The Effect Of R-Cadherin And Pax6 To Commissural Axon Projection In Spinal Cord During Chicken Embryonic Development

BackgroundR-cadherin and Pax6 play important roles in the development of the central nervous system.As a member of the type I classic cadherins,Retinal cadherin(R-cadherin)can affect neural cell location,migration,as well as axon growth and pathway finding through mediating intercellular adhesion.Pax6 is a kind of important transcription and regulation factor,involved in cell proliferation,differentiation,migration and adhesion,it has different functions in different tissues and cells.Studying the function of R-cadherin and Pax6 help us to better understand the mechanism of nervous system development.Objective1.To develop the method of simultaneous tracing the development of commissural axons from bilateral chick spinal cord using dual-color fluorescent.2.To study the expression pattern of R-cadherin and Pax6 in chick spinal cord and the effect of R-cadherin and Pax6 to commissural axon projection.Methods1.The p CAGGS-GFP plasmid which carried the report gene of green fluorescent protein was transfected into the chick spinal cord using ovo electroporation after incubation of fertilized eggs for E2.5~E3.GFP-positive chick embryos were obtained at E6.At least three samples were collected each group.Subsequently,the specimens were respectively undergone either transverse section or “Open-book” processing.In “Openbook” processing,the spinal cord was unfolded and placed on the glass slide.After injecting Di I solution into the contralateral side of transfected region,the samples were incubated for 3d at 4?,and commissural axons were observed under a fluorescent microscopy.2.Different periods of the chicken spinal cords were collected and cut into frozen sections during chicken embryonic development.The expression patterns of R-cadherin and Pax6 were detected with in situ hybridization and by immunofluorescence respectively.3.The abnormal plasmids of R-cadherin were transfected into the HEK293 T cells and The target gene Pax6 was cloned into the plasmid of p CAGIG,and confirmed by colony PCR,double digestion and sequence.After it was confirmed that the abnormal plasmids of R-cadherin and Pax6 were useful,the abnormal plasmids of R-cadherin and Pax6 were transfected into chicken spinal cords using ovo electroporation.At E2.5-E3,for control group,The p CAGGS-GFP plasmid was transfected into the chick spinal cord using ovo electroporation.For experimental group,the abnormal plasmids of R-cadherin and Pax6 were transfected by the same way.positive embryos were collected and sliced at E4?E5 and E6 respectively.At least three samples were collected in every group.Then the expression of R-cadherin and Pax6 were detected using immunofluorescence histochemistry,and commissural axons projection affected by the abnormal plasmids of R-cadherin and Pax6 were detected by Open-book technology.The relationship between R-cadherin and Pax6 was studied using immunofluorescence histochemistry.Results1.The results of GFP labeling from both transverse section and Open-book collectively showed that commissural axons crossed the midline region through the floor plate to the contralateral side of the spinal cord,then they projected rostrally and caudally along the antero-caudal(A-P)axis within ventral fasciculus and lateral fasciculus.Di I tracing showed similar trajectory of commissural axons.2.The results of in situ hybridization showed that R-cadherin had different expression at different periods in the chicken embryonic spinal cord.The expression of R-cadherin was increased with the development of the chicken spinal cord.The rsults of immunofluorescence histochemistry showed that Pax6 was expressed in ventricular zoneand reduced in the late development of the chicken spinal cord.3.Cell immunofluorescence results showed the abnormal plasmids of R-cadherin were useful.The overexpression and inhibited expression model of R-cadherin were built successfully in chicken spinal cord.Compared with the control group,After R-cadherin overexpression,the commissural axons could across the floor plate and project stereotypically both in slice level and whole mount level,After the expression of Rcadherin was inhibited,the commissural axons guidance was abnormal.the p CAGIG-Pax6 vector was built successfully.After Pax6 overexpression,the commissural axons couldn't across the floor plate and project stereotypically both in slice level and whole mount level.The results of fluorescent immunohistochemical showed that the overexpression of Rcadherin and Pax6 had no effect to the expression of each other.Conclusion1.Dual-color fluorescent tracing was established,which could provide a new method for the study of development of commissural axons of the spinal cord.2.Different expression patterns of R-cadherin and Pax6 mean diverse roles for them in the spinal cord during chicken embryonic development.The overexpression of Rcadherin had no effect to commissural axon projection,The inhibitted expression of Rcadherin resulted in misguidance of commissural axon;Overexpression Pax6 could inhibit the projection of commissural axon,which suggested that R-cadherin and Pax6 play an important role for commissural axon projection in chicken embryonic spinal cord.