br Results br Discussion Notch signaling has
Discussion Notch signaling has been reported to play multiple roles during the CNS development. In progenitor cells, Notch signaling inhibits the differentiation of NSCs into INPs (Gao et al., 2009; Mizutani et al., 2007; Pierfelice et al., 2011). There are at least three types of neural progenitor antimalaria medication in embryonic neocortex, including NSCs in the VZ, INPs in the VZ, and INPs in the SVZ. While a majority of INPs are in the SVZ, a fraction of INPs coexists with NSCs in the VZ. The Notch pathway is differentially used by these different types of progenitor cells. NSCs in the VZ, which have self-renewal and multipotent differentiation characteristics, exhibit RBP-J dependence. INPs in the VZ, in contrast, display attenuated RBP-J activity and are predominantly neurogenic. In addition, INPs in the SVZ signal to adjacent cells by expressing Notch ligands. A major unanswered question about the role of Notch signaling in NSCs-INPs is the downstream molecules controlling NSC differentiation. In this study, we have identified miR-342-5p as one of the downstream miRNAs of Notch signaling during NSC differentiation based on the following findings. The expression of miR-342-5p is inversely correlated with Notch signaling in the tissues and cells examined. The in situ hybridization results of miR-342-5p showed that its expression gradually elevated from the VZ to the SVZ, which was complementary to the pattern of attenuated RBP-J-dependent Notch signaling from the VZ to the SVZ (Mizutani et al., 2007). In addition, modulations of Notch signaling led to changes in the expression of miR-342-5p. Blocking Notch signaling genetically or pharmaceutically in NS/PCs induced increased miR342-5p expression, whereas the activation of Notch signaling by NSC-specific NICD overexpression led to decreased miR342-5p expression. Since INPs increased in Notch blockade spheres (Gao et al., 2009), and miR-342-5p expression was upregulated in INPs compared with NSCs, it was difficult to judge whether the increase of miR-342-5p expression in Rbp-j KO spheres was only the result of INP enrichment or that miR-342-5p was directly regulated by Notch signaling, which then regulated the differentiation of NS/PCs in turn. To clarify this, we tested the expression profiles of miR-342-5p and INP marker Tα1 in neurospheres treated with GSI for different periods of time, and found that miR-342-5p was upregulated prior to the increase of Tα1, indicating that the increase of miR-342-5p expression after Notch blockade was not only a consequence of INP enrichment. Therefore, Notch signaling might directly regulate miR-342-5p expression. Although the intronic miRNA expression often occurs independent of host gene transcription, previous research has shown that the methylation of the Evl promoter region silenced the expression of both Evl and miR-342 together (Grady et al., 2008), indicating that the expression of miR-342 might be regulated by the same promoter of its host gene. Therefore, we investigated the regulation of the Evl promoter by Notch signaling. By using reporter assays, we have demonstrated that Notch signaling could directly regulate the transactivation of the promoter of Evl that harbors miR-342-5p and miR-342-3p genes. Because NICD repressed a truncated reporter in which the putative RBP-J-binding site was deleted, we propose that Notch signaling likely modulates the Evl promoter through both RBP-J and HES proteins. Indeed, ChIP assays have shown that HES1 and HES5 could bind to multiple N boxes and at least one E box in the Evl promoter. However, further investigations on NSC-specifically NICD-overexpressed mice showed that Hes5 expression was elevated, whereas Hes1 expression showed different changes in the cortex and GE, and miR-342-5p and Evl expressions were decreased on Notch activation. In addition, the knockdown of Hes5 by siRNA transfection into NS/PCs resulted in upregulated miR-342-5p and Evl expressions. Therefore, we thought that HES5 might directly regulate miR-342-5p expression through the Evl promoter instead of HES1 in vivo.