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  The Annals of Thoracic Surgery  Volume 85, Issue 6, Pages 2090-2098 (June 2008) DOI: 10.1016/j.athoracsur.2008.02.027 Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

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. The Annals of Thoracic Surgery 2008 85, 2090-2098DOI: (10.1016/j.athoracsur.2008.02.027) Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

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. The Annals of Thoracic Surgery 2008 85, 2090-2098DOI: (10.1016/j.athoracsur.2008.02.027) Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

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. The Annals of Thoracic Surgery 2008 85, 2090-2098DOI: (10.1016/j.athoracsur.2008.02.027) Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

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.) The Annals of Thoracic Surgery 2008 85, 2090-2098DOI: (10.1016/j.athoracsur.2008.02.027) Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

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.) The Annals of Thoracic Surgery 2008 85, 2090-2098DOI: (10.1016/j.athoracsur.2008.02.027) Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

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.) The Annals of Thoracic Surgery 2008 85, 2090-2098DOI: (10.1016/j.athoracsur.2008.02.027) Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions

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.) The Annals of Thoracic Surgery 2008 85, 2090-2098DOI: (10.1016/j.athoracsur.2008.02.027) Copyright © 2008 The Society of Thoracic Surgeons Terms and Conditions