8 ± 0.7-fold) and decreased significantly in the cytoplasm (by 60.4% ± 6.2%) in differentiating NPCs (Figure 3C). Therefore, these findings indicate that Axin accumulates in the nuclei of NPCs in response to differentiation signals. The nucleocytoplasmic shuttling of Axin is tightly controlled by the nuclear localization signal (NLS) and nuclear export signal (NES) of the protein (Cong and Varmus, 2004). To elucidate the specific roles of cytoplasmic and nuclear Axin, we generated two point mutants of Axin, allowing the protein to be expressed specifically in the cytoplasm (Axin-NLSm) or nucleus (Axin-NESm) (Figure 3D). Like wild-type Axin, the overexpression

of cytoplasmic Axin (Axin-NLSm) at E13.5 increased FK228 mw the proportion of GFP+ cells in the VZ/SVZ at E15.5 (Figures 3E and 3F), suggesting that cytoplasmic Axin enhances NPC expansion. Furthermore, the re-expression of Axin-NLSm in Axin-knockdown NPCs also led to NPC BKM120 pool expansion (Figures 3G–3L) specifically through the enlargement of the IP population (Figures 3H, 3J, and 3L). In contrast, the expression of nuclear Axin (Axin-NESm) (Figures 3E and 3F) or re-expression of the protein in Axin-knockdown NPCs depleted the GFP+ NPCs in the VZ/SVZ and promoted the differentiation of NPCs into neurons (Figures 3G–3L). Together with the nuclear accumulation of Axin in cultured NPCs upon differentiation (Figures 3A–3C), these findings strongly suggest

Rolziracetam that Axin in different subcellular compartments of NPCs specifically regulates the amplification and differentiation of NPCs; cytoplasmic Axin in RGs enhances IP amplification,

whereas Axin in the nucleus of IPs promotes neuronal differentiation of IPs. Next, we investigated the molecular mechanism that controls the trafficking of Axin between the cytoplasm and nucleus. Treating RGs with leptomycin B led to the nuclear accumulation of Axin (Cong and Varmus, 2004) (Figure S4A), suggesting that the nuclear enrichment of Axin is regulated by nuclear export. It was noted that the Cdk5-dependent phosphorylation site (Thr485) is located close to the NES of Axin (amino acids 413–423) (Fang et al., 2011) (Figure 4A). Although Axin phosphorylation at Thr485 (p-Axin) could be detected in wild-type mouse neocortices at E13.5, this specific phosphorylation was markedly reduced in cdk5−/− littermates (by 45.5% ± 4.3%; Figures 4B, S4B, and S4C), indicating that Cdk5 is a major kinase that phosphorylates Axin during neurogenesis in vivo. Importantly, the nuclear level of Axin was reduced in cdk5−/− neocortices (by 68.2% ± 5.1%) accompanied by an increased level of cytoplasmic Axin (2.0 ± 0.3-fold; Figure 4B). These results suggest that Cdk5-dependent Axin phosphorylation is critical for controlling the nuclear localization of Axin in the embryonic cerebral cortex. To explore the role of Cdk5-mediated Axin phosphorylation, we examined how Axin phosphorylation is regulated in NPCs.