1 Department of Embryology, Carnegie Institution of Washington 115 West
University Parkway, Baltimore, MD 21210, USA
2 Department of Molecular and Medical Genetics, University of Toronto and
Program in Developmental Biology, The Hospital for Sick Children, 555
University Avenue, Toronto, Ontario M5G 1X8, Canada
* Author for correspondence (e-mail: fan{at}ciwemb.edu)
Accepted 8 September 2003
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SUMMARY |
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Key words: Sonic hedgehog, Somite, Mouse, Patterning, Gli
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Introduction |
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The secreted signaling molecule Sonic hedgehog (Shh), expressed in the
notochord and floorplate, is crucial for sclerotome development as
Shh mutant mice lack vertebral columns, and form only a few
rudimentary rib cartilages (Chiang et al.,
1996). Consistent with this, Shh induces expression of sclerotomal
markers including the paired-box containing transcription factors
Pax1 and Pax9, and the HMG-box containing transcription
factor Sox9 in PSM in vitro (Fan
and Tessier-Lavigne, 1994
;
Murtaugh et al., 1999
;
Zeng et al., 2002
). These
target genes are essential for sclerotome development as Pax1/Pax9
double mutants have severe defects in formation of ribs and vertebrae
(Peters et al., 1999
), and
Sox9 is required for the transcription of collagen
2, an
extracellular component necessary for cartilage formation
(Bell et al., 1997
;
Bi et al., 1999
). In addition
to the sclerotomal markers, Shh induces proliferation of the somitic mesoderm
(Fan et al., 1995
), possibly
by upregulation of G1 cyclins, which are Shh targets in other tissues
(Kenney and Rowitch, 2000
;
Mill et al., 2003
). Shh also
negatively regulates its own signaling by upregulation of its own binding
receptor patched 1 (Ptch) and a decoy receptor hedgehog interacting
protein (Hhip) (Briscoe et al.,
2001
; Chuang and McMahon,
1999
; Goodrich et al.,
1996
). Both the proliferation and negative feedback induced by Shh
may help define the shape and size of sclerotome-derived skeletal components.
Thus, the roles of Shh in the somite can be divided into three categories,
patterning, proliferation and negative feedback.
Induction of Shh targets in the somite is thought to be carried out through
the conserved Hedgehog (Hh) signaling pathway first described in
Drosophila (reviewed by McMahon,
2000). In this pathway, Hh binds to its receptor Patched (Ptc) and
relieves Ptc inhibition of the signaling component Smoothened (Smo). Smo then
signals to the transcription factor Cubitus interruptus (Ci) to activate gene
expression. Ci acts as a bipotential transcription factor, repressing some of
the same target genes in the absence of Hh
(Methot and Basler, 2001
;
Muller and Basler, 2000
). In
the mouse, there are three Ci homologs, Gli1, Gli2 and Gli3
(Hui et al., 1994
). Gli1 and
Gli2 are thought to act primarily as activators, while Gli3 acts primarily as
a repressor (Bai et al., 2002
;
Lee et al., 1997
;
Ruiz i Altaba, 1998
;
Sasaki et al., 1997
;
Sasaki et al., 1999
;
Shin et al., 1999
).
Bipotential functions of Gli2 and Gli3 on reporter genes in cultured cells has
been demonstrated (Sasaki et al.,
1999
), but in vivo evidence of bipotential Gli2 activity is
lacking.
Genetic data supports a crucial role for Gli2 and Gli3 in
formation of the axial skeleton as Gli2 and Gli3 mutants
exhibit distinct vertebral and rib defects late in development, whereas
Gli1 mutant mice exhibit no developmental defects.
Gli2/Gli3+/ mice
exhibit more severe defects than either single mutant, indicating some
overlapping functions in skeletogenesis
(Mo et al., 1997). These mice
exhibit defects similar to Pax1/Pax9 double mutants, suggestive of
defects early in sclerotome induction. However, the molecular basis for
Gli2 and Gli3 mutant phenotypes and the role of each Gli in
sclerotome development have not been investigated.
To determine whether Gli2 and Gli3 are required for Shh-dependent sclerotome induction, we examined Shh target gene expression in Gli2/Gli3 compound mutants. We find that sclerotomal gene expression is severely reduced in Gli2/Gli3/ mice and that at least one copy of either Gli2 or Gli3 is required in the somitic mesoderm to confer Shh-responsiveness. We also investigated the specific role of each Gli in activating Shh target genes by overexpression in the PSM in vitro. We find that each Gli displays preferential activation of different sclerotomal targets involved in Shh-directed patterning, proliferation and negative feedback.
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Materials and methods |
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Radioactive section in situ hybridization
Mice at embryonic day 9.5 (E9.5) were fixed overnight in 4%
paraformaldehyde and cryosectioned at 12 µm. Riboprobes were labeled with
[-35S]UTP (Amersham Pharmacia) using T3 or T7 RNA Polymerase
(Promega). Radioactive section in situ hybridization was performed as
described (Frohman et al.,
1990
). Slides were exposed to NBT emulsion (Kodak), developed,
stained by Hematoxylin, and mounted in Permount (VWR). Images were
photographed under dark field illumination to visualize silver granules and
presented with the corresponding bright-field images.
Whole-mount in situ hybridization
Whole-mount in situ hybridization using digoxigenin (DIG)-UTP (Roche)
labeled riboprobes was performed as described
(Buttitta et al., 2003). Probes
for Pax1, Sim1 and Pax3 have been described previously
(Fan and Tessier-Lavigne,
1994
). Myf5 probe was a gift from Dr M. Buckingham.
Pax9 and Sox9 probes were generated by reverse transcription
coupled with PCR (RT-PCR) of 0.45kb and 1kb regions of the respective
transcripts. Primers used for RT-PCR are available upon request. Embryos
hybridized to Pax9, Shh and Sox9 probes exhibited higher
background and were destained in methanol resulting in a bluish signal.
Embryos were photographed using an Axiocam camera. Selected embryos were
cryosectioned at 20 µm and mounted in Crystal Mount (Biomeda).
Explant induction assays
E9.5 mouse PSM explants from CD1 mice, or Gli2/Gli3 progeny, were
cultured in collagen gels as described
(Fan and Tessier-Lavigne,
1994). For inductions in the presence of cycloheximide, PSM was
cultured for 10 hours prior to exposure to Shh-N conditioned media, 1 µg/ml
cycloheximide (Sigma), or the combination for 8 hours. Shh-N and control
conditioned media were collected from COS cells as described
(Fan et al., 1995
) and used at
500 µg/ml Shh-N. RNA was isolated from explants by RNAsol and used for
RT-PCR with 30 cycles of amplification. Primer sequences are available at
http://www.ciwemb.edu/labs/fan/index.html.
PCR products were resolved on 2% agarose gels and visualized by ethidium
bromide staining. Images of gels were captured using a UVP 7500 Gel
Documentation System and quantified using Image-Quant v.1.2 (Amersham
Pharmacia). All RT-PCR assays were performed within the linear range of
amplification for each product as determined by quantitative real-time
PCR.
Adenovirus production and explant infection
Adenoviruses carrying full-length mouse Gli1, mouse Gli2, human Gli3 or
N-terminally truncated Gli2 as C-terminal EGFP (Clontech) fusion proteins;
wild-type Smoothened (Smo); an activated form of Smo (Smo-M2 designated here
as Smo*) (Xie et al.,
1998); or EGFP driven by a CMV promoter were constructed using the
AdenoX system (Clontech). Production of fusion proteins was confirmed by
western blot using anti-EGFP (Molecular probes) or anti-Gli1, Gli3 and Smo
antibodies (Santa Cruz). For infection, explants were cultured in the presence
of 0.5-1.0x108 plaque-forming units (pfu) of adenovirus for
100% infection. Protein expression levels appeared similar as assessed
visually by GFP fluorescence. Infected explants were used for RT-PCR or fixed
and processed for immunofluorescence as described
(Lee et al., 2001
).
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Results |
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Expression of Gli1 is absent throughout the PSM. Upon formation of the somites, Gli1 is expressed in the ventral domain (Fig. 1A,A'). Transverse sections through the early somites reveal expression in the most ventromedial domain of the sclerotome (Fig. 1D,D'). By comparison, Gli2 is more widely expressed. Gli2 is weak in the posterior two-thirds of the PSM, but becomes stronger in the anterior domain (Fig. 1B,B'). Gli2 expression is absent from the dermomyotome but found in the sclerotome, expanding more dorsally and laterally than Gli1 (Fig. 1E,E'). Gli3 exhibits the broadest expression of all three genes. Gli3 is expressed throughout the anterior two-thirds of the PSM as well as both dorsal and ventral domains of the early somites (Fig. 1C,C'). Transverse sections reveal that Gli3 is expressed throughout the sclerotome, extending laterally like Gli2, but has stronger expression in the dermomyotome (Fig. 1F,F'). Thus, all three Gli genes are expressed in the ventral domain of the early somites. As Gli2 and Gli3 are expressed in the anterior PSM, they may act earliest in sclerotome patterning.
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Somite morphology is abnormal in
Gli2/Gli3/
embryos
The severe loss of sclerotomal gene expression in
Gli2/Gli3/
embryos prompted us to investigate the somite morphology in these embryos. In
wild-type,
Gli2+/Gli3/ and
Gli2/Gli3+/
embryos, the dermomyotome (dm), myotome (my) and sclerotome (scl) are visible
and organized (Fig. 3A, see
Fig. S1C at
http://dev.biologists.org/supplemental/).
By contrast, in
Gli2/Gli3/
somites, the dermomyotome had an abnormal upside down U-shape, owing to
ectopic epithelium that extends ventromedially adjacent to the neural tube
(Fig. 3B, open arrowheads). We
also frequently observed a closed sphere of epithelium in the trunk somites of
these embryos (indicated by broken lines in
Fig. 3H,J,L,P). In
Gli2/Gli3/
embryos, mesenchyme resembling the sclerotome was present but reduced in size,
and the myotome was not clearly distinguishable.
|
We next examined the expression of the myotomal marker and Gli
target gene Myf5 (Gustafsson et
al., 2002) by whole-mount in situ hybridization. Myf5
expression is normally restricted to the dorsomedial lip of the dermomyotome
and the developing myotome. In
Gli2/Gli3/
embryos, Myf5 expression appeared diffuse and laterally expanded
throughout the somites (arrowheads in Fig.
3E,F). In contrast to Pax3 and Myf5, expression
of the lateral somite marker Sim1 appeared normal in
Gli2/Gli3/
embryos (arrowheads in Fig.
3I,J).
Somites of
Gli2/Gli3/
embryos exhibit more severe defects than somites of
Shh/Gli3/
embryos
The somite defects we observed in
Gli2/Gli3/
mutants appear less severe than those described for Shh mutants
(Chiang et al., 1996). Loss of
Gli3 in a Shh mutant background rescues specific aspects of
neural tube patterning due to the removal of Gli3 repressor function
(Litingtung and Chiang, 2000
),
but whether Gli3 also acts as a repressor in the somites has not been
examined. To test whether
Gli2/Gli3/
embryos more closely resemble those in which both the Shh signal and Gli3
repressor activity is lost, we compared the expression of Pax1 and
Myf5 in
Gli2/Gli3/,
Shh/ and
Shh/Gli3/
embryos.
Transverse sections through the trunk reveal that the weak Pax1 expression observed in Gli2/Gli3/ embryos is restricted to a group of medially located mesenchymal cells within an epithelial sphere (Fig. 3L). In Shh/ embryos Pax1 expression in the trunk was undetectable in the ventral mesenchyme (Fig. 3M). Strikingly, strong Pax1 expression was restored in Shh/Gli3/ embryos (Fig. 3N). In wild-type embryos, Myf5 expression is restricted to the developing myotome (Fig. 3O). In Gli2/Gli3/ embryos, Myf5 expression was observed throughout mesenchymal cells within the epithelial spheres without forming a distinct layer (Fig. 3P). Expression of Myf5 in Shh/ embryos was not as tightly organized as in the wild-type and dorsomedial expression was reduced (arrowhead in Fig. 3Q). By contrast, in Shh/Gli3/ embryos strong dorsomedial expression of Myf5 was restored, but remained less organized than in the wild type (Fig. 3R). These findings demonstrate that Gli2/Gli3/ embryos exhibit more severe sclerotomal and myotomal phenotypes than Shh/Gli3/ embryos, and that similar to the developing neural tube, loss of Gli3 function ameliorates the sclerotomal phenotype of Shh mutants.
At least one copy of either Gli2 or Gli3 is
required for Shh-dependent sclerotome induction tissue autonomously
Motoyama et al. (Motoyama et al.,
2003) have demonstrated that
Gli2/Gli3/
embryos have severely reduced expression of Shh in the brain and
brachial regions. Thus, the reduced sclerotomal gene expression in these
embryos might be due to a loss of Shh expression. As shown in
Fig. 4A, Shh
expression in the notochord and floorplate of double mutants is significantly
weaker than in wild type. This finding questions whether the reduction in
sclerotome gene expression in
Gli2/Gli3/
embryos is due to a reduction of Shh, or an inability to transcriptionally
activate Shh targets in the somites. To distinguish between these
possibilities, we tested whether PSM isolated from
Gli2/Gli3/
embryos can activate sclerotomal genes in response to exogenously provided
Shh. If loss of Shh is the cause of the sclerotomal defects, PSM from
Gli2/Gli3/
embryos will be able respond to exogenous Shh. If an inability to
transcriptionally activate Shh target genes is the cause of the sclerotomal
defects, exogenous Shh will not be able to induce Shh target genes in the PSM
from
Gli2/Gli3/
embryos.
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NG2 mimics Shh and Smo* signaling in the somitic
mesoderm
To further dissect the function of each Gli in mediating specific target
gene expression, we generated adenoviral vectors for overexpression of the Gli
genes in the PSM. Adenoviral vectors contained either full-length Gli1, Gli2,
Gli3 or an activated N-terminally truncated form of Gli2 (NG2)
(Sasaki et al., 1999
), as
C-terminal fusions to EGFP driven by a cytomegalovirus (CMV) promoter (see
Fig. S2A at
http://dev.biologists.org/supplemental/).
Additional vectors serving as negative and positive controls contained EGFP
alone, wild-type Smo or a constitutively active form of Smo (Smo*)
(Xie et al., 1998
). Production
of the desired protein products upon adenoviral infection was confirmed by
western analysis (see Fig. S2B-D at
http://dev.biologists.org/supplemental/)
and functionality of Gli-EGFPs was tested using a Shh-responsive cell line and
a luciferase reporter downstream of eight Gli binding sites (see Fig. S2E at
http://dev.biologists.org/supplemental/).
Virally expressed Gli genes, Smo and Smo* functioned as predicted
from previous studies (Sasaki et al.,
1997
; Sasaki et al.,
1999
; Shin et al.,
1999
; Taipale et al.,
2000
).
As Shh signaling in the somite is thought to occur via the conserved
HH-PTC-Smo signaling pathway (Zhang et
al., 2001), we first wanted to establish whether overexpression of
Smo* by adenovirus can activate Shh target genes in cultured PSM.
To test this, PSM from E9.5 mice was cultured alone, with Shh-N (500 ng/ml),
or infected with adenovirus carrying EGFP, Smo or Smo* for 24 hours
and analyzed for induction of the Shh target genes Ptch, Ccnd2, Hhip,
Pax1, Pax9 and Sox9. As shown by RT-PCR in
Fig. 5A, treatment with Shh-N
induces all Shh target genes tested, while infection with EGFP adenovirus does
not affect any Shh targets. Expression of Smo induces Ptch, Ccnd2,
Hhip and weak Pax1, while expression of Smo* induces
all Shh target genes tested. In conclusion, Shh target gene induction in the
somite can be recapitulated by overexpression of Smo* in the
PSM.
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We next tested the activity of full-length Gli genes in the presence of Shh-N to determine whether the Gli genes can also exhibit repressive effects when overexpressed. PSM was cultured for 24 hours with each Gli adenovirus in the absence or presence of 500 ng/ml Shh-N, and expression of Ptch and Pax1 was assessed by RT-PCR. As shown in Fig. 5C, infection with EGFP and Gli1 adenoviruses does not affect induction of Pax1 and Ptch genes by Shh, while infection with Gli2 seems to have a moderate inhibitory affect (threefold) specifically on Pax1. By contrast, infection with Gli3 strongly represses Shh induction of both Ptch and Pax1. These results confirm the repressive ability of Gli3, and suggest that although full-length Gli2 can activate some Shh targets, full-length Gli2 expressed at high levels can also repress specific targets.
In other tissues Gli2 has been shown to be critical for induction
of Gli1 expression (Bai et al.,
2002; Ding et al.,
1998
). Similarly, we found that Gli2 and
NG2 can induce the
expression of Gli1 in the PSM, although neither Gli1 nor Gli3 induce
Gli1 expression (Fig.
5D and data not shown), demonstrating that Gli1 and Gli2 can
preferentially activate different target genes.
Ptch, Pax1 and Pax9 are direct transcriptional targets of
Shh signaling in the PSM that can be induced in the absence of protein
synthesis (Dockter, 2000)
(C.M.F., unpublished). We next tested whether Gli1, Ccnd2 and
Hhip are also direct targets of Shh signaling in the PSM. PSM
explants were treated with Shh-N in the absence or presence of 1 µg/ml
cycloheximide to inhibit protein synthesis. In the absence of protein
synthesis Shh-N induces only the expression of Gli1 and
Ccnd2, but not of Hhip
(Fig. 5E). Together with the
Gli2 and
NG2 overexpression, these data support a linear pathway where
Gli2 may be activated upon Shh signaling to induce Gli1, which in
turn can induce the secondary target Hhip.
Gli1 cannot restore Shh-responsiveness in
Gli2/
Gli3/ PSM
Although PSM from
Gli2/Gli3/
embryos does not activate Shh target genes in response to exogenous Shh-N,
there is a low level of Pax1 and Pax9 expressed in
Gli2/Gli3/
somites (Fig. 2E,E'). It
is possible that Gli1 expressed in the somites of
Gli2/Gli3/
embryos can mediate some Shh target gene expression. We measured the level of
Gli1 expression in
Gli2/Gli3/
embryos by quantitative real-time PCR and found it was reduced to 9% of
wild-type levels (data not shown). To address whether this low level of
Gli1 is responsible for the Pax1 and Pax9
expression observed we tested whether expression of Gli1 in the
Gli2/Gli3/
PSM could restore Shh-responsiveness. PSM explants from wild-type or
Gli2/Gli3/
embryos were cultured in the presence of 500 ng/ml Shh-N and infected with
either EGFP or Gli1 adenovirus for 24 hours. As shown in
Fig. 6, infection with Gli1
adenovirus did not restore Shh-responsiveness in
Gli2/Gli3/
PSM. We then extended these results by testing the Shh-responsiveness of
somites from
Gli2/Gli3/
embryos expressing endogenous Gli1 (data not shown).
Gli2/Gli3/
somites cultured with Shh-N also did not exhibit any Shh-responsiveness (data
not shown). These findings demonstrate that in the absence of Gli2
and Gli3, Gli1 cannot restore Shh-responsiveness in the somitic
mesoderm. Surprisingly, we found that overexpression of Gli1 failed to
increase Ptch and Hhip expression in
Gli2/Gli3/
PSM, as it did in wild-type tissues (3.2- and 2.0-fold respectively),
suggesting that Gli1 somehow requires Gli2 or Gli3 for
transcriptional activity.
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Discussion |
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Skeletal defects in Gli2/,
Gli3/ and
Gli2/Gli3+/ mice
are likely due to loss of Sox9 expression
We demonstrate that Gli2/,
Gli3/ and
Gli2/Gli3+/
embryos have reduced expression of Sox9 in the sclerotome of anterior
somites at E9.5 and E10.5. A small reduction in Sox9 expression can
result in severe chondrogenic defects, as Sox9+/
cells cannot contribute to cartilage or bone in chimeric animals
(Bi et al., 1999). We suggest
that the loss of Sox9 is thus a likely cause of the skeletal
phenotypes observed in these mutants (Mo
et al., 1997
), as the domains of Sox9 loss largely
correspond to the specific skeletal abnormalities observed in each mutant. The
normal Sox9 expression in the early somites of these mutants suggests
that the defect is due to an inability to maintain proper levels of
Sox9 expression in the mature somites. Recent work has shown that
although Shh initiates Sox9 expression, BMP signals maintain a
positive regulatory loop leading to sustained Sox9 expression
(Zeng et al., 2002
). Our data
is consistent with either the establishment or maintenance of this positive
regulatory loop being affected in these mutants.
Gli2 can act as a repressor and Gli3 can act as an
activator for Shh target genes in the somites
Although Gli3 has been shown to activate Gli1 and Ptch
promoters in cultured cells (Dai et al.,
1999; Shin et al.,
1999
), the physiological role of this activator function is not
clear. We provide two lines of evidence supporting a Shh-induced activator
function for Gli3 in the somites. First,
Gli2/Gli3+/
embryos express levels of Pax1 and Pax9 comparable with
wild-type levels. Second,
Gli2/Gli3+/ PSM
can activate expression of Shh target genes while
Gli2/Gli3/
PSM cannot. This indicates that one copy of Gli3 is sufficient to
mediate Shh-dependent gene induction.
Although Gli2 is generally considered to be an activator of Shh signaling,
in vitro studies suggest that it may also possess repressor function
(Sasaki et al., 1999). Recent
studies in zebrafish embryos have uncovered a possible repressor function for
Gli2 specifically in telencephalon and muscle development
(Karlstrom et al., 2003
;
Wolff et al., 2003
). We
provide evidence of a repressor function for Gli2 on direct targets
of Shh in the sclerotome. Adenoviral overexpression of Gli2 in PSM can repress
Shh induction of Pax1 (Fig.
5C), and
Gli2+/Gli3/ PSM
does not exhibit the same level of Shh target de-repression as
Gli2/Gli3/
PSM (Fig. 4B; see Fig. S1D at
http://dev.biologists.org/supplemental/),
indicating that one copy of Gli2 is sufficient to mediate some
repressive functions similar to those carried out by Gli3. The
finding that the activator Gli1 can replace Gli2 in vivo
supports the notion that Gli2 functions solely as an activator
(Bai and Joyner, 2001
).
However, our data illustrate that Gli2 repressor function is probably masked
by the strong Gli3 repressor and is revealed only in the absence of Gli3, or
when overexpressed in vitro. Thus, in addition to their overlapping activator
functions, we suggest Gli2 and Gli3 also share repressor functions during
somite development in mice.
Gli genes and somite patterning
The temporal and spatial expression of Gli genes in the developing paraxial
mesoderm is consistent with Gli2 and Gli3 mediating initial Shh-induced
sclerotomal gene expression. Our overexpression and mutant analysis together
support a model for Gli function in the somite as illustrated in
Fig. 7A. In wild-type somites,
Gli2 is modified to a strong activator (similar to NG2) upon Shh
signaling, and functions as the primary mediator of Shh signaling to promote
both proliferation and patterning programs. Gli1 then acts downstream of Gli2,
primarily activating Shh-dependent negative feedback mechanisms. However, as
loss of Gli1 does not affect somite development
(Park et al., 2000
), this
function of Gli1 may be compensated by the activated form of Gli2. In the
absence of Shh or when overexpressed, Gli3 represses Shh-induced programs. If
Gli2 is lost, Gli3 activator function is revealed that compensates for the
loss of Gli2. Conversely, in the absence of Gli3, Gli2 repressor function is
revealed which compensates for the loss of Gli3. In the absence of both Gli2
and Gli3, the low level of Gli1 is non-functional and Shh responsiveness, as
well as target repression, are lost, resulting in a low level of
Shh-independent target gene expression.
|
Shh functions are divided preferentially amongst different Gli genes
in the somite
Although previous analyses of Gli2 and Gli3 have revealed
cooperative functions in several contexts, including the neural tube, lung,
trachea and teeth (Hardcastle et al.,
1998; Motoyama et al.,
1998
; Motoyama et al.,
2003
; Persson et al.,
2002
), how multiple Gli proteins cooperate together in mediating
Shh response is not well understood. Our analysis of Gli function extends the
previous studies by addressing the tissue autonomous contributions of each Gli
gene to regulation of several direct Shh targets in the somitic mesoderm.
Using mutant explants, we find that Shh targets display different
sensitivities to loss of each Gli gene. For example, Pax9 is the most
sensitive to loss of Gli repressor function, as it is abnormally expressed in
the absence of Shh in Gli3/ and
Gli2/Gli3/
PSM (Fig. 4B). By comparison,
Ptch is the most sensitive to loss of Gli2 activator function, as it
cannot be induced in Gli2/ PSM by Shh. Thus,
Gli genes may mediate Shh induction of target genes in at least two ways. One
way is for Shh to inhibit formation of Gli repressors, which may be the
primary method of Shh induction of Pax9. A second way is for Shh to
stabilize formation of Gli activators, which compete with Gli repressors for
target gene activation. This is consistent with the regulation we observe for
Ptch, which cannot be activated in the absence of Gli2 until
one copy of Gli3 is lost, reducing the concentration of Gli
repressor. For Pax1 and Gli1 a small amount of activator,
such as that possibly formed by Gli3 in the presence of Shh in
Gli2/Gli3+/
embryos, is sufficient to mediate gene expression. This demonstrates
additional distinctions in the functional output of each Gli and suggests a
previously unappreciated ability of Gli genes to distinguish between different
promoter/enhancer contexts.
Mis-patterning of the
Gli2/Gli3/
somite results from a complete loss of responsiveness to Hedgehog
signaling
Our data suggest that the somite phenotypes of
Gli2/Gli3/
embryos result from a loss of Shh responsiveness and Gli3 repressor
function. However, the somitic phenotypes observed in these embryos are more
severe than those of
Shh/Gli3/
embryos. Comparisons of Shh and Smo mutants have revealed a
compensatory role in the somite for another HH family member, Indian hedgehog
(Ihh). As Smo mutants lose all Hh-responsiveness, they cannot be
compensated by Ihh, and thus exhibit more severe phenotypes than Shh
mutants (Zhang et al., 2001).
We suggest that
Gli2/Gli3/
embryos also lose all Hh-responsiveness based upon the following evidence.
First, Pax1 expression in
Gli2/Gli3/
somites is weaker than that in
Shh/Gli3/
somites, suggesting a lack of rescue by Ihh. Second, we observe ectopic
Pax3 in the ventromedial somite of
Gli2/Gli3/
embryos, consistent with recent studies of neural tube patterning in
Smo/ chimeras where dorsal markers are
ectopically expressed in the most ventral regions
(Wijgerde et al., 2002
). Last,
we find Myf5 and Pax1 both expressed in the mesenchymal
cells of
Gli2/Gli3/
somites, suggesting mixing of Pax1-positive and
Myf5-positive cells (illustrated in
Fig. 7B). This is consistent
with the mixing of ventral cell types observed in the neural tube of
Smo/Gli3/
embryos (Wijgerde et al.,
2002
). Thus, the patterning and gene expression observed in
Gli2/Gli3/
somites probably represents a default state, independent of all Hh signaling
and Gli repressive activity.
How does transcriptional activation by Gli1 occur?
We present the unexpected result that in the absence of Gli2 and
Gli3. Gli1 cannot transcriptionally activate its downstream targets
Ptch and Hhip in the PSM. This suggests that Gli1
transcriptional activation somehow requires either Gli2 or
Gli3. This is surprising in light of previous studies that strongly
suggest direct transcriptional activation by Gli1. For example, Gli1 has been
shown to bind a conserved Gli enhancer sequence in vitro
(Sasaki et al., 1997).
Furthermore, Gli1 can largely replace Gli2 function in vivo,
suggesting a level of functional equivalency between the two Gli genes
(Bai and Joyner, 2001
).
However, our result does not necessarily contradict these findings. One
possibility is that Gli1 activation requires physical interaction with Gli2 or
Gli3. Alternatively, Gli2 or Gli3 is required to provide a downstream factor
necessary for Gli1 activator function. Further molecular and biochemical
analyses of the Gli1 transcriptional activation mechanism will help to explain
our finding.
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ACKNOWLEDGMENTS |
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Footnotes |
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