Fig. 9. E10.5 AVC explants from Hhex^–/– hearts show increased EMT that is blocked by inhibiting Vegf signaling. (A) Top six panels show light microscopic images of E10.5 AVC explants from C57Bl/6J mice, wild-type mice from the Hhex^+/– intercrosses, and Hhex^–/– mice cultured for 72 hours showing increased numbers of transformed mesenchymal cells in the Hhex^–/– explants. The bottom two panels are representative confocal images of endocardial cell outgrowths immunstained for -SMA. In the wild-type explant (left panel), the cells are more epitheliod in morphology with relatively few mesenchymal cells. In the Hhex^–/– explants, most of the cells are spindle-shaped mesenchymal cells that exhibit little cell-cell contact. Thus, there is ongoing EMT of AVC explants in the absence of Hhex at E10.5, a gestational age when EMT is usually complete. (B) Confocal images of E10.5 AVC explants immunostained for -SMA in untreated wild-type explants (left panel), untreated Hhex^–/– explants (middle panel), and in Hhex^–/– explants treated with 25 µg/ml s-Flt (right panel). Below each panel is a corresponding z-plane image showing the distance cells have moved into the collagen gel, which is indicative of the invasive capacity of the cells. The broken line represents the top of the collagen gel. These panels show that wild-type cells at E10.5 are epithelioid in morphology, maintain close cell-cell contact, and do not invade the collagen gel. Hhex^–/– cells undergo extensive transformation into spindle-shaped mesenchymal cells, maintain little cell-cell contact, and show extensive migration into the collage gel (arrows). Treatment of Hhex^–/– explants with s-Flt reverts the cell morphology to the wild-type phenotype as evidenced by rounded cells with extensive cell-cell contact and no invasion of the collagen gel.