The Skirball Institute and Department of Cell Biology, NYU School of Medicine, 540 First Avenue, New York, NY 10016, USA
Author for correspondence (e-mail:
ria{at}saturn.med.nyu.edu)
Accepted 17 October 2003
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SUMMARY |
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Key words: Hedgehog, GLI, Neocortical stem cells, Mouse
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Introduction |
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To address this question, we have investigated the role of SHH-GLI signaling in the developing mouse neocortex, analyzing the mid- and late-gestation brain phenotypes of Shh, Gli2 and Gli3 mutants. The results of these experiments, together with those testing for the function and requirement of SHH signaling in vivo as well as in vitro, carried out under conditions in which self-renewal and multipotentiality can be tested, indicate that the SHH-GLI pathway controls the number of cells with stem cell properties in the developing neocortex. In addition, we find synergism between SHH and EGF signaling. We discuss the implications of these findings for brain development, for the ability to manipulate stem cell lineages and for tumorigenesis.
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Materials and methods |
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BrdU incorporation, histology, immunofluorescence and in situ hybridization
BrdU treatment (20 mg/kg, IP injection), microtome (12 µm) or cryostat
(10-18 µm) sections, immunofluorescence, in situ hybridization, and
Hematoxylin and Eosin staining was performed as described
(Dahmane et al., 2001). BrdU
was added to cortical nsp cultures, at 3 µM, 7 hours prior to culture
fixation. The following antibodies were used: beta III tubulin TuJ1 antibodies
(1/300; Babco), Nestin (1/200, PharmigenBecton Dickinson), activated caspase 3
(1/50, R&D Systems), GFAP (1/500, Sigma), O4 (1/40; Roche). Pyknotic cells
were identified by intense DAPI labeling. Cells undergoing apoptosis were
identified by TUNEL reactivity and/or activated caspase 3 expression. Clone 53
derives from a screen for neocortical genes with patterned expression, and was
used as a marker of vz/svz cells. Sense probes confirmed the specificity of in
situ hybridization reactions. All probability values were obtained using the
Student's t-test. Cells were quantified by counting the number of
marker-positive cells as a percentage of DAPI-positive cells per ocular grid
area at x40 magnification (5-12 random areas per experiment, from at
least three independent samples).
RT-PCR and genotyping
Conditions and sequences were as described previously
(Dahmane et al., 2001). Other
primers were: mGLI2-1 (forward), gca gct ggt gca tca ta; and mGLI2-2
(reverse), cgg tgc tca tgt gtt tg. Reactions conditions were Tm=55°C,
producing an 828 bp band for the wild-type allele and a 913 bp band for the
Gli2 mutant allele. Primers for Ihh and Dhh were
used at Tm=58°C, and gave expected band sizes of 267 bp for Ihh
and 311 bp for Dhh
(Tekki-Kessaris et al., 2001
).
Egfr and Emx1/2 primers were used at Tm=55°C and gave
the expected band sizes of 459 bp for Egfr, 183 bp for Emx1
and 247 bp for Emx2 (Represa et
al., 2001
; Yoshida et al.,
1997
).
Neurospheres
Mid and late embryonic cortical nsps were obtained by standard procedures
(see Doetsch et al., 2002).
Cells were incubated in neurosphere medium [Neurobasal Medium (GIBCO),
containing N2 (GIBCO), 2 mM glutamine, 0.6% (w/v) glucose, 0.02 mg/ml insulin,
antibiotics and 15 mM HEPES] with 10 ng/ml of EGF (human recombinant, GIBCO)
and 10ng/ml of bFGF (Upstate Biotech) unless otherwise noted. For
proliferation assays, nsps were plated at 3000 cells/well onto
polyornithine/laminin coated Lab-Tek chamber slides (Nunc) in the presence of
EGF and FGF, and grown for 1 week. For differentiation assays, growing nsps
were plated at 20,000 cells/well onto polyornithine/laminin coated (10 mg/ml)
Lab-Tek chamber slides without growth factors and incubated for 5-7 days. For
cloning assays, cells were plated either by dilution at 1 cell/well in 96-well
plates (Nunclon), with 50% conditioned media [50% nsp-defined media containing
EGF (10 ng/ml) and bFGF (10 ng/ml)], or by assessing clonal sphere colony
formation at low cell densities in 1/3 conditioned media documented to yield
clonal cultures (Reynolds and Weiss,
1996
; Tropepe et al.,
2000
; Seaberg et al.,
2002
). The number and size of cloned nsps was counted after one
week in culture. For cloning assays with Gli2-/- and
wild-type littermate neurospheres at E18.5, healthy cells from the first or
second passage were visually chosen for their round morphology and homogeneous
appearance under the microscope, and manually transferred to single wells in
96-well plates (Nunclon). This was to avoid the potential problem of damaged
Gli2-/- cells, which are fragile, after dissociation of
pelleted neurospheres.
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Results |
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SHH often acts in a concentration-dependent manner (reviewed by
Ingham and McMahon, 2001).
Therefore, to test for a concentration-dependent effect of inhibition of HH
signaling by cyc, wild-type E15.5 and postnatal day 2 (P2) neocortical
EGF-responsive nsps were selected and treated with varying concentrations of
cyc. Treated and control nsps were assayed for BrdU incorporation and cloned.
Increasing concentrations of cyc lead to a progressive decrease in the level
of BrdU incorporation and in the number of secondary clones, as compared with
non-treated nsps (Fig. 1E-H).
SHH pathway activity thus regulates the size of the pool of nsp cells with
self-renewal properties in a concentration-dependent manner.
Pharmacological inhibition of HH signaling in vivo
To test whether an in vivo effect of cyc could be discerned,
cyclodextrin-conjugated cyc, or just cyclodextrin (HBC) as control, was
injected intraperitoneally, daily, into pregnant mothers
(van den Brink et al., 2001)
carrying E10.5-E12.5 embryos, and these were collected five days later. By
E10.5, the ventral forebrain has already received HH signaling and the eyes
have formed. Consequently, cyc treatment did not cause cyclopia and the
treated E15.5-E17.5 embryos appeared normal. Analyses of fresh neocortical
tissue dissected
12 hours after the last cyc injection showed that in
vivo cyc treatment decreased the expression of Gli1, a loyal marker
of SHH signaling (Lee et al.,
1997
), and abolished that of Egfr
(Fig. 1I), whereas expression
of Gli2 and Gli3 was unchanged (not shown). Acute
dissociation of neocortical tissue at this time, and its subsequent culture
without growth factors, confirmed that in vivo cyc treatment inhibits cell
proliferation (Fig. 1J). To
expand this finding, we analyzed neocortical cell behavior from mice mutant
for Shh, Gli2 and Gli3, as Gli1 null mice appear
normal (Park et al.,
2000
).
Shh mutant mice show deficits in cells able to form neurospheres
Shh-/- mice die at birth showing overt signs of
cyclopia and lacking all ventral CNS cell types
(Chiang et al., 1996). Their
dorsal-only CNS comprises an Emx1+,
Tbr1+ forebrain cortex
(Chiang et al., 1996
;
Dahmane et al., 2001
)
(Fig. 2A). The
Shh-/- cortex produced nsp cultures in full media, but
these were fewer and smaller than those from wild-type cortices, and contained
fewer BrdU+ cells (Fig.
2B-G,M-O). Analyses of gene expression confirmed the loss of
Shh transcripts in the few Shh-/- nsps that
formed (representing a small pool of viable cells). A decrease in
Ptch1 and Dhh expression was detected, whereas the
expression of Ihh, Gli1 and Gli2 were unchanged, and the
expression of Gli3 expression was slightly higher
(Fig. 2L). These
Shh-/- nsps expressed nestin
(Fig. 2H) and were
tripotential, as judged by the ability to differentiate as Tuj1+
neurons, GFAP+ astrocytes or O4+ oligodendrocytes
(Fig. 2I-K and not shown).
Cloning assays showed that Shh-/- nsps contain
approximately one quarter of the number of nsp-forming stem cells of wild-type
nsps at E15.5 (Fig. 2O). At
E18.5, there were very few, if any, mutant nsps
(Fig. 2O).
Gli2 mutant mice display a mid and late embryonic dorsal brain phenotype
Gli2-/- mice also die at birth, displaying defects in
multiple organs (Mo et al.,
1997; Ding et al.,
1998
; Matise et al.,
1998
). We have found novel dorsal brain phenotypes of
Gli2-/- mice at mid and late gestation stages in an
outbred background. Gli2 null embryos present a variably penetrant
severe phenotype, displaying excencephaly by E13.5 (also seen at E17-E18.5;
not shown), and a consistent milder phenotype characterized by expanded but
thinner telencephalic vesicles, most clearly seen posteriorly, and an overtly
reduced tectum and cerebellum (Fig.
3A-C). We have focused here on the non-exencephalic
Gli2-/- mice. Histological analyses showed that E18.5
Gli2-/- telencephalic vesicles have a thinner
proliferative zone (an
30-50% reduction of the vz/svz;
Fig. 3D). Gli2-/- mice have fewer BrdU+ precursors in the
cortex at mid and late gestation periods
(Fig. 1E-G), suggesting defects
in neuronal as well as glial cell populations. The decrease is most notable in
the deeper proliferative area (the svz). Local variations without a clear
pattern in the density of BrdU+ nuclei were also observed (not
shown), indicating an additional degree of neocortical disorder in these
mutant mice. TUNEL and activated caspase 3 analyses did not show an increase
in apoptosis (not shown).
|
Neocortical cells from Gli2 mutant mice show compromised neurosphere-forming abilities
Gli2-/- neocortices gave rise to nsps
(Fig. 4A-D), containing
Nestin+ cells (Fig.
4F) that were tripotential
(Fig. 4G-I). However, at late
embryonic stages mutant nsps progressively became smaller, more delicate, and
showed more blebbing than wild-type nsps
(Fig. 4A-D,J,K). Gli2-/- nsps decreased in numbers during culture and,
after a few passes (6), they were rare, and all died soon after. The
Gli2-/- nsps surviving at passage
2-4 lacked
Gli1 expression and showed downregulation of Ihh and
Dhh expression. Shh expression was unchanged, whereas
Gli3 and Ptch1 expression was reduced
(Fig. 4E). The expression of
Egfr was also reduced. Cloning assays in the presence of EGF and FGF
showed that there was an
10-fold decrease in the number of
Gli2-/- cells able to form secondary nsps, as compared
with wild-type cells (Fig.
4L,M).
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Discussion |
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Our data suggest that cells with stem cell properties increase their
numbers in response to higher than normal endogenous HH levels; normal HH
levels equate to homeostasis, and reduction of HH signaling below a critical
threshold decreases their numbers. In this case, our present and previous data
(Dahmane et al., 2001)
suggests that HH signaling affects both precursors and neocortical stem cells,
although definitive proof requires the prospective identification of stem
cells in vivo and their direct analyses in terms of response to SHH-GLI
function, which is not yet feasible. The scenario we propose parallels the
multiple effects of SHH signaling on adjacent cell types in other parts of the
brain (Dahmane and Ruiz i Altaba,
1999
), and the separate effects of SHH signaling on the
differentiation of a single cell type at different times
(Ericson et al., 1996
). Our
results are also consistent with the finding that Smoothened, a
crucial component of the HH-GLI pathway, is expressed in populations enriched
for SOX2+ neural stem cells, as well as in derived nsps from the
midgestation mouse telencephalon (D'Amour
and Gage, 2003
). It remains possible that the smaller size of nsps
that we observe after inhibiting SHH-GLI function could also reflect changes
in cell identity or cell populations, as wild-type nsps of different sizes can
express partially different subsets of genes
(Suslov et al., 2002
) and SHH
might also affect precursor maturation
(Viti et al., 2003
). However,
the loss of virtually all nsps from E18.5 Shh and Gli3 null
mice suggests the general requirement of the HH-GLI pathway.
The phenotype of Gli2, Gli3 and Shh mutant mice is likely
to include a decrease in the number of neocortical cells with stem cell
properties. The existence of severe (exencephalic) and milder Gli2
and Gli3 null phenotypes indicates that genetic modifiers affect GLI
function. Functional compensation between GLI3 and GLI2 may alleviate the
phenotypes, and also explain the maintained response of
Gli2-/- neocortical explants to SHH (data not shown).
Nevertheless, such compensation is likely to be only partial as
Gli2-/- nsps are severely affected, and GLI2 and GLI3
regulate gene expression differently. For example, loss of either one results
in lower Gli1 levels, but Dhh is downregulated in
Gli2-/- mice whereas it is upregulated in Gli3
mutants. It remains possible that GLI2 and GLI3 respond to, and/or mediate,
non-HH inputs (Brewster et al.,
2000).
HH signals that control neocortical expansion may derive from the
precursors themselves, as nsps express HH genes, and from differentiated cells
located at a distance (Dahmane et al.,
2001), possibly paralleling the axonal transport of HH
(Huang and Kunes, 1996
;
Traiffort et al., 2001
). A
similar situation is likely to occur in the cerebellum, where early external
germinal zone precursors transiently express Shh
(Dahmane and Ruiz i Altaba,
1999
) in addition to its strong expression by Purkinje neurons
(Dahmane and Ruiz i Altaba,
1999
; Wallace,
1999
; Weschler-Reya and Scott, 1999;
Dahmane et al., 2001
). In the
hippocampus, SHH is produced by cells in the hilus of the dentate gyrus
(Dahmane et al., 2001
), but
there is also an effect from Shh+ septal cells located at
a distance (Lai et al.,
2003
).
The findings that SHH affects the behavior of cells treated with low doses
of EGF, and that high doses of EGF cannot efficiently induce nsp cultures from
Shh-/- mice, indicates that SHH acts on EGF-responsive
cells and that it is unlikely to be only a stem cell survival factor. It is
possible that the few nsps observed from Shh null mice derive from
the complementary action of IHH and DHH. How the EGF and SHH pathways interact
is not clear, but it is interesting to note that neocortical precursors
express EGFR, and that changes in EGFR expression have been associated with
changes in progenitor cell behavior (see
Burrows et al., 1997). This
raises the possibility that changes in the responsiveness to EGF signaling
could modulate or alter the response to SHH signaling or vice versa. Here we
provide evidence that SHH signaling may synergize with EGF signaling in the
brain through its regulation of Egfr expression, paralleling its
regulation in the early neural tube and fly brain
(Amin et al., 1999
;
Viti et al., 2003
).
HH signaling in defined brain domains may be involved in maintaining niches
in which stem cells exist and proliferate. Such niches may be defined, in
part, by a critical concentration range at which SHH acts, possibly
cooperating with EGF. This idea is consistent with the finding that
Shh and GLI genes are expressed in other stem cell niches, such as
the adult forebrain subventricular zone (SVZ) (V.P., N. Dahmane, D. Lim, A.
Álvarez-Buylla and A.R.A., unpublished) and the dentate gyrus
(Dahmane et al., 2001), and
with the requirement of HH signaling in the the subgranular layer of the
hippocampus (Lai et al.,
2003
), in the SVZ (V.P., D. Lim, N. Dahmane, P. Sánchez, Y.
Gitton, A. Álvarez-Buylla and A.R.A., unpublished) and for somatic stem
cell proliferation in the fly ovary and brain
(Zhang and Kalderon, 2001
;
Park et al., 2003
). Together,
these findings suggest an unexpected general role of SHH-GLI signaling in the
control of the behavior of stem cell lineages throughout the brain and in
different species. How some niches, such as the developing neocortex, are
extinguished in adulthood, while others persist is unclear but it is possible
that alterations in SHH-GLI pathway activity might underlie these changes.
How SHH signaling is integrated with other niche factors is also not known.
For example, in the adult SVZ, a niche of persistent stem cell-derived
neurogenesis (see Doetsh et al., 1999), BMP signaling inhibits neuronal
differentiation and promotes gliogenesis
(Lim et al., 2000), whereas
endogenous SHH signaling enhances neurogenesis and the number of cells with
stem cell properties (V.P., D. Lim, N. Dahmane, P. Sánchez, Y. Gitton,
A. Álvarez-Buylla and A.R.A., unpublished). Here, ependymal cells
secrete the BMP antagonist noggin, thereby creating a niche in which
neurogenesis can occur (Lim et al.,
2000
). In the developing neocortex, noggin inhibits whereas BMPs
enhance differentiation (Li et al.,
1998
; Li and LoTurco,
2000
; Mabie et al.,
1999
), and BMPs are also proposed to inhibit the transition of
stem cells to an EGF-responsive state
(Lillien and Raphael, 2000
).
In the neocortex, as in the SVZ and neural tube
(Liem et al., 1995
), BMPs and
SHH may also therefore act in opposite manners. This raises the possibility
that the synergism we report here between EGF and SHH signaling may take
place, not only directly through the activation of Egfr by SHH, but
also indirectly through the inhibition of BMP signaling. This may be supported
by the finding that activation of ERKs by EGF leads to the phosphorylation of
SMAD1 in the linker region, rendering it unable to respond to activation by
activated BMP receptors (Kretzchmar et al., 1997) and thus silencing BMP
signaling. The antagonism between SHH and BMP signaling, possibly also
integrated at the level of SMAD-GLI interactions
(Liu et al., 1998
), may thus
underlie many aspects of stem cell and neurogenic niches, being a common
target for regulators of stem cell properties and neurogenesis.
The present findings support a crucial role of SHH signaling in building
the vertebrate brain, by modulating its size through the regulation of the
number of cells with stem cell properties, in addition to controlling
precursor proliferation (Dahmane and Ruiz i
Altaba, 1999; Wallace,
1999
; Weschler-Reya and Scott, 1999;
Dahmane et al., 2001
;
Lai et al., 2003
). Overall or
local changes in SHH signaling, or its reception, during evolution may have
contributed to the evolving sizes and shapes of the brain, including the
expansion of the neocortex in primates, of the tectum in birds and of the
cerebellum in electrosensitive fish. The price for such plasticity may be
tumorigenesis. Our data, together with previous results on the involvement of
the SHH-GLI pathway in tumorigenesis (reviewed by
Ruiz i Altaba et al., 2002b
),
suggest that the many cancers that arise from constitutive HH signaling in
various tissues, such as brain, skin, muscle and lung, may derive from cells
with stem cell properties that inappropriately maintain an active response to
HH signaling, with continued signaling being required for tumor maintenance.
In the brain, the synergism between EGF and SHH signaling raises the
possibility that enhancement or inappropriate activation of either pathway,
such as through EGFR amplification or upregulation of GLI1 function, could
give stem cells an advantage to initiate cancer.
In contrast to tumor development resulting from unregulated activity, the controlled modulation of SHH and EGF signaling in vitro and in vivo is likely to lead to the development of protocols to increase the number of cells with stem cell properties in an effort to ameliorate the effects of degenerative diseases.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Department of Genetic Medicine, Faculty of Medicine,
University of Geneva, CMU, 1 rue Michel Servet, CH-1211, Geneva 4,
Switzerland
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