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in sodium dodecyl sulfate-polyacrylamidegels. Evidence for a protein structure con-sisting of multiple identical phosphoryl-atable subunits. J Biol Chem 259:1834–1841.

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function in vivo. J Biol Chem 275:10,538–10,544.

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PII S1050-1738(03)00037-9 TCM

Vertebrate species display an externalbilateral symmetry. However, severalorgans—such as the gut, the heart, andthe spleen—have an asymmetric dispo-sition. The left–right positioning of thedifferent handed organs is identicalwithin a species, a condition dubbed situssolitus. In rare occasions, the entire bodyplan is inverted, a condition dubbed si-tus inversus, which is fully compatiblewith normal life. However, in othercases, some organs display the normalleft–right arrangement whereas othersdisplay the mirror-image arrangement,a condition termed situs ambiguus (het-erotaxia). In the heterotaxia syndromes,the heart displays a wide spectrum ofanomalies ranging from atrial septal de-fects (ASD) or ventricular septal defects(VSD) to life-threatening malformationssuch as double outlet right ventricle(DORV), common atrioventricular canal(CAVC), double inlet left ventricle (DILV),or atrial isomerism (AI). These malfor-mations may appear singly or in combi-nation (Icardo and Sanchez de la Vega1991). For example, CAVC often is ac-companied by DORV, AI, persistence ofthe sinus venosus, and anomalous venousreturn (Seo et al. 1992). Over the lastyears, new insights have been gainedabout the molecular mechanisms under-lying the spectrum of cardiac abnormal-ities associated with heterotaxia (Suppet al. 1997). This review highlights thearguments in favor of impaired left–right signaling as a putative moleculardeterminant of common cardiac con-genital malformations.

• The Left–Right Signaling Cascade

The heart is the first organ to displaymorphologic asymmetry. However, a left–right differential gene expression pro-gram starts earlier in development thandoes any visible cardiac asymmetry (Bur-dine and Schier 2000, Wright 2001, Yost1999). Regardless of the site and themethod of asymmetry initiation (Boor-man and Shimeld 2002), the intermedi-ate pathway converges in all speciesexamined on the activation of the TGF-�superfamily member nodal in the leftlateral plate mesoderm (LPM) at earlysomitogenesis (Collingnon et al. 1996,Lowe et al. 1996, Meno et al. 1996 and1998). A direct downstream target ofnodal is the bicoid-related homedomaintranscription factor Pitx2 (Campione et

The Role of Pitx2 during Cardiac DevelopmentLinking Left–Right Signaling and Congenital Heart DiseasesDiego Franco* and Marina Campione

Pitx2 is a bicoid-related homeodomain transcription factor that plays acritical role in directing cardiac asymmetric morphogenesis. EctopicPitx2c expression in the developing myocardium correlates withdouble outlet right ventricle (DORV) in laterality mutants. Pitx2 loss offunction experiments cause severe cardiovascular defects, such asatrial isomerism (AI), double inlet left ventricle, transposition of thegreat arteries (TGA), persistent truncus arteriosus (PTA), and abnormalaortic arch (AAA) remodeling. Current studies suggest that Pitx2-mediatedsignaling during cardiogenesis is conducted within three different celltypes: the myocardium, the cardiac neural crest (CNC) cells, and thepharyngeal arch mesenchyme. Impaired Pitx2 function in discretemyocardial regions seems to lead to DORV, AI, and possibly TGA. Onthe other hand, impaired Pitx2 expression in the CNC leads preferen-tially to PTA. AAA remodeling is likely to occur owing to impairedcross-talk of the CNC cells with the pharyngeal arch mesenchyme.Thus, Pitx2 appears to be directing left–right identity to the cardiacvenous components (e.g., the atria), whereas it appears to be modelingthe morphologic arrangement of distinct myocardial components inthe arterial pole. These data suggest that altered left–right signalingunderlies the etiology of several common congenital cardiac malfor-mations. (Trends Cardiovasc Med 2003;13:157–163) © 2003, ElsevierInc.

Diego Franco is at the Department of Experimental Biology, University of Jaén, Jaén, Spain.Marina Campione is at the National Research Council, Institute of Neurosciences, Departmentof Biomedical Sciences, University of Padova, Padova, Italy.

* Address correspondence to: Prof. Dr. Diego Franco, Department of Experimental Biology,Faculty of Health and Experimental Sciences, University of Jaén 23071, Jaén, Spain. Tel.: (�34)953-002763; fax: (�34) 953-012141; e-mail: dfranco@ujaen.es.

© 2003, Elsevier Inc. All rights reserved. 1050-1738/03/$-see front matter

158 TCM Vol. 13, No. 4, 2003

Figure 1

Figure 1. Pitx2 isoform expression during cardiogenesis. In the car-diac crescent stage (E7.5 mouse), Pitx2c is expressed only in the leftcardiac crescent (a). At the tubular heart stage (E8.0 mouse), expres-sion of Pitx2c is restricted to the left part of the tubular heart, span-ning beyond the myocardial boundaries at both poles (b). At the loop-ing heart stage (E8.5), Pitx2c remains expressed in the left-derivedtubular heart components, which are becoming located in an anterior–ventral position in the prospective ventricular chambers, whereas itmaintains its leftward position in the prospective atrial chambers (c).Pitx2c also is asymmetrically expressed in the left pharyngeal archmesenchyme at this stage (c). At E10.5, expression of Pitx2c is ob-served in the left inflow tract, the left atrial appendage myocardium,and the left atrioventricular canal myocardium, whereas it spans to-ward the ventral aspects of the prospective ventricles, coming backprogressively to the left-ventral aspect of the outflow tract myocar-dium (d). Pitx2c expression also is observed in the left pharyngealarch mesenchyme. Furthermore, expression of Pitx2 (most likely allthree isoforms) is observed in the migrating cardiac neural crest(CNC) cells in the pharyngeal mesenchyme. At fetal stages, Pitx2c be-comes restricted to the left atrioventricular canal and left atrium (e),including the left superior caval vein and the primary and secondary atrial septa (f). aa, aortic arches; ias, interatrial septum; in, inflow region;ivs, interventricular septum; la, left atrium; lcc, left cardiac crescent; lscv, left superior caval vein; lv, left ventricle; oft, outflow tract; out, outflowregion; pv, pulmonary veins; ra, right atrium; rcc, right cardiac crescent; rscv, right superior caval vein; rv, right ventricle.

Figure 2

Figure 2. Tissue-specific role of Pitx2 isoforms during cardiogenesis.The expression and the proposed role of the different Pitx2 isoforms inthe myocardium, cardiac neural crest (CNC) cells, and pharyngeal archmesenchyme is illustrated in this schematic representation. On the ba-sis of published data, we hypothesize that abnormal Pitx2 signaling inthe CNC leads to persistent truncus arteriosus (PTA) and abnormal aor-tic arch (AAA) remodeling, whereas in the pharyngeal arch mesen-chyme, Pitx2 seems to play a role exclusively in the AAA remodeling.Impaired cross-talk between CNC and non-CNC cells in the pharyngealarches may underlie the etiology of AAA remodeling. Within the myo-cardium, regional impairment of Pitx2 expression would lead to a widerange of cardiac malformations, such as double outlet right ventricle(DORV), transposition of the great arteries (TGA), double inlet left ven-tricle (DILV), atrial isomerism (AI), and abnormal venous return (AVR).Conditions such as atrial septal defects (ASD) and ventricular septal de-fects (VSD) might be secondary only to some of the previously men-tioned cardiac defects. It seems that all Pitx2 isoforms are expressed inthe CNC, whereas only Pitx2c plays a role in the non-CNC cells of thepharyngeal arches as well as in the developing myocardium. AA, aorticarches; as, aortic sac; avc/AVC, atrioventricular canal; endo, endoderm;IFT, inflow tract; la, left atrium; lv, left ventricle; OFT, outflow tract;phm, pharyngeal mesenchyme; ra, right atrium; rv, right ventricle.

Figure 3

TCM Vol. 13, No. 4, 2003 159

al. 1999, Logan et al. 1998, Piedra et al.1998, Ryan et al. 1998, Yoshioka et al.1998). The asymmetric cascade of geneexpression must be translated into asym-metric morphogenetic processes at theorgan level. Notably, nodal expressionis transient, whereas Pitx2 expression ismaintained during morphogenesis ofhanded organs such as the heart and thegut (Campione et al. 1999 and 2001).Thus, Pitx2 seems to be the moleculartransducer of embryonic left–right sig-naling at the organ level. Mutations inthe Pitx2 gene have been associated withAxenfeld-Rieger syndrome, which is char-acterized by ocular, craniofacial, andumbilical anomalies (Semina et al.1996). Curiously, cardiac abnormalitiesare uncommon in this syndrome, proba-bly due to the fact that only low levels ofPitx2 are required for correct cardiac de-velopment (Liu et al. 2001).

Molecular studies have demonstratedthat three different Pitx2 isoforms (Pitx2a,Pitx2b, and Pitx2c) are expressed through-out development, and are generated byalternative splicing and promoter usage(Schweickert et al. 2000). Recently, afourth isoform (Pitx2d) has been de-scribed, although it appears to occuronly in humans (Cox et al. 2002). Amongall isoforms, only Pitx2c is expressedasymmetrically within the LPM and thedeveloping heart (Campione et al. 2001,Schweickert et al. 2000).

• Pitx2c and Cardiac Looping

The distinct expression pattern of Pitx2cin the LPM at early stages of cardiac de-velopment suggested that Pitx2c mightplay a critical role in establishing thedirection of cardiac looping. A first setof Pitx2 overexpression experiments inchicken and Xenopus embryos resultedin abnormal cardiac looping (Campione

et al. 1999, Logan et al. 1998, Piedra etal. 1998, Ryan et al. 1998, Yoshioka et al.1998). However, expression of Pitx2c inthe LPM of distinct laterality mutantmodels with abnormal cardiac morpho-genesis, such as iv and inv mice, is ran-domly distributed, suggesting a role ofPitx2c in cardiac morphogenesis ratherthan merely in cardiac looping (Campi-one et al. 1999, Piedra et al. 1998). Infact, Pitx2abc null mutant mice andPitx2c isoform-specific null mutants re-vealed normal rightward looping, cast-ing doubts on the specific role of Pitx2 inthis process (Gage et al. 1999, Kitamuraet al. 1999, Liu et al. 2001 and 2002, Luet al. 1999).

• Functional Role of Pitx2 Isoforms duringCardiovascular Development

Pitx2abc null mice displayed normalcardiac looping, but developed severeembryonic malformations such as ab-normal body wall closure. Furthermore,severe cardiac malformations such asright AI (RAI), persistent truncus arterio-sus (PTA), DORV, ASD, and VSD (Gageet al. 1999, Kitamura et al. 1999, Liu etal. 2001, Lu et al. 1999) have been ob-served. Some of these cardiac defectsalso are observed in the Pitx2c isoform-specific knockout (DORV, RAI, ASD, andVSD), suggesting a critical role of Pitx2cduring cardiac morphogenesis that is notcompensated for by other isoforms—thatis, Pitx2a or Pitx2b (Liu et al. 2001 and2002). Notably, PTA has not been ob-served in the Pitx2c null mutants, sug-gesting that Pitx2a or Pitx2b can com-pensate for the lack of Pitx2c in specificcell types. Intriguingly, abnormal aorticarch (AAA) remodeling has been re-ported exclusively in the Pitx2c null mice.Overall, impaired Pitx2 signaling in dis-

tinct cell types seems to be an underly-ing etiology of those cardiac congenitalmalformations reported above.

• Pitx2 Tissue Distribution during Normal Cardiogenesis

In situ hybridization analyses and celllineage studies have provided detailedinformation on Pitx2 isoform distributionduring cardiogenesis, which has contrib-uted greatly to the understanding of theetiology of cardiac abnormalities foundin Pitx2 loss of function mice.

Cardiac development is a complex anddynamic process (Fishman and Chien1997). The first myocardial expressionof Pitx2c is observed at the cardiac cres-cent stage confined to the left cardiaccrescent (Figure 1), both in mouse andchicken. Subsequently, as the primitiveheart tube is formed, expression ofPitx2c remains confined to the left partof the forming cardiac tube (Campioneet al. 1999 and 2001). Soon after, theheart tube bends rightward, displayingthe first morphologic asymmetry ob-served in the developing embryo (Stals-berg 1969). Pitx2c transcript expressionfollows the torsion of the looping heartat the ventricular side but not at itsatrial position, acquiring progressively amore anterior–ventral location in theprospective ventricular chambers (Fig-ure 1). Fate mapping experiments inchicken embryos have demonstratedthat Pitx2c expressing cells emergingfrom the left side of the heart tube locateto the most ventral region of the ventric-ular chambers, whereas the cells derivedfrom the right of the straight cardiactube appear in the dorsal aspect of theventricular chambers (Campione et al.2001). The expression of Pitx2c is not re-stricted to the myocardial boundariesbut also is projected asymmetrically into

Figure 3. Molecular mechanisms mediating Pitx2-induced congenital heart diseases. Pitx2 plays a key role during cardiac morphogenesis in dif-ferent tissues such as the myocardium, cardiac neural crest cells (CNC), and pharyngeal arch mesenchyme. Asymmetric expression of Pitx2c ismediated by nodal expression in the lateral plate mesoderm (LPM). Therefore, laterality mouse models such as iv, inv, legless, and gene knock-outs for distinct components of the left–right signaling (such as lefty-1 and activin RII) display randomized expression of Pitx2 in the LPM,which in turn will be translated into abnormal expression of Pitx2 in discrete regions of developing myocardium. Aberrant expression of Pitx2in distinct regions of developing myocardium might thus lead to local impairment of the proliferation, and, therefore, generate a wide range ofdistinct cardiac congenital malformations, such as double outlet right ventricle (DORV), transposition of the great arteries (TGA), double inletleft ventricle (DILV), and atrial isomerism (AI). Within the CNC, the Wnt-1/Dvl2/�-catenin signaling pathway induces Pitx2 expression. Lack ofPitx2 in the CNC abolishes proliferation of these cells and apparently blocks CNC invasion of the outflow tract (OFT) endocardial cushions, lead-ing to persistent truncus arteriosus (PTA). Notably, Dvl2 null mice developed PTA at a rather low frequency (1/16), whereas DORV was more abun-dant (6/16) (Hamblet et al. 2002). These data suggest that Dvl2 affects the Pitx2 myocardial signaling. The role of Pitx2 in the non-CNC pharyn-geal arch mesenchyme is more enigmatic. It is not clear which molecular mechanisms underlie normal aortic arch remodeling. Interestingly,these arterial pole abnormalities are similar to those observed in CNC ablation experiments, suggesting that a cross-talk between non-CNC andCNC in the pharyngeal arches is necessary for normal development.

160 TCM Vol. 13, No. 4, 2003

the forming pharyngeal arches (Figure1) (Liu et al. 2002). With further devel-opment, Pitx2c expression is confined tothe left part of the inflow tract and atrio-ventricular canal, whereas Pitx2c ex-pression in the outflow tract smoothlyshifts from a ventral to a left position,spanning from the ventricular junction tothe aortic arches (Campione et al. 2001).Recently, it has been demonstrated thata subpopulation of the cardiac neuralcrest (CNC) cells in the pharyngeal archesalso displays Pitx2 expression (Figure 1)(Hamblet et al. 2002, Kioussi et al.2002), although the isoform distributionhas not been investigated yet. Expres-sion of Pitx2c declines during fetal stagesin the myocardium, and is not detect-able from E16.5 onward.

• Multiple Tissue Targets of Pitx2 during Cardiovascular Development

The development of the heart is a com-plex process in which distinct celltypes are involved. The promyocardialsheets of the cardiac crescents, giveraise to the myocardial and endocardialcomponents of the tubular heart (Fish-man and Chien 1997). Subsequently theheart loops and chamber myocardiumdevelops at discrete regions of the outercurvature of the heart (Christoffels et al.2000). With further development, a sub-set of migrating neural crest cells in-vades the endocardial cushions of theoutflow tract, playing a critical role innormal outflow tract (OFT) septation(Kirby et al. 1999). At approximately thesame stage, the heart recruits epicardiallyderived cells that eventually form theconnective tissue of the adult ventricularmyocardium and its vasculature (Perez-Pomares et al. 2002). The etiology of thedifferent congenital malformations in thePitx2 mutant mice can be dissected in lightof the available expression data on Pitx2distribution in three essential componentsof the cardiovascular system during devel-opment: the myocardium, the CNC, andpharyngeal arch mesenchyme (Figure 2).

• Pitx2 Signaling in the Developing Myocardium

The first experiments of gene deletionresulted in the absence of all Pitx2 iso-forms (Pitx2abc) during embryogenesis.In Pitx2abc null mice, the heart invari-

ably displayed RAI, abnormal venousreturn (AVR), and DORV. In a fraction ofPitx2abc null mutants, DILV, ASD, andVSD also were reported (Table 1) (Gageet al. 1999, Kitamura et al. 1999, Lin etal. 1999, Lu et al. 1999). More recently, aseries of Pitx2 hypomorphic mutants weregenerated (Liu et al. 2001). Interestingly,the hearts of Pitx2ab null mice devel-oped normally, suggesting that only Pitx2cis required for correct cardiac develop-ment. In fact, Pitx2c null mice recapitu-late most of the cardiac defects observedin the Pitx2abc null mice, such as RAIand DORV (Liu et al. 2002). Overallthese data suggest the asymmetrical Pitx2expression in the myocardium as thesource of the defective signal leading tothese cardiac congenital malformations.

Additional analysis of Pitx2c cardiacexpression in the iv mouse laterality mu-tant (Hummel and Chapman 1959) sup-ports this hypothesis. In iv mice, Pitx2cexpression was expressed normally or ina mirror-image arrangement. In othercases, and regardless of the direction ofcardiac looping, Pitx2 either was absentfrom the atria or bilaterally expressed(Campione et al. 2001). In those cases,aberrant atrial expression correlated withthe presence of AI, a situation in whichboth atria are morphologically identical.Additionally, we (Campione et al. 2001)showed that left–right differences areconverted into dorso–ventral in the de-veloping ventricles. In iv mice, we ob-served extended Pitx2c expression in the

dorsal and ventral portions of the ventri-cles in 48% of the hearts examined. This,to our knowledge, constitutes the first ev-idence of ventricular isomerism detect-able at the molecular level (Campione etal. 2001). More importantly, abnormalPitx2c expression in the atrial and ven-tricular chambers significantly correlatedwith abnormal outflow tract development,in particular with DORV (82%) (Figure 3)(Campione et al. 2001). It is importantto realize that ectopic atrial and/or ven-tricular Pitx2c expression, correlatingwith DORV, converges into a single anddiscrete myocardial region, the innercurvature of the heart. Thus, the asym-metric expression of Pitx2c in the devel-oping myocardium and its altered ex-pression profile in iv mice, has led us tohypothesize that ectopic expression ofthis transcription factor in the innercurvature of the heart is responsible, perse, for the development of certain out-flow tract abnormalities, such as DORV(Figure 2) (Campione et al. 2001 and 2002).

• Pitx2 and CNC Proliferation

A recent re-examination of the knockoutphenotype has revealed that almost allof the Pitx2abc null mice developed PTA(Kioussi et al. 2002), which is considereda hallmark of CNC deficiency (Kirby etal. 1999). Indeed, Pitx2 is expressed in asubpopulation of (Pax3 positive) CNCcells at E10.5 (Kioussi et al. 2002). Pitx2expression in the CNC seems to depend

Table 1. Cardiac phenotypes in Pitx2 null mutantsa

PTA DORV TGA AAA

Pitx2abc�/�b �100% Yes Yes ?

Pitx2ab�/�c No No No ?

Pitx2c�/�d No �100% No �100%

Wnte (� catenin�/�) �80% No No ?

Dvl2�/�f 8% 35% 8% ?

CNC ablationg �80% Yes Yes �80%

iv/ivh Yes �80% Yes ?

AAA, abnormal aortic arch; CNC, cardiac neural crest cells; DORV, double outlet right ventricle; PTA, persis-tent truncus arteriosus; TGA, transposition of the great arteries. a Cardiac malformations of the arterial pole observed in distinct Pitx2 null mutants, Wnt/Dvl/� catenin sig-naling pathway mutants, and CNC ablation experiments and the iv/iv laterality mice. Yes means that such amalformation is reported but there are no quantitative assessments.b Data from Gage et al. 1999, Kitamura et al. 1999, Liu et al. 2001, Lu et al. 1999.c Data from Liu et al. 2001.d Data from Liu et al. 2002.e Data from Kioussi et al. 2002.f Data from Hamblet et al. 2002.g Data from Kirby et al. 1999.h Data from Icardo and Sanchez de la Vega, 1991.

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on the Wnt/Dvl/�-catenin signaling path-way. Null mutants for the distinct com-ponents of this signaling pathway (Dvl2or Wnt-mediated recombinase of floxed� catenin) all lack Pitx2 expression inthe CNC and concomitantly display similarcardiac phenotypes, namely PTA, DORV,or TGA. It has been proposed that de-creased Pitx2 signaling in the CNC leadsto arrest of cell proliferation, resultingin PTA. These data are in line with thosederived from CNC ablation experiments,in which PTA is the most prominentphenotype (Kirby 1999). Interestingly,loss of Pitx2 expression in the CNC cellscorrelates with reduced proliferation inthe CNC cells, as assessed by BrdU stain-ing. It remains unclear, however, whetherlack of Pitx2 function in the CNC is re-sponsible for all the malformations atthe arterial pole of the heart. Along thisline, tracing the Pitx2 daughter cellsmight shed light on their role in arterialpole remodeling.

However, the phenotypes observed inthese two models are not fully compati-ble with just a CNC dysfunction (Figure2). Dvl2�/� mice exhibited predominantlya DORV phenotype, with merely a singlecase of PTA, whereas CNC-specific de-pletion of � catenin leads predominantlyto a PTA phenotype. As we understandit, Dvl2 function might impair Pitx2 ex-pression in the CNC cells, leading to PTA;but, most importantly, it impairs the myo-cardial Pitx2 expression, leading to DORV.On the other hand, depletion of � cate-nin in the CNC exclusively impairs theexpression of Pitx2 in the CNC cells, lead-ing to PTA (Figure 3).

• Pitx2c and Pharyngeal Arch Remodeling

In addition to its prominent functionduring myocardial development, a rolefor Pitx2c in the developing pharyngealarch mesenchyme has been postulatedrecently (Liu et al. 2002). Pitx2c is ex-pressed asymmetrically in the developingpharyngeal arches (Liu et al. 2002). NullPitx2c mutants have revealed a widerange of aortic arch anomalies, such asright aortic arch or double aortic arch(Liu et al. 2002). These cardiac pheno-types are rather similar to those observedin selective CNC ablation experiments inchicken. Intriguingly, CNC accumulatenormally in Pitx2c null embryos at E10.5,and invasion of the CNC cells into the

arterial pole seems to proceed normally(Liu et al. 2002), as assessed at E12.5 bycrossing the mutant mice into the Wnt-lacZ transgene background (Jiang et al.2000). Furthermore, no PTA develops inPitx2c null mice, which is considered thehallmark of CNC deficiency (Kirby et al.1999). Although differential isoform dis-tribution has not been investigated, it ispossible that the expression of Pitx2aand/or Pitx2b in the CNC cells can com-pensate for the lack of Pitx2c transcripts,thereby permitting normal cell migra-tion. Normal remodeling in the pharyn-geal arches requires cross-talk betweenmesenchymal cells and CNC cells, be-cause alteration of any of these cell com-ponents invariably leads to similar phe-notypical alterations (Figure 3). Atpresent, there is scarce information aboutthe molecular mechanisms underlyingthe cross-talk in the pharyngeal archmesenchyme. We hypothesize that themorphogenetic mechanisms leading toAAA remodeling in Pitx2c knockout micedo not depend on impaired CNC contri-bution to normal aortic arch remodeling,but on the absence of Pitx2c signaling inthe pharyngeal arch mesenchyme.

• Abnormal Laterality and Congenital Heart Disease: The Pitx2 Contribution

Taking into account all the above, wecan reasonably argue that Pitx2 playsa critical role during cardiogenesis in atissue-specific and isoform-specific man-ner. Overall, Pitx2 appears to be direct-ing left–right identity to the cardiacvenous components, whereas it appearsto be modeling the morphologic arrange-ment of distinct myocardial componentsin the arterial pole. These data suggestthat altered left–right signaling under-lies the etiology of several common con-genital cardiac malformations. As illus-trated in Figure 2, altered expression ofPitx2c in discrete regions of the develop-ing myocardium invariably leads toDORV (inner curvature) and AI (atrialappendages), respectively. These malfor-mations, therefore, can be consideredcharacteristic of dysfunction of asym-metric Pitx2c-mediated myocardial sig-naling. The abnormal expression of Pitx2cin distinct cardiac compartments corre-lates with a high incidence of DORV inabnormal laterality mutant mice. Thisfact sheds light on the relationship be-

tween impaired left–right signaling andcardiac septation. This hypothesis is fur-ther sustained by the fact that the mostfrequent cardiac malformation observedin Pitx2c null mice is DORV. However,the molecular mechanisms underlyingthe role of Pitx2 during myocardial de-velopment remains to be established. Aworking hypothesis has been proposedrecently (Campione et al. 2002) that sug-gests abnormal remodeling of the innercurvature as a mechanistic source forimpaired cardiac septation, leading toDORV, DILV, or both. For this to occur,Pitx2 would have to play a critical role incell proliferation, as it was postulatedrecently (Kioussi et al. 2002), becausenormal development of the inner curva-ture is mediated by a rightward shift ofthe atrioventricular canal myocardium(Kim et al. 2001). In the iv mutant micealtered atrial expression does not alwayscorrelate with altered Pitx2c expressionin the ventricles, suggesting a modularbase of Pitx2 action in discrete regionsof the developing myocardium, such asthe outflow tract/ventricles, the atrio-ventricular canal/atria, and the inflowtract (taking into account distinct cavaland mediastinal myocardial domains(Franco et al. 2000) (Figure 2). AlteredPitx2c signaling in any of those regionswould underlie the etiology of the wideheterogeneity of cardiac phenotypes ob-served in laterality syndrome patients.We would suggest that abnormal Pitx2expression in the caval/pulmonary myo-cardium would lead to abnormal venousreturn, whereas abnormal expression inthe atrioventricular canal would lead toeither DORV or DILV. Altered expressionin the outflow tract can underlie eitherTGA or DORV. Other cardiac malforma-tions, such as VSD and ASD, might beonly secondary. On the other hand, PTAresults from abnormal Pitx2 signaling inthe CNC (Figure 2). It seems likely thatany Pitx2 isoform—Pitx2a, Pitx2b, orPitx2c—would be sufficient for propersignaling in the CNC, because Pitx2cnull mice do not display PTA, nor doPitx2ab null mutants; a full PTA pene-trance, however, is observed in Pitx2abcnull mice. Normal aortic arch remodel-ing seems to be dependent on Pitx2c sig-naling in the developing pharyngealmesenchyme (Figure 2), although thereare scarce data with regard to the mor-phogenetic and molecular mechanismsunderlying these processes.

162 TCM Vol. 13, No. 4, 2003

• Perspectives

We have revised herein evidence thatPitx2 plays a critical role in distinct celltypes during cardiogenesis. Several as-pects, however, remain elusive. First, itis not clear why Pitx2 overexpressionexperiments lead to abnormal cardiaclooping, whereas lack of Pitx2 does notalter cardiac turning. Along the same line,it has been reported recently that ec-topic expression of Pitx2 correlates withectopic activation of flectin, an extracel-lular matrix protein asymmetrically ex-pressed during early cardiogenesis(Tsuda et al. 1996), which leads to abnor-mal cardiac looping (Linask et al. 2002).

Second, there is unequivocal evidencethat abnormal expression of Pitx2c in themyocardium invariably leads to congeni-tal heart diseases such as DORV, DILV,and RAI. However, limited informationis available with regard to the moleculareffectors of Pitx2 in the myocardial cells,whether such downstream genes are ac-tivated locally, or whether they are dis-tributed in a compartment-specific fash-ion. The goal of future research shouldbe to dissect genetically the local contri-bution of Pitx2 signaling in discrete re-gions of the developing cardiovascularsystem as well as to identify those Pitx2downstream genes in the myocardium.

Third, the involvement of Pitx2 dur-ing CNC population of the embryonicheart should be explored in more detail.It remains unclear whether all isoforms(Pitx2abc) are expressed in the CNCcells. Moreover, clarification is neededto understand whether CNC migrateinto the developing heart in absence ofPitx2 signaling, and what are the molec-ular and morphologic mechanisms bywhich PTA is generated.

• Acknowledgments

The authors would like to thank AmeliaAránega and Francisco Navarro for theircritical reading of the manuscript. D.F.is supported by a grant from the Minis-try of Science and Technology of theSpanish Government (grant #BCM2000-0118-C02-01).

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PII S1050-1738(03)00039-2 TCM

Analysis of Gene Expression (SAGE) tech-nique can be applied to cardiovascularmedicine, based on some of the lessonslearned from successful microarraysstudies. For additional information onother differential gene expression meth-ods, the reader is referred to more com-prehensive technical reviews (Carulli etal. 1998, Fryer et al. 2002).

The basic principle of the SAGE tech-nique is that a short stretch of DNAsequence (tag) is of sufficient complexityto identify a unique gene transcript(Polyak and Riggins 2001, Velculescuet al. 1995). These sequence tags are ob-tained from mRNA as schematized inFigure 1, and the tags are serially concat-enated and sequenced for high through-put. The SAGE technique involves multi-ple steps with potential pitfalls; therefore,the reader is referred to more detailedSAGE reviews (Fryer et al. 2002, Patinoet al. 2002, Ye et al. 2002) and resourceWebsites (www.sagenet.org; www.ncbi.nlm.nih.gov/SAGE/). SAGE data analy-sis also requires an extensive genetic se-quence database for gene identification;thus, the studied organism’s genome needsto have been characterized to some ex-tent. Some discussions on the bioinfor-matic aspects of the SAGE techniquecan be found in several excellent reviews(Clark et al. 2002, Fryer et al. 2002,Stollberg et al. 2000).

Since its first description, the SAGEtechnique has undergone a number ofmodifications to simplify it and to in-crease its efficiency for examining verysmall numbers of cells or even a singlecell. One notable modification, calledLongSAGE, uses a different tagging en-zyme to create a longer 17-base pair(bp) SAGE tag (Saha et al. 2002). Theadditional complexity of this longer tagis sufficient to directly map it to the hu-man genome sequence rather than tothe expressed gene database as withthe original SAGE method. The Long-SAGE modification significantly allevi-ates the problem of multiple gene asso-ciations with a single tag, but it doesnot eliminate entirely all gene assign-ment ambiguities.

• Comparison of DNA Microarray and SAGE Techniques

The salient features of the microarrayand SAGE techniques are summarizedin Table 1. The previous differences in

Current and Future Applications of SAGE to Cardiovascular MedicineWillmar D. Patino, Omar Y. Mian, Yukitaka Shizukuda, and Paul M. Hwang*

The recently sequenced mammalian genomes represent unprecedentedresources for advancing our understanding of human diseases. Character-izing gene expression is an important step in translating genomicsequences into clinically useful information. Currently, gene expressionstudies are revolutionizing the approaches taken to address both basic sci-ence and clinical questions. Two major methods have emerged for the glo-bal examination of the transcriptome: microarrays and Serial Analysis ofGene Expression (SAGE). The SAGE technique comprehensively mapsgene transcription by using the genomic database, yet it remains relativelyunderutilized for studying cardiovascular biology. This review describescurrent cardiovascular studies using the SAGE technique and outlinessome potential strategies for employing this powerful tool to further ourunderstanding of the cardiovascular system in health and in disease.(Trends Cardiovasc Med 2003;13:163–168) © 2003, Elsevier Inc.

Willmar D. Patino, Omar Y. Mian, YukitakaShizukuda, and Paul M. Hwang are at theCardiovascular Branch, National Heart, Lung,and Blood Institute, The National Institutesof Health, Bethesda, Maryland, USA

* Address correspondence to: P.M. Hwang,NHLBI-NIH, Building 10, Room 7B15, 10Center Drive, Bethesda, MD 20892, USA. Tel.:(�1) 301-435-3068; fax: (�1) 301-402-0888;e-mail: hwangp@nhlbi.nih.gov.

1050-1738/03/$-see front matter. Publishedby Elsevier Inc.

• Serial Analysis of Gene Expression

Although there are several well-establishedtechniques for evaluating differences ingene expression—such as subtractive hy-bridization and differential-display reversetranscriptase-polymerase chain reaction—microarrays have gained widespread usein translational medical research, mostnotably in cancer genetic studies. Thisreview focuses on determining ways inwhich the unique strengths of the Serial