Effect Of Shh On Neuronal Migration And Axon Growth In The Central Nervous System

Background The brain and spinal cord are important components of the central nervous system,and their development is strictly regulated.During the development of the spinal cord and cerebral cortex,the morphology and migration of neurons play an important role.To play their roles,they must be properly regulated.Studies have shown that Shh can promote the proliferation of neural precursor cells and then affect the development of the spinal cord and cerebral cortex.However,it is unclear whether Shh affects neuronal migration and neurite outgrowth.In this experiment,Shh was overexpressed to study the function of Shh on neuronal development and migration in the spinal cord and cerebral cortex.Objective1.To establish of tissue slice culture technique following mouse embryonic spinal cord and cerebral cortex electroporation in vitro2.To study the effect of Shh on neuronal axon growth and migration during spinal cord development3.To study the effect of Shh overexpression on neuronal migration in the cerebral cortex.Methods1.E12 and E13.5 embryonic mice were taken,and pCAGGS-GFP plasmid was injected into the spinal cavity and lateral ventricle in vitro,and then electroporated,sliced,and cultured.After 48 h,fixed staining was performed to observe the morphological structure and migration of individual neurons in tissue slices.2.The Shh overexpression vector was transfected into SH-SY5 Y cells,E12 spinalcord slices,and E13.5 cerebral cortex,respectively,and it was examined whether the overexpression vector can work at the three levels: cell,tissue,and in vivo.3.The GFP and Shh plasmids were transferred into E12 mouse embryo spinal cord by electroporation,sliced and cultured for 48 h,and the morphology of neurons was observed by staining.The GFP and Shh plasmids were transferred into the cerebral cortex of E13.5 mouse embryos using in utero electroporation.Positive embryos were collected at E16.5,sliced and stained to study whether Shh overexpression affects neuronal migration and morphology.4.The GFP and Shh plasmids were transfected into SH-SY5 Y cells,and the protrusions of GFP-positive cells were observed.Then the proteins were extracted,and the expression of related proteins was detected by Western blot.Results1.The transfected GFP-positive cells can be observed in the cultured spinal cord slices and brain slices,indicating that this method can successfully transfer exogenous genes into the neural precursor cells of the spinal cord and cerebral cortex.2.According to the Western blot results of SH-SY5 Y cells and the results of immunofluorescence staining of spinal cord slices and cerebral cortex showed that Shh overexpression vector can overexpress Shh in cell,tissue,and in vivo.3.Shh was overexpressed in spinal cord slices after 48 h of culture,and it was found that Shh overexpression inhibited the outgrowth of neuronal processes by MAP2 staining and measuring the length of the processes.4.The proportion of GFP positive cells in each zone,MAP2 and DCX staining showed that Shh overexpression inhibits neuronal migration of E13.5 cerebral cortex.5.Shh overexpression vector transfected SH-SY5 Y cells and found that most of the cell protrusions shortened or became spherical.Western blot results showed that the expression levels of CyclinD1,c-Myc and PCNA in the experimental group increased significantly,while the expression levels of N-cadherin decreased.Conclusion1.Combining mouse embryonic spinal cord and cerebral cortex in vitro electroporation and tissue slice culture provides an effective method for studying mouse spinal cord and cerebral cortex development.2.Shh overexpression affects the growth of spinal neuron processes.3.Shh overexpression inhibits the development of the cerebral cortex by inhibiting the migration of neurons.