Ephrin-A1 is a prototypic ligand for class A Eph receptor tyrosine kinases. Originally discovered as a TNF-α-inducible gene (16
), it was subsequently found that ephrin-A1 induced corneal angiogenesis and tumor neovascularization (3
). During embryonic development, ephrin-A1 is expressed in the endothelium of the developing blood vessels and in the endocardium as early as E8.5 (13
). At E12.5, ephrin-A1 expression is restricted in the endothelial lining of the endocardium, whereas EphA3 receptor is expressed in a complementary pattern within endocardial cushion mesenchyme (36
). EphA3 knockout mice are perinatal lethal and exhibit abnormal AV valves and hypoplastic septum (36
). If ephrin-A1 only functions as a ligand to activate EphA3, loss of ephrin-A1 would be expected to result in a phenotype similar to EphA3-null mice. However, ephrin-A1 knockout mice are viable and have thickened valves, raising the possibility that ephrin-A1 regulates heart valve morphogenesis through independent or additional mechanisms. Interestingly, mice homozygous for the cytoplasmic domain deletion mutant of ephrin-B2 are embryonic lethal with thickened cardiac valves, a phenotype reminiscent of the ephrin-A1 knockout mice (9
). It remains to be determined whether the relatively mild phenotype of ephrin-A1-null mice is due to functional compensation by ephrin-B2.
Heart valve morphogenesis initiates with endocardial cushion transformation in which endocardial cells delaminate, transdifferentiate into mesenchymal cells, and invade into cardiac jelly. Transformed endocardial cushions then undergo subsequent remodeling through proliferation and apoptosis, giving rise to precisely formed cardiac valves that direct blood flow in heart. Three lines of evidence suggest that ephrin-A1 may regulate heart valve formation through, at least in part, inhibition of endocardial cushion transformation. First, outflow tract endocardial cushion cellularity is significantly increased in ephrin-A1 knockout embryos in the absence of changes in neural crest cell contribution. Second, increased cellularity in ephrin-A1-deficient endocardial cushions is accompanied by upregulation of the expression of mesenchymal markers in the embryonic heart. Third, exogenous ephrin-A1-Fc or overexpression of ephrin-A1 inhibited EMT in chick explant collagen gel assays, consistent with an inhibitory role of ephrin-A1 on EMT in vivo.
In addition to a role in inhibiting EMT, ephrin-A1 may also regulate valve morphogenesis through other mechanisms. After completion of EMT, endocardial cushions undergo remodeling, a process that is dependent upon proliferation and apoptosis, cellular migration and reorganization of the extracellular matrix. We observed a moderate reduction in apoptosis in AV cushion mesenchyme and an increase in cellularity in E14.5 Efna1−/− animals (data not shown), suggesting an additional role of ephrin-A1 in regulation of mesenchymal cell survival in the endocardial cushion. Furthermore, Movat's pentachrome staining failed to reveal any gross abnormalities in the distribution of collagen, elastin and glycoproteins within aortic and mitral valves (data not shown), indicating that valve thickness is not due to abnormal ECM deposition or abnormal development of valve layers.
What are the molecular mechanisms of ephrin-A1 action in heart valve development? As ephrin-A1 and EphA3 are expressed complementarily in juxtaposed tissue in endocardial cushion and EphA-ephrin-A interactions are known to mediate repulsive signal during neural development (39
), one possibility is that the EphA3-expressing cells undergoing EMT may be repelled by the ephrin-A1-expressing endocardial cells. However, our data does not support this hypothesis as EphA3-null animals exhibited hypoplastic endocardial cushion (36
) whereas ephrin-A1 knockout mice display hyperplastic valves. Alternatively, ephrin-A1 activation has been shown to increase cell adhesiveness (6
). Ephrin-A1 could positively regulate cell-cell adhesion. Therefore, loss of ephrin-A1 would decrease cell adhesiveness and allow cell detachment from the endothelial layer. In addition, ephrin-A1 could also inhibit the expression of EphA3 receptor, as ephrin-A1 has been shown to negatively regulate the levels of EphA2 receptor in tumor cells (19
). Ablation of ephrin-A1 would then upregulate expression of EphA3, resulting in increased EMT. Our data derived from overexpression of EphA3 in chick heart tube is consistent with this hypothesis.
In summary, we have shown that ephrin-A1 is critical in regulating proper formation of the mammalian heart valve. As ephrin-A1 is also expressed in blood vessels, the role of ephrin-A1 in vascular development and adult angiogenesis remains to be determined. Thus, the ephrin-A1 knockout mouse provides a valuable animal model for studying congenital heart defects as well as angiogenesis in adult.