cycle. An uncoupling of Pitx2expression and the direction of ventricular bending hasalso been observed in other models of disturbed laterality,such as in chick embryos in which N-cadherin has beenblocked and in IV/IV-mutated mice132,139.

The apparent paradox in the Pitx2-mutant pheno-type raises an interesting and important issue in theL/R field. Randomization of heart looping has beenassociated with abnormalities in the L/R pathway inmany systems. On the basis of influential theories onthe origins of laterality140,141, this is often taken to meanthat laterality confers only a bias in the otherwise ran-dom generation of molecular and morphologicalasymmetries135, rather than determine the nature ofheart looping directly. This stance implies that all thenecessary morphogenetic processes for looping areintrinsic to the heart. It is now evident that the situa-tion is more complex, highlighting the necessity toconsider separately the initial breaking of symmetry inthe early embryo and the interpretation of laterality byorgan precursors135. In many models of laterality per-turbation in different organisms, the normally reliableleft-sided expression of laterality effector genes such asNodal and Pitx2 is destabilized, and transcripts arefound sometimes on the left, right, both sides or not atall. There is little wonder that complex laterality distur-bances occur under these circumstances. However, inmouse embryos that are mutant for some of the genesthat act directly or indirectly in the laterality pathway,including T (brachyury), Gdf1 (growth differentiationfactor 1), Cfc1 (cripto, FRL1, cryptic family 1), Acvr2b(activin receptor 2b) and compound heterozygotes forNodal-mutant alleles, the flow of laterality informationto the left is blocked, rather than scrambled asdescribed above for the other models38. In the variousmutant strains, ventricular bending has been scored asrandom, only to the right, or absent. Importantly,however, where scored, embryos from these strainsshow right morphological isomerism of the atria andlungs, but, interestingly, no true reversal of heart loop-ing. Instead, they have either leftward- or rightward-heart looping, and a C-shaped ventricular bendingthat is highly abnormal and retains left and right ven-tricles in a cranio-caudal arrangement (FIG. 6c). Theabnormal laterality in these mutants leads to varioussevere cardiac abnormalities.

Since the first discovery of L/R molecular asymme-tries in the peri-node/organizer region of chick gas-trulae133, rapid progress has been made in unravellingthe molecular pathways that support L/R body asym-metry134,135. How bilateral symmetry in the embryo isfirst broken might differ between species. However, inall vertebrate models examined, one or more genesthat encode the Tgf-β family member, Nodal, areexpressed in the left LATERAL-PLATE MESODERM (LPM)extending into the left side of the heart field134 (FIG. 6a).Nodal is regarded as the principal transducer of later-ality information to, and in, left-sided progenitors ofvisceral organs, and recent progress begins to definethe exquisitely tight regulatory influences on theNodal system135. For example, Nodal signallingdirectly induces genes that encode the Nodal inhibitorproteins Lefty1 and Lefty2. Nodal can diffuse overmany cell diameters in the embryo, although the rapidinduction of Lefty proteins limits its signalling rangeand duration135. In the forming heart, Nodal andLefty2 are expressed along the left dorsal meso-cardium and pericardial mesoderm, encroaching intomyocardium only in the sinuatrial region136.

LATERAL-PLATE MESODERM

The mesoderm that is located inthe lateral region of the earlysomite-stage embryo.

JB3Sna/SnR

RV

LV

Left-sided Left-enriched

Nodal–Cryptic

Lefty2

SnR

Nkx2.5

Bmp2

Hand1

FlectinhLAMPPitx2

Right-enriched

Ckb

?Heart tube

a

b c

Cr

Ca

R L

Figure 6 | Left–right asymmetry pathways in the heart. a | Schematic of a heart tube showingthe essential components of the Nodal pathway, which is expressed in the left side of the heartfield. Also shown are genes reported to be enriched in the left or right sides of the heart in varioussystems (see REFS 38,134,135). b,c | Wild-type embryonic stage (E)8.5 mouse embryo andequivalent stage embryo mutant for the cryptic gene (Cfc1). Ventral views. Arrows in b indicate in-line relationship between left (LV) and right (RV) ventricles. Note these chambers are arranged ina cranio-caudal fashion in the mutant. Reproduced with permission from REF. 148 © (1999) ColdSpring Harbor Laboratory Press. BMP, bone morphogenetic protein; Ca, caudal; Ckb, creatinekinase, brain; Cr, cranial; hLAMP, heart lectin-associated myocardial proteins; JB3, fibrillin 2; L,left; Pitx, paired-like homedomain transcription factor 2; R, right; Sna, homologue of DrosophilaSnail; SnR, Snf1-related.


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mechanism integrates with the other patterning sys-tems that are active in the heart.

Summary and perspectivesAs discussed in this review, the migration, induction,patterning and morphogenesis of heart progenitorcells can now be described in some detail and manykey genes have been discovered. However, Bateson’sview of the metapattern (a higher-order pattern) wasone of a dance of interacting parts. To understandheart development further, we must first complete ourdefinition of its building blocks, then seek the points ofintegration between the various molecular systemsimpinging on the heart field and the forming hearttube. Many biological processes are likely to be nonlin-ear, and to see (and to appreciate) such events dynami-cally is perhaps one of the greatest challenges in thefield. Linking heart morphology and molecular mech-anism to congenital heart disease is also an importantgoal. The recent discovery that dominant mutations insingle cardiac transcription-factor genes in humanscan give rise to a whole spectrum of cardiac abnormal-ities as diverse as ATRIAL SEPTAL DEFECT and TETRALOGY OF

FALLOT143, underscores the deficiencies in our knowledgeof heart morphogenesis. Finally, the recent discoveryof substantial regenerative capacity in the heart144–146

identifies new dimensions in cardiac developmentalbiology that were previously unimagined, and high-lights the fact that the developmental blueprint, oncewritten, is maintained to some degree in the structureof the completed organ.

Are there any key messages buried in these complexdata? Furthermore, can it be said that ventricularbending is controlled by the laterality pathway? We cansafely say that laterality is not just a biasing mecha-nism, but contributes profoundly to heart morpho-genesis. It seems that heart looping has co-evolvedwith the L/R axial system and L/R signalling is a dedi-cated architect of cardiac structure. However, theuncoupling of Pitx2 expression and the perceiveddirection of ventricular bending raises doubts as to thevalidity of this scoring system as a method of assessingthe effects of disturbed laterality on the heart. It alsoraises questions concerning the extent to which lateral-ity might contribute to morphogenesis in the ventricu-lar region of the heart. On the one hand, the directionof ventricular bending could become destabilized iflaterality in the caudal heart is indeterminate or iso-merized. Alternatively, the grossly abnormal ventricu-lar bending that occurs in embryos that lack left-sidedNodal signalling indicates that laterality probably con-tributed to ventricular morphology. Indeed, theexpression of several genes in addition to Nodal, Lefty2and Pitx2 is enriched in the left or right sides of theventricles during looping in different species (FIG. 6a). Itis also feasible that a graded series of cell states is estab-lished across the ventricles in response to lateralityinformation, with signalling from the right potentiallybeing a key feature of the system142. These questionsremain unanswered; however, intense research in thisarea will soon produce new markers and insights intothe molecular basis of cardiac looping and how this

ATRIAL SEPTAL DEFECT

Abnormal development of theatrial septum in humans, whichleads to a persistentcommunication between leftand right atria, generallyprogressing to right heart failurein the middle years of life.

TETRALOGY OF FALLOT

Complex congenital heartabnormality showing ventricularseptal defect (hole in theinterventricular septum), anaorta that communicates withboth left and right ventricles,narrowing of the pulmonaryartery and right ventricularhypertrophy.

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AcknowledgementsI thank N. Rosenthal for the artwork in the boxes and in figure 3,and N. Groves and C. Biben for help in preparing the manu-script.

Online links

DATABASESThe following terms in this article are linked online to:FlyBase: http://flybase.bio.indiana.eduDelta | hairy/enhancer of split | Heartless | Hey | NotchLocusLink: http://www.ncbi.nlm.nih.gov/LocusLinkAcvr2b | Aldh1a7 | Anf | Atf2 | Atp2a2 | Bmp4 | CD44 | Cfc1 |Chisel | Cited1 | E-cadherin | EGF receptor | Erbb2 | Erbb4 | Fast2| Fgf4 | Fgf8 | Fgf10 | Fgfr1 | Fn1 | Fog2 | Foxh1a | Gata4 | Gata5 |Gata6 | Gdf1 | Gja5 | Hand1 | Hand2 | Has2 | Indian hedgehog |Irx1 | Irx3 | Irx5 | Jagged1 | Mef2b | Mef2c | Mesp1 | Mesp2 |neuregulin | Nkx2-5 | Nodal | Pitx2 | Pln | RALDH2 | retinoic acidreceptor | retinoid-X receptor | serotonin 2B receptor | Serrate |Smad family | Smo | Snail | T | Tak1 | Tbx5 | Tbx20 | Tgf-β | Wnt OMIM: http://www.ncbi.nlm.nih.gov/Omimcardia bifida | Holt–Oram syndrome | Rieger syndromeZFIN: http://zfin.orgmiles apartAccess to this interactive links box is free online.

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Organogenesis: Patterning the vertebrate heart