Radial glia and neural stem cells

Cell Tissue Res (2008) 331:165–178 DOI 10.1007/s00441-007-0481-8

REVIEW

Radial glia and neural stem cells Paolo Malatesta & Irene Appolloni & Filippo Calzolari

Received: 21 May 2007 / Accepted: 17 July 2007 / Published online: 11 September 2007 # Springer-Verlag 2007

Abstract During the last decade, the role of radial glia has been radically revisited. Rather than being considered a mere structural component serving to guide newborn neurons towards their final destinations, radial glia is now known to be the main source of neurons in several regions of the central nervous system, notably in the cerebral cortex. Radial glial cells differentiate from neuroepithelial progenitors at the beginning of neurogenesis and share with their ancestors the bipolar shape and the expression of some molecular markers. Radial glia, however, can be distinguished from neuroepithelial progenitors by the expression of astroglial markers. Clonal analyses showed that radial glia is a heterogeneous population, comprising both pluripotent and different lineage-restricted neural progenitors. At late-embryonic and postnatal stages, radial glial cells give rise to the neural stem cells responsible for adult neurogenesis. Embryonic pluripotent radial glia and adult neural stem cells may be clonally linked, thus representing

This work was supported by AIRC (Associazione Italiana per la Ricerca sul Cancro) NUSUG grant (In vivo screening for genes implicated in glioma formation and development of new animal models of glial tumors) and by Fondazione CARIGE grant (Basi molecolari e cellulari dei gliomi: individuazione di marcatori diagnostici e di nuovi bersagli terapeutici). P. Malatesta : I. Appolloni : F. Calzolari Dipartimento di Oncologia, Biologia e Genetica, Università degli Studi di Genova, Largo Rosanna Benzi 10, 16132 Genoa, Italy P. Malatesta (*) : I. Appolloni : F. Calzolari Istituto Nazionale per la Ricerca sul Cancro (IST), IRCCS, Largo Rosanna Benzi 10, 16132 Genoa, Italy e-mail: [email protected]

a lineage displaying stem cell features in both the developing and mature central nervous system. Keywords Neural progenitors . Ventricular zone . Lineage tracing . Neurogenesis . Gliogenesis

Introduction Radial glial cells are ubiquitously present during the neurogenic phases in all vertebrates. The dominant view of their function until the last decade was that they serve as a scaffold for neuronal migration. It is now clear that in many regions of the central nervous system (CNS), radial glia represents the main population of neural progenitors and that their progeny includes all the main lineages of the CNS: neurons, astrocytes, oligodendrocytes, ependymocytes and adult neural stem cells (Malatesta et al. 2003; Merkle et al. 2004; Spassky et al. 2005). In this review, we will examine the role of radial glia during development, with particular emphasis on the mammalian telencephalon. The reason for selecting the telencephalon is because it is the best characterized area of the CNS and is the only region in the mammalian CNS where stem cells are known to remain throughout the entire life of the organism. We will also discuss the relationship between radial glial cells and their ancestors, the neuroepithelial progenitor cells. Since radial glial cells undergo both symmetric and asymmetric cell divisions, we will discuss the possible mechanisms that are involved in this process. Subsequently, we will examine the fate of radial glia during development and the molecular cues responsible for the maintenance of their phenotype and the determination of their fate. We will also discuss the conversion of radial glia into adult neural stem cells and finally the

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acquisition of radial glia-like phenotypes by embryonic stem cells differentiating towards neural lineages. Terminological premise A strong definition of “stem cell” is based on the ability of a cell to give rise to multiple lineages, ideally to all the cell types of a given organ, and to self-renew in vivo or in vitro an indefinite number of times without any significant changes in its phenotype and differentiation capabilities. Following this definition, in this review we will refer to “progenitor” cells when talking about cells that undergo limited rounds of self-renewing divisions during the development of the central nervous system, and then progress toward more lineage-restricted precursors, or terminally differentiate. We will reserve the term “stem cells” for the populations of cells that persist for the entire life of the organism (adult stem cells), like those present in the adult subependymal zone of the lateral ventricle.

Neuroepithelial cells and early phase of neurogenesis At the end of neurulation, the CNS is made up of a pseudostratified epithelium where radially arranged bipolar cells span the entire thickness of the neural tube (Fig. 1). Neural progenitors undergo a characteristic alternate movement of the nucleus (interkinetic nuclear migration) between the basal and the apical surface, recognized as early as 1935 (Sauer 1935). This migration is synchronized

Fig. 1 Neuroepithelial to radial glia transition. During neurogenesis, neuroepithelial cells progressively convert to radial glial cells that elongate following the thickening of the neural tube wall. Basal progenitors (red outlined) are generated at early stages by neuroepithelial cells, and at later stages by radial glia, and accumulate in the SVZ. Preplate neurons (green) are generated at early stages by basal progenitors. At later stages neurons derive from both radial glia (blue) and basal precursors (red). CP cortical plate; SVZ subventricular zone; VZ ventricular zone

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with the cell cycle: at mitosis, the nucleus stands at the apical surface, near the ventricle, moving towards the basal surface during phase G1, reaching the most basal location in S phase, and then migrating back apically during the G2 phase. Since the progenitor cells are not synchronized and at any given time there are cells in all possible phases of the cycle, the epithelium results in being pseudostratified (Fig. 1). Before neurogenesis, epithelial cells divide symmetrically, originating two identical progenitors and thereby increasing their number (Rakic 1995). As development proceeds, epithelial cells undergo some changes in their gene expression pattern, cytological characteristics and differentiation potential. The first phenotypic change, which in the mouse telencephalon occurs at around E9/E10, consists of the induction of the intermediate filament nestin (Frederiksen and McKay 1988) and of the related antigen recognized by the antibody RC2 (Edwards et al. 1990; Misson et al. 1988). Moreover, at this stage, intercellular junctional coupling is loosened, following down-regulation of occludin, a tight junction component. The apical junctional complex thus loses its previous role as a permeability barrier, as demonstrated by injection of horseradish peroxidase (HRP) into the amniotic cavity of developing mouse embryos (Aaku-Saraste et al. 1996). These experiments showed that whereas tight junctions inhibit the flow of HRP into the lateral intercellular space at E8, soon after they become non-functional, allowing the transit of HRP. It is however not clear whether these functional changes occur in all the species, since an early

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study suggested that a permeability barrier persists during neurogenesis in sheep and in primates (Mollgoard and Saunders 1975). Concomitantly to the permeability changes, the dependency of junctions on Notch signaling becomes evident. Mutant mice lacking the Notch effectors Hes1, 3, 5 do not show clear phenotypes before E8, whereas at later stages they show disorganization of the entire neuroepithelium, which loses its polarity and fails to properly differentiate into radial glia (Hatakeyama et al. 2004). Starting from this stage, the cells can be considered properly “neuroepithelial” and are characterized by the distinctive traits summarized in Fig. 2. Neuroepithelial progenitors (NEPs), as earlier progenitors, maintain the interkinetic nuclear migration, which involves their entire soma, and divide mainly symmetrically on the luminal surface of the neural tube (Kosodo et al. 2004; Rakic 1995). From this stage, however, an increasing number of cells start to divide asymmetrically, giving rise to another neuroepithelial cell and either to a neuron or, alternatively, to a progenitor cell that will undergo mitosis at a significant distance from the ventricular surface (basal progenitor; Fig. 1) and which will generate neurons via a symmetric division (Haubensak et al. 2004). Basal progenitors will be discussed in greater detail in Chap. 3. Since NEPs are the only cells present in the neural tube at early stages, it is clear that, as a population, they generate, directly or indirectly, all of the neurons and glial

Fig. 2 Summary of the features of neuroepithelial (NE), radial glia (RG) and basal progenitor (BP) cells. Neuroepithelial (NEPs) and radial glia cells have a similar shape, but are distinguished by the expression of molecular markers and peculiar characteristics

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cells that compose the adult CNS. It makes sense, however, to ask how broad the differentiation potential of single NEPs is, since the apparent homogeneity in the expression of known molecular markers may mask an early commitment towards specific fates. The analysis of the cell fate of NEPs performed by isolating them in very low density culture (Qian et al. 2000) or by labeling them with retroviral vectors in high density culture (Williams and Price 1995) and in vivo (McCarthy et al. 2001) showed that a percentage between 10% and 20% of NEPs are bipotent, as they are able to generate both neurons and glia. Surprisingly, even at E9.5, progenitor cells restricted to glial fates are already present (McCarthy et al. 2001), underscoring the functional heterogeneity of NEPs despite the absence of molecular markers.

Radial glia and its relationship with neuroepithelial cells Definition of radial glia cells Shortly after the appearance of the first neurons, neuroepithelial cells undergo a second change in their characteristics and acquire molecular and cytological features typical of the astroglial lineage, as they give rise to the radial glial cells (Figs. 1, 3a–c). Among these features (summarized in Fig. 2), there is the expression of genes typical of mature or reactive astrocytes, like the lipid-binding protein BLBP (Feng et al. 1994; Hartfuss et al. 2001), the astrocytic glutamate transporter GLAST (Hartfuss et al. 2001; Malatesta et al. 2000; Shibata et al. 1997), the adhesion molecule TN-C (Bartsch et al. 1992; Gotz et al. 1997), the enzyme glutamine synthase (Akimoto et al. 1993), the calcium-binding protein S100β (Vives et al. 2003), the intermediate filament vimentin (Schnitzer et al. 1981) and, in some species (but not in rodents), GFAP (Levitt and Rakic 1980; Sancho-Tello et al. 1995). Moreover, glycogen granules start to accumulate in the radial glia cytoplasm, which becomes electron-lucent, another characteristic of astrocytes (Choi 1981). Notably, apart from the peculiar shape, in many species no markers are known that allow discrimination of radial glia from astrocytes. This is the case, for instance, in primates, where the immunoreactivity for GFAP is shown by both cell types (whereas in rodents it is limited to astrocytes), and the mouse-specific antibody RC2 is unsuitable. In contrast to neuroepithelial cells, radial glial cells lack tight junctions, while maintaining an adherens junctions-mediated intercellular coupling (AakuSaraste et al. 1996). Although rodent radial glia does not express GFAP, the 2.2 kb element of the human promoter of the GFAP gene, termed gfa2, is transcriptionally active also in murine radial glia (Malatesta et al. 2000). This suggests that the transcriptional cues provided by the radial

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Fig. 3 Examples of radial glial cells and radial glia lineage. a–c Microphotographs of embryonic E14 sections derived from a mouse expressing the reporter gene eGFP from the human promoter of GFAP (gfa2 promoter). a Cerebral cortex; b ganglionic eminences (modified from Malatesta et al. 2003); c diencephalon; d lineage tracing of radial glia by detection of β-galactosidase in frontal sections of the gfa2-Cre/R26R telencephalon (P21) by X-Gal histochemical staining (modified from Malatesta et al. 2003). Blue cells are derived by radial glia expressing Cre recombinase under the gfa2 promoter. v Ventricle, p pial surface, ctx cortex, hip hippocampus, thl thalamus, bg basal ganglia, ic inner capsule. Scale bar 50 μm (a, b); 100 μm (c); 500 μm (d)

glia nucleoplasm in the two species are similar, and the difference in GFAP expression may be due to the divergence between the human and the murine GFAP promoter (Brenner and Messing 1996). Time of appearance of radial glia It is important to mention that the exact point of radial glia appearance is still a debated topic. Some authors tend to consider as a fundamental landmark the induction of RC2 and the expression of BLBP mRNA (Anthony et al. 2004), while others refer to the induction of the astroglial features (Gotz et al. 2002; Malatesta et al. 2003). These different views are probably at the root of the discrepancy between different observations about the fate of radial glial progeny (Anthony et al. 2004; Malatesta et al. 2003), as will be discussed in Chap. 4. Radial glial cells maintain a shape very similar to NEPs, with a long basal process reaching the outer (pial) surface and a shorter apical process in contact with the lumen of the ventricle. Like NEPs, radial glial cells undergo interkinetic

nuclear migration, but their somata do not move across the entire thickness of the neural tube; rather they remain confined to an apical region, which, therefore, is densely packed with cell nuclei and which is referred to as the ventricular zone (VZ; Fig. 1). At the time of radial glia appearance, the telencephalon starts to be a pluristratified epithelium due to the basal accumulation, within the preplate, of the postmitotic neurons generated by the early neuroepithelial cells, and to the increasing number of basal progenitors. These cells now form a layer at the border between the VZ and the preplate, known as the subventricular zone (SVZ; Fig. 1). Following the thickening of the neural tube wall, the basal processes of the radial glia cells elongate in such a way that their “mushroom-shaped” basal end-feet maintain contact with the pial surface, where they form a membrane known as the glia limitans (Rakic 2003). Notably, the basal process of the radial glia is not retracted when cells undergo mitosis (Miyata et al. 2001, 2004; Noctor et al. 2001, 2004), though it becomes extremely thin, because of basal-to-apical “flows” of cytoplasm and plasma membrane, as shown in

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time-lapse videomicroscopy (Miyata et al. 2001). After mitosis, the basal fiber is inherited by one of the two daughter cells, while the other extends a new process. In agreement with their postulated allocation to the glial lineage, radial glial cells have for a long time been described as a sort of rail track used by newly generated neurons moving from the ventricular surface to their definitive destinations in the cortical plate. Meticulous electron-microscopic reconstructions of the developing cerebral cortex of primates clearly showed that during neurogenesis many young bipolar neurons are associated with the basal process of radial glial cells (Rakic 1972). As described in Chap. 4, however, more recent observations have compelled scholars to change this view (Anthony et al. 2004; Gotz et al. 2002; Malatesta et al. 2000, 2003; Miyata et al. 2001; Noctor et al. 2001, 2004), and it is now clear that radial glia is the main source of neurons in many CNS districts.

Basal progenitors As previously mentioned, not all proliferating cells undergo mitosis at the ventricular surface of the neural tube as the first wave of neurogenesis starts. As early as E9.5 in the mouse telencephalon, an increasing number of progenitor cells appear that divide in the basal side of the ventricular zone. Video time-lapse studies and the analysis of a knockin mouse model where the GFP is expressed from the locus of Tis21, a marker of neurogenic cell divisions (Iacopetti et al. 1999), showed that at early stages, apically dividing NEPs may undergo asymmetric divisions, generating a novel apically dividing NEP, which shows a low level of Tis-21-driven GFP expression, and a basal progenitor with high GFP levels that migrates basally (Haubensak et al. 2004). These basal progenitors, in turn, divide symmetrically at a significant distance from the ventricular surface and generate two daughter cells showing identical levels of GFP, which exit from the VZ differentiating into neurons (Fig. 1; Haubensak et al. 2004). Before the differentiation of radial glia, basal progenitors are the main source of neurons. At later stages, when the transition from NEPs to radial glia has occurred, the population of basal progenitors (SVZ) is maintained by the continuous supply from asymmetric cell divisions of the radial glial cells (Miyata et al. 2004; Fig. 1). At this stage, the expression of the bHLH transcription factor Ngn2 (Sommer et al. 1996) seems to play a fundamental role in committing a cell to becoming a basal progenitor, as shown by the fact that the transcription factor is expressed mainly by this type of precursor and that retrovirus-driven expression of Ngn2 converts apically dividing cells into basally-dividing cells (Miyata et al.

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2004). The role of Ngn2 in the transition between apical to basal progenitor cells is however not completely understood since a recent report showed that in the Ngn2−/− knock-out mouse, the percentage of basal mitoses increases starting from E15.5 (Britz et al. 2006). This unexpected effect might be due to the interplay between Ngn2 and another proneural gene, Mash1, which is upregulated following the loss of Ngn2 (Fode et al. 2000; Schuurmans et al. 2004) and whose overexpression promotes the transit of apical progenitor to the SVZ (Britz et al. 2006). Basal progenitors differ from apical progenitors in terms of gene expression: they do not express astroglial markers, nor the transcription factors Pax6 and Hes (Cappello et al. 2006; Englund et al. 2005), while they can be positively identified by the expression of Tbr2 (Englund et al. 2005), Cux1 and 2 (Conti et al. 2005), VGlut2 (Schuurmans et al. 2004), Satb2 (Britanova et al. 2005) and the non-coding RNA Svet1 (Tarabykin et al. 2001). In some regions of the CNS, as for instance in the ventral telencephalon, basal progenitor cells outnumber radial glial cells (Smart 1976) and are the main source of neurons (Malatesta et al. 2003). However, at later fetal and early postnatal stages, these ventral basal progenitors become restricted to the astroglial and oligodendroglial lineage (Malatesta et al. 2003; Schmid et al. 2003; Voigt 1989). Basal progenitors may be considered as a sort of committed transit amplifying population deriving from radial glial cells (or NEPs, at early stages). The different proportions between apical and basal precursors in different areas could depend on the rounds of amplification (i.e., the number of cell cycles) that basal progenitors undergo in each area before differentiation. Notably, the adult stem cells that populate the subependymal zone of the lateral wall of the lateral ventricle in the telencephalon do not derive from the basal progenitors of the fetal subventricular zone, but rather from radial glial cells, as will be discussed below.

Radial glia fate during neurogenesis The former notion that radial glia merely serves as a scaffold for neuronal migration started to be challenged when it was recognized that radial glial cells proliferate (Misson et al. 1988) and that they represent, at least in some districts (notably in the cerebral cortex), the main proliferating population (Hartfuss et al. 2001). A series of observations both in vitro and in vivo showed that, during neurogenesis, radial glia generate mostly neurons (Anthony et al. 2004; Malatesta et al. 2000, 2003; Miyata et al. 2001, 2004; Mo et al. 2007; Noctor et al. 2001, 2004). The first direct observation in this sense came from the analysis of the differentiation of radial glia isolated by

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fluorescence-activated cell sorting (Malatesta et al. 2000). Cells were labeled by exploiting two independent characteristics of radial glia: the ability to express GFP from the human GFAP (hGFAP) promoter (Fig. 3a–c) and the presence of a contact with the basal membrane, allowing them to be stained with a lipophilic dye applied to the pial surface (Malatesta et al. 2000). The analysis demonstrated that the majority of radial glial cells generate homogeneous clones, composed either of neurons or of non-neuronal cells (glia and/or progenitor cells) exclusively. The percentage of mixed neuronal/non-neuronal clones was extremely low, showing that radial glia is a heterogeneous population of already committed neuronal and glial progenitor cells (Malatesta et al. 2000). The generation of neurons from radial glia in the cerebral cortex was corroborated by video time-lapse analyses showing the generation of a “radial unit” by the asymmetric division of a single radial glial cell (Noctor et al. 2001). Such radial units were composed of the radial glial cell itself plus multiple postmitotic neurons. Subsequently, lineage-tracing studies in vivo based on the Cre/LoxP recombination system showed that radial glia is the main source of postmitotic neurons in the cerebral cortex (Malatesta et al. 2003) and, possibly, in the entire telencephalon (Anthony et al. 2004). Both of these studies were based on transgenic mouse lines where Cre-recombinase expression was driven by radial glial specific promoters: the hGFAP- (Malatesta et al. 2003) and the BLBP-promoter (Anthony et al. 2004). Cre-expressing mice were crossed with reporter mouse lines, in which Cre-induced recombination caused the permanent induction of a reporter gene (Lobe et al. 1999; Novak et al. 2000; Soriano 1999). Whereas both studies agreed that radial glia is responsible for the generation of all projection neurons of the cerebral cortex (Fig. 3d), discrepancies were found regarding the role of radial glia in the generation of ventral telecephalic neurons. In the case of the h-GFAP promoterbased system, most of the neurons of the basal ganglia and most of the cortical interneurons, which derive from the ganglionic eminences, were found to be unlabeled, suggesting that they do not derive from radial glia (Fig. 3d). In contrast, by employing the BLBP promoter, almost all the neurons across the entire telencephalon and midbrain were labeled. The probable reason for these discrepancies lies in the different timing of Cre appearance. While the hGFAP-driven Cre was absent both in the ganglionic eminences and in the cerebral cortex before E12.5, and appeared in correlation with the astroglial markers (Malatesta et al. 2003), the induction of the BLBP-driven Cre occurred at the same time as that of RC2 immunoreactivity, and some recombined cells were identifiable as early as E10.5 (Anthony et al. 2004). It is therefore conceivable that the recombination events in the

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latter mouse line occur in neuroepithelial cells, rather than exclusively in radial glia.

Mechanics and mode of radial glia cell division Basal process inheritance Since newborn neurons migrate using the radial glial processes as scaffolds, and since the end feet of radial glia form a stable membrane at the outer surface of the CNS (Rakic 2003), the finding that radial glia actively proliferate during neurogenesis posed puzzling issues about the fate of the long basal process during cytokinesis. A series of video time-lapse analyses of cultured brain sections provided answers to this question and showed that different patterns of radial glia mitosis may exist (Miyata et al. 2001; Nadarajah et al. 2001; Noctor et al. 2001, 2004). By injecting GFP-expressing retrovirus into the telencephalic ventricles (Noctor et al. 2001, 2004) or by applying the lipophilic dye DiI to the ventricular surface (Miyata et al. 2001), it was possible to follow divisions of radial glia in organotypic slices. The analyses consistently revealed that the basal process persists during mitosis, though it becomes extremely thin. However, the studies differed in their conclusions regarding the identity of the cell that inherits the process after mitosis. Noctor and colleagues (Noctor et al. 2004) reported the occurrence of asymmetric divisions in which one daughter cell retains the basal process and maintains a radial glia phenotype, while the other daughter cell uses it as a guide to migrate by “locomotion” towards the cortical plate, differentiating then into a neuron. This division pattern is responsible for the generation of radial units, where many migrating neurons are associated to a single radial glial cell. On the contrary, Miyata and colleagues found that, especially at early stages, the basal process is in most cases inherited by the cell that starts to migrate basally by “somal translocation,” differentiating into a neuron possibly after a second round of division in the SVZ (Miyata et al. 2001). Taken together, these observations show that radial glia probably uses two alternative patterns of cell division, possibly in a stagedependent manner (Nadarajah et al. 2001). Interestingly, the inheritance of the basal process seems not to correlate to cell fate choice, as cells are as likely to maintain a radial glia phenotype as they are to differentiate into neurons, upon fiber inheritance. This observation is consistent with the results obtained by the analysis of mouse lines defective for basal membrane components. Neurogenesis is unaffected in these mutants, despite the severe disorganization of the radial glial scaffold, which results only in neuronal mispositioning, consistent with the known role of the radial fiber in neuronal migration support (Haubst et al. 2006).

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When the process is inherited by the differentiating neuron, the ventricular progenitor cells elaborate new basal processes. Indeed radial glial cells with short processes, sometimes ending with a growth cone-like structure, have been identified in vivo, but their percentage is relatively low (about 20% at E14; Hartfuss et al. 2003). This could indicate either that process regeneration is very fast or that the basal fiber is inherited by the migrating neuroblast in only few cases. Since the speed of process elongation has been estimated in vitro to be about 20 μm/ h (Miyata et al. 2001), and the total length of a basal process at E14 is on average 300 μm, the regeneration of the process should take more than 10 h, a time comparable to an entire cell cycle at the stage considered (Cai et al. 1997). Taken together, these considerations suggest that at E14 the basal process is predominantly inherited by the progenitor cell. Apical membrane inheritance While the inheritance of the basal process does not seem to be related with the fate of the daughter cells, the differential inheritance of apical aspects might be a key determinant of asymmetric cell divisions. The possibility that, as in Drosophila, the orientation of the cleavage plane could cause the asymmetric partitioning of determinants, thus influencing the fate choice of two daughter cells after mitosis, has been proposed since a long time ago. A first report suggested that the basal localization of Notch and its differential distribution following horizontal mitoses (i.e., parallel to the ventricular surface), resulted in asymmetric cell divisions of ferret neuroblasts (Chenn and McConnell 1995). This finding, however, could not be confirmed by subsequent studies. Furthermore, the observation that horizontal mitoses represent only a small fraction of all cell divisions at any given stage during CNS development suggests that they cannot account for all the asymmetric divisions occurring during neurogenesis (Haydar et al. 2003; Noctor et al. 2002; Smart 1973; Stricker et al. 2006). By using the knock-in mouse model where the GFP is expressed from the locus Tis21, Kosodo and colleagues showed that the switch between symmetrical and asymmetrical divisions is likely to depend on the mode of segregation of a relatively tiny portion of the NEP basal membrane, lacking cadherin-E and containing prominin-1, as a result of the positioning of the mitotic cleavage plane (Kosodo et al. 2004, reviewed in Gotz and Huttner 2005). This model reconciles the previous observations from Chen and McConnell and other authors showing a correlation between the orientation of the cleavage furrow and the mode of cell division (Chenn and McConnell 1995; Chenn et al. 1998; Landrieu and Goffinet 1979; Zhong et al. 1996).

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Radial glia contains different subpopulations of progenitor cells restricted to individual fates Though radial glia is able to generate neurons, astrocytes and oligodendrocytes, clonal analyses showed that the majority of progenitors are very early committed to neuronal or glial fates (Malatesta et al. 2000; McCarthy et al. 2001; Price and Thurlow 1988; Qian et al. 2000; Williams and Price 1995). This restriction is partly present already in neuroepithelial cells (McCarthy et al. 2001; Qian et al. 2000). The occurrence of early commitments to late cell fates suggests that already committed progenitors can remain quiescent even for long periods. This seems to be the case, for instance, with ependymal cells. Recent observations showed that their radial glia progenitors undergo the last mitosis mostly between E12 and E16, but nevertheless they maintain radial glia characteristics until postnatal stages, when they acquire the typical multiciliated and cuboid phenotype of ependymocytes (Spassky et al. 2005). The apparent progressive lineage switch shown by radial glia could therefore partly be due to the differential expansion of distinct populations of already-committed progenitors in response to extrinsic and intrinsic signals, rather than only to a change in the differentiation pattern of multipotent progenitors. This conclusion would acknowledge the identification of progenitor cells restricted to a late fate already at early stages. It is, however, also well established that bi- or tri-potent progenitors, sometimes referred to as stem cells, are present at all developmental stages, though their number is relatively low (McCarthy et al. 2001; Qian et al. 2000; Shen et al. 2006; Williams and Price 1995). It is therefore possible that such stem cells supply, throughout development, fate-restricted transitamplifying progenitors, which, at all stages, largely outnumber the stem cells. The finding that radial glia contains progenitor cells already committed to different fates prompts the obvious question of whether it is possible to distinguish progenitors with distinct differentiation capabilities by means of appropriate molecular markers. An important contribution to the understanding of radial glia heterogeneity was the analysis of the co-expression of the markers RC2, BLBP and GLAST during telecephalic development (Hartfuss et al. 2001). The study showed that telecephalic cortical precursor cells, identified by the expression of the proliferation marker ki67 (Scholzen and Gerdes 2000), can be subdivided into subpopulations co-expressing radial glial markers in different combinations. The study also showed that the proportion of the different subpopulations varies between stages and regions. The different populations show heterogeneity in their cell cycle length, a parameter that is thought to be related to the pattern of differentiation (Calegari et al. 2005; Calegari and Huttner 2003). The

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changes in the expression of markers are likely to be related to a restriction in the differentiation potential of radial glia, but it is not clear whether this reflects the maturation of single progenitor cells or, rather, the differential expansion of pre-existing subpopulations. Confirmation that heterogeneity in the co-expression of the radial glial markers is correlated to the differentiation potential can be found by comparing the clonal analyses of radial glia isolated by fluorescence-activated cell sorting based on GFP expression driven either by the BLBP or the hGFAP promoter. Notably, the activity of the latter is closely correlated to the expression of GLAST (Malatesta et al. 2000). The comparison shows a clear enrichment of bipotent progenitor cells in the BLBP-GFP-based sorting versus the hGFAP one (Anthony et al. 2004; Malatesta et al. 2000). Interestingly, the level of GFP expression driven by the hGFAP promoter seems, by itself, to allow discrimination between glial-restricted and neuronalrestricted radial glia, since highly GFP-expressing cells are enriched in gliogenic precursors (P.M. unpublished results, Magdalena Goetz, personal communication). It is worth reminding that some caution should be taken when interpreting the results about the differentiation potential of progenitor cells based exclusively on in vitro observations. Possible biases may be imposed by the culture conditions, which can favor particular cell types at the expenses of others. The lack of extrinsic signals and of the appropriate cytoarchitectural niche may also alter, to some extent, the pattern of proliferation and differentiation.

Molecular mechanisms of radial glia establishment, maintenance and differentiation One molecular signal known to be essential for the establishment and maintenance of the radial glia phenotype during neural development is the activation of Notch. Notch is a well-known proneural gene product, which in Drosophila implements the lateral inhibition process, preventing neurogenesis (reviewed in Gaiano and Fishell 2002). During neurogenesis in mammals, Notch-1 is expressed by radial glia and is activated by Notch ligands, in particular Delta-1, which is expressed by newborn neurons. A lack of the Notch downstream effectors HES13-5 causes disruption of the neuroepithelium and inhibits the proper differentiation of radial glial cells (Hatakeyama et al. 2004). On the contrary, the retrovirus-mediated overexpression of a constitutively active form of Notch (NotchICD) forces radial glia to maintain its phenotype (Gaiano et al. 2000). This does not result, however, in the permanent loss of the neurogenic potential of the radial glial cell affected, as elegantly shown in experiments where a floxed Notch-ICD was removed before the end of neurogenesis by

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employing a Cre-expressing vector. These experiments showed that radial glia can resume generating neurons, within an appropriate time window, when Notch-signaling over-activation ceases (Mizutani and Saito 2005). This finding reveals that during neurogenic stages, Notch signaling promotes the maintenance of an undifferentiated phenotype, rather than inducing gliogenesis. As will be described in detail in a following section, the role of Notch signaling changes at the end of neurogenesis, when it becomes progliogenic (Hermanson et al. 2002). A second signaling pathway important for the establishment and maintenance of the radial glia phenotype depends on the paracrine factor neuregulin-1 (NRG-1) and its tyrosine kinase receptor ErbB2 (Schmid et al. 2003). In NRG-1 knock-out mice, radial glia generation is severely impaired, but it can be rescued by the reintroduction of exogenous NRG-1. Moreover, the down-regulation of the receptor ErbB2 leads to the premature differentiation of radial glia into astrocytes, while the reintroduction of ErbB2 converts mature astrocytes back to radial glial cells, although it is not clear whether they re-acquire a neurogenic potential (Schmid et al. 2003). Notably, ErbB2 expression is upregulated by an activated form of Notch, showing that the two pathways largely crosstalk (Patten et al. 2006; Schmid et al. 2003). Possible cell autonomous cues involved in the proliferation of radial glia and in the maintenance of their undifferentiated phenotype are the transcription factor Six3 and Emx2 (Appolloni et al. 2007; Heins et al. 2001). Retrovirus-mediated over-expression of the homeoboxcontaining transcription factor Six3 in E14 progenitor cells increases their proliferation rate, whereas its functional ablation reduces it. In vivo, the over-expression of Six3 enriches progenitor cells that remain in contact with the ventricular surface and maintain expression of radial glial markers (Appolloni et al. 2007). A similar effect is induced by the homeobox transcription factor Emx2, which, when over-expressed in high density cultures of cortical progenitor cells, enhances their proliferation, forcing them to divide symmetrically. Consistently with this, cortical precursors derived from Emx2−/− knockout mouse, show reduced proliferation (Heins et al. 2001). Notably, the role of Emx2 in promoting symmetrical divisions of neural stem cells is still debated, since its gain and loss of function in progenitor cells cultured as neurospheres show the opposite effect compared with the above findings. In these conditions, the over-expression of Emx2 in neural stem cells decreases their proliferation rate and their clonogenicity, whereas progenitor cells derived from Emx2−/− mice prove to be more clonogenic than wild-type progenitors (Galli et al. 2002; Gangemi et al. 2001, 2006). Whereas the previously described extrinsic and intrinsic signals play a role in the induction and maintenance of the

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radial glial phenotypes, the bHLH transcription factors Ngn1 and 2 and the paired domain containing transcription factor Pax6 promote the acquisition of the neurogenic fate. Ngn1 and 2 have been demonstrated to activate neurogenic genes directly, including β-III-tubulin (Nieto et al. 2001; Schuurmans et al. 2004; Sun et al. 2001), and to promote a particular type of asymmetric cell division. Ngn2-expressing radial glia divisions generate a new radial glial cell and a basal progenitor that undergoes neuronal differentiation (Miyata et al. 2004). In the cerebral cortex, neurogenesis from radial glia depends on the transcription factor Pax6. Mice lacking Pax6 have a reduced cortical plate and show a disorganized radial glial scaffold (Gotz et al. 1998). Clonal analysis of cortical radial glia isolated from mice lacking Pax6 showed a dramatic reduction in neuronal clones, while neurogenesis was still possible from non-radial glial precursors (Heins et al. 2002). Conversely, the transduction of Pax6 into cortical progenitors increases the number of neuronal clones. Notably, the transduction of Pax6 is sufficient to redirect even neonatal astrocytes towards the neuronal lineage, showing that their differentiation status is reversible (Heins et al. 2002). Switch from neurogenesis to gliogenesis Possible mechanisms involved in the switch between neurogenesis and gliogenesis of multipotent progenitor cells have been identified in recent years and involve extrinsic and intrinsic cues. Among the extrinsic signals implicated in this process, there are members of the IL6 family of cytokines, in particular cartiotrophin-1 (CT-1) [reviewed in Miller and Gauthier (2007)]. The activation of the receptors gp130 and LifRβ by these paracrine factors stimulates gliogenesis via the Jak/STAT pathway (Barnabe-Heider et al. 2005). Activated STAT1/3 play a double role: on the one hand, they act together with BMP-2 effectors SMAD1/5/8 and with the coactivator P300 to upregulate the expression of glial specific genes directly, including s100β and GFAP (Bonni et al. 1997; Nakashima et al. 1999); on the other hand, they allow Notch signaling via RBP-Jk to become gliogenic, by promoting the exit of the Notch corepressor NCoR from the nucleus (Hermanson et al. 2002). Moreover, STAT1/3 potentiates its own pathway via a positive autoregulatory loop (He et al. 2005). Notably, newborn neurons express the Notch ligand Delta-1 and the factor CT-1. The latter reaches the critical concentration to stimulate gliogenesis via the pathway described only around E17.5 in the mouse (Barnabe-Heider et al. 2005), which provides an example of a cell signaling network that contributes to the establishment of the timetable of cell differentiation in the CNS.

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Intrinsic signals involved in the neurogenic-to-gliogenic switch are less well understood; however, b-HLH transcription factors are thought to play a role inhibiting the aforementioned pathway during neurogenesis. Transcription factors Ngn1 and Ngn2 are able to bind to the SMADs/ P300 complex, thus inhibiting its binding to active STAT1/3 and maintaining neural progenitors unresponsive to the IL6 gliogenic signals during neurogenesis (Sun et al. 2001). The acquisition of responsiveness to gliogenic signals relies, in addition, on demethylation of the CpG island within the promoter of glial specific genes, making them accessible to the STAT/SMAD/P300 complex (Namihira et al. 2004; Takizawa et al. 2001).

Adult subependymal stem cells Widespread neurogenesis in mammals is restricted to the embryonic period, but a limited number of neural stem cells (NSCs) persist during the entire life of the organism. The areas of the CNS known to contain adult NSCs are the subependymal layer of the lateral wall of the lateral ventricle, sometimes referred to as the “adult subventricular zone,” and the subgranular layer of the hippocampus. The neural stem cells of the subependymal layer, also referred to as TypeB cells, are embedded in a peculiar niche containing also fast-cycling transit-amplifying progenitor cells (TypeC cells) and migrating neuroblasts (TypeA cells), which are guided towards the olfactory bulb thanks to ensheathing glial tubes, and give rise to the rostral migratory stream (RMS; Fig. 4; Doetsch 2003b). TypeB cells have astroglial characteristics and express GFAP, Nestin, vimentin and GLAST, accumulate glycogen granules in their cytoplasm and are relatively quiescent (Bolteus and Bordey 2004; Doetsch 2003a, b; Doetsch et al. 1997; Jankovski and Sotelo 1996; Peretto et al. 1997). Following pharmacological depletion of TypeA and TypeC cells with Ara-C, TypeB cells can repopulate the niche (Doetsch et al. 1999). In addition to subependymal TypeB, some authors presented data suggesting that ependymocytes might represent neural stem cells (Johansson et al. 1999). This view is not generally accepted since other reports demonstrated, on the one hand, that although ependymal cells can proliferate in vitro they do not generate neurons (Chiasson et al. 1999), and, on the other hand, that ependymocytes do not proliferate in vivo where the only proliferating cells in contact with the ventricle are TypeB astrocytes (Spassky et al. 2005). The question about the embryonic origin of TypeB cells has been answered only recently, showing that adult subventricular stem cells are not related to embryonic subventricular precursors (basal precursors), but rather to embryonic ventricular radial glia. The direct evidence for this embryonic derivation comes from in vivo lineage tracing

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Fig. 4 Adult neural stem cell niche. Astrocyte-like cells (TypeB; blue), lining the ventricle, give rise to progenitor cells (TypeC; orange) that subsequently generate migrating neuroblasts (TypeA cells; green). Early generated neurons reach the olfactory bulb migrating along the rostral migratory stream (RMS)

experiments, where ventral radial glia was labeled at postnatal day 0 (P0) by stereotaxic injections of Cre-expressing adenovirus close to the border with the pyriform cortex. Labeled cells were later found lining the ventricle as ependymocytes, in the subependymal layer, in the RMS and in the olfactory bulb. An accurate analysis at different stages showed that radial glial cells convert their morphology by retracting their basal process (Merkle et al. 2004). More recently, a similar analysis of lineage-tracing in vivo showed that also dorsal radial glia, labeled at P1 or P2, give rise to subependymal stem cells (Ventura and Goldman 2007). The conversion of radial glia into TypeB cells involves the loss of the radial morphology and a slowing down of the cell cycle. These changes might be due to alterations in the neurogenic requirements of the mammalian telencephalon at postnatal stages. Since, apparently, in physiological conditions, adult mammals need to generate only olfactory bulb interneurons, which in all vertebrates use a specialized form of glial tracks (Adolf et al. 2006; Curtis et al. 2007; Doetsch and Scharff 2001; Perez-Canellas et al. 1997); progenitor cells no longer need a long basal process to guide the migration of newly-generated neurons. Notably, in vertebrates that undergo widespread adult neurogenesis, like fish, reptiles and amphibians, radial glia persists during adult life (Adolf et al. 2006; Alvarez-Buylla 1990; Chapouton et al. 2006; Echeverri and Tanaka 2002; Garcia-Verdugo et al. 2002; Grandel et al. 2006). Consistently with the idea that the conversion of radial glia into TypeB cells is mainly a morphological change, the two cell types share molecular regulatory mechanisms, like the neurogenic role of the transcription factor Pax6. When retroviral vectors were used in vivo to transduce Pax6 in adult NSCs, the number of migrating neuroblasts increased, whereas the use of a Pax6 dominant negative-expressing virus or a Cre-expressing virus in a Pax6-floxed background caused a reduction in the neurogenic activity of adult NSCs (Hack et al. 2005). Also the role of the Notch signaling pathway is shared between radial glia and NSCs. Activated Notch overexpression in the TypeB cells diminishes their neurogenic

potential and forces them to maintain their astroglial phenotype in a quiescent state (Chambers et al. 2001). Taken together, these findings show that despite the clear morphological transformations occurring during the radial glia-to-adult NSC transition, adult NSCs appear to share regulatory mechanisms with their embryonic ancestors.

Stem cells acquire radial glia features during neural differentiation In the last decade, numerous efforts have been directed at the in vitro generation of neurogenic stem cells, aiming to provide the basis for possible future therapeutic applications. In view of their pluripotency, embryonic stem cells (ES) have been considered as particularly promising candidates (Glaser et al. 2007; Smith 2001). Other natural candidates are the adult neural stem cells of the subependymal layer,

a Blastocyst

ES cells

NE-like cells

RG-like cells

b Embryonic VZ/SVZ

Embryonic NPCs

c Adult SVZ

Adult NSCs

Fig. 5 In vitro cultures of neural stem cells. Radial glia-like cells appear when embryonic (a), fetal (b) and adult (c) stem cells are induced to generate neurons. NE Neuroepithelial cells; NPCs neural progenitor cells; RG radial glia

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which can be extensively maintained in vitro and are able to differentiate towards the three main neural lineages (Reynolds and Weiss 1996). For a long time, however, the preferentially gliogenic activity of these cell types precluded the generation of appreciably high numbers of neurons in vitro (Englund et al. 2002; Winkler et al. 1998). Recently, this situation has changed, and new protocols allowing an almost homogeneous differentiation towards specific neuronal subtypes have been established (Bibel et al. 2004; Conti et al. 2005). ES cells cultured as embryoid bodies for a short time, and then exposed to retinoic acid (RA), differentiate homogeneously, eventually up-regulating neuronal markers like β-III-tubulin, synaptophysin, and vescicular glutamate transporter vGlut1, and acquiring a typical pyramidal neuron morphology and the characteristic electrophysiological activity (Bibel et al. 2004). Notably, during the progression from ES cells to mature neurons, the cells pass through a phase in which they acquire both morphological and molecular features of radial glial cells. Nearly all cells show a spindle-shaped morphology and become positive for Nestin, RC2 and BLBP (Bibel et al. 2004; Liour and Yu 2003; Plachta et al. 2004; Fig. 5). In addition, these radial glia-like precursor cells up-regulate the expression of the transcription factor Pax6, before differentiating into neurons, showing that they obey the same differentiation cues as radial glia of the dorsal telencephalon and adult subependymal NSCs (Bibel et al. 2004). A similar acquisition of a radial glia-like phenotype is also shown by murine fetal- and adult- and human fetalstem cells expanded as a monolayer in the presence of EGF and bFGF (Conti et al. 2005; Pollard et al. 2006; Fig. 5). This culture method was originally designed to expand populations of neurally induced ES cells in vitro, but it proved suitable for the propagation of somatic neural stem cells as well. In all the aforementioned cases, cultured cells were found to be immunopositive for the main radial glial markers and for the transcription factor Pax6 (Conti et al. 2005; Pollard et al. 2006). Interestingly, time-lapse video microscopy showed that adherent cultures of radial glia-like cells undergo interkinetic nuclear migration, suggesting a cell-autonomous nature for this process. Unlike observations in physiological conditions, however, the nuclear migration is not coordinated to the cell cycle (Conti et al. 2005), consistently with the notion that cell cycle and interkinetic nuclear migration rely on independent molecular mechanisms, and can be uncoupled by pharmacological treatment (Messier and Auclair 1974; Murciano et al. 2002). Taken together, these data suggest that the acquisition of the radial glia phenotype is a sort of mandatory pathway towards neuronal differentiation. Furthermore, the faithful reproduction of a complex developmental program in vitro suggests that most of the necessary steps along this process might be cell-autonomously encoded.

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Concluding remarks Since the first discovery of the neurogenic role of radial glia, significant advances have been made. Radial glia has been recognized as a heterogeneous population of progenitor cells variably committed to different neural fates, and some molecular cues directing their differentiation have been identified. Up to now, however, we have only limited information allowing a clear distinction between differently committed subsets of radial glia cells. Moreover, considering that radial glia has been shown to eventually give rise to multipotent adult neural stem cells, it is a primary issue to understand whether the observed developmental fate restrictions are reversible, or rather if early neuroepithelial cells/radial glia contain a specific subpopulation of unrestricted progenitors that is fated to be maintained during the entire life of the organism. Notably, much recent evidence suggests that the radial glial phenotype is a mandatory intermediate between any neural stem cells and their differentiated progeny. Acknowledgment We would like to acknowledge Prof. M. Götz for helpful comments on the manuscript.

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