By comparing rat CSF from several ages, we determined that the effects of CSF on survival and proliferation are strikingly age dependent and mimicked the temporal profile of CSF-Igf2 expression (Figure 3C). E17 CSF (near the middle of neurogenesis) maintained the healthiest explants and produced the maximal increase in the frequency of PH3-labeled proliferating cells in E16 cortical explants compared to explants cultured with E13 (early in neurogenesis), P6, or adult CSF
(Figures 4D, 4E, S2C, and data not shown). Many mitotic cells were identified as proliferating neuroepithelial progenitor cells by their immunoreactivity for phospho-Vimentin (4A4; Figures 4F and S2C). In contrast, no differences were Angiogenesis inhibitor seen in Tbr2-positive basal progenitors, which do not contact the CSF directly (data not shown). Together, these data suggest that age-dependent differences in CSF signals are both supportive selleck kinase inhibitor and instructive for neuroepithelial
precursor proliferation in the developing cortex. The CSF effects may be specific to neuroepithelial progenitors, which contact the ventricle through the apical complex, without affecting the intermediate progenitors of the SVZ. We tested directly whether CSF-borne Igf2 was necessary to explain the effects of age-specific CSF on rat cortical explants. The frequency of proliferating cells declined in explants grown in E17 CSF in the presence of Igf2 neutralizing antibodies (Igf2 Nab; Figure 4G). Igf2 neutralization with Igf2 NAb did not interfere with
Igf1 levels in CSF compared to control as assayed by ELISA (data not shown). While Igf signaling is known to promote neuronal survival (Popken et al., 2004), we did not observe differences in ventricular progenitor cell survival in these explant experiments (data not shown), suggesting that Igf actions on neural cell survival likely depends on the cell type, developmental stage, and microenvironment. These data confirm the important role for CSF borne Igf2 in regulating cerebral cortical progenitor cells but do not rule out roles of other CSF borne factors as well. Neural stem cells ADAMTS5 cultured as neurospheres confirmed the age-dependent capacity of CSF to maintain neural stem cells (Reynolds and Weiss, 1996) and provided additional evidence suggesting that Igf2-mediated signaling is an essential determinant of CSF activity on neural stem cells. CSF from any age supported the proliferation and maintenance of isolated cortical stem cells cultured as primary or secondary neurospheres (Figure 4H and data not shown; Vescovi et al., 1993). However, E17 CSF was maximally effective in generating increased numbers of neurospheres, larger neurospheres, and maintained neurospheres even in long-term cultures for up to 44 days in vitro (Figures 4H, S2D–S2G, and data not shown).