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First published online January 12, 2006
doi: 10.1242/10.1242/dev.02228
1 Instituto de Biología Molecular de Barcelona, CSIC, Parc
Científic de Barcelona, C/Josep Samitier 1-5, Barcelona, Spain.
2 National Institute for Medical Research, The Ridgeway, Mill Hill, London
NW71AA, UK.
* Authors for correspondence (e-mail: emgbmc{at}ibmb.csic.es; james.briscoe{at}nimr.mrc.ac.uk)
Accepted 29 November 2005
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Spinal cord, Neural development, Pattern formation, Cell proliferation, Cell survival, Hedgehog signaling, Gli proteins, Chick
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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).
Distinct neuronal subtypes emerge in a precise spatial order from progenitor
cells, resulting in the partition of the dorsoventral axis of the neural tube
into discrete regions occupied by different neuronal subtypes
(Jessell, 2000). At the same
time, a balance between differentiation and proliferation of progenitors
ensures the growth of the neural tube and replenishes the pool of progenitors,
allowing continued rounds of neuronal and glial differentiation.
Much attention has focused on the mechanisms that control the identity of
differentiating neurons. Particularly well characterized is the regulation of
neuronal subtype identity in caudal regions of the neural tube that comprise
the spinal cord and hindbrain. In ventral regions, Sonic hedgehog (Shh)
secreted from the floor plate and notochord acts as a long-range graded signal
that controls the pattern of neurogenesis
(Jessell, 2000;
Briscoe and Ericson, 2001).
Dorsally, members of the Bmp and Wnt families of signaling molecules expressed
by the roof plate and overlying ectoderm have been implicated in determining
neuronal subtype identity (Helms and
Johnson, 2003). These signals act by regulating the spatial
pattern of expression, in progenitor cells, of transcription factors that
include members of the homeodomain protein (HD) and basic helix-loop-helix
(bHLH) families (Helms and Johnson,
2003; Briscoe et al.,
2000; Ericson et al.,
1997; Muhr et al.,
2001; Novitch et al.,
2001; Pierani et al.,
2001; Vallstedt et al.,
2001) and the experimental evidence indicates that changing the
progenitor transcription factor code alters neuronal subtype in a predictable
manner. Thus, the profile of transcription factor expression, established by
the secreted signals, determines the subtype identity of the neurons generated
from a progenitor cell (Briscoe et al.,
2000).
Less well understood is how the growth and proliferation of progenitors in
the neural tube is controlled and how this is coordinated with the mechanisms
that control the pattern of neurogenesis. A number of studies have suggested
an important role for Wnt signaling in promoting the proliferation and
survival of neural progenitors at least in dorsal regions of the caudal neural
tube (Dickinson et al., 1994;
Megason and McMahon, 2002;
Panhuysen et al., 2004).
However, increasing experimental data supports the idea that Shh may also play
a major role. The development of a number of anterior neural structures,
including the cerebellum (Dahmane and Ruiz
i Altaba, 1999; Pons et al.,
2001; Wallace,
1999; Wechsler-Reya and Scott,
1999), neocortex and tectum
(Dahmane et al., 2001;
Palma and Ruiz i Altaba, 2004)
depends on a mitogenic response to Shh signaling; moreover, Shh signaling is
also required for the maintenance of neural stem cells during late development
and in adult CNS (Lai et al.,
2003; Machold et al.,
2003). In caudal regions, notochord removal causes a decrease in
the size of the neural tube (Charrier et
al., 2001; van Straaten and
Hekking, 1991) and notochord-derived signals can regulate
neuroepithelial cell proliferation (van
Straaten et al., 1989; Placzek
et al., 1993). Consistent with the idea that this signal
corresponds to Shh, the ectopic expression of Shh
(Rowitch et al., 1999) or the
ectopic activation of the Shh signaling pathway
(Goodrich et al., 1997;
Epstein et al., 1996;
Hynes et al., 2000) results in
hyper-proliferation of progenitors. Furthermore, genetic manipulation of the
dose of two endogenous inhibitors of the pathway, Patched1 and Hip1, results
in a noticeable and dose-dependent enlargement of the neural tube
(Jeong and McMahon, 2005).
Conversely, blockade of Shh signaling either by the genetic removal of Shh or
components of the Shh pathway (Chiang et
al., 1996; Litingtung and
Chiang, 2000; Wijgerde et al.,
2002) results in a decrease in both the proliferation and survival
of progenitor cells within the neuroepithelium.
Shh signals by binding its receptor Patched (Ptc), a multi-pass
transmembrane protein. In the absence of Shh, Ptc acts to suppress the
activity of a second transmembrane protein, Smoothened (Smo) (for a review,
see Lum and Beachy, 2004).
Liganding of Ptc by Shh relieves repression of Smo, then, through a mechanism
yet to be fully elucidated, Smo signals intracellularly to
zinc-finger-containing transcription factors of the Gli family – highly
conserved transcriptional mediators of the Shh pathway that can activate or
repress transcription of specific target genes (reviewed by
Jacob and Briscoe, 2003).
Despite evidence of the role for Shh in regulating the survival and
proliferation of progenitors in the neural tube, how Shh mediates these
activities remained unclear. Here, by manipulating components of the Shh
signaling pathway in vivo in chick embryos, we provide evidence that Shh acts
directly on progenitor cells to promote neural progenitor proliferation and
survival. Moreover, our data indicate that the regulation of Gli activity by
Shh signaling is responsible for controlling progenitor proliferation and
survival. Finally, we provide evidence that the Shh regulated patterning,
proliferation and survival of progenitors are separable activities, suggesting
that each of these cellular properties is an independently regulated response
to Shh/Gli signaling. Thus, Shh signaling appears to coordinate directly the
growth and patterning of developing neural tube through Gli mediated
transcriptional regulation of discrete sets of target genes.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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loop2) (Briscoe et
al., 2001), a deleted form of human Gli3 encoding amino acids
1-768 (Gli3R) (Persson et al.,
2002), the complementary deleted form of human Gli3 encoding amino
acids 468-1580 (Gli3AHIGH)
(Stamataki et al., 2005), the
sequence encoding amino acids 471-645 of the human Gli3 zinc-finger (Gli-ZnF)
and a dominant negative form of protein kinase A (dnPKA)
(Epstein et al., 1996) were
all inserted into pCIG (Megason and
McMahon, 2002) upstream of an internal ribosomal entry site (IRES)
and three nuclear localization sequences tagged EGFP in pCAGGS expression
vector (Niwa et al., 1991). A
human BCL2-coding sequence inserted into pcDNA3 expression vector was
used for co-electroporation.
Chick in ovo electroporation
Eggs from White-Leghorn chickens were incubated at 38.5°C in an
atmosphere of 70% humidity. Embryos were staged according to Hamburger and
Hamilton (HH) (Hamburger and Hamilton,
1951).
Chick embryos were electroporated with Clontech purified plasmid DNA at 2-3 µg/µl in H2O with 50 ng/ml Fast Green. Briefly, plasmid DNA was injected into the lumen of HH stage 10-12 neural tubes, electrodes were placed either side of the neural tube and electroporation carried out using and Intracel Dual Pulse (TSS10) electroporator delivering five 50 msecond square pulses of 30-40 V.
Transfected embryos were allowed to develop to the specific stages, then dissected, fixed and processed for immunohistochemistry or in situ hybridization. For bromodeoxyuridine (BrdU) labeling, 5 µg/µl BrdU was injected into the neural tubes 1 or 4 hours prior fixation.
Immunohistochemistry
Embryos were fixed 2-4 hours at 4°C in 4% paraformaldehyde in PB,
rinsed, sunk in 30% sucrose solution, embedded in OCT and sectioned in a Leica
cryostat (CM 1900). Alternatively, embryos were section in a Leica vibratome
(VT 1000S). Immunostaining was performed following standard procedures.
For BrdU detection, sections were incubated in 2N HCl for 30 minutes followed by 0.1 M Na2B4O7 (pH 8.5) rinses further PBT rinses and anti-BrdU incubation.
Antibodies against the following proteins were used; green fluorescence protein (GFP) (Molecular Probes), anti-myc (9E10, Santa Cruz), a unique ßIII-Tubulin (Tuj-1) (Medpass), phospho-Histone 3 (p-H3) (Upstate Biochemicals), NeuN (Chemicon), caspase 3 (BD), Bcl2 (Santa Cruz). Monoclonal antibodies to BrdU (G3G4), Pax7, Mnr2 (81.5C10), Nkx2.2 (74.5A5) were all obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.
Alexa488- and Alexa555-conjugated anti-mouse or anti-rabbit antibodies (Molecular Probes) were used. After single or double staining, sections were mounted, analyzed and photographed using a Leica Confocal microscope.
Cell counting was carried out on 10-40 different sections of at least four different embryos after each experimental condition (n>4).
In situ hybridization
Embryos were fixed overnight at 4°C in 4% paraformaldehyde in PB,
rinsed and processed for whole-mount RNA in situ hybridization following
standard procedures using probes for chick N-myc and
cyclinD1 (from the chicken EST project, UK-HGMP RC).
Hybridization was revealed by alkaline phosphatase-coupled anti-digoxigenin Fab fragments (Boehringer Mannheim). Hybridized embryos were postfixed in 4% paraformaldehyde, rinsed in PBT and vibratome sectioned.
TUNEL assay
Detection and quantification of apoptosis (programmed cell death) was
performed using the `In Situ Cell Death Detection Kit-POD conjugated' from
Roche, following manufacturer's instructions.
Western blot
HH stage 11-12 embryos were electroporated with Gli3AHIGH,
neural tubes dissected at 12 and 24 hours post electroporation were collected
in PBS and directly lysed in 1xSDS loading buffer [10% glycerol, 2% SDS,
100 mM DTT and 60 mM Tris-HCl (pH 6.8)] and the DNA disrupted by sonication.
Samples were normalized by total protein content, separated by SDS-PAGE gel
electrophoresis and transferred to nitrocellulose membranes, blocked with 8%
non fat dry milk in TTBS [137 mM NaCl; 0.05 Tween 20 and 20 mM Tris-HCl (pH
7.4)], and probed with anti-Bcl2 antibody (Santa Cruz). The blots were
developed using anti-rabbit coupled peroxidase plus the ECL system (Amersham).
Quantifications were performed using a Biorad Chemiluminiscence Analyser
(VERSADOC 5000).
Fluorescence associated cell sorting (FACS)
EGFP-containing plasmid DNA was injected into the lumen of HH stage 11-12
neural tube, embryos were electroporated as described above, and neural tubes
dissected out 12-24 hours later. Single cell suspension was obtained by 10-15
minutes incubation in Trypsin-EDTA (Sigma). At least three independent
experiments were analyzed by FACS for each experimental condition.
Hoescht and GFP fluorescence were determined by flow cytometry using a MoFlo flow cytometer (DakoCytomation, Fort Collins, CO). Excitation of the sample was carried out using a Coherent Enterprise II argon-ion laser. Excitation with the blue line of the laser (488 nm) permits the acquisition of forward-scatter (FS), side-scatter (SS) and green (530 nm) fluorescence from GFP. UV emission (40 mW) was used to excite Hoescht blue fluorescence (450 nm). Doublets were discriminated using an integral/peak dotplot of Hoechst fluorescence. Optical alignment was based on optimized signal from 10 µm fluorescent beads (Flowcheck, Coulter Corporation, Miami, FL). DNA analysis (Ploidy analysis) on single fluorescence histograms was done using Multicycle software (Phoenix Flow Systems, San Diego, CA).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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loop2). This version of Ptc1
lacks most of the second large extracellular loop and has lost the capacity to
bind Shh but retains the ability to inhibit Smoothened or a downstream
Smoothened effector, and inhibits Shh signaling in a cell-autonomous manner
(Briscoe et al., 2001). To
allow informative comparisons between embryos and constructs, we standardized
the conditions used in all experiments: HH11-12 embryos were electroporated
with each construct and following incubation and processing, analysis was
restricted to the forelimb and the anterior thoracic regions.
In embryos expressing Ptc1loop2 the transfected side of
the neural tubes was noticeably reduced at 24 hours post-electroporation (PE)
(Fig. 1A-D), suggesting that
blocking Shh signaling inhibits cell proliferation and/or increases cell
death. We first analyzed proliferation in transfected embryos. The number of
mitotic cells, assessed by the marker phospho-Histone3 (pH3), was reduced by
33% in the Ptc1loop2 transfected side of the neural
tube compared with the non-transfected control side of the neural tube [ratio
of pH3+ cells EP versus non-EP neural tube: pCIG 24h-PE 1.00±0.09 pH3+
cells (n=4); Ptc1loop2 24h-PE 0.68±0.1 pH3+
cells (n=3) (P=0.0012)]
(Fig. 1A,M). Moreover, pulse
labeling electroporated embryos with bromodeoxyuridine (BrdU) indicated that
entry of neuroepithelial cells into S-phase of the cell cycle, was also
decreased (46% reduction) in cells transfected with
Ptc1loop2 compared with control cells transfected with the
empty vector pCIG [24h-PE 29.77±5.75% of GFP+ cells in pCIG embryos
incorporate BrdU ventrally and 27.14±1.24% dorsally (n=2);
12.87±3.49% of Ptc1loop2 transfected cells
incorporate BrdU ventrally (P=0.0024) and 17.14±6.57% dorsally
(n=2) (P=0.0057)] (Fig.
1B,N). The combined visualization of GFP, to assess cell
transfection, with anti-BrdU immunostaining indicated that the blockade of
cell cycle progression was cell autonomous
(Fig. 1B,F), while adjacent
untransfected cells continued to incorporate BrdU
(Fig. 1F, inset). We next
analyzed apoptosis in transfected embryos. Comparing the side of the neural
tube transfected with Ptc1loop2 with the control
non-transfected side indicated a significant increase in TUNEL staining
(Fig. 1C) and immunoreactivity
for the apoptotic marker caspase 3 [24h-PE pCIG embryos contain
1.02±0.64 caspase3+ cells/section (n=4),
Ptc1loop2 embryos contain 11.99±5.74 caspase
3+ cells/section (n=4) (P=0.0002)]
(Fig. 1D,O). Thus, by 24 hours
PE, blockade of Shh signaling in neuroepithelial cells inhibits both cell
cycle progression and cell survival.
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loop2 12h-PE
0.86±0.2 pH3+ cells (n=4)]
(Fig. 1E,I,M). Quantitation
indicated a decrease of 15% in ventral regions and 50% in dorsal
regions in the number of transfected cells incorporating BrdU [12h-PE
30.35±5.03% of GFP+ cells in pCIG embryos incorporate BrdU ventrally
and 34.91±1.05% dorsally (n=2); by contrast,
26.17±11.95% of Ptc1loop2 transfected cells
incorporate BrdU ventrally (P=0.0024) and 17.51±6.89% dorsally
(n=2) (P=0.017)] (Fig.
1F,J,N). These data indicate that blockade of Shh signaling
results in a decreased rate of neuroepithelial cell cycle progression within
12 hours PE and this occurs prior to onset of apoptotic cell death. These data prompted us to ask whether Shh signaling is required to maintain neural precursor cell proliferation at later stages of neural development. Embryos electroporated at stage HH14 and analyzed 48 hours PE also showed a reduction in the size of the electroporated neural tube and a significant cell-autonomous reduction in the number of GFP/BrdU double-labeled cells (see Fig. S1A-D in the supplementary material). Similar results were obtained with embryos electroporated at stage HH18 and analyzed by 48 hours PE (see Fig. S1E-H in the supplementary material). Increased levels of apoptosis were also apparent on the transfected side of these embryos (see Fig. S1I,J in the supplementary material).
Together, the data indicate that active Shh signaling is required for cell cycle progression and cell survival and suggest that Ptc1 acts directly on cells to regulate these properties. To test for a role of the canonical Shh/Gli pathway on these effects, we next analyze the effects of introducing different version of Gli proteins.
Gli activator protein promotes neural cell survival
Although the evidence suggests that all patterning activities of Shh are
Gli dependent (Jeong and McMahon,
2005; Lei et al.,
2004; Bai et al.,
2004; Meyer and Roelink,
2003; Persson et al.,
2002), it is unclear whether Gli dependent transcription is
required for the Shh mediated survival of cells. Thibert et al.
(Thibert et al., 2003) have
provided evidence that, in the absence of the ligand Shh, the receptor Ptc1
acts as a transmembrane death receptor able to activate the caspase cascade
independently of Smoothened signaling and Gli-mediated transcription. However,
in the developing limb, Gli-dependent transcription has been suggested to
mediate cell survival/apoptosis (Bastida et
al., 2004).
To test for an involvement of Gli-mediated transcription in the
apoptosis/survival of neural progenitors, we first examined whether
introducing a dominant inhibitor of Gli dependent transcription affected cell
survival. We transfected a deleted form of Gli3 (Gli3R) that contains the
N-terminal repressor and the zinc-finger, DNA-binding domains that are
equivalent to the proposed proteolytically processed form of Gli3
(Wang et al., 2000). In vitro,
Gli3R blocks all Shh/Gli-mediated transcriptional activation acting as a
dominant inhibitory Gli protein, and in vivo Gli3R blocks the Shh-mediated
ventralization of the neural tube (Meyer
and Roelink, 2003; Persson et
al., 2002). Stage HH11/12 chick embryos electroporated with Gli3R
and assayed 12 hours PE showed the expected ectopic ventral activation of Pax7
(Fig. 2A,C). Moreover,
transfection of Gli3R caused a marked and very rapid induction of cell death
(by 6 hours PE) (Fig. 2B) that
reached a maximum 12 hours PE [ratio of apoptotic cells at 12h-PE, EP versus
non-EP sides, in pCIG embryos is 1.24±0.34 cells (n=4), the
ratio in Gli3R embryos is 8.35±4.57 (n=5)
(P<0.0001)] (Fig.
2D,S). These data indicate that, similar to the ectopic expression
Ptc1loop2, blocking Gli dependent transcription induces
neuroepithelial cell apoptosis. This increase in apoptosis was also apparent
at 6 hours PE and occurred prior to detectable changes in the regulation of
progenitor expressed transcription factors that respond to Shh signaling
(Fig. 2A,B). Together, these
data raise the possibility that the apoptosis induced by
Ptc1loop2 is a consequence of increased Gli repressor
activity.
To address this, we took advantage of a deleted form of Gli3 protein that
contains only the DNA-binding zinc-finger-domain (Gli-ZnF,
Fig. 2E). In vivo, Gli-ZnF is
unable to activate transcription of class II homeodomain (HD) proteins such as
Nkx2.2 (Fig. 2F) or to
de-repress transcription of class I HD proteins such as Pax7
(Fig. 2G); in vitro it blocks
both positive and negative Gli activity (data not shown). This indicates that
Gli-ZnF lacks both Gli-mediated transcriptional activation and repression
activities. In Gli-ZnF-transfected embryos analyzed 24 hours PE, neural tubes
contained increased caspase 3 immunostaining [ratio of apoptotic cells, EP
versus non-EP sides, 8.66±3.76 (n=4) compared with
1.02±1.55 (n=6) (P=0.0002)]
(Fig. 2H,S). This suggests the
possibility that the increased apoptosis in Ptc1loop2
transfected embryos might be due to a reduction in positive Gli
transcriptional activity.
We next asked whether Ptc- and Gli-mediated apoptosis were two independent
events. To test whether Ptc1loop2 mediated apoptosis of
neuroepithelial cells was dependent on Gli activity, we analyzed embryos that
were co-electroporated with Ptc1loop2 and the Gli-ZnF
constructs. Co-expression of both constructs was monitored by immunostaining
with the anti-Myc antibody as a reporter of the Gli-ZnF expression and GFP as
a reporter of Ptc1 expression (Fig.
2I). Lack of Gli transcriptional activity inhibited the
Ptc1loop2-induced ventral expansion of Pax7
(Fig. 2J), suggesting that it
was dependent on Gli-repressor activity
(Briscoe et al., 2001).
However, the level and kinetics of apoptosis assessed by caspase 3
immunostaining was similar in co-electroporated embryos
(Fig. 2K,S) to that seen in
embryos electroporated independently with Ptc1loop2 and with
Gli-ZnF [At 12 hours PE in Gli-ZnF the ratio (EP versus non-EP side) of
apoptotic cells is 1.58±0.45 cells (n=4); in
Ptc1loop2+ZnF the ratio is 2.50±1.55 (n=6).
At 24 hours PE, in Gli-ZnF the ratio is 8.66±3.76 (n=4)
(P=0.0002); and in Ptc1loop2+Gli-ZnF the ratio is
7.86±0.08 (n=4).] (Fig.
1D, Fig. 2H,S). The
absence of a synergistic effect in co-electroporated embryos is consistent
with the idea that the induction of apoptosis in Ptc1loop2
transfected cells is due to a decrease in Gli transcriptional activity.
Moreover, the fact that the Gli-ZnF construct rescues changes in cell fate but
not the increased apoptosis, suggests that Shh signaling regulates the
survival of neural progenitors independent of patterning.
To test whether Ptc1loop2-mediated apoptosis was
dependent on Gli transcriptional repression, we examined whether providing Gli
transcriptional activity was sufficient to promote the survival of cells
expressing Ptc1loop2. To achieve this, we took advantage of
a dominant active form of Gli3 from which the entire N-terminal/repressor
domain was deleted (hGli3N2)
(Stamataki et al., 2005). This
construct provides strong transcriptional activation in vitro and in vivo
(Gli3AHIGH) (Stamataki et al.,
2005). HH stage 11-12 chick embryos co-electroporated with
Ptc1loop2 and Gli3AHIGH, assayed 24 hours PE
displayed the expected cell autonomous downregulation of the dorsal marker
Pax7 together with the suppression of the ventral expansion of Pax7 induced by
Ptc1loop2 (Fig.
2M). Moreover, expression of Gli3AHIGH was sufficient
to inhibit Ptc1loop2-induced apoptosis. The transfected side
of the neural tube of co-electroporated embryos no longer displayed the
shortening characteristic of Ptc1loop2 transfectants and
assays for caspase 3 immunostaining (Fig.
2N) and TUNEL (not shown) indicated that co-electroporated embryos
had wild-type levels of apoptosis [the ratio of apoptotic cells in the neural
tube co-electroporated with Ptc1loop2 + Gli3AHIGH
is 3.61±1.88 (n=4) compared with 11.99±5.74
(n=4) in Ptc1loop2-alone embryos]
(Fig. 2S). Together, these data
suggest that Gli dependent transcription mediates Ptc1-induced apoptosis of
neural tube progenitors, and indicate that Gli3 is epistatic to
Ptc1loop2 for both activities.
In keratinocytes, the anti-apoptotic factor Bcl2 has been shown to be
induced by Gli-dependent transcription
(Bigelow et al., 2004;
Regl et al., 2004). We
therefore asked whether Bcl2 is induced by Gli-transcriptional activation in
neuroepithelial cells. Indeed, electroporation of Gli3AHIGH was
sufficient to induce Bcl2 expression cell autonomously, as detected by
immunohistochemistry (Fig. 2L).
Furthermore, western blot analysis revealed low levels of Bcl2 expression on
control side and a two-fold increase in Bcl2 protein levels in neural tubes 24
hours after electroporation of Gli3AHIGH
(Fig. 2O). Thus, Bcl2 seems to
be a conserved Gli-transcriptional survival target, functioning also in
neuroepithelial cells. We next reasoned that as Bcl2 was a target of
Gli-transactivation, overexpression of Bcl2 may rescue
Ptc1loop2 and Gli3R induced apoptosis. Consistent with this
idea, forced expression of human BCL2, together with
Ptc1loop2 (Fig.
2S) or with Gli3R (Fig.
2P,Q,S) reduced the levels of apoptosis in transfected neural
tubes [the ratio of apoptotic cells in the neural tube of embryos
co-electroporated with Ptc1loop2 +human BCL2 is
3.14±1.60 cells (n=4) and in Gli3R+BCL2 is 3.72±2.04
(n=4)]. Thus, these results indicate that Bcl2 overexpression is
sufficient for the survival of neuroepithelial cells and suggests that basal
levels of Gli-mediated induction of Bcl2 may be required for survival of
neuroepithelial cells.
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loop2, there was
no increase in the number of post-mitotic neurons at 24 hours PE, thus the
decrease in progenitor proliferation could not be accounted for by precocious
differentiation of neuroepithelial cells. Moreover, by 24 hours PE there were
fewer differentiated neurons on the Ptc1loop2 transfected
side of the embryo compared with the control side
(Fig. 3A,B). This most likely
reflects the general reduction in tissue size caused by the
Ptc1loop2 expression: the lack of Shh signaling did not
appear to inhibit neural differentiation in a cell-autonomous manner, as many
GFP-positive transfected cells were also NeuN and Tuj1 positive
(Fig. 3A,B). Moreover, the
decrease in proliferation was apparent prior to changes in the expression of
transcription factors regulated by Shh signaling, that control progenitor cell
identity. For example, in embryos transfected with
Ptc1loop2, the changes in the expression of Pax7 were
observed at 24 hours PE (Fig.
3D) but at 12 hours PE, a time point at which a decrease in
progenitor proliferation was apparent, the expression of Pax7 was comparable
with the non-transfected control side of the neural tube
(Fig. 3C). Together, these data
raise the possibility that Shh/Ptc signaling directly regulates cell cycle
progression.
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)
left open the possibility that Ptc1 inhibits the cell cycle independent of Gli
proteins. We first transfected the dominant inhibitory Gli protein, Gli3R,
into stage HH11/12 chick embryos. Assays at 12 hours PE showed a reduction in
the total number of mitotic cells (Fig.
4A) and a reduction in the number of transfected cells that can
incorporate BrdU to 50% [Gli3R expression reduced the proportion of
transfected cells incorporating BrdU to 13.13±0.36% (n=4)
(P=0.001) in the ventral neural tube and to 12.68±0.01%
(n=4) (P<0.0001) dorsally compared with
26.25±10.19% ventrally and 26.18±8.02% dorsally in pCIG embryos]
(Fig. 4B,M). Furthermore,
embryos analyzed 24 hours PE exhibited a dramatic reduction in the number of
proliferating cells and size of the neural tube
(Fig. 4C; data not shown). The
severity of this phenotype most probably reflects the combined consequence of
lack of proliferation together with enhanced cell death seen in embryos
expressing Gli3R (Fig. 2B,D). Therefore, in order to analyze whether Gli3R regulated cell cycle progression
independently of apoptosis, we co-electroporated Gli3R and human BCL2.
Co-transfection of both plasmids was monitored by immunostaining with
anti-Bcl2 and GFP fluorescence as a reporter of Gli3R (not shown). Embryos
transfected with Gli3R+human BCL2, analyzed 12 hours PE had a significantly
decreased proliferation rate, as assessed by pH3 immunostaining
(Fig. 4D) and by BrdU
incorporation (Fig. 4E,M). We
observed a 75% reduction in BrdU incorporation in Gli3R+human BCL2
transfected cells when compared with control pCIG-transfected progenitors
(Fig. 4M) [6.69±0.62%
cells in Gli3R+human BCL2 embryos are double labelled for GFP and BrdU
(n=7) (P<0.0001) in the ventral and 5.92±0.82%
(n=7) (P<0.0001) in the dorsal neural tube]. The enhanced
blockade of cell cycle progression compared with embryos transfected with
Gli3R alone, is probably the result of the survival of additional
Gli3R-expressing cells and the fact that these cells do not enter S phase
(Fig. 4M). In order to test whether cell cycle arrest is mediated through active Gli repression or through the lack of Gli-transactivation, we blocked all Gli transcriptional activities using Gli-ZnF (Fig. 2E-H). Embryos analyzed either 12 or 24 hours PE of Gli-ZnF, showed normal rates of proliferation as assessed by either pH3 immunostaining (Fig. 4G) or BrdU incorporation (Fig. 4H,I). Furthermore, both halves of the neural tube were the same size, reflecting the normal growth of these neural tubes. These data indicate that the observed Gli3R-dependent cell cycle arrest might be due to active Gli-repression.
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loop2 and Gli-ZnF and assayed at
12 and 24 hours PE. Total number of mitotic cells, assessed by immunostaining
to pH3 was restored in co-electroporated embryos at both time points
(Fig. 4J, data not shown).
Furthermore, lack of Gli repression restored BrdU incorporation in a
cell-autonomous manner, both by 12 hours PE [28.16±6.86% of
Ptc1loop2 +Gli-ZnF transfected cells incorporate BrdU
(n=4) in the ventral and 31.11±3.11% cells (n=4) in
the dorsal neural tube] (Fig.
4K,M) and 24 hours PE [in Ptc1loop2 +Gli-ZnF
embryos 19.65±3.28% cells are double-labelled of GFP/BrdU
(n=4) in the ventral and 23.52±4.65% in the dorsal neural tube
(n=4)] (Fig. 4L,N),
strongly suggesting that Ptc1loop2-dependent cell cycle
arrest is mediated through Gli-repression.
Inhibiting Shh/Gli signaling lengths the G1 phase of the cell cycle
We next tested how transfection of Gli3-R modulated cell cycle phase
distribution. For this purpose, neuroepithelial cells electroporated in ovo
with the control vector pCIG±human BCL2
(Fig. 5A), with GLi3R
(Fig. 5B) or with Gli3R+human
BCL2 (Fig. 5C) were analyzed by
flow cytometry. This analysis indicated that in control conditions, 12 hours
PE, 59% of cells were in the G1 phase of the cell cycle (1N DNA content), 23%
in S phase (intermediate DNA content) and 18% in G2/M phase (2N DNA content)
(Fig. 5D). Gli3R transfection
caused a dramatic increase in apoptosis and generated a very abnormal cell
cycle profile, with a sub-G1 population that probably corresponds to dead and
dying cells (Fig. 5B).
Therefore, we analyzed the cell cycle of neuroepithelial cells that were
co-transfected with Gli3R+human BCL2. The restoration of the cell cycle
profile in these cells confirmed the anti-apoptotic effect of Bcl2
(Fig. 5C) and in these
conditions the proportion of transfected cells in G1 had increased to 71%,
while only 15% of cells were in S phase and 14% cells were in G2/M phase
(Fig. 5D). These data suggest
that transfection of Gli3R prevents entry of neuroepithelial cells into the S
phase of the cell cycle and results in cells accumulating in G1.
Consistent with a G1 block in cell cycle progression, the expression of
cyclin D1, a cyclin characteristic of G1 progression, was repressed, at least
in ventral regions of Gli3R-transfected neural tubes
(Oliver et al., 2003;
Kenney and Rowitch, 2002)
(Fig. 5E). Noteworthy, however,
was the continued cyclin D1 expression in dorsal neural progenitors,
suggesting that the cell cycle block initiated by Gli3R may involve the
regulation of genes in addition to cyclin D1. Expression of N-myc, a
Shh-target gene promoting proliferation of cerebellar granule precursors
(Kenney et al., 2003) was also
repressed in Gli3R+human BCL2 electroporated neural tubes
(Fig. 5F). Together these data
suggest that Gli-mediated repression of genes required for completion of the
G1 phase of the cell cycle, cause cell cycle arrest of neuroepithelial
cells.
Activator Gli-proteins induce overproliferation
It has previously been shown that dorsal activation of the Shh-pathway by
either ectopic expression of the ligand
(Rowitch et al., 1999) or by
the introduction of a dominant-negative form of PKA
(Epstein et al., 1996) caused
a prominent overgrowth of the dorsal neural tube. HH11-12 embryos
electroporated with this same dnPKA construct show the expected changes in
patterning 24 and 48 hours PE (see Fig. S2A-C in the supplementary material),
together with increased proliferation on neural tubes (see Fig. S2D-I in the
supplementary material).
|
To further examine how transfection of Gli3AHIGH modulated cell cycle phase distribution, neuroepithelial cells electroporated with the control vector pCIG (Fig. 6F) or with Gli3AHIGH (Fig. 6G) were analyzed by flow cytometry. Twenty-four hours PE with pCIG-transfection, 61% of neuroepithelial cells were present in the G1 phase of the cell cycle (1N DNA content), 33% of cells were in the S phase (intermediate DNA content) and 6% of cells were in the G2/M phase (2N DNA content) (Fig. 6H). However, Gli3A transfection decreased the proportion of cells in G1 to 49% and increased the number in S phase to 36% and to 16% cells for in G2/M phase (Fig. 6H). These results support a model in which Gli transcriptional activation shortens the length of G1 for neuroepithelial cells, resulting in a higher proportion of cells residing in S or G2/M at the time of analysis.
Consistent with this idea, transfection of Gli3AHIGH increased
the expression of genes involved in progression of the G1 phase of the cell
cycle, such as cyclin D1 (Fig.
6I) and N-myc (data not shown) in the neural tube. Thus, both
cyclin D1 and N-myc seem to be conserved Gli-transcriptional target genes
(Oliver et al., 2003;
Kenney and Rowitch, 2002;
Kenney et al., 2003;
Lobjois et al., 2004).
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
During neural development, Shh, which is produced by the axial midline
structures of the notochord and floor plate
(Martí et al., 1995),
functions as a long-range signal to direct the dorsoventral patterning of
neural progenitors and control neuronal subtype identity
(Jessell, 2000;
Briscoe and Ericson, 2001). In
addition, Shh signaling is required for growth and survival of neural
progenitors. We provide evidence that this requirement for Shh signaling is
cell-autonomous. Thus, decreases in proliferation observed on blockade of Shh
signaling is restricted to those progenitors transfected with the inhibitory
constructs (Ptc1loop2 and Gli3R), while increases in
proliferation is restricted to cells transfected with the activating
constructs (dnPKA and Gli3AHIGH). This indicates a direct influence
of Shh on the control of proliferation of cells and, although the data do not
exclude the possibility that other factors, in conjunction with Shh signaling,
influence the proliferation and survival of progenitors, this study argues
against an exclusively indirect role for Shh in which Shh signaling induces
the expression of another secreted molecule that acts at long range to promote
the growth of the neural progenitors.
Notably, blockade of Shh signaling in both dorsal and ventral regions of
the neural tube affects the survival and proliferation of progenitors. One
possibility is that a tonic, ligand-independent low-level activation of the
pathway in dorsal regions may be sufficient to maintain progenitor
proliferation and survival. Alternatively, it is possible that there is an
extended range of influence of Shh that includes progenitors throughout most
of the neural tube. This would be consistent with the observation that
elevated levels of the Shh responsive gene, Ptc1, are present in a broad
domain of progenitors that includes the dorsal neural tube
(Goodrich et al., 1997).
Moreover in embryos lacking Shh signaling
(Chiang et al., 1996;
Litingtung and Chiang, 2000;
Thibert et al., 2003;
Wijgerde et al., 2002) the
entire neural tube, not just the ventral regions appear decreased in size.
Thus, the long-range action of Shh could be required not only for the
patterning of progenitors but also for their survival and proliferation,
providing a means to couple these attributes of neural cells.
The canonical transduction of Shh signals is initiated by Shh binding to
Patched and culminates with the regulation of activity of transcription
factors of the Gli family, which control Shh regulated gene expression
(reviewed by Jacob and Briscoe,
2003). Although evidence of non-canonical signaling mechanisms has
emerged (Thibert et al., 2003;
Barnes et al., 2001;
Testaz et al., 2001), raising
the possibility that the regulation of cell survival and proliferation by Shh
precedes via a non canonical pathway independent of Gli activity, our data
suggest that this is not the case. Blockade of Shh signaling at either the
receptor level, using Ptc1loop2, or at the transcriptional
effector level using the dominant inhibitory Gli protein Gli3R had similar
effects: promoting apoptosis and decreasing proliferation. Moreover, the
pro-apoptotic and anti-mitogenic activity of Ptc1loop2 could
be counteracted by the simultaneous transfection of a dominant-active Gli
protein, Gli3AHIGH, suggesting that Gli activity is epistatic to
Ptc1. The anti-proliferative effect of Ptc1loop2 was also
blocked by co-expression of Gli-ZnF, a construct consisting only of the
DNA-binding domain of Gli3 that is capable of blocking both the repressor and
activator functions of Gli. This suggests that in
Ptc1loop2-expressing cells, it is the increase in Gli
repressor activity that is responsible for the reduced proliferation rate. By
contrast, the expression of Gli-ZnF alone promoted apoptosis in neural cells.
This level of apoptosis was not increased by the co-expression of
Ptc1loop2 with Gli-ZnF, as would be expected if the two
proteins induced apoptosis via distinct pathways. Rather, the proapoptotic
effect of Gli-ZnF suggests that a tonic level of Gli activator protein is
required for the survival of neuroepithelial cells. Titration of this
activator protein either using Gli-ZnF or by expression of
Ptc1loop2 can decrease the survival of neuroepithelial
cells.
Consistent with these data, the growth defects observed in the neural tubes
of mouse embryos lacking Shh or Smo are rescued in double mutant embryos that
also lack Gli3, suggesting that, in the absence of Shh signaling, Gli3
functions to repress proliferation and cell survival
(Litingtung and Chiang, 2000).
Inhibition of the repressive activity of Gli3 either by increasing Shh
signaling or genetic removal of Gli3 relieves this repression, allowing
progenitors to survive and grow. Together with the data presented here,
therefore, the evidence supports a central role for the regulation of Gli
activity in the control of neural cell proliferation and survival. What
contributions non-canonical mechanisms make to the control of these properties
and whether these non-canonical pathways ultimately converge on the regulation
of Gli activity remain to be determined.
Our data suggest that the regulation of cell survival and proliferation by
Shh are independent of one another. Inhibition of Shh signaling by
Ptc1loop2 results in decreases in the proliferation rate of
neural cells prior to the onset of apoptosis. Thus, delayed apoptosis could be
interpreted as a consequence of cell cycle arrest and the induction of
`mitotic collapse'. However, several lines of evidence argue against this.
First, the expression of Gli-ZnF promotes apoptosis without affecting the
proliferation rate of neuroepithelial cells. Second, the expression of Gli3R
is able to induce high levels of apoptosis within 6 hours of transfection,
prior to any detectable change in proliferation, suggesting a direct effect on
a gene or genes necessary for the survival of neuroepithelial cells. Thus, the
decrease in cell survival in the absence of Shh signaling does not appear to
be exclusively a consequence of cell cycle arrest; instead, the data suggest
that Shh signaling directly regulates cell survival, independently of
proliferation rate. Conversely, ectopic activation of Shh signaling with
Gli3AHIGH increases the rate of proliferation without affecting the
survival of neural cells. Moreover, a decrease in mitogenesis is also observed
in cells unable to respond to Shh in which apoptosis has been blocked by
forced expression of Bcl2. Together these data suggest that the lower levels
of proliferation observed on blockade of Shh signaling are not a simply a
consequence of an increase in apoptosis, but instead a consequence of Gli
mediated regulation of cell cycle progression.
Previous studies have suggested that canonical Wnt signaling from the
dorsal aspect of the neural tube via activation of ß-catenin has an
important role in the control of precursor cell proliferation
(Megason and McMahon, 2002).
The data showing that Shh also influences the proliferation and survival of
neural progenitors raise the issue of how these two mitogenic factors interact
to ensure the normal growth of progenitors. It is possible that the two
factors regulate the growth of the neural tube via distinct mechanisms
involving different transcriptional responses. Alternatively, it is possible
that both factors converge on the same set of target genes; indeed, the
possibility of cross-talk between the two should not be ruled out given the
number of shared components between the two pathways
(Meng et al., 2001;
Jia et al., 2002;
Price and Kalderon, 2002) and
the number of common target genes (Megason
and McMahon, 2002; Panhuysen
et al., 2004). Accordingly, cross-talk between the two pathways
may act to integrate the mitogenic and survival responses of cells to ensure
the well regulated growth and morphogenesis of the neural tube.
The identity of the target genes that control the survival and
proliferation of neural progenitors remains to be clarified. The regulation of
dorsoventral position by Shh signaling involves a group of transcription
factors that control neuronal subtype identity by partitioning neural
progenitors into a series of domains
(Jessell, 2000;
Briscoe and Ericson, 2001).
These transcription factors do not, however, appear to be involved in the
regulation of the proliferation or survival of progenitors. There are no
reported changes in the growth or survival of neural progenitors in gain- or
loss-of-function experiments that change the expression of the homeodomain
proteins (Briscoe et al., 2000;
Ericson et al., 1997;
Muhr et al., 2001;
Novitch et al., 2001;
Pierani et al., 2001;
Vallstedt et al., 2001),
despite their ability to change the dorsoventral organization of the neural
tube and the pattern of neuronal subtype generation. Moreover, in the current
study, we provide evidence that the changes in the proliferation rate and
survival of cells elicited by the blockade of Shh signaling occur rapidly,
prior to evident changes in the expression of homeodomain protein expression.
Indeed, Gli3R appears to induce high levels of neural apoptosis within 6 hours
of transfection, precluding the presence of multiple steps between Gli
repression and a proapoptotic response. Furthermore, activated Gli constructs
that mimic different levels of Shh signaling and induce different sets of
homeodomain proteins (Stamataki et al.,
2005) have similar effects on promoting the proliferation of
progenitors (data not shown). Together, these data dissociate the expression
of the transcription factors that pattern progenitors from the control of
neural progenitor survival and proliferation, raising the possibility that Gli
activity may directly regulate the genes that mediate these distinct
responses.
A number of candidate genes have been previously identified as Shh
responsive and involved in cell survival and advancing the cell cycle. Bcl2 is
induced in keratinocytes (Bigelow et al.,
2004; Regl et al.,
2004) and we demonstrate an upregulation of Bcl2 in neural cells
expressing an activated Gli construct, thus the regulation of Bcl2 may ensure
the survival of neural progenitors. Our analysis of the cell cycle indicates
that Shh signaling affects the length of G1, consistent with this idea N-myc
(Kenney et al., 2003) and
cyclin D1 (Oliver et al.,
2003; Kenney and Rowitch,
2002; Lobjois et al.,
2004) have been found to be responsive to Shh signaling in the
developing cerebellum and in the neural tube. Indeed our analysis indicates
that alteration of Shh signaling in the neural tube affects the expression of
both N-myc and cyclin D1; however, the changes in the expression of these two
genes does not appear to be sufficient to account for the observed changes in
the cell cycle. For example, Gli3R expression represses the ventral expression
of cyclin D1; however, cyclin D1 expression is maintained in dorsal
progenitors, despite the proliferation rate of these cells being retarded to
the same extent as ventral progenitors. Conversely, the expression of N-myc is
observed in intermediate and dorsal progenitors but is excluded from ventral
regions, suggesting that other genes must promote proliferation ventrally.
Other candidates for affecting the proliferation of neural progenitors include
genes such as the polycomb group protein Bmi1
(Leung et al., 2004).
Identifying the genes regulated by Shh signaling that mediate the
proliferative responses and survival of neural cells may shed light on how Shh
signaling coordinates progenitor growth and survival with the patterning of
the neural tube and cell fate specification.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/3/517/DC1
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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