4% ± 4.1%; Figure 5K). The observation that most p-Axin+ cells in the upper VZ and lower SVZ were IPs (Figure 4D) suggests that p-Axin+ IPs exit cell cycling at the G1 phase (Dehay and Kennedy, 2007). Therefore, we conclude that the Cdk5-dependent phosphorylation of Axin at Thr485 maintains the nuclear accumulation of Axin in IPs and promotes neuronal differentiation. How does cytoplasmic Axin amplify IPs? The size of the IP pool is negatively regulated by multiple pathways including Wnt (Munji et al., 2011), Notch (Mizutani et al., 2007), and FGF (Kang et al., 2009) signaling; each of these pathways can be modulated by a key

regulator, GSK-3 (Kim et al., 2009b). Axin colocalized and interacted with GSK-3β in the cytoplasmic compartment of NPCs at E13.5 (Figures 6A, 6B, and S6A). Notably, the re-expression of an Axin point mutant that failed to bind GSK-3β (GIDm) (Fang et al., 2011) in Axin-knockdown cortices abolished the ability of cytoplasmic Axin www.selleckchem.com/products/Gefitinib.html to enhance NPC amplification, resulting in early neuronal differentiation (Figures 6C–6F, S6B, and S6C).

Therefore, our findings suggest that cytoplasmic Axin expands the NPC (i.e., IP) population in a GSK-3β-dependent manner. To confirm this finding, we utilized small peptides, FRATtide (Bax et al., 2001) and GID peptide (Hedgepeth et al., 1997), which can enhance and block Axin-GSK-3β interaction, respectively (Figures S6D and S6E), and examined their effects on the regulation of the fate of NPCs. FRATtide expression led to the enlargement of the NPC pool (Figures 6G and 6H) and promoted the generation of IPs from RGs (Figures 6I, Epacadostat 6J, S6F, and S6G); meanwhile, GID peptide depleted the NPC pool (Figures 6G–6H) and promoted the direct neuronal differentiation of RGs (Figures 6G–6J, S6F, and S6G). Thus, Axin in the cytoplasm of RGs enhances IP amplification via a mechanism dependent on its interaction with GSK-3β. Next, we investigated how nuclear Axin promotes neuronal differentiation. Axin was progressively

enriched in the nuclei tuclazepam of NPCs upon neuronal differentiation (Figures 3C and 7A). The neuronal differentiation of progenitors was marked by the prominent upregulation of proneural target genes of β-catenin (including Ngn1 and NeuroD1) (Hirabayashi et al., 2004 and Kuwabara et al., 2009) together with reduced levels of antineural β-catenin targets (e.g., Cyclin D1 and N-myc) (Clevers, 2006 and Kuwahara et al., 2010) (Figure S7A). Axin interacted with β-catenin in the nuclear compartments of differentiating NPCs (Figure 7A). Although the nuclear accumulation of β-catenin is important for its transcriptional activity, the nuclear accumulation of Axin and hence its interaction with β-catenin were not prerequisites for the nuclear localization of β-catenin; this is because Axin level was significantly reduced (Figure 4B), whereas β-catenin level remained unchanged (Figures S7B and S7C), in the nuclei of cdk5−/− cells.