Plays an Intrinsic Role in Regulating Proliferation of Mesenchymal Cells in the Developing Palate.

Cleft palate is a common congenital abnormality that results from defective secondary palate (SP) formation. The () gene has been linked to abnormalities of craniofacial and kidney development. Our current study examined, for the first time, the specific role of in embryonic mouse SP development. mRNA and protein expression were identified in the palatal shelves from embryonic days (E)12.5 to E15.5, with peak levels during early stages of palatal shelf outgrowth. Immunohistochemical staining (IHC) showed that Six2 protein is abundant throughout the mesenchyme in the oral half of each palatal shelf, whereas there is a pronounced decline in Six2 expression by mesenchyme cells in the nasal half of the palatal shelf by stages E14.5-15.5. An opposite pattern was observed in the surface epithelium of the palatal shelf. Six2 expression was prominent at all stages in the epithelial cell layer located on the nasal side of each palatal shelf but absent from the epithelium located on the oral side of the palatal shelf. is a putative downstream target of transcription factor and we previously demonstrated that plays an intrinsic role in embryonic palate formation. We therefore investigated whether expression was altered in the developing SP of null mice. Reverse transcriptase PCR and Western blot analyses revealed that mRNA and protein levels were upregulated in palatal shelves at stages E12.5-14.5. Moreover, the domain of Six2 protein expression in the palatal mesenchyme of embryos was expanded to include the entire nasal half of the palatal shelf in addition to the oral half. The palatal shelves of embryos displayed a higher density of proliferating, Ki-67 positive palatal mesenchyme cells, as well as a higher density of Six2/Ki-67 double-positive cells. Furthermore, palatal mesenchyme cells in culture displayed both increased proliferation and elevated expression relative to wild-type cultures. Conversely, siRNA-mediated knockdown restored proliferation and expression in palatal mesenchyme cultures to near wild-type levels. Our findings demonstrate that functions downstream of as a positive regulator of mesenchymal cell proliferation during SP development.