BMP4 is required in the anterior heart field and its derivatives for endocardial cushion remodeling, outflow tract septation, and semilunar valve development

Abstract

The endocardial cushions play a critical role in septation of the four-chambered mammalian heart and in the formation of the valve leaflets that control blood flow through the heart. Within the outflow tract (OFT), both cardiac neural crest and endocardial-derived mesenchymal cells contribute to the endocardial cushions. Bone morphogenetic protein 4 (BMP4) is required for endocardial cushion development and for normal septation of the OFT. In the present study, we show that anterior heart field (AHF)-derived myocardium is an essential source of BMP4 required for normal endocardial cushion expansion and remodeling. Loss of BMP4 from the AHF in mice results in an insufficient number of cells in the developing OFT endocardial cushions, defective cushion remodeling, ventricular septal defects, persistent truncus arteriosus, and abnormal semilunar valve formation.

Fig. 1. Bmp4 expression overlaps the activity of Mef2c-AHF-Cre in the myocardial layer of the outflow tract (OFT) and is abolished in the OFT in Bmp4 AHF conditional knockout mice

(A) Strategy for inactivation of Bmp4 in the Mef2c-AHF-Cre expression domain. (B, C) The activity of the Mef2c-AHF-Cre transgene is specific to the developing pharyngeal mesoderm, OFT, and RV at E10.5 as determined by crossing to ROSA26R Cre-dependent LacZ reporter mice (Rosa) stained with X-gal. (D, E) In situ hybridization for Bmp4 transcripts in control mice (ctrl) demonstrates Bmp4 expression in the pharyngeal endoderm (PE), OFT, and forelimb bud (LB) at E10.5. Within the heart, Bmp4 expression is limited to the myocardial layer (myo) (E) of the OFT and overlaps the activity of the Mef2c-AHF-Cre transgene only in this region. (F, G) In situ hybridization in Bmp4 AHF knockout embryos (ko) shows a complete loss of Bmp4 expression from the myocardial layer (myo) of the OFT at E10.5. Note that expression of Bmp4 in the PE and LB, which are outside the Mef2c-AHF-Cre expression domain, is retained. Genotypes are indicated above panels B and C, D and E, and F and G. The bars in the transverse sections in C, E, and G are equal to 100 μm.

Fig. 2. BMP4 is required in the AHF for septation of the ventricles and outflow tract

(A, B) Transverse sections of neonatal hearts taken from control (ctrl) (A) and Bmp4 AHF knockout (ko) (B) neonatal mice showed a normal interventricular septum (IVS) in control mice and a ventricular septal defect (VSD) in knockout mice. (C, D) Transverse sections through the outflow tracts (OFT) of neonatal mice showed a normally septated pulmonary artery (PA) and aorta (Ao) in control mice (C), while Bmp4 AHF knockout mice exhibited persistent truncus arteriosus (PTA) (D). (E, F) High magnification images of the normally formed membranous IVS in control mice (E) compared to the absence of a membranous septum and a large VSD in Bmp4 AHF knockout mice (F). Note also the misshapen valve leaflets in Bmp4 AHF knockout mice (F). LV, left ventricle; RV, right ventricle. The bars are equal to 100 μm in all panels.

Fig. 3. BMP4 is required in the AHF for proper formation of the semilunar valve leaflets

(A, B) Control neonatal mice (ctrl) have normal formation of the aortic valve (A) and pulmonary valve (B) leaflets with normal leaflet architecture and number. (C-F) Bmp4 AHF knockout (ko) mice have a single common outflow tract (OFT) valve with abnormal formation of the semilunar valve leaflets. The neonatal ko valve leaflets have irregular morphology and disrupted architecture. Note the single outflow valve in (E), which leads to the persistent truncus arteriosus (PTA) that then divides into the aorta (Ao) and pulmonary artery (PA). Modified Movat’s pentachrome staining of ctrl (G) and ko (H) neonatal valves shows the trilaminar extracellular matrix (ECM) and cellular organization of the neonatal leaflets. The cellular component of the control leaflet (G) has become condensed within the ventricularis (V) layer and the proteoglycan is focused within the spongiosa (S) layer. By contrast, the ko leaflet has cells and proteoglycan distributed throughout the leaflet. F, fibrosa layer. Asterisks mark the valve leaflets in all panels. The bars in all panels are equal to 100 μm.

Fig. 4. BMP4 is required in the AHF for outflow tract endocardial cushion expansion

(A, B) Alcian blue staining was used to detect the presence of acidic glycosaminoglycans, such as hyaluronic acid within the endocardial cushions. In comparison to control (ctrl) embryos (A), Bmp4 AHF knockout (ko) embryos (B) had a similar amount of alcian blue staining in both the atrioventricular (AVC) and outflow tract (OFT) endocardial cushions at E10.5. (C) Control embryos showed normal expansion of the AVC and OFT endocardial cushions at E10.5. (D) Bmp4 AHF knockout embryos showed deficient expansion of the OFT endocardial cushions at E10.5. Note the normal expansion of the AVC endocardial cushions, which do not develop adjacent to the OFT myocardium. (E, F) The difference in OFT endocardial cushion expansion was also evident at E12.5 in Bmp4 AHF knockout embryos (F) compared to control embryos (E). LA, left atrium; PA, pulmonary artery; RV, right ventricle; VSD, ventricular septal defect. Asterisks in panels E and F denote the endocardial cushions. The bars in all panels are equal to 100 μm.

Fig. 5. Bmp4 AHF knockout embryos have fewer proximal outflow tract (OFT) endocardial cushion cells than littermate controls at E10.5 and E12.5

(A, B) Quantification of DAPI-stained nuclei in the proximal OFT endocardial cushions of control (ctrl) and Bmp4 AHF knockout (ko) embryos showed a significant reduction in the number of cells within the OFT endocardial cushions of knockout embryos at both E10.5 (A) and E12.5 (B) (p < 0.001 at E10.5 and p < 0.0001 at E12.5, n=10 at each stage). (C, D) Quantification of BrdU-labeled cells as a percentage of total DAPI-stained nuclei showed no difference in the rate of proliferation of proximal OFT endocardial cushion mesenchymal cells at E10.5 (C) and E12.5 (D) (p = 0.7172 at E10.5 and p= 0.6967 at E12.5, n=10 at each stage). (E, F) TUNEL staining of the endocardial cushions in the proximal OFT showed no difference in the number of apoptotic cells between control and Bmp4 AHF knockout embryos at E10.5 (E) and E12.5 (F). LA, left atrium; PA, pulmonary artery; RV, right ventricle. Asterisks mark the endocardial cushions in panels E and F. The bars in panels E and F are equal to 100 μm.