Ephrin Reverse Signaling Rescued Palatal Growth at Early Stages

Objectives: The secondary palate forms from mesenchymal shelves covered with epithelium, which adhere to form the midline epithelial seam (MES). Complete disintegration of the MES is essential to form a confluent structure. Failure of palatal fusion causes cleft palate. Studies suggest that the epithelial cells undergo epithelial-to-mesenchymal transition (EMT), apoptosis, or both. Previous experiments demonstrate that TGF-β3 signaling causes the transformation of the MES into mesenchymal cells as well as apoptosis which leads to the completion of palatogenesis. Eph receptor tyrosine kinases and their ephrin ligands are responsible for many developmental processes. Binding of ephrins causes receptor activation in Eph-bearing cells (forward signaling), and intracellular signaling inside ephrin-bearing cells (reverse signaling). We demonstrated that ephrin-B reverse signaling is required for palatal fusion and is sufficient to cause fusion in chicken palates without Tgfβ3 (San Miguel et al., 2011). We asked if ephrin reverse signaling can rescue growth and fusion in palates at early stages without functional TGF-β receptors.
Methods: E13.5 mouse palatal shelves were cultured in the following conditions: control IgG Fc, TGF-β RI kinase inhibitor (SB 431542) (Calbioquem), with or without clustered recombinant EphB2/Fc protein for 36 and 48 hours. In addition, TUNEL assay was performed in the palates and limbs using an apoptosis detection kit (Millipore).
Results: Histological analysis of palatal fusion showed that the control IgG Fc group and the TGF-β RI inhibitor with EphB2 group showed similar growth, apoptosis rate and partial fusion. Palates treated with the TGF-β RI inhibitor showed less growth, had fewer apoptotic cells and did not fuse. Also, the limbs showed interdigital apoptotic cells. The experiments were performed 2 times with n=6 to n=8 per group.
Conclusions: These results demonstrate that ephrin reverse signaling rescues growth and fusion of the palate without TGF-β3 signaling at early stages of development.