ABSTRACT: We report that the dominant human missense mutations G303E and G296S in GATA4, a cardiac-specific transcription factor gene, cause atrioventricular septal defects and valve abnormalities by disrupting a signaling cascade involved in endocardial cushion development. These GATA4 missense mutations, but not a mutation causing secundum atrial septal defects (S52F), demonstrated impaired protein interactions with SMAD4, a transcription factor required for canonical bone morphogenetic protein/transforming growth factor-? (BMP/TGF-?) signaling. Gata4 and Smad4 genetically interact in vivo: atrioventricular septal defects result from endothelial-specific Gata4 and Smad4 compound haploinsufficiency. Endothelial-specific knockout of Smad4 caused an absence of valve-forming activity: Smad4-deficient endocardium was associated with acellular endocardial cushions, absent epithelial-to-mesenchymal transformation, reduced endocardial proliferation, and loss of Id2 expression in valve-forming regions. We show that Gata4 and Smad4 cooperatively activated the Id2 promoter, that human GATA4 mutations abrogated this activity, and that Id2 deficiency in mice could cause atrioventricular septal defects. We suggest that one determinant of the phenotypic spectrum caused by human GATA4 mutations is differential effects on GATA4/SMAD4 interactions required for endocardial cushion development.
Project description:NADPH oxidases (NOX) are a major source of reactive oxygen species (ROS) production in the heart. ROS signaling regulates gene expression, cell proliferation, apoptosis, and migration. However, the role of NOX2 in embryonic heart development remains elusive. We hypothesized that deficiency of Nox2 disrupts endocardial to mesenchymal transition (EndMT) and results in congenital septal and valvular defects. Our data show that 34% of Nox2-/- neonatal mice had various congenital heart defects (CHDs) including atrial septal defects (ASD), ventricular septal defects (VSD), atrioventricular canal defects (AVCD), and malformation of atrioventricular and aortic valves. Notably, Nox2-/- embryonic hearts show abnormal development of the endocardial cushion as evidenced by decreased cell proliferation and an increased rate of apoptosis. Additionally, Nox2 deficiency disrupted EndMT of atrioventricular cushion explants ex vivo. Furthermore, treatment with N-acetylcysteine (NAC) to reduce ROS levels in the wild-type endocardial cushion explants decreased the number of cells undergoing EndMT. Importantly, deficiency of Nox2 was associated with reduced expression of Gata4, Tgf?2, Bmp2, Bmp4, and Snail1, which are critical to endocardial cushion and valvoseptal development. We conclude that NOX2 is critical to EndMT, endocardial cushion cell proliferation, and normal embryonic heart development.
Project description:Septal defects and coronary vessel anomalies are common congenital heart defects, yet their ontogeny and the underlying genetic mechanisms are not well understood. Here, we investigated the role of chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII, NR2F2) in cardiac organogenesis.We analyzed embryos deficient in COUP-TFII and observed a spectrum of cardiac defects, including atrioventricular septal defect, thin-walled myocardium, and abnormal coronary morphogenesis. We show by expression analysis that COUP-TFII is expressed in the endocardium and the epicardium but not in the myocardium of the ventricle. Using endothelial-specific COUP-TFII mutants and molecular approaches, we show that COUP-TFII deficiency resulted in endocardial cushion hypoplasia. This was attributed to the reduced growth and survival of atrioventricular cushion mesenchymal cells and defective epithelial-mesenchymal transformation (EMT) in the underlying endocardium. In addition, the endocardial EMT defect was accompanied by downregulation of Snai1, one of the master regulators of EMT, and upregulation of vascular endothelial-cadherin. Furthermore, we show that although COUP-TFII does not play a major role in the formation of epicardial cell cysts, it is critically important for the formation of epicardium. Ablation of COUP-TFII impairs epicardial EMT and coronary plexus formation.Our results reveal that COUP-TFII plays cell-autonomous roles in the endocardium and the epicardium for endocardial and epicardial EMT, which are required for proper valve and coronary vessel formation during heart development.
Project description:Wnt signaling mediated by beta-catenin has been implicated in early endocardial cushion development, but its roles in later stages of heart valve maturation and homeostasis have not been identified. Multiple Wnt ligands and pathway genes are differentially expressed during heart valve development. At E12.5, Wnt2 is expressed in cushion mesenchyme, whereas Wnt4 and Wnt9b are predominant in overlying endothelial cells. At E17.5, both Wnt3a and Wnt7b are expressed in the remodeling atrioventricular (AV) and semilunar valves. In addition, the TOPGAL Wnt reporter transgene is active throughout the developing AV and semilunar valves at E16.5, with more localized expression in the stratified valve leaflets after birth. In chicken embryo aortic valves, genes characteristic of osteogenic cell lineages including periostin, osteonectin, and Id2 are expressed specifically in the collagen-rich fibrosa layer at E14. Treatment of E14 aortic valve interstitial cells (VICs) in culture with osteogenic media results in increased expression of multiple genes associated with bone formation. Treatment of VIC with Wnt3a leads to nuclear localization of beta-catenin and induction of periostin and matrix gla protein but does not induce genes associated with later stages of osteogenesis. Together, these studies provide evidence for Wnt signaling as a regulator of endocardial cushion maturation as well as valve leaflet stratification, homeostasis, and pathogenesis.
Project description:Congenital heart defects affect ?2% of live births and often involve malformations of the semilunar (aortic and pulmonic) valves. We previously reported a highly penetrant <i>GATA4</i> p.Gly296Ser mutation in familial, congenital atrial septal defects and pulmonic valve stenosis and showed that mice harboring the orthologous G295S disease-causing mutation display not only atrial septal defects, but also semilunar valve stenosis. Here, we aimed to characterize the role of Gata4 in semilunar valve development and stenosis using the <i>Gata4<sup>G295Ski/wt</sup></i> mouse model. GATA4 is highly expressed in developing valve endothelial and interstitial cells. Echocardiographic examination of <i>Gata4<sup>G295Ski/wt</sup></i> mice at 2?months and 1?year of age identified functional semilunar valve stenosis predominantly affecting the aortic valve with distal leaflet thickening and severe extracellular matrix (ECM) disorganization. Examination of the aortic valve at earlier postnatal timepoints demonstrated similar ECM abnormalities consistent with congenital disease. Analysis at embryonic timepoints showed a reduction in aortic valve cushion volume at embryonic day (E)13.5, predominantly affecting the non-coronary cusp (NCC). Although total cusp volume recovered by E15.5, the NCC cusp remained statistically smaller. As endothelial to mesenchymal transition (EMT)-derived cells contribute significantly to the NCC, we performed proximal outflow tract cushion explant assays and found EMT deficits in <i>Gata4<sup>G295Ski/wt</sup></i> embryos along with deficits in cell proliferation. RNA-seq analysis of E15.5 outflow tracts of mutant embryos suggested a disease state and identified changes in genes involved in ECM and cell migration as well as dysregulation of Wnt signaling. By utilizing a mouse model harboring a human disease-causing mutation, we demonstrate a novel role for GATA4 in congenital semilunar valve stenosis.This article has an associated First Person interview with the joint first authors of the paper.
Project description:Distal outgrowth, maturation and remodeling of the endocardial cushion mesenchyme in the atrioventricular (AV) canal are the essential morphogenetic events during four-chambered heart formation. Mesenchymalized AV endocardial cushions give rise to the AV valves and the membranous ventricular septum (VS). Failure of these processes results in several human congenital heart defects. Despite this clinical relevance, the mechanisms governing how mesenchymalized AV endocardial cushions mature and remodel into the membranous VS and AV valves have only begun to be elucidated. The role of BMP signaling in the myocardial and secondary heart forming lineage has been well studied; however, little is known about the role of BMP2 expression in the endocardial lineage. To fill this knowledge gap, we generated Bmp2 endocardial lineage-specific conditional knockouts (referred to as Bmp2 cKOEndo) by crossing conditionally-targeted Bmp2flox/flox mice with a Cre-driver line, Nfatc1Cre, wherein Cre-mediated recombination was restricted to the endocardial cells and their mesenchymal progeny. Bmp2 cKOEndo mouse embryos did not exhibit failure or delay in the initial AV endocardial cushion formation at embryonic day (ED) 9.5-11.5; however, significant reductions in AV cushion size were detected in Bmp2 cKOEndo mouse embryos when compared to control embryos at ED13.5 and ED16.5. Moreover, deletion of Bmp2 from the endocardial lineage consistently resulted in membranous ventricular septal defects (VSDs), and mitral valve deficiencies, as evidenced by the absence of stratification of mitral valves at birth. Muscular VSDs were not found in Bmp2 cKOEndo mouse hearts. To understand the underlying morphogenetic mechanisms leading to a decrease in cushion size, cell proliferation and cell death were examined for AV endocardial cushions. Phospho-histone H3 analyses for cell proliferation and TUNEL assays for apoptotic cell death did not reveal significant differences between control and Bmp2 cKOEndo in AV endocardial cushions. However, mRNA expression of the extracellular matrix components, versican, Has2, collagen 9a1, and periostin was significantly reduced in Bmp2 cKOEndo AV cushions. Expression of transcription factors implicated in the cardiac valvulogenesis, Snail2, Twist1 and Sox9, was also significantly reduced in Bmp2 cKOEndo AV cushions. These data provide evidence that BMP2 expression in the endocardial lineage is essential for the distal outgrowth, maturation and remodeling of AV endocardial cushions into the normal membranous VS and the stratified AV valves.
Project description:Noonan syndrome (NS) is the most common nonchromosomal genetic disorder associated with cardiovascular malformations. The most prominent cardiac defects in NS are pulmonary valve stenosis and hypertrophic cardiomyopathy. Gain-of-function mutations in the protein tyrosine phosphatase Shp2 have been identified in 50% of NS families. We created a NS mouse model with selective overexpression of mutant Shp2 (Q79R-Shp2) in the developing endocardial cushions. In our model, Cre recombinase driven by the Tie2 promoter irreversibly activates transgenic Q79R-Shp2 expression in the endothelial-derived cell lineage. Q79R-Shp2 expression resulted in embryonic lethality by embryonic day 14.5. Importantly, mutant embryos showed significantly enlarged endocardial cushions in the atrioventricular canal and in the outflow tract. In contrast, overexpression of wild-type Shp2 protein at comparable levels did not enhance endocardial cushion growth or alter the morphology of the mature adult valves. Expression of Q79R-Shp2 was accompanied by increased ERK1/2 activation in a subset of cells within the cushion mesenchyme, suggesting that hyperactivation of this signaling pathway may play a pathogenic role. To test this hypothesis in vivo, Q79R-Shp2-expressing mice were crossed with mice carrying either a homozygous ERK1 or a heterozygous ERK2 deletion. Deletion of ERK1 completely rescued the endocardial cushion phenotype, whereas ERK2 protein reduction did not affect endocardial cushion size. Constitutive hyperactivation of ERK1/2 signaling alone with a transgenic approach resulted in a phenocopy of the valvular phenotype. The data demonstrate both necessity and sufficiency of increased ERK activation downstream of Shp2 in mediating abnormal valve development in a NS mouse model.
Project description:Mutations in developmental regulatory genes have been found to be responsible for some cases of congenital heart defects. One such regulatory gene is Gata4, a zinc finger transcription factor. In order to circumvent the early embryonic lethality of Gata4-null embryos and to investigate the role of myocardial Gata4 expression in cardiac development, we used Cre/loxP technology to conditionally delete Gata4 in the myocardium of mice at an early and a late time point in cardiac morphogenesis. Early deletion of Gata4 by Nkx2-5Cre resulted in hearts with striking myocardial thinning, absence of mesenchymal cells within the endocardial cushions, and selective hypoplasia of the RV. RV hypoplasia was associated with downregulation of Hand2, a transcription factor previously shown to regulate formation of the RV. Cardiomyocyte proliferation was reduced, with a greater degree of reduction in the RV than in the LV. Late deletion of Gata4 by Cre recombinase driven by the alpha myosin heavy chain promoter did not selectively affect RV development or generation of endocardial cushion mesenchyme but did result in marked myocardial thinning with decreased cardiomyocyte proliferation, as well as double-outlet RV. Our results demonstrate a general role of myocardial Gata4 in regulating cardiomyocyte proliferation and a specific, stage-dependent role in regulating the morphogenesis of the RV and the atrioventricular canal.
Project description:Congenital heart disease is the most common type of birth defect with an incidence of 1%. Previously, we described a point mutation in GATA4 that segregated with cardiac defects in a family with autosomal dominant disease. The mutation (G296S) exhibited biochemical deficits and disrupted a novel interaction between Gata4 and Tbx5. To determine if Gata4 and Tbx5 genetically interact in vivo, we generated mice heterozygous for both alleles. We found that nearly 100% of mice heterozygous for Gata4 and Tbx5 were embryonic or neonatal lethal and had complete atrioventricular (AV) septal defects with a single AV valve and myocardial thinning. Consistent with this phenotype, Gata4 and Tbx5 are co-expressed in the developing endocardial cushions and myocardium. In mutant embryos, cardiomyocyte proliferation deficits were identified compatible with the myocardial hypoplasia. Similar to Gata4, Gata6 and Tbx5 are co-expressed in the embryonic heart, and the transcription factors synergistically activate the atrial natiuretic factor promoter. We demonstrate a genetic interaction between Gata6 and Tbx5 with an incompletely penetrant phenotype of neonatal lethality and thin myocardium. Gene expression analyses were performed on both sets of compound heterozygotes and demonstrated downregulation of alpha-myosin heavy chain only in Gata4/Tbx5 heterozygotes. These findings highlight the unique genetic interactions of Gata4 and Gata6 with Tbx5 for normal cardiac morphogenesis in vivo.
Project description:The transcription factor GATA4 is essential for heart morphogenesis. Heterozygous mutation of GATA4 causes familial septal defects. However, the phenotypic spectrum of heterozygous GATA4 mutation is not known. In this study, we defined the cardiac phenotypes that result from heterozygous mutation of murine Gata4. We then asked if GATA4 mutation occurs in humans with these forms of congenital heart disease (CHD). In mice, heterozygous Gata4 mutation was associated with atrial and ventricular septal defect (ASD, VSD), endocardial cushion defect (ECD), RV hypoplasia, and cardiomyopathy. Genetic background strongly influenced the expression of ECD and cardiomyopathy, indicating the presence of important genetic modifiers. In humans, non-synonymous GATA4 sequence variants were associated with ECD (2/43), ASD (1/8), and RV hypoplasia in the context of double inlet left ventricle (1/9), forms of CHD that overlapped with abnormalities seen in the mouse model. These variants were not found in at least 500 control chromosomes, and encode proteins with non-conservative amino acid substitutions at phylogenetically conserved positions, suggesting that they are disease-causing mutations. Cardiomyopathy was not associated with GATA4 mutation in humans. These data establish the phenotypic spectrum of heterozygous Gata4 mutation in mice, and suggest that heterozygous GATA4 mutation leads to partially overlapping phenotypes in humans. Additional studies will be required to determine the degree to which GATA4 mutation contributes to human CHD characterized by ECD or RV hypoplasia.
Project description:Valvular heart disease is a major cause of mortality and morbidity. Revealing the cellular processes and molecules that regulate valve formation and remodeling is required to develop effective therapies. A key step in valve formation during heart development is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endocardial cells in the atrioventricular cushion (AVC). The type III transforming growth factor-? receptor (TGF?R3) regulates AVC endocardial cell EMT in vitro and mesenchymal cell differentiation in vivo. Little is known concerning the signaling mechanisms downstream of TGF?R3. Here we use endocardial cell EMT in vitro to determine the role of 2 well-characterized downstream TGF? signaling pathways in TGF?R3-dependent endocardial cell EMT. Targeting of Smad4, the common mediator Smad, demonstrated that Smad signaling is required for EMT in the AVC and TGF?R3-dependent EMT stimulated by TGF?2 or BMP-2. Although we show that Smads 1, 2, 3, and 5 are required for AVC EMT, overexpression of Smad1 or Smad3 is not sufficient to induce EMT. Consistent with the activation of the Par6/Smurf1 pathway downstream of TGF?R3, targeting ALK5, Par6, or Smurf1 significantly inhibited EMT in response to either TGF?2 or BMP-2. The requirement for ALK5 activity, Par6, and Smurf1 for TGF?R3-dependent endocardial cell EMT is consistent with the documented role of this pathway in the dissolution of tight junctions. Taken together, our data demonstrate that TGF?R3-dependent endocardial cell EMT stimulated by either TGF?2 or BMP-2 requires Smad4 and the activation of the Par6/Smurf1 pathway.