Inhibition Of Hedgehog Signalling Pathway Vismodegib Establishes Mice Model Of Median Cleft Lip And Its Related Mechanisms

Background:Median cleft in upper lip is a rare congenital malformation,seriously affecting patients’ daily lives.At present it has been identified as a complex disease caused by the mix of genetic and environmental factors.The mechanisms that cause median cleft lip are still not completely understood,likely due to the lack of stable experimental animal models.The Hh signaling pathway mediates the normal development of facial midline tissue.Interventions in Hh signaling pathway are known to result in abnormal development of upper lip.Obejective:Vismodegib(GDC-0449)is a selective Hh signalling pathway inhibitor.This research was designed to establish a stable mouse model of median cleft in upper lip by using Vismodegib in early stage of lip development.Further explores were made to investigate the development process、possible pathogenesis and the changes of signaling pathway of median cleft lip.Methods:1.Pregnant SPF grade ICR mice were exposed to 150 mg/kg Vismodegib by single-dose oral gavage respectively at different times.We collected E15.5 embryos to observe the phenotype under stereoscope and count the incidence of median cleft lip,trying to confirm the best time point of the model.2.E11.5 embryos were collected to observe the fusion of median nasal process、lateral nasal process and maxillary process by electron microscope and HE staining technique.E10.5-E11.5 embryos were collected to observe the dynamic development of median cleft lip and morphological changes of mnp by electron microscope and stereoscope.3.E16.5-E17.5 embryos were collected to observe other malformations in midface by HE staining technique and Alcian Blue – Alizarin Red staining.4.E12.5 embryos were collected to count midface distance and biocular distance to analyze the difference between experimental group and control group.5.E10.5 embryos were collected to detect cell proliferation and cell death of maxillary process and median nasal process by immunofluorescence staining of PHH3 and Caspase3.6.Hh signaling activity was detected by immunofluorescence staining and RT-q PCR.Results:1.E9.5 mice exposed to 150 mg/kg Vismodegib by single-dose oral gavage exhibited higher incidence rate,lower fetal mortality and more serious malformation.So E9.5 was considered as the best time to establish mice model.This mice model we made was proved to be an animal model of median cleft lip.3.The mice model exhibited other malformations in median face,including shorter upper lip 、primary palate missing with obvious or submucous cleft palate 、abnormal development of premaxilla、upper incisors missing、Hypoglossia or aglossia、abnormal development of nasal septum 、 shorter nose and morphological abnormality of sphenoid bone.4.Vismodegib-exposed mice showed abnormal morphology and angle in caudal median nasal process in early stage of lip development with widen midface distance and shorten biocular distance.The ratio of PHH3 positive cells/total cells increased and Caspase3 immunoreactivity diminished in median nasal process and maxillary process24 h after exposure.5.Ptch1 and Gli1 gene were down-regulated in median nasal process and maxillary process of experimental groups 24 h after exposure.Ptch1 gene showed no significant difference between experimental groups control groups 48 h after exposure.Gli1 were up-regulated in experimental groups 48 h after exposure.Conclusion:The use of 150 mg/kg Vismodegb in early stage of lip development can establish a stable mouse model of median cleft in upper lip with other malformations in median face.Hh signaling pathway was inhibited transiently in the next 24 h,which caused median cleft in upper lip.Morphological abnormality in median nasal process and widen midface distance may be the possible pathogenesis.Cell proliferation increasing and cell death diminishing in median nasal process and maxillary process may be related to widen midface distance but not the pathogenesis.The critical period to form median cleft lip is E9.5-E10.5.