Expression Pattern And Functional Redulation Of N-Cadherin In The Central Nervous System During Chicken Embryonic Development

During embryonic development, there is a large number of neuronal generations andmigration regulated by numerous molecules, among of which, the cell adhesion molecule ofcadherin family members play a critical role in the central nervous system (CNS). Neuralcadherin (N-Cadherin), one of cadherin family members, was first discovered in the chickneural tube, and plays a vital role during the CNS development. Disrupted differentiation andmigration of newborn neurons can cause nervous system abnormalities during the CNSdevelopment. Studies on the molecular mechanism leading to abnormality of neuronaldifferentiation or migration has widespreadly become focus of neurobiology. In the presentstudy, by using in vivo electroporation we konckdown or overexpression N-Cadherin in theCNS during chicken embryo development, to analyze the effects of N-Cadherin on the axonalpathfinding and neuronal migration, and further to explore the functional regulation ofN-Cadherin during the nervous system development. The results were as follows:1. In ovo and ex ovo culture of chicken embryos were developed successfully, meanwhileestablishing and optimizing in vivo electroporation, and the exogenous gene expression wasobtained in the spinal cord and tectum using above methods. The optimal stage for spinal cordtransfection is at E2.5-3(HH stage18-24), and for tectum optimal transfection is E5(HHstage27). The efficiency of transfection in spinal cord can reach54.5%, and in tectum60%.This method therefore can meet the requirement of experimental studies.2. Three plasmid vectors: pCAGGS-GFP, pEGFP and pCL-GFP carrying differentpromoters were selected, and their driving strength was compared in developing chickenembryos. The3promoters included:(1) the CAG promoter (containing the CMV immediateearly enhancer and the chicken β-actin promoter),(2) the CMV promoter (the humancytomegalovirus immediate early region enhancer), and (3) the SV40promoter (simian virus40). The results clearly showed that the expression intensity of the reporter gene differedsignificantly among the3promoters: chicken β-actin promoter induced the highest intensityof GFP expression, while SV40promoter induced the lowest intensity. In order to obtain highexpression of exogenous genes during the chicken embryo development, CAG promotercould be the best choice. The expression of GFP does not affect the expression of α-smooth muscle actin (α-SMA) and neurofilament (NF), as well as the morphology of chickenembryos after chicken embryos were transfected with pCAGGS-GFP, so GFP can well serveas a report gene for chicken embryo in vivo electroporation.3. The rate of positive expression of pCAGGS-GFP after liposomes transfected293Tcells in vitro was67.8%. We selected the most striking one of interference among the threedesigned interferential sequences of shRNA by western blot. The sequence isGCAAATGAAGGTGAAGCTAAC. Immunohistochemical results showed that the rate ofpositive expression of overexpression vector pCAGGS-N-Cadherin was44.6%. After theoverexpression of N-Cadherin, the expression of cellular associated proteins was influencedin different degrees. The most noticeable effect was the change of catenins, especially theβ-catenin, whose expression quantity and the expression sites all happened to change. Theexpression of β-catenin increased significantly when the N-Cadherin was overexpressed. Theβ-catenin expression was reduced in the group co-transfected with pCAGGS-N-Cadherin andN-Cadherin-shRNA vector compared to that in overexpression group, and β-cateninaccumulated into nuclei. The results suggest that there is a close connection betweenN-Cadherin and β-catenin, and therefore β-catenin could be used as an important index in vivolater.4. The expression patterns of N-Cadherin in the spinal cord and tectum were examinedbefore the experiment in vivo for analyzing the co-expression pattern of NF (neurofilament)and N-Cadherin. The results showed that the expression patterns of N-Cadherinspatiotemporally changed during chicken embryonic development, and N-Cadherin wasdetected at E3in spinal cord and E4in tectum in the early development of embryo andreached a peak in E6-E8, then its expression reduced from E10but maintained at a relativelow level at the late stage of embryonic development. The expression patterns of N-Cadherinand NF showed similar pattern, such as the spatiotemporal change, but also some difference,such as regional expression. After demonstrating the expression pattern of N-Cadherin, wefurther conducted the experiment of N-Cadherin overexpression or konckdown in spinal cordand tectum during the chicken embryo development.5. Knockdown or overexpression of N-Cadherin perturbed the projection of commissuralfibers in the chicken spinal cord. In the control group, the commissural fibers have two modes:commissural axons turn rostrally into the longi-tudinal plane and project alongside the FP[medial longitudinal commissural (MLC)] and grow into an intermediate region of the spinalcord [intermediate longitudinal commissural (ILC)]. In the case of N-Cadherinoverexpression only MLC was observed and ILC was not obvious. In the case of N-Cadherindown-regulated the MLC and ILC disappeared, similar results was observed in the group of β-catenin down-regulated, and in the case of double down-regulated of N-Cadherin andβ-catenin, this phenomenon was more obvious. The results suggested that N-Cadherin couldaffect on projection of commissural fibers through β-catenin signaling pathway.6. Overexpression or down-regualtion of N-Cadherin could inhibit the migration oftransfected cells during the development of chicken embryo, causing the residence of a largenumber of cells in NE (Neuroepithelium) layer. Under the overexpression of N-Cadherin theβ-catenin expression increased, but conversely the expression of β-catenin reduced.In summary, this study analyzed the expression pattern of N-Cadherin in the centralnervous system during chicken embryonic development, and we established a method of genefunctional studies by in vivo electroporation. In addition, it first demonstrated the abnormalexpression of N-Cadherin would perturb the commissural axon projection in the chickenspinal cord and also inhibit the migration of neural precursor in tectum. In a word, our studyprovides a new method and reference for N-Cadherin functional research in vivo.