Biomarker Screening For Cleft Palate In Amniotic Fluid Of Pregnant Mice

Part 1:Biomarker screening for cleft palate in amniotic fluid of pregnant mice Objective:Cleft lip and palate(CLP),a congenital defect in the oral and maxillofacial regions,is usually diagnosed through prenatal ultrasonographic examination.However,the accuracy of ultrasonography for prenatal diagnosis of CLP is highly variable and dependent on the experience of the sonographer,fetal position,the amount of AF,and the type of cleft.The objective of the study was to screen biomarkers for the prenatal diagnosis of cleft palate in amniotic fluid(AF)of pregnant mice and to characterize the expression level of the specific proteins with the palatogenesis.Methods:Amniotic-fluid samples(AFS)obtained from pregnant mice were divided into three groups:healthy control,cleft palate induced by all-trans retinoic acid(atRA),and cleft palate induced by TCDD.Label-based mouse antibody array was used to compare proteins in AFS of embryonic day(E)16.5 fetuses between control and atRA groups.ELISA and immunohistochemistry were respectively performed to assess the variations of the proteins in AFS and palatal tissue from E13.5 to E16.5 among the three groups.Results:Using label-based mouse antibody array,seven proteins were identified differentially expressed in AFS obtained from pregnant mice from atRA groups,including three down-regulated proteins(RAGE,epiregulin,and LIF),and four up-regulated proteins(EL-10,IL12-p40/p70,IFN-?,and IFN-?).ELISA further revealed that atRA group and TCDD group had decreased expression of RAGE and epiregulin at E16.5.Furthermore,RAGE was observed present in the palate at stages E13.5 to E16.5 in control group by immunohistochemistry.However,an obvious decrease of RAGE immunostaining was detected in the palatal tissues sectioned from E14.5 to E16.5 both in atRA group and TCDD group.Conclusion:RAGE and epiregulin may serve as valuable AF biomarkers for the prenatal diagnosis of cleft palate.RAGE may play a role in the development of nonnal palate and cleft palate.Part 2:Lithium inhibits palatal fusion and osteogenic differentiation in palatal shelves in vitroObjective:Glycogen synthase kinase-3 3?(Gsk-3?)/?-catenin signaling regulates development of the secondary palate.It has been unclear about the effects of Gsk-3?/?-catenin signaling on palatal fusion and osteogenic differentiation in palatal shelves.Methods:In this study,palatal shelves from mouse embryonic day 13(E13)were cultured in vitro with or without lithium chloride(LiCl).Palatal fusion was evaluated by hematoxylin-eosin staining.The expression of osteogenic markers in palatal shelves was measured by quantitative PCR,and immunohistochemical staining.Cell proliferation and apoptosis were examined by Ki-67 immunohistochemical and TUNEL staining,respectively.Gsk-3? expression was evaluated by quantitative PCR and Western blotting.?-catenin protein expression was evaluated by Western blotting.Results:After the treatment with 10 mM LiCl,palatal shelves failed to fuse,and the mRNA and protein levels of osteogenic markers were reduced compared with controls.The number of Ki67-positive cell in the palatal osteoid was significantly higher in the LiCl group than in the controls.The apoptotic cells in the midline epithelial seam were reduced by LiCl.Gsk-3? mRNA and protein expression levels decreased and?-catenin protein expression levels increased by treatment of LiCl.Conclusion:Our findings show that LiCl-mediated GSK3? inhibition prevents palatal fusion and osteogenic differentiation in palatal shelves by increased ?-catenin signaling.It indicated that overactivation of canonical Wnt signaling might impair the fusion of the secondary palate.