The Molecular Mechanism Of Rap1 Effect On Neuronal Migration During Central Nervous System Development

Background The development of nervous system is a very complicated and sophiscated process,especially the cortical development.During the cortical architechture,neural migration plays vital roles since only neurons migrate to the right place on the right time,then forming the correct neural circuit and consequently preforming its conresponding functions.There are three models of neural migration: multipolar migration,glia-dependent locomotion and somal translocation.Neurons born in early stage mainly adopt somal translocation,while the later born neurons preform these three types of migration as discribed above successively.Undoubtedly,there are many molecules and signialing pathways involved in this process.It has been reported that Rap1,a kind of GTPase,participated in somal translocation via Reelin sigaling pathway during the early development of embryo.However,some researches indicated that the activity of Rap1 was also detected in the later born neurons but the phosphorylation sites are different with the ones in Reelin signaling pathway.Here,we proposed that Rap1 is involved in the migration of later born neurons through other signaling pathways.Rap1 gap,an inhibitor of Rap1,was overexpressed or down-regulated in our study to investigate the roles of Rap1 during the cortical development.Objective 1.To explore whether Rap participates in regulating the migration of later born neurons.2.To investigate through which signaling pathway Rap1 affects the migration of later born neuronsMethods 1.Rap1 overexpression construct(p CAGGS-Rap1gap)and interfering constructs(Rap1gap-sh RNA1 and Rap1gap-sh RNA2)were tranfected into CHO cells via Lipofectamine 3000 Reagent.Total proteins extracted from CHO after transfection were detected by Western blot to investigate whether the overexpression and down-regulation of Rap1 gap were achieved.Besides,in ovo electroporation was preformed to transform Rap1 overexpression construct and interfering constructs into the spinal cord of E2.7 chick.Collect samples at E5 and preform spinal transverse section.Immunofluorescent staining was conducted to detected whether Rap1 gap was overexpessed or down-regulated.2.By in ovo electroporation,p CAGGS-Rap1 gap,Rap1gap-sh RNA1 and Rap1 gapsh RNA2 were transfected into optic tectum of E4 chick,and the samples were obtained at E9.In addition,in utero electroporation was preformed to transfect p CAGGS-Rap1 gap into the ventricular zone of E16 mouse embryo and samples were collected at E19.All samples were preformed frozen section after fixation of 4% PFA,dehydration of 18% sucrose and OCT embedding.The transfected neurons were traced by GFP to investigate whether neural migration and morphology can be influenced by the aberrant expression of Rap1 gap.3.The transfected CHO cells were preformed immunofluorescent staining to detect the expression of Rap1 gap,Tubulin,Vinculin,F-actin and NF,and observe the alterations of neural morphology.4.To study which molecules and signaling pathways Rap1 regulate to affect neural migration,our research detected the expression of some molecules such as ?-catenin,Ncadherin,PI3 K,PKC,AKT,Tubulin,vinculin and F-actin after the abnormal expression of Rap1 gap.5.To explore whether Rap1 affects neural migration through regulating N-cadherin,in utero electroporation was preformed to knock down the expression of N-cadherin in ventricular zone of E16 mouse.Observe the neural migration and morphology,and detect the expression of Tubulin,Vinculin and F-actin.Results 1.After the transfection of p CAGGS-Rap1 gap,Rap1gap-sh RNA1 and Rap1 gapsh RNA2 into CHO cells and chick spinal cords,the results of Western blot and immunofluorescent staining indicated that p CAGGS-Rap1 gap was successfully overexpressed in CHO cells and chick spinal cord,while both Rap1gap-sh RNA1 and Rap1gap-sh RNA2 can down-regulate the expression of Rap1 gap and the former is more efficient..2.Overexpression and down-regulation of Rap1 gap were achieved in chick optic tectum and mouse cortex by in vivo electroporation.Immunochemistry indicated that overexpression of Rap1 gap in E9 chick optic tectum,which is inactivation of Rap1,result in the abberrance of neural migration and alteration of neural morphology.Besides,most of transfected neurons can not protrude axons or extend very short axons.However,there was no significant difference after the down-regulation of Rap1 gap,compared with the control group.Consistent with it,neurons cannot migrate outwardly and settle at the upper layer of ventricular zone after the overexpression of Rap1 gap in E19 mouse cortex.What's more,the neural shapes were transformed and most of them didn't have axons or had very tiny axons.3.After the transfection of Rap1 gap into CHO cells,the results of immunofluorescent staining showed that Rap1 gap was overexpressed in CHO cells and cellular morphology has been altered by marking the cytoskeletal proteins.Most of them presented round shape.4.When Rap1 gap was overexpressed in CHO cells,it was detected by Western blot that ?-catenin and N-Caderin were significantly affected,some cyctoskeletal proteins such as Tubulin and F-actin were down-regulated and many associated kinase such as PI3 K and PKC detected decrease.5.Consistent with the inactivation of Rap1,the majority of neurons settled at the ventricular zone after the inhibition of N-cadherin in mouse embryonic cortex and the neural morphology has been transformed,besides,many cytoskeletal proteins such as Tubulin,F-actin were detected reduction.In addition,the expression of N-cadherin was detected decrease in ?-catenin knock out cells by Western blot.Conclusion 1.Rap1 is involved in the regulation of glia-dependent locomotion during the later neural migration 2.Rap1 could regulate the neural morphology via N-cadherin to influnce neural migration.