The Funtions Of Sufu In The Development Of Neural Crest Cells-derived Skull Bones

Craniofacial malformation accounted for 3/4 of all the human congenital diseases, which primarily influences on head, neck and facial development. Most of the skull bones are derived from the cranial neural crest cells (CNCCs). Many clinical symptoms of neurocristopathy are embodied as craniofacial malformations. Further studies on the pathogenesis of some important neurocristopathy syndromes and the critical signaling pathways and regulators during embryo development will contribute to find the targets and theoretical base for the prenatal diagnosis and intervention of these diseases.. Genetic studies with mouse models have revealed important signaling pathways involved in the skull bone development, including Hh, Wnt, BMP,FGF, and Notch pathways. This project mainly employed the conditional knockout mouse of Hh pathway inhibition factor Sufu gene as a model to study its function in the development of the CNCCs-derived skull bones. We found that conditional knockout of Sufu gene in CNCCs with Wntl-Cre transgenic mice leaded to failure formation of all the CNCCs-derived skull bones, suggesting that Sufu may be an essential regulator for skull bone development. We used the R26R mouse which expresses the LacZ gene to mark and trace the CNCCs, and found that they could migrate to their destinations. Apoptosis of osteogenic cells did not appear obvious increase, but their proliferation was significantly reduced. Furthermore, the expression of critical regulatory factors of osteoblast differentiation including Osterix and Runx2 was completely inhibited, indicating that the developmental defects of skull bones in Sufu knockout mice were due to the inhibition of proliferation and differentiation of osteogenic cells. To further reveal the underlying molecular mechanism, we used in situ hybridization technique to detect the gene expression of transcription factors upstream of Runx2 including Msx1, Msx2 and Dlx5, and found that the expression of these genes was significantly reduced in the Sufu mutants. Meanwhile, the expression of Hh pathway target molecule Gli1 and Notch pathway molecule Jag1 was significantly up-regulated, but FGF, BMP pathway molecules including Fgfr2, Bmp2 were significantly down-regulated. Western Blot analysis showed that the repressor form of Gli3 was significantly down-regulated, while the expression of full-length Gli2 was significantly up-regulated. Genetic study revealed that double mutant of Sufu and Gli2 in CNCCs could rescue the phenotype resulted from Sufu mutation. These results suggested that during skull bones development Sufu can repress the activity of Hh pathway by inhibiting the production of full-length Gli2 protein and promoting the processing of Ghi3 protein, which further lead to the activation of FGF and BMP pathways, and inhibition of Notch pathway. The abnormity of these proliferation-and differentiation-related signaling pathways leads to the inhibition of osteoblast differentiation and proliferation, and ultimately leads to the failure formation of CNCCs-derived skull bones in Sufu mutant.