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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.
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