Defining Critical Periods For Hedgehog Pathway Antagonist-induced Frontal Bone Dysplasia Associated With Micrognathia And Limb Defects In The Mouse

Background:The flat bones of the skull is an intricate developmental process,which mesenchymal cells first condense and subsequently differentiate into osteoblasts.Hedgehog(Hh)signaling has an indisputable role in embryonic development and organ formation.A growing body of evidence suggests that Hedgehog(Hh)signaling pathway plays an important role in craniofacial development.However a lot of research is based on knockout experiments,there is few research model that trough the exogenous inhibitors to study the development of skull.Objective:The purpose of this research is to determine the critical Periods for Hedgehog Pathway Antagonist-Induced the frontal bone hypoplasia.And further explore the etiology of the frontal bone hypoplasia from pharmacological exogenous inhibition of Hedgehog signaling.Material and Methods:Timed-pregnant wildtype ICR mice were administered a single dose of the potent pathway antagonist GDC-0449(100mg/kg,150mg/kg)at discrete time points,including:embryonic day 8.5,9.5,10.5,11.5 and 12.5.Embryos were analyzed in details using histology,Alcian blue alizarin red skeletal staining.Through the above results to determine the optimal concentration.And then Timed-pregnant wildtype ICR mice were treated with GDC-0449 at the optimal concentration as experimental group.Embryos of treated and control groups were collected at E12.5,E13.5,E14.5,E15.5,E16.5 and E18.5for histology,immunohistochemical staining,Aniline blue staining,Micro-CT.Moreover,we performed a lineage tracing experiment using the neural crest specific driver Wnt1-Cre combined with Rosa26 m Tm G mice.Result:150mg/kg GDC-0449 administrated on E9.5 and E10.5 result in the frontal bone hypoplasia,but E10.5 is more ideal.This model showed a decreased ossification in the region of frontal bone with the downregulation of HH protein.Osteoid thickness of the frontal bone was significantly reduced.Meanwhile,the neural crest derived frontal bone primordial of GDC-0449-treated is reduced,a decreased rate of cell proliferation and increased cell death in the frontal bone primordial,And the early function of the Hedgehog is likely independent of the Bmp signaling pathway.Conclusion:Our date suggest that GDC–0449,the Hedgehog signaling pathway inhibitors,can specifically to suppress Hedgehog signals to induce the frontal developmental defects at a specific time point.Thus affect the migration of CNC cells into the frontal primordium,as well as the proliferation and differentiation of CNC-derived mesenchymal precursors in the frontal bone region.