1 The Skirball Institute, NYU School of Medicine, 540 First Avenue, New York, NY
10016, USA
2 Neurosurgery Research, UCSF, 10 Kirkham Street, San Francisco, CA 94143,
USA
3 University of Geneva Medical School, 8242 CMU, 1 rue Michel Servet, 1211
Geneva 4, Switzerland
4 Brain and Mind Institute, EPFL, Bat AAB, 1015 Lausanne, Switzerland
** Author for correspondence (e-mail: ariel.ruizaltaba{at}medecine.unige.ch)
Accepted 8 November 2004
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SUMMARY |
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Key words: Mouse, Stem cell, Brain, Hedgehog, Gli, Subventricular zone
![]() |
Introduction |
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The secreted factor Shh plays multiple roles in the formation of the CNS,
including the regulation of early ventral pattern in the neural tube
(Jessell and Sanes, 2000) and
of later precursor proliferation in the dorsal brain
(Dahmane and Ruiz i Altaba,
1999
; Weschler-Reya and Scott, 1999;
Wallace, 1999
;
Dahmane et al., 2001
) (reviewed
by Ruiz i Altaba et al.,
2002
). This and the finding that Shh regulates the behavior of
cells with stem cell properties in the developing embryonic neocortex
(Palma and Ruiz i Altaba,
2004
) led us to investigate its possible involvement in the
control of stem cell lineages in the postnatal and adult forebrain SVZ, the
best studied stem cell niche in the adult mammalian brain
(Alvarez-Buylla et al.,
2002
).
Here we show that SVZ cells express Shh and Gli1 and that
blockage of hedgehog (Hh) signaling in adult and perinatal mice results in
diminished expression of Gli1 and deficits in SVZ cell proliferation
in vivo. Our data are consistent with the phenotype of conditional Shh
signaling mutants (Machold et al.,
2003) and with the increase in Ptch1 expression observed
after injection of Shh into the striatum
(Charytoniuk et al., 2002
).
However, we further show that in vitro addition of Shh results in an increase
in SVZ cell proliferation and in the number of cells with stem cell properties
and resulting neurons, while blocking its function decreases their number. We
provide evidence that Shh synergizes with EGF signaling in the modulation of
SVZ cell proliferation. Moreover, cell-sorting and single-cell assays identify
periventricular GFAP+ astrocytes and GFAP- early
precursors, but not ependymal cells or migrating neuroblasts, as
Gli1+ in vivo responders to endogenous Shh signaling. Our
results thus show that Shh signaling is a critical mechanism for the
maintenance of stem cell lineages and neurogenesis in the postnatal and adult
brain. Together with the involvement of Shh signaling in the control of the
behavior of stem cells in the embryonic neocortex
(Palma and Ruiz i Altaba,
2004
) and in the adult hippocampus
(Lai et al., 2003
;
Machold et al., 2003
), the
present results demonstrate that Shh signaling regulates stem cell behavior in
multiple brain niches throughout life. Moreover, we identify for the first
time a cell type previously characterized as being a bona fide stem cell, the
periventricular astrocyte (Doetsch et al.,
1999
), as an in vivo target of Shh signaling.
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Materials and methods |
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SVZ neurospheres
To make SVZ neurospheres (Reynolds and
Weiss, 1992; Doetsch et al.,
1999
) the lateral walls of the lateral ventricle of postnatal or
adult mice were dissected, collected in PBS, and incubated in PBS containing
0.3% activated papain (Worthington Biochemical). After 30 min incubation,
ovomucoid inhibitor (Worthington Biochemical) and DNAse (Sigma) were added in
Neurobasal Medium (GIBCO) and the cells dissociated using a fire-polished
pasteur pipette. After centrifugation, the cellular pellets were resuspended
in neurosphere medium [Neurobasal Medium (GIBCO) supplemented with N2 (GIBCO),
2 mM glutamine, 0.6% (w/v) glucose, 0.02 mg/ml insulin, antibiotics and 15 mM
Hepes]. The cells were counted and plated in uncoated 25 ml culture flasks
(1x106 cells per bottle) in the neurosphere medium containing
10 ng/ml epidermal growth factor (EGF; human recombinant, GIBCO). For
passaging, neurospheres were centrifuged, triturated in 100 µl of medium
with a P200 pipette and replated. For proliferation assays, neurospheres were
plated at 4000 cells/well onto polyornithine/laminin-coated labtech chambers
(Nunc) in the presence of 10 ng/ml EGF and grown for 1 week, unless otherwise
indicated. For dose-response experiments, EGF was used at 1 ng/ml in
combination with 5 and 0.5 nM Shh or 5 nM Shh with 5 and 0.5 µg/ml EGF.
Clonal dilution assays were performed by plating at low cell densities
documented to yield clonal cultures in one-third of conditioned media
(Seaberg and van der Kooy,
2002
).
RNA, RT-PCR and in situ hybridization
RNA was isolated from whole SVZ dissections or from purified cells
(Lim et al., 2000).
5000-10,000 selected cells were collected directly into RNeasy lysis buffer.
Total RNA was isolated with RNeasy columns and concentrated by vacuum
centrifugation to 12 µl; 9 µl of total RNA was combined with 1 µl of
100 mM T7LD3' primer, heated to 70°C for 5 minutes, then placed on
ice. Forty units of RNasin, 4 µl of 5x first strand buffer, 2 µl
of 0.1 M DTT, 1 µl of 20 µM SMART III oligo (Clontech), and 1 µl of
10 mM dNTPs were added, and tubes were incubated at 42°C for 5 minutes.
Four-hundred units of Superscript II was then added, and reactions incubated
for another 60 minutes at 42°C. Two units of RNaseH were added and tubes
incubated at 37°C for 20 minutes; 3 µl of total RNA was used in
parallel reactions without Superscript as RT-minus controls. PCR was then
performed for Gli1, Gli2, Gli3, Shh, Ptch1 and Hprt as
previously described (Dahmane and Ruiz i
Altaba, 1999
; Dahmane et al.,
2001
). In situ hybridizations with anti-sense digoxygenin-labeled
anti-Shh or anti-Gli1 probes were performed on fresh-frozen
or perfused sections (Dahmane and Ruiz i
Altaba, 1999
; Dahmane et al.,
2001
).
BrdU incorporation and immunohistochemical analyses
Incorporation of BrdU and immunochemical detection was performed as
described (Lim et al., 2000;
Dahmane et al., 2001
) with
anti-BrdU mouse monoclonal antibodies (Abs) (1:200; Roche). BrdU was added to
cultures at 3 µM 16 hours prior to culture fixation except in the
dose-response assays where 6-hour incubations were used. Neuronal phenotype
was determined by immunolabeling with anti-beta III tubulin TuJ1 antibodies
(1:1000; Babco). Nuclei were counterstained with Hoechst 33258 (Molecular
Probes).
Postnatal SVZ cell cultures
For the thymidine incorporation assay, 300,000 P3 SVZ cells were plated
into uncoated wells into a 96-well plate in DMEM/F12/N2/B27/Gln/15 mM Hepes,
pH 7.4 (Gibco) in the presence or absence of 5E1 or IgG (R&D systems)
antibody at 4-5 µg/ml and cultured for a total of 44 hours. At this cell
density, aggregates of SVZ cells form. At 27 hours, 2 µCi of
[3H]-thymidine was added to each well. Cells were collected onto
glass filters with a Tomtec 96 cell harvester, and [3H]-thymidine
incorporation measured with a betaplate filter counter. Postnatal SVZ cultures
for neurosphere growth were prepared as follows: 100,000 P7 SVZ cells were
plated into 96-well plates in DMEM with 10% FCS for 3 days; at this point,
cultures contain GFAP+ and Tuj1+ cells. The cultures were then washed and the
medium changed to DMEM/F12/N2 (Gibco). In DMEM/F12/N2, these cultures
proliferate to produce type A cells. After incubation with or without Shh,
cultures were then washed with PBS three times, dissociated with papain
(Lim and Alvarez-Buylla,
1999), and 30,000 cells cultured in neurosphere medium in six-well
plates.
In vivo and in vitro cyclopamine treatments
Cyclopamine (cyc, Toronto Research Biochemicals) was used at 1 mg/ml
conjugated with 2-hydropropyl-ß-cyclodextrin [HBC (Sigma)]; prepared as a
45% solution in PBS (van den Brink et al.,
2001). Five- to ten-week-old inbred C57Bl6/j mice were injected
intraperitoneally for one week with HBC alone as control or cyc at 10
mg/kg/day. The day following the last injection, the mice were pulsed for 2
hours with BrdU (20 mg/kg, IP injection). Immunofluorescence of cryostat
sections was as described (Dahmane et al.,
2001
). The stainings were digitally recorded using a cooled CCD
camera-equipped Axiophot (Zeiss) and the BrdU+/DAPI+
nuclei counted within the lateral wall of the lateral ventricles. For the in
vivo cyc treatment followed by the preparation of neurospheres, P4 pups or
adult animals were injected for 5 days with HBC alone or cyc at 10 mg/kg/day
and neurospheres prepared as above from the SVZ of P9 or adult animals.
Cyclopamine for in vitro use was dissolved in ethanol (10 mM) and used at 5
µM final concentration.
For measurements in the olfactory bulb, ten (2 months old) C57Bl6/j mice
were injected intraperitoneally for one week with HBC alone as control or
cyc at 10 mg/kg/day (Palma and
Ruiz i Altaba, 2004) and were injected intraperitoneally with BrdU
(15 mg/ml dissolved in 0.7 mM NaOH with 0.9% NaCl; Sigma) at 100 mg/kg body
weight, four times, two hours apart on the day before the last HBC or
cyc injection. Mice were perfused 30 days later, first with saline
(0.9% NaCl) containing heparin (5 units/ml) at 37°C then with 4% PFA in
0.1 M PB at 4°C. Brains were harvested and post-fixed for 48 hours in 4%
PFA at 4°C then cut in 40 µm coronal sections with a vibratome (Leica)
and stored in 0.1 M PBS. Free-floating sections were washed in 0.1 M TBS with
0.1% Tween (TBST), pretreated in TBST with 0.7% H2O2 and
0.1% Triton X-100, then pretreated in 2 M HCl in TBS to denature DNA, blocked
with rabbit serum (10% in TBST; Vector) and incubated overnight at 4°C
with rat monoclonal anti-BrdU antibody (1:600 in blocking solution; Accurate
Scientific). Detection was performed with rabbit anti-rat biotinylated
secondary antibody (1:200; Vector) followed by ABC kit (Vector) and revelation
with DAB (175 µg/ml; Sigma). Sections were mounted on slides and
counterstained with cresyl violet acetate (0.5%; Sigma). One in three
sections, 120 µm apart, were selected for BrdU quantitation. An area of
interest comprising the granule cell, internal plexiform and mitral cell
layers was measured by computer-assisted microscopy (MicroBrightField). The
number of BrdU-positive nuclei in this area of interest was related to its
sectional volume to obtain a density per millimeter cubed.
SVZ slice preparation and cell harvesting
C57/Bl6j mice (5-6 weeks old) were anesthetized by isoflurane inhalation
before decapitation. The brain was rapidly removed and immediately placed in a
4°C normal artificial cerebrospinal fluid (ACSF) solution. The ACSF
contained (mM): 124 NaCl, 3 KCl, 2 CaCl2, 1.3 MgSO4, 25
NaHCO3, 1.2 NaH2PO4, 10 D-glucose with pH=7.3
when bubbled with 95% O2-5% CO2. Coronal slices (150-200
µm) were cut with a vibrating microslicer (Leica), kept in normal
oxygenated ACSF at 34°C for about 20 min, and then stored at 20°C
before the experiment. For cell harvesting, the slices were placed under a
microscope in a chamber superfused with oxygenated ACSF warmed at 37°C.
Cells close to the lateral ventricle were optically identified. Patch clamp
pipettes filled with 6 µl RNase free intracellular solution (in mM: 100
K-gluconate, 20 KCl, 10 HEPES, 4 ATP, 0.3 GTP, 10 phosphocreatine) were used
to capture the cells. The seal of the pipette was monitored using a
patch-clamp amplifier (Axoclamp 2B, Axon Instruments). The cell was then
harvested from the tissue by applying negative pressure to the pipette and
removing it from the tissue. The cell and pipette content were then expelled
into a reaction tube.
Primer design and multiplex single-cell RT-PCR
Primer design and single-cell RT-PCR were performed based on methods
described previously (Dulac,
1998; Toledo-Rodriguez et al.,
2004
; Wang et al.,
2002
). In brief, primers were designed using MacVector. Possible
interactions between primers were then tested using Amplify 2.1. In addition,
primers were subjected to a nucleotide database search to check for sequence
specificity. To perform the reverse transcription, 5 pmol of each antisense
primer, 0.3 µl (40 U/µl) recombinant RNasin ribonuclease inhibitor
(Promega, Madison/WI, USA), 1 µl 6.15% NP-40, and RNase-free water were
added to the cell content (final volume 12.3 µl) and incubated for 3
minutes at 65°C. The tube was put on ice and the content spinned down. For
a final volume of 20 µl, 0.4 µl ribonuclease inhibitor, 4 µl X5
First-Strand Buffer, 2 µl 0.1 M DTT (both Invitrogen), and 1 µl (10 mM)
dNTP mix were added. The mixture was incubated for 5 minutes at room
temperature, placed at 42°C and 0.3 µl (200 U/µl) SuperScript II
RNase H- reverse transcriptase (Invitrogen) were added. After
incubation for 1 hour at 42°C and 10 minutes at 65°C, the cDNA
containing mix was aliquoted into 2x10 µl. One aliquot was kept at
4°C for a maximum of three days until use, the other one was stored at
-20°C. The amplification and analysis of the single cell cDNA was
performed in two distinct steps. For the first step, the ten cDNAs of interest
were amplified in a single tube containing (in a final volume of 50 µl): 10
µl RT product, 0.2 mM dNTPs (Promega), 0.1 µM of each primer, 1x
PCR buffer, 1x solution Q, and 1.25 U HotStarTaq DNA polymerase (all
Qiagen). As positive control 1 ng of whole brain total RNA was subjected to
reverse transcription and 1/10 of the product was used for the first round of
cDNA amplification; the negative control contained water. The first
amplification consisted of 10 minutes hot start at 95°C followed by 28
cycles of (40 seconds 94°C, 40 seconds at 56°C, 1 minute at 72°C),
10 minutes at 72°C. The PCR was performed in a Mastercycler gradient
(Eppendorf, Hamburg, Germany). The second round of PCR consisted of 3 minutes
at 95°C followed by 35 cycles of (40 seconds at 95°C, 40 seconds at
56°C, 1 minute at 72°C), 10 minutes at 72°C. The amplification was
performed in a total volume of 20 µl containing water, 2 µl first PCR
product, 0.2 mM dNTPs, 1 M Betaine, 1 µM of corresponding primer pair,
1x ThermoPol Reaction buffer, and 1 U Taq DNA polymerase (both New
England BioLabs). After amplification, the PCR products were analyzed on 1.5%
agarose gels. Used primers were as follows: GAPDHsense:
5'-TGACATCAAGAAGGTGGTGAAGC-3' and GAPDHantisense:
5'-CCCTGTTGCTGTAGCCGTATTC-3', amplifying a 203 bp fragment.
GFAPsense: 5'-ATCCGCTCAGGTCATCTTACCC-3' and GFAPantisense:
5'-TGTCTGCTCAATGTCTTCCCTACC-3', amplifying a 287 bp fragment.
Gli1sense: 5'-CAGCCTCTGTTTTCACATCATCC-3' and Gli1antisense:
5'-CGGTTTCTTCCCTCCCACAAC-3', amplifying a 215 bp fragment.
![]() |
Results |
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|
|
In order to confirm that B cells do indeed express Gli1, we
performed single cell multiplex RT-PCR from single randomly collected SVZ
cells with a patch-clamp pipette after recording their position
(Fig. 3A). This method allows
analysis of the expression of several genes at a single cell resolution. We
found that 32.3% and 27.7% out of 65 sampled cells expressed GFAP and
Gli1, respectively (Fig.
3B,C). Importantly, 17% of the cells co-expressed both genes,
confirming that one population of GFAP+ cells, known to be
stem cells (Doetsch et al.,
1999), express Gli1
(Fig. 3C). Not all
GFAP+ cells expressed Gli1, possibly reflecting
the choice of non-SVZ astrocytes in some cases, the existence of two
subpopulations of GFAP+ SVZ astrocytes or the activation by Shh of
only a fraction of all SVZ B cell astrocytes at any one point. Moreover, many
Gli1+ cells did not express GFAP, suggesting that
more abundant precursors (C cells) also respond, or may have recently
responded, to Shh signaling as purified A or E cells do not express
Gli1 (Fig. 2).
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The differences between control and Shh-treated samples are likely to reflect a cumulative effect of Shh on neurogenesis, as seen by the number of TuJ1+/BrdU+ cells (Fig. 6E,F), derived from a direct effect on early progenitors (B or C cells). Since Shh is added only at the beginning of the culture period, the observed increases in proliferation and neurogenesis after several days in vitro could be explained by an initial amplification of these early progenitors. A transient increase in stem/precursor cells by acute Shh treatment would explain the later increase in production of neuroblasts derived from such expanded progenitor pools.
Exogenous Shh increases the number of SVZ neurospheres
To directly test for an effect of Shh on SVZ early progenitors, postnatal
SVZ cell cultures (see Materials and methods) were grown with or without Shh
(5 nM) for four days. Cells were then dissociated and washed, and equal
numbers of cells cultured without additional Shh in the presence of EGF (10
ng/ml). This latter treatment induces the formation of neurospheres, floating
colonies derived from single cells with stem cell properties exhibiting
self-renewal and multipotentiality
(Reynolds and Weiss, 1992).
After 1 week in culture, the number of neurospheres was counted. Shh treatment
was found to increase the number of neurospheres 2.5-fold over that obtained
in untreated control samples (Fig.
7A,B).
|
At lower EGF concentrations, however, Shh had a synergistic effect on the
proliferation of neurosphere cells. Adult SVZ neurospheres were prepared by
standard methods using 10 ng/ml EGF and passaged twice. Such neurospheres were
then plated on an adhesive substrate in the presence of 1 ng/ml EGF plus 5 or
0.5 nM recombinant Shh. Analyses of BrdU incorporation after 48 hours showed
enhanced proliferation with 5 versus 0.5 nM Shh (2.5-fold;
Fig. 7C). Conversely, adult
neurospheres plated on adhesive substrate in the presence of 5 nM Shh plus 5
or 0.5 nl/ml EGF resulted in enhanced proliferation with 5 versus 0.5 ng/ml
EGF (
2-fold; Fig. 7C).
Interestingly, the percentage of BrdU incorporation was higher with 5 nM Shh
plus 1 ng/ml EGF than with 5 nM Shh plus 5 ng/ml EGF. This difference suggests
that higher doses of EGF negate any proliferative effects of Shh, explaining
the lack of effect of Shh with full media containing 10 ng/ml EGF (not shown)
(Machold et al., 2003
;
Palma and Ruiz i Altaba,
2004
). Because Shh on its own, without EGF, is unable to sustain
growth of neurospheres (not shown)
(Machold et al., 2003
;
Palma and Ruiz i Altaba,
2004
), our results suggest that Shh acts as a mitogenic cofactor
when other growth factors are present. The lower synergistic effect of 5 ng/ml
versus 1 ng/ml of EGF with Shh suggests that such synergism occurs within a
limited concentration range, paralleling the effects we described with
embryonic neocortical neurospheres (Palma
and Ruiz i Altaba, 2004
).
Inhibition of Shh signaling decreases SVZ cell proliferation and the number of neurosphere-forming stem cells
To directly test for the requirement of endogenous Shh signaling on adult
SVZ cell proliferation and self-renewal using the neurosphere assay, we have
utilized cyclopamine on floating neurosphere cultures. Treatment of adult SVZ
neurospheres with cyclopamine (5 µM) led to an inhibition of proliferation
as measured by BrdU incorporation (Fig.
7D), showing that adult SVZ progenitors require Hh signaling for
normal proliferation. In addition, cyclopamine treatment also decreased the
number of neurospheres obtained in cloning assays
(Fig. 7E), indicating that Hh
signaling controls the number of neurosphere-forming adult SVZ cells. The
lower relative decrease in clone number versus BrdU incorporation
(Fig. 7D,E), together with the
in vivo data, suggests a differential effect on two targets:
GFAP-/Gli1+ C cells
(Fig. 3), and
GFAP+/Gli1+ B stem cells
(Fig. 3) as the former account
for the bulk of BrdU+ cells while the latter can form neurospheres
in clonogenic assays.
![]() |
Discussion |
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Our in vivo single cell analyses indicate that Shh acts directly on
GFAP+ periventricular astrocytes (B cells) and more abundant
GFAP- early precursors (C cells). In vitro results are consistent
with this conclusion, although responsiveness to Shh, present in stem (B)
cells, could also be induced in precursors in vitro. Indeed, transit
amplifying precursors (C cells) can give rise to neurospheres in vitro under
the influence of EGF, which may induce them to display stem cell properties
(Doetsch et al., 2002).
Ependymal (E) cells, which were also proposed to behave as stem cells
(Johansson et al., 1999
) (see
Doetsch et al., 1999
;
Capela and Temple, 2002
), and
migrating neuroblasts (A cells) expressed Gli2 and Ptch1,
but not Gli1, suggesting that these cells do not show the canonical
response to Shh if they respond at all. Consistently, A cells did not increase
their proliferation in vitro in response to Shh.
In the developing cerebral cortex, Shh acts in cooperation with EGF but Shh
on its own is not sufficient to alter neurosphere size or number
(Palma and Ruiz i Altaba,
2004). We show here that Shh similarly has a proliferative effect
on SVZ neurospheres in cooperation with low doses of EGF, demonstrating the
conserved action of Shh as a mitogen that synergizes with EGF. The selective
expression of EGFR in C cells (Doetsch et
al., 2002
), and thus the ability to respond to EGF, provides
additional evidence that Shh and EGF synergize in the proliferation of these
early precursors. Moreover, the inhibition of adult cell proliferation and
neurosphere formation by cyclopamine further proves the requirement of Shh
signaling in adult stem cell lineages.
In contrast to our results in the SVZ, Shh is sufficient to induce
proliferation of hippocampal precursors (Lai et al., 1993). Such a difference
could suggest the endogenous production of cofactors, such as EGF, by
hippocampal cells at sufficient quantities in vitro. In addition here, and in
contrast to other data (Machold et al.,
2003), we show that Shh is sufficient to increase the number of
neurospheres derived from SVZ cultures grown over quiescent astrocytes,
indicating that in this case, such cultures may also produce sufficient levels
of EGF or other cofactors. This difference might relate to the method used: it
is possible that the astrocytes in the feeder layer produce enough cofactors
but at low enough levels for Shh to act, whereas saturating levels of EGF mask
the effects of Shh (Palma and Ruiz i
Altaba, 2004
).
Interestingly, our present data show that Shh and EGF synergize maximally
within a narrow concentration range, in a manner similar to that we described
in the embryonic neocortex (Palma and Ruiz
i Altaba, 2004). It is therefore possible that in both cases, the
range of effective Shh and EGF concentrations determines the neurogenic niche
where stem cells exist and where the population of early precursors can
expand.
The finding that Shh mRNA is detected in the walls of the lateral
ventricles supports the idea that this molecule contributes to the definition
of stem cell niches in the adult brain. However, since we have not yet been
able to detect expression of Shh in sorted SVZ cell populations or in isolated
single cells (not shown), it remains unclear which cells are the signaling
cells. One can therefore not reject the possibility that Shh may be produced
at a distance and transported through axonal terminals or dendritic arbors
that reach the SVZ from afar. This possibility is also suggested, in part, by
the finding that dopaminergic neurons in the ventral midbrain control SVZ cell
proliferation at a distance
(Höglinger et al., 2004).
Whether these cells exert their action through the secretion of Shh remains to
be determined. A similar scenario has been proposed for the control of cell
proliferation in the hippocampal stem cell niche, where Shh has been proposed
to be transported from the septum to the subgranular layer (Lai et al., 1993).
However, we note that there are cells that express Shh in the hilus
(Dahmane et al., 2001
), which
could also exert a local effect. Shh has also been shown to be axonally
transported in the fly visual system
(Huang and Kunes, 1996
), and
it is possible that it is secreted from Purkinje cell dendrites to affect
external germinal layer cell proliferation in the cerebellum
(Dahmane and Ruiz i Altaba,
1999
; Weschler-Reya and Scott, 1999;
Wallace, 1999
).
How Shh signaling is integrated with other niche factors, such as APP
(Caillé et al., 2004),
is not yet clear. For instance, BMP and Shh signaling show an antagonistic
relationship in the neural tube patterning
(Liem et al., 1995
). In the
postnatal brain SVZ, BMP signaling inhibits neurogenesis and promotes glial
differentiation: ependymal cells secrete the BMP antagonist Noggin, thereby
creating a favorable microenvironment for neurogenesis to occur
(Lim et al., 2000
). It is
possible that in the postnatal and adult SVZ, as in the embryonic neural tube,
BMP and Shh signaling act antagonistically, and inhibition of the former is
necessary for the latter to act.
Our results showing that Shh is a niche factor that regulates the number of
SVZ cells with stem cell properties and neurogenesis, parallel those in other
CNS regions: Shh is expressed in the septum and the hilus of the
hippocampus (Dahmane et al.,
2001; Lai et al.,
2003
; Machold et al.,
2003
), it regulates cell proliferation in the subgranular layer
(Lai et al., 2003
;
Machold et al., 2003
), and is
also involved in the control of stem cell behavior in the developing neocortex
(Palma and Ruiz i Altaba,
2004
). Shh-Gli signaling may thus be a general mechanism for the
regulation of the number of stem cells and the number of precursors derived
from primary progenitors. Moreover, it is interesting to propose that the
control of the production of new adult neurons, from stem cell astrocytes in
the SVZ and hippocampus (Doetsch et al.,
1999
; Seri et al.,
2001
), is largely regulated by already existing cells, located
nearby or at a distance, through the action of Shh (see also
Dahmane et al., 2001
),
providing a mechanism for the homeostatic regulation of neuronal number and
perhaps a mechanism for response to injury and disease.
Finally, our findings also suggest a method for manipulating stem cell lineages for the generation of new neurons through the regulation of Shh signaling. Such a method may help develop new strategies for the treatment of neurodegenerative diseases, such as Parkinson's disease (reviewed by McKay et al., 2004), by expanding stem/precursor cell population in vitro, prior to reintroduction in vivo or by the activation of dormant endogenous stem cell activity in vivo.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Laboratorio de Biología del Desarrollo,
Departamento de Biología, Universidad de Chile, Las Encinas 3370,
Edificio Milenio piso 3, MACUL, Santiago, Chile
Present address: The Wistar Institute, 3601 Spruce Street, Philadelphia, PA
19104, USA
Present address: Departament de Biologia Cel.lular, Universitat de
València, Facultat de Ciències Biológiques, Ed B, 4
planta, c/Dr Moliner, s/n 46100 Burjassot, València, Spain
¶ Present address: Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris
Cedex 05 Paris, France
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