An essential role of Bmp4 in the atrioventricular septation of the mouse heart

Abstract

Proper septation and valvulogenesis during cardiogenesis depend on interactions between the myocardium and the endocardium. By combining use of a hypomorphic Bone morphogenetic protein 4 (Bmp4) allele with conditional gene inactivation, we here identify Bmp4 as a signal from the myocardium directly mediating atrioventricular septation. Defects in this process cause one of the most common human congenital heart abnormalities, atrioventricular canal defect (AVCD). The spectrum of defects obtained through altering Bmp4 expression in the myocardium recapitulates the range of AVCDs diagnosed in patients, thus providing a useful genetic model with AVCD as the primary defect.

Figure 1.

Expression of Bmp4 during cardiogenesis. (a-e) Embryonic hearts of Bmp4^lacZ/+ embryos on ICR background were collected at stages labeled in each panel, and were stained with X-gal. In the Bmp4^lacZ allele, the lacZ reporter gene was knocked into the Bmp4 locus, and lacZ expression faithfully recapitulates endogenous Bmp4 transcription in Bmp4^lacZ/+ embryos. (f-o) Transverse sections of whole embryos (f-g) or frontal sections of hearts (h-o) after whole-mount X-gal staining. All sections were counterstained with eosin. In addition to expression in the muscle layer of the OT (g,j,l,n,o) in cardiomyocytes overlying the IEC (f,h) and in muscular portions of ASP (i,k) described in the text, positive signals were also detected in the mesenchyme of the truncus cushion at E10.5 (green arrowhead in j), in the dorsal wall of the atrium (f,h,i), in venous valves (i,k), and in the annulus of mitral and tricuspid valves (m). The Bmp4 expression pattern was confirmed with in situ hybridization analysis (data not shown). (at) Aorta trunk; (ca) common atrium; (la) left atrium; (lv) left ventricle; (nb) newborn; (ot) outflow tract; (pt) pulmonary trunk; (ra) right atrium; (rv) right ventricle; (vv) venous valve; (^*) AV endocardial cushions; (black arrow) atrial septum primum. Bars, 0.2 mm.

Figure 2.

Recombination induced by cTnT-Cre. (a,b) The cTnT Cre;R26R^+/- embryos at E7.5 (a) and E10.5 (b) were stained with X-gal. (c) The heart dissected from the embryo in panel b. (d,e) Sagittal sections of a cTnT-Cre; R26R^+/- embryo at E10.5 stained with X-gal. Panel d represents the view in bright field and panel e corresponds to the dark-field view. X-gal staining products appear pink, and are more easily detected in the dark field. Sections were counterstained with eosin. (f) Embryonic hearts were collected from Bmp4^{loxp-lacZ/tm1} (-Cre) or Bmp4^{cre;loxp-lacZ/tm1} (+Cre) embryos from stages E9.5 to E11.5. RT-PCR analysis was performed to examine the expression of Bmp4, and β-Actin was used as a positive control. No PCR products were obtained if reverses transcriptase was omitted (data not shown). (H) Heart; (hf) head fold; (la) left atrium; (ot) outflow tract; (pc) precardiac crescent; (a) atrium; (ra) right atrium; (v) ventricle; (lv) left ventricle. Bars, 0.4 mm.

Figure 3.

Dosage requirement of Bmp4 for AV septation. (a-f) Neonatal hearts from cTnT-Cre;Bmp4^loxp-lacZ/+ (a,b), Bmp4^{loxp-lacZ/tm1} (c,d), and Bmp4^{cre;loxp-lacZ/loxp-lacZ} (e,f) mice were stained with X-gal. cTnT-Cre;Bmp4^loxp-lacZ/+ mice are indistinguishable from wild-type mice (data not shown) and are used as positive controls. Bmp4^{loxp-lacZ/tm1} and Bmp4^{cre;loxp-lacZ/loxp-lacZ} hearts display partial AVCD with normal AV and ventricular septa. (g-h) Sagittal sections of an E11.0 Bmp4^{cre;loxp-lacZ/tm1i} (Cko) embryo (g) or an E11.0 cTnT-Cre;Bmp4^loxp-lacZ/+ (Contr) embryo (h) stained with X-gal. (i-q) Frontal sections of Bmp4^{cre;loxp-lacZ/tm1} (Cko) embryonic hearts (i,k,m,n,p,q) and cTnT-Cre;Bmp4^loxp-lacZ/+ (Contr) embryonic hearts (j,l,o) at stages labeled in each panel. Panels n, p, and q are from the same heart shown in panel m. Panel n shows the defect in ASP, which is not obvious in panel m. Panels p and q show that both aorta and pulmonary trunk are connected to the right ventricle. (r) An ultrasound scan of a heart from a human patient with the complete form of AVCD. The + indicates the common AVC. All sections were counterstained with eosin except sections of panels m, n, p, and q, which were stained with hematoxylin. (a) Atrium; (at) aorta trunk; (la) left atrium; (lv) left ventricle; (ot) outflow tract; (pt) pulmonary trunk; (ra) right atrium; (rv) right ventricle; (v) ventricle; (arrowhead) atrial septum primum; (^*) AV septum in panels b, d, and f, and AV cushions in panels g, i, and j. Bars, 0.2 mm.

Figure 4.

Phenotype of AV region of embryos of different Bmp4 genotypes. (a,b,c) Normal formation of AV cushions in Bmp4^lacZ/lacZ embryonic hearts. A heart from a Bmp4^lacZ/lacZ embryo with 30 somites is grossly normal when compared with a Bmp4^lacZ/+ heart at the same stage (a). Formation of AV cushions in a Bmp4^lacZ/lacZ embryo (b) parallels that of a Bmp4^lacZ/+ embryonic heart (c). (d,e) Results of section in situ hybridization using a TGFβ2 probe. Expression of TGFβ2 in the AVC of a Bmp4^{cre;loxp-lacZ/tm1} embryonic heart at E12.5 (d) is not altered when compared with the results observed from a wild-type embryonic heart at the same stage (e). (f-i) Frontal sections of Bmp4^{cre;loxp-lacZ/tm1} (f,g) and cTnTCre;Bmp4^loxp-lacZ/+ (h,i) embryonic hearts at E12.5 are stained with DAPI to visualize the nuclei (f,h) and with anti-Ki67 antibody to visualize proliferating cells (g,i). Panels f and g correspond to the boxed regions of Figure 3c; panels h and i correspond to the boxed regions of Figure 3d. (j) The number of Ki67-positive nuclei as a percentage of total nuclei (mean ± S.E.) of Bmp4^{cre;loxp-lacZ/tm1} embryonic hearts (Cko) is reduced to about 80% of cTnTCre;Bmp4^loxp-lacZ/+ embryonic hearts (Contr). Data were averaged from four embryonic hearts of each strain, and at least 1500 nuclei were counted for each heart. (ca) Common atrium; (v) ventricle; (^*) AV cushions; (#) P = 0.047 < 0.05.