The Effect Of Cadherin6B On The Projection Of Spinal Neural Fibers During The Chicken Embryonic Development

BackgroundCadherins,one family of Ca2+ dependent transmembrane glycoproteins,mainly mediate the adhesion among cells.During the formation and development of neural system,neurons accumulate to form a nucleus through the same kind of adhesion molecules.The projections of neurons extend toward the positive section of same kind of molecules,which generate a complicated neural circuit.Cadherin family have numerous members,Cdh6B mediate cell adhesion and affect the cellular sorting,migrationn,location and the determination of growth pathway of axons.Based on the studyof these members,the connections among the members of Cadherins have been founded,and these enable us to get a further study on the role of cadherins during the development of neural system.ObjectiveBuild the techniques of Chicken embryo in vivo electroporation and Open-book.Then use these methods to overexpress and surpress the Cdh6B expression to study the effect of Cdh6B to the neural growth,migrateon and location of axons in spinal cord during the development of Chicken embryo.Methods1.Overexpression and inhibition vector of Cadherin6B were transfected into HEK293T cells by liposomes.Examine whether there are some plasmids that expressed abnormally.2.Overexpression and down-regulation vectors of Cadherin6B were respectively transformed into the cavities of spinal cords in the chicken embryo electroporated around 2.5 days.Spinal cords respectively obtained from 24h,48h,and 72h after electroporation,were fixed with 4%polyoxymethylene overnight and then was embedded and fixed by low temperature after being dehydrated by 18%sucrose.The frozen sections made of the fixed materials were stained by immunofluorescence dye and hybridized in ovo for examining their change.3.Overexpression and down-regulation vectors of Cadherin6B were respectively transformed into the cavities of spinal cords in the chicken embryo electroporated around 2.5 days.The entire spinal cord derived from the entire chicken embryo removed after electroporation 6 days,was placed on the slide with the technique of Open-book and stained with DAPI 30 min after being fixed by 4%polyoxymethelene.With the fluorescent microscopy,we observe whether the projection of neurons is normal.Results1.The overexpression vector of Cdh6B,pCAGGS-Cdh6B,was transfected into the HEK293 cells.The result of cell immunofluorensence and western blot showed that Cdh6B was really overexpressed successfully.Meanwhile,the supression vectors pGPU/GFP/Neo-Cdh6B-shRNAl and shRNA2 were tested using the same way.The results revealed that pGPU/GFP/Neo-Cdh6B-shRNA1 worked well while shRNA2 didn't work.2.Built the chicken embryo electroporation technology successfully.For chicke-n embryos,the best date is E2.5.And 37? normal saline containing antibiotic was dropwise added during experiment.The most proper condition for electroporation with the CUY-21 machine is voltage 18V,pulse 60mA,interval 100mA,pulse 6 times totally.After transfer theGFPplasmid 24 h,survival rate of chicken embryo was 80%(8/10),and GFP positive expression rate was 85.7%(18/21)for survival embryos.Do slices for GFP positive embryos and find that GFP positive cells exsited just at one side.This result revealed the the abnormal model has been built successfully using chicken embryo electroporation technology.3.The overexpression and supression plasmids were transfected into chicken spinal cord.The results of immunofluorescence showed that the overexpression modelhas been built successfully while the supression model failed because of the particular positon of Cdh6B expression.4.The chicken embryo after electroporation 2.5days,was divided into three groups,respectively control group only transfected with pCAGG-GFP,overexpression group,and interference group.The entire spinal cord removed at 6 or 7days was tiled and stained with DAPI.With the fluorescent microscopy,the increase of projection of neural fibers in overexpression group was observed,while in interference group the projection of neural fibers was inhibited and could not cross the base plate to project to the offside.ConclusionThe unnormal expression of Cdh6B in chicken embryo spinal cord has an improtant effect for nerve fiber projection.This reveals that Cdh6B takes part in neurons axon growth and path selection in chicken spinal cord.