Summary The Wnt / β-catenin pathway directs neuronal differentiation of cortical neural precursor cells

Neurons and glia in the central nervous system originate from common precursor cells (neural precursor cells, or NPCs) that proliferate in the ventricular zone (VZ) of the fetal brain and spinal cord (Temple, 2001). In the developing mouse brain, NPCs give rise to neurons mostly between embryonic day (E) 10 and E17, and to astroglia after birth (Qian et al., 2000). The balance between proliferation and differentiation of NPCs is essential in determining the size of each region within the brain. This fate decision is made by a complex interplay between extrinsic signals and intrinsic genetic mechanisms. Growth factors such as fibroblast growth factor (Fgf) 2 and epidermal growth factor (Egf), as well as activation of the transmembrane receptor Notch, inhibit neuronal differentiation and promote the self-renewal capacity of NPCs (Johe et al., 1996; Panchision and McKay, 2002; Temple and Qian, 1996). Neuronal differentiation involves the proneural basic helixloop-helix (bHLH) transcription factors. For example, the bHLH proteins neurogenin (Ngn) 1 and Ngn2 are essential for neurogenesis in the neocortex (Schuurmans and Guillemot, 2002). The Notch signaling inhibits neuronal differentiation through a mechanism mediated by the bHLH proteins Hes1 and Hes5, which inhibit neurogenesis by antagonizing the proneural bHLH proteins (Ohtsuka et al., 2001). However, relatively little is known about the extrinsic cues that ‘trigger’ neuronal differentiation during cortical development. Previous studies have suggested that platelet-derived growth factor (Pdgf) cause an increase in neuronal cell number in cortical cultures, but this effect was shown to be the result of selective expansion of immature neurons (neuronal progenitors), rather than from instruction of neuronal fate (Erlandsson et al., 2001; Johe et al., 1996; Williams et al., 1997). Although erythropoietin appears to induce de novo neurogenesis in the adult brain in response to hypoxic insults (Shingo et al., 2001), its involvement in embryonic neurogenesis has not been demonstrated. Wnt genes encode secreted factors that regulate various cell fate decisions depending on the cellular context. In mammals, 19 Wnt genes have been identified to date, and several of these (such as Wnt7a, Wnt7b, Wnt2b and Wnt8b) are expressed during cortical development in complex spatiotemporal patterns (Fougerousse et al., 2000; Grove et al., 1998; Kim et al., 2001a; Lee et al., 2000). Wnt proteins signal through a receptor complex composed of members of the Frizzled (Fz) and low-density lipoprotein receptor-related protein (Lrp) families, and activate a number of intracellular signaling pathways including the β-catenin/TCF pathway (known as the canonical Wnt pathway) (Brantjes et al., 2002; Wodarz and Neural precursor cells (NPCs) have the ability to self-renew and to give rise to neuronal and glial lineages. The fate decision of NPCs between proliferation and differentiation determines the number of differentiated cells and the size of each region of the brain. However, the signals that regulate the timing of neuronal differentiation remain unclear. Here, we show that Wnt signaling inhibits the selfrenewal capacity of mouse cortical NPCs, and instructively promotes their neuronal differentiation. Overexpression of Wnt7a or of a stabilized form of β-catenin in mouse cortical NPC cultures induced neuronal differentiation even in the presence of Fgf2, a self-renewal-promoting factor in this system. Moreover, blockade of Wnt signaling led to inhibition of neuronal differentiation of cortical NPCs in vitro and in the developing mouse neocortex. Furthermore, the β-catenin/TCF complex appears to directly regulate the promoter of neurogenin 1, a gene implicated in cortical neuronal differentiation. Importantly, stabilized β-catenin did not induce neuronal differentiation of cortical NPCs at earlier developmental stages, consistent with previous reports indicating self-renewal-promoting functions of Wnts in early NPCs. These findings may reveal broader and stage-specific physiological roles of Wnt signaling during neural development.