The Role Study Of Notch Signaling Pathway In The Neural Tube Defects Genesis In Mouse

ObjectiveTo study the role of Notch signaling in the development of neuroepithelium and the closure of the neural tube,trying to reveal the function of this pathway and the possible molecular mechanism in the NTDsMethodIn the present study,the expression and the change of Notch mRNA were observed during theembryonic development using the whole-mount embryo in situ hybridization,especially in the neurulation stage;the change of Notch mRNA were observed in the NTDs mouse by establishing a model of all transretinoic acid(ATRA)-induced NTDs through administrating ATRA(50mg/kg)to pregnant mouse.Meanwhile,we detected the expression and the change of related development gene of the Notch signaling in controls and cases using RT-PCR.And the expression and the change of the intracellular domain of Notch(NICD)were observed by western blots.ResultsⅠ.The expressing of Notch I in the neurulation stage of mouseThe expression of Notch mRNA was detected from E9.5d to E11.5d by the whole-mount embryo in situ hybridization staining.At E9.5d,Notch mRNA was slightly detected in the forebrain vesicle and tail end of embryo with the closureof neural tube;at E10.5d,Notch mRNA increased in midbtain vesicle and the whole spinal column;at E11.5d,Notch mRNA was observed in the whole cerebral vesicle and the spinal column,but the expression magnitude is weaker than E10.5d.Ⅱ.The relationship between Notch and RA-iuduced NTDs of mouse1.compared with the control group,the embryo with the malformation can be found through administrating RA(50mg/kg)to pregnant mouse.At E9.5d and E10.5d,the malformation mainly was an unclosed prosencephalon and an unclosed metencephalon,and the entirely unclosed neural tube was also found in a few of embryos.Therefore this indicated that RA-treated mice could cause NTD malformation.2.The changes of Notch mRNA expression in RA-induced NTD embryos were detected by whole-mount embryo in situ hybridization staining.At E9.5d,in contrast to the normal embryos,the expression of Notch mRNA was dramatically increased,not only in forebrain vesicle but also in incomplete spinal column;at E10.5d,the expression of Notch mRNA was less than control;at E11.5d,with the closure of neural tube,the expression of Notch mRNA is similar to that of the normal embryo.Ⅲ.The expression of the related development gene of the Notch signalingData obtained from RT-PCR showed that,at E9.5d,the expression of Notch1 gene in NTDs mouse was higher than controls(P＜0.05),and the RBP-Jk,PS1 and Hes1 gene is not significant (P＞0.05);at E10.5,the expression of Notch1 and RBP-Jk gene was not statistically significant between NTDs cases and normal cases(P＞0.05),but the expression of PS1 and Hes1 gene in NTDs cases was lower than controls(P＜0.05);at E11.5d,the expression of four gene was not obviously changed compared with normal cases(P＞0.05).In normal neural tube tisse,the expression of Notch1 mRNA at E10.5d was higher than E9.5d and is similar to E11.5;the expression of RPBP-Jk and Hes1 at E10.5d was higher than E9.5d at E11.5d;the expression of PS1 was incressing in this days.In NTDs mouse,the change of Notch1 and RBP-Jk was not conspicuous;the expression of PS1 and Hes1 was decressed at E10.5d.Ⅳ.The expression of the intracellular domain of Notch(NICD)proteinWestern blots showed that,the expression of NICD protein was not significant between NTDs cases and controls at E9.5d(P＞0.05);at E10.5d the expression of NICD protein in NTDs is higher than normal(P＜0.05);the results was similar to controls and was not obviously changed at E11.5d(P＞0.05).The expression of NICD was observed in neurepithelium around neural tube using immunohistochemistry. Objective: It is controwersial on whether the gene methylenetetrahydrofolate reductase(MTHFR) and reduce folate carrier-1 (RFC-1) are the susceptible genes of neural tube defects(NTDs).Methods: The polymorphism of MTHFR C677T and RFC-1A80G gene were analyzed by acombination of polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) in 38 mothers with a previous child with NTDs and 80 female health control. Thelevel of defect was assigned according to Van Allen's classification, we stratified our cases intotwo groups, i.e. 'upper defect' which included anencephaly and occipital encephalocele, and the'lower defect' included thoracolumbar or lumbosacral spina bifida.Results:1.There was no statistically significant difference between the genotype and allele frequency of677C→T polymorphism in mothers with a previous child with NTDs and controls. Thefrequency of 677C→T homozygotes was higher in mother who had a previous child with a lowerdefect type, compared to controls (OR= 0.59 (95% CI 0.46-0.76), p=0.08), but the difference wasnot statistically significant. The frequency of T alleles was significantly higher among motherswith a previous child with a lower defect type compared to controls (OR=0.32 (95% CI 0.11-0.9),p=0.027).2.The frequency of RFC-1 80A→G homozygotes was higher than that in corresponding controls(4 vs. 13%), and the difference was statistically significant. OR was 8.85 (95%CI 1.70-44.3),p=0.008. The frequency of the mutated G allele was significantly higher than controls(OR=1.97(95% CI 1.12-3.44), p=0.021), and the difference was statistically significant. For theupper defect type of NTDs, there was no statistically significant difference between frenquenciesof genotypes and alleles. However, the frequency of RFC-1 80A/G and 80G/G was higher inmother who had a previous child with a upper defect type compared to controls. The differencewas statistically significant respectively (p=0.009 and p=0.005). The frequency of G alleles wassignificantly higher among mothers with a previous child with a upper defect type compared tocontrols (OR- 2.42 (95% CI 1.28-4.58), p=0.008).Conclusions: Our data showed that the 677-T allele frequency was not significant in cases andcontrols. It was not considered as a risk factor in shanxi NTDs . But the RFC-1 was a geneticalrisk factor of NTDs .There is remarkably different in genotypes frequency and allele frequencyof the mothers with a previous child with NTDs and controls.