Mechanism Of Deformed Mandible Induced By Overexpression Of Fgf18 In Neural Crest Cells

The fibroblast growth factors(Fgfs)signalings are important for the development of craniofacial bone and muscle by modulating cell proliferation,differentiation and apoptosis.Single nucleotide polymorphisms(SNPs)in FGF18,a member of the fibroblast growth factor family,are associated with cleft lip and palate.Although previous studies have shown that Fgf18 deficient mice exhibit cleft palate,micrognathism and reduction in ossification of skull,there are no reports on the effect of Fgf18 activation on craniofacial development.So we activated Fgf18 signaling in cranial neural crest cells by crossing Wnt1-Cre mice with pMes-Fgf18 mice generated in our laboratory.Wnt1-Cre;pMes-Fgf18 mice exhibited cleft palate,malformed tongue,micrognathia and cranial dysplasia.Previous study indicated that cleft palate in Wnt1-Cre;pMes-Fgf18 mice was associated with failure of palatal shelf elevation caused by tongue malposition and malformed mandible.However,the mechanism of mandibular deformity in Wnt1-Cre;pMes-Fgf18 mice remains unclear.To investigate the cause of mandibular deformity in Wnt1-Cre;pMes-Fgf18 mice,we collected a large number of mouse embryos.Results from Alizarin red and Alcian blue staining showed that Wnt1-Cre;pMes-Fgf18 mice exhibited the length of the mandible was significantly reduced and Meckel cartilage deformation.And the expression of Ki67,a cell proliferation marker,Cleaved-Caspase3,the apoptosis marker,Sp7,an osteoblast marker,and the chondrocyte marker of Sox9 in mandible and Meckel cartilage of E13.5 control and Wnt1-Cre;pMes-Fgf18 mice was detected by immunofluorescence.Results from IF showed the expression of Sp7,Sox9 and Ki67 increased in Wnt1-Cre;pMes-Fgf18 mice,while the number of CleavedCaspase3 positive cells did not change significantly.These results indicated that the proliferation of chondrocytes in Meckel cartilage and osteoblasts of the mandible was increased,but apoptosis was not affected.The temporomandibular joint(TMJ),consisting of mandibular condyle,glenoid fossa and articular disc,is a synovial joint and and plays an important role in the functioning of the craniofacial bone.Histological analysis showed that Wnt1-Cre;pMes-Fgf18 mice exhibited stunted glenoid fossa and disorder of cell arrangement in condyle as well as lacked of articular disc with abnormal expression of collagen.Then,we detected the expression of bone related markers in mandibular condyle using immunofluorescence(IF).IF results showed decreased level of biomarker of chondrocyte hypertrophy Col10 and prohypertrophic chondrocyte marker Ihh in E16.5Wnt1-Cre;pMes-Fgf18 indicating that the process of cell hypertrophy in condyle of Wnt1-Cre;pMes-Fgf18 mice was impaired.And IF results showed abnormal expression of chondrocyte marker Sox9 and chondroblast associated protein Col2 in Wnt1-Cre;pMes-Fgf18 mice while expression of Sp7(the osteoblastic key transcription factor),Col1(osteogenic differentiation marker)and Runx2(skeletal progenitor cells marker)were increased.These results indicated that the number of preflattened chondrocytes in Wnt1-Cre;pMes-Fgf18 mice condyle significantly increased because co-expression of Sox9,Sp7,Col2,and Col1 was detected only in preflattened chondrocyte layers of wild-type mice.Finally,we detected signaling pathways affected by FGF,cell apoptosis and cell proliferation in condylar cartilage.We found that unaffected apoptosis and increased cell proliferation with lower level of p21 in Wnt1-Cre;pMes-Fgf18 mice compared to controls while expression of Erk1/2,pp38,Akt and β-catenin were increased.These results indicated that FGF18 can affect the differentiation and proliferation of condylar chondrocytes causing abnormal endochondral ossification by regulate the expression of Runx2 and P21 by activating these signaling pathways.Meckel’s and condylar cartilages anomalies further resulted in defective development of the mandible in Wnt1-Cre;pMes-Fgf18 mice.Therefore,our study is important to further understand the molecular regulatory mechanisms of mandibular development and provide a theoretical basis for the studies of mandibular malformation.