Fate of Medial Edge Epithelium in Mouse Palatogenesis in vitro : Apoptosis, Migration, and Epithelial-mesenchymal Transformation

Abstract

The present study aimed to investigate the pathways and fates of medial edge epithelial (MEE) cells in organ culture of the mouse palatal shelve in combination with vital-dye labelings and in situ confocal observation of labeled MEE cells. Embryos at E13.5–13.7 were used to collect palatal shelves for organ culture. Epithelia of the palatal shelves were labeled with two fluorochromes, CCFSE and DiI. The organ culture was validated histomorphologically to assimilate in vivo palatogenesis at least with respect to sequential events at the midline, i.e., the formation and discontinuity of the epithelial seam and the mesenchymal confluence. Immunohistochemistry showed that extensive cell death was evoked at the midline seam and triangles over the entire fusion processes. Alexa-tagged ssDNA immunostaining showed that, by 6 h after the MEE cells were placed in contact, the first sign of cell death was observed within the intact epithelial seam, distributing along the whole length of the midline seam as well as the triangle. A majority of the labeled MEE cells exhibited motility and followed pathways toward the oral and nasal ends, where the triangular morphology was generated. A relatively small number of MEE cells crossed over the basal lamina and executed phenotypic changes in the mesenchyme. The combined localization of the fluorescence and cytokeratin markers confirmed a loss of epithelial phenotype in fractions of the MEE cells after their migration into the mesenchyme. Their histological appearance indicated that they remained viable after leaving the epithelial population. These results support the theory that multiple cellular events, namely apoptosis, migration, and epithelial-mesenchymal transformation (EMT), most likely contribute to palatal shelf fusion and that apoptosis may be a major contributor for the removal of MEE cells and activation of adjacent MEE cells.