Effect Of N-Cadherin On Neuron Migration In The Developing Mouse Cerebral Cortex With In Utero Electroporation

BackgroundThe cerebral cortex of higher animals is a highly organized complex consisting of multilayer neural cells. This process is strictly regulated to ensure that an accurate and functional cerebral cortex can be formed. Previous studies have showed that when N-Cadherin was conditional knocked out in mouse embryo, the delamination of cerebral cortex became disordered and lost the normal structure completely. The results indicated that N-Cadherin plays an important role during mouse brain development. Recently, mouse in utero electroporation have been used to investigate gene function in vivo, especially for the genes in the central nervous system.ObjectiveSet up mouse in utero electroporation technology, and combined with RNA interference to investigate the effect of N-Cadherin on neuron migration during mouse cerebral cortex development.Methods1. Mouse in utero electroporation technology: the pregnant mouse at embryonic15day (E15) was anesthesia with chloral hydrate, and embryos were taken out, then2μg pCAGGS-GFP plasmid was accurately injected into paracele of embryos through proper condition of electroporation. After24h electroporation, the positive embryo brains were collected, fixed by4%PFA and sliced. DAPI was used to label nuclears. Then observe the change of morphological structure and detect the expression of a-SMA by immunofluorescence.2. Construction and verification of misexpression plasmids. The overexpression plasmid of N-Cadherin was purchased from Addgene, and RNAi plasmid was firstly designed using online design software, then constructed by company. After the overexpression and suppression plasmids were transfected into HEK293T cells, the immunofluorescence and western blotting were used to examine the expression of N-Cadherin.3. Screening for the most proper plasmid as the report gene for mouse in utero electroporation. pCAGGS-GFP, pEGFP-Nl and pCL-GFP were electroporated into cerebral cortex of E15mouse embryos at the same molar concentration. The positive mouse embryos were collected, observed and imaged using Stereo Fluorescence Microscope after24h electroporation. After selecting the most strong expression plasmid as the reporter gene, the most proper concentration was determined also.4. N-Cadherin was overexpressed and suppressed by mouse in utero electroporation technology. In Situ Hybridization and immunofluorescence were used to examine the result of abnormal expression. The change of neuron migration can be traced by GFP during the development of cerebral cortex.5. In order to study whether N-Cadherin plays an important role during stem cells differentiate into nerve cells, the P6adipose tissue-derived mesenchymal stem cells(ADMSCs) was induced into nerve-like cells. Subsequently, the gene expressions of N-Cadherin, β-cadherin, ADAM17and so on, were detected by RT-PCR, and the cellular morphology was observed.Results1. Mouse in utero electroporation technology was successfully constructed through testifying various transfection conditions. Then the GFP positive embryonic brains were sliced and observed. We found that there was no difference between the gene transfection area and normal area both in tissue morphology structure and the expression of a-SMA.2. The fluorescence intensity triggered by pCAGGS-GFP is the strongest among pCAGGS-GFP、pEGFP-N1and pCL-GFP. And the most suitable concentration as reporter gene is0.25μg/μL～0.5μg/μL.3. The overexpression plasmid of mouse N-cadherin, pEGFP-N-Cad, was transfected into HEK293T cells, and the immunofluorescence and western blotting results showed that pEGFP-N-Cadherin plasmid can ensure the overexpression of N-cadherin. Then, N-Cadherin interference plasmids, shRNA1and shRNA2were also examined using the same methods, and the results showed that both of them can suppress the expression of N-cadherin, but the former was better.4. Mouse in utero electroporation technology successfully ensures the suppression and overexpression in mouse cerebral cortex. And we found that when N-Cadherin was suppressed, neuron in cerebral cortex would stay in VZ and SVZ region, and could not migrate normally. Meanwhile, the overexpression of N-cadherin did not affect the neuron migration significantly.5. After ADSCs was induced into neuron-like cells in vitro, GFAP expression was positive. Meanwhile, the result of RT-PCR showed a significant increase in N-Cadherin expression and a prominent reduction in β-catenin, ADAM9and ADAM17.Conclusion1. Set up the mouse in utero electroporation technology successfully.2. Select an interference secquence, which can strongly suppress the expression of mouse N-cadherin, and construct the relative interference plasmid.3. Select a suitable reporter gene for in utero electroporation and its proper concentration is0.25μg/μL～0.5μg/μL.4. When N-Cadherin in cerebral cortex was knocked down, neuron migration was supressed, while the overexpression of N-Cadherin didn’t affect the neuron migraton significantly.5. After ADSCs was induced into neuron-like cells in vitro, GFAP expression was positive. Meanwhile, the result of RT-PCR showed a significant increase in N-Cadherin expression and a prominent reduction in β-catenin, ADAM9and ADAM17. These results suggest that N-Cadherin plays an important role during the differentiation process from stem cells to neuron cells.