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J. Rivera-Feliciano, C.J. Tabin / Developmental Biology 295 (2006) 580588


the yolk sacs of the Bmp2Nkx2.5del

embryos show normal

vasculature and blood formation (data not shown). We have demonstrated that Bmp2 is indispensable for extracellular matrix deposition, endocardial swelling and heart-valve mesenchyme formation.

Bmp2Nkx2.5del atrioventricular endothelium fails to initiate


Since Bmp2Nkx2.5del

mutants live past stages of EC spe-

cification, we can examine the proximal tissue interactions between myocardium and endocardium during cushion morphogenesis at the AVC. When cultured in a collagen lattice, AVC explants reiterate EMT (Runyan and Markwald, 1983). Using this assay we determined the stage at which valve specification arrested in Bmp2Nkx2.5del hearts. After 48 h in culture, control explants produced a halo of mesenchymal

cells (Fig. 2A, bright field), while Bmp2Nkx2.5del

explants did

not (Fig. 2B, bright field), indicating that Bmp2 is required to produce invasive mesenchyme. The absence of EMT in mutant explants at 48 h in culture verifies the fact that the AV canal is incapable of undergoing normal morphogenesis in these animals.

BMP2 rescues EMT from AV explants deficient for Bmp2 by inducing cellcell separation and invasive mesenchymal cell formation

As cardiac conditioned media can replace the requirement for myocardium to achieve EMT in culture (Mjaatvedt and Markwald, 1989), it has been proposed that myocardium secretes a molecule(s) that elicits endocardial cell differentia- tion. Bmp2 is thought to be this factor as it is only expressed in myocardium adjoining cushion-forming segments and induces EMT in monolayer cultures of AV endothelium (Sugi et al., 2004). To address physiological sufficiency, a biochemical replacement experiment was performed in Bmp2-deficient explants. We found that addition of 200 ng/ml BMP2 to Bmp2Nkx2.5del explants rescued cellcell separation and invasive mesenchymal cell formation (Fig. 2C, bright field), indicating BMP2 is sufficient for EMT. To confirm that invasive mesenchymal cells form, we analyzed PECAM1 and smooth muscle alpha actin (SMA) expression to discern endothelial from mesenchymal cells (Sugi et al., 2004). The cell adhesion molecule PECAM1 is expressed in endothelial cells and downregulated during EMT in the AVC (Sugi et al., 2004). PECAM1 was maintained in endocardial cells of control,

Fig. 2. Bmp2 is necessary and sufficient for epithelial to mesenchymal transformation of the atrioventricular cushions. (A) Bright field and confocal micrographs of a representative control explant. Immunohistochemical detection of platelet cell adhesion molecule (PECAM, green) and anti-smooth muscle actin (a-SMA, red). Nuclei are stained with DAPI in blue. The merged images are on the right. Black arrowheads indicate cells with mesenchymal morphology. Yellow arrowheads show transformed mesenchymal cells, and asterisks show activated untransformed endothelial cells. e, Endothelium; m, myocardium. Magnification for bright field 6.3×;

magnification for confocal micrographs 400×. (B) Bmp2Nkx2.5del

representative explant, note the absence of cells with mesenchymal morphology (bright field) and the

lack of activated and transformed endothelium (Merge). (C) BMP2 protein can rescue invasive mesenchyme formation (bright field), activation and transformation of the endothelium (Merge) in Bmp2Nkx2.5del explants.

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