Crk and Crkl Are Required in the Endocardial Lineage for Heart Valve Development.

Background Endocardial cells are a major progenitor population that gives rise to heart valves through endocardial cushion formation by endocardial to mesenchymal transformation and the subsequent endocardial cushion remodeling. Genetic variants that affect these developmental processes can lead to congenital heart valve defects. Crk and Crkl are ubiquitously expressed genes encoding cytoplasmic adaptors essential for cell signaling. This study aims to explore the specific role of Crk and Crkl in the endocardial lineage during heart valve development. Methods and Results We deleted Crk and Crkl specifically in the endocardial lineage. The resultant heart valve morphology was evaluated by histological analysis, and the underlying cellular and molecular mechanisms were investigated by immunostaining and quantitative reverse transcription polymerase chain reaction. We found that the targeted deletion of Crk and Crkl impeded the remodeling of endocardial cushions at the atrioventricular canal into the atrioventricular valves. We showed that apoptosis was temporally increased in the remodeling atrioventricular endocardial cushions, and this developmentally upregulated apoptosis was repressed by deletion of Crk and Crkl. Loss of Crk and Crkl also resulted in altered extracellular matrix production and organization in the remodeling atrioventricular endocardial cushions. These morphogenic defects were associated with altered expression of genes in BMP (bone morphogenetic protein), connective tissue growth factor, and WNT signaling pathways, and reduced extracellular signal-regulated kinase signaling activities. Conclusions Our findings support that Crk and Crkl have shared functions in the endocardial lineage that critically regulate atrioventricular valve development; together, they likely coordinate the morphogenic signals involved in the remodeling of the atrioventricular endocardial cushions.

ß-Catenin regulates endocardial cushion growth by suppressing p21.

Endocardial cushion formation is essential for heart valve development and heart chamber separation. Abnormal endocardial cushion formation often causes congenital heart defects. ß-Catenin is known to be essential for endocardial cushion formation; however, the underlying cellular and molecular mechanisms remain incompletely understood. Here, we show that endothelial-specific deletion of ß-catenin in mice resulted in formation of hypoplastic endocardial cushions due to reduced cell proliferation and impaired cell migration. By using a ß-catenin DM allele in which the transcriptional function of ß-catenin is selectively disrupted, we further reveal that ß-catenin regulated cell proliferation and migration through its transcriptional and non-transcriptional function, respectively. At the molecular level, loss of ß-catenin resulted in increased expression of cell cycle inhibitor p21 in cushion endocardial and mesenchymal cells in vivo. In vitro rescue experiments with HUVECs and pig aortic valve interstitial cells confirmed that ß-catenin promoted cell proliferation by suppressing p21. In addition, one savvy negative observation is that ß-catenin was dispensable for endocardial-to-mesenchymal fate change. Taken together, our findings demonstrate that ß-catenin is essential for cell proliferation and migration but dispensable for endocardial cells to gain mesenchymal fate during endocardial cushion formation. Mechanistically, ß-catenin promotes cell proliferation by suppressing p21. These findings inform the potential role of ß-catenin in the etiology of congenital heart defects.