1 Department of Genetic Medicine and Development, 8242 CMU, 1 rue Michel Servet,
University of Geneva Medical School, 1211 Geneva 4, Switzerland
2 Skirball Institute, NYU School of Medicine, 540 First Avenue, New York, NY
10016, USA
Author for correspondence (e-mail:
ariel.ruizaltaba{at}medecine.unige.ch)
Accepted 13 May 2005
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SUMMARY |
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Key words: Gli, Neurogenesis, Tumor, Morpholino, Neural plate, Xenopus, Antisense
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Introduction |
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Several intercellular signaling pathways have been implicated in the
control of neurogenesis in vertebrates, including the bHLH/Notch and retinoic
acid pathways (e.g. Ruiz i Altaba and
Jessell, 1991; Chitnis et al.,
1995
; Franco et al.,
1999
; Pierani et al.,
1999
; Sharp and Goldstone, 2000), as well as the Sonic
hedgehog-Gli (Shh-Gli) pathway (Lee et
al., 1997
; Hynes et al.,
1997
; Brewster et al.,
1998
; Ruiz i Altaba et al., 1998;
Franco et al., 1999
;
Lai et al., 2003
;
Machold et al., 2003
;
Palma and Ruiz i Altaba, 2004
;
Palma et al., 2005
). The Gli
proteins are obligatory mediators of Hh signals, but it is not clear how they
act. It has been proposed that the crucial step is the overall read out of
positive and negative Gli functions: the Gli code
(Ruiz i Altaba, 1997
;
Ruiz i Altaba, 1998
) (reviewed
by Ruiz i Altaba et al.,
2003
).
Gain- and loss-of-function analyses in different species show: that the Gli
proteins function in a context-dependent manner; that Gli2 and Gli3, unlike
Gli1, harbor strong dominant-negative function in C-terminally truncated
forms; and that there is partial redundancy (reviewed by
Ruiz i Altaba et al., 2003).
Dominant-negative assays in the frog neural plate and the chick neural tube
indicate the requirement of overall positive Gli function for normal
patterning (Brewster et al.,
1998
; Persson et al.,
2002
; Meyer and Roelink,
2003
). Similarly, in mice, the phenotype of the double Gli2/Gli3
null, which does not seem to express Gli1, indicates a requirement of Gli
proteins for proper neurogenesis and patterning
(Bai et al., 2004
), consistent
with the absolute requirement in flies for Ci, the Gli homologue, in
Hh-mediated patterning (Methot and Basler,
2001
).
A problem in understanding Gli function resides in part on the difficulty of examining similar alterations for the three Gli proteins in any given system, as their functions are context dependent. The following summarizes current knowledge.
(1) Gli1 behaves as a positive activator. In frog embryos, Gli1
gain of function mimics Shh signaling (Lee
et al., 1997; Ruiz i Altaba,
1998
; Ruiz i Altaba,
1999
). This result contrasts with the reported normality of
Gli1/mice
(Park et al., 2000
;
Bai et al., 2002
), although
minor embryonic defects are seen when compounded with mutation in Gli2
(Park et al., 2000
). In
zebrafish, however, Gli1 mutants have a phenotype
(Karlstrom et al., 2003
). Gli1
has been also reported to be superfluous for mouse tumorigenesis
(Weiner et al., 2002
), but it
is required for the proliferation of human tumor cells
(Sanchez et al., 2004
).
(2) Gli2 can act as a positive or negative element in Hh signaling. In
frogs, it can induce motoneurons while inhibiting floor plate and neural crest
differentiation (Ruiz i Altaba,
1998; Brewster et al.,
1998
). In mice, it is mostly a weak activator of floor plate
differentiation in the early CNS (Matise
et al., 1998
; Ding et al.,
1998
). Here, its function can be replaced by that of Gli1
(Bai and Joyner, 2001
) but in
zebrafish it harbors mostly negative function
(Karlstrom et al., 1999
;
Karlstrom et al., 2003
),
although there is also evidence for limited positive effects
(Tyurina et al., 2005
). At
late CNS stages, mouse Gli2 is required for normal brain growth and stem cell
maintenance (Palma and Ruiz i Altaba,
2004
). In humans, GLI2 is required for proper midline development
with its loss of function leading to holoprosencephaly and pituitary
deficiencies (Roessler et al.,
2003
), a phenotype with similarities to that of loss of
Shh in mice and humans (Chiang et
al., 1996
; Belloni et al.,
1996
; Roessler et al.,
1996
).
(3) Gli3 can induce neurogenesis and repress neural crest and floor plate
differentiation in the frog neural plate
(Brewster et al., 1998;
Ruiz i Altaba, 1998
). In
zebrafish, it can act as both an activator and a repressor in vivo, and can
cooperate with or inhibit Gli1 functions in vitro
(Tyurina et al., 2005
). Mouse
mutants for Gli3 display neural tube closure defects and polydactyly
(Johnson, 1967
;
Schimmang et al., 1992
;
Hui and Joyner, 1993
;
Theil et al., 1999
;
Tole et al., 2000
), the latter
much like humans with a defective GLI3 gene
(Vortkamp et al., 1991
;
Kang et al., 1997
;
Radhakrishna et al., 1997
).
Gli3 harbors strong repressive function, possibly in C'
forms
(such as Gli2 and Ci, but not Gli1) detected in vivo
(Aza-Blanc et al., 1997
;
Ruiz i Altaba, 1999
;
Shin et al., 1999
;
von Mering and Basler, 1999
;
Aza-Blanc et al., 2000
;
Wang et al., 2000
). Gli3 plays
a critical role in Shh signaling in the early neural tube as Shh inhibits
Gli3 transcription (Ruiz i Altaba
1998
) and, importantly, loss of Gli3 can partially rescue
the phenotype of Shh mouse mutants
(Litingtung and Chiang, 2000
).
In the brain, Gli3 is also required for growth and the regulation of stem cell
behavior (Palma and Ruiz i Altaba,
2004
).
Together, these data on patterning and cell type specification by the three
Gli genes in different species demonstrate the importance of Gli function. The
Gli code is thus thought to regulate the CNS homeodomain code, the latter
being required for specification of different neuronal subtypes
(Briscoe et al., 2000).
However, the variable results make a unifying interpretation difficult.
Similarly, their function in primary neurogenesis is not completely clear.
Pan-Gli dominant-negative assays and gain-of-function analyses with individual
Gli genes indicate that the Gli proteins are key components of neurogenesis
(Brewster et al., 1998
), but
their individual requirement in the neurogenic program has not been
determined. Here, we have used a knockdown antisense approach to determine the
requirement of each Gli protein in cell fate specification, using primary
neurogenesis as a model system.
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Materials and methods |
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In vitro translation
Gli1, Gli2, Gli3 and GLI1 cDNAs in pCS2 vectors (0.5 to
1.0 µg) and 1 µM of MO-C, MO-1, MO-2 and MO-3 were used to test for
protein production using the TNT Coupled Reticulocyte Lysate Systems Kit
(Promega) in the presence of 35S-methionine. The levels of specific
Gli proteins were measured by autoradiography after 7% SDS-PAGE, immersion in
Enhance (Amersham) and drying.
In situ hybridization and ß-gal staining
Whole-mount in situ hybridization was performed with digoxigenin-labeled
single stranded RNA probes followed by nitroblue tetrazolium (NBT) plus
5-bromo-Y-chloro-3-indolyl phosphate (BCIP) (purple) substrates. Gli1,
Gli2 and Gli3 (Lee et al.,
1997; Ruiz i Altaba,
1998
); Pintallavis
(Ruiz i Altaba and Jessell,
1992
); Sonic hedgehog
(Ruiz i Altaba et al., 1995
);
Zic2, Slug, Snail and N-tubulin
(Brewster et al., 1998
) probes
were as described. Other probes were made with the following enzymes:
Xash3 (Ferreiro et al.,
1994
), NotI and T3; Xmyt
(Bellefroid et al., 1996
),
BamH1 and T7; Xaml
(Tracey et al., 1998
),
SalI and T7; and Sox3
(Penzel et al., 1997
),
EcoRI and T3. For ß-gal staining, fixed embryos were incubated
with X-gal substrates (purple red/pink precipitates) before dehydration with
methanol.
BrdU incorporation
5-bromo-deoxyuridine (BrdU 10 nl of 3 mg/ml; Sigma) were injected into
three areas of stage 13 embryos previously injected with MO-3. Incorporated
BrdU was detected immunocytochemically with diaminobenzidine
(Hardcastle and Papalopulu,
2000; Dahmane et al.,
2001
). Mouse anti-BrdU antibodies (mouse from Becton Dickinson)
were used at 1:100. Horseradish peroxidase-coupled secondary anti-IgG
antibodies (Amersham) were used at 1:200.
Quantitative real-time PCR
Total RNA extracted from animal caps was denatured for 10 minutes at
65°C in the presence of random hexamer primers, immediately cooled in ice
water, then reverse transcribed using SuperScript II reverse-transcriptase.
Real-time PCR was performed with an OpticonTM machine (MJR). Reactions
were carried out in 30 µl containing 1 µl of template, 200 nM of each
forward and reverse primer, and 1 x IQTM SYBR Green Supermix
(BioRad). To quantify transcripts, dilutions of cDNA controls (from sibling
embryos non injected) were run in parallel. All experiments were performed in
triplicate.
Cell transfection and immunocytochemistry
Gli1, Gli2, GLI3 and Zic2 cDNAs were subcloned into
Flag-tagged or myc-tagged vectors either as full-length clones or deletion
mutants (Lee et al., 1997;
Brewster et al., 1998
; Ruiz i
Altaba et al., 1999; Liu et al., 1998). Deletion mutations were carried out
using restriction sites within the gene, resulting in the following
constructs: GLI3N'
StuI (amino acids 1-390 deleted),
GLI3C'
ClaI (amino acids 745-1596 deleted),
GLI3C'
XhoI (amino acids 558-1596 deleted), Gli2C'
(amino acids 559-1468 deleted), Gli1C'
PstI (amino acids 551-1397
deleted). Other deletion mutations were carried out using PCR:
Zic2N'
(amino acids 1-260 deleted), Zic2C'
(amino
acids 451-501 deleted), VP16Zic2ZF
(Brewster et al., 1998
),
GLI3ZF1,2 (amino acids 473-547) and Zic2ZF1,2 (amino acids 270-351).
Immunocytochemistry of transfected COS-7 cells was as described
(Ruiz i Altaba, 1999
).
Co-immunoprecipitation and western analyses
COS-7 cells were transfected with indicated plasmids at 60% confluency with
lipofectamine. Forty-eight hours post-transfection, cells were washed with
PBS, lysed with RIPA buffer [150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS, 50 mM
Tris pH 7.5, protease inhibitor cocktail (Sigma P-2714) plus 100 mM PMSF] and
incubated with either 2 µl Flag Ab (Sigma F3165) or 10 µl Myc antibody
(Santa Cruz, sc-789) for 1 hour at 4°C. ProteinA/G beads (20 µl; Santa
Cruz) were added and incubated for 1 hour at 4°C. Beads were then washed
four times with ice-cold RIPA buffer, sample buffer added, the mixture boiled
and subjected to 10% SDS-PAGE, transferred onto membranes and probed with
opposing antibody to immunoprecipitation. For immunoblotting, anti-Myc and
anti-Flag antibodies were used at concentration of 1/250 and 0.5 µg/ml,
respectively. Binding was visualized using ECL reagents (Amersham). Total
protein from each experiment was run separately on SDS-PAGE gels for
confirmation of protein expression and lack of degradation.
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Results |
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Injection of MO-C had minor effects, comparable with injection of tracer alone, on the development of primary neurons assayed at stage 14 through the expression of the neuronal-specific marker N-tubulin by whole-mount in situ hybridization (MO-C: 1% embryos with reduction of N-tubulin, n=16; Fig. 1F). By contrast, injection of morpholinos specific for Gli1 (MO-1), Gli2 (MO-2) or Gli3 (MO-3) inhibited primary neuron differentiation [MO-1: 66.7%, n=12 (Fig. 1C); MO-2: 50%, n=19 (Fig. 1D); MO-3: 83.9%, n=26 (Fig. 1E) embryos with fewer primary neurons than MO-C injected embryos] in all three populations. Quantification of the number of N-tubulin+ neurons per embryo in the injected half versus the uninjected half in each population is given in Fig. 1B.
The specificity of MO action was further proven by rescue experiments. Co-injection of 10, 5 or 2.5 ng of MO plus 1 ng of the corresponding Gli RNA resulted in an increasing number of rescued embryos with normal neuronal pattern: 16, 46 and 50% rescue for Gli1; 44 and 56% for the two lower amounts of MO for Gli2; and 25, 42 and 67% rescue for Gli3 (Fig. 1G,H; data not shown). Only one embryo injected with 1 ng Gli3 RNA plus 2.5 ng MO-3 showed ectopic neurogenesis, indicating that the MO rescues ectopic neurogenesis by the appropriate injected Gli RNA (and not by the other two Gli genes, see below) and Gli RNA rescues the suppression of endogenous neurogenesis by the appropriate MO.
|
Knockdown of Gli3 did not alter the expression of Gli2 (n=33) or of the midline markers Pintallavis (n=19) and Sonic hedgehog (n=20) at stage 14 (Fig. 2). It inhibited Gli1 expression adjacent to the midline (58%, n=19) and minimally its own transcription (6%, n=26), but not that of Zic2 (n=29) near the edges of the neural plate (Fig. 2). However it impaired the expression of the neural crest markers Slug (51%, n=29) and Snail (48%, n=33; Fig. 2). In addition, MO-3 inhibited the expression of the early bHLH neurogenic cascade genes Xash3 (43%, n=25) and Xmyt1 (26%, n=38; Fig. 2). The inhibition of neuronal populations was confirmed by the loss of Xaml (60%, n=28) expression, a marker of primary sensory neurons (Fig. 2). MO-3 slightly increased the size of the neural plate as judged by Sox3+ staining (65%, n=43), but did not appreciably change BrdU+ incorporation (n=35 embryos tested; Fig. 2 and not shown). This was evident comparing the injected and control halves of the neural plate and also in cases where there was cell mixing in the neural plate between expressing and non-expressing cells (Fig. 2).
The three Gli proteins are required for neuronal differentiation induced by the ectopic activation of neurogenic pathways
Neurogenesis can be triggered by the activation of the bHLH neurogenic
pathways, which specify neuronal fate, and by activated Notch signaling, which
is involved in lateral inhibition (reviewed by
Chitnis and Kintner, 1995;
Kintner, 2002
). Injection of
neurogenin 1 (Ngn1a) RNA resulted in massive
N-tubulin+ neuronal differentiation
(Ma et al., 1996
). Similarly,
expression of NeuroD (Lee et al.,
1995
), another bHLH gene that acts downstream of Ngn1a
also resulted in a strong neurogenic phenotype (stage 14-15;
Fig. 3; Ngn1a+MO-C,
94% ectopic induction, n=34; NeuroD+MO-C, 79% ectopic
induction, n=33). These phenotypes were reversed by MO-3
(Fig. 3; Ngn1a+MO-3,
89% reduction, n=28; NeuroD+MO-3, 86% reduction,
n=29). In a second set of experiments, each Gli MO inhibited
neurogenesis by co-injected Ngn1a RNA (stage 13;
Fig. 3, right panels;
Ngn1a+MO-C, 86% ectopic N-tubulin, n=70;
Ngn1a+MO-1, 100% reduction, n=21; Ngn1a+MO-2, 100%
reduction, n=43; Ngn1a+MO-3, 100% reduction, n=22,
not shown). In addition, neurogenesis induced by a mutant form of
Delta (Delta1Stu), which cell-autonomously
inhibits Notch signaling and thus bypassing normal lateral inhibition
(Chitnis et al., 1995
;
Chitnis and Kintner, 1996
)
(Fig. 3;
Delta1Stu+MO-C, 75% ectopic induction, n=29), was
reversed by MO-3 (Fig. 3;
Delta1Stu+MO-3, 76% reduction, n=34).
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GLI1-induced tumorigenesis requires endogenous Gli1 and Gli3 function
As a second test for the requirement of endogenous Gli function in
Gli-induced processes we have used the induction of tadpole skin tumors by
GLI1 (Dahmane et al., 1997).
We have previously shown that expression of human GLI1 induces epidermal
hyperplasias or tumors which are marked by the expression of ß-gal from
the co-injected lacZ tracer
(Dahmane et al., 1997
), and
that co-injection with MO-1 inhibited tumor formation
(Dahmane et al., 2001
). As
expected, injection of GLI1 and MO-C resulted in massive tumor
induction and co-injection with MO-1 decreased the tumorigenic phenotype
(Fig. 5A,B: GLI1, 88%
tumor formation, n=59; GLI1+MO-1, 23% tumor formation,
n=56). Paralleling the neurogenic results, co-injection of MO-3, but
not MO-2, also reduced tumor formation
(Fig. 5A,B: GLI1+MO-3,
19% tumor induction, n=51; GLI1+MO-2, 90% tumor induction,
n=50).
Gli proteins cooperate in a dynamic fashion to regulate gene expression in a target-and context-specific manner
To extend the results with whole embryos, we have used the animal cap assay
to further test and quantify the specific requirements of each Gli gene in
target gene induction. Gene expression was tested in pooled animal cap samples
from sibling embryos after injection of each Gli RNA along with MO-C or MOs
for the other Gli genes. The analyses were carried out in triplicate and
quantified with real-time PCR. Animal caps expressing frog Gli RNAs plus MOs
were collected at stage 12 and stage 14 to allow for a temporal comparison,
while those expressing full-length synthetic human GLI RNAs plus MOs were
collected at stage 14 to allow for a species of origin comparison. Tabulation
of the results is shown for Gli plus MO-C as the ratio of the value of Gli
plus MO-C over that in uninjected animal caps (asterisks in
Table 1). The value of these
numbers is not important, as it depends on the kinetics of the exact primer
pairs used. However, a positive value means induction and a negative one
repression. The values given for Gli plus MO-1, MO-2 or MO-3 is the ratio of
the number for the specific combination of Gli plus MO over that of the same
Gli plus MO-C, i.e. 1 means no change and therefore no effect after gene
knockdown. As an arbitrary threshold, we have chosen to highlight changes of
at least 40% with green representing activation or activator function and red
representing repression or repressor function
(Table 1). It is not known if
the majority of these genes are direct targets or whether the Gli proteins act
directly as bona fide transcriptional activators or repressors in all cases.
The color of the values indicated with asterisks rows refer to the action of
the exogenous Gli protein injected, the others refer to the inferred function
of the endogenous Gli protein inhibited by the specific MO used. The results
are summarized for Gli and neurogenic genes in
Fig. 6.
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Gli3
Gli3 was repressed by injected Gli1 at stage 12. At stage 14, injected Gli1
and GLI1 did not affect its expression. However, endogenous Gli1 acted as a
repressor in the presence of GLI1 as here MO-1 induced Gli3
activation. Injected Gli2 repressed Gli3 at stage 12, but at stage 14
injected Gli2 and GLI2 induced it. However, injected Gli2 did so in a
Gli1-dependent manner, while endogenous Gli2 acted as a repressor with
injected GLI2. Finally, induction of Gli3 by injected GLI3 required
Gli1 and Gli2.
|
Ngn1a
Ngn1a was induced by all injected Gli proteins. However, at stage
14 but not stage 12, endogenous Gli3 acted as a repressor in the presence of
injected Gli1. By contrast, endogenous Gli3 acted as a repressor at stage 12
and an activator at stage 14 in the presence of injected Gli2. Endogenous Gli1
was required for induction of Ngn1a by injected Gli2 at stages 12 and
14. Induction of Ngn1a by injected Gli3 required endogenous Gli1 at
stages 12 and 14 but only endogenous Gli2 at stage 14. Its induction by
injected GLI1 required endogenous Gli1 but Gli2 acted as a repressor. By
contrast, endogenous Gli2 and Gli3 acted as repressors in the presence of
injected GLI2. Finally, all three endogenous Gli proteins were required for
induction by injected GLI3.
Cooperative effects of Gli proteins in nuclear localization
The results presented above suggest cooperative functional interactions in
target gene regulation. To begin to investigate the basis of such
cooperativity, we first probed the ability of Gli proteins to influence the
subcellular localization of each other. Here, we took advantage of the finding
that human GLI1 localizes to the nucleus, while frog Gli2 localized to the
cytoplasm in transfected COS-7 cells (Fig.
7A,C,D) (Ruiz i Altaba,
1998; Ruiz i Altaba,
1999
). Co-transfection of both GLI1 and Gli2 resulted in the
preferential localization of Gli2 in the nucleus only when co-expressed with
GLI1 (Fig. 7A,C). A similar
result was obtained with human GLI3, which normally localized to the cytoplasm
(Ruiz i Altaba, 1999
) but
became nuclear when co-expressed with GLI1
(Fig. 7C,D).
Zic proteins are members of the Gli superfamily that harbor a very similar
five zinc-finger DNA-binding domain
(Brewster et al., 1998;
Nakata et al., 1998
). Zic1 has
been reported to enhance the nuclear localization of Gli1 and Gli3, and to
affect protein function in vitro (Koyabu
et al., 2001
; Mizugishi et
al., 2001
). In vivo, Gli proteins and Zic2 interact to establish
the pattern of the neural plate where Zic2 functions as an anti-neurogenic
factor in primary neurogenesis (Brewster
et al., 1998
). We therefore tested whether Zic2 could also affect
Gli localization in transfected COS-7 cells. Zic2 protein was nuclear
(Brewster et al., 1998
)
(Fig. 7B) and its co-expression
with frog Gli1, which is also heavily cytoplasmic
(Lee et al., 1997
;
Ruiz i Altaba, 1999
), Gli2 or
GLI3 resulted in an increased nuclear localization of each of these Gli
proteins (Fig. 7B,D).
Physical interactions through the first two fingers of the five zinc-finger domain in the Gli superfamily
Given the colocalization of co-expressed Gli/Zic2 proteins, we tested
whether GLI3 could bind Zic2. Immunoprecipitation and western blot analyses of
co-transfected Zic2 and different forms of GLI3 showed that full-length Zic2
can bind N- and C-terminally deleted forms of GLI3
(Ruiz i Altaba, 1999), as well
as Zic2 itself (Fig. 7E). This
focused our attention on the central zinc-finger domain. Zic1 and Gli proteins
bind each other through their last three zinc fingers
(Koyabu et al., 2001
), a
domain that represents the most highly conserved region in these proteins.
This interaction may render the proteins unable to bind DNA as the last three
fingers also represent the DNA-binding domain
(Pavletich and Pabo, 1993
).
Unlike with Zic1 (Koyabu et al.,
2001
), we found that the first two fingers of Zic2 or GLI3 are
sufficient to bind Zic2 and Gli proteins
(Fig. 7F,G). Here, we used
C-terminally deleted forms, which are expressed at higher levels than
full-length Gli proteins and are therefore easier to detect
(Ruiz i Altaba, 1999
). The
first two zinc fingers of GLI3 specifically recognized Zic2, Gli2 and Gli3,
but not Gli1 (Fig. 7F).
Similarly, the first two fingers of Zic2 recognized Gli2 and GLI3, but not
Gli1 (Fig. 7G). Although other
binding sites may exist, this specificity correlated with the ability of Gli2
and Gli3, but not Gli1, to harbor potent dominant-negative function, an
activity shared with Zic2 (Ruiz i Altaba,
1999
; Brewster et al.,
1998
).
|
![]() |
Discussion |
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We have previously shown that there is a critical involvement of Gli
function in the patterning of the neural plate, the CNS primordium
(Lee et al., 1997;
Brewster et al., 1998
;
Ruiz i Altaba, 1998
;
Ruiz i Altaba, 1999
). The
present data crucially extends this work by showing an unexpected requirement
for each Gli protein in the induction of all primary neurons: motor, sensory
and interneurons. Our results appear strikingly different from those obtained
in mice, as loss of the function of any single or all Gli proteins in mouse
embryos does not abolish neural tube neurogenesis
(Bai et al., 2004
). This
difference could be due to the species-and context-dependent function of Gli
proteins (e.g. Ruiz i Altaba,
1998
; Ruiz i Altaba,
1999
; McDermott et al.,
2005
). Indeed, Gli1 is not required in mice for development or
tumorigenesis (Park et al.,
2000
; Weiner et al.,
2002
), but it is essential for frog embryo tumors
(Dahmane et al., 1997
;
Dahmane et al., 2001
) (this
work) and human cancer (Sanchez et al.,
2004
). Similarly, Gli3 has a major negative function in the mouse
neural tube, which must be suppressed by Shh signaling in order to allow
ventral neuronal differentiation
(Litingtung and Chiang, 2000
),
but here we demonstrate a sweeping positive role for Gli3 as a required
component of primary neurogenesis. Evidence for positive Gli3 activity has
been also described in gain-of-function analyses on amphibian primary
neurogenesis (Ruiz i Altaba et al., 1998;
Brewster et al., 1998
) and in
a limited fashion in the zebrafish, chick and mouse neural tube
(Persson et al., 2002
;
Meyer and Roelink, 2003
;
Bai et al., 2004
;
Tyurina et al., 2005
).
|
Mechanistically, we show that the Gli proteins regulate neurogenic bHLH
genes (Brewster et al., 1998)
(this work), indicating that they act upstream of neuronal specification,
consistent with their expression patterns: the Gli genes are co-expressed at
low levels throughout the animal cap, the neural plate primordium, preceding
the expression of neurogenic bHLH genes, and are later expressed in partially
overlapping domains throughout the mature neural plate
(Lee et al., 1997
;
Mullor et al., 2001
). Primary
neurogenesis therefore requires Gli function in different neural plate areas
that display high and low expression levels, and/or in the neural plate
primordium where Gli expression is low and ubiquitous. However, the Gli
proteins are also required for neurogenesis directly induced by the ectopic
expression of the neurogenic bHLH proteins and the Notch-Delta pathway, which
modulates bHLH gene expression (Chitnis et
al., 1995
; Chitnis and
Kintner, 1996
), suggesting sustained function of the Gli proteins
is required at distinct steps in the neurogenic cascade. This is also
consistent with results with the involvement of Zic2 upstream and downstream
of the bHLH neurogenic cascade (Brewster
et al., 1998
). Therefore, although it remains unclear exactly how
Gli proteins regulate bHLH protein function for example, by affecting
co-factors or additional components, such as Id (e.g.
Liu and Harland, 2003
) or
homeodomain proteins (Briscoe et al.,
2000
; Gershon et al.,
2000
), or by interacting with multiple bHLH proteins the
data highlight a multistep link between bHLH/Notch and Gli activities that is
essential for neurogenesis.
In addition to neurogenic function, the Gli proteins have anti-neural crest
activity (Brewster et al.,
1998). Our previous work has shown that ectopic Gli2 or Gli3
function suppresses neural crest differentiation whereas Zic2 or
dominant-negative GLI function induces it
(Brewster et al., 1998
),
consistent with a general positive involvement of Zic proteins in neural crest
development (Nakata et al.,
1998
). Unexpectedly, we demonstrate here that knockdown of Gli3,
the Gli gene most prominently expressed in the neural folds, also suppresses
neural crest differentiation. An explanation for this apparent discrepancy may
reside in the fact that the MOs interfere with the action of both positive
(activator) and negative (repressor) forms of Gli2 and Gli3, as the latter
form post-transcriptionally. We propose that Gli repressor function at the
edges of the neural plate, far from midline sources of Shh, is required to
inhibit any positive neurogenic Gli function and thus allow for neural crest
differentiation.
The quantification of target gene expression we present indicates that the
Gli proteins act in a cooperative fashion that is dynamic, target gene and
species dependent. The larger number of functional interactions observed after
expression of the human proteins in comparison with those detected after
expression of frog Gli proteins, would appear to result from the interplay of
four Gli proteins (the three endogenous ones plus the exogenous one injected)
versus the interplay of only three players (the injected plus the other two
endogenous ones). This cooperative function of Gli proteins and their ability
to influence each other's subcellular localization raise the possibility of
the existence of protein complexes. In support of this idea, we show that the
first two Gli zinc fingers can act as a docking site for other Gli proteins,
possibly through the same domain. These findings offer a role for an
evolutionarily conserved subdomain that had so far remained functionally
orphaned and that shows a higher degree of variability within the Gli
superfamily (Lee et al., 1997;
Mizugishi et al., 2001
) than
the last three zinc fingers, which bind DNA
(Pavletich and Pabo, 1993
). In
contrast to Gli-Gli and Gli-Zic interactions, Zic2 forms homocomplexes through
the N-terminal region and not through the zinc-finger domain, showing
specificity and suggesting the possibility of hetero-oligomers. Together, the
data thus suggest the existence of a Gli protein network.
The ability of this protein network to act in a context-dependent manner is
most probably reliant upon the availability of interacting co-factors. For
example, the binding of Zic2 to C' forms of Gli2 and Gli3, but
not of Gli1, demonstrates specificity and suggests functional relevance, as
Zic2 and C'
forms of Gli2 and Gli3, but not Gli1, have dominant
repressive function (Ruiz i Altaba,
1999
; Shin et al.,
1999
; Aza-Blanc et al.,
2000
). The overall read out of positive and negative Gli function,
the combinatorial Gli code, may thus depend on the type of cooperative
interactions present the network and the types of factors that
dock on or interact with the network. These factors include Zic proteins
(Brewster et al., 1998
;
Koyabu et al., 2001
); Ski,
which recruits the histone deacetylation complex
(Dai et al., 2002
); Suppressor
of Fused (Kogerman et al.,
1999
; Ding et al.,
1999
; Dunaeva et al.,
2003
); Dyrk1 (Mao et al.,
2002
); and Hox proteins, some of which can impart activator
function to Gli3C'
repressors
(Chen et al., 2004
). Shh
signaling, and other signaling inputs
(Brewster et al., 2000
), may
thus act on the Gli code by modifying the combination of Gli proteins that
interact in a cooperative network and thus the possible factors that
associate.
The cooperative and combinatorial function of Gli proteins may also be
critical in cancer (e.g. Ruiz i Altaba et
al., 2004) as endogenous Gli1 has been shown to be required for
the induction of epidermal and neural tumors by exogenous GLI1
(Dahmane et al., 1997
;
Dahmane et al., 2001
). Here,
we extend these data and show that endogenous Gli3 is also required for
GLI1-induced tumorigenesis. This finding is consistent with the ability of
Gli3, as Gli1 and Gli2, to act as an activator in different contexts
(Brewster et al., 1998
;
Persson et al., 2002
;
Meyer and Roelink, 2003
;
Bai et al., 2004
;
Zuñiga and Zeller,
1999
; Zakany et al.,
2004
). However, the data also reveal a differential requirement
for Gli2 and Gli3 as knockdown of Gli2, like that of the other Gli proteins,
has drastic effects on target gene expression but it is not required for
GLI1-mediated neurogenesis or tumorigenesis. Sustained Gli activity necessary
for tumor growth thus appears to require the cooperative function of Gli1 and
Gli3. This is interesting as ectopic expression of Gli1, but not Gli3, leads
to tumor formation. Instead, ectopic Gli3 leads to supernumerary tail
development (Brewster et al.,
2000
). As ectopic expression occurs at higher than endogenous
levels, this result further suggests that the Gli code is both qualitative and
quantitative.
We propose that the context-specific cooperative (this work) and
combinatorial (Ruiz i Altaba,
1998) action of Gli proteins, forming a Gli network, is a general
property and underlies a number of previous unexplained observations in
different systems. In frogs, these include: the ability of co-injected Gli2 or
Gli3 to inhibit ventral forebrain neuronal and floor plate inductions by
exogenous Gli1; and the ability of exogenous Gli3 to inhibit motoneuron
induction by co-injected Gli2 (Ruiz i
Altaba, 1998
). In the mouse neural tube, a Gli network could
explain the requirement of Gli1 for positive Gli3 function
(Bai et al., 2004
). Similarly,
in zebrafish and cell culture (Tyurina et
al., 2005
) a combinatorial and cooperative Gli network could
account for the findings that: Gli3 cooperates with Gli1 to induce ventral CNS
targets; Gli3 repressor function affects a number of targets differently; Gli3
and Gli2 inhibit Gli1-induced Gli reporter activation; but Gli3 enhances it in
the presence of Shh while Gli3 alone is ineffective.
Finally, the finding that amphibian primary neurogenesis and tumorigenesis
(Dahmane et al., 1997;
Brewster et al., 1998
) (this
work), on the one hand, and adult mammalian stem cell neurogenesis
(Lai et al., 2003
;
Machold et al., 2003
;
Palma and Ruiz i Altaba, 2004
;
Palma et al., 2005
) and
cancers of different organs (e.g. Dahmane
et al., 2001
; Thayer et al.,
2003
; Watkins et al.,
2003
; Sanchez et al.,
2004
) (reviewed by Ruiz i
Altaba et al., 2004
), on the other hand, depend on Hh-Gli
signaling, further suggests the extension of a cooperative Gli network to
other scenarios. These include stem cells, adult neurogenesis and cancer.
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ACKNOWLEDGMENTS |
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Footnotes |
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