Failure of the secondary palate to fuse at midline causes cleft palate, a frighteningly common birth defect that can only be treated with surgery and poses a tremendous health burden. Our long-term goal is to understand the mechanism of palatal fusion so as to better treat cleft palate. Transforming growth factor beta-3 (Tgfss3) is thought to be required to form a fused palate of confluent mesenchyme by causing degradation of the midline epithelial seam (MES) between palatal shelves. However, we recently discovered that activated ephrin-B signaling is necessary and sufficient to cause fusion in the absence of Tgfss3. Ephrin reverse signaling causes epithelial-to-mesenchymal transition (EMT) in cultured palate epithelial cells via a mechanism involving phosphotidyl inositol-3 kinase (PI3K). Because ephrins are also known to mediate programmed cell death, and apoptosis is a major part of the fusion mechanism, we propose the hypothesis that ephrin signaling causes palatal fusion through induction of EMT and apoptosis in palatal epithelia. We will test this hypothesis through pursuit of three aims. First, we will use our discovery that ephrin-B2 marks MES cells in fusing palates to track the fate of these cells in real time with confocal imaging. Second, we will determine how the known ephrins in the palate contribute to the epithelial-mesenchymal interactions that govern MES cell migration and apoptosis. Third, we will learn the molecular mechanism of ephrin reverse signaling in fusion by discovering: 1) the signaling proteins that interact with the ephrin cytodomain, 2) the specific PI3K pathway intermediates that transduce the ephrin signal, and 3) the intersection points in the cytoplasm and the nucleus between the ephrin and the known Tgfss3 signaling pathways in palatal epithelium.
This project will answer fundamental questions of palate development and describe a previously unknown mechanism of ephrin signaling, thus having a broad impact on the study of development in general. Because ephrin signaling is associated with carcinoma metastasis, this project will also advance the understand of cancer cell EMT and migration. This work will thus reveal targets for molecular-based therapies for craniofacial deformities and metastatic cancers.