PatterningHeartNatReviewsGen02.pdf

REVIEWSBox 2 | Heart developmentAMNIOTEA reptile, bird or mammal, inwhich a membrane, called theamnion, separates the conceptusfrom its environment.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 ventriclesand the inter-ventricularseptum. During theremodelling phase of heartdevelopment (panels g andh), division of the heartRchambers 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 onRthe 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 theRendocardial cushions of theatrioventricular canal,therefore completing theseparation of the chambers.Ca, caudal (inferior); Cr,cranial (superior); L, left; R, right.CrCracCaCreCaCrgCaCardiac cresent E7.75CaLinear heart tube E8.25LLooping heart E10.5LRemodelling heart E12.5LbdForegutDorsalmesocardiumfInter-ventricularseptumhECPrimitive foregutendodermRARVTLAECLVTNeuralepitheliumHead mesodermIntra-embryoniccoelomHeartprogenitorsDorsal pericardialmesodermMyocardiumEndocardiumOutflow tractCommon atriumAV canalForming rightventricleForming leftventricleInter-atrialseptumAV septumInter-ventricularseptum546 | JULY 2002 | VOLUME 3 www.nature.com/reviews/genetics© 2002 Nature Publishing Group

REVIEWSabNPcYSCCPEIECCCFPSMEPCMFigure 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 reporter 48 . 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.cardiogenesis 29 . Wnt inhibitors of the Frizzled-like andDickkopf families are also expressed in the endodermthat is juxtaposed to heart mesoderm and innodal/organizer tissues 11,12 (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 endoderm 11,12 . Remarkably, however, the cardiomyogenicprogramme can be activated in posteriornon-cardiac mesoderm of chick and frog embryos inthe presence of both BMP and a Wnt inhibitor 11,12,30 ;and, in frogs, such conditions can lead to formation ofectopic-beating myogenic heart tubes that are lined withendothelial cells 12 .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 tissues 30,31 (FIG. 2). BMPs might also actin a concentration-dependent manner to induce orrepress cardiogenesis 29 . Likewise, the balance of Wntsand Wnt antagonists might be crucial for determing cellfate 12 . In response to inductive signals, the cardiac crescentactivates 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 established 38 . Several studies now indicatethat signalling inputs in addition to BMPs are requiredfor proper expression of these transcription-factorgenes 14,19,20,39–42 . For example, the expression of Nkx2-5seems to require several, temporally distinct signals. InXenopus, blocking BMP-mediated cardiogenesisby overexpressing dominant-negative forms ofBMP receptors inhibits only the maintenance not theinduction of Nkx2-5 expression 20 . Conversely, in themouse, an early but not a late phase of Nkx2-5 expressionis inhibited in embryos that are mutant for theSmoothened gene (Smo), which encodes a membraneboundsignalling protein for Hedgehog-relatedligands 42 . This latter finding indicates that Hedgehogsignalling might be involved in the initiation of the cardiogenicprogramme in mammals. One Hedgehog ligand,Indian hedgehog, is expressed during gastrulationin the endodermal layers that will make contact with thecardiac mesoderm 43 . Indian hedgehog can also induceexpression of the Bmp4 gene 43 , 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 pericardialmesoderm dorsally — is formed (FIG. 1c). At aroundthis time, a further lineage split occurs in the heart mesodermallayer that separates myocardium — the muscularlayer of the heart — from endocardium, its endotheliallining 44,45 . Endocardial cells first appear between themyocardial and endodermal layers (FIG. 1b,c) and, in thechick, they express both myocardial and endocardialmarkers 46,47 . The underlying endoderm is required fortheir formation 46 . 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 formation48,49 . By contrast, however, experiments using retrovirus-mediatedlineage tagging of single cells in chickembryos failed to reveal a common myocardial andNATURE REVIEWS | GENETICS VOLUME 3 | JULY 2002 | 547© 2002 Nature Publishing Group

REVIEWSAnteriorectodermalsignal(BMP)Anteriorendodermalsignal (BMP, Fgf8and Wnt inhibitors)Gradeddistributionof BMPactivityInhibitory signalsfrom neuralplate (Wnts)HeadmesodermAxialmesendodermalsignals (BMPinhibitors)Neural plateEctodermCardiacmesodermFigure 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.endocardial precursor cell 45 . Whenever endothelial progenitorswere 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 distinctpopulation of endothelial precursor cells migratesinto the forming heart tube 44,50 .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) transcriptionfactors Mesp1 and Mesp2 (mesoderm posterior1 and 2) (REFS 51,52). Fgf4 is downregulated in mice thatare mutant for the Mesp1 and Mesp2 genes 51,52 , indicatingthat 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 activatesthe transcription-factor gene Snail, which theninduces an epithelial–mesenchymal transition in epiblastcells by repressing the expression of the gene encodingthe calcium-dependent cell-adhesion moleculeE-cadherin 53 . This might be a conserved mechanism forcell migration, because the FGF receptor Heartless isrequired for migration of heart mesoderm in flies 54,55 ,and FGFs are broadly implicated in cell migration inother developing systems 56 . A second wave of migrationbrings established cardiac progenitor cells ventrally toform the heart tube 57 . This movement depends onthe graded distribution of fibronectin along the craniocaudalaxis, and this fibronectin gradient is deposited inthe extracellular matrix at the mesodermal–endodermalinterface. Blocking the interaction between fibronectinand integrin in avian embryos — for example, with antibodies58 — or in mice, by mutation of the fibronectin(Fn1) gene 59 , 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 normally,but never migrate ventrally to form a heart tube 59 .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, leadingto various degrees of severity of cardia bifida 60,61 .The mouse Gata4 and zebrafish Gata5 genes, for example,which encode transcription factors of the zincfingerfamily, are required in endoderm for migration ofheart precursor cells to the midline, as well as for transcriptionof a host of cardiac genes 62,63 . 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 mesoderm 64 . The zebrafish mutationmiles apart, which also causes cardia bifida, has beenmapped to a gene encoding a G-protein-coupled receptorfor sphingosine-1-phosphate, a bioactive lipid 65 .Members of this receptor family are known to mediatechemotaxis of human endothelial and aortic smoothmusclecells, as well as many other biological processes 66 .Miles apart is required in cells other than cardiac precursors,indicating that its gene product probably indirectlycapacitates heart cells for migration 65 .The primary heart fieldDuring the migrations discussed above, heart progenitorsexist in a morphoregulatory field. Recent advancesin our understanding of signalling in the heart field haveuncovered new and important aspects of heart development.A morphoregulatory field has been described 67 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 structurein vitro. A key finding is that fields are often largerthan the area that is fated to a specific tissue and are regulative,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 cranioventraland lateral mesoderm that closely matches the548 | JULY 2002 | VOLUME 3 www.nature.com/reviews/genetics© 2002 Nature Publishing Group

REVIEWSPrimary heartfieldSecondaryheart fieldRCrCaBRANCHIAL ARCHESA 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.LFormingheart tubeRightventricleE7.75 E8.0 E8.25 E9.5Formingoutflow tractLeft ventricleAtrioventricularcanalCaudal heartprogenitorsFigure 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), fromcardiac 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.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 pericardialmesoderm (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 foregut 68 . The timing of myogenic differentiationand restriction of myogenic potential in lateral(regulatory) cells of the field seems to be mediated byNotch signalling 69 , which acts in cell-fate decisions inmany organisms 70 . 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 interpretationof Notch signalling is central to the resolution ofmyogenic and non-myogenic domains 69 .The secondary heart fieldRegulative heart fields in amniotes have not beendefined. In chick embryos, for example, removing partor the entire heart field causes corresponding deficienciesin the formed heart 71,72 . Even head mesoderm,which can autonomously differentiate into heart musclein vitro when removed from inhibitory tissues 30 ,isincapable of regulation in vivo 71 . However, the dorsalmesocardium and pericardial mesoderm — tissuesare regulatory for the frog heart — express the cardiactranscription-factor genes Nkx2-5 and Gata4(REFS 48,73), indicating that they could constitute a persisting(or secondary) heart field. Indeed, cells from thisregion of the embryo are now known to contribute toformation of the outflow tract 72–74 , a defined cardiacsegment that is added to the heart some time after theventricular and inflow regions have been formed 75 .Studies using vital dyes, as well as retroviral and transgenicmarkers, have shown that cells that are dorsal tothe heart — which could potentially include dorsalmesocardium and pericardium — as well as BRANCHIAL-ARCH mesenchyme, migrate into the heart 72–74 ; they areused to build the outflow tract, and possibly, in mouse,the right ventricle 74 (FIG. 3). Explant assays show that theproximity of dorsal cells to right ventricular tissueunmasks their latent myogenic potency 72 . The cells ofthe secondary heart field express Fgf8 and Fgf10, whichmight confer on them migratory properties and/ormaintain myogenic potential 29,73,74 . 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 underliethe 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 outflowtractabnormalities account for about one-third of congenitalheart defects 79 . These data also account for thedelayed time of formation of the outflow tract 75 ,andmight relate, in part, to the apparent differences in themode of transcriptional regulation of several individualgenes in different heart compartments 24,80,81 . 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 contributesto formation of the atria 82 , indicating that secondaryheart-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 profoundimpact 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 correspondsto the precursors of dorsal mesocardium andpericardial mesoderm — cells that constitute part ofthe secondary heart field 74 . 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 controlledby a specific enhancer of the chick GATA6 gene— is expressed in cells of the cardiac crescent thatare furthermost from the midline 81 (FIG. 4a,b). In laterdevelopment, expression of this transgene predominantlymarks cells that differentiate into the cardiacconductionsystem, which is responsible for appropriatelydistributing the electrical impulse during theheart beat (FIG. 4c). These studies and others cited belowshow that unique regulatory domains are already establishedin the cardiac crescent, and strongly reinforce theview that it is during this period that the initial elementsof cardiac pattern are laid down.NATURE REVIEWS | GENETICS VOLUME 3 | JULY 2002 | 549© 2002 Nature Publishing Group

REVIEWSTERATOGENICAble to cause birth defects.METAMERICComposed of similar segments(metameres), as in the body planof segmented animals such asarthropods, and in embryonicstructures such as somites andrhombomeres of the hindbrain.aEERHeart-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 field 83 . Thisfunctional behaviour might relate to the opposingexpression gradients of Atp2a2 (ATPase, Ca 2+ transporting,cardiac muscle, slow twitch 2) and Pln (phospholamban)— genes that encode proteins that regulateintracellular calcium flux and the timing of excitationand contraction 84 . Furthermore, only myocytes that areexplanted from the caudal region of the heart field activatea myosin gene that is normally expressed only incaudal (sinuatrial) myocytes of the heart tube 85 .It has long been known that excess retinoids cause arange of birth defects in vertebrates 86 . 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 development 86–88 . 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 stages 89 .Recent studies confirm a key role for RA signalling at theearliest stages of heart patterning. The distribution of retinaldehydedehydrogenase 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 hearttube 89,90 (FIG. 5a,b). Furthermore, hearts from vitamin-Adeficientquail embryos are closed caudally and seem tolack sinuatrial tissue, including its venous tributaries thatclater contribute to the caval veins 91 . Mouse embryos thatare treated with a RA synthesis inhibitor or a pan-RARantagonist also lack sinuatrial tissue 89,92 . 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 region 93 (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 region 33,95 . Because Tbx5 isRA inducible 93,96 and its graded distribution in the heartis flattened in Aldh1a7-mutant embryos 93 , 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 genes 94 , and gradedexpression patterns, as seen for Tbx5, might be crucial topatterning. Indeed, in homozygous Tbx5-knockoutmice, sinuatrial development is severely curtailed 94 andtransgenic overexpression of Tbx5 in the ventricles leadsto abnormal ventricle morphology and downregulationof ventricle-specific genes 96 . 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 characteristics 89,96 (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 gastrulation 88 .Interestingly, abnormalities in the timing of developmentalevents 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.bCCRAVRFigure 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.AVBEstablishing boundaries on the cranio-caudal axisThe heart is often regarded and depicted as a segmentedstructure 97 , 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 signalling?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/Notchpathway 98 . Notably, Hey1 and Hey2 are also amongthose genes that are regionally expressed in heart progenitorsat or before heart-tube fusion. At later stages,expression of Hey1 is restricted to the atria and outflowtract, and that of Hey2 to the ventricles 98 — a complementaritythat is also evident in other tissues 99 .Hey proteins,like their Drosophila relatives that are active in theNotch pathway, seem to be transcriptional repressors100,101 . Furthermore, Hey1 and Hey2 have beenshown to act downstream of Delta/Notch signalling550 | JULY 2002 | VOLUME 3 www.nature.com/reviews/genetics© 2002 Nature Publishing Group

REVIEWSacbdduring SOMITOGENESIS and in vitro 101–103 . Hey proteins canheterodimerize with each other 103 , as well as with distinctbHLH proteins, including Hand1 and Hand2 (REF. 104),which might also act as transcriptional repressors 104,105 .Genetic studies in mice and zebrafish have shown thatHand genes are essential for specification and/or survivalof cardiomyocytes that are associated with specificchambers 78,106–109 . 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 specified 106 .The Notch signalling system can amplify and stabilizedifferences between cells — even small differencesbetween otherwise identical cells 70 — 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 boundariesbetween them. The congenital heart defects seen inhuman patients that carry mutations in Jagged1, whichencodes one of the Notch ligands, hint at direct involvementof upstream as well as downstream elements of theNotch pathway in the heart 110 .SOMITOGENESISThe process of progressiveformation, duringembryogenesis, of metamericmesodermal units (somites) thatrepresent the precursorstructures of dermis, skeletalmuscles and the axial skeleton.eFigure 5 | Role of retinoic acid in heart development.a,b | Chick embryos (stage 9) showing expression of the atrialspecificmyosin 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) Companyof 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 expressionof 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 theRA-treated heart. Reproduced with permission from REF. 89 ©(1999) Company of Biologists Ltd. All views are ventral.fChamber 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 tube 84,95 . These dataindicate that working myocardium might form inresponse to the integration of cranio-caudal and dorsal–ventral(D/V) patterning information in the formingheart.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 highlyordered 111 and this intriguing feature of heart architectureis 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 familyand the muscle-specific cytoskeletal proteinChisel 38,95,112,113 . We can now define the outer curvature,and subdomains in it, as distinct transcriptional compartmentsin the heart. Furthermore, it is evident thatchamber myocardium forms as a specialization of amore primitive form of muscle that is present in the primaryheart tube 95 , and activates conduction proteinsand cytoskeletal elements that are appropriate for thedemands of its specialized function.NATURE REVIEWS | GENETICS VOLUME 3 | JULY 2002 | 551© 2002 Nature Publishing Group

REVIEWSHow are these patterns established? The neuregulin/Erbbsignalling system is essential for the formationof trabeculae 114–116 . Neuregulin1 is expressed inthe endocardium and signals to its membrane-boundtyrosine kinase co-receptors Erbb2 and Erbb4, whichare expressed in myocardium. Endocardium is separatedfrom myocardium by a complex proteoglycanand glycosamino-glycan-rich matrix called cardiacjelly, and endocardium makes contact withmyocardium in numerous places through an invasiveprocess 111 . 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 governedby localized expression of the neuregulin coreceptorErbb2, because the global expression of theErbb2 gene in myocardium through targeted integrationinto the Nkx2-5 locus does not seem to expandthe zone of working myocardium 117 . The associationand functional coupling of Erbb2 with CD44,a membranesignalling protein that uses cardiac-jelly componentssuch as hyaluronan as ligands 118 , 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 expression 120 . This receptor actsthrough the heterotrimeric GTPases Gα qand Gα 11,and embryos that are doubly mutant for their encodinggenes also show a ventricular defect 121 . Studies onthe epidermal growth factor (EGF) receptor, anothermember of the Erbb family, indicate that the intracellulardomain of Erbb receptors can act as an integrativeplatform for signalling through variousligand/receptor types, including G-protein-coupledreceptors, which activate the mitogen-activatedprotein kinase pathway 122 . Understanding theseinteractions 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–OramT-box factor, Tbx5, interacts directly with the homeodomainfactor Nkx2-5 and, in vitro, these factorscooperatively activate expression of the Anf (Nppa) 123and connexin 40 (Gja5) genes — markers of workingmyocardium in the embryo 94,95 . 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 abolishedor severely downregulated 38,77,112,124–126 . 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 hypoplastichearts, and expression of the Nppa gene is abolished94 . We have recently shown in the mouse that theneuregulin1 signalling pathway maintains expressionof Nkx2-5 and Tbx5, as well as numerous other cardiactranscription factor and downstream genes,in the developing left and possibly right ventricles(D. Li, M. Zhou and R.P.H., unpublished observations).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 pathway 127,128 .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 homozygous-mutantembryonic stem cells (I. von Both andJ. 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 shown 129 .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 integrateswith these pathways is yet to be explored.The left–right frontierOne of the most vexing aspects of heart biology concernsthe contribution of the embryonic left–right(L/R) axial system to heart patterning and morphogenesis.This system specifies the lateral asymmetriesof the body and its organs. Its precise contributions toheart morphogenesis are poorly characterized; however,on the basis of outcomes of disturbed laterality inhumans and animal models, we can clearly say that thepathway determines L/R differences in the morphologyof 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 mirrorimagereversals 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 characteristics 130,131 . In the heart,only the atrial appendages (BOX 1) can be truly isomerized,as they derive from left and right cardiac primordia,respectively 132 . By contrast, the left (systemic) andright (pulmonary) ventricles arise in development in acranio-caudal arrangement, and receive contributionsfrom both left and right cardiac progenitor populations132 . The act of looping itself brings the ventriclesinto a L/R configuration. These fundamental differencesin L/R development of the atria and ventriclesare explored further below.552 | JULY 2002 | VOLUME 3 www.nature.com/reviews/genetics© 2002 Nature Publishing Group

REVIEWSRight-enrichedJB3Sna/SnRRabCrCaLHeart tubeLATERAL-PLATE MESODERMThe mesoderm that is located inthe lateral region of the earlysomite-stage embryo.Left-sidedLefty2SnRNodal–CrypticcPitx2Nkx2.5RVLVLeft-enrichedBmp2?Hand1CkbFlectinhLAMPFigure 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 indicatein-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.Since the first discovery of L/R molecular asymmetriesin the peri-node/organizer region of chick gastrulae133 , rapid progress has been made in unravellingthe molecular pathways that support L/R body asymmetry134,135 . 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 field 134 (FIG. 6a).Nodal is regarded as the principal transducer of lateralityinformation to, and in, left-sided progenitors ofvisceral organs, and recent progress begins to definethe exquisitely tight regulatory influences on theNodal system 135 . 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 duration 135 . In the forming heart, Nodal andLefty2 are expressed along the left dorsal mesocardiumand pericardial mesoderm, encroaching intomyocardium only in the sinuatrial region 136 .Laterality and the Pitx2 gene. A key target of Nodal signallingin 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 syndrome 134 .Thefunction of Pitx2 is not known, although overexpressionof Pitx2 in the right LPM of chick embryos induces formationof symmetrical unlooped hearts 137 , 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 isomerismthat is indicative of loss of left-sided identity 138 .Over-proliferation in trunk and body-wall mesoderm inPitx2 mutants indicates a possible role for this genein 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 mice 132,139 .The apparent paradox in the Pitx2-mutant phenotyperaises 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 laterality 140,141 , this is often taken to meanthat laterality confers only a bias in the otherwise randomgeneration of molecular and morphologicalasymmetries 135 , 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 situationis more complex, highlighting the necessity toconsider separately the initial breaking of symmetry inthe early embryo and the interpretation of laterality byorgan precursors 135 . In many models of laterality perturbationin 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 disturbancesoccur 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 models 38 . 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 looping.Instead, they have either leftward- or rightwardheartlooping, and a C-shaped ventricular bendingthat is highly abnormal and retains left and right ventriclesin a cranio-caudal arrangement (FIG. 6c).Theabnormal laterality in these mutants leads to varioussevere cardiac abnormalities.NATURE REVIEWS | GENETICS VOLUME 3 | JULY 2002 | 553© 2002 Nature Publishing Group

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Biben for help in preparing the manuscript.Online linksDATABASESThe 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 | WntOMIM: 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.556 | JULY 2002 | VOLUME 3 www.nature.com/reviews/genetics© 2002 Nature Publishing Group