Ephrin-B Reverse Signaling for Palatal EMT Requires Contact

Objective: We previously demonstrated that ephrin-B reverse signaling is necessary and sufficient to cause palate EMT and fusion when palatal shelves were cultured with their medial edge epithelia (MEE) in contact.  Furthermore, this signaling was PI-3 kinase dependent.  Palatal shelves depend on a contact with apposing shelf MEE to initiate fusion.  Since Ephs and ephrins are membrane-bound molecules that control contact-mediated events in development, we asked whether they supply the contact-dependent fusion initiation signal.

Method: Embryonic day eight (E8) chicken palatal shelves were cultured on nucleopore polycarbonate membranes in isolation from each other for 24, 48, or 72 h in 37^oC with 5% CO₂.  Medium was replaced every 24 h with fresh TGFß3 (50 ng/ml), clustered EphB2/Fc (4 µg/ml), or control IgG Fc (also 4 µg/ml).  TGFß3 or EphB2/Fc at these concentrations normally cause complete fusion within 72 h when shelves are in contact.

Result: We predicted that clustered (bioactive) Eph/B2/Fc would cause EMT and epithelial seem degradation even in the absence of shelf-to-shelf MEE contact.  Instead, we observed an unusual hypertrophy of the epithelial layer.  The cells in this layer lacked the polarity and organization of a normal epithelium, but neither did they display the fibroblastic morphology of the underlying mesenchyme.

Conclusion: Application of clustered, soluble EphB2/Fc is not sufficient to cause palatal EMT and epithelial layer degradation without shelf-shelf contact.  Instead, ephrin signaling in the absence of the contact-mediated signal appears to initiate a growth/proliferation program in the epithelium.  We are currently characterizing the consequences of this signaling and its underlying mechanism.

Supported by Baylor Oral Health Foundation