Davee Department of Neurology, Northwestern University's Feinberg School of Medicine, Chicago, IL 60611, USA
* Author for correspondence (e-mail: m-bonaguidi{at}northwestern.edu)
Accepted 13 October 2005
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SUMMARY |
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Key words: GFAP, Glia, Astrocyte, Neural stem cell, Leukemia inhibitory factor, Bone morphogenetic protein, Noggin
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Introduction |
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NSCs express GFAP in response to several signaling molecules, including the
leukemia inhibitory factor (LIF)/ciliary neurotrophic factor (CNTF) and BMP
families (Gross et al., 1996;
Johe et al., 1996
).
Canonically, LIF/CNTF activates the JAK/STAT pathways, whereas BMPs signal
primarily through SMAD pathways. Nevertheless, their signaling pathways have
points of convergence in the regulation of GFAP, leading to suggestions that
these cytokine families activate astrogliogenesis through the same mechanisms
(Nakashima et al., 1999a
;
Sun et al., 2001
). However,
whereas BMP signaling promotes the generation of astrocytes from SVZ forebrain
stem cells both in vitro and in vivo
(Gomes et al., 2003
;
Gross et al., 1996
), LIF
signaling inhibits the restriction of early embryonic forebrain stem cells to
a glial lineage and helps to maintain a stem cell phenotype
(Shimazaki et al., 2001
).
Furthermore, BMP2 treatment of progenitor cells cultured from
LIFR-/- animals induces astrogliogenesis
(Koblar et al., 1998
),
indicating that signaling from this receptor is not necessary for the
generation of astrocytes. It is currently unclear whether LIF and BMP
signaling generate GFAP-expressing cells with similar characteristics and
developmental potential. We therefore used a combined in vitro and in vivo
approach to compare the properties of GFAP-expressing cells that are generated
in response to LIF versus BMP signaling. Our findings suggest that LIF
signaling induces GFAP+ progenitor cells, whereas BMP signaling
promotes a mature astrocyte phenotype that lacks stem/progenitor cell
potential.
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Materials and methods |
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Immunochemistry
Postnatal day 15 (P15) brains were fresh-frozen on dry ice, cut into 10
µm coronal sections, fixed in 4% paraformadehyde (PFA), and blocked with
10% goat serum for 1 hour. Primary antibodies diluted in PBS containing 1% BSA
and 0.25% Triton X-100 were applied overnight at 4°C. Antibodies were as
follows: Ki67 (rabbit polyclonal, 1:1000; Novocastra), GFAP (mouse IgG1 or
rabbit polyclonal, 1:400; Sigma), vimentin (mouse IgM, 1:4; Developmental
Studies Hybridoma Bank). Primary antibodies were visualized with mouse or
rabbit Cy2- or Cy3-conjugated secondary antibodies (Jackson Laboratories).
Nuclei were counterstained with Hoechst 33342 (Sigma). Cells were counted in
the dentate SGZ (a two-nucleus-wide band below the apparent border between the
GCL and the hilus and inner third of the GCL) and ML (superior/dorsal to the
GCL), and normalized to the area analyzed in mm2
(Kempermann et al., 2003).
Bromodeoxyuridine (BrdU) labeling
BrdU (10 mM) was added to differentiating neural cells on day 6, and
processed on day 7 after 16 hours. Cells were fixed with 4% PFA and processed
with 2N HCl for 45 minutes, then 0.1 M Borax (pH 8.5) for 15 minutes before
immunochemistry.
Immunochemistry of cultures
Prior to PFA fixation, 15 µl/ml O4 (mouse IgM, Chemicon) and 5 µg/ml
LeX/CD15 (mouse IgM, clone MMA; BD Biosciences) were added to cells for 30
minutes at 4°C. Fixed coverslips were blocked with serum for 45 minutes
and incubated with primary antibodies at room temperature for 2-3 hours.
Antibodies were as follows: BrdU (mouse IgG2a, 1:1000; clone BU-1, Chemicon),
ßIII-tubulin (mouse IgG2b, 1:400; Sigma), SOX1 (rabbit polyclonal,
1:1000; a kind gift from Dr Hisato Kondoh, Osaka University). Primary
antibodies were visualized with Alexa 647-(infrared) Alexa 555/594-(red),
Alexa 488-(green) and Alexa 350-(blue) conjugated secondary antibodies
(Molecular Probes). Cells were counted in seven alternate fields of each
coverslip and verified in a minimum of three independent experiments.
Generation of progenitor cell neurospheres and differentiation cultures
The ganglionic eminences of E18.5 mice were dissociated and grown in
serum-free medium (SFM) with EGF (20 ng/ml, human recombinant, Biosource) for
7 days, as previously described, to generate neurospheres
(Mehler et al., 2000;
Zhu et al., 1999
). Primary
spheres were grown for 3-4 days in vitro (DIV), and then passaged by
dissociating with 0.25% trypsin (Invitrogen) for 2 minutes followed by
incubation with a soybean trypsin inhibitor (Sigma), a 5-minute spin, and
repeated trituration. Secondary spheres were grown for an additional 3-4 DIV
and used for subsequent studies. For differentiation studies, neurospheres
were dissociated and plated at a density of 1x104
cells/cm2 onto poly-D-lysine-coated (PDL, Sigma, 20 µg/ml for
>1 hour) coverslips within 24-well culture plates, and then grown for 7 DIV
in SFM plus 2 ng/ml EGF and 250 ng/ml Noggin (R&D Systems), 20 ng/ml LIF
(Chemicon) or LIF+Noggin, or 20 ng/ml BMP4 (R&D Systems). Cells were
re-fed on day 3.
Retrovirus production and neurosphere infection
The EGFP-N1 cassette (Clontech) was cloned into the BglII and
BstBI sites on the pLXRN retrovirus shuttle vector (Clontech) and
replaced the G418r cassette. The rat 1.9 kb GFAP promoter
(Sun et al, 2001) was
partially digested and inserted into the HpaI and BglII
shuttle sites, which excised the PRSV. Virus was packaged by
co-transfecting the shuttle (rGFAPp-EGFP or control PRSV-EGFP) with
VSVG into GP2-293 cells (Clontech), using Lipofectamine 2000 (Invitrogen).
Supernatant collected on days 2, 3 and 6 was concentrated 1000x and
stored at -80°C until use. Secondary neurospheres were passaged, infected
with 15 µl virus in 10 ml medium the day following dissociation, cultured
and passaged once more before plating for differentiation.
Fluorescent-activated cell sorting (FACS) and neurosphere-forming assay
Plated cells were harvested, resuspended at a density of
1x106 cells/ml in SFM, and sorted on the basis of
forward-side scatter and GFP expression at 1000 events/second. Sorted cells
were plated at a density of 1000 cells/well into non-adherent 96-well plates
containing SFM plus 20 ng/ml EGF, or 2 ng/ml EGF plus 20 ng/ml LIF where
denoted. Cell survival was >85% by Trypan Blue exclusion analysis. The
numbers of free-floating spheres were counted at day 7 in a minimum of three
independent experiments.
RT-QPCR (reverse transcriptase-quantitative polymerase chain reaction)
Plated cells were treated with cytokines for 20 hours before harvesting RNA
using RNeasy, according to the manufacturer's protocol (Qiagen). Reverse
transcriptase (RT) was performed using Thermoscript (Invitrogen), and QPCR
using Platinum SYBR Green (Stratagene). Specificity of the PCR reaction was
confirmed by running PCR products on a 2% agarose gel. Two replicates were run
for each cDNA sample with the test and control primers. An amplification plot
showing cycle number versus the change in fluorescent intensity was generated
by the Sequence Detector program (Applied Biosystems).
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Results |
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Although treatment with LIF or BMP4 each increases the number of cells expressing GFAP, the morphologies of those cells differ drastically (Fig. 1D-F,H-J). Treatment with LIF alone promotes a mixture of elongated bipolar/tripolar and stellate morphologies (Fig. 1E). However, when noggin is included to inhibit endogenous BMP signaling, LIF treatment promotes the bipolar/tripolar morphology at the expense of a stellate one (Fig. 1F,I). By contrast, treatment with BMP4 leads to cells with a stellate morphology characteristic of some mature astrocytes (Fig. 1D,H). GFAP+ cells generated after BMP4 treatment had an average of six major processes per cell, whereas GFAP+ cells generated in the presence of LIF and noggin have an average of slightly more than two major processes per cell (Fig. 1J).
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LIF increases, whereas BMP4 decreases, neural precursor markers in GFAP+ cells
The foregoing observations suggested that the GFAP+ cells
generated by LIF signaling might represent stem/progenitor cells, whereas the
quiescent GFAP+ cells generated by BMP signaling might represent
more differentiated astrocytes. We therefore compared these populations of
cells with respect to the expression of neural stem/progenitor cell markers.
We first examined the SRY transcription factor SOX1, which is expressed by
both early and adult progenitors cells, but not by astrocytes
(Bylund et al., 2003). SOX1 was
expressed by just 7.0±1.1% of the control GFAP+ cells
(Fig. 3A,F). However noggin,
LIF and LIF plus noggin all increased the number of
SOX1+GFAP+ precursors by approximately 200%
(Fig. 3B-D,F;
P<0.005). Conversely, BMP4 decreased the number of
SOX1+GFAP+ precursors by 43%
(Fig. 3E,F;
P<0.05). The glycoprotein LeX (CD15/SSEA1) is also expressed by
neural stem/progenitor cells (Capela and
Temple, 2002
; Kim and
Morshead, 2003
). In control cultures, 11.7±1.5% of
GFAP+ cells expressed LeX (Fig.
3G,L). Inhibition of endogenous BMP by noggin did not alter LeX
expression by GFAP+ cells (Fig.
3H,L). However, LIF treatment increased the number of
LeX+GFAP+ cells by 81% (20.2±2.2%,
P<0.005; Fig.
3I,L), and LIF plus noggin increased
LeX+GFAP+ cells by 127% (25.1±2.0%,
P<0.005; Fig.
3J,L). By contrast, BMP4 treatment decreased the number of
LeX+GFAP+ cells by 92% (1.0±0.8%,
P<0.005; Fig.
3K,L), and virtually no LeX+ cells were present in the
BMP4-treated cultures. Additionally, the same pattern of findings was observed
with a third neural progenitor marker, the intermediate filament vimentin
(M.A.B., unpublished).
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LIF increases neuron production from GFAP-expressing cells
To determine whether exposure to LIF alters the potential of
GFAP+ cells, control or cytokine-treated GFAP+-derived
neurospheres (derived from the FACS-sorted cells) were dissociated and plated
for 4-5 days of differentiation. The resultant cells were processed for
ßIII-tubulin to determine neuron numbers. Control spheres produced a
small number of neurons (3.8±2.5%;
Fig. 4I,M). BMP inhibition
(noggin) did not significantly change the number of neurons generated,
although there was a trend towards an increase (6.8±1.5%;
Fig. 4J,M). However, LIF
increased neuron numbers by 90% (11.0±1.1%, P<0.005;
Fig. 4K,M). Moreover, LIF plus
noggin further increased neurogenesis by 55% relative to LIF alone
(17.1±2.8%, P<0.005;
Fig. 4L,M). These observations
demonstrate that LIF-induced GFAP+ cells have an increased ability
to produce neurons that is further enhanced by suppressing BMP signaling.
LIF maintains GFAP-expressing multipotent progenitors for prolonged periods of time
We then investigated whether LIF could maintain the GFAP+
progenitor cell state for prolonged periods of time. Because high mitogen
levels may reprogram more committed cells to exhibit progenitor
characteristics that they might otherwise not display
(Kondo and Raff, 2000;
Anderson, 2001
), neural
progenitors were plated with only low (1-2 ng/ml) levels of EGF with LIF, and
were passaged repeatedly. Neural cells could be propagated this way as a
monolayer for at least 10 passages and for several months
(Fig. 5A). By contrast, in the
absence of LIF, the cells became progressively sparser with attempted
passaging and could only be passaged a few times. We then examined whether
GFAP-expressing cells generated in the presence of LIF could still proliferate
(incorporate BrdU) and express the immunocytochemical characteristics of
progenitor cells at the higher passages. In the LIF-treated cultures,
GFAP-expressing cells at passage 7 (p7) still incorporated BrdU (2-hour pulse)
and expressed the progenitor marker LeX
(Fig. 5B). LIF-treated cultures
were also able to generate neurons at high passages (>p7), whereas nearby
GFAP-expressing cells remained in cell cycle, as assayed by Ki67 expression
(Fig. 5C). To directly assess
the self-renewal and multipotentiality of GFAP+ cells maintained by
LIF, higher passage (>p7) cells were infected with the rGFAPp-EGFP
retrovirus, selected by FACS, plated in a neurosphere-forming assay (2 ng/ml
EGF plus 20 ng/ml LIF) and subsequent spheres plated for differentiation.
GFAP-expressing cells maintained by LIF were able to self-renew at high
passages, as evidenced by neurosphere formation, and they retained
multipotentiality and, specifically, the ability to produce neurons
(Fig. 5E). Thus, LIF maintains
GFAP-expressing cells as multipotential stem/progenitor cells for prolonged
periods of time, independent of exposure to high levels of other mitogens.
BMPs regulate the morphology of GFAP-expressing cells in vivo, and are necessary and sufficient for cell-cycle exit
In vivo, GFAP-expressing astrocytes are derived from radial glia during the
postnatal period (Schmechel and Rakic,
1979; Voigt,
1989
). Most of these cells lose progenitor function, but radial
glia also give rise to GFAP+ adult stem cells in the SVZ and
hippocampus (Eckenhoff and Rakic,
1984
; Merkle et al.,
2004
; Rickmann et al.,
1987
; Voigt,
1989
). To test the hypothesis that BMPs developmentally regulate
the maturation of GFAP+ cells in vivo, we generated transgenic
animals that overexpress either BMP4 or its antagonist noggin under the
control of the neuron-specific enolase (NSE) promoter
(Gomes et al., 2003
;
Guha et al., 2004
). Transgene
expression begins before gliogenesis at embryonic day 16 (E16), peaks
postnatally, and persists into adult life
(Gomes et al., 2003
).
Therefore, these animals serve as an excellent model to study the development
of GFAP-expressing cells. BMP4 overexpressing animals have an increased number
of GFAP+ cells in the brain, whereas the noggin overexpressing
animals conversely have significantly reduced numbers of GFAP+
cells (Gomes et al., 2003
)
(see also Fig. S2 in the supplementary material). Similarly, BMP4
overexpressing animals have increased numbers of S100ß-expressing cells,
whereas numbers of these cells are significantly reduced in the noggin
transgenic animals (Gomes et al.,
2003
) (Fig. S2 in the supplementary material). In the adult brain,
GFAP+ progenitor cells in neurogenic regions including the
hippocampal SGZ remain in cell cycle (Seri
et al., 2001
; Garcia et al.,
2004
). As the NSE transgene is expressed at the highest levels in
the hippocampus (Gomes et al,
2003
), we examined the effects of BMP signaling on the
proliferation of GFAP+ cells in the SGZ using Ki67 and GFAP double
labeling. Overexpression of BMP4 depleted the SGZ of
Ki67+GFAP+ cells, whereas inhibition of BMP signaling by
noggin significantly increased the number of these cells
(Fig. 6). Specifically, at P15,
60.6±3.8 GFAP-expressing cells/mm2 (in 10 µm sections)
remained in cell cycle in the SGZ of wild-type animals
(Fig. 6B,L). Inhibition of BMP
signaling significantly increased the number of
Ki67+GFAP+ cells by nearly 100% (118.4±2.4
cells/mm2, P<0.03;
Fig. 6A,L). Conversely,
overexpression of BMP4 reduced the number of these cells by 92%
(10.4±1.3 cells/mm2, P<0.03;
Fig. 6C,L). High magnification
confocal images demonstrated the co-localization of Ki67 and GFAP in the SGZ
(Fig. 6D, and Fig. S3 in the
supplementary material). To determine whether BMP signaling is necessary for
promoting the cell-cycle exit of GFAP+ cells, we investigated
co-labeling with Ki67 in the hippocampal molecular layer (ML), an area that
does not normally contain proliferative GFAP+ cells in the adult
(Garcia et al., 2004
). We
found only rare Ki67+GFAP+ cells in the ML in P15
wild-type mice (6.2±0.3 cells/mm2;
Fig. 6F,L), and BMP4
overexpression almost completely depleted this small population of cells
(1.1±0.2 cells/mm2, P<0.04;
Fig. 6G,L). However, noggin
overexpression markedly increased the number of cycling GFAP+ cells
remaining in the ML (27.4±1.5 cells/mm2, P<0.01;
Fig. 6F,H,L), demonstrating
that BMP is necessary for the normal exit of GFAP+ cells from the
cell cycle in this region.
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|
BMPs regulate the maturation of GFAP-expressing cells in vivo
The foregoing observations suggested that BMPs regulate the maturation of
GFAP-expressing cells in vivo by promoting cell-cycle exit and increasing
process ramification. Consequently, we further investigated whether BMP
signaling regulates the maturation of GFAP+ progenitor cells into
astrocytes in vivo by examining the expression of progenitor cell markers in
the hippocampal SGZ and ML. Because LeX staining was difficult to quantitate
in vivo, we instead used the progenitor marker vimentin, in addition to SOX1
(Garcia et al., 2004;
Seri et al., 2004
). The
wild-type SGZ contained 32.5±0.7 cells/mm2 that were
GFAP+SOX1+ and 157.6±0.2 cells/mm2
that were GFAP+vimentin+
(Fig. 7B,I-J). In noggin
overexpressing animals, the number of GFAP+ progenitors was
significantly increased, as assayed both by SOX1 (83.9±4.6
cells/mm2, P<0.02,
Fig. 7A,D,I) and by vimentin
(209.8±4.4 cells/mm2, P<0.03;
Fig. 7J, see also Fig. S4 in
the supplementary material). By contrast, BMP4 overexpression in the
developing SGZ reduced the number of GFAP+ progenitors, as assessed
by SOX1 (18.2±1.5 cells/mm2, P<0.02;
Fig. 7C,I) and vimentin
(101.1±1.2 cells/mm2, P<0.03,
Fig. 7J, Fig. S4). Thus, BMP
signaling promotes the loss of progenitor markers, as well as a quiescent
state in GFAP+ cells in neurogenic areas. To determine whether BMPs
are necessary for the maturation of GFAP+ cells in non-neurogenic
regions, we analyzed SOX1 and vimentin expression in the ML of the
noggin-overexpressing animals. GFAP+ cells in the ML rarely
co-express SOX1 or vimentin in wild-type mice (SOX1, 3.9±0.2
cells/mm2; vimentin, 10.5±3.1 cells/mm2;
Fig. 7F,I,J), or in BMP4
overexpressing mice (SOX1, 3.0±0.2 cells/mm2; vimentin,
8.7±5.0 cells/mm2; Fig.
7G,I,J; Fig. S5 in the supplementary material). However, BMP
inhibition (noggin overexpression) in the ML increased the number of
GFAP+ cells maintaining progenitor cell markers, suggesting that
BMP signaling is necessary for astrocyte maturation in this region (SOX1,
7.3±0.2 cells/mm2, P<0.05; vimentin,
37.9.±5.1 cells/mm2, P<0.01;
Fig. 7E,H-J and Fig. S4). Thus,
BMP signaling in vivo causes GFAP+ cells to exit the cell cycle and
lose progenitor markers in normally neurogenic regions, similar to the
observations in vitro. Conversely, inhibiting BMP signaling in non-neurogenic
regions in vivo prevents GFAP+ cells from maturing, as assessed by
exit from the cell cycle and the loss of progenitor markers that typically
occur in this region (Hutchins and
Casagrande, 1989
; Bylund et
al., 2003
).
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Discussion |
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Substantial evidence has been generated previously indicating that LIF
signaling exerts effects on re-entry into cell cycle and on the maintenance of
stem/progenitor cell traits. Null mutation of the LIF receptor (LIFR)
decreases the number of progenitors derived from E14 brain in vitro, and a
reduction in the levels of gp130 decreases progenitor cell re-entry into cell
cycle in vivo (Hatta et al.,
2002; Shimazaki et al.,
2001
). Conversely, LIF injection in vivo or treatment of E14
progenitors in vitro increases re-entry into cell cycle
(Hatta et al., 2002
;
Pitman et al., 2004
). In the
adult, LIFR+/- mice show a loss of EGF-responsive progenitors
derived from the SVZ, whereas CNTF injection increases the number of
multipotential adult progenitors in vivo and in vitro at the expense of
glial-restricted cells (Shimazaki et al.,
2001
). These observations are fully concordant with our finding
that LIF signaling promotes a stem/progenitor cell state. In most neurosphere
cultures, only about 2% of the cells are typically `stem' cells that have the
capacity to self-renew; the remainders are multipotent progenitor cells. This
same principle probably applies to the GFAP+ cells in the LIF plus
noggin conditions. These cells are definitely not terminally differentiated
astrocytes, as they proliferate, express progenitor cell markers, and display
multipotentiality. In fact, when LIF-generated GFAP+ cells were
subsequently treated with BMP4, they adopted the morphology and
characteristics of BMP4-generated astrocytes (M.A.B., unpublished). These
findings are consistent with a hypothesis that LIF signaling generates a
GFAP+ progenitor cell that differentiates into an astrocyte under
the influence of BMP signaling (Fig.
8).
Mechanisms for astrocyte generation
Astrocyte generation has been postulated to occur through the convergence
of LIF/CNTF and BMP signaling. The transcriptional co-activator p300 bridges
the LIF and BMP signaling targets STAT3 and SMAD1, and mediates their
cooperative effects on GFAP expression
(Nakashima et al., 1999a). Our
studies do not address the question of whether BMP signaling can promote
astrogliogenesis without some prior activation of STAT signaling. BMP2
treatment of progenitor cells cultured from LIFR-/- animals induces
astrogliogenesis (Koblar et al.,
1998
), indicating that the LIFR is not required, but it is
possible that STAT signaling is activated by cytokines that do not utilize the
LIFR such as EGF (Shuai et al.,
1993
; Zhong et al.,
1994
). In this regard, it is noteworthy that BMP signaling does
not promote astroglial differentiation of early embryonic neural stem cells,
but rather promotes neuronal differentiation
(Mabie et al., 1999
). This may
reflect both the presence of high levels of neurogenin1/2 (NGN1/2) that
sequester the CBP-SMAD1 transcription complex away from astrocyte
differentiation genes (Sun et al.,
2001
), and the absence of signaling from the EGFR
(Eagleson et al., 1996
;
Gross et al., 1996
;
Zhu et al., 1999
). EGFR is
associated with a switch in bias of the cells from neurogenesis to gliogenesis
(Burrows et al., 1997
), and
EGFR regulates the ability of stem cells to interpret LIF, but not BMP, as a
GFAP+-inducing agent (Viti et
al., 2003
). EGFR expression is upregulated in neural stem cells
between E13 and E16 in mice (Burrows et
al., 1997
), and GFAP expression in response to LIF and BMP can
first be observed at approximately E14.5
(Eagleson et al., 1996
;
Gross et al., 1996
). FGF2
signaling also primes neural stem cells for gliogenesis, at least in part, by
the removal of histone methylation at the STAT-binding site on the rat GFAP
promoter (Song and Ghosh,
2004
). This may reflect an FGF2-mediated increase of EGFR
expression (Lillien and Raphael,
2000
). Concurrent with increased EGFR expression levels, there is
a decrease in the expression of genes that inhibit gliogenesis
(Ngn1/2) (Sun et al.,
2001
), and an increase in the expression of other putative
proglial genes, such as hairy-enhancer of split 1/5 (Hes1/5)
and Hes-related genes 1 and 2 (Takizawa et
al., 2003
). Thus, the ability of neural stem cells to interpret
BMPs as pro-astrocytic differentiation factors depends upon the status of
other signaling pathways and the intrinsic regulation in the cell, but may not
depend upon the LIF/CNTF-mediated conversion of GFAP- NSCs into
GFAP+ progenitor cells (Fig.
8).
BMP4 signaling generates mature astrocytes
The ability of noggin to prevent the maturation of GFAP+
progenitor cells into astrocytes in vivo indicates that BMP signaling normally
regulates astrocytic lineage commitment. However, BMP4 and other BMPs are
abundantly expressed throughout the nervous system
(Furuta et al., 1997;
Mehler et al., 1997
). How then
is the progenitor cell phenotype maintained in the adult brain? Noggin is
normally expressed in the SVZ and SGZ of adult animals, and helps to maintain
a niche for adult neurogenesis (Chmielnicki
et al., 2004
; Lim et al.,
2000
). Furthermore, antisense noggin reduces proliferation in the
adult dentate gyrus (Fan et al.,
2004
). These observations are consistent with our findings that
noggin preserves the GFAP+ progenitor cell phenotype and prevents
the astrocytic differentiation of these cells. Thus, noggin not only maintains
the proliferation of cells within the niche, but more generally maintains a
multipotent progenitor cell phenotype by inhibiting BMP-directed
differentiation. Our data further suggest that the number of GFAP+
progenitor cells is inversely proportional to the amount of BMP signaling in
the developing hippocampus. Because noggin is expressed in the anterior
subiculum in neonates, and in the dentate gyrus from one week of age into
adulthood (Fan et al., 2003
),
it was not clear whether noggin overexpression in this area would have much
effect. However, we found that noggin overexpression markedly increased the
number of GFAP+ progenitor cells in the SGZ, indicating that the
levels of endogenous noggin expression are insufficient to fully inhibit BMP
signaling in this area. The almost complete depletion of GFAP+
progenitor cells from the SGZ of BMP-overproducing animals highlights the
essential role played by BMP inhibitors such as noggin in maintaining the
progenitor cell phenotype.
Mature astrocytes and adult progenitors are separate cell populations
The molecular characterization of GFAP and the relatively limited number of
cell types that express the protein led to its use as a surrogate marker for
the astrocyte phenotype. The lack of an unambiguous biochemical marker has
complicated the precise definition of astrocyte identity and of the astrocytic
lineage (Gotz and Steindler,
2003; Kimelberg,
2004
). Although it is clear that some GFAP+ cells in
the adult brain have stem cell potential
(Doetsch et al., 1999
;
Garcia et al., 2004
;
Imura et al., 2003
;
Morshead et al., 2003
;
Seri et al., 2001
), only a
morphologically distinct subpopulation of GFAP+ cells produce new
neurons (Garcia et al., 2004
).
This has led to terms such as radial astrocyte and horizontal astrocyte, which
are based on morphological criteria in vivo, to help to distinguish the unique
subsets of GFAP+ cells in the brain that display progenitor cell
traits (Seri et al., 2004
).
The lineage relationship between adult progenitor cells and other astrocytes
has been unclear. Our findings suggest a lineage relationship in the rodent
brain in which GFAP+ progenitors generate mature astrocytes in
response to BMP signaling (Fig.
8), and, further, that these represent distinct and separable cell
types. It may therefore be inappropriate to continue to use the same term -
astrocyte - for these disparate cell types, particularly as GFAP+
progenitors cells also generate other lineages in the normal adult brain
(Garcia et al., 2004
). It
might be more accurate to use terms such as radial progenitor or horizontal
progenitor, and to reserve the use of the term `astrocyte' for more terminally
differentiated phenotypes, as these cells differ in their morphology,
molecular characteristics and potentiality
(Morest and Silver, 2003
).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/24/5503/DC1
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ACKNOWLEDGMENTS |
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