olution, we MedChemExpress GW 501516 quantified the percentage of retrovirus-labeled cells that expressed either DCX or incorporated BrdU. Compared with control retrovirus-infected cells, neither shmiR-195 retrovirus, nor miR-195-sponge virus had significant effect on aNSCs BrdU incorporation. On the other hand, although shmiR-195 retrovirus did not alter neuronal differentiation, a higher percentage of miR-195-sponge retrovirus-infected cells were DCXpositive. Therefore, inhibition of endogenous miR-195 levels promotes neuronal differentiation of aNSCs. These results suggest that the level of endogenous miR-195 in aNSCs is an important regulatory mechanism for neuronal 
differentiation. MBD1 is a Target of miR-195 in aNSCs miRNAs are known to function at least in part by repressing protein translation of their mRNA targets. We therefore searched for potential mRNA targets of miR-195 by cross-referencing two widely used miRNA target prediction programs, TargetScan and miRanda. Several miR-195 targets were predicted by both software programs, including Mbd1, Bdnf, Wee1, Mib1, and CyclinD1, and we have validated that miR-195 can indeed repress Renilla luciferase expression via the 39 UTRs of some of these predicted targets. Surprisingly, we found that MBD1 was among the list of potential targets of miR-195 predicted by both programs, and the Mbd1 39 UTR contained a classic miR-195 target seed sequence, suggesting that MBD1 and miR-195 might form an important regulatory loop in aNSC regulation. Indeed, high levels of miR-195 could repress the expression of R-Luc through the Mbd1 39 UTR, whereas anti-195 enhanced R-Luc expression. In addition, retro-shmiR-195 could also repress the expression of R-Luc through the Mbd1 39 UTR, whereas retromiR-195-sponge also enhanced R-Luc expression, further supporting a regulatory role for miR-195 on MBD1 expression. We then mutated the miR-195 seed sequence within the Mbd1 39 UTR. As predicted by our previous data, the mutation abolished both miR-195mediated suppression and anti-195mediated enhancement of R-Luc activities. These results strongly suggest that miR-195 directly represses MBD1 expression through the predicted target seed sequence located in the Mbd1 39 UTR. Next we determined the effect of miR-195 on endogenous MBD1 expression in aNSCs. We found that miR-195transfected aNSCs showed a reduction in MBD1 protein levels, whereas anti-195 transfection rescued the levels of MBD1 expression in Mbd1 KO aNSCs. Taken together, these data argue that MBD1 is a direct target of miR-195, therefore MBD1 and miR-195 form a negative regulatory loop in aNSCs. miR-195 Regulates the Differentiation of aNSCs in vivo To assess the functions of miR-195 in aNSCs in vivo, we decided to use a retrovirus-based single cell genetics approach . We constructed a retroviral vector that expressed both short hairpin pre-miR-195 driven by a U6 promoter and GFP by a chicken actin promoter for our gain-of-function assay. We also created a miR-195-sponge to inhibit endogenous miR-195 for our loss-of-function assay. We confirmed that shmiR195virus-infected aNSCs exhibited increased miR-195 levels and reduced neuronal differentiation, whereas miR-195-sponge virus infected aNSCs enhanced neuronal differentiation DG-aNSCs were transfected with either synthetic miR-195 mimics or control miRNA mimics, and aNSC differentiation was analyzed using neuronal lineage marker Tuj1 and astrocyte lineage marker GFAP. DAPI, blue. DG-aNSCs transfected with miR-195 mimics