Download Radial glia and neural stem cells | SpringerLink

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
166
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
Cell Tissue Res (2008) 331:165–178
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
Cell Tissue Res (2008) 331:165–178
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
167
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
168
Cell Tissue Res (2008) 331:165–178
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
Cell Tissue Res (2008) 331:165–178
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.
169
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
170
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
Cell Tissue Res (2008) 331:165–178
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).
Cell Tissue Res (2008) 331:165–178
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).
171
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
172
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
Cell Tissue Res (2008) 331:165–178
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
Cell Tissue Res (2008) 331:165–178
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.
173
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
174
Cell Tissue Res (2008) 331:165–178
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
Cell Tissue Res (2008) 331:165–178
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.
175
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.
References
Aaku-Saraste E, Hellwig A, Huttner WB (1996) Loss of occludin and
functional tight junctions, but not ZO-1, during neural tube
closure–remodeling of the neuroepithelium prior to neurogenesis.
Dev Biol 180:664–679
Adolf B, Chapouton P, Lam CS, Topp S, Tannhauser B, Strahle U,
Gotz M, Bally-Cuif L (2006) Conserved and acquired features of
adult neurogenesis in the zebrafish telencephalon. Dev Biol
295:278–293
Akimoto J, Itoh H, Miwa T, Ikeda K (1993) Immunohistochemical
study of glutamine synthetase expression in early glial development. Brain Res Dev Brain Res 72:9–14
Alvarez-Buylla A (1990) Mechanism of neurogenesis in adult avian
brain. Experientia 46:948–955
Anthony TE, Klein C, Fishell G, Heintz N (2004) Radial glia serve as
neuronal progenitors in all regions of the central nervous system.
Neuron 41:881–890
Appolloni I, Calzolari F, Corte G, Perris R, Malatesta P (2007) Six3
controls the neural progenitor status in the murine CNS. Cereb
Cortex DOI 10.1093/cercor/bhm092
Barnabe-Heider F, Wasylnka JA, Fernandes KJ, Porsche C, Sendtner M,
Kaplan DR, Miller FD (2005) Evidence that embryonic neurons
regulate the onset of cortical gliogenesis via cardiotrophin-1. Neuron
48:253–265
Bartsch S, Bartsch U, Dorries U, Faissner A, Weller A, Ekblom P,
Schachner M (1992) Expression of tenascin in the developing
and adult cerebellar cortex. J Neurosci 12:736–749
Bibel M, Richter J, Schrenk K, Tucker KL, Staiger V, Korte M, Goetz M,
Barde YA (2004) Differentiation of mouse embryonic stem cells into
a defined neuronal lineage. Nat Neurosci 7:1003–1009
Bolteus AJ, Bordey A (2004) GABA release and uptake regulate
neuronal precursor migration in the postnatal subventricular
zone. J Neurosci 24:7623–7631
176
Bonni A, Sun Y, Nadal-Vicens M, Bhatt A, Frank DA, Rozovsky I,
Stahl N, Yancopoulos GD, Greenberg ME (1997) Regulation of
gliogenesis in the central nervous system by the JAK-STAT
signaling pathway. Science 278:477–483
Brenner M, Messing A (1996) GFAP Transgenic Mice. Methods
10:351–364
Britanova O, Akopov S, Lukyanov S, Gruss P, Tarabykin V (2005)
Novel transcription factor Satb2 interacts with matrix attachment
region DNA elements in a tissue-specific manner and demonstrates cell-type-dependent expression in the developing mouse
CNS. Eur J Neurosci 21:658–668
Britz O, Mattar P, Nguyen L, Langevin LM, Zimmer C, Alam S,
Guillemot F, Schuurmans C (2006) A role for proneural genes in
the maturation of cortical progenitor cells. Cereb Cortex 16
(Suppl 1):i138–i151
Cai L, Hayes NL, Nowakowski RS (1997) Local homogeneity of
cell cycle length in developing mouse cortex. J Neurosci 17:
2079–2087
Calegari F, Huttner WB (2003) An inhibition of cyclin-dependent
kinases that lengthens, but does not arrest, neuroepithelial cell
cycle induces premature neurogenesis. J Cell Sci 116:4947–4955
Calegari F, Haubensak W, Haffner C, Huttner WB (2005) Selective
lengthening of the cell cycle in the neurogenic subpopulation of
neural progenitor cells during mouse brain development. J
Neurosci 25:6533–6538
Cappello S, Attardo A, Wu X, Iwasato T, Itohara S, WilschBrauninger M, Eilken HM, Rieger MA, Schroeder TT, Huttner
WB, Brakebusch C, Gotz M (2006) The Rho-GTPase cdc42
regulates neural progenitor fate at the apical surface. Nat
Neurosci 9:1099–1107
Chambers CB, Peng Y, Nguyen H, Gaiano N, Fishell G, Nye JS
(2001) Spatiotemporal selectivity of response to Notch1 signals
in mammalian forebrain precursors. Development 128:689–702
Chapouton P, Adolf B, Leucht C, Tannhauser B, Ryu S, Driever W,
Bally-Cuif L (2006) her5 expression reveals a pool of neural stem
cells in the adult zebrafish midbrain. Development 133:4293–4303
Chenn A, McConnell SK (1995) Cleavage orientation and the
asymmetric inheritance of Notch1 immunoreactivity in mammalian neurogenesis. Cell 82:631–641
Chenn A, Zhang YA, Chang BT, McConnell SK (1998) Intrinsic
polarity of mammalian neuroepithelial cells. Mol Cell Neurosci
11:183–193
Chiasson BJ, Tropepe V, Morshead CM, van der Kooy D (1999)
Adult mammalian forebrain ependymal and subependymal cells
demonstrate proliferative potential, but only subependymal cells
have neural stem cell characteristics. J Neurosci 19:4462–4471
Choi BH (1981) Radial glia of developing human fetal spinal cord:
Golgi, immunohistochemical and electron microscopic study.
Brain Res 227:249–267
Conti L, Pollard SM, Gorba T, Reitano E, Toselli M, Biella G, Sun Y,
Sanzone S, Ying QL, Cattaneo E, Smith A (2005) Nicheindependent symmetrical self-renewal of a mammalian tissue
stem cell. PLoS Biol 3:e283
Curtis MA, Kam M, Nannmark U, Anderson MF, Axell MZ,
Wikkelso C, Holtas S, van Roon-Mom WM, Bjork-Eriksson T,
Nordborg C, Frisen J, Dragunow M, Faull RL, Eriksson PS
(2007) Human neuroblasts migrate to the olfactory bulb via a
lateral ventricular extension. Science 315:1243–1249
Doetsch F (2003a) The glial identity of neural stem cells. Nat Neurosci
6:1127–1134
Doetsch F (2003b) A niche for adult neural stem cells. Curr Opin
Genet Dev 13:543–550
Doetsch F, Scharff C (2001) Challenges for brain repair: insights from
adult neurogenesis in birds and mammals. Brain Behav Evol
58:306–322
Cell Tissue Res (2008) 331:165–178
Doetsch F, Garcia-Verdugo JM, Alvarez-Buylla A (1997) Cellular
composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J Neurosci
17:5046–5061
Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A
(1999) Subventricular zone astrocytes are neural stem cells in the
adult mammalian brain. Cell 97:703–716
Echeverri K, Tanaka EM (2002) Ectoderm to mesoderm lineage
switching during axolotl tail regeneration. Science 298:1993–1996
Edwards MA, Yamamoto M, Caviness VS Jr (1990) Organization of
radial glia and related cells in the developing murine CNS. An
analysis based upon a new monoclonal antibody marker.
Neuroscience 36:121–144
Englund U, Bjorklund A, Wictorin K (2002) Migration patterns and
phenotypic differentiation of long-term expanded human neural
progenitor cells after transplantation into the adult rat brain. Brain
Res Dev Brain Res 134:123–141
Englund C, Fink A, Lau C, Pham D, Daza RA, Bulfone A,
Kowalczyk T, Hevner RF (2005) Pax6, Tbr2, and Tbr1 are
expressed sequentially by radial glia, intermediate progenitor cells,
and postmitotic neurons in developing neocortex. J Neurosci
25:247–251
Feng L, Hatten ME, Heintz N (1994) Brain lipid-binding protein
(BLBP): a novel signaling system in the developing mammalian
CNS. Neuron 12:895–908
Fode C, Ma Q, Casarosa S, Ang SL, Anderson DJ, Guillemot F
(2000) A role for neural determination genes in specifying
the dorsoventral identity of telencephalic neurons. Genes Dev
14:67–80
Frederiksen K, McKay RD (1988) Proliferation and differentiation of
rat neuroepithelial precursor cells in vivo. J Neurosci 8:1144–1151
Gaiano N, Fishell G (2002) The role of notch in promoting glial and
neural stem cell fates. Annu Rev Neurosci 25:471–490
Gaiano N, Nye JS, Fishell G (2000) Radial glial identity is promoted
by Notch1 signaling in the murine forebrain. Neuron 26:395–404
Galli R, Fiocco R, De Filippis L, Muzio L, Gritti A, Mercurio S,
Broccoli V, Pellegrini M, Mallamaci A, Vescovi AL (2002) Emx2
regulates the proliferation of stem cells of the adult mammalian
central nervous system. Development 129:1633–1644
Gangemi RM, Daga A, Marubbi D, Rosatto N, Capra MC, Corte G
(2001) Emx2 in adult neural precursor cells. Mech Dev 109:323–329
Gangemi RM, Daga A, Muzio L, Marubbi D, Cocozza S, Perera M,
Verardo S, Bordo D, Griffero F, Capra MC, Mallamaci A, Corte
G (2006) Effects of Emx2 inactivation on the gene expression
profile of neural precursors. Eur J Neurosci 23:325–334
Garcia-Verdugo JM, Ferron S, Flames N, Collado L, Desfilis E, Font
E (2002) The proliferative ventricular zone in adult vertebrates: a
comparative study using reptiles, birds, and mammals. Brain Res
Bull 57:765–775
Glaser T, Pollard SM, Smith A, Brustle O (2007) Tripotential
differentiation of adherently expandable neural stem (NS) cells.
PLoS ONE 2:e298
Gotz M, Huttner WB (2005) The cell biology of neurogenesis. Nat
Rev Mol Cell Biol 6:777–788
Gotz M, Bolz J, Joester A, Faissner A (1997) Tenascin-C synthesis
and influence on axonal growth during rat cortical development.
Eur J Neurosci 9:496–506
Gotz M, Stoykova A, Gruss P (1998) Pax6 controls radial glia
differentiation in the cerebral cortex. Neuron 21:1031–1044
Gotz M, Hartfuss E, Malatesta P (2002) Radial glial cells as neuronal
precursors: a new perspective on the correlation of morphology
and lineage restriction in the developing cerebral cortex of mice.
Brain Res Bull 57:777–788
Grandel H, Kaslin J, Ganz J, Wenzel I, Brand M (2006) Neural stem
cells and neurogenesis in the adult zebrafish brain: origin,
Cell Tissue Res (2008) 331:165–178
proliferation dynamics, migration and cell fate. Dev Biol
295:263–277
Hack MA, Saghatelyan A, de Chevigny A, Pfeifer A, Ashery-Padan R,
Lledo PM, Gotz M (2005) Neuronal fate determinants of adult
olfactory bulb neurogenesis. Nat Neurosci 8:865–872
Hartfuss E, Galli R, Heins N, Gotz M (2001) Characterization of CNS
precursor subtypes and radial glia. Dev Biol 229:15–30
Hartfuss E, Forster E, Bock HH, Hack MA, Leprince P, Luque JM,
Herz J, Frotscher M, Gotz M (2003) Reelin signaling directly affects
radial glia morphology and biochemical maturation. Development
130:4597–4609
Hatakeyama J, Bessho Y, Katoh K, Ookawara S, Fujioka M,
Guillemot F, Kageyama R (2004) Hes genes regulate size, shape
and histogenesis of the nervous system by control of the timing
of neural stem cell differentiation. Development 131:5539–5550
Haubensak W, Attardo A, Denk W, Huttner WB (2004) Neurons
arise in the basal neuroepithelium of the early mammalian
telencephalon: a major site of neurogenesis. Proc Natl Acad
Sci USA 101:3196–3201
Haubst N, Georges-Labouesse E, De Arcangelis A, Mayer U, Gotz M
(2006) Basement membrane attachment is dispensable for radial
glial cell fate and for proliferation, but affects positioning of
neuronal subtypes. Development 133:3245–3254
Haydar TF, Ang E Jr, Rakic P (2003) Mitotic spindle rotation and
mode of cell division in the developing telencephalon. Proc Natl
Acad Sci USA 100:2890–2895
He F, Ge W, Martinowich K, Becker-Catania S, Coskun V, Zhu W,
Wu H, Castro D, Guillemot F, Fan G, de Vellis J, Sun YE (2005)
A positive autoregulatory loop of Jak-STAT signaling controls
the onset of astrogliogenesis. Nat Neurosci 8:616–625
Heins N, Cremisi F, Malatesta P, Gangemi RM, Corte G, Price J,
Goudreau G, Gruss P, Gotz M (2001) Emx2 promotes symmetric
cell divisions and a multipotential fate in precursors from the
cerebral cortex. Mol Cell Neurosci 18:485–502
Heins N, Malatesta P, Cecconi F, Nakafuku M, Tucker KL, Hack MA,
Chapouton P, Barde YA, Gotz M (2002) Glial cells generate
neurons: the role of the transcription factor Pax6. Nat Neurosci
5:308–315
Hermanson O, Jepsen K, Rosenfeld MG (2002) N-CoR controls
differentiation of neural stem cells into astrocytes. Nature
419:934–939
Iacopetti P, Michelini M, Stuckmann I, Oback B, Aaku-Saraste E,
Huttner WB (1999) Expression of the antiproliferative gene
TIS21 at the onset of neurogenesis identifies single neuroepithelial cells that switch from proliferative to neuron-generating
division. Proc Natl Acad Sci USA 96:4639–4644
Jankovski A, Sotelo C (1996) Subventricular zone-olfactory bulb
migratory pathway in the adult mouse: cellular composition and
specificity as determined by heterochronic and heterotopic
transplantation. J Comp Neurol 371:376–396
Johansson CB, Momma S, Clarke DL, Risling M, Lendahl U, Frisen J
(1999) Identification of a neural stem cell in the adult mammalian
central nervous system. Cell 96:25–34
Kosodo Y, Roper K, Haubensak W, Marzesco AM, Corbeil D, Huttner WB
(2004) Asymmetric distribution of the apical plasma membrane
during neurogenic divisions of mammalian neuroepithelial cells.
Embo J 23:2314–2324
Landrieu P, Goffinet A (1979) Mitotic spindle fiber orientation in
relation to cell migration in the neo-cortex of normal and reeler
mouse. Neurosci Lett 13:69–72
Levitt P, Rakic P (1980) Immunoperoxidase localization of glial
fibrillary acidic protein in radial glial cells and astrocytes of the
developing rhesus monkey brain. J Comp Neurol 193:815–840
Liour SS, Yu RK (2003) Differentiation of radial glia-like cells from
embryonic stem cells. Glia 42:109–117
177
Lobe CG, Koop KE, Kreppner W, Lomeli H, Gertsenstein M, Nagy A
(1999) Z/AP, a double reporter for cre-mediated recombination.
Dev Biol 208:281–292
Malatesta P, Hartfuss E, Gotz M (2000) Isolation of radial glial cells
by fluorescent-activated cell sorting reveals a neuronal lineage.
Development 127:5253–5263
Malatesta P, Hack MA, Hartfuss E, Kettenmann H, Klinkert W,
Kirchhoff F, Gotz M (2003) Neuronal or glial progeny: regional
differences in radial glia fate. Neuron 37:751–764
McCarthy M, Turnbull DH, Walsh CA, Fishell G (2001) Telencephalic
neural progenitors appear to be restricted to regional and glial fates
before the onset of neurogenesis. J Neurosci 21:6772–6781
Merkle FT, Tramontin AD, Garcia-Verdugo JM, Alvarez-Buylla A
(2004) Radial glia give rise to adult neural stem cells in the
subventricular zone. Proc Natl Acad Sci USA 101:17528–
17532
Messier PE, Auclair C (1974) Effect of cytochalasin B on interkinetic
nuclear migration in the chick embryo. Dev Biol 36:218–223
Miller FD, Gauthier AS (2007) Timing is everything: making neurons
versus glia in the developing cortex. Neuron 54:357–369
Misson JP, Edwards MA, Yamamoto M, Caviness VS Jr (1988)
Mitotic cycling of radial glial cells of the fetal murine cerebral
wall: a combined autoradiographic and immunohistochemical
study. Brain Res 466:183–190
Miyata T, Kawaguchi A, Okano H, Ogawa M (2001) Asymmetric
inheritance of radial glial fibers by cortical neurons. Neuron
31:727–741
Miyata T, Kawaguchi A, Saito K, Kawano M, Muto T, Ogawa M
(2004) Asymmetric production of surface-dividing and non-surfacedividing cortical progenitor cells. Development 131:3133–3145
Mizutani K, Saito T (2005) Progenitors resume generating neurons
after temporary inhibition of neurogenesis by Notch activation in
the mammalian cerebral cortex. Development 132:1295–1304
Mo Z, Moore AR, Filipovic R, Ogawa Y, Kazuhiro I, Antic SD,
Zecevic N (2007) Human cortical neurons originate from radial
glia and neuron-restricted progenitors. J Neurosci 27:4132–4145
Mollgoard K, Saunders NR (1975) Complex tight junctions of epithelial
and of endothelial cells in early foetal brain. J Neurocytol 4:453–468
Murciano A, Zamora J, Lopez-Sanchez J, Frade JM (2002) Interkinetic nuclear movement may provide spatial clues to the
regulation of neurogenesis. Mol Cell Neurosci 21:285–300
Nadarajah B, Brunstrom JE, Grutzendler J, Wong RO, Pearlman AL
(2001) Two modes of radial migration in early development of
the cerebral cortex. Nat Neurosci 4:143–150
Nakashima K, Yanagisawa M, Arakawa H, Kimura N, Hisatsune T,
Kawabata M, Miyazono K, Taga T (1999) Synergistic signaling in
fetal brain by STAT3-Smad1 complex bridged by p300. Science
284:479–482
Namihira M, Nakashima K, Taga T (2004) Developmental stage
dependent regulation of DNA methylation and chromatin
modification in a immature astrocyte specific gene promoter.
FEBS Lett 572:184–188
Nieto M, Schuurmans C, Britz O, Guillemot F (2001) Neural bHLH
genes control the neuronal versus glial fate decision in cortical
progenitors. Neuron 29:401–413
Noctor SC, Flint AC, Weissman TA, Dammerman RS, Kriegstein AR
(2001) Neurons derived from radial glial cells establish radial
units in neocortex. Nature 409:714–720
Noctor SC, Flint AC, Weissman TA, Wong WS, Clinton BK,
Kriegstein AR (2002) Dividing precursor cells of the embryonic
cortical ventricular zone have morphological and molecular
characteristics of radial glia. J Neurosci 22:3161–3173
Noctor SC, Martinez-Cerdeno V, Ivic L, Kriegstein AR (2004)
Cortical neurons arise in symmetric and asymmetric division zones
and migrate through specific phases. Nat Neurosci 7:136–144
178
Novak A, Guo C, Yang W, Nagy A, Lobe CG (2000) Z/EG, a double
reporter mouse line that expresses enhanced green fluorescent
protein upon Cre-mediated excision. Genesis 28:147–155
Patten BA, Sardi SP, Koirala S, Nakafuku M, Corfas G (2006) Notch1
signaling regulates radial glia differentiation through multiple
transcriptional mechanisms. J Neurosci 26:3102–3108
Peretto P, Merighi A, Fasolo A, Bonfanti L (1997) Glial tubes in the
rostral migratory stream of the adult rat. Brain Res Bull 42:9–21
Perez-Canellas MM, Font E, Garcia-Verdugo JM (1997) Postnatal
neurogenesis in the telencephalon of turtles: evidence for
nonradial migration of new neurons from distant proliferative
ventricular zones to the olfactory bulbs. Brain Res Dev Brain Res
101:125–137
Plachta N, Bibel M, Tucker KL, Barde YA (2004) Developmental
potential of defined neural progenitors derived from mouse
embryonic stem cells. Development 131:5449–5456
Pollard SM, Conti L, Sun Y, Goffredo D, Smith A (2006) Adherent
neural stem (NS) cells from fetal and adult forebrain. Cereb
Cortex 16(Suppl 1):i112–i120
Price J, Thurlow L (1988) Cell lineage in the rat cerebral cortex: a
study using retroviral-mediated gene transfer. Development
104:473–482
Qian X, Shen Q, Goderie SK, He W, Capela A, Davis AA, Temple S
(2000) Timing of CNS cell generation: a programmed sequence
of neuron and glial cell production from isolated murine cortical
stem cells. Neuron 28:69–80
Rakic P (1972) Mode of cell migration to the superficial layers of fetal
monkey neocortex. J Comp Neurol 145:61–83
Rakic P (1995) A small step for the cell, a giant leap for mankind: a
hypothesis of neocortical expansion during evolution. Trends
Neurosci 18:383–388
Rakic P (2003) Developmental and evolutionary adaptations of
cortical radial glia. Cereb Cortex 13:541–549
Reynolds BA, Weiss S (1996) Clonal and population analyses
demonstrate that an EGF-responsive mammalian embryonic
CNS precursor is a stem cell. Dev Biol 175:1–13
Sancho-Tello M, Valles S, Montoliu C, Renau-Piqueras J, Guerri C
(1995) Developmental pattern of GFAP and vimentin gene
expression in rat brain and in radial glial cultures. Glia 15:
157–166
Sauer FC (1935) Mitosis in the neural tube. J Comp Neurol 62:377–405
Schmid RS, McGrath B, Berechid BE, Boyles B, Marchionni M,
Sestan N, Anton ES (2003) Neuregulin 1-erbB2 signaling is
required for the establishment of radial glia and their transformation
into astrocytes in cerebral cortex. Proc Natl Acad Sci USA
100:4251–4256
Schnitzer J, Franke WW, Schachner M (1981) Immunocytochemical
demonstration of vimentin in astrocytes and ependymal cells of
developing and adult mouse nervous system. J Cell Biol
90:435–447
Scholzen T, Gerdes J (2000) The Ki-67 protein: from the known and
the unknown. J Cell Physiol 182:311–322
Schuurmans C, Armant O, Nieto M, Stenman JM, Britz O, Klenin N,
Brown C, Langevin LM, Seibt J, Tang H, Cunningham JM, Dyck
R, Walsh C, Campbell K, Polleux F, Guillemot F (2004)
Sequential phases of cortical specification involve Neurogenindependent and -independent pathways. Embo J 23:2892–2902
Shen Q, Wang Y, Dimos JT, Fasano CA, Phoenix TN, Lemischka IR,
Ivanova NB, Stifani S, Morrisey EE, Temple S (2006) The
Cell Tissue Res (2008) 331:165–178
timing of cortical neurogenesis is encoded within lineages of
individual progenitor cells. Nat Neurosci 9:743–751
Shibata T, Yamada K, Watanabe M, Ikenaka K, Wada K, Tanaka K,
Inoue Y (1997) Glutamate transporter GLAST is expressed in the
radial glia-astrocyte lineage of developing mouse spinal cord. J
Neurosci 17:9212–9219
Smart IH (1973) Proliferative characteristics of the ependymal layer
during the early development of the mouse neocortex: a pilot
study based on recording the number, location and plane of
cleavage of mitotic figures. J Anat 116:67–91
Smart IH (1976) A pilot study of cell production by the ganglionic
eminences of the developing mouse brain. J Anat 121:71–84
Smith AG (2001) Embryo-derived stem cells: of mice and men. Annu
Rev Cell Dev Biol 17:435–462
Sommer L, Ma Q, Anderson DJ (1996) Neurogenins, a novel family
of atonal-related bHLH transcription factors, are putative mammalian neuronal determination genes that reveal progenitor cell
heterogeneity in the developing CNS and PNS. Mol Cell
Neurosci 8:221–241
Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre
reporter strain. Nat Genet 21:70–71
Spassky N, Merkle FT, Flames N, Tramontin AD, Garcia-Verdugo
JM, Alvarez-Buylla A (2005) Adult ependymal cells are
postmitotic and are derived from radial glial cells during
embryogenesis. J Neurosci 25:10–18
Stricker SH, Meiri K, Gotz M (2006) P-GAP-43 is enriched in
horizontal cell divisions throughout rat cortical development.
Cereb Cortex 16(Suppl 1):i121–i131
Sun Y, Nadal-Vicens M, Misono S, Lin MZ, Zubiaga A, Hua X, Fan G,
Greenberg ME (2001) Neurogenin promotes neurogenesis and
inhibits glial differentiation by independent mechanisms. Cell
104:365–376
Takizawa T, Nakashima K, Namihira M, Ochiai W, Uemura A,
Yanagisawa M, Fujita N, Nakao M, Taga T (2001) DNA
methylation is a critical cell-intrinsic determinant of astrocyte
differentiation in the fetal brain. Dev Cell 1:749–758
Tarabykin V, Stoykova A, Usman N, Gruss P (2001) Cortical upper
layer neurons derive from the subventricular zone as indicated by
Svet1 gene expression. Development 128:1983–1993
Ventura RE, Goldman JE (2007) Dorsal radial glia generate olfactory
bulb interneurons in the postnatal murine brain. J Neurosci
27:4297–4302
Vives V, Alonso G, Solal AC, Joubert D, Legraverend C (2003)
Visualization of S100B-positive neurons and glia in the central
nervous system of EGFP transgenic mice. J Comp Neurol
457:404–419
Voigt T (1989) Development of glial cells in the cerebral wall of
ferrets: direct tracing of their transformation from radial glia into
astrocytes. J Comp Neurol 289:74–88
Williams BP, Price J (1995) Evidence for multiple precursor cell types
in the embryonic rat cerebral cortex. Neuron 14:1181–1188
Winkler C, Fricker RA, Gates MA, Olsson M, Hammang JP,
Carpenter MK, Bjorklund A (1998) Incorporation and glial
differentiation of mouse EGF-responsive neural progenitor cells
after transplantation into the embryonic rat brain. Mol Cell
Neurosci 11:99–116
Zhong W, Feder JN, Jiang MM, Jan LY, Jan YN (1996) Asymmetric
localization of a mammalian numb homolog during mouse
cortical neurogenesis. Neuron 17:43–53