Effects Of Pax3/Pax7 On Differentiation,Migration And Axon Formation Of Neural Progenitor Cells

BackgroundPax gene(Paired box gene)is an important family of transcription factors.The growth and development of vertebrates is closely related to the functional regulation of Pax gene.Normally expressed Pax gene dynamically regulates the transcription of developmentrelated genes,promotes cell proliferation and self-renewal,inhibits apoptosis,and guides the directional migration and differentiation of precursor cells.Pax3 and Pax7 are the only two members of the Pax protein family that contain intact PD(Paired DNA-binding domain),HD(DNA-binding homeodomain)and OP(Octapeptide)domains,which are closely related to neural development.The previous research work on Pax3 and Pax7 is mainly focused on muscle genesis and development,but there is no in-depth discussion on its function in the central nervous system,especially on the construction of neural network.Objective1.Construction the model of abnormal expression of Pax3 and Pax7 in the cortex of mouse embryo.2.Construction the model of abnormal expression of Pax3 and Pax7 in the optic tectum of chicken embryo.3.To study the effects of abnormal expression of Pax3 and Pax7 on neuronal migration and axonal growth.Methods1.To construct mouse Pax3 and Pax7 overexpression vectors.RNA is extracted from brain tissue of C57 BL/6N mouse embryo for reverse transcription.Pax3 and Pax7 genes are amplified by PCR using c DNA as template.Pax3 and Pax7 genes are amplified into multi-clone sites of p CAGGS-MCS-EGFP vector by restriction endonuclease digestion and ligation,and p CAGGS-EGFP-Pax3 and p CAGGS-EGFP-Pax7 vectors were obtained.The inserted gene is identified by colony PCR and endonuclease digestion,and the gene is sequenced.2.Verification of mouse Pax3 and Pax7 overexpression vectors.PCAGGS-EGFPPax3 and p CAGGS-EGFP-Pax7 vectors are transferred into N2 a cells and E13.5 mouse embryonic cerebral cortex by liposome transfection technique and mouse uterine electroporation technique,respectively.Western blot and immunofluorescence are used to detect the expression effect of mouse Pax3 and Pax7 overexpression vectors at the cellular and in vivo level,respectively.3.The abnormal expression plasmids of chicken Pax3 and Pax7 are transferred into the optic tectum neuroepithelium of chicken embryo at E4 by chicken embryo in ovo electroporation technique,and the samples are collected at E12 d.Frozen sections are used to detect the expression of Pax7,DCX,Map2 and other proteins by immunofluorescence technique,and the morphological structure and distribution of nerve cells are observed.4.The total protein is extracted from the electroporation positive tissue,and the expression of Pax7,Pax6,DCX after Pax3 overexpression is detected by Western blot and Pax3 overexpression.The overexpression plasmids of p MIWIII-Pax3 HA and p MES-Pax7-GFP are transferred into the optic tectum neuroepithelium of chicken embryo E4 by in situ electroporation technique,and the samples are taken at E12 d.Frozen section,immunofluorescence technique is used to detect the expression of DCX and Map2 protein,and the morphological structure and distribution of nerve cells are observed.Results1.PCR restriction endonuclease digestion and gene sequencing shown that p CAGGSEGFP-Pax3 and p CAGGS-EGFP-Pax7 vectors are successfully constructed.2.The immunofluorescence results of Western blot and mouse brain sections of N2 a cells transfected with p CAGGS-EGFP-Pax3 and p CAGGS-EGFP-Pax7 showen that the expression of Pax3 and Pax7 genes increased in N2 a cells and mouse cerebral cortex.3.In the optic tectum of E4 chicken embryo,the nerve cells could not protrude or protrude short processes after Pax3 overexpression and the interference of Pax7 gene.The immunofluorescence results shown that the expression intensity of DCX decreased in the experimental group,and in the positive transfection area,the expression of DCX is disordered and shown irregular pore shape.4.The protein is extracted from the positive site of Pax3 overexpression in chicken embryo optic tectum by electroporation,and the effect of Pax3 overexpression on Pax7,Pax6,DCX expression is identified by Western blot.The results shown that the overexpression of Pax3 interfered with the expression of Pax7 and interfered with DCX to some extent.Conclusion1.Overexpression of Pax3 inhibits the expression of endogenous Pax7;2.Overexpression of Pax3 and knock down of Pax7 can inhibit synaptic formation of nerve cells;3.Overexpression of Pax3 and knock down of Pax7 reduce the number of axons and synaptic length of multipolar nerve cells.