PI-3 kinase activity is required for epithelial-mesenchymal transformation during palate fusion.
Epithelial-mesenchymal transformation (EMT) is the primary mechanism for the disappearance of medial edge epithelia (MEE) during palate fusion. This phenotype transition is highly regulated by growth factors, extracellular matrix, cell surface receptors, and a variety of intracellular signaling. Phosphatidylinositol-3 (PI-3) kinase regulates cytoskeleton reorganization, cell migration, and transforming growth factor (TGF) beta-regulated EMT. Therefore, we investigated the role of PI-3 kinase in EMT during palatal fusion in vitro. Palatal shelves from embryonic (E) 13.5 day mouse embryos were collected and cultured for up to 72 hr. A specific PI-3 kinase inhibitor, LY294002, was added to the medium at concentrations of 100 etaM, 1 microM, and 10 microM. The fate of midline epithelia was traced by carboxyfluorescence labeling and analyzed by confocal microscopy. Harvested tissues were also processed for immunohistochemical analysis of a specific marker for basal lamina (laminin). Palatal fusion stages were scored on a scale of 1 to 5, with 1 equal to complete nonfusion and 5 equal to complete fusion. The mean fusion score (MFS) was calculated for each treatment group. Palatal shelves fused after 72 hr of culture in control and 100 etaM LY294002 inhibitor-treated groups, with MFS of 4.67 and 4.5, respectively. Laminin was absent in the midline and epithelia transformed into mesenchyme. However, when cultured palates were treated with 1 and 10 microM LY294002, MEE persisted in the midline and the basal lamina remained intact after 72 hr. The MFS was significantly less in the 1 and 10 microM LY294002-treated tissues at 2.08 and 1.33, respectively. Our results demonstrate that EMT during palatal fusion in vitro is dependent on PI-3 kinase activity.
Kang
Pei
P
Biomedical Sciences, Texas A&M University System, Baylor College of Dentistry, Dallas, Texas 75246, USA.
Svoboda
Kathy K H
KK
D000818:Animals; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004848:Epithelium / Q000196:embryology / Q000201:enzymology; D066298:In Vitro Techniques; D008648:Mesoderm / Q000166:cytology / Q000201:enzymology; D051379:Mice; D008815:Mice, Inbred Strains; D018613:Microscopy, Confocal; D009025:Morpholines / Q000494:pharmacology; D010159:Palate / Q000166:cytology / Q000196:embryology* / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors
D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one