Research On The Developmental Deformity Of Mouse Cranial Base Induced By Hedgehog Pathway Inhibitor Vismodegib

Background:Cranial base development is a delicate morphogenesis process that requires the migration,aggregation,proliferation and chondrogenic differentiation of mesenchymal cells from the cranial neural crest and paraxial mesoderm,and then bone is formed through endochondral ossification.Hedgehog(Hh)signaling pathway is essential for the bone development.The lack of Hedgehog leads to the premature death of most embryos or the failure formation of the cranial base cartilage.Therefore,the specific mechanism of Hh signaling in early development of cranial base is still unclear.Objective:Inhibition of Hh signaling by Hh pathway antagonist Vismodegib in the early embryonic development of mice,a malformation model of cranial base in mice was established and the regulatory mechanism of Hh signaling in the early development of the cranial base was further explored.Material and Methods:Timed-pregnant wildtype ICR mice were treated with a single dose of the Hh inhibitor Vismodegib 150mg/kg by oral gavage from Embryonic day(E)7.5 to E13.5.Skulls at E17.5 were collected for alcian blue and alizarin red skeletal preparations,HE,alcian blue and nuclear fast red staining,and the establishment of experimental group according to the incidence of cranial base deformity.By detecting the Hh pathway target,proliferation,cartilage differentiation,osteogenic marker and the regulatory factor of cartilage differentiation in the cranial base,the dynamic development of the cranial base deformity was analyzed from the levels of morphology,histology and molecular biology,and the corresponding hypotheses were proposed and verified.By detecting the protein of the Wnt pathway,that the interaction of Hh and other signaling pathways to regulate the normal development of the cranial base was explored.Results:Embryos that treated to 150mg/kg Vismodegib at E9.5 and E10.5 demonstrated the highest percentage of cranial base deformity,but the Vismodegib-induced stillbirth rate was much lower in E10.5-exposure,so it was selected as the experimental group.The cranial base synchondrosis of experimental group displayed that the Col IIpositive cartilage was prematurely replaced by Col X-positive cartilage after E14.5,whereas the expression of Col II and Col X was relatively normal at E11.5-E12.5.At E10.5,Shh can be detected in the vicinity of cranial base,whereas Ihh is not detected.The expression of Ptch1 in the cranial base of experimental group was significantly reduced at E11.5,and the expression of proliferation marker PHH3 at E14.5 and the expression of PTHr P in the cranial base were not significantly weakened at E14.5-E15.5,but the β-catenin expression in the vicinity of cranial base was increased at E11.5-E12.5.Conclusions:E9.5 and E10.5 are the critical period for Hh signaling to regulate the early development of the cranial base,and significant inhibition of Shh by Vismodegib can lead to accelerating terminal differentiation and premature osteogenesis in cranial base synchondrosis.The cranial base craniosynostosis is not caused by proliferation reduction or PTHr P inhibition,but may be related to abnormal activation of Wnt signals after the Hedgehog pathway inhibition in the early development.