The Role And Mechanism Of Bmp2 And Fgf8 Signalings In The Development Of Palate And Tooth In Mice

BMP and FGF signalings play crucial roles in the development of craniofacial organs.Mutations in numerous members of BMP and FGF signaling pathways may lead to several severe human syndromes,including Pierre Robin sequence?PRS?caused by heterozygous loss of BMP2.In this study,we generated mice carrying Bmp2 specific deletion in cranial neural crest cells using floxed Bmp2 and Wnt1-Cre alleles to mimic PRS in humans.Mutant mice exhibit severe PRS with significantly reduced size of craniofacial bones,cleft palate,malformed tongue,and micrognathia.To further elucidate the pathogenic mechanism of PRS and the role of Bmp2 in craniofacial development,we collected E13.4,E14.5 and E16.5 mouse embryos for paraffin section.We found that E13.5 head revealed comparable morphology between control and mutant.However,palatal shelves of the control have elevated to upon the tongue and were undergoing fusion at the midline at E14.5.Besides,the height of the tongue in Wnt1-Cre;Bmp2^f/f/f mice is significantly higher than that of wild type mice.Then we performed in vitro roller culture and palate fusion assay to test if elevation and fusion of palate could happen in the mutant mouse.The results showed that the palates of mutants were able to elevate and fused after culturing.Next,by comparing the neural crest cells migration,cell proliferation and cell apoptosis of palatal mesenchymal cells and the expression of key genes in the development palatal shelves between wild type and Wnt1-Cre;Bmp2^f/f/f mice,we found no significant difference between them.Based on the results above,it is suggested that cleft palate in Wnt1-Cre;Bmp2^f/f/f mice is a secondary defect that caused by the malformed tongue.However,the tongue in Wnt1-Cre;Bmp2^f/f/f mice does not exhibit altered rates of cell proliferation and apoptosis,as well as abnormal muscle differentiation,suggesting contribution of extrinsic defects to the failure of tongue descent.Further studies revealed obvious reduction in cell proliferation and differentiation of osteogenic progenitors in the mandible of the mutants,attributing to the micrognathia phenotype.Interestingly,deletion of Bmp2 in neural crest cells seems to have no effect on early tooth development.The results showed the morphology of teeth,enamel differentiation and the intensity of BMP signal in Wnt1-Cre;Bmp2^f/f/f mice are all comparable compared with those of wild type.After further examination of the expression of two other important ligands of BMP pathway,Bmp4 and Bmp7,we found that the expression of Bmp7 in the teeth of Wnt1-Cre;Bmp2^f/f/f mice was significantly increased.Therefore,we conclude that BMP ligands have a functional redundancy and compensate for the absence of Bmp2 in early tooth development.The tooth development is not affected in Wnt1-Cre;Bmp2^f/f/f mice,probably due to compensation of Bmp7.Clinical data shows that gain of function mutations in FGF signaling are associated with several human syndromes,including craniosynostosis,cancer,and tooth dysplasia.In our study,we generated mice carrying Fgf8 specific overexpression in epithelial cells using a conditional Fgf8 overexpression allele and K14-Cre allele to study the mechanism of Fgf8 in mouse tooth development.K14-Cre;R26R-Fgf8^/+mice exhibit several malformations including hairless,polydactyly.Supernumerary incisors were observed in both maxillary and mandible in K14-Cre;R26R-Fgf8^/+mice.At E15.5,the development of K14-Cre;R26R-Fgf8^/+mouse incisor is consistent with wild type controls,forming the labial and lingual cervical loop.At E16.5,the lingual cervical loop of K14-Cre;R26R-Fgf8^/+mouse incisor became larger and lingual epithelial abnormality extended to the distal end.At E17.5,the second new incisor formed in K14-Cre;R26R-Fgf8^/+mice.In our result,overexpression of Fgf8 in epithelial cells appeared to alter the fate of lingual cervical loop,inducing them to regain stemness,ultimately leading to the formation of an incisor-like structure.Further study on signaling pathway suggested that Fgf8 regulated the development of incisor mainly through Erk1/2and p38 intracellular pathways,and inhibited the expression of Bmp4 and?-catenin signaling.In addition,K14-Cre;R26R-Fgf8^/+mice exhibited delayed developing and fused molars.Further study has found that Fgf8 signal entered the nucleus via downstream Erk1/2 and p38 signaling pathways.At E16.5,Shh was expressed in advance in the second molar of K14-Cre;R26R-Fgf8^/+mice,and Bmp4 as well as?-catenin were ectopically expressed in the mesenchyme at the junction of the first molar and the second molar.Our study demonstrates that the Fgf8 promotes cell proliferation,inhibits cell differentiation,thereby delaying the rate of development of teeth.Besides,overexpressed Fgf8 has influence on the temporal and spatial expression of BMP and Wnt/?-catenin signals,preventing the absence of a differentiated inner enamel epithelium in the junction region of the first molar and second molar in mutant mice at E16.5,and eventually leading to the formation of fusion teeth.In a word,our study illustrates the pathogenesis of PRS caused by Bmp2 mutation,highlights the crucial role of BMP2 in the development of craniofacial bones,and emphasizes precise coordination in the morphogenesis of palate,tongue,and mandible during embryonic development.And elucidated the molecular mechanism by which Fgf8promotes cell proliferation,inhibits cell differentiation,and ultimately delays the rate of tooth development.It emphasizes that the pathways of FGF,BMP,Wnt/?-catenin form a complex signal network,which interacts,cooperates and precisely regulates teeth development.