Fig. 4.

Ablation of Rere in the endocardium leads to the development of hypocellular AV endocardial cushions and defective EMT. (A-C) At E10.5, the mesenchymal cell density in the AV endocardial cushions of Rere^flox/flox;Tie2-Cre embryos is significantly reduced in comparison with that of Rere^flox/flox embryos. Black arrows point to the AV endocardial cushions. Quantification was performed using eight slides containing at least three sections from five independent littermates. Scale bars: 200 µm. (D-F) The AV canal explants isolated from the hearts of each of the Rere^flox/flox;Tie2-Cre and Rere^flox/flox embryos were cultured on collagen gels. Migrating mesenchymal cells were counted at 24 h and 48 h. The number of migrating mesenchymal cells generated from AV canal explants of Rere^flox/flox;Tie2 Cre embryos is significantly reduced in comparison with that of Rere^flox/flox embryos. Red arrows indicate migrating mesenchymal cells. AV canal explants prepared from five independent littermates were used for collagen gel culture and quantification. AVC, atrioventricular canal explants. Scale bars: 100 µm. (G-I) At E12.5, the percentage of mitotic cells in the AV endocardial cushions of Rere^flox/flox;Tie2 Cre embryos was comparable to that of Rere^flox/flox embryos. Yellow arrows indicate mitotic cells in the AV endocardial cushions. Twenty-one sections obtained from each of three embryos were used for quantification. Scale bars: 100 µm. Data are mean±s.d. Unpaired two-tailed Student's t-test was used to determine P-values (*P<0.05).