Palatal Epithelial Cells Migrate Independently of Ephrin Signaling during Fusion

Abstract

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 showed 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. In order to observe this migration, we performed real time imaging of fusing embryonic mouse palates in culture using two‐photon confocal microscopy. The palates came from mice expressing an ephrin‐B2‐driven green fluorescent protein (GFP) that specifically labels the nuclei of epithelial cells in the palate. Immediately after 48 h of imaging, tissues were fixed, sectioned, and examined for fusion. The motion of individual MES cells was tracked over the time course of culture using Imaris software. MES cells migrated in a surprisingly unidirectional fashion 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 confirmed histologically, thus demonstrating that our culture time frame encompassed the period required for palatal fusion. Addition of unclustered EphA4/Fc recombinant protein during the culture period to block ephrin reverse signaling inhibited fusion, as we previously established, but did not change the observed migration of GFP‐labeled cells. 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.Support or Funding InformationNIH/NIDCR R01DE022804This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.