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Ann Thorac Surg 2008;85:2090-2098. doi:10.1016/j.athoracsur.2008.02.027
© 2008 The Society of Thoracic Surgeons
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Original Articles: Pediatric Cardiac

Endothelial Expression of Bone Morphogenetic Protein Receptor Type 1a is Required for Atrioventricular Valve Formation

Kan Kaneko, MD, PhD, Xiaodong Li, MD, PhD*, Xiaoxue Zhang, MD*, John J. Lamberti, MD, Stuart W. Jamieson, MB, FRCS, Patricia A. Thistlethwaite, MD, PhD*

Division of Cardiothoracic Surgery, University of California San Diego, San Diego, California

Accepted for publication February 1, 2008.

* Address correspondence to Dr Thistlethwaite, Division of Cardiothoracic Surgery, University of California San Diego, 200 W Arbor Dr, MC Suite 8892, San Diego, CA 92103-8892 (Email: pthistlethwaite{at}ucsd.edu).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 Footnotes
 Acknowledgments
 References
 
Background: Atrioventricular canal defects account for 4% of all congenital heart anomalies. They arise from failure of endocardial cushion formation, a process dependent on transition of endothelial cells into clustered mesenchymal cells in the mid–atrioventricular septum. To date, the genetic signals necessary for atrioventricular canal defects are poorly understood. We hypothesized that bone morphogenetic protein signaling in cardiac endothelial cells may be crucial to this process.

Methods: To study the role of bone morphogenetic protein receptors (Bmpr) in the developing heart, we created knockout mice with inactivation of Bmpr1a selectively in endocardium. Two strains of null mice were created: one with constitutive endothelial-specific knockout of Bmpr1a and one with time-inducible, endothelial-specific knockout of Bmpr1a. Embryos and animals were analyzed by microscopy, RNA in situ hybridization, and microangiography.

Results: Animals with null mutation of Bmpr1a in endothelium were embryonic lethal at E11.5 to 12.0 and demonstrated absence of endocardial cushion formation. Embryos failed to form atrioventricular valves and adjacent septa. Endocardial knockout of Bmpr1a did not affect development of the outflow tract or aortic arches. Using time-inducible, cell-specific knockout mice, we show that Bmpr1a has two functions in the developing atrioventricular canal: to induce endocardial endothelial-mesenchymal transition, and to pattern the septal mesenchyme into endocardial cushions. We demonstrate that these processes are temporally linked to expression of the transcription factors Id1 and Id3.

Conclusions: Endocardial cushion formation is dependent on cell-specific expression of Bmpr1a. Our results suggest that Bmpr1a-mediated signaling is a crucial pathway involved in pathogenesis of atrioventricular septal and valve malformations, which are among the most common congenital heart defects in humans.


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Comment
 Footnotes
 Acknowledgments
 References
 
In humans, abnormalities in the atrioventricular (AV) valves are among the most frequent congenital heart defects. The first visible manifestation of this process is the formation of the endocardial cushions that separate the forming atria and ventricles. Atrioventricular endocardial cushion formation occurs in humans when embryonic size reaches approximately 5 mm at 3 weeks' gestation, and is first seen in the mouse at day 9.5 (E9.5) of a 21-day gestational period.

Formation of AV endocardial cushions during cardiac morphogenesis is required for normal valvular and septal morphogenesis. Five critical morphologic steps (Fig 1) are requisite for normal endocardial cushion formation and maturation [1]: (1) the specification of endocardial endothelial cells overlying the atrioventricular canal region to a lineage destined for valve tissue; (2) the delamination of AV endothelial cells away from underlying myocardium with separation of endocardium and myocardium by cardiac jelly; (3) the differentiation of AV endocardial endothelial cells into mesenchymal cells that invade the cardiac jelly, a process termed "endocardial-to-mesenchymal transition" (EMT); (4) the fusion of cellularized endocardial cushions to form the AV valvuloseptal complex that divides the ventricular inflow into right and left AV annuli; and (5) the further remodeling of mesenchymal cells into fibrous tissue that forms the central core of mature valve leaflet tissue. Although detailed morphologic assessment of embryologic valve formation has been widely studied, the study of genetic signals that govern endocardial cushion formation is a newly emerging field.


Figure 1
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  		Fig 1. Endocardial cushion formation and maturation occurs through discrete steps, including (1) the specification of endocardial cells overlying the area connecting the common atria and ventricles (atrioventricular canal [AVC]) to a lineage destined valve tissue; (2) the separation of atrioventricular (AV) endothelial cells (EC) from underlying myocardial cells (My); (3) the deposition of cardiac matrix jelly between the AV endothelium and myocardium; (4) endothelial-to-mesenchymal transition (EMT) of endocardial cells (responding to bone morphogenetic protein [Bmp] stimulation from myocardial AV cells); and (5) the remodeling of mesenchymal cells into fibrous tissue that forms the core of the valve leaflets. The E8.0 to E12.5 represent postcoital gestational days 8 to 12.5.

 
Recently, single gene disruption in mice and large-scale mutagenesis screens in zebrafish have begun to delineate genes that govern early cardiac valve development. Bone morphogenetic proteins (Bmps) have been shown to play a role in mammalian heart valve development [2], and to contribute to cardiac looping in frogs, and cardiac myogenesis and valve formation in chicken and fish [3]. Mouse and avian explant studies, using collagen gel invasion assays, have demonstrated the role of Bmps as an essential paracrine signal in EMT in endocardial cushion formation [2, 4].

In other cell systems, Bmp signaling has been found to be mediated by two types of serine/threonine kinase receptors, type 1 and type 2. There are three Type 1 receptors: bone morphogenetic protein receptor type 1a (Bmpr1a), bone morphogenetic protein receptor type 1b (Bmpr1b), and activin A receptor, type 1 (Acvr1). There is a single Type 2 receptor: bone morphogenetic receptor type 2 (Bmpr2). In the presence of Bmp ligand, Bmpr2 phosphorylates a type 1 receptor in a heteromeric cell surface complex, which activates signaling by intracellular Smad transcription factors [5]. In a variety of cell types, Bmpr signaling through Smad proteins has been found to increase the transcription of Id ("inhibitor of differentiation") genes [6]. The Id proteins act by sequestering basic helix loop helix (bHLH) transcription factor-E proteins, preventing them from binding DNA. The Ids thus block bHLH-mediated transcription, and they exert their effect through a dominant-negative mode of regulation [7].

Germline mutation in mice in either Bmpr1a or Bmpr2 leads to death during gastrulation [8]. Thus, conventional null alleles cannot be used to test whether, where, or how Bmpr signaling functions during cardiac morphogenesis. Conditional deletion of Bmpr receptors, by targeted expression of Cre recombinase, has provided a means to circumvent early lethality and study the role of these receptors in mammalian heart development.

The goal of our research was to understand the role of Bmpr receptors in heart morphogenesis, specifically their role in myocardial endocardium and valve patterning. To this end, we studied the endothelial derivation of cardiac valve structures using lacZ reporter mice, and created cell-specific and time-inducible Bmpr1a null mice and studied their phenotype.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Comment
 Footnotes
 Acknowledgments
 References
 
Generation of Mutant Mice
Four mouse lines were used to generate mouse mutants that were studied: Floxed Bmpr1a mice (Bmpr1af/f) [9], Tie2-Cre mice [10], Tie2-CreERT2 mice [11], and Rosa26R (R26R) reporter mice [12]. Homozygously floxed animals exhibit gene knockdown in all cells expressing the Cre transgene. The Tie2 promoter directs the expression of the Cre transgene in rodents uniquely and abundantly to endothelial cells, allowing for knockdown of specific genes in this cell type. The Tie2-CreERT2 line is a tamoxifen-inducible, endothelial cell–specific Cre-expressing mouse line that was created to allow gestational timed, cell-specific knockdown of specific genes. To generate endothelial-specific knockout mice, we created Tie2-Cre–/+::Bmpr1af/f embryos. To generate time-inducible, endothelial-specific knockout mice, we made Tie2-CreERT2–/+::Bmpr1af/f mice. To induce knockout of Bmpr1a in Tie2-CreERT2–/+::Bmpr1af/f embryos, pregnant mothers were injected intramuscularly with 3 mg tamoxifen on the gestational day of desired gene knockdown. This dose of tamoxifen has been previously shown to be nonteratogenic in mice and induces Cre-dependent gene knockdown in Tie2-CreERT2–/+ endothelium within 24 hours [11]. Genomic DNA for polymerase chain reaction genotyping was derived from tail clips or embryonic yolk sacs. Mice received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" (available at: www.nap.edu/catalog/5140.html). All experiments performed were approved by the University of California, San Diego, Institutional Animal Use and Care Committee.

Whole-Mount RNA in Situ Hybridization
Whole-mount RNA in situ hybridization was done as previously described [13], with at least 6 mutants and 6 controls analyzed for each gestational timepoint. Probes for Id1 (890 base pairs incorporating exons 1 and 2), Id2 (914 base pairs incorporating exons 1 to 3), and Id3 (938 base pairs incorporating exons 1 to 3) were used.

LacZ Staining and Histology
For histologic sectioning, embryos were fixed in 4% paraformaldehyde, dehydrated in ethanol and embedded in paraffin. Transverse 5-µm sections were stained with hematoxylin-eosin, and examined by microscopy. For Cre-dependent LacZ expression, whole embryos of age E12.5 or less and isolated neonatal hearts were whole-mount stained for LacZ activity, cut into 5-µm transverse sections, and examined by microscopy. In all hematoxylin-eosin and LacZ staining experiments, at least 6 mutants and 6 controls were analyzed for each gestational timepoint using each technique.

Microfil Polymer Injection
Microfil was injected into the left ventricle of E10.5 Tie2-Cre–/+::Bmpr1a+/+ (control) and Tie2-Cre–/+::Bmpr1af/f (mutant) embryos (4 embryos examined for each group) using 30 gauge glass pipettes and a Harvard (South Natick, Massachusetts) micropump. After circulation of the polymer, embryos were fixed in 4% paraformaldehyde, dehydrated in increasing series of ethanol, and cleared in methyl salicylate.


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 Footnotes
 Acknowledgments
 References
 
Endocardial Endothelial Cells of the Atrioventricular Canal Are the Origin of the Atrioventricular Valves and Annulus Fibrosus
To study the fate of endocardial endothelial cells in the AV canal, we used mice expressing Cre under the control of the Tie2 endothelial-specific promoter (Tie2-Cre) and LacZ-reporter mice for Cre activity (R26R mice). Cells in the R26R mice that have undergone Cre-mediated recombination express LacZ irreversibly, allowing for the mapping of their fate. Because all progeny of cells expressing LacZ will also express LacZ, the lineage fate of these cells in determining contribution to valve maturation can be determined. Previous studies have indicated that Tie2 is an endothelial-specific promoter, and the activity of the Tie2 promoter can be detected in a subset of endocardial endothelial cells in the common atrial chamber as early as E8.5 in mouse embryos [14]. We found that Tie2 promoter activity localizes to cardiac cushion cells of the AV canal at E10.5, when common atrial and ventricular cavities are present (Fig 2A). By E10.5, Tie2-Cre::R26R double heterozygote animals express LacZ homogenously throughout the mesenchymal cells that populate endocardial cushions of the AV canal (Fig 2A). The high levels of LacZ staining seen in AV cushions and valve primordia are indicative of extensive endothelial contribution to these structures.


Figure 2
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  		Fig 2. Endothelial origins of valve leaflets and supporting structures in Tie2-Cre::R26R double heterozygote mice. The LacZ staining of Tie2-Cre–/+::R26R–/+ mouse hearts from different gestational days demonstrates the fate of endocardial cushion cells in the tricuspid and mitral valves. (A) Gestational day E10.5 heart section stained for LacZ (blue) and counterstained with eosin, showing endothelial origin of atrioventricular (AV) valve mesenchyme (arrow points to AV cushion) (LA = left atrium; LV = left ventricle; RA = right atrium; RV = right ventricle.) (B) LacZ expression (blue) in neonatal bisected heart with the atria removed, showing endothelial derivation of tricuspid and mitral valves. (ant = anterior leaflet; Ao = aorta; PA = pulmonary artery; post = posterior leaflet; sep = septal leaflet.) (C) Fresh adult bisected heart with atria removed, showing tricuspid and mitral valves for anatomic comparison with B. (D) Section of neonatal heart demonstrating whole mount LacZ staining (blue) in neonatal tricuspid and mitral valves, and fibrous septum connecting the two valves. The AV canal region, including tricuspid (box E) and mitral (box G) valve leaflets and the fibrous supporting apparatus (box F) is visualized. (E, F, G) Higher magnification of the indicated boxed areas in D, showing the tricuspid valve leaflets (E), the fibrous supporting apparatus (F), and the mitral leaflets (G). Arrowheads in E and G point to tricuspid and mitral leaflets respectively; arrowhead in F points to the fibrous septum connecting mitral and tricuspid valves.

 
In the neonate, high levels of LacZ expression were detected in the mature valve leaflets, chordae tendinae, and myotendinous junctions, demonstrating their endothelial origin (Fig 2B–G). The majority of cells in these structures were LacZ positive, although localized areas of less staining were noted (Fig 2E and G). An additional area of LacZ staining was seen in the supporting valvuloseptal fibrous continuity that encompasses the ventricular septum between the leaflets of the mitral and tricuspid valves (Fig 2F). In contrast, LacZ staining was not seen in muscular components of the atrial or ventricular septa, suggesting alternate cell origins for these structures.

Bmpr1a Is Required for Cardiac Septation and Normal Cushion Morphogenesis
No newborn pups were found with the Tie2-Cre–/+::Bmpr1af/f genotype of 20 litters examined (Fig 3A). All three other potential genotypes were present in a Mendelian ratio. Endothelial-restricted deletion of Bmpr1a (Tie2-Cre–/+::Bmpr1af/f) was invariably lethal by E11.5 to E12.0, with embryos showing growth retardation or hemorrhage, or both, and resorption between E11.5 and E12.5. By E13.5, marked resorption of nonviable embryos was seen, and fewer than 5% of nonviable embryos were recovered (Fig 3B).


Figure 3
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  		Fig 3. Bone morphogenetic protein receptor type 1a (Bmpr1a) is essential for atrioventricular (AV) endocardial cushion morphogenesis. (A) Recovery of embryos with the Tie2-Cre–/+::Bmpr1af/f genotype. Mendelian frequencies of Tie2-Cre–/+::Bmpr1af/f were recovered at gestational day E11.5, but began to be lost by E12.5. No mutants were recoverable at E14.5. In total, 140 embryos were collected with 20 embryos at each stage. (B) Morphology of Tie2-Cre–/+::Bmpr1af/f embryos (mutant) during development, compared with Tie2-Cre–/+::Bmpr1a+/+ (controls). Note, hemorrhage and death by E11.5 to E12.0 and smaller embryonic size from resorption by E12.5 in Bmpr1a mutant embryos.

 
Endothelial-specific deletion of Bmpr1a (Tie2-Cre–/+::Bmpr1af/f) invariably produced an absence of AV cushion (Fig 4). Histologic examination of hearts showed that these mutant embryos displayed a paucity of mesenchymal cells within the area of the AV cushions. At E10.5, animal hearts demonstrated endocardial endothelium separation (delamination) from underlying myocardium in the AV canal in the usual manner; however, no EMT or invasion of mesenchymal cells into the cardiac jelly between endothelium and myocardium was seen. By this gestational age, a complete absence of mesenchymal cells was seen in the area separating endocardium and myocardium. The hypocellular AV cushion phenotype was 100% penetrant in homozygous endothelial-specific Bmpr1a knockout animals. Failure of endocardial cushion formation in Bmpr1a mutants led to complete AV canal defect by E9.5 to E11.5 and death by E11.5 to E12.0.


Figure 4
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  		Fig 4. Endothelial knockout of bone morphogenetic protein receptor type 1a (Bmpr1a) results in failure to form endocardial cushions, leading to absence of tricuspid and mitral valves. Histologic analysis of atrioventricular (AV) cushion development in Tie2-Cre–/+::Bmpr1af/f mutants (right column) compared with Tie2-Cre–/+::Bmpr1a+/+ controls (left column) from gestational days E10.5 to E11.5. Atrioventricular endothelium in Bmpr1a mutant animal hearts failed to undergo endothelial-to-mesenchymal transition, resulting in absence of mesenchymal cells in the AV cushion region between endothelial and myocardial layers. Red arrows indicate AV endothelium; black arrows indicate AV myocardium; blue arrows indicate mesenchymal cells populating the cardiac jelly. (LA = left atrium; LV = left ventricle; RA = right atrium; RV = right ventricle.)

 
Outflow Tract and Aortic Arch Development Is Not Affected by Null Mutation of Bmpr1a
The absence of AV cushion formation and presence of persistent interatrial/interventricular communication in Tie2-Cre–/+::Bmpr1af/f animals was not the result of aberrant or obstructive outflow tract and aortic arch formation. Microangiography performed on E10.5 embryos demonstrated normal conal and truncal outflow tract development as well as normal development of aortic arches III (carotid arteries), IV (aortic arch and subclavian arteries), and VI (ductus arteriosus and right/left pulmonary arteries) in Bmpr1a mutant and control Bmpr1a-expressing mice (Fig 5).


Figure 5
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  		Fig 5. Microfil polymer injection of Tie2-Cre–/+::Bmpr1a+/+ (control: left panel) and Tie2-Cre–/+::Bmpr1af/f (mutant: right panel) animals at E10.5 reveals normal outflow tract and aortic arch development in bone morphogenetic protein receptor type 1a (Bmpr1a) mutants. Outflow tract (OFT) will form ascending aorta and main pulmonary artery; aortic arch III will form external and internal carotid arteries; aortic arch IV will form aortic arch and subclavian arteries; aortic arch VI will form ductus arteriosis and right and left pulmonary arteries. (Upper panel = left lateral view; lower panel = posterior view.)

 
Bmpr1a Regulates Expression of Id1 and Id3
Since Id proteins have been implicated as downstream effectors of Bmpr1a signaling, we examined the expression of Id1 to Id3 in the developing mouse heart and in Tie2-Cre–/+::Bmpr1af/f mutant mouse hearts. By RNA in situ hybridization, we found that Id1 and Id 3 are highly expressed in the AV cushion region, particularly in AV endocardium and mesenchymal cells in E9.5 to E11.5 control Tie2-Cre–/+::Bmpr1a+/+ embryos (Fig 6). In contrast, Id2 staining was only weakly detected in Bmpr1a control hearts in the AV cushions, implying that Bmpr1a signaling may not be linked to Id2 expression in cells in this region (data not shown). No Id1 and Id3 staining was detected in the AV canal region in Bmpr1a null embryos (Tie2-Cre–/+::Bmpr1af/f embryos), suggesting that Bmpr1a expression is requisite for Id1/3 expression in this area.


Figure 6
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  		Fig 6. The "inhibitor of differentiation" genes Id 1 and Id 3 are expressed in the atrioventricular (AV) cushions of control Tie2-Cre–/+::Bmpr1a+/+ mouse embryos. Top panel: whole mount in situ hybridization of gestational day E10.5 embryos with Id1 and Id3 probes, showing staining of AV cushions separating atria and ventricles. Arrows point to AV cushions. Bottom panel: sections of hearts seen in top panel. Arrows point to mesenchymal cells in the AV cushions. (Bmpr1a = bone morphogenetic protein receptor type 1a; LA = left atrium; LV = left ventricle; RA = right atrium; RV = right ventricle.)

 
Timing of Abrogation of Bmpr1a Endocardial Expression Determines Phenotypic Outcome of AV Canal
Using tamoxifen-inducible Tie2-Cre mice (Tie2-CreERT2), we investigated when expression of Bmpr1a was essential for AV valve formation during embryogenesis. Animals with induced knockout of Bmpr1a (Tie2-CreERT2–/+::Bmpr1af/f) at E8.0 demonstrated absence of AV cushion formation, suggesting that expression of this protein was necessary as early as day 8 of gestation (during heart looping and segmentation into chambers) for AV cushion commitment (Fig 7). In contrast, animals with induced knockout of Bmpr1a at E10.5 developed normal AV cushions and had normal AV valves at birth. The molecular patterning responsible for AV valve cushion morphogenesis is committed before E10.5 in the mouse.


Figure 7
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  		Fig 7. Endothelial commitment to atrioventricular (AV) cushion formation occurs by gestational day E8.0. Sections of eosin-stained mouse hearts from control (Tie2-CreERT2–/+::Bmpr1a+/+: left panel) and mutant (Tie2-CreERT2–/+::Bmpr1af/f: right panel) embryos harvested at E11.5. Mothers of both sets of embryos were treated with tamoxifen at E8.0. Tamoxifen-induced endothelial knockout of bone morphogenetic protein receptor type 1a (Bmpr1a) in the homozygously floxed Bmpr1a mutants. Endothelial-specific deletion of Bmpr1a at E8.0 resulted in absence of cushion mesenchymal cells in Tie2-CreERT2–/+::Bmpr1af/f embryo hearts (arrows indicate endocardial cushions). That suggests that expression of Bmpr1a is necessary as early E8.0 for AV cushion and valve formation. (LA = left atrium; LV = left ventricle; RA = right atrium; RV = right ventricle.)

 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
Footnotes
 Acknowledgments
 References
 
The heart is the first organ that forms in a mammalian fetus. In the human fetal heart, a complex dialogue of regulated signals is required for valve development. Because of this complexity, the process often derails, resulting in atrioventricular canal defects that account for approximately 4% of all congenital heart anomalies. Valve defects have even a higher prevalence in fetal death before term [15]. Multiple transcription and growth factors have been implicated in valve formation, but the precise hierarchical molecular code regulating this process remains unknown.

We have several major conclusions from this study. First, using cell lineage mapping, we show that the progeny of cells derived from endocardial endothelium become the cells that comprise the mature tricuspid and mitral valves (leaflets and chordae) as well as the fibrous septum that divides these two valves in the mouse. Second, we demonstrate that endothelial-specific expression of Bmpr1a is required for population of AV cushions with mesenchymal cells. Inactivation of Bmpr1a within endothelial-derived cells blocked EMT, resulting in a paucity of mesenchymal cells within the AV cushions. The requirement for Bmpr1a endothelial function occurred in a gestational timeframe from E8.0 to E10.5. Animals with knockout of the Bmpr1a gene, as early as the eighth day of gestation, fail to develop AV cushions, whereas mice with knockout of this gene after the tenth day of gestation were born with morphologically normal tricuspid and mitral valves. Third, our analysis of Bmpr1a-deficient embryos indicates that the processes of delamination and mesenchymal transformation can be distinguished. Endothelial expression of Bmpr1a does not appear to be necessary for separation of cardiac endothelial cells from myocardial cells in early AV cushion formation. Fourth, we show that endothelial-specific knockout of Bmpr1a causes pathology restricted to the AV cushion region and does not affect the common outflow tract or aortic arch formation. Atrioventricular and left-to-right communication between the forming heart chambers is not the result distal obstructive pathology in the outflow trunk or forming great vessels. Finally, expression of Bmpr1a was associated with expression of Id1 and Id3 in endothelial and mesenchymal cells within the developing AV cushions. We suspect that loss of Id1 and Id3 expression in Bmpr1a null animals affects the transcription of bHLH-activated sets of genes that pattern the morphogenesis of AV valve leaflets. Collectively, these results suggest that temporal and cell-specific expression of a single gene (Bmpr1a) plays a major role in endocardial cushion development and processing, which is requisite to the formation of mature mitral and tricuspid valves.

Previous experiments have shown that Bmps secreted in the AV region serve as a ligand stimulus for AV cushion formation [16, 17]. Bone morphogenetic protein 2 and 4 (Bmp2 and 4) have been found to be expressed in cardiac myocytes adjacent to the AV cushions, Bmp5 and 7 more homogeneously in atrial and ventricular cardiac myocytes, and Bmp10 in trabeculae [7]. It has been postulated that Bmp2 is released from myocardium as a chemical gradient for induction of EMT. Knockout studies using Bmp2 have shown that this ligand has three functions in the AV myocardium of mice: to stimulate the formation of cardiac jelly by regulating the production of Has2, an extracellular matrix protein; to induce EMT (presumably through Bmpr1a signaling in endocardial endothelial cells); and to regulate AV myocardial gene expression. It has been found that Bmp2 is required for myocardial Tbx2 expression, which normally represses expression of chamber-specific genes (namely, Anf, chisel, connexin 40) in the AV canal. Interestingly, mice mutant for an intracellular inhibitor of Bmp signaling, Smad6, display the converse phenotype of enlarged endocardial cushions [5]. Smad6 is an inhibitor of signal transduction downstream of Bmp receptors, whose function is thought to be to control and limit the duration of the ligand-induced signaling cascade through Smad1, 5, and 8 [18].

Several recent papers have shown that Bmpr1a and 1b expression may be essential for normal atrioventricular valve formation in the chicken [19] and the mouse [20, 21]. Using a viral gene transfer approach, Okagawa and colleagues [19] have shown that overexpression of a dominant-negative form of Bmpr1b alters AV valve morphogenesis in the developing avian embryo at stage 14–, the period in which EMT occurs in the chick. Similarly, conditional knockout of Bmpr1a in the mouse in flt1-expressing cells resulted in embryonic lethality between E10.5 and E11.5 [20]; while conditional knockout of Bmpr1a in the mouse in Tie1-expressing cells resulted in embryonic lethality at E12.5 [21].

Our research differs from these studies in two major ways. First, in our work, conditional knockout was done for Bmpr1a in Tie2-expressing cells. The Tie2 receptor is expressed in all endothelial cells in the mammalian heart and great vessels, whereas Tie1 has expression restricted to certain endothelial cell subsets in the mediastinum [22], and flt1 is expressed not only in endothelial cells but also in other cell types, particularly smooth muscle progenitor cells and myocytes [23]. Restricting conditional knockdown to endothelium allowed our focus to be inclusive but not restrictive of the endothelial cell lineage in the heart and great vessels. Second, although Id expression has been shown to be linked to Bmpr signaling in other organs, this is the first report of Id1 and Id3 expression in AV cushion cells. Our results do not necessarily prove that Bmpr1a signaling is requisite for Id1/3 expression; rather, we show a temporal and spatial link between expression of these two gene families. Finally, our work has demonstrated that AV canal defects caused by single gene inactivation are not linked to (or are the result of) downstream obstruction, like right ventricular outflow tract or aortic arch aberrant/obstructive development.

It may be that Bmpr1a may also have pleomorphic effects in AV valve formation after AV cushion formation. It has recently been shown that in utero conditional myocyte deletion of Bmpr1a, using a cGATA6-Cre transgene, results in term-born animals that have normal AV cushion development [24]. However, myocyte Bmpr1a knockdown resulted in animals with downward displacement of the posterior tricuspid leaflet, a defect similar to that seen in Ebstein's anomaly, suggesting that valve leaflet and chordal remodeling may also be influenced by expression of this gene.

A growing number of single-gene mutations have also been associated with abnormal development of cardiac valves and with defects in endocardial cushion formation. These include mutations in Gata4, fibroblast growth factor 4 (Fgf4), Notch, hesr2, Nfatc1, neuregulin, ErbB3, type1 neurofibromatosis 1 (Nf1), platelet-derived growth factor receptor-{alpha}, Shp2, TGFs, sox9-10, hesr2, foxp1, and others [1]. However, to date, little progress has been made in assembling these individual genes into genetic or molecular cascades that control AV cushion development or subsequent valve remodeling.

In summary, the most frequent congenital heart defects in humans affect the cardiac valves and adjacent septa, and the genetics of this process is being revealed. Our results demonstrate that signaling through the type-I receptor Bmpr1a is required for appropriate transcriptional activation of Id1 and Id3, and induction of EMT during formation of mouse AV endocardial cushions in vivo, an early step in leading to development and maturation of AV-valve leaflets and septa. This study provides conclusive evidence that knockout of Bmpr1a signaling can disrupt cardiac morphogenesis in mammals. The invariable defects resulting from conditional deletion of Bmpr1a in endothelium provide strong support for its assessment as a candidate gene in human congenital heart disease.


   Acknowledgments
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Footnotes
 Acknowledgments
References
 
Dr Thistlethwaite is supported by grants from the National Institutes of Health (NIH R01 HL 70852) and the Center for Medical Research and Education Fund (CMREF). Floxed Bmpr1a mice were provided by Richard Behringer (M.D. Anderson Cancer Center); Tie2-Cre mice were a gift from Masashi Yanagisawa (University of Texas Southwestern Medical Center), and Tie2-CreERT2 mice were provided by Bernd Arnold (University of Heidelberg). Rosa26R (R26R) reporter mice were obtained from Jackson Labs.


   Footnotes
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Footnotes
Acknowledgments
 References
 
* These authors contributed equally to this manuscript. Back


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 Footnotes
 Acknowledgments
 References
 

  1. Schroeder JA, Jackson LF, Lee DC, Camenisch TD. Form and function of developing heart valves: coordination by extracellular matrix and growth factor signaling J Mol Med 2003;81:392-403.[Medline]
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  3. Lincoln J, Alfieri CM, Yutzey KE. BMP and FGF regulatory pathways control cell lineage diversification of heart valve precursor cells Dev Biol 2006;15:292-302.
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Richard W. Kim
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