Roles Of Brn4 In Rat Radial Glial Cells Differentiation And Astrocytes Reprogramming

Part I Role of Brn4 in rat radial glial cells differentiationBrain transcription factor 4(Brain 4,Brn4),also known as POU3F4,belongs to the POU protein family with Brn1 and Brn2.Previous studies have found that Brn4 can promote the differentiation of neural stem cells(NSCs)into neurons in the hippocampus of adult rats.In this process,the expression of Gli family zinc finger protein 1(Gli1)and C-terminal binding protein 2(Ct BP2)genes change significantly,suggesting that Gli1 and Ct BP2 genes may participate in the regulation of Brn4 on NSCs differentiation.Radial glial cells(RGCs)belong to NSCs in early mammalian development.Whether Brn4 can regulate the differentiation of RGCs in embryonic stage is still unclear.ObjectiveTo detect the role of Brn4 in the differentiation of RGCs derived from rat embryonic cortices and explore its related mechanisms.Methods1.RGCs were obtained from embryonic cortices of 14 d SD rats and amplified in vitro.The expression of specific markers of RGCs was detected by immunofluorescence assay to determine its purity;2.After being infected by Brn4 overexpression lentiviruses,RGCs were cultured in the differentiation medium for 7 d.The expression of markers of neurons or glial cells was detected by immunofluorescence assay.The expression of Gli1 and Ct BP2 was detected by Western blotting to clarify the effect of Brn4 on Gli1 and Ct BP2;3.Gli1 overexpression lentiviruses or its sh RNA interfering lentiviruses infected RGCs respectively,and the cells were induced to differentiate for 7 d.Cellular immunofluorescence assay was used to detect the expression of specific markers of neurons or glial cells to determine the effect of Gli1 on the differentiation of RGCs.In addition,the expression of Ct BP2 was detected by Western blotting to determine whether Gli1 could act on Ct BP2;4.Ct BP2 overexpression lentiviruses or its sh RNA interfering lentiviruses infected RGCs respectively,and the cells were subsequently induced to differentiate for 7 d.Then,the differentiation of RGCs was detected by cellular immunofluorescence assay,and the expression of Gli1 was detected by Western blotting to determine the effect of Ct BP2 on Gli1.Results1.The RGCs markers of Brain lipid binding protein(BLBP),Sex-determining region Y-box 2(Sox2),Vimentin and Nestin were expressed in a large number of cells obtained from 14 d rat embryonic cerebral cortices.A small number of cells expressed glial fibrillary acidic protein(GFAP).β-Tubulin III(Tuj1)and Microtubule associated protein 2(MAP2)were almost not expressed.These results suggested that the cultured cells were mainly RGCs with purity of about 90%,which could be used in subsequent experiments.2.After RGCs were infected with Brn4 overexpression lentiviruses and induced to differentiate,the proportion of Tuj1 and MAP2 positive cells increased,and the proportion of GFAP positive cells decreased.Meanwhile,the expression of Gli1 increased,while the expression of Ct BP2 decreased significantly.3.The percentage of Tuj1 or MAP2 positive cells in the Gli1 overexpression group increased,and the percentage of GFAP positive cells decreased significantly.When Gli1 interfering lentiviruses infected cells to reduce the expression of Gli1,the results were contrary.No matter increasing or decreasing the expressioin of Gli1,Ct BP2 expression had no significant difference compared with the respective control groups.4.The percentage of Tuj1 or MAP2 positive cells in the Ct BP2 overexpression group decreased,while the percentage of GFAP positive cells increased.However,when the expression of Ct BP2 was decreased by its interfering lentiviruses,the results were contrary.Neither increasing nor decreasing the expression of Ct BP2 could significantly change the expression of Gli1.Part II Role of Brn4 in rat astrocytes reprogrammingObjectiveThe purpose of this study is to verify whether Brn4 can be used as a reprogramming factor to promote astrocytes to transdifferentiate into neurons or dopaminergic neurons.Methods1.Astrocytes derived from cerebral cortices of neonatal 1 d SD rats were cultured in vitro.The expression of astrocyte markers GFAP and S100β was detected by immunofluorescence assay to determine the purity of the cultured cells.2.Astrocytes were infected with lentiviruses carrying human Achaete-scutehomolog 1(Ascl1)gene or human Brn 4 gene alone or in combination.After 28 days of culture with the induction medium,the expression of neuronal markers Tuj1 and MAP2 was detected by cellular immunofluorescence assay to determine whether Brn4 could promote astrocytes reprogramming to neurons.3.Rat astrocytes were infected with the lentiviruses carrying human Nuclear receptor-related factor 1(Nurr1)gene,the lentiviruses carrying human Ascl1 gene and the lentiviruses carrying human Brn4 gene according to different combinations(the Mock group,the negativecontrol group,the Ascl1+Nurr1 group and the Ascl1+Nurr1+Brn4 group).The expression of Tyrosine hydroxylase(TH)and Dopamine transporter(DAT)was detected by cellular immunofluorescence assay to determine whether Brn4 could promote astrocytes to transdifferentiate into dopaminergic neurons.Results1.The percentage of GFAP and S100β positive astrocytes obtained from the cerebral cortices of neonatal 1 day SD rats was more than 95%,which indicated that the purity of the obtained astrocytes was high and could be used in subsequent experiments.2.After the astrocytes were infected with Ascl1 overexpression lentiviruses alone,some of them could express Tuj1,but MAP2 could not be detected.Neither Tuj1 nor MAP2 could be detected in the astrocytes which were infected by Brn4 overexpression lentiviruses alone.However,Tuj1 and MAP2 could be detected in the astrocytes being co-infected with these two lentiviruses.3.After 28 days of induction,TH positive cells in the three-viruses infection group(Ascl1+Nurr1+ Brn4)were significantly more than those in the two-viruses infection group(Ascl1+Nurr1),and the TH positive cells had obvious protuberances.However,DAT positive cells were not detected in all groups.Conclusions1.Brn4 can promote the differentiation of RGCs into neurons.In this process,the expression of Gli1 increases and that of Ct BP2 decreases;2.Both increasing Gli1 expression and decreasing Ct BP2 expression can make RGCs tend to differentiate into neurons;3.Brn4 may promote the differentiation of RGCs into neurons by activating Gli1 and inhibiting Ct BP2,but there is no direct effect between the two proteins;4.Under the condition of combined use of transcription factor Ascl1,Brn4 can promote astrocytes reprogramming toward neurons,while Brn4 alone can not induce such transdifferentiation;5.Brn4 can promote astrocytes reprogramming into TH positive cells induced by Ascl1 and Nurr1,but the expression of DAT is negative,suggesting that the induced dopaminergic neurons are not mature.