Inactivation of Bmp4 from the Tbx1 expression domain causes abnormal pharyngeal arch artery and cardiac outflow tract remodeling

Abstract

Maldevelopment of outflow tract and aortic arch arteries is among the most common forms of human congenital heart diseases. Both Bmp4 and Tbx1 are known to play critical roles during cardiovascular development. Expression of these two genes partially overlaps in pharyngeal arch areas in mouse embryos. In this study, we applied a conditional gene inactivation approach to test the hypothesis that Bmp4 expressed from the Tbx1 expression domain plays a critical role for normal development of outflow tract and pharyngeal arch arteries. We showed that inactivation of Bmp4 from Tbx1-expressing cells leads to the spectrum of deformities resembling the cardiovascular defects observed in human DiGeorge syndrome patients. Inactivation of Bmp4 from the Tbx1 expression domain did not cause patterning defects, but affected remodeling of outflow tract and pharyngeal arch arteries. Our further examination revealed that Bmp4 is required for normal recruitment/differentiation of smooth muscle cells surrounding the PAA4 and survival of outflow tract cushion mesenchymal cells.

Fig. 1

Effective inactivation of Bmp4 by Tbx1-Cre. a–d Tbx1-Cre male mice were crossed with Bmp4^{loxP-lacZ/loxP-lacZ} female mice to obtain Tbx1-Cre;Bmp4^loxP-lacZ/+ embryos at different stages. Cre-mediated recombination on the Bmp4^loxP-lacZ allele will lead to lacZ knocked into the Bmp4 locus, and therefore expression of the lacZ reporter will be under the control of endogenous Bmp4 regulatory elements. An embryo at E9.0 was wholemount stained with X-gal (a), and was sagittally sectioned (b). A heart isolated from an E12.5 embryo was stained with X-gal (c) and further sectioned (d). The red arrows indicate examples of positively stained cells. e, f Tbx1-Cre;Bmp4^tm1/+ male mice were crossed with Bmp4^{loxP-lacZ/loxP-lacZ} female mice to get mutant embryos (Tbx1-Cre;Bmp4^{tm1/loxP-lacZ}) and their littermate controls at E10.5. Wholemount in situ hybridization analysis was performed using a probe corresponding to the exon 4 of Bmp4, which is expected to be removed upon Cre-mediated recombination. The red arrows indicate the region where the signal was dramatically reduced in the mutant embryo (f) compared to the control (e). Scale bar = 200 μm.

Fig. 2

Spectrum of cardiovascular defects in Tbx1-cre;Bmp4^{tm1/loxP-lacZ} embryos. Tbx1-Cre;Bmp4^tm1/+ male mice were crossed with Bmp4^{loxP-lacZ/loxP-lacZ} female mice to obtain mutant embryos (Tbx1-Cre;Bmp4^{tm1/loxP-lacZ}) and their littermate controls at different stages. a–d Gross examination of the outflow tract and pharyngeal arch artery regions of control (a) and mutant embryos (c, d) at E14.5. b Example of a mutant embryo with the outflow tract septation defect observed in the proximal region, but not in the distal region. c Example of a mutant embryo with complete persistent truncus arteriosus. d Embryo with IAA-B and retroesophageal right subclavian artery, which are commonly observed aortic arch artery defects in DS patients. All embryos were subsequently sectioned, revealing that they all possess a ventricular septum defect (data not shown). e, f A mutant and a control embryonic heart at E19.5 were sectioned and HE stained. A ventricular septal defect is identified in the mutant heart (f). Scale bars = 500 μm.

Fig. 3

Defective pharyngeal arch artery remodeling in Tbx1-cre;Bmp4^{tm1/loxP-lacZ} embryos. a, b The 3rd and 4th pharyngeal arch artery derivatives were patent in both wild-type (a) and mutant (b) E10.5 embryos. c–f Cardiac ink injection was performed on E11.5 (c, d) and E12.5 (e, f) embryos. The remodeling of pharyngeal arch arteries was comparable between wild-type (c) and mutant (d) embryos at E11.5. While at E12.5, the remodeling defect became apparent in mutant embryos (f). The arrowhead in f indicates the IAA-B defect. g, h Sagittal sections of a wild-type (g) and a mutant (h) embryo at E11.0 were immunostained with an antibody against SMA. A group of SMA-positive cells surrounding the endothelial tube of the right 4th pharyngeal arch artery derivative were observed in the wild-type embryo but not in the mutant one. Expression of SMA was detected in the atrial wall of both control and mutant embryos. Scale bars = 200 μm (a–f); 30 μm (g, h).

Fig. 4

Hypoplastic outflow tract cushion defect in Tbx1-cre;Bmp4^{tm1/loxP-lacZ} embryos. Mutant (b, d) and littermate control (a, c) embryos were isolated at E10.5 (a, b) and 11.5 (c, d), and cross-sectioned followed by HE staining. The arrows indicate outflow tract cushions. Scale bars = 200 μm.

Fig. 5

Normal expression of NCC markers in the pharyngeal region. Mutant (b, d) and control (a, c) embryos were isolated at E9.5 and subjected to wholemount in situ hybridization analysis using AP-2 (a, b) and Crabp1 (c, d) probes. The arrows indicate NCCs between the 4th and 6th pharyngeal arch artery derivatives, where cardiac NCCs are localized. Scale bars = 200 μm.

Fig. 6

Cell proliferation and apoptosis in the outflow tract cushion of control (a, c, e, g) and mutant (b, d, f, h) embryos. a–d E11.5 embryos were cross-sectioned and immunostained with an antibody against phospho-histone H3 (PH3), which stains cells at the M phase. c and d correspond to a and b, respectively. The arrows indicate examples of positively stained nuclei. No visible reduction in cell proliferation rate was detected. Similar results were observed at other stages (data not shown). e–h E12.5 embryos were cross-sectioned and subjected to the TUNEL assay. g and h correspond to e and f, respectively. Arrows indicate examples of apoptotic cells in outflow tract cushions. More apoptotic cells were observed in mutant outflow tract cushions. i, j Quantitative analysis of cell proliferation (i) and apoptosis (j) in outflow tract cushions of control and mutant embryos from E10.5 to E12.5. Data were averaged from at least 3 independent embryos with error bars indicating standard derivation. * p < 0.01 (Student's t test). Scale bars = 200 μm (a, b, e, f); 80 μm (c, d, g, h).