Purified rat E17 CSF directly stimulated Igf1R mediated signaling activity, reflected by Igf1Rβ phosphorylation as well as phosphorylation of Akt and MAPK (Figure 3G), two downstream targets of Igf signaling as well as other growth factors that may be present in CSF. Igf2 treatment by itself induced Igf signaling similar to embryonic CSF (Figure 3G). Igf2 binding to progenitors, the localization of the Igf1R, its phosphorylation, as well as the phosphorylation of its downstream targets Akt and MAPK in response to CSF, strongly suggest that the CSF is a primary source of

LDK378 nmr Igf ligands for cerebral cortical neuroepithelial cells, although additional sources cannot be completely excluded. We next tested whether Igf2 supports progenitor proliferation in a cerebral cortical explant system. In this system, rat embryonic cortex dissected from the lateral pallium is placed on polycarbonate membranes and floated on defined media (Figure 3H). We found that Igf2 added to neurobasal medium (NBM) with 20% artificial CSF (ACSF) stimulated the proliferation buy Alectinib of progenitor cells marked by phospho-Vimentin 4A4 in rat cortical explants (Figure 3I; Noctor et al., 2002). In addition, Igf2 treatment alone maintained GLAST-positive neurospheres, an in vitro model of neural stem cells,

even in the absence of Fgf2 (fibroblast growth factor 2) and Egf (epidermal growth factor) (Figure 3J; Vescovi et al., 1993). Finally, pharmacologic activation Ergoloid of the signaling pathway with insulin demonstrated that activation of Igf signaling by ligands other than Igf2 is sufficient to stimulate proliferation (PH3-positive cells/100 μm VZ ± SEM in E16 rat explant: control mean, 5.6 ± 0.7; insulin (10 μg/ml) mean, 11.2 ± 0.4; Mann-Whitney, p < 0.05; n = 6). Therefore, Igf signaling modulates proliferation of isolated cortical precursors or those maintained in their pallial environment in vitro. Since the CSF is a complex fluid

containing many factors including Igf binding proteins that may modulate Igf2 bioavailability and signaling (Figures 4A and 4B; Table S1; Clemmons, 1997 and Zappaterra et al., 2007), we tested whether native CSF alone could support cortical tissue growth. We used a heterochronic “mix-and-match” approach for exposing cortical tissue to CSF collected at different ages. E16 rat cortical explants with intact meninges and vasculature cultured with 100% E17 rat CSF for 24 hr, without any additional exogenous media or factors, retained remarkable tissue architecture, cell viability, and proliferation, approximating in vivo E17 rat cortex (Figure 4C). In contrast, E16 explants cultured with 100% artificial CSF failed to thrive, had decreased mitotic activity, disorganized neuronal morphology, and increased cell death (Figures 4C, S2A, and S2B). Filtration analysis of E17 CSF showed that the sizes of CSF factors that support stem cells likely range from 10 kDa–100 kDa, suggesting that they are proteins (Table S2 and data not shown).