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Vol. 55. Issue 9.
Pages 962-974 (September 2002)
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Congenital Heart Disease, Heterotaxia and Laterality
Malformaciones cardíacas, heterotaxia y lateralidad
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José Manuel Icardoa, Juan Manuel García Rincóna, María Ángeles Rosa
a Departamento de Anatomía y Departamento deBiología Celular. Facultad de Medicina. Universidad de Cantabria. Santander. España.
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Fig. 1. Scanning electron microscopy. Mouse hearts, E10.5. a) situs solitus; b) situs inversus. Both hearts are morphologically normal and appear as mirror images. RA indicates right atrium; LV, left ventricle; CT, cono-trunk. The arrows in a and b indicate the interventricular furrow. a) b:×95. (Reproduced from Icardo, 1997.)
Fig. 2. In the chick embryo, the initially symmetrical expression of Shh in the nodule of Hensen (NH) is restricted to the right side by the activity of activinß mediated by Bmp4. Shh induces the expression of Lefty1 in the left half of the midline as well as Nodal and Car. The expression of Nodal in the left lateral mesoderm (LLM) is facilitated by the expression of CFC under the control of BMP. On the side right, the activin pathway induces Fgf8, which in turn induces cSnR and prevents the expression of Nodal.
Fig. 3. a: Expression of Nodal in a) 4-somite chick embryo; b) expression of Lefty1 in a 4-somite chick embryo; c: expression of Pitx2 in a 4-somite chick embryo; d) expression of mPitx2 in a mouse embryo in E8.25; e) expression of mPitx2 in the heart of an iv/iv mouse with right loop in E11.5; f) expression of mPitx2 in the heart of an iv/iv mouse with left loop in E11.5; g) bilateral atrial expression of mPitx2 in E11.5 with a loop to the right; h) absence of atrial expression of mPitx2 in a heart with a loop to the left in E11.5; i) right view of the heart of an iv/iv mouse in E11.5 which shows expansion of the expression of mPitx2 on the dorsal wall of the right ventricle; the right atrium (asterisk) is also positive; j) scanning electron microphotograph showing DORV (double date) in a heart that showed dorsal expression of mPitx2 in the right ventricle in E11.5. RA indicates right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle. (Figures e-j) reproduced from Campione et al, 2001.)
Fig. 4. Scanning electron microscopy showing the internal aspect of a heart (E18.5) with common atrioventricular canal and double outlet right ventricle. Situs solitus. The heart has been sectioned in the frontal plane and the ventral a) and dorsal b) halves are shown. The two ventricles communicate through a large ventricular septal defect that occupies approximately one-half of the ventricular height. The anterior leaflet (arrows in b) of the common atrioventricular valve is joined to the right side of the ventricular septum by a single papillary muscle. The two great vessels arise from the right ventricle by separate outflow chambers (arrows in a). A muscular band separates the origin of both chambers. The atria are symmetrical, even exhibiting symmetrical anterior prolongations (stars in b). Ao indicates aorta; P, pulmonary artery; a, b, ×45. (Reproduced from Icardo and Sánchez de Vega, 1991.)
Fig. 5. E10.5. Situs inversus. This heart is displaced toward the side right of the embryo. The right ventricle (RV) and cono-trunk (CT) form a U with no clear separation. The right ventricle is cephalad with respect to left ventricle (LV). The arrow indicates the interventricular furrow. ×45.
Fig. 6. E10.5. Situs solitus. The two ventricles do not maintain the characteristic lateral relation. The interventricular (arrow) and conoventricular (arrowhead) furrows appear abnormally marked. Compare the morphology of the hearts shown in Figures 5 and 6 with those that appear in Figure 1, which are in the same stage of development. ×110.
Fig. 7. This photographs shows the ventral (a) and dorsal (b) halves of a malformed heart (E12.5), which has been sectioned in the frontal plane Situs inversus. The ventricles and interventricular septum are sectioned tangentally, indicating an abnormal spatial position. The right ventricle is much more anterior than the left ventricle. The septum primum (Sp) and septum secundum (Ss) appear in b. Note the abnormal spatial disposition of the atrial and ventricular septa. The dorsal cushion (in b) appears bifurcated. The two great arteries arise from the right ventricle (arrow in a). a and b: ×85.
Fig. 8. Normal heart (E13.5). Situs solitus. The heart has been dissected in the parasagittal plane. The dissection plane passes through the left side of the atrioventricular region. The right half is observed after eliminating the left fragment. The atrioventricular cushions are fusing and the septum primum (arrow) continues into the septal cushion. The ventricular septal defect (arrowhead) remains open. P indicates pulmonary artery. ×90.
Fig. 9. Malformed heart (E.13.5). Situs solitus. The preparation is similar to that in Figure 8. The ventral (v) and dorsal (d) atrioventricular cushions have not fused. The septum primum (arrow) is normal, but does not contact the cushions. The arrowhead indicates the ventricular septal defect. P indicates pulmonary artery. ×90.
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Congenital heart disease occurs in about 0,8% of all newborns. Many cardiac malformations occur among relatives and have a polymorphic presentation. The origin of most congenital heart disease is thought to be multifactorial, implying both anomalous expression of genes and the influence of epigenetic factors. However, in a small number of cases, the origin of congenital heart disease has been directly related to chromosomal anomalies or to defects in a single gene. Curiously, defects in a single gene can explain a polymorphic presentation if the anomalous gene controls a basic embryonic process that affects different organs in time and space. Some of these genes appear to control the establishment of laterality. The establishment of the left-right asymmetry starts at the Hensen node. Here, the initial embryonic symmetry is broken by cascades of gene activation that confer specific properties on the left and right sides of the embryo. Although there are variations between species, some basic patterns of gene expression (Nodal, Pitx2) appear to be maintained along the phylogenetic scale. Anomalous expression of these genes induces the heterotaxia syndrome, which usually courses with congenital heart disease. The development of heart malformations is illustrated with the mouse mutant iv/iv, which is a model for the heterotaxia syndrome and the associated congenital heart disease.
Keywords:
Congenital heart disease
Heterotaxia
Left-right asymmetry
Las malformaciones cardíacas ocurren en aproximadamente el 0,8% de todos los nacidos vivos. Muchas de estas malformaciones se presentan en grupos familiares y muestran un tremendo polimorfismo. El origen de la mayor parte de las malformaciones cardíacas se desconoce, estableciéndose lo que se ha llamado un origen multifactorial. Aunque este término implica la expresión anómala de genes y la intervención de factores epigenéticos, el desarrollo de las malformaciones cardíacas se asocia en algunos casos a anomalías cromosómicas o a defectos de un único gen. Curiosamente, defectos de un único gen pueden explicar gran parte de las presentaciones polimórficas si este gen controla procesos embrionarios básicos que afectan, en tiempo y espacio diferentes, a distintos órganos. Algunos de estos genes parecen estar implicados en el establecimiento de la lateralidad embrionaria. El establecimiento izquierda-derecha del eje embrionario comienza en el nódulo de Hensen donde se rompe la simetría inicial y se inducen cascadas de expresión génica que confieren a cada lado del embrión propiedades específicas. Aunque los desencadenantes de la ruptura inicial de la simetría varían entre las diferentes especies, existen patrones de expresión génica (Nodal, Pitx2) conservados a lo largo de la escala filogenética. La expresión anormal de estos genes induce la aparición del síndrome de heterotaxia, que se acompaña de malformaciones cardíacas. El desarrollo de estas malformaciones se ilustra con la mutante de ratón iv/iv, que constituye un modelo del síndrome de heterotaxia y las malformaciones cardíacas asociadas.
Palabras clave:
Malformaciones cardíacas
Heterotaxia
Asimetría izquierda-derecha
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INTRODUCTION

Cardiacmalformations represent almost half of the malformationsencountered at birth. Approximately 0.8% of all live births presentthis type of malformations.1-3 The high incidence ofcongenital malformations of the heart has originated an intensivesearch to identify the factors involved in the development of thesemalformations. However, the results of this search have beendisappointing and 90% of all the malformations continue to be ofunknown origin. In these cases we refer to a multifactorial origin,a term that indicates more about our ignorance of the topic ratherthan what we really understand. It seems logical, however, that thedevelopment of these malformations could be due as much to geneticas to environmental factors. In fact, it has been postulated thatenvironmental factors, which act in genetically predisposedindividuals, activate the anomalous expression of genes until thethreshold of normality is breached, which is when they induce thedevelopment of a given malformation. The abnormal expression of allthe genes involved would result in the production of a severedefect, often incompatible with life, while a disturbance in onlypart of these genes would cause much milder defects (or theirabsence). This would explain the presence of intermediate orsubclinical forms that can be considered frustrated forms of thebasic hereditary defect.5-8 The variable phenotypicalexpression occurs in both family groups3,4 and in animalmodels of cardiac malformations.5,9,10

CHROMOSOMAL ANOMALIES. SINGLE-GENE DEFECTS

Although ourreal knowledge of the origin of most of the cardiac malformationsis fairly imprecise, it should not be overlooked that a geneticorigin has been clearly established in a small number of cases. Therelation between the presence of chromosomal anomalies and cardiacmalformations is well known. These anomalies can be numerical, dueto the absence of chromosomal disjunction, or structural, due tochromosomal breaks and loss of the broken fragment or itstranslocation to another chromosome. Among the numerical anomalies,trisomy 21 is associated in one-half of the cases with complexmalformations, especially common atrioventricular canal andventriculoarterial discordance.2 The study of cases withpartial trisomy of this chromosome has shown that the origin of theabnormal cardiac phenotype resides in band q22 of the long arm ofthis chromosome.11 Trisomy 18 is accompanied by a largenumber of cases of atrial and ventricular septal defect, as well asvalvular dysplasia in 100% of cases.12 Trisomy 13 isassociated with a high percentage of dextrocardia and tetrasomy ofthe short arm of chromosome 22 (cat´s eye syndrome) isassociated with anomalous pulmonary venous return.13 Thesame occurs in cases of failure of the disjunction of the sexualchromosomes, such as the Turner syndrome (which is associated withaortic coarctation and aortic stenosis) and Klinefelter syndrome(which is associated with tetralogy of Fallot and Ebsteinanomaly).14

An importantgroup of clinical syndromes that include cardiac malformations havebeen associated with specific deletions in different chromosomes.Deletion of the short arm of chromosome 5 (cat´s crysyndrome) or chromosome 4 (Wolf-Hirschhorn syndrome) are alsoaccompanied by cardiac malformations. The development of newtechniques like high resolution chromosomal banding andfluorescence in situ hybridization (FISH) has allowed thepresence of minimal deletions in contiguous genes to beestablished, and has lead to the recognition of new syndromes likedeletion of chromosome 22q11 (CATCH 22, velo-cardio-facialsyndrome) and the Miller-Dieker (17p13.3) and Williams (17q11.23)syndromes, among others.15

The recognitionof new syndromes does not directly explain the development ofspecific cardiac malformations or the severity of the syndrome. Thefact that the search for anomalous genes now centers onincreasingly smaller chromosomal segments has not yet made possiblethe massive identification of candidate genes. In fact, themechanisms by which a gene or group of genes produce a specificsyndrome vary widely. For example, it has been assumed that theloss of function of a dominant allele leads to specific syndromes.However, increased gene function, with the consequent increment inthe amount of product of that gene (or anomalous production), caninterfere with normal developmental mechanisms to produce a givensyndrome.

Anotheralternative is that only the paternal or maternal allele of acertain gene is active in development (genomic impression). Adefect in the maternal copy can be transmitted as an autosomaldominant defect, while the same defect in the paternal copy doesnot produce disturbances. Likewise, a defect of maternal origin canproduce a certain syndrome, whereas the same defect of paternalorigin produces a totally different syndrome, as occurs with thedeletion of band q12 of chromosome 15 (15q12). In a similar way,the cardiac phenotype in Turner syndrome (45,X) seems to depend onthe parental origin of the anomalous X chromosome.

Three percent ofall cardiac malformations seem to be due to the action of a singlegene. Within this group are included atrial septal defectassociated with defects in cardiac conduction and hypertrophicsubaortic stenosis.4,8 The genetic origin is also clearin other anomalies like right ventricular dysplasia, some dilatedcardiomyopathies, and complex atrioventricular septation defects,which seem to have an autosomal dominanttransmission.16-18 Likewise, the presence of cardiacmalformations is frequent in coagulation disorders like vonWillebrand syndrome or hemophilia.8

The existence ofcardiac phenotypes originated by the loss of function of a singlegene constitutes an attractive hypothesis for the study of cardiacdevelopment. In these cases, can the so-called multifactorialorigin and polymorphic presentation be explained adequately? Recentevidence indicates that many polymorphic presentations are due tothe action of a single gene. The concept of parsimony has been usedto explain it. Throughout development, a single gene can control abasic morphogenetic process, such as the synthesis or degradationof a protein. That protein could be fundamental for the developmentof organs as different as the brain and kidney, so the gene has tobe activated during embryogenesis at different times and indifferent places. Its deactivation would result in a series ofdefects in distant organs, and the severity of the presentationwould depend on the capacity of each organ to supplement orcompensate the genetic defect. In humans, various forms of defectswith autosomal transmission and polymorphic presentation areapparently caused by deactivation of a single gene.19Some of these genes seem to be involved in establishing embryonallaterality. Their deactivation results in visceral spatial positionanomalies and in a wide array of cardiac malformations.

SYMMETRY, ASYMMETRY, AND CARDIAC MALFORMATIONS

The design ofthe human body, like that of most vertebrates, has an evidentbilateral symmetry with respect to the midline. However, thissymmetry is not conserved inside the body since organ dispositionis clearly asymmetrical. It is said that our body has apseudobilateral symmetry. The visceral asymmetry is not limited tothe thoracic and abdominal organs, it extends to the brain andnervous system organization. This is important in the functionalspecialization of the cerebral hemispheres and in behavioralaspects like the preferential use of one hand. The establishment ofasymmetry in the nervous system seems to occur independently fromthat of the trunk, a question that we will not attempt to addressin this article.

Among the firsttasks of an embryo is to define the corporal plan, that is, toestablish the primary embryonal axes. An anteroposterior orcephalocaudad axis is defined that will distinguish the cephalicand caudad ends, and a dorsoventral axis will distinguish thedorsal and ventral sides of the embryo. The left-right axis isautomatically defined after these axes are formed.

The normaldisposition of the heart and organs is called situs solitus(Figure 1). Although there is some confusion in the literature, situs inversus designates the perfect inversion of situssolitus, with the heart toward the right. Any differentdisposition is denominated heterotaxia or situs ambiguus(see below). The incidence of situs inversus is estimated at1 in 10 000 births. The incidence of heterotaxia is generally muchlower and usually accompanied by complex cardiovascularmalformations.20

Fig.1. Scanning electron microscopy. Mouse hearts, E10.5. a)situs solitus; b) situs inversus. Both hearts aremorphologically normal and appear as mirror images. RA indicatesright atrium; LV, left ventricle; CT, cono-trunk. The arrows in aand b indicate the interventricular furrow. a)b:×95.(Reproduced from Icardo, 1997.)

The relationbetween the presence of cardiac malformations and lateralitydefects has long been known.21 An important number ofheart diseases are accompanied by anomalies in the cardiacposition, atrial isomerism, anomalous venous drainage,abnormalities in the form and position of the spleen, and anomaliesin the position of the thoracic and/or abdominal viscera.Consequently, the asplenia-polysplenia syndrome has beendescribed,21-24 characterized basically by the tendencyto visceral symmetry in organs that are normally asymmetrical.Another fundamental aspect of this syndrome is its markedpolymorphism, given that it is defined by the existence of a commoncause with different final expressions.25 Therecognition of the existence of a single syndrome lead to a moregeneral definition of heterotaxia, implying the presence of more orless complex anomalies in visceral and/or venous laterality.Heterotaxia also includes the absence of visceral asymmetry, asituation known as isomerism or sequence isomerism, which mainlyinvolves the bronchi, lungs, and atria in the thorax.

Although thedescriptions of heterotaxia were initially made in isolated cases,the study of large series has shown that in many cases there is aclear familial relation. The clearest example may be that of anAmish family with a high degree of consanguinity, in which variousmembers had visceral situs inversus and cardiacmalformations.26 The study of this and other familygroups27-29 has revealed the presence of a geneticdefect with an autosomal recessive, autosomal dominant, or even Xchromosome-linked transmission.30 Different degrees ofheterotaxia are also observed in midline syndromes like the Meckelsyndrome (situs inversus and polysplenia),31 orKartagener syndrome,32 a primary ciliary anomalycharacterized by bronchiectasia and, in 50% of cases, situsinversus. The latter syndrome is due to ciliary hypomotilitycaused by the absence of the external arms of microtubulardyneine.33,34

ANIMAL MODELS OF HETEROTAXIA

The existence ofa mutant race of mice with the heterotaxia syndrome has opened newareas of investigation. The iv/iv (inversus viscerum) mutantstrain has long been known,35 but only recently was theexistence of cardiac malformations in the embryonal productsdiscovered.36,37 Adult mice exhibit inversion of thecardiac site in 50% of the cases and a percentage close to 30% ofvisceral and/or venous heterotaxia. The heterotaxia includesanomalous venous return, portal vein in a ventral position, hepaticand pulmonary isomerism, atrial isomerism, polysplenia, andthoracoabdominal visceral discordance. In addition, the embryoshave cardiac malformations in 45% of cases.9,10,36,38,39A careful characterization of these hearts has made it possible torecognize the existence of a typical malformation, the so-calledbulboventricular loop, characterized by persistence of the sinusvenosus, common atrioventricular canal (CAVC), and double outletright ventricle (DORV).9,10 This is the basic defectinherited, which normally courses with atrial isomerism. As occursin human syndromes, the presentation is polymorphic, with simpleatrial or ventricular septal defect occupying the opposite extremeof the phenotypical spectrum.9,10,39 In addition,presentation is independent of sex. The same as in humans, the iv gene seems to exhibit complete dominance in such a way that,in absence of its function, the visceral site is determinedrandomly. The absence of this genetic control explains thedifferent patterns of heterotaxia35 as well asvariations in the cardiac phenotypes.40,41

Another mutantstrain of mouse, the so-called legless strain obtained bytransgenic insertion, shows craniofacial and limb anomalies and situs inversus in 50% of cases.42 The inv/invmice, another mutant strain obtained by transgenicinsertion,43 show total situs inversus in 90% ofcases, venous heterotaxia, anomalies of the right cardiac outflowtract and ventricular septal defect.44

The ivgene seems to be found 3 centimorgans from the gene of the heavychain of immunoglobulin Igh-C in chromosome 12 of themouse,45,46 which is the equivalent of human chromosome14. Interestingly, the transgenic insertion in the leglessmouse is also located in chromosome 12, close to the site of theiv gene,47 suggesting that the mutation couldhave affected the iv locus. While the iv and lgl mutations produce randomization of the visceral site, themutated gene in the inv strain, which encodes inversin, islocated on chromosome 4 and seems to direct the visceralsite.48 However, it could also be a mutation due to lossof function. The reason why genetic controls for the establishmentof visceral site are located in such different positions is notknown, but it suggests a close and complex regulation.

Theidentification of the different mutated genes in these strains ofmice has clarified important aspects of their function. Themodified gene in the iv and lgl strains encodes adyneine associated with ciliary microtubules,49 which iswhy it has come to be known as LRD (left-right dyneine). When thisprotein is deactivated in transgenic mice, the laterality anomaliesfound in iv and lgl mice are reproduced.50Other strains of mutant mice with alterations in the morphogenesisof the cilia 51,52 also present laterality anomalies.Curiously, the identification of inversin as the product of themutated gene in the inv/inv strain does not explain anyaspect of its function.

Many of thesemutant mice strains do not show structural ciliary anomalies. Thissuggested that there was no relation with human Kartagenersyndrome, where these structural anomalies do exist. However,patients with Kartagener syndrome also exhibit mutations in thedyneine proteins,53 which suggests that many syndromeswith laterality abnormalities have a common origin. More recentadvances in molecular biology and new detection techniques havemade it possible to prepare a complex picture, as yet incomplete,which includes the expression in cascade of a series of genes, theconcurrent expression of other genes with the cascade, and ciliaryactivity in the node (of Hensen) or organizer during embryonalgastrulation stages. All these factors are involved in establishingnormal laterality and their disruption causes laterality defects inboth humans and animal models.

ESTABLISHMENT OF LATERALITY

In the initialstages of development, the embryo appears symmetrical with respectto the midline. Although a mild transitory asymmetry in themorphology of the Hensen nodule has been described in chickembryos,54 the first clear evidence of morphologicalasymmetry emerges with the formation of the cardiacloop.55 The heart, which at first is tubular and medial,invariably curves to form a loop to the right. Upon continuingembryonal development, the rest of the organs progressively acquiretheir characteristic asymmetrical distribution.

Conceptually,establishment of the left-right axis takes place in threephases.56,57 In the first phase, the initial symmetry ofthe embryo disappears, specifying two unequal halves, one right andone left. The initial break in symmetry takes place duringgastrulation, in relation with the nodule of Hensen. In a secondphase, and as a consequence of the previous phase, numerous genesare expressed asymmetrically, to the left or to the right, thusgiving identity to each embryonal side. Most of these genes encodesignaling molecules that interact to establish signaling cascades.These cascades of asymmetrical expression start around the noduleand eventually end by establishing wide domains of asymmetricalgene expression in the lateral mesoderm. Finally, this geneexpression translates into the normal asymmetrical morphology ofthe organs.

The factorsinvolved in the initial break in symmetry are still largelyunknown. In the mouse and, by extension, in mammals, the novel andattractive model of «nodal flow» has beenproposed.58-60 The cells of the node have a singlecilium on their ventral surface. These cilia have a vorticalmovement that, in conjunction with other cells, produce a leftwardflow of the perinodal fluid. It is postulated this flow causes anasymmetrical distribution of a postulated, but as yet notidentified morphogen, which is responsible for initiating the pathof left-right signaling. In fact, the flow of perinodal fluid tothe left is very weak in inv/inv mice and does not takeplace in iv/iv mice due to the absence of ciliary motility.The ciliary flow could also be altered in other mutant strains ofmouse characterized by the abnormal morphogenesis of nodalcilia,50,58 or their absence.51 The humansyndromes with which site alterations are associated with primaryciliary anomalies61,62 constitute an important supportfor this model.

However, themodel of nodal flow may not be valid in other species. In the chickembryo, monociliary cells are irregularly distributed on theventral and dorsal surface of the embryo, constituting only part ofthe cells of the nodule of Hensen.63 Thus, thehypothesis of nodal flow cannot easily be applied to the chickembryo. In addition, it has been demonstrated that some genes areexpressed asymmetrically before formation of the node.64It has thus been proposed that in birds and amphibians the break insymmetry originates in the tissues that surround thenode.65-68

A hypothesisthat is currently under study involves the cellular gap typejunctions that are established in the tissues that surround thenode. If one small molecule were capable of circulating throughthose junctions in single direction, molecules would accumulate onone side of the midline, thus disrupting symmetry and triggering aresponse of asymmetrical gene activation in the node.67In any case it seems that in all the species studied, the initialbreak in symmetry is related to the nodule of Hensen.64Parting from the node, in a second phase the specific gene cascadesof laterality are activated, in such a way that the asymmetricalinformation is reinforced and transmitted to the lateral mesoderm(Figures 2 and 3). The derivatives of the lateral mesoderm willform the asymmetrical organs.

Fig.2. In the chick embryo, the initially symmetrical expression ofShh in the nodule of Hensen (NH) is restricted to the rightside by the activity of activinβ mediated by Bmp4. Shhinduces the expression of Lefty1 in the left half of the midline aswell as Nodal and Car. The expression of Nodal in theleft lateral mesoderm (LLM) is facilitated by the expression of CFCunder the control of BMP. On the side right, the activinpathway induces Fgf8, which in turn induces cSnR and prevents theexpression of Nodal.

Fig.3. a: Expression of Nodal in a) 4-somite chick embryo;b) expression of Lefty1 in a 4-somite chick embryo;c: expression of Pitx2 in a 4-somite chick embryo; d)expression of mPitx2 in a mouse embryo in E8.25; e)expression of mPitx2 in the heart of an iv/iv mouse withright loop in E11.5; f) expression of mPitx2 in theheart of an iv/iv mouse with left loop in E11.5; g)bilateral atrial expression of mPitx2 in E11.5 with a loopto the right; h) absence of atrial expression of mPitx2 in a heart with a loop to the left in E11.5; i)right view of the heart of an iv/iv mouse in E11.5 which showsexpansion of the expression of mPitx2 on the dorsal wall ofthe right ventricle; the right atrium (asterisk) is also positive;j) scanning electron microphotograph showing DORV (doubledate) in a heart that showed dorsal expression of mPitx2 in theright ventricle in E11.5. RA indicates right atrium; LA, leftatrium; RV, right ventricle; LV, left ventricle. (Figures e-j)reproduced from Campione et al, 2001.)

In the chickembryo it has been demonstrated that various signaling moleculesshow small domains of asymmetrical expression in the nodule ofHensen. Among these molecules are established regulatory loops thatcontrol the asymmetry of laterality (Figure 2). For example, theasymmetrical expression of Sonic hedgehog (Shh) on the leftside of the node is essential for the correct development oflaterality. At first, Shh is expressed symmetrically in thenode but, due to negative signaling mediated byActivinβB and Bmp4, its expression is repressed on the rightside and remains confined to the left side. The asymmetricalexpression of Shh on the left side of the node induces theasymmetrical expression of Nodal in the left lateralmesoderm.67,69-76 Nodal, in turn, induces theexpression of Pitx2.

The induction ofNodal by Shh is not direct, but mediated by anintermediate factor recently identified as Caronte(Car).64,77-79 Car is included in a group ofmolecules (the family of Cerberus/DAN) that act as antagonists ofbone morphogenetic proteins (BMP). Various BMP are expressed in thelateral mesoderm in a lateral domain of Nodal. Since Car blocks the activity of these BMP, it has beenproposed77,78 that the function of Car is toantagonize the repressive effect of the BMP on Nodalexpression (Figure 2). However, we have recently demonstrated thatBMP signaling positively regulates Nodalexpression.80 BMP activity induces the expression of CFC(Cripto/FRL-1/Cryptic), which is the only member of thefamily of EGF (epidermal growth factor)-CFC identified in chickembryo to date.81,82 CFC is an essential extracellularfactor for Nodal signaling.83 Although morestudies are needed to clearly establish the role that the BMP playin left-right specification, the existing discrepancies demonstratethe complexity of the regulation established between the differentmolecular pathways that control laterality.

In the chickembryo, activation of the activin pathway on the side right of thenodule of Hensen results in the right expression of Bmp4. Inturn, Bmp4 inhibits the right expression of Shh andinduces the expression of Fgf8 which, in turn, induces theexpression of the Snail (cSnR) transcription factor in theright lateral mesoderm. These activation sequences are specific tothe right side.

Since specificsignaling pathways are established on each embryonal side, it isimportant that the information on one side not pass to the oppositeside. In this respect, the embryonal midline has a critical barrierrole. The mutations that course with morphological or biochemicaldefects of the midline are accompanied by laterality disturbances.The expression of Lefty1 on the midline (on the left half ofthe ground plate precursors; Figure 3) has been proposed as themolecule responsible for this barrier.84 Lefty1is a member of the transforming growth factor-β (TGF-β) family and may carryout its function by blocking Nodal. Another member of thesame family, Lefty2, seems to control the temporal extensionof the expression of Nodal.84 An example thatillustrates the importance of the midline in humans are thealterations of visceral site observed in Siamese twins. It has longbeen recognized that in twins joined by the trunk the twin on theright often presents visceral site alterations. This is interpretedas the influence of the left signaling cascade of the twin locatedon the left side over the twin on the right side.

We commentedearlier that there seems to be a notable divergence between thedifferent species in the beginning of left-right specification.However, very recent evidence indicates that the latterlateralization signals, as well as the pathways that modulate them,function similarly in humans. For example, mutations in the gene ofthe activin receptor85 cause laterality disturbances andcardiac malformations. In particular, the pattern of expression ofNodal is markedly conserved in all the species studied todate, from the zebrafish to humans.86 Nodalbelongs to the TGF-β superfamily, is transitorilyexpressed in the left lateral mesoderm, and is considered a leftdeterminant given that its expression correlates directly with thelaterality of the heart and other organs.69,87 Theconservation of the pattern of expression of Nodal and itstarget gene Pitx2 (Figure 3) has meant that the stage inwhich they are expressed is denominated the left-right phylotypicalstage of asymmetry.88,89

The third phasein the establishment of asymmetry is the translation of theprevious gene expressions as the normal asymmetrical morphogenesisof the organs. Pitx2 is the main target gene of Nodalto be identified to date.90-94 Pitx2 is expressedinitially in a domain very similar to that of Nodal, but itsexpression continues while the asymmetrical morphogenesis of theviscera progresses, when the expression of Nodal has alreadybeen repressed. Pitx2 is a transcription factor with ahomeodomain of the bicoid type,95 which has important functions duringembryonal development in addition to its participation inasymmetry. Pitx2 presents three isoforms denominated a, band c. The asymmetrical expression of Pitx2 correspondsexclusively to the Pitx2c isoform.96,97 Thestudy of transgenic mice in which the expression of Pitx2has been eliminated or reduced (hypomorphic mutations)98indicates that the different organs have a variable sensitivity tothe presence of Pitx2.

In order tounderstand the phenotypical variability in heterotaxia, it hassuggested that the thresholds of Pitx2 necessary for correctmorphogenesis vary for each organ. In fact, different levels ofexpression in various segments of the lateral mesoderm could berelated with thoracoabdominal discordance.91,98 If thiswere to be the case, it could also be speculated that somethingsimilar could occur in different segments of the heart. This wouldhelp to explain the different cardiac phenotypes.

Pitx2 isinitially expressed on the left side of the tubular heart andcardiac loop, and then is confined to the left atrium, anteriorface of the ventricles, and left side of the outflow chamber(Figure 3). In the last phase, the expression of Pitx2 islimited to the left atrium, until it ends updisappearing.99 The patterns of expression of Pitx2 are equivalent in the chick and mouse, and are invertedin mice with situs inversus. In malformed hearts, bilateralatrial expression (or the bilateral absence of expression)accompanies atrial isomerism, with expression also being able toexist on the posterior side of the right ventricle (Figure 3). Iniv/iv mouse, this anomalous expression seems to be relateddirectly with the development of double outlet rightventricle.99 Thus, although there are clear indicationsthat Pitx2 intervenes directly in cardiac morphogenesis, theexact relation is unknown since the possible target genes have notyet been identified. Procollagen hydroxylysine could be one ofthese targets,100 but its exact role isunknown.

CARDIAC MALFORMATIONS IN HETEROTAXIA

The organs reachtheir definitive form through a series of basic activities thatinclude division and cell death, cellular emigration, cellularaggregation in tissues that specialize in different functions, thesecretion of extracellular materials and, in the heart, thepossible interaction of all these factors with hemodynamic forces.At present, it is not known which of these activities dependdirectly on Pitx2 expression. To date, the only asymmetrydetected at these levels is in the distribution of flectin, anextracellular glycoprotein that is expressed on the left side ofthe ventricle and on the right side of the outflow chamber duringthe formation of the cardiac loop. The differential expression offlectin is inverted in iv/iv mice, which is why it has beensuggested that this protein is involved in the direction of thecardiac loop.101

As mentionedabove, the iv/iv mutant is an excellent model for the studyof the heterotaxia syndrome. Although we do not know how thepatterns of gene expression are resolved in specific patterns ofcellular conduct, the iv/iv mutant has also been shown to bea model for the study of the cardiac malformations found inheterotaxia.9,37 The sequential study of cardiacdevelopment in iv mutants helps us to understand not onlynormal cardiac development, but also the mistakes in developmentthat result in the production ofmalformations.9,38-40

The basiccardiac malformation or type corresponds to the so-calledbulboventricular tube. Within the cardiac phenotypical syndromethere is, however, a regular combination consisting of CAVC andDORV (Figure 4).102 CAVC is a complex malformation thatincludes atrial septal defect, ventricular septal defect, andcommon atrioventricular valve.103-106

Fig.4. Scanning electron microscopy showing the internal aspect ofa heart (E18.5) with common atrioventricular canal and doubleoutlet right ventricle. Situs solitus. The heart has been sectionedin the frontal plane and the ventral a) and dorsal b)halves are shown. The two ventricles communicate through a largeventricular septal defect that occupies approximately one-half ofthe ventricular height. The anterior leaflet (arrows in b) of thecommon atrioventricular valve is joined to the right side of theventricular septum by a single papillary muscle. The two greatvessels arise from the right ventricle by separate outflow chambers(arrows in a). A muscular band separates the origin of bothchambers. The atria are symmetrical, even exhibiting symmetricalanterior prolongations (stars in b). Ao indicates aorta; P,pulmonary artery; a, b, ×45. (Reproduced from Icardo andSánchez de Vega, 1991.)

In normaldevelopment, the two ventral and dorsal atrioventricular cushionsfuse to form the so-called septal cushion. This mesenchymal massconstitutes the center of the developing heart, on which the septum primum that initially divides the atria,interventricular septum, and conal partitionconverge.107 In malformed hearts, the firstmorphological deviations appear during the formation of the cardiacloop,37 but they become clearer in stageE10.5,40 being characterized by the presence of abnormalspatial relations between the different cardiac segments (Figures 5and 6). The structure of the heart is normal although theendocardial cushions begin to show anomalies of position and form.In stages E11.5 and E12.5, these anomalies become more noticeable(Figure 7). The cushions appear hypoplastic, adopt a triangularform, and may be divided or present abnormal relations. In somecases, one of the lateral cushions is enormously enlarged. In stageE13.5, the atrioventricular cushions fuse in normal hearts (Figure8). In malformed hearts they do not fuse and they remain separatedby a wide space (Figure 9). At the same time, th e septumprimum does not contact the cushions and does not close the foramen primum (Figure 7). The interventricular septum, whichhas to contact the right side of the septal cushion, does not do soand is left in an intermediate position or even deviated to theleft side. The septation of the cardiac outflow chamber, thecono-trunk, can be normal. However, the abnormal spatialdisposition of the interventricular septum and undividedatrioventricular channel prevents the two partitions fromcontacting. The two ventricular outflow chambers do not becomeindependent and originate abnormally. The later development of theatrioventricular cushions (dorsal, ventral, and lateral) willdetermine the final morphology of the common atrioventricularvalve.

Figs.5-9.Scanning electron microscopy showing some morphologicalaspects of normal and malformed hearts. (Reproduced from Icardo etal, 1995.)

Fig.5. E10.5. Situs inversus. This heart is displaced toward theside right of the embryo. The right ventricle (RV) and cono-trunk(CT) form a U with no clear separation. The right ventricle iscephalad with respect to left ventricle (LV). The arrow indicatesthe interventricular furrow. ×45.

Fig. 6. E10.5.Situs solitus. The two ventricles do not maintain thecharacteristic lateral relation. The interventricular (arrow) andconoventricular (arrowhead) furrows appear abnormally marked.Compare the morphology of the hearts shown in Figures 5 and 6 withthose that appear in Figure 1, which are in the same stage ofdevelopment. ×110.

Fig.7. This photographs shows the ventral (a) and dorsal (b) halvesof a malformed heart (E12.5), which has been sectioned in thefrontal plane Situs inversus. The ventricles and interventricularseptum are sectioned tangentally, indicating an abnormal spatialposition. The right ventricle is much more anterior than the leftventricle. The septum primum (Sp) and septum secundum (Ss) appearin b. Note the abnormal spatial disposition of the atrial andventricular septa. The dorsal cushion (in b) appears bifurcated.The two great arteries arise from the right ventricle (arrow in a).a and b: ×85.

Fig.8. Normal heart (E13.5). Situs solitus. The heart has beendissected in the parasagittal plane. The dissection plane passesthrough the left side of the atrioventricular region. The righthalf is observed after eliminating the left fragment. Theatrioventricular cushions are fusing and the septum primum (arrow)continues into the septal cushion. The ventricular septal defect(arrowhead) remains open. P indicates pulmonary artery.×90.

Fig. 9.Malformed heart (E.13.5). Situs solitus. The preparation is similarto that in Figure 8. The ventral (v) and dorsal (d)atrioventricular cushions have not fused. The septum primum (arrow)is normal, but does not contact the cushions. The arrowheadindicates the ventricular septal defect. P indicates pulmonaryartery. ×90.

It is clear thatthe main anomaly in the development of CAVC in the heterotaxiasyndrome is the non-fusion of the atrioventricular cushions.However, other structures, such as interatrial and interventricularsepta, can exhibit an anomalous development, contributing in avariable way to the abnormal phenotype. The development of anyorgan is due to the close association established between thecomponent parts. All the components must coincide in time and spacefor the organs to acquire their definitive form. When one or moreof these components fail, the general morphogenetic mechanismscontinue their course, but the organ in question has a deficiencythat will be potentiated throughout development.

An importantquestion that should be considered is whether all the cardiacphenotypes found in the heterotaxia syndrome can be explained bythe action of a single gene. It is clear that in the initial stagesof development anomalies of position and rotation of the cardiacloop take place. These anomalies are not corrected, but carriedinto later stages. The lack of alignment between the interatrialand interventricular septa can be explained by the loss of spatialsignals in the primitive atrium and ventricle, and between thesechambers and the atrioventricular canal. The same thing must occurin atrioventricular canal. Once the spatial signals are modified,control over the formation of the cushions is lost. This wouldexplain both their abnormal position and variations in form andsize.40 In any case, the development of thesemalformations cannot simply be explained by spatial anomalies.Laterality anomalies should be considered among the basicmorphogenetic mechanisms. For example, iv/iv hearts oftenshow a lateral deviation of the interventricular septum withrespect to the atrioventricular cushions. The growth of theinterventricular septum is closely associated to the growth of theventricles. The growth of the ventricles depends in great measureon the presence of active centers and cell proliferation. If thesecenters do not receive or produce suitable signals, or are locatedin an abnormal apposition, ventricular development will be abnormaland the septum will have an altered apposition. Hypotheticalmechanisms of malformation, such as that which has just beendescribed, are perfectly compatible with the action of a singlegene. However, the possible influence of the iv gene on cellproliferation and other basic morphogenetic mechanisms is notknown. It can be anticipated that some of these relations will bedeciphered in the near future.

ACKNOWLEDGMENTS

Subsidized bygrants PB98-1418-C02-02, BMC2000-0118-CO2-01 (JMI), andDGICYT-PM-98-0151 (MAR) of the Spanish Ministry of Science andTechnology.


Correspondence: Dr. J.M. Icardo.
Departamento de Anatomía y Biología Celular.
Facultad de Medicina. Universidad de Cantabria.
Polígono de Cazoña, s/n.
39011 Santander. España.
E-mail: icardojm@unican.es

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Revista Española de Cardiología (English Edition)

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