Msx1 and Msx2 are essential for myocardial patterning and morphogenesis of the outflow tract and atrioventricular cushions

Combined deficiencies of the homeobox genes Msx1 and Msx2 in mice have been shown to cause a broad spectrum of cardiac anomalies including malalignment of the outflow tract (OFT), atrial and ventricular septal defects, and hypoplastic atrioventricular (AV) valves. Here we investigate the cellular mechanisms that contribute to these cardiac defects. In the Msx1-/-; Msx2-/- mutant secondary heart field (SHF) progenitor cells, we found normal expression of early markers Isl1 and Mef2c but reduced expression of later markers Hand1, Hand2, Fgf10 and Pitx2, which regulate differentiation, specification and migration of SHF myocardial precursors destined to incorporate into the OFT. Disruption of asymmetric Pitx2 expression in the Msx1-/-; Msx2-/- mutant SHF and myocardium is associated with left-right mispatteming of the OFT and atrial myocardium as well as atrial septal defect. Reduced bone morphogenetic protein (Bmp) signaling in the Msx1-/-; Msx2-/- mutant atrial and AV myocardium is associated with impaired endothelial-to-mesenchymal transdifferentiation of AV cushions and perturbed expression of chamber-specific genes, such as decreased expression of Tbx2 in the AV myocardium and ectopic expression of Anf in the AV and right ventricular myocardium.; Interestingly, in the Msx1-/-; Msx2-/- mutant pharyngeal arch mesoderm, there was increased instead of decreased Bmp signaling compared with the wild-type embryo. The abnormal increase of Bmp signaling in Msx1-/-; Msx2-/- mutants extends to the outflow tract (OFT) between E10.5 and E11.5 and contributes, together with the premature down-regulation of p27KIP1, to the significant increase of proliferation in the double mutant OFT between E10 and E11. The ultimate consequence is an excessive number of mesenchymal cells accumulating in the double mutant OFT cushion mesenchyme by E12.5, which may obstruct the developing OFT and lead to pulmonary stenosis or atresia, or disrupt OFT rotation and lead to double outlet right ventricle,...