Atrioventricular cushion transformation is mediated by ALK2 in the developing mouse heart

Abstract

Developmental abnormalities in endocardial cushions frequently contribute to congenital heart malformations including septal and valvular defects. While compelling evidence has been presented to demonstrate that members of the TGF-beta superfamily are capable of inducing endothelial-to-mesenchymal transdifferentiation in the atrioventricular canal, and thus play a key role in formation of endocardial cushions, the detailed signaling mechanisms of this important developmental process, especially in vivo, are still poorly known. Several type I receptors (ALKs) for members of the TGF-beta superfamily are expressed in the myocardium and endocardium of the developing heart, including the atrioventricular canal. However, analysis of their functional role during mammalian development has been significantly complicated by the fact that deletion of the type I receptors in mouse embryos often leads to early embryonal lethality. Here, we used the Cre/loxP system for endothelial-specific deletion of the type I receptor Alk2 in mouse embryos. The endothelial-specific Alk2 mutant mice display defects in atrioventricular septa and valves, which result from a failure of endocardial cells to appropriately transdifferentiate into the mesenchyme in the AV canal. Endocardial cells deficient in Alk2 demonstrate decreased expression of Msx1 and Snail, and reduced phosphorylation of BMP and TGF-beta Smads. Moreover, we show that endocardial cells lacking Alk2 fail to delaminate from AV canal explants. Collectively, these results indicate that the BMP type I receptor ALK2 in endothelial cells plays a critical non-redundant role in early phases of endocardial cushion formation during cardiac morphogenesis.

Co-ordinating Notch, BMP, and TGF-β signaling during heart valve development

“…For example, BMP2 and TGFβ2 can bind to Betaglycan to induce EMT in chick ventricular and AVC explant assays [174,175], which suggests this interaction plays an important role in AVC cushion formation. Loss of ALK2 in endothelium leads to reduced phosphorylation of TGFβ and BMP Smads [45] and indicates either that loss of BMP signalling reduces TGFβs expression (as mentioned earlier) or that BMP signalling can induce TGFβ Smads specifically via different combinations of receptor-ligand complexes or vice versa. In support of this, TGFβs have been shown to induce both TGFβ and BMP Smads in keratinocytes and authors suggest that TGFβ activation of BMP Smads likely occurs via ALK2 (not ALK1, although this is also seen in other cell types) and is dependent on ALK5 [176].…”

“…Interestingly, if ALK3 is specifically deleted from the AV myocardium only, mutant mice exhibit defects in AV valve leaflets and disruption of the annulus fibrosis [62], indicating that ALK3 is required for later stages of valve remodelling. Similar to the BMP ligands, no OFT cushion defects were seen in ALK2 and ALK3 knockout mice [45,60,61]. Thus, tissuespecific loss of BMP type I receptors in the developing heart indicates that they are essential for cardiac cushion formation and EMT in the AVC.…”

“…BMP ligands 2, 4, 5, 6, and 7 are all expressed in AVC and OFT myocardium during valve formation [39][40][41][42][43][44]. The BMP type I receptor, ALK2 is expressed in endocardium and a subset of mesenchymal cells in the heart at E10 [45]. During mouse embryonic development, ALK3 and BMPRII are ubiquitously expressed [46,47].…”

“…Endocardial cells lacking ALK2 fail to transform into mesenchymal cells and populate the cardiac cushions and embryos that do survive to E14.5 have ventricular septal defects and valve abnormalities [45]. Endocardial loss of ALK2 leads to decreased phosphorylation of both BMP-and TGFβ-specific Smads and reduction of Snail, a transcriptional repressor required for EMT [45].…”

“…Endocardial loss of ALK2 leads to decreased phosphorylation of both BMP-and TGFβ-specific Smads and reduction of Snail, a transcriptional repressor required for EMT [45]. The reduction in TGFβ-Smad phosphorylation was surprising and suggest that BMP signalling allows endocardial cells to be responsive to TGFβ signalling [45]. This endocardial responsiveness could be attributable to ALK2-mediated BMP signalling that leads to activation of Notch signalling resulting in TGFβ2 expression and subsequently induction of Smad2/3 signalling [51].…”