Abstract
Neurosphere formation is commonly used as a surrogate for neural stem cell (NSC) function but the relationship between neurosphere-initiating cells (NICs) and NSCs remains unclear. We prospectively identified, and isolated by flow cytometry, adult mouse lateral ventricle subventricular zone (SVZ) NICs as Glast(mid)EGFR(high)PlexinB2(high)CD24(-/low)O4/PSA-NCAM(-/low)Ter119/CD45(-) (GEPCOT) cells. They were highly mitotic and short-lived in vivo based on fate-mapping with Ascl1(CreERT2) and Dlx1(CreERT2). In contrast, pre-GEPCOT cells were quiescent, expressed higher Glast, and lower EGFR and PlexinB2. Pre-GEPCOT cells could not form neurospheres but expressed the stem cell markers Slc1a3-CreER(T), GFAP-CreER(T2), Sox2(CreERT2), and Gli1(CreERT2) and were long-lived in vivo. While GEPCOT NICs were ablated by temozolomide, pre-GEPCOT cells survived and repopulated the SVZ. Conditional deletion of the Bmi-1 polycomb protein depleted pre-GEPCOT and GEPCOT cells, though pre-GEPCOT cells were more dependent upon Bmi-1 for Cdkn2a (p16(Ink4a)) repression. Our data distinguish quiescent NSCs from NICs and make it possible to study their properties in vivo.DOI: http://dx.doi.org/10.7554/eLife.02669.001.
Highlights
Neural stem cells (NSCs) reside in two regions of the adult mammalian forebrain: the subgranular zone in the dentate gyrus and the subventricular zone in the lateral wall of the lateral ventricle (SVZ)
We examined the localization of pre-GlastmidEGFRhighPlexinB2highCD24−/lowO4/PSA-NCAM−/lowTer119/ CD45− (GEPCOT) and GEPCOT cells in whole mount stains of the SVZ. pre-GEPCOTs were distinguished by GFAP expression, a marker of type B cells (Doetsch et al, 1999a) and GEPCOTs were distinguished by Epidermal Growth Factor Receptor (EGFR) expression, a marker of type C cells (Doetsch et al, 2002)
To test whether qNSCs depend upon Bmi-1 for their maintenance in vivo we examined the frequency of pre-GEPCOT cells in tamoxifen-treated Nestin-CreERT2; Bmi-1fl/fl mice and littermate controls. pre-GEPCOT cells were not depleted at 2 weeks or 6 months after tamoxifen treatment (Figure 7P)
Summary
Neural stem cells (NSCs) reside in two regions of the adult mammalian forebrain: the subgranular zone in the dentate gyrus and the subventricular zone in the lateral wall of the lateral ventricle (SVZ). SVZ NSCs persist throughout adult life (Maslov et al, 2004; Molofsky et al, 2006; Imayoshi et al, 2008), giving rise primarily to neurons in the olfactory bulb as well as some astrocytes in the olfactory bulb (Lois and Alvarez-Buylla, 1994; Lois et al, 1996; Doetsch et al, 1999b; Ahn and Joyner, 2005; Kuo et al, 2006; Lagace et al, 2007; Merkle et al, 2007; Imayoshi et al, 2008; Chen et al, 2009) and oligodendrocytes in the corpus callosum and cortex (Nait-Oumesmar et al, 1999; Menn et al, 2006).
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