The Mechanism Study Of Soft Palate Agenesis Caused By Over-expression Of Fgf8 In The Soft Palate Mesenchyme

Background: Cleft palate is the most common congenital malformation that affects important physiological functions,such as breathing,swallowing and speech.At present,the cleft hard palate can be closed by surgery,but the treatment of the cleft soft palate is still difficult as the restoration of the normal arrangement of soft palate muscle fibers,and the poor tissue regeneration ability after surgery,There is still a lack of ideal cleft palate repair,partly because the molecular mechanisms regulating the development of soft palate are not clear.Mouse is an excellent model for studying human soft palate development,as the anatomy and developmental process of mouse soft palate are similar to those of humans.The development of the mouse soft palate involves growth,elevation and fusion of palatal shelves,any abnormalities during which will lead to cleft palate.FGF signaling is expressed in cranial neural crest-derived mesenchymal cells,myogenic cells and epithelial cells in the soft palate,suggesting a role of FGF signaling in the development of the soft palate.In FGF family,the expression of Fgf18 was significantly stronger in the posterior palate than in the anterior palate,indicating that it may regulate the development of the soft palate.Previous studies have reported that Fgf18 conditional knockout in mouse palatal mesenchyme which used Osr2-cre tool mouse didn’t cause any abnormalities,but it is unclear that whether activation of Fgf18 in the palatal mesenchyme affects soft palate development.Objective: Since previous studies have explored the effect of loss-of-function on palate development by knocking out Fgf18 in palatal mesenchyme,while there was no mouse model overexpressing Fgf18,in this study,Fgf18 homologous gene Fgf8 was used instead of Fgf18,and Osr2-cre tool mouse was used to further explore the the mechanism of Fgf18 in palate development by activating Fgf18 in the palatal mesenchyme.Methods:(1)Osr2-cre mice and Rosa26R-Fgf8 mice were mated for Osr2-cre;Rosa26R-Fgf8 mice,activating Fgf18 homologous gene Fgf8 in the palatal mesenchyme,and the remaining normal littermates(Wide Type,WT)were regarded as controls.Tissues were fixed,dehydrated,embedded and sectioned;(2)Morphological changes of the mouse soft palate were observed by Masson staining;(3)Cell proliferation and apoptosis in the soft palate were tested by Brd U and TUNEL experiments,respectively;(4)Immunohistochemistry was used to check the expression level and distribution range of Sox9,Myo D,Myosin,FGFR1,p-Smad1/5/8 and p-Erk1/2 in the soft palate,as well as Fibronectin and β-integrin in the anterior,middle and posterior palate;(5)In situ hybridization experiments detected the expression pattern of Scx,Tnc,and Col I m RNA in the soft palate;(6)The migration of palatal mesenchymal cells was observed by fluorescently labeled phalloidin.Results:(1)Masson staining showed that Osr2-cre;Rosa26R-Fgf8 mice express no obvious soft palate structure and abnormal cartilage hyperplasia in the TVP level compared to WT mice in E13.5,E14.5 and E16.5;(2)Immunohistochemistry of Sox9 and in situ hybridization results of Scx,Tnc and Col I showed that,in E14.5,Sox9 distribution range was extended in the mesenchyme below the pterygoid process but interrupted at the site where aponeurosis should be formed;Scx expression was not significantly different from WT mice;Tnc expression was concentrated in the mesenchyme below the epithelium of the palatal process but decreased in the tendon around TVP;the expression of Col I decreased at the site where aponeurosis should be formed.These results implicate that the development of the soft palate tendon and aponeurosis in Osr2-cre;Rosa26R-Fgf8 mice have been inhibited,and soft palate mesenchymal cells below the pterygoid process were converted to chondrogenic fate;(3)Myo D and Myosin immunohistochemistry showed that there was no significant change in myogenic determinant Myo D expression in Osr2-cre;Rosa26R-Fgf8 mice soft palate at E13.5,while the expression of mature muscle fiber marker Myosin was obviously decreased at E16.5,implying that the development of soft palate muscle fibers in Osr2-cre;Rosa26R-Fgf8 mice was inhibited;(4)The results of Brd U and TUNEL showed that both of proliferation and apoptosis in Osr2-cre;Rosa26R-Fgf8 mice soft palate have no clear difference compared with WT mice at E13.5,indicating that agenesis of the soft palate in Osr2-cre;Rosa26R-Fgf8 mice was not due to abnormal cell proliferation and apoptosis;(5)Fluorescently labeled phalloidin staining on both coronal and transverse section views at E13.5 showed altered cytoskeletal arrangement orientation of Osr2-cre;Rosa26R-Fgf8 mice palate mesenchymal cells,suggesting abnormal migration direction of palatal mesenchymal cells;(6)Immunohistochemistry results of E13.5 fibronectin and β-integrin showed that there is no obvious difference of fibronectin expression in Osr2-cre;Rosa26R-Fgf8 mice anterior,middle and posterior palate compared with WT mice,while β-integrin expressed stronger in lateral mesenchyme than medial mesenchyme in anterior palate and more widespread than WT mice in the middle and posterior palate,which suggested altered traction force between the cell skeleton and the extracellular matrix and the abnormal signaling networks that regulate cell migration,may eventually lead to abnormal cell migration(7)Immunohistochemistry results of FGFR1,p-Smad1/5/8 and p-Erk1/2 at E14.5 showed that,FGFR1 expression was elevated in Osr2-cre;Rosa26R-Fgf8 mice soft palate mesenchyme below the pterygoid process,which suggests that FGF signaling pathway is up-regulated after activating Fgf8 in the soft palate mesenchyme;greater expression range of p-Smad1/5/8 in the soft palate mesenchyme and increased expression of p-Erk1/2 in the mesenchyme below the pterygoid process indicate up-regulation of BMP signaling pathway in Osr2-cre;Rosa26R-Fgf8 mice soft palate mesenchyme,whose abnormal cartilage hyperplasia on the TVP level may be caused by the up-regulation of BMP signaling pathway.Conclusion:(1)The agenesis of the soft palate in Osr2-cre;Rosa26R-Fgf8 mice resulted from the abnormal migration direction of the palatal mesenchymal cells;(2)Abnormal chondrohyperplasia in TVP plane of Osr2-cre;Rosa26R-Fgf8 mice was caused by abnormal activation of BMP signaling pathway in soft palate mesenchyme;(3)The mesenchyme in Osr2-cre;Rosa26R-Fgf8 mice soft palate was convered to chondrogenic fate owing to the activation of Fgf8.