Self-renewing and differentiating properties of cortical neural stem cells are selectively regulated by basic fibroblast growth factor (FGF) signaling via specific FGF receptors.

Developmental processes mediating the initiation of lineage commitment from self-renewing neural stem cells (NSCs) remain mostly unclear because of the persisting ambiguity in identifying true NSCs from proliferative lineage-restricted progenitors (LRPs), which are directly or indirectly derived from NSCs. Our multilineage immunohistochemical analyses of early embryonic rat telencephalon at the onset of neurogenesis revealed clear dorsoventral gradients in the emergence of two types of neuronal progenitors (NPs) from multilineage-negative NSCs. Enumeration of NSCs using comprehensive flow cytometric analysis demonstrated that their precipitous decline in vivo involved both active differentiation into NPs and an increased propensity toward apoptosis. Both processes paralleled the dorsoventral changes in fibroblast growth factor receptor (FGFR) expressions. NSCs residing in the dorsal telencephalon coexpressed FGFR1 and FGFR3, whereas those residing in the ventral telencephalon also expressed FGFR2. NSCs exposed to basic fibroblast growth factor (bFGF) in vitro generated four stereotypical clonal expansion states: efficiently self-renewing, inefficiently self-renewing limited by apoptosis, exclusively neurogenic, and multipotential, generating up to five types of LRPs. The plasticity among these expansion states depended on ambient [bFGF], telencephalic developmental stage, and differential activation/inactivation of specific FGFRs. Coactivation of FGFR1 and FGFR3 promoted symmetrical divisions of NSCs (self-renewal), whereas inactivation of either triggered asymmetrical divisions and neurogenesis from these cells. Developmental upregulation of FGFR2 expression correlated with a shift of NSCs into a multipotential state or apoptosis. These results provide new insights regarding the roles of FGFRs in diversification of NSC properties and initiation of neural lineage-restricted differentiation.

Canonical transient receptor potential 1 plays a role in basic fibroblast growth factor (bFGF)/FGF receptor-1-induced Ca2+ entry and embryonic rat neural stem cell proliferation.

Basic fibroblast growth factor (bFGF) and its major receptor FGF receptor-1 (FGFR-1) play an important role in the development of the cortex. The mechanisms underlying the mitogenic role of bFGF/FGFR-1 signaling have not been elucidated. Intracellular Ca2+ concentrations ([Ca2+]i) in proliferating cortical neuroepithelial cells are markedly dependent on Ca2+ entry (Maric et al., 2000a). The absence of voltage-dependent Ca2+ entry channels, which emerge later, indicates that other membrane mechanisms regulate [Ca2+]i during proliferation. Canonical transient receptor potential (TRPC) family channels are candidates because they are voltage independent and are expressed during CNS development (Strübing et al., 2003). Here, we investigated the involvement of TRPC1 in bFGF-mediated Ca2+ entry and proliferation of embryonic rat neural stem cells (NSCs). Both TRPC1 and FGFR-1 are expressed in the embryonic rat telencephalon and coimmunoprecipitate. Quantitative fluorescence-activated cell sorting analyses of phenotyped telencephalic dissociates show that approximately 80% of NSCs are TRPC1+, proliferating, and express FGFR-1. Like NSCs profiled ex vivo, NSC-derived progeny proliferating in vitro coexpress TRPC1 and FGFR1. Antisense knock-down of TRPC1 significantly decreases bFGF-mediated proliferation of NSC progeny, reduces the Ca2+ entry component of the Cai2+ response to bFGF without affecting Ca2+ release from intracellular stores or 1-oleoyl-2-acetyl-sn-glycerol-induced Ca2+ entry, and significantly blocks an inward cation current evoked by bFGF in proliferating NSCs. Both Ca2+ influx evoked by bFGF and NSC proliferation are attenuated by Gd3+ and SKF96365 two antagonists of agonist-stimulated Ca2+ entry. Together, these results show that TRPC1 contributes to bFGF/FGFR-1-induced Ca2+ influx, which is involved in self-renewal of embryonic rat NSCs.

Prospective cell sorting of embryonic rat neural stem cells and neuronal and glial progenitors reveals selective effects of basic fibroblast growth factor and epidermal growth factor on self-renewal and differentiation.

We directly isolated neural stem cells and lineage-restricted neuronal and glial progenitors from the embryonic rat telencephalon using a novel strategy of surface labeling and fluorescence-activated cell sorting. Neural stem cells, which did not express surface epitopes characteristic of differentiation or apoptosis, were sorted by negative selection. These cells predominantly expressed fibroblast growth factor receptor type 1 (FGFR-1), and a minority exhibited basic fibroblast growth factor (bFGF), whereas few expressed epidermal growth factor receptor (EGFR) or EGF. Clonal analyses revealed that these cells primarily self-renewed without differentiating in bFGF-containing medium, whereas few survived or expanded in EGF-containing medium. Culturing of neural stem cells in bFGF- and EGF-containing medium permitted both self-renewal and differentiation into neuronal, astroglial, and oligodendroglial phenotypes. In contrast, lineage-restricted progenitors were directly sorted by positive selection using a combination of surface epitopes identifying neuronal or glial phenotypes or both. These cells were also primarily FGFR-1(+), with few EGFR(+), and most expanded and progressed along their expected lineages in bFGF-containing medium but not in EGF-containing medium. Ca(2+) imaging of self-renewing neural stem cells cultured in bFGF-containing medium revealed that bFGF, but not EGF, induced cytosolic Ca(2+) (Ca(2+)c) responses in these cells, whereas in bFGF- and EGF-containing medium, both bFGF and EGF evoked Ca(2+)c signals only in differentiating progeny of these cells. The results demonstrate that bFGF, but not EGF, sustains a calcium-dependent self-renewal of neural stem cells and early expansion of lineage-restricted progenitors, whereas together the two growth factors permit the initial commitment of neural stem cells into neuronal and glial phenotypes.