Neural stem cells, which exhibit self-renewal and multipotentiality, are generated in early embryonic brains and maintained throughout the lifespan. The mechanisms of their generation and maintenance are largely unknown. Here, we show that neural stem cells are generated independent of RBP-J , a key molecule in Notch signaling, by using RBP-J −/− embryonic stem cells in an embryonic stem cell-derived neurosphere assay. However, Notch pathway molecules are essential for the maintenance of neural stem cells; they are depleted in the early embryonic brains of RBP-J −/− or Notch1 −/− mice. Neural stem cells also are depleted in embryonic brains deficient for the presenilin1 (PS1) gene, a key regulator in Notch signaling, and are reduced in PS1 +/− adult brains. Both neuronal and glial differentiation in vitro were enhanced by attenuation of Notch signaling and suppressed by expressing an active form of Notch1. These data are consistent with a role for Notch signaling in the maintenance of the neural stem cell, and inconsistent with a role in a neuronal/glial fate switch.[Key Words: Presenilin; RBP-J ; embryonic stem cell; self-renewal; multipotentiality; cell cycle time]Received August 31, 2001; revised version accepted February 11, 2002. Neural stem cells, which are considered the ultimate lineage precursors to all neuronal and glial cells in the mammalian nervous system, are present not only in the developing brain but also in the adult brain Gage 2000). Although neural stem cells have a fundamental role in generating cellular diversity in the developing mammalian nervous system and in maintaining normal brain functions in adult brains (Lois and Alvarez-Buylla 1994;Tropepe et al. 1999;Shors et al. 2001), little is known concerning molecular mechanisms regulating the generation and maintenance of neural stem cells. In vitro, single neural stem cells proliferate to form clonally derived floating sphere colonies (neurospheres), which contain cells that, upon dissociation into single cells, give rise to new sphere colonies (self-renewal) and cells that can differentiate into neurons or glia (multipotentiality). Fibroblast growth factor-2 (FGF2)-responsive neural stem cells first appear in vivo at embryonic day (E) 8.5 and a separate and additive population of epidermal growth factor (EGF)-responsive neural stem cells arises from the earlier born FGF2-responsive stem cells by asymmetric division between E11 and E13 (Burrows et al. 1997;Mayer-Proschel et al. 1997;Tropepe et al. 1999). Both FGF2-responsive and EGF-responsive neural stem cells expand their populations and extend their cell cycle times during later embryogenesis (Martens et al. 2000). In the adult forebrain, neural stem cells are present as a relatively quiescent subpopulation in the subependyma, a remnant of the embryonic germinal zone (Morshead et al. 1994). This population persists into senescence, and the number is maintained throughout life (Tropepe et al. 1997). Thus, the generation and the size of the neural stem-cell population are tightly regul...

Basic fibroblast growth factor (FGF2)-responsive definitive neural stem cells first appear in embryonic day 8.5 (E8.5) mouse embryos, but not in earlier embryos, although neural tissue exists at E7.5. Here, we demonstrate that leukemia inhibitory factor-dependent (but not FGF2-dependent) sphere-forming cells are present in the earlier (E5.5-E7.5) mouse embryo. The resultant clonal sphere cells possess self-renewal capacity and neural multipotentiality, cardinal features of the neural stem cell. However, they also retain some nonneural properties, suggesting that they are the in vivo cells' equivalent of the primitive neural stem cells that form in vitro from embryonic stem cells. The generation of the in vivo primitive neural stem cell was independent of Notch signaling, but the activation of the Notch pathway was important for the transition from the primitive to full definitive neural stem cell properties and for the maintenance of the definitive neural stem cell state. Received March 31, 2004; revised version accepted May 26, 2004. The epiblast is endowed with positional information along its anteroposterior axis by the primitive streak stage at embryonic day 6.5 (E6.5), and the anteromedial part of the epiblast is further specified to form neural plate by E7.5 in the mouse embryo. Expression of neural tissue-specific marker genes, such as Nestin and Sox1, first becomes detectable at E7.0-E8.0 (Wood and Episkopou 1999; Kawaguchi et al. 2001). This happens before the first appearance of basic fibroblast growth factor (FGF2)-responsive neural stem cells at E8.5 (Tropepe et al. 1999). Thus, the first neurally specified cell in the epiblast could be a transient neural progenitor cell that is induced (by signals from mesoderm or endoderm) to yield neural stem cells, or perhaps, more interestingly, a primitive neural stem cell itself that builds the neural plate before giving rise to the definitive neural stem cell. Here, we define the "definitive" neural stem cell as the neural stem cell that is present in the late embryonic or adult brain and proliferates in response to FGF2 (and epidermal growth factor [EGF], or has the potential to acquire EGF responsiveness) to form clonal floating sphere colonies in vitro.We developed a colony-forming ES sphere assay, in which embryonic stem ( Historically, Notch signaling in Drosophila was thought to maintain cells in an undifferentiated state through a lateral inhibition mechanism (Artavanis- Tsakonas et al. 1995;Kimble and Simpson 1997). The Notch signaling also plays significant roles in mammalian neurogenesis: disruption of Notch pathway genes results in the reduction of the neural stem cell pool size (Nakamura et al. 2000;Hitoshi et al. 2002). The activation of this signaling promotes the symmetrical divisions of neural stem cells, and thereby enhances the self-renewal ability of the neural stem cells. However, little is known about molecular mechanisms underlying the generation of primitive and definitive neural stem cells in vivo. In this study, we used an in vitr...

Here we show a novel function for Retinoblastoma family member, p107 in controlling stem cell expansion in the mammalian brain. Adult p107-null mice had elevated numbers of proliferating progenitor cells in their lateral ventricles. In vitro neurosphere assays revealed striking increases in the number of neurosphere forming cells from p107−/− brains that exhibited enhanced capacity for self-renewal. An expanded stem cell population in p107-deficient mice was shown in vivo by (a) increased numbers of slowly cycling cells in the lateral ventricles; and (b) accelerated rates of neural precursor repopulation after progenitor ablation. Notch1 was up-regulated in p107−/− neurospheres in vitro and brains in vivo. Chromatin immunoprecipitation and p107 overexpression suggest that p107 may modulate the Notch1 pathway. These results demonstrate a novel function for p107 that is distinct from Rb, which is to negatively regulate the number of neural stem cells in the developing and adult brain.

Nicotine, the primary psychoactive component of tobacco smoke, is known to possess potent rewarding and aversive stimulus properties. The mammalian ventral tegmental area (VTA) is involved importantly in the mediation of the motivational effects of nicotine. However, the neural outputs from the VTA that may be involved in the transmission of the rewarding and aversive motivational effects of nicotine are not well understood. We report that bilateral lesions of the tegmental pedunculopontine nucleus (TPP) double dissociate the rewarding and aversive motivational effects of nicotine. Using a conditioned place preference paradigm, bilateral TPP lesions blocked a nicotine reward signal and revealed the aversive motivational properties of intra-VTA nicotine. These same TPP lesions did not block an aversive nicotine signal, as measured in a conditioned taste aversion paradigm. TPP lesions also produce an attenuation in nicotine-induced locomotor activity; however, neither learning nor performance deficits can account for these observed effects, because TPP-lesioned animals still showed clear aversive nicotine conditioning in two separate behavioral paradigms. Our results suggest that the rewarding effects of nicotine in the VTA are dependent on a nondopaminergic, descending reward pathway to the brainstem TPP.

Recently, Notch signaling has been reported to underscore the ability of neural stem cells (NSCs) to self-renew. Utilizing mice deficient in presenilin-1(PS1), we asked whether the function of Notch signaling in NSC maintenance was conserved. At embryonic day 14.5, all NSCs – both similar (cortex-, ganglionic eminence- and hindbrain-derived) and distinct (retinal stem cell) – require Notch signaling in a gene-dosage-sensitive manner to undergo expansionary symmetric divisions, as assessed by the clonal, in vitro neurosphere assay. Within the adult, however, Notch signaling modulates cell cycle time in order to ensure brain-derived NSCs retain their self-renewal property. At face value, the effects in the embryo and adult appear different. We propose potential hypotheses, including the ability of cell cycle to modify the mode of division, in order to resolve this discrepancy. Regardless, these findings demonstrate that PS1, and presumably Notch signaling, is required to maintain all NSCs.