The Role Of Foxg1 In The Postmitotic Specification Of Cortical Projection Neurons

The cerebral cortex is the most complex structure in the mammalian brain,consisting of hundreds of distinct neuronal subtypes.The neocortex has a typical six layer structure,which contains specific subtypes of neurons.Neurons of each layer have unique morphologies,electrophysiological features and connectivity.During the process of neural development,the precise generation,laminar position and connectivity of specific subtypes of neurons are at the core of cortical function and underlies its most sophisticated tasks.Callosal-projecting neurons,whose axons cross the midline in the brain,project interhemispherically,and function to integrate information between the two cerebral hemispheres,whereas Corticofugal neurons are located in the deep layers of the cortex(layers 5 and6)and mainly send their axons to subcortical structures,function to complete information feedback and behavior adjustment.A variety of neurodevelopmental disorders,such as schizophrenia,autism,and Rett syndrome,have been found to be associated with defects of cortical neuron subtypes,but the mechanisms underlying subtype specification still need to be further studied.During the process of cortical neurogenesis,the transcriptional factor Foxg1 continues to be expressed in the postmitotic neurons until adulthood,suggesting that it plays an important role in the specification of postmitotic neurons.Foxg1 mutations are associated with a variety of human neuropathy showing corpus callosum abnormalities and motor stereotyping,which is called the Foxg1syndrome.In this study,in order to explore the role of Foxg1 in the specification of postmitotic neurons,we use the conditional knockout strategy to delete Foxg1 in the postmitotic neurons.This study will put insigt to understanding the pathogenesis of Foxg1 syndrome.We first examined the effects of Foxg1 deletion on the development of three kinds of neurons.Compared with the control mouse,Satb2~+CPN was sharply reduced in mutant mice,Ctip2 and Tbr1were continuously expressed in CFuPN which were usually expressed in SCPN and CThPN respectively,unable to form a layer specific expression pattern.The numbers of Ctip2~+cells and Tbr1~+cells are increased,and both the proportion of Ctip2~+Tbr1~+cells among Tbr1~+cells or Ctip2~+cells increased significantly.Secondly,we performed birthdating test by intraperitoneal Brdu injection at E12.5,E13.5 and E15.5,the time window when CThPN,SCPN and CPN generated,respectively.We found that the specification of CFuPNs were abnormal,CFuPNs continued express high level of Ctip2 and Fezf2,can’t differentiate into Ctip2~+SCPN and Tbr1~+CThPN.The Brdu~+neurons that born in E15.5 were stacked beneath the CP,unable to migrate into CP,expressing Brn2 normally and expressing Satb2weakly.Then we also detected the feedback effect of Foxg1 CKO on the precursor cells,the results showed that both E12.5 and E15.5 precursor cell banks and their proliferation and division were not significantly changed.Then we detected the effects of the deletion of Foxg1 on the projection of cortical neurons through the Ai9-RFP reporter gene.The results showed that the axons of CPN projecting to the contralateral hemisphere were missing,the projection fibers of the RFP~+deep neurons were abnormal in the ventral telencephalon,and the guidance of L1~+axon was abnormal.Finally,we detected the regulatory molecules that might interact with Foxg1.The results showed that the expression of Sox5 at protein level and mRNA level both decreased significantly.In situ hybridization showed that the expression of Sox4 and Sox11 in CP and proliferating areas was obviously down regulated.The results showed that after the deletion of Foxg1,the number of CPN decreased sharply and the specification of CPN and CFuPN was abnormal.The precursor pool and its proliferation and division ability did not change,that is to say,the abnormality of specification caused the severe reduction of CPN.The axons of CPN projecting to the contralateral hemisphere developed defective,the subcortical projection fibers were reduced,and the distribution of axons in the ventral telencephalon were disorderly,indicating that Foxg1 is necessary for the correct establishment of the cortical neural circuit.The expression of Sox family factor(Sox5,Sox4,Sox11)is reduced,suggesting that Foxg1 may interact with the Sox family to regulate the specification of postmitotic neurons.Our study are helpful to understand the mechanisms of Foxg1 syndrome and the role of Foxg1 in the development of cortical neurons.