TGFβ3 promotes transformation of chicken palate medial edge epithelium to mesenchyme in vitro

Epithelial-mesenchymal transformation plays an important role in the disappearance of the midline line epithelial seam in rodent palate, leading to confluence of the palate. The aim of this study was to test the potential of the naturally cleft chicken palate to become confluent under the influence of growth factors, such as TGFβ3, which are known to promote epithelial-mesenchymal transformation. After labeling medial edge epithelia with carboxyfluorescein, palatal shelves (E8-9) with or without beak were dissected and cultured on agar gels. TGFβ1, TGFβ2 or TGFβ3 was added to the chemically defined medium. By 24 hours in culture, medial edge epithelia form adherent midline seams in all paired groups without intact beaks. After 72 hours, seams in the TGFβ3 groups disappear and palates become confluent due to epithelialmesenchymal transformation, while seams remain mainly epithelial in control, TGFβ1 and TGFβ2 groups. Epithelium-derived mesenchymal cells are identified by carboxyfluorescein fluorescence with confocal microscopy and by membrane-bound carboxyfluorescein isolation bodies with electron microscopy. Labeled fibroblasts completely replace the labeled epithelia of origin in TGFβ3treated palates without beaks. Single palates are unable to undergo transformation, and paired palatal shelves with intact beaks do not adhere or undergo transformation, even when treated with TGFβ3. Thus, physical contact of medial edge epithelia and formation of the midline seam are necessary for epithelial-mesenchymal transformation to be triggered. We conclude that there may be no fundamental difference in developmental potential of the medial edge epithelium for transformation to mesenchyme among reptiles, birds and mammals. The bird differs from other amniotes in having developed a beak and associated craniofacial structures that seemingly keep palatal processes separated in vivo. Even control medial edge epithelia partly transform to mesenchyme if placed in close contact. However, exogenous TGFβ3 is required to achieve complete confluence of the chicken palate.