The Role Of Fibronectin During Palate Development

Objective:Abnormal palatogenesis cause cleft palate,a common birth defect in humans However,little is known about the pathogenesis of cleft palate.Palatal shelves are composed of the epithelium and underlining mesenchyme mainly derived from neural crest cells Previously,mouse genetical studies have shown that epithelial-mesenchymal interaction is important for the palatal shelf outgrowth.We aim to investigate how Fibronectin,an essential and abundant component of the extracellular matrix regulating cell-ECM and cell-cell interaction,modulates the palatal epithelial-mesenchymal interaction by regulating Shh signaling during palatogenesisMethods:We conditionally deleted Fibronectin in the neural crest cells by mating Fnflox/+;Wnt1-Cre2 male and Fnflox/flox;RosamTmG/mTmG female.Pregnant female mice were sacrificed,and the embryos were dissected at E13.5-E18.5.Fnflox/+;RosamTmG/mTmG;Wnt1-Cre2 was designated as the controls and Fnflox/flox;RosamTmG/mTmG;Wnt1-Cre2 as the mutants.Embryonic heads were collected for whole mount skeletal staining,or paraffin section used for immunostaining,H&E staining,Edu staining and in situ hybridizationResults:By using the Cre/Loxp system to conditionally knock out the fibronectin in the neural crest cells of the mouse embryos,cleft palate was observed in mutant embryos(100%,n=42)1.H&E&Alicn blue stained sections showed that the morphology of mutant palate shelves was similar to the controls at E13.5,both of them extending downwards along the tongue After E15.5,the palate shelves along both sides of the tongue in the controls could elevate and fuse at the midline,but the elevation of palatal shelves were delayed in the mutants and unable to fuse at the midline,resulting in cleft palate2.Edu proliferation experiments suggested that during the palate growth and elevation,the proliferation of the palatal mesenchymal cells in the mutants were decreased,indicating that fibronectin may affect the palate growth and development by regulating the proliferation of neural crest cells3.Comparing the skeletal staining of the embryos in the controls and the mutants,we found that the bony palates of the mutant embryos were defective4.Immunohistochemical staining(using anti-Runx2,Sox9,and Osterix antibodies)also suggested that the differentiation of neural crest derived palatal mesenchymal cells into osteoblasts was significantly reduced in the mutants,indicating that fibronectin may affect the formation of hard palate by regulating the differentiation of neural crest cells into osteoblasts.5.In situ hybridization suggested that the expression of Gli1,Ptach1,Bmp4,and Fgf10 were significantly decreased in the mesenchymal cells in the mutants compared with those in the controls.Loss of fibronectin in the neural crest cells leads to the decreased expression of Gli1,Ptach1(both are key factors in Shh signaling),Bmp4,and Fgf10,which suggested that fibronectin may affect the palate development by regulating Shh signalingConclusions:1.Fibronectin derived from neural crest cells is involved in the development of the palatal process.2.Fibronectin derived from neural crest cells may be involved in regulating the proliferation and differentiation of palatal mesenchymal cells3.Fibronectin derived from neural crest cells may regulate palatal development by regulating and integrating with Shh,BMP and FGF signaling pathways.