The Effect Of Wnt3a Overexpression To The Projection Of Commissural Axons And Neuronal Migration In Spinal Cord

BackgroundDuring spinal cord development of chicken embryo,commissural axons projected toward the contralateral side.In addition,only if neurons migrated to certain position which can form the normal tissue structure and nerve circuit,information transmission of spinal cord can function correctly.Wnt3 a,a member of Wnt family,is involved in multiple cellular functions,including self-renewal,proliferation,differentiation,and motility.Even though many studies demonstrated that Wnt3 a could potentially repair the injuried spinal cord,the function of Wnt3 a on the developing spinal cord remains elusive.Objective1.To investigate the effect of Wnt3 a ectopic expression on commissural axonal projection and neuronal migration.2.To explore the effect of Wnt3 a overexpression on neuronal morphology and the expression of associated molecules.Methods1.Wnt3 a cDNA was cloned into pCAGGS-EGFP vector by using some molecular and biological techniques such as RNA extract,PCR amplification,enzymatic digestion and ligation to construct Wnt3 a overexpression construct.And then colony PCR and double-digestion was preformed to verify whether Wnt3 a recombinant was successfully constructed.Finally,gene sequencing was performed.2.To investigate whether this recombinant could overexpress Wnt3 a,Wnt3a overexpression construct(pCAGGS-EGFP-Wnt3a)was first transfected into SH-SY5 Y cells.Western blot was conducted to detect whether Wnt3 a was overexpressed.Besides,pCAGGS-EGFP-Wnt3 a was transfected into E2.8 chicken spinal cord using in vivo electroporation.Samples were collected at E4.Then was preformed transverse section and the expression of Wnt3 a was detected by immunostaining.3.The pCAGGS-EGFP-Wnt3 a or pCAGGS-EGFP was transfected into E2.8 chicken spinal cord using in vivo electroporation.Samples were collected at E6 and each group contained more than 5 samples.On the one hand,samples were conducted frozen section for immunofluorescent staining to detect the expression level of Wnt3 a,Pax7,Map2 and MNR2,meanwhile,neuronal morphology and migration were observed by GFP tracing after Wnt3 a ectopic expression;on the other hand,spinal cord neurons were extracted for samples and cultured to observe neuronal morphology in vitro.4.The pCAGGS-EGFP-Wnt3 a or pCAGGS-EGFP was transfected into SH-SY5 Y cells using Liposome transfection.Immunostaining against Tubulin was preformed to observe the alteration of neuronal morphology.Besides,total proteins of SH-SY5 Y after Wnt3 a transfection were extracted and then the expression levels of ?-catenin,CyclinD1,C-myc,PCNA,Caspase3,Bcl-2 were detected by Western blot.Results1.The results of gene sequencing and PCR showed that the pCAGGS-EGFP-Wnt3 a vector was successfully constructed.2.After pCAGGS-EGFP-Wnt3 a vector was transfected in SH-SY5 Y and chicken spinal cord,the results of Western blot in SH-SY5 Y cells and immunostaining in transverse section of spinal cord both indicated that Wnt3 a was successfully overexpressed in SH-SY5 Y and chicken spinal cord.3.After Wnt3 a ectopic expression in E6 spinal cord,commissural axons failed to project toward the contralateral sides while the majority of GFP positive neurons resided at the ventricular zone(VZ),and neuronal morphology has been transformed and most neurons did not outgrow axons and only a few extend very short axons.After Wnt3 a ectopic expression,the result of immunofluorescent staining showed that Map2 positiveexpression area was decreased;while the number of MNR2 positive cells were significantly decreased and most MNR2 positive neurons were settled at VZ;besides,Pax7 expression had no obvious change.Most neurons extracted from chicken spinal cord after Wnt3 a overexpression presented in round shape,and only a few extended very tiny processes.4.The pCAGGS-EGFP-Wnt3 a vector was transfected into SH-SY5 Y cells and then immunostaining against Tubulin was conducted to observe the alteration of cell morphology.Most transfected cells presented in round shape,and only a few extended very tiny processes.The result of Western blot showed the levels of ?-catenin,C-myc and PCNA significantly increased after Wnt3 a over-expression while Bcl-2 was down-regulated.However,the expression of CyclinD1 and Caspase3 did not show any change.Conclusion1.Wnt3 a ectopic expression inhibits commissural axonal projection.2.Wnt3 a ectopic expression regulates cell morphology,which then affects neuronal migration.3.Wnt3 a overexpression regulates cell proliferation and apoptosis via ?-catenin signalling pathway.