Relationship Between Wnt Signaling Pathway And Neural Tube Defects Induced By Excess Retinoic Acid In Mouse

PrefaceBecause of the severe environmental pollution accompanied by the development of the industry,the incidence rate of congenital malformation was rising gradually and mote and more scientists began to study the congenital malformation.Neural tube defect(NTD)was one of the most common and pernicious congenital malformation, and therefore,the study on the morphogenetic mechanism of the NTD have become popular in teratologic research.NTDs are generally considered to exhibit a multifactorial etiology invoking multiple interacting genes and environmental factors.The rate of recurrence in a sibship of NTDs is about 2-5%and the risk of incidence in the parents who have had NTDs fetus ever is three times as ligh as that in the normal parents.In addition,the environmental factors,such as heavy metals,antibiotics,X-ray,smoking,drinking, excess vitamin A and its derivant-retinoic acid(RA),are all important teratogenetic factors the incidence of NTDs.As the common phenotypes of NTDs,anencephaly and spina bifida are all called open NTDs.Spina bifida is a birth defect that involves an incomplete development of the spinal cord and its coverings.Anencephaly is the most serious NTD,in which the brain and cranial vault are grossly malformed and the cerebrum and cerebellum are reduced or absent.Furthermore,because exencephaly fetuses always died in uterus, the elucidation of the mechanism underlying development of exencephaly is hampered by the relative paucity of information regarding the cause in humans. Murine exencephaly can provide an ideal model for investigating the pathophysiology of human exencephaly.However,the established mouse models are either not induced at a high frequency in live fetuses or always accompanyed with many other malformations,even in the case of SELH/Bc mice which is a strain genetically liable to exencephaly,the exencephaly rate is only 53%.Along with the development of molecular biological techniques,it was proved that retinoic acid is a necessary morphogen during embryonic development.It not only determines the establishment of embryonic cephalocaudal axis,but also take a role in the differentiations of the prosencephalon,mesencephalon,epencephalon and spinal cord during the development of central nervous system.The receptor theory is popular for the mechanism of action of retinoic acid.Retinoic acid receptors(RARs) belong to the superfamily of intranuclear receptors and include three subtypes,α,βandγ.After binding the ligands,RARs form dimmer with retinoid X receptors,then bind to the retinoic acid response elements in the promoters of the target genes,and regulate the transcription of the target genes at last.It is known that normal embryonic morphogenesis,especially development of the central nervous system is related to the developmental regulation genes regulated by retinoic acid.It is considered that teratogenic effect of excess retinoic acid is probably owing to that retinoic acid unduly activate or inhibit some downstream morphogenetic regulation genes.Caspase-3 is a key executant in the downstream of cell apoptosis.Some apoptosis marks,such as chromosome condense and fragmentation of DNA,have direct correlations with Caspase-3.Appropriate apoptosis is indispensable for the normal morphogenisis of the neural tube and the exess or deficiency of apoptosis both will induce NTDs.The Wnt proteins are a kind of secretory glycoproteins and activate the Wnt signal transduction by binding with the receptor superfamily of Frizzlled and low-density lipoprotein associating proteins.Wnt signaling pathway is correlated closely with the embryonic development and cell proliferation,cell differentiation and cell apoptosis.B-catenin is an indispensable effector in the canonical Wnt signaling pathway and controls the embryonic development by activate the transcription factor of LEF/TCF(T cell factor/lymphoid enhancer factor).During the developmental process of central nervous system,β-catenin takes part in a series of biological events such as cell growth,cell differentiation,cell apoptosis and the morphogenesis of neural tube.Its misexpresion can result in neural tube defects,facial cranial defects and dyspoiesis of optic cup and prosomo.Dishevelled is all adaptor of many Wnt signaling pathways and will be activated when the protein binds the Frizzled.The activated Dishevelled can activate the canonical Wnt pathway by stabilizing the plasmosinβ-catenin or the noncanonical Wnt pathway by using its structural domain.PCP(planar cell polarity)signaling pathway needs the participation of Frizzled and Dishevelled proteins but not needs theβ-catenin.The recent researches showed that PCP signaling pathway plays key roles in the neural tube closure by regulating the convergent extension movement during the intestinal phase and neurula period. Vangl is one of the core members of PCP signaling pathway,and its gene mutation would induce NTD during embryoal development process.In order to clarify the relationship between Wnt signaling pathway and NTD induced by excess RA in mouse,and to provide scientific basis for the prevention and treatment of NTD,we plan to establish a mouse model for exencephaly and detect the expression and distribution changes of RARα,RARβ,Caspase-3,Dishevelled,β-catenin and Vang12 genes in the teratogenenic neural tube induced by excess RA with the techniques of immunohistochemistry,in situ hybridization,Western-blot and RT-PCR.Methods1.Establishing the mouse model for exencephaly and detecting the RARα, RARβand Caspase-3 proteins:(1)Establishing the mouse model for exencephaly: Virginal female KM mice weighting25-35g mated with the same strain of males. The day of detection of the vaginal plug was considered embryonic day(E)0.The pregnant mice(n=80)were randomly divided into different research(RA)groups and control group.The mice of research groups were orally treated respectively with a single dose of 20,30,40 or 50mg/kg of all-trans-RA dissolved in peanut oil at a selective time of E7.5,E7.75,E8.0 or E8.5.The controlled mice were administrated with a volume of peanut oil,equal to the highest dose of RA.All animals were killed by cervical dislocation on E10.5 or E16.5 and the numbers of implantation,resorption, dead embryos and exencephaly embryo were counted.Some of the fetuses of El0.5 and E16.5 were weighed individually by electronic balance,the lengthes of crown-rump and tail were measured by vernier caliper.According to the analyzing results,the best dose and best time of RA administration for the mouse model were determined.Microscopic changes in nervous tissues were evaluated in the specimens, sectioned routinely in 5μm thickness at a transverse plane with the paraffin microtome and stained with heamatoxylin and eosin(HE).(2)Detecting the RARα, RWRβand Caspase-3 proteins:The RARα,RARβand Caspase-3 proteins in the fetal neural tube tissue at 18,42,66 and 72h after RA(E7.75,30mg/ke)or vehicle treatment were detedted with Western-blot method.Results were corrected byβ-actin and arc expressed as means±standard error.2.Detection of Dishevelled1/2/3,β-catenin and Vang12 genes:The level of mRNA of Dishevelled1/2/3,β-catenin and vang12 were detected with RT-PCR and in situ hybridization and the level of protein of Dishevelled1/2/3,β-catenin and Vang12 were detected with Western-blot and immuhistochemistry in both experimental and control groups.Results1.Animal models:(1)With the increasing dose of RA,the incidence of RA-induced exencephaly became higher,and with the progress of development,the teratogenic sensitivity of the murin embryos to RA became declined.The best animal model of exencephaly induced by RA was established with the maternal administration of 30mg/kg RA on E7.75 that results in 94.34%exencephaly rate on E10.5 and 76.92%on E16.5.2.Protein expression of the RARα,RARβand Caspase-3 genes:(1)In the normal fetal neural tube tissue,the expressive level of RARαand RARβprotein increased first at 18h after treatment and achieve the peak value at 42h,then decreased slowly to the virgin level.RA treated groups resulted in a decreased expression of RARαand RARβat 18 and 42h(P<0.05)after treatment and an increased expression in RARβat 66 and 90h(P＜0.05).No significant difference in RARαprotein was noted at 66h and 90h between RA and vehicle-treated groups.(2) Expression of Caspase-3 protein displayed a ruled change with the embryonic development.In the normal fetal neural tube tissue,the expressive level of Caspase-3 protein increased first at 18h after treatment and achieved the peak value at 42 and 66h,then decreased slowly to the virgin level at 90h.No significant difference in the expression of Caspase-3 protein was observed between RA treatment and vehicle-treated groups at 18h after maternal treatment,but the expressive levels of RA-treated group were much lower at 42h(P＜0.05),and much higher at 66h and 90h (P＜0.05)than those of the vehicle-treated group.3.The detection of expression of the Dishevelled1/2/3,β-catenin and Vang12 genes:(1)Expression of Dishevelled1/2/3 genes displayed a ruled change with the embryonic development.The expression of Dishevelled1/2/3 mRNA increased firstly at 4h and achieved the peak value at 18h,then decreased slowly to the virgin level at 42 and 66h in the control groups.RA treatment groups resulted in a significant decrease in Dishevelled1/2/3 mRNA in fetuses at 4 and 18h(P＜0.05)after maternal treatment and a significant increase at 42h(P＜0.05),no difference was noted in Dishevelled1/2 mRNA at 66h between RA and vehicle-treated groups except a significant increase in Dishevelled3(P＜0.05).The expression of Dishevelled2 protein displayed a decreased trend after the treatment in the controll groups.RA treated groups resulted in a significant decrease in Dishevelled2 protein in fetuses at 18h (P＜0.05)after maternal treatment and a significant increase at 42h(P＜0.05),no difference was noted in Dishevelled2 protein at 66 and 90h between RA and vehicle-treated groups.The results of immunohistochemistry displayed that in control group,Dishevelled2 protein is positive in the cytoplasm of the neuroepithelial cells, and with the embryo development,the expression of Dishevelled2 protein declined gradually in the neural tube tissure.In RA-treated group,the expression of Dishevelled2 protein presents a trend of decreasing first and then increasing.(2) Expression ofβ-catenin gene displayed a ruled change with the embryonic development.The expression ofβ-catenin mRNA and protein all displayed a decreased trend after the treatment in the control group;RA treated groups resulted in a significant decrease inβ-catenin mRNA at 4 and 18h(p＜0.05)after treatment and a significant increase at 66h(P<0.05),no significant difference inβ-catenin mRNA was noted at 42h between RA and vehicle-treated groups.In RA-treated group,the expression ofβ-catenin protein were significantly decreased at 18 and 42h(P＜0.05) and significantly increased at 90h(P＜0.05),no significant difference was noted at 66h between RA and vehicle-treated groups.The staining of in situ hybridization and immunohistochemistry displayed thatβ-catenin was mainly expressed in the cytoplasmum of the neuroepithelial cells,and showed a time-dependent change trend.The expression ofβ-catenin mRNA and protein degraded continuously during embryo development in the control groups.B-catenin mRNA and protein showed a decreased expression first and then an increased expression in the RA treated groups.(3)The expression of Vang12 mRNA increased first at 18h and achieved the peak value at 42h,then decreased slowly to the virgin level in the control groups.The expression of Vang12 protein increased at 18h and achive the peak value at 42h,then decreased slowly to the virgin level at 66h and 90h in the control groups.RA treatment groups resulted in a significant decrease in vang12 mRNA at 4h and 18h (P＜0.05)after maternal treatment and a significant increase at 66h,no difference was noted at 42h between RA and vehicle-treated groups.RA-treated groups resulted in a significantly decreased expression of Vang12 protein at 18 and 42h(P＜0.05)and a significantly increased expression at 66 and 90h(P＜0.05).The staining of in situ hybridization and immunohistochemistry displayed that Vang12 was expressed in the neural tube tissure and showed a time-dependent change trend.The expression of Vang12 mRNA and protein rised first and then degrade during embryo development in the control groups.In RA-treated groups,vang12 mRNA and protein showed a decreased expression first and then an increased expression.Conclusions1.The animal model of exencephaly induced by RA was established successfully with maternal administration of 30mg/kg RA on E7.75 that results in 94.34%exencephaly rate on E10.5 and 76.92%exenccphaly rate on E16.5.1.RA is necessary for normal differentiation of the nervous systcm.2.The modulation of RARαand RARβgenes by excess RA may affect the expression of their downstream genes associating with embryonic development,and then interfere the closure of the neural tube,causing neural tube defect(NTD).4.Apoptosis takes part in the normal differentiation of the nervous system.Excess RA may interfere the normal apoptosis,which may play a role in the development of NTD induced by excess RA.5.Wnt signaling pathway takes part in the normal differentiation of the nervous system.The modulation of Dishevelled1/2/3,β-catenin and Vang12 genes by RA may take part in the development of RA-induced NTDs.