Pbx/Meis Deficiencies Demonstrate Multigenetic Origins of Congenital Heart Disease
Congenital heart diseases are traditionally considered to be multifactorial in pathogenesis resulting from environmental and genetic interactions that determine penetrance and expressivity within a genetically predisposed family. Recent evidence suggests that genetic contributions have been significantly underestimated. However, single gene defects occur only in a minority of cases, and multigenetic causes of congenital heart diseases have not been fully demonstrated. Here, we show that interactions between alleles of 3 Pbx genes, which encode homeodomain transcription factors, are sufficient to determine the phenotypic presentation of congenital heart diseases in mice. A major role is served by Pbx1, whose inactivation results in persistent truncus arteriosus. Reduction or absence of Pbx2 or Pbx3 leads to Pbx1 haploinsufficiency and specific malformations that resemble tetralogy of Fallot, overriding aorta with ventricular septal defect, and bicuspid aortic valves. Disruption of Meis1, which encodes a Pbx DNA-binding partner, results in cardiac anomalies that resemble those caused by Pbx mutations. Each of the observed cardiac defects represents developmental abnormalities affecting distinct stages of cardiac outflow tract development and corresponds to specific types of human congenital heart disease. Thus, varied deficiencies in the Pbx gene family produce a full spectrum of cardiac defects involving the outflow tract, providing a framework for determining multigenetic causes of congenital heart anomalies.
Congenital anomalies of the heart occur in up to 5% of live births1 and are the leading cause of birth defect–related death in the United States.1 Defects in cardiac outflow tract (OFT) formation account for 20% to 30% of congenital heart defects (CHDs).2 Isolated malformations of the semilunar valves of the OFT, including bicuspid aortic valve, occur in an additional 2% to 3% of the population.3 Formation of the OFT is a complex developmental process that involves the division of the arterial trunk into aortic and pulmonic arteries, alignment of these 2 arteries with the cardiac chambers, and development of the aortic and pulmonic valves.4 Failures of arterial division, alignment, or valve formation lead to specific types of congenital heart disease in humans.4 For example, the absence of arterial septation results in persistent truncus arteriosus (PTA), whereas unequal septation of the OFT may lead to narrowing of the right ventricular OFT accompanied by ventricular septal defect and other associated defects (tetralogy of Fallot). Furthermore, cardiac defects may arise from misalignment of the aorta to the left ventricle, resulting in an overriding aorta straddling both right and left ventricles. Malformations of semilunar valves can cause bicuspid aortic valve or pulmonic valve stenosis. These different forms of cardiac OFT defects have distinct clinical manifestations and implications for treatment and long-term care.5
A long standing clinical view holds that CHDs are determined by individual genetic predisposition interacting with influential environmental factors.1,6–8 This traditional model, although underestimating genetic contributions,1,9 has been used to explain a low coincidence rate (2% to 3%) for most cardiac anomalies among siblings and to explain the variety of anomalies observed within a single pedigree. Although single gene or chromosomal abnormalities cause certain cardiac lesions or syndromes,1,9–11 they represent only a minority of cases and do not account for the complex inheritance of most CHDs. As alternative explanations to environmental effects or single gene inheritance, the display of CHDs may be determined by multiple alleles of a single major gene, by interaction of a major gene with several minor genes, or by the interactions of several minor genes acting together. Distinguishing among these scenarios, however, is challenging to study in the human population. The availability of targeted mouse mutations provides an experimental approach to study multigenetic etiology of CHDs.
Here, we describe how interactions between alleles of a new class of transcriptional regulators of heart development, the Pbx gene family, provide a mouse model for the complex inheritance of CHD. The Pbx genes, including Pbx1, -2, and -3, which encode TALE class homeodomain transcription factors,12–14 form heterooligomeric complexes with Hox and Meis proteins.15,16 We have examined the cardiac phenotypes of all possible combinations of null alleles of the Pbx genes. Embryos with different combinations of Pbx mutations display a spectrum of cardiac malformations in the OFT that include PTA, tetralogy of Fallot, overriding aorta, and bicuspid aortic valves. Each of the phenotypes produced by specific Pbx compound mutations represent developmental aberrations at distinct stages during OFT development, correlate with Pbx gene dosage, and correspond to specific types of CHDs in humans. Our studies also suggest that Pbx-governed OFT patterning requires interactions with their DNA-binding partner Meis proteins16 because Meis1 disruption results in cardiac defects that resemble Pbx mutations. These results provide a framework to consider the complex inheritance of human CHD as multigenetic interactions between paralogous genes.
Materials and Methods
Pbx-Deficient and Meis1-Deficient Mice
Targeted disruption of the Pbx and Meis1 genes and generation of the respective knockout mice have been described previously.17–19 Heterozygous knockout parental lines were backcrossed for at least 8 generations onto a C57BL/6 genetic background before intercrossing to obtain homozygous or compound mutant embryos. Phenotypes were generally analyzed in embryos (embryonic day [E]10.5 to 15.5) or neonates. Gestational age was determined by the date of observing a vaginal plug (set as E0.5) and by ultrasonography before harvesting embryos.20 For embryonic/perinatal lethality determination, 4 to 5 littermate controls were observed per mutant mouse. For cardiac structure analysis, 1 to 2 littermate controls were analyzed per mutant mouse. The care and use of mice were according to the Stanford University and NIH guidelines.
The chest wall of mice was opened under microscopic visualization. A 33-gauge needle (Hamilton) mounted on a 1-mL tuberculin syringe was used to inject an acrylic resin (Batson no. 17) containing blue dye (Methyl Methacrylate Casting Kit, Polyscience Inc) into the right ventricle to perfuse the great arteries. Mice were then held at 4°C for 2 to 6 hours to allow the resin to polymerize and cast the vasculature.
Paraffin sections of mouse embryos were prepared as described previously.21,22 Consecutive sections through the chest (5 to 7 μm) were collected, stained with hematoxylin/eosin, and analyzed by light microscopy.
Immunohistochemistry using anti-Pbx1b,16,23 anti-Pbx2 (clone 2.1), or anti-Pbx3a19 monoclonal antibodies was performed on paraffin sections of embryonic tissues. Paraffin sections (7 μm) from E9.5 and E12.5 mouse embryos were prepared and rehydrated. For antigen retrieval, the slides were immersed in citric acid–based antigen unmasking solution (Vector Laboratories) and boiled in a pressure cooker for 10 minutes, then cooled down at 4°C for 20 minutes. Endogenous peroxidase activities were blocked by treating in 3% H2O2 for 10 minutes. The slides were then blocked in 5% normal goat serum for 30 minutes. The primary mouse monoclonal antibodies were used at the following dilutions: anti-Pbx1b, 1:200; anti-Pbx2, 1:400; and anti-Pbx3a, 1:400. The antibodies were then incubated for 2 hours at room temperature. The biotinylated antimouse IgG secondary antibody (Vector Laboratories) was used at 1:250 for 30 minutes at room temperature. The signal was amplified using the VECTASTAIN Elite ABC kit (Vector Laboratories) for 30 minutes and developed with DAB (3,3′-diaminobenzidine, Vector Laboratory). Finally, the slides were counterstained with hematoxylin for nuclei and mounted with Permount (ThermoFisher).
Pbx Proteins Are Widely Present in Tissues Essential for Heart Development
The Pbx genes (Pbx1, -2, and -3) regulate a variety of developmental processes,17,24–31 suggesting that they may also be important for heart development. To investigate this hypothesis, we first examined the distribution of Pbx proteins in tissues involved in heart development. In the heart, Pbx1b, the predominant isoform of Pbx1 during mouse embryogenesis,23 was present in both endocardial and myocardial cells of the OFT at E9.5 (Figure 1A). At E12.5, Pbx1b was present in endocardial cells and the mesenchyme of endocardial cushions that septate the OFT and give rise to the semilunar valves (Figure 1D). Pbx1b was also detected in endothelial and vascular smooth muscle cells of the aorta and main pulmonary arteries (Figure 1D). Furthermore, at E8.75 and E9.5, Pbx1b was widely present in cells within the neural tube, where the premigratory and newly delaminated cardiac neural crest cells (CNCCs) are located (Figure 1A and Figure I in the online data supplement, available at http://circres.ahajournals.org). CNCCs migrate to the OFT, form the aorticopulmonary septum, and contribute to OFT septation.32 Pbx1b was also present in mesenchymal cells that surround CNCCs and influence their migration (Figure 1A).33,34 Similarly to Pbx1, Pbx2 was broadly present at E9.5 in cells that included premigratory CNCCs within the neural tube and branchial mesenchyme (Figure 1B). Within the heart, Pbx2 was present in endocardial, myocardial, and cushion mesenchymal cells (Figure 1B and 1E). In the aorta and main pulmonary arteries, Pbx2 was detected in both endothelial and vascular smooth muscle cells (Figure 1E). Pbx3a was widely present at E9.5, including in premigratory CNCCs as well as all cell types of the heart (Figure 1C). However, by E12.5, Pbx3a was downregulated in myocardial cells but remained present in endocardial cells and mesenchymal cells of the endocardial cushions (Figure 1F). Additionally, Pbx3 was present in endothelial but not vascular smooth muscle cells of the great arteries (Figure 1F), distinguishing it from Pbx1b and Pbx2. The widespread and overlapping presence of Pbx proteins in embryonic sites of cardiac development prompted us to investigate heart development in mice lacking individual or multiple Pbx genes.
Pbx1 Is Required for Septation of the Cardiac OFT
Embryos deficient for Pbx1 manifested a failure in septation of the cardiac OFT, resulting in a single arterial trunk (termed persistent truncus arteriosus or PTA) that emerged from the right ventricle (N=28) (Figure 2A through 2D). The PTA in Pbx1-null embryos gave rise to left and right coronary arteries anteriorly and a main pulmonary artery posteriorly (Figure 2B, 2E through 2G, and 2K). The main pulmonary artery branched into left and right pulmonary arteries as in wild-type embryos. A large ventricular septal defect was present in Pbx1−/− hearts (data not shown). Furthermore, a single heart valve was present at the base of the truncus (Figure 2H to 2K) and contained 3 valve leaflets and cusps: the right anterior, left anterior, and posterior cusps, representing the most common valve configuration seen in human PTA.5 Overall, the cardiac abnormalities of Pbx1−/− embryos resemble type II PTA in humans (Collett and Edwards classification),5 a severe form of cardiac OFT developmental failure. Besides cardiac defects, Pbx1−/− embryos exhibited anomalous patterning of the great arteries, including cervical aortic arch, absent or residual common carotid artery, aberrant origins of the subclavian arteries, and occasional right-sided aorta.34a Vascular patterning defects were not observed in mice with any of the compound Pbx mutations described below.
Pbx2 Contributes to the Alignment of Cardiac OFTs and Semilunar Valve Morphogenesis
To investigate potential multigenic Pbx contributions to OFT development, mice bearing null alleles17–19 for Pbx1, Pbx2, and Pbx3 were intercrossed to produce embryos with 10 distinct Pbx allelotypes. These 10 allelic Pbx combinations represented all possible genotypes from the 27 theoretical combinations of 3 Pbx genes because of early lethality of mice of certain genotypes. For example, Pbx1+/−;Pbx3−/− mouse embryos could not be generated because the required parental Pbx1+/−;Pbx3+/− mice died neonatally (Table). Furthermore, to circumvent strain background effects, phenotypes were analyzed in embryos or neonates derived from parental mice that had been backcrossed onto the C57BL/6 genetic background for at least 8 generations. Most combinations of Pbx mutations yielded no detectable cardiac abnormalities, including mice homozygous for Pbx2- or Pbx3-null alleles (Table).18 However, when Pbx1 gene dosage was reduced by half, Pbx2 nullizygosity resulted in cardiac defects in the alignment of the aorta to the left ventricle in addition to specific semilunar valve malformations. Pbx1+/−;Pbx2−/− mice died within 24 hours after birth. Angiography of newborn mice showed an abnormal connection of the right ventricle with the ascending aorta (Figure 3A) and an overriding aorta that straddled both the right and left ventricles (Figure 3B), defects that were also evident by histological analysis (N=17) (Figure 3C and 3D). A ventricular septal defect underlying the aortic valve was present in Pbx1+/−;Pbx2−/− hearts (Figures 3C and 5⇓H). Furthermore, in contrast to the normal trileaflet valves (Figure 4A, 4C, and 4G), the aortic valve was bicuspid in Pbx1+/−;Pbx2−/− mice, containing only the right and left coronary cusps (Figures 4B, 4D, 4H, and 5⇓K) that gave rise to the right and left coronary arteries, respectively (Figure 4B). The bicuspid valve leaflets did not form commissures at their anchoring points along the aortic wall (Figure 4D), indicating that these valves could not properly occlude the aortic lumen and thus would lead to significant aortic regurgitation. Similarly, the pulmonic valve was also bicuspid containing right and left posterior leaflets in the absence of an anterior leaflet (Figure 4F and 4H). These cardiac defects resemble anomalies in human patients with overriding aorta and bicuspid aortic valve, functionally important anomalies of less severity than PTA. Thus, heterozygosity of Pbx1 reveals roles for Pbx2 in the alignment of the left ventricular OFT and semilunar valve morphogenesis.
Pbx3 Participates in the Formation of Cardiac OFT and Semilunar Valves
To further investigate the roles of Pbx3 in heart development, we intercrossed a Pbx3-null allele19 onto Pbx1- and/or Pbx2-deficient backgrounds. We found that embryos with compound Pbx1+/−;Pbx2−/−;Pbx3+/− mutations exhibited serious cardiac anomalies characteristic of tetralogy of Fallot. Pbx1+/−; Pbx2−/−;Pbx3+/− embryos, unlike Pbx1+/−;Pbx2−/− embryos (Figure 5B), displayed generalized edema (Figure 5C) similar to that reported previously in Pbx1−/− embryos.17 Pbx1+/−; Pbx2−/−;Pbx3+/− embryos showed unequal division of the truncus arteriosus at the expense of the right ventricular OFT (RVOT), leading to narrowing of RVOT and malformations of pulmonic valves (N=8) (Figure 5F). This RVOT obstruction, together with pulmonic valve malformations (Figure 5F), resulted in hypertrophy of the right ventricular wall (Figure 5I). Mild left ventricular hypertrophy was also present, likely reflecting volume overload of the left ventricle as a result of increased right to left cardiac shunting caused by RVOT obstruction and the presence of a large ventricular septal defect (Figure 5I), as well as possible pressure overload caused by the presence of malformed aortic valves (Figure 5L). The aorta of Pbx1+/−;Pbx2−/−;Pbx3+/− embryos arose mainly from the right ventricle, but it overrode both right and left ventricles (Figure 5L and data not shown). Malformed aortic valves were present between the misaligned aorta and ventricles (Figure 5L). The constellation of RVOT obstruction, right ventricular hypertrophy, ventricular septal defect, and overriding aorta in Pbx1+/−;Pbx2−/−;Pbx3+/− embryos mimics tetralogy of Fallot in humans. Thus, reduction of Pbx1 gene dosage uncovers the contributions of Pbx2 and Pbx3 toward equal division of truncus arteriosus into the left and right outflow channels.
Semilunar valve formation depends on both Pbx2 and Pbx3. Although Pbx1+/−;Pbx2+/− mice displayed no valve defects, Pbx1+/−;Pbx2+/−;Pbx3+/− mice had isolated bicuspid aortic and pulmonic valves as their only cardiac malformations (N=11) (data not shown), indicating that Pbx3 participates in trileaflet valve formation. Similarly, comparison of Pbx1+/−;Pbx2+/−;Pbx3+/− embryos, which had distinct bicuspid semilunar valves, with the normal valves in Pbx1+/−; Pbx3+/− mutants, further indicates that Pbx2 contributes to semilunar valve development. In addition, the necessity of Pbx1 haploinsufficiency for the development of semilunar valve malformations in Pbx2 and Pbx3 mutants, because Pbx2−/−;Pbx3+/− mice had no semilunar valve defects (Table), demonstrates that all 3 Pbx genes contribute to semilunar valve morphogenesis.
Meis1 Is Essential for Cardiac OFT Development
Given that Meis proteins are major in vivo DNA-binding partners of Pbx proteins,16 we hypothesized that development of the cardiac OFT would require Pbx and Meis interactions. To test this idea, we examined cardiac development in mice lacking Meis1.35 Meis1−/− embryos displayed subcutaneous hemorrhage and died between E14.5 and E15.5 (supplemental Figure IIB). A further analysis of Meis1−/− embryos revealed that the aorta was septated from the main pulmonary artery (supplemental Figure IID) but overrode both right and left ventricles accompanied by a ventricular septal defect (N=9) (Figure 6B and 6D and supplemental Figure IIE and IIF). This phenotype resembles Pbx1+/−Pbx2−/− heart defects and falls within the spectrum of OFT anomalies defined by Pbx deficiencies (Figure 7). This suggests that Pbx proteins function in concert with Meis1 partners during heart development. Because Meis1 is also part of a multigene family,36 compound mutations of Meis genes may also recapitulate a broad spectrum of cardiac defects similar to those induced by Pbx deficiencies.
Many CHDs arise from malformations of the cardiac OFT and failure at specific steps in its formation results in distinctive cardiac anomalies of varying severity. These steps involve the division of the OFT into aorta and pulmonary arteries, alignment of these arteries to cardiac chambers, and formation of heart valves.4 Our results demonstrate how multigenetic interactions between alleles of related genes can determine the specific type of heart defect that develops.
The different cardiac phenotypes observed in Pbx allelic combinations suggest Pbx genes contribute to multiple steps of OFT development (Figure 7). Reduction of total Pbx1 to -3 gene dosage to half of wild-type levels reached a threshold where Pbx availability became limiting for heart development and manifested by isolated bicuspid semilunar valves in triple heterozygous Pbx1+/−;Pbx2+/−;Pbx3+/− mice. More severe phenotypes were observed in Pbx1+/−;Pbx2−/− mice, which displayed defects in the alignment of the left ventricular OFT, with resultant overriding aorta. Even more dramatic phenotypes occurred with additional compound alleles in Pbx1+/−; Pbx2−/−;Pbx3+/− mutants, where the cardiac OFT was divided unequally between the right and left hearts, leading to an obstruction in the right ventricular OFT. These biased divisions, compared with equal divisions in Pbx1+/−;Pbx2+/−; Pbx3+/− and Pbx1+/−;Pbx2−/− embryos, suggest that both Pbx2 and Pbx3 are essential, but functionally overlap, in fine-tuning the septation of the OFT and alignment of its left and right sides. The most severe phenotype was observed in Pbx1-null embryos, in which the OFT failed to complete the initial septation phase, suggesting that Pbx1 provides the greatest amount of combined Pbx function among the 3 proteins. Our results are most consistent with overlapping contributions of the 3 Pbx family members to common downstream molecular pathways that ultimately are required for OFT septation and alignment, as well as heart valve development. In support of an overlapping function, Pbx1, Pbx2, and Pbx3 have similar biochemical activities in forming transcriptional complexes with Hox and Meis proteins.15,16 The observations that certain members of these gene families, such as Hoxa337 and Meis1 (present study), are required for cardiac OFT development suggest that Pbx proteins likely heterodimerize with Hox and/or Meis proteins to control a subset of target genes to regulate OFT formation.
A functional-overlap model for Pbx genes in OFT development suggests that Pbx proteins act in cells in which the 3 proteins are simultaneously present. The roles of Pbx genes could occur early in development to establish correct differentiation or localization of relevant cells that later contribute to cardiac septation. Alternatively, Pbx proteins may function during active septation of the OFT. Pbx1, Pbx2, and Pbx3 are broadly present in E9.5 embryos, when the various cell types involved in OFT septation are being established. All 3 Pbx proteins are present in CNCCs before their delamination from the neural tube and during their migration to the heart to form the aortopulmonary septum.32 Additionally, Pbx proteins are present in branchial mesenchymal cells, surface ectoderm and endoderm cells that neighbor the migrating CNCCs. Pbx proteins may function in these cells to regulate CNCC differentiation and/or migration.4,38–41 Furthermore, all Pbx proteins are present in endocardial and myocardial cells of the OFT at E9.5. Derived from secondary heart fields,42 these OFT cells are sites of action for genes, such as endothelin43 and semaphorin 3C,44 that regulate OFT development. Later, at E12.5, during the active process of OFT septation, Pbx1, Pbx2, and Pbx3 are commonly present in endocardial and cushion mesenchymal cells but not in myocardial or smooth muscle cells. Thus, the partially redundant functions of Pbx genes are, in part, a result of their overlapping expression in cells that contribute to cardiac OFT development.
The Pbx-defined cardiac malformations support a model in which the total level of Pbx gene function correlates with phenotype severity (Figure 7). A major gene (Pbx1) contributes to OFT development in the context of subsidiary roles for paralogous genes (Pbx2 and Pbx3). The spectrum of OFT defects observed in different combinations of Pbx gene mutations indicates that progressive reductions of Pbx gene dosage below a critical threshold correlate with increasingly severe anomalies, each corresponding to specific heart defects in humans. Conversely, there is no manifestation of congenital heart disease once combined Pbx expression is above a minimum level. Thus, the cardiac malformations observed in 10 distinct combinations of loss-of-function alleles of Pbx1, Pbx2, and Pbx3 demonstrate that genetic interactions and gene dosage are sufficient to determine the penetrance and expression of a range of specific cardiac lesions. Analogously in humans, environmental factors may not always be necessary to account for the penetrance or variable degrees of severity of cardiac defects within a genetically predisposed family. An examination for polymorphic alleles of paralogous or interacting genes of an identified predisposing gene within human pedigrees may contribute to understanding the complex inheritance of congenital heart disease and ultimately improve the accuracy of genetic counseling.
We thank M. Ambrus and C. Nicolas for excellent technical assistance. We are indebted to M. Rabinovitch, D. Bernstein, and members of the laboratory of C.-P.C. for support and advice.
Sources of Funding
This work was supported by National Cancer Institute grants CA90735 and CA42971 (to M.L.C.); and National Heart, Lung, and Blood Institute grant HL085345, the American Heart Association, the Children’s Heart Foundation, the March of Dimes Birth Defects Foundation, the Baxter Foundation, and a Tobacco-Related Disease Research Program award (to C.P.C.). K.S. is supported by an American Heart Association Postdoctoral Fellowship; C.S. is supported by an NIH National Research Service Award fellowship; and K.Y.T. is supported by Stanford University Dean’s, McCormick, and NIH National Research Service Award fellowships.
↵*Both authors contributed equally to this work.
Original received March 11, 2008; revision received August 4, 2008; accepted August 11, 2008.
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