Tracking MEE with b-gal and DiI Labeling during Epithelial-Mesenchymal Transformation in Palatogenesis

Medial edge epithelial (MEE) cells undergo epithelial-mesenchymal transformation coincident with palatal fusion and disappearance of the midline epithelial seam. Tracking the fate of transdifferentiated MEE has been complicated by the absence of markers to distinguish the cranial neural crest (CNC) mesenchyme from the mesenchymal MEE. Objective: To use the Wnt1-cre/R26R mouse model and vital cell dye labeling in palatal organ culture to observe the MEE during transdifferentiation from epithelium to mesenchyme. Methods: Wnt1-cre and R26R mouse strains were bred to generate embryos with b-galactosidase (b-gal) specifically activated in CNC. Vital cell dye (DiI) labeling was used to mark the MEE in palatal organ cultures of E14 palatal shelves isolated from the Wnt1-cre/R26R cross breeding. TGF-b3 and TGF-b type III receptor (TbR-III) immunostaining was used to characterize the differentiation of the MEE in vitro. Palate tissue was examined after 12, 24, 36 and 48 hours of E14 palatal organ culture. Results: Palate mesenchyme had two groups of mesenchymal cells defined by b-gal expression, b-gal (+) and b-gal (-) and two cell populations defined by DiI labeling, DiI (+) and DiI (-). The DiI (+) mesenchymal cells were all b-gal (-) thus transdifferentiated MEE could be distinguished based on the two labeling methods. Another population of mesenchymal cells of underdetermined origin existed that were DiI (-) and b-gal (-). The b-gal (-) /DiI (+) mesenchymal cells continued to express TGF-b3 however TbR-III was only expressed in b-gal (-)/DiI (+) MEE that had an epithelial phenotype. Conclusion: These results demonstrate that use of heritable b-gal cell marking and vital cell DiI labeling can be used to distinguish different populations of mesenchymal cells in the developing palate. (Supported by NIDCR grants PO1DE-12941 and RO1DE-12711)