Effects Of RNA Interference And Point Mutations Of Fyn On Neuronal Migration And Its Molecular Mechanism

The formation of the six-layer structure of mammalian cerebral cortex depends on the correct neuronal migration,which is regulated by various molecules.Fyn is a non-receptor tyrosine kinase.The cerebral cortex has defects in Fyn knockout mice: the number of neurons decreased in the superficial layers and late-born neurons mislocate in the deeper layers of cortex.Fyn plays an important role in neuronal migration.However,the molecular mechanism remains unclear.In order to study the effects of Fyn on neuronal migration and the molecular mechanisms,we explored the role of Fyn in neuronal migration and its regulation effects in combination with the results of our previous studies.In utero electroporation was performed combined with RNA interference(RNAi)and point mutations,to explore the effects of Fyn knockdown and mutations of N-terminal modification sites on neuronal migration and morphology.Western blot and immunofluorescence staining were performed to detect signaling molecules that may act downstream of Fyn.Then,different point mutant vectors of these molecules were constructed and were co-transfected with the expression and RNAi vector of Fyn,to explore Fyn signaling pathways in regulating neuronal migration.The results are as follows:1.The combination of in utero electroporation and RNAi revealed that decrease of Fyn expression inhibited migration of early-and late-born neurons.In utero electroporation was performed at embryonic day 15.5(E15.5)to label the late-born neurons,and mice were scarified at postanal day 1(P1).Results showed that most neurons reached the maginal zone(MZ)in control group,whereas the majority of neurons transfected with RNAi vectors accumulated in the intermediate zone(IZ).Neurons were also arrested in the IZ and cortical plate(CP)at P7,indicating that knockdown of Fyn inhibited rather than delayed neuronal migration.The early-born neurons were transfected at E12.5 and migrated into the CP in the control at E16.5,while neurons were clustered in the IZ in RNAi group.Knockdown of Fyn impaired the formation of leading processes of migrating neurons in early phase of migration.Percentage of bipolar neurons was lower at the boundary between the IZ and CP.Most neurons transfected with Fyn RNAi vector were round without leading processes.Fyn-deficent neurons had longer leading process compared with the control when they migrated into the CP.The leading processes of some neurons were inverted.Futhermore,Fyn knockdown increased the lenghth of leading processes and reduced the branching number of neurons when they contacted the MZ.2.The eukaryotic expression vectors with different point mutants in N-terminal of Fyn were constructed,including myristoylation site,palmitoylation site and the methylation modification sites.The results of in utero electroporation showed that overexpression of Fyn-WT inhibited neuronal migration,and the neurons formed cluster aggregations in the ventricular zone(VZ)and IZ.Only a few cells reached the MZ.The majority of neurons transfected with the mutants contacted the MZ,which is similar with the control group,but had significant differences with Fyn-WT transfection group.The percentage of the neurons forming aggregations decreased,indicating that these modification sites play important roles for Fyn in regulating neuronal migration.3.The results of western blot and immunofluorescence showed that the expression of Fyn induced phosphorylation of FAK-Y925 and PI3K-p85.In utero electroporation showed that FAK-Y925F(non-phosphorylation form)rescued the defects of neuronal migration and aggregation caused by overexpression of Fyn-WT.Rho protein GTPase is important for neuronal migration.In utero electroporation showed that the distribution of transfected neurons in Rac1-DN+Fyn-WT and Rac1-CA+shFyn groups was significantly different compared with that of Fyn-WT transfected group and control,indicating that Rac1 is involved in neuronal migration regulated by Fyn,but it could rescue neuronal migration defects caused by Fyn only to a certain extent.In RhoA-DN+Fyn-WT and RhoA-CA+shFyn transfected groups,the distribution of the transfected neurons was similar to that in the control group.The percentage of neurons forming aggregations was lower in RhoA-DN+Fyn-WT group compared with that in Fyn-WT transfected group.In shFyn+RhoA-CA transfected group,most neurons transformed into the typical bipolar morphology in the IZ-CP border,and the length of leading processes of neurons in the CP was as same as that in the control.However,neurons reached the MZ also had longer leading processes and less branches compared with the control,suggested that RhoA may be a key element for Fyn in regulating neuronal migration.In conclusion,Fyn is critical for migration of both early-born and late-born neurons,and has a great influence on neuronal morphology.The defect of Fyn expression leads to abnormal morphology of leading processes.FAK and RhoA may be the key molecules in neuronal migration regulated by Fyn and Rac1 is also involved in Fyn signal.