Effect Of Notchl On Neural Tube Defects And Neural Stem Cell Differentiation Induced By All-trans Retinoic Acid

BackgroundNeural tube defects(NTDs)are a severe congenital brain defect caused by abnormal closure of the neural tube during fetal development.The defects are mainly characterized by brain loss,cerebral dilatation,meningeal dilatation and recessive spina bifida.Neural stem cells(NSCs)are involved in neural tube closure,and their proliferation,differentiation and migration are key to normal neural tube closure.Abnormal differentiation of NSCs leads to abnormal brain development.NSCs are the most unstable cells in the nervous system.They are self-renewing and pluripotent,and can differentiate into three basic neuroectodermal lineages.NSCs produce neurons,astrocytes and oligodendrocytes in a region-and stage-appropriate manner throughout their life cycle.Notch signaling plays a sophisticated and complex role in cell proliferation,differentiation and embryonic development among various cell fate regulatory pathways.Notch signaling is an evolutionically highly conserved pathway that plays arole in multiple cell differentiation processes in multicellular animals and humans.Notch signaling pathway in vertebrates is composed of Notch receptor[Notch 1(N1)-Notch4],Notch ligand(DHL,Delta 1-Delta4)and jagged 1-Jagged2,as well as DNA binding protein RBPJK/CBF1.The extracellular end of the Notch receptor and the ligand binding after activation,have two consecutive protein hydrolysis process,the first is the extracellular domain S2 cutting locus[1],the second was the cut of the film in S3 site,then Notch intracellular period(NICD)are released into the nucleus,then forming complexes with DNA binding proteins,which further regulates downstream target genes to regulate cell proliferation and differentiation.Presenilin 1(PS1)is the catalytic activity center of the γ-endocrinase complex,which is involved in Notch receptor cleavage site in S3[2].All-trans Retinoic Acid(atRA)is a normal metabolite of Retinoic Acid in human body and is an important factor in embryonic development.It has been found that excessive atRA can lead to the occurrence of NTDs,but its specific molecular mechanism has not been clearly studied.This study confirmed the role of Notchl in atRA treatment of mouse embryonic brain tissue(in vivo)and NSCs(in vitro).In addition,we demonstrate for the first time that atRA can inhibit PS1 and thus reduce Notchl signaling pathway activity,thereby promoting NSCs differentiation,and further reveal the molecular mechanism of atRA induced NTDs.This study provides a new direction for the application of neural stem cells and the clinical prevention of birth defects.PartⅠ Estabishment of neural tube defect model in mice and the mechanism of Notchl in atRA induced neural tube defectsObjectiveThis section aims to establish a neural tube defect model in mice and explore the mechanism of Notch1(N1)in neural tube defects induced by all-trans retinoic acid on the basis of the model.MethodFemale C57BL/6 mice were randomly divided into experimental group and control group.The experimental group was given 100 mg/kg atRA by gavage on the 7th day of pregnancy(E7),and the control group was given the same volume of corn oil by gavage on the 7th day of pregnancy(E7).All pregnant mice were sacrificed via cervical dislocation on E11,E13,E15 and E17.Embryo brains were obtained for hematoxylin and eosin(H&E)staining to detect abnormal brain development.The expression pattern of Notchl in mouse embryonic brain was detected by immunolocalization and Western Blot.ResultsatRA can induce abnormal brain development of mouse embryos.Compared with the control group,embryos exposed to atRA group showed symmetrical brain material formation earlier,and the growth of brain material was more obvious.Notchl was present in the neurocortex of the brain tissue of embryonic mice.Immunofluorescence and Western blot showed that the expression of Notchl in the brain tissue of embryonic mice treated with atRA was significantly decreased compared with the control group.ConclusionsThe neural tube defect model of mouse embryo can be established by atRA.atRA promotes the differentiation of neural tube by inhibiting the activation of Notchl,leading to NTDs.Part II The role of Notchl in the differentiation of NSCs induced by atRAObjective1.This part aims at the isolation and culture of neural stem cells,identification of neural stem cells and their differentiation neurons,astrocytes and oligodendrocytes,and the distribution of Notchl in these cells was detected.2.NSCs were treated with atRA to detect the expression of Notchl,PS1 and various cell markers of differentiation cells.To investigate the molecular mechanism of Notch1,PS1 and differentiation of neural stem cells treated with atRA.Method1.Primary neural stem cells were extracted from the brains of fetal mice at 18.5 days of gestation and 10%fetal bovine serum differentiation medium was used to induce differentiation of neural stem cells.2.Co-expression of Notchl,Nestin,NF,GFAP and GALC in NSCs as detected using co-immunofluorescence staining.To confirm the expression of neurofilament protein(NF),astrocyte marker-glial fibrillary acidic protein(GFAP)and oligodendrocyte marker-galactoencephaloside(GALC)in neurons,astrocytes and oligodendrocytes by immunofluorescence staining.3.The protein expression of Notch1、PS1、NF、GFAP、GALC on Day 1,35,7 from atRA-treated or control cultured NSCs were analyzed using Western blot analysis.Results1.Primary neural stem cell culture shows the whole process of nerve cells differentiation.Differentiation culture 0 day,we can see the nerve bulb attached to the bottom of the bottle.After 1 day of culture,the volume of the nerve bulb increased,and a small amount of NSCs were released from the nerve bulb and began to replicate and divide.After 3 days of culture,different types of cells appeared around the nerve globules,and individual cells protruded like cords.Different shapes of cells,including neuronal cells,astrocytes and oligodendrocytes,were detected at 7 days of culture2.Cell marker proteins Nestin,NF,GFAP and GALC fluoresceed positively in immunofluorescence staining,indicating that neural stem cells differentiated into neuron cells,astrocytes and oligodendrocytes.Meanwhile,Notchl was co-expressed with Nestin,NF,GFAP and GALC in neural stem cells and differentiated cells.3.The expression levels of Notch1 and PS1 decreased regularly in the control group,but the expression levels of NF,GFAP and GALC increased gradually,suggesting that the decrease of Notchl expression significantly promoted the differentiation of neural stem cells to a certain extent.In the atRA treatment group,the expression levels of Notchl and PS1 were gradually decreased,while the expression levels of NF,GALC and GFAP were significantly increased,suggesting that atRA enhanced the differentiation ability of NSCs,and atRA decreased the expression of Notchl by inhibiting PS 1 after NSCs treatment.Conclusions1.NSCs and their differentiated neuronal cells,astrocytes and oligodendrocytes were observed Notchl expression.2.After atRA treatment of neural stem cells,it can reduce the activity of Notch1 signaling pathway by inhibiting PS1,thereby promoting NSCs differentiation.