Palatal Epithelial Cells Migrate Independently of Ephrin Signaling During Fusion

Objectives: The mammalian secondary palate forms from two shelves of mesenchyme sheathed in a single layered epithelium. Failure of the midline epithelial seam (MES) to degrade when these shelves meet at midline prevents mesenchymal confluence and results in a cleft palate. Our previous studies indicate that cultured MES cells undergo features of epithelial-to-mesenchymal transition (EMT) when ephrin reverse signaling is activated. We therefore hypothesized that midline epithelial cells are removed during fusion by migration away from the seam in response to ephrin action.
Methods: We performed real time imaging of fusing embryonic mouse palates in culture using confocal microscopy. The palates came from mice expressing an ephrin-B2-driven green fluorescent protein (GFP) that specifically labels epithelial cells in the palate. Immediately after 48 h of imaging, tissues were fixed and examined for fusion.
Results: MES cells moved in a sheet toward the oral surface over 48 h of imaging, regardless of tissue orientation on the culture dish, and maintained their GFP expression for the duration of the experiment. Fusion of the imaged palates was observed histologically. Addition of unclustered EphA4/Fc recombinant protein to block ephrin reverse signaling inhibited fusion, as we previously reported, but did not change the observed migration of GFP-labeled cells.
Conclusions: MES cells migrated in a surprisingly unidirectional fashion over the time interval necessary for palatal fusion. Because these cells maintained GFP expression, we conclude that they remained alive and that MES cell death by apoptosis is therefore not required for fusion, as was previously thought. Although ephrin reverse signaling is required for palate fusion, we found that it is not necessary for MES cell migration.