Max-Planck-Institute for Molecular Genetics, Berlin, Germany and Department for Developmental Biology, Center for Medical Biotechnology, University Duisburg-Essen, Essen, Germany
* Author for correspondence (e-mail: andrea.vortkamp{at}uni-due.de)
Accepted 26 September 2005
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SUMMARY |
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Careful analysis of chondrocyte differentiation in Gli3/ mutants revealed that Gli3 negatively regulates the differentiation of distal, low proliferating chondrocytes into columnar, high proliferating cells. Our results suggest a model in which the Ihh/Gli3 system regulates two distinct steps of chondrocyte differentiation: (1) the switch from distal into columnar chondrocytes is repressed by Gli3 in a PTHrP-independent mechanism; (2) the transition from proliferating into hypertrophic chondrocytes is regulated by Gli3-dependent expression of PTHrP. Furthermore, by regulating distal chondrocyte differentiation, Gli3 seems to position the domain of PTHrP expression.
Key words: Indian hedgehog (Ihh), Gli3, Gli2, Gli1, PTHrP (Pthlh), Chondrocyte, Cartilage, Bone
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Introduction |
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Ihh is one of the key regulators of endochondral ossification controlling
at least three distinct differentiation steps. (1) To regulate the onset of
hypertrophic differentiation, Ihh interacts with a second secreted growth
factor, Parathyroid hormone related protein (PTHrP; Pthlh Mouse Genome
Informatics). Ihh signals to the distal periarticular chondrocytes to
upregulate the expression of PTHrP. PTHrP in turn signals back to the
proliferating chondrocytes and inhibits the differentiation of proliferating
cells into the Ihh expressing early hypertrophic cell type. (2) In
addition, Ihh regulates chondrocyte proliferation and (3) induces the
ossification of the perichondrium in PTHrP independent mechanisms
(Kronenberg, 2003).
Whereas the regulation of chondrocyte proliferation and the ossification
process can be explained by Ihh acting on neighboring tissues, it has been an
open question as to how the Ihh signal reaches the joint region to induce the
expression of PTHrP. Initially, secondary factors have been
hypothesized to mediate the Ihh signal. Recent analyses of mice carrying a
hypomorphic allele of exostosin 1 (Ext1), a glycosyltransferase
necessary for the synthesis of heparan sulfate (HS), have, however, shown that
HS negatively regulates the propagation of Ihh in the cartilage anlagen. In
addition, these investigations strongly support a model in which Ihh acts as a
long range morphogen, directly inducing the expression of PTHrP
(Koziel et al., 2004).
To further understand Ihh signaling it is important to investigate how the
signal is translated in the different skeletal target tissues. The molecular
mechanism of hedgehog signaling has been best analyzed in Drosophila.
In short, in the receiving cells, Hedgehog (Hh) is bound by a receptor complex
consisting of the 12-transmembrane receptor Patched (Ptc) and the
7-transmembrane receptor Smoothened (Smo). Binding of Hh to Ptc releases the
repression of Smo, which, via a complex signaling cascade, alters the activity
of the zinc finger transcription factor Cubitus interruptus (Ci)
(Lum and Beachy, 2004). Ci
belongs to the Gli family of transcription factors. These proteins contain a
domain of five conserved zinc fingers of the C2H2 type and a conserved
C-terminal transactivation domain. In the absence of Hh signals the 155 kDa Ci
protein is phosphorylated and proteolytically processed into a truncated
N-terminal repressor protein of 75 kDa containing the zinc fingers, which
inhibits the expression of Hh target genes. Upon Hh signaling, phosphorylation
and thus proteolytic processing is blocked and full length Ci protein acts as
a transcriptional activator of Hh target genes
(Aza-Blanc et al., 1997
;
Chen et al., 1998
;
Jia et al., 2002
;
Methot and Basler, 2001
;
Price and Kalderon, 2002
).
In vertebrates three Ci homologues have been identified: Gli1, Gli2 and
Gli3. Biochemical investigations indicate that, similar to Ci, Gli2 and Gli3
can be proteolytically processed into a truncated repressor form, whereas Gli1
lacks the protein kinase A recognition site necessary for phosphorylation and
subsequent cleavage. Gli1 is therefore likely to function exclusively as an
activator (Aza-Blanc et al.,
2000; Price and Kalderon,
2002
; von Mering and Basler,
1999
).
The role of Gli genes in vertebrates has been mainly analyzed in relation
to sonic hedgehog (Shh) signaling. In the neural tube, Shh signaling from the
floor plate controls the differentiation of six classes of neurons in a
concentration-dependent manner. Gli1 and Gli2 are expressed
in a gradient from ventral to dorsal flanking the expression domain of Shh. In
contrast Gli3 is expressed in an inverse gradient from dorsal to
ventral. Gain- and loss-of-function experiments carried out in different
laboratories have revealed complex, overlapping functions of these
transcription factors: Gli3 seems to act mainly as a repressor of Shh target
genes, whereas Gli2 and Gli1 act mainly as activators
(Jacob and Briscoe, 2003).
However, replacing Gli2 with Gli3 demonstrated that Gli3 can
also act as an activator (Bai et al.,
2004
). Interestingly, Gli1 is not directly regulated by the Shh
signaling cascade but requires activation by either Gli2 or Gli3
(Bai et al., 2002
;
Dai et al., 1999
). In summary,
Shh signaling in the neural tube is translated into gradients of decreasing
Gli activator and increasing repressor activity from ventral to dorsal,
thereby determining the graded differentiation of distinct neuronal cell types
(Bai et al., 2004
;
Persson et al., 2002
;
Wijgerde et al., 2002
).
The biological importance of Gli3 acting as a repressor has become
strikingly evident by the analysis of
Shh/;Gli3/
double mutants. Limbs of Shh mutants lack anterior-posterior polarity
and develop only one digit. Loss of Gli3 converts the Shh
phenotype into the polydactylous limb phenotype of
Gli3/ mutants
(Litingtung et al., 2002;
te Welscher et al., 2002
). Shh
seems thus to act mainly by opposing the repressive activities of Gli3.
Mutations in vertebrate Gli genes result in a range of different
phenotypes, however the process of endochondral ossification is only mildly
affected (Hui and Joyner,
1993; Mo et al.,
1997
; Park et al.,
2000
; Schimmang et al.,
1992
). Whereas no bone phenotype has been detected in
Gli1/ mutants, loss of Gli2 or
Gli3 results in a slight reduction in bone length. Analysis of
Gli2/,Gli3+/
compound mutants revealed a more severe phenotype indicating functional
redundancy of Gli2 and Gli3 in controlling endochondral bone
formation (Mo et al.,
1997
).
To obtain insights into the function of the Gli transcription factors downstream of Ihh signaling, we investigated the role of Gli3 in regulating chondrocyte differentiation. Our analysis revealed that in the absence of Ihh signaling Gli3 acts as a strong repressor, negatively controlling chondrocyte proliferation and inhibiting the expression of the two Ihh target genes, Ptch (Ptch1 Mouse Genome Informatics) and PTHrP. Interestingly, loss of Gli3 function in mice, which overexpress Ihh in chondrocytes, rescues the delayed onset of hypertrophic differentiation, strongly suggesting an activating role of Gli3 downstream of Ihh in activating PTHrP expression. Furthermore our analysis revealed Gli3 as key regulator of the differentiation from distal into columnar chondrocytes.
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Materials and methods |
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Histology
Paraformaldehyde-fixed tissue was embedded in paraffin wax and stained with
Hematoxylin and Eosin (H&E), Toluidine Blue (TB) or with Safranin Weigert
(SW) for histological analysis. To identify mineralized cartilage and bone,
limbs were stained with 1% silver nitrate according to the method of van
Kossa, and counterstained with nuclear fast red.
In situ hybridization analysis
Embryonic limbs were fixed overnight in 4% paraformaldehyde at 4°C and
embedded in paraffin wax. Serial sections of 5 µm were processed for
radioactive in situ hybridization using [33P]UTP-labeled antisense
riboprobes. Hybridization was carried out at 70°C in 50% formamide as
previously described (Minina et al.,
2002). Sections were counterstained with Toluidine Blue. Probes
for in situ hybridization were as follows: Col10a1
(Minina et al., 2001
),
Ihh (Bitgood and McMahon,
1995
); Ptch (Goodrich
et al., 1996
), Pthr1
(Abou-Samra et al., 1994
);
PTHrP (Koziel et al.,
2004
), Fgfr1 and Fgfr3
(Minina et al., 2005
),
Gli1 and Gli3 (Hui et al.,
1994
); Gli2
(Niedermaier et al.,
2005
).
To analyze the size of expression domains, photographs of two different
sections (of approximately 60 µm distance) per limb were taken at 50x
magnification. All pictures were printed at the same resolution, the borders
of the expression domains were defined by an independent investigator and
measured in a double blind test (relative units). For all measurements, an
unpaired two-tailed Student's t-test was performed (a P
value 0.05 represents a significant difference). Original measurements are
summarized in Table S1 in supplementary material.
BrdU labeling
Mice were sacrificed 2 hours after receiving an intra-peritoneal injection
of 50 µg/g body weight of 5-bromo-2'-deoxyuridine (BrdU) (BrdU
labeling and detection kit II; Roche). Limbs were fixed in 4% paraformaldehyde
at 4°C and embedded in paraffin wax. Proliferating cells, in 5 µm
sections, were detected by antibody staining performed according to the
manufacturer's instructions.
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Results |
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Gli3 acts as a repressor downstream of Ihh during endochondral ossification
Loss of any single Gli gene does not result in an obvious bone
phenotype. As Gli3 has been shown to act as a strong repressor of Shh target
genes, we decided to focus our analysis on this transcription factor. To
determine, whether Gli3 similarly represses Ihh target genes during
endochondral ossification, we examined the genetic interaction between Ihh and
Gli3. Ihh-deficient mice are characterized by severely reduced
chondrocyte proliferation, an accelerated onset of hypertrophic
differentiation and lack of ossification in endochondral bones. Furthermore,
hypertrophic differentiation is not initiated perpendicular to the
longitudinal axis of the cartilage elements but starts in their center
spreading into all directions. The resulting skeletal elements thus display a
central, Col10a1-expressing hypertrophic region that is surrounded by
non-hypertrophic cells expressing procollagen type II 1
(Col2a1). In addition, chondrocytes of Ihh-deficient mice
fail to express PTHrP and Ptch
(Karp et al., 2000
;
St-Jacques et al., 1999
). In
contrast Gli3-deficient mice display only a mild endochondral
ossification phenotype with a slight delay in the overall differentiation
process (Mo et al., 1997
).
|
To test if the increased size of the cartilage elements in
Ihh/;Gli3/
mutants results from an increased rate of chondrocyte proliferation we
performed BrdU labeling in wild-type, Ihh/
and
Ihh/;Gli3/
double mutant mice. Consistent with previous investigations
(St-Jacques et al., 1999), the
proliferation rate in Ihh/ mutants is
severely reduced throughout the cartilage anlagen at E16.5. Strikingly,
additional loss of Gli3 dramatically increases the number of
BrdU-positive chondrocytes (Fig.
2M-O).
On the molecular level, the expression of the hypertrophic marker Col10a1, which is expressed throughout the cartilage anlagen in Ihh/ mice, is restricted to the center of the cartilage elements in Ihh/;Gli3/ mice. The Col10a1-expressing hypertrophic cells are flanked by distinct domains of non-hypertrophic chondrocytes on either side. Remarkably, the onset of hypertrophic differentiation occurs perpendicular to the longitudinal axis of the bone (Fig. 3A-C). As Gli3 seems to act as a strong repressor downstream of Ihh in proliferating cells, we analyzed the expression of the Ihh target genes Ptch and Gli1, which are not expressed in Ihh/ mutants. We found low, but significant, levels of Ptch expression throughout the proliferating chondrocytes in Ihh/;Gli3/ double mutants (Fig. 3D-F). In contrast, Gli1 is not expressed in Ihh/;Gli3/ mutants (Fig. 3G-I), suggesting that, as in the neural tube, activation of Gli1 transcription depends on an activating Ihh signal.
|
In summary loss of Gli3 function rescues the chondrocyte phenotype of Ihh/ mice to a significant degree, suggesting that Gli3 acts as a strong repressor of Ihh target genes.
Loss of Gli3 does not rescue bone collar formation in Ihh-deficient mice
Another important role of Ihh is the induction of the ossification process
in the perichondrium (Long et al.,
2004; Long et al.,
2001
; St-Jacques et al.,
1999
). In wild-type limbs the bone collar forms adjacent to the
prehypertrophic and hypertrophic chondrocytes. We performed van Kossa staining
to analyze matrix mineralization and bone collar formation in skeletal
elements of Ihh/ and
Ihh/;Gli3/
mutants. Although in Ihh/ mutants
hypertrophic differentiation is accelerated, perichondrial cells do not
develop into osteoblasts. In
Ihh/;Gli3/
mutants hypertrophic chondrocytes in the center of the skeletal elements are
mineralized, however, no bone collar is formed surrounding the hypertrophic
cells. Instead bone collar-like structures are found intermittently in areas
adjacent to the mineralized region (Fig.
2P-U). Loss of Gli3 is thus not sufficient to rescue
perichondrial ossification.
Gli3 mutant mice display reduced zones of proliferating chondrocytes
Given our demonstration that Gli3 is an important effector molecule of Ihh
in regulating chondrogenesis, we carefully analyzed chondrocyte
differentiation in Gli3/ mice using a panel
of chondrocyte differentiation markers. At E14.5 and E16.5 the skeletal
elements of Gli3/ mutants are shorter than
those of wild-type mice (92%, n=6, P=0,0036; Table S1A in
supplementary material, and data not shown) and the zone of ossification in
their center is reduced (Fig.
4A,B). Hybridization with Ihh and Col10a1
revealed normal regions of hypertrophic cells in
Gli3/ limbs
(Fig. 4A-D). Similarly no
alteration in the expression level and domain of Ptch and
Gli1, the direct targets of Ihh signaling, could be detected
(Fig. 4E,F and data not
shown).
|
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As PTHrP is the effective regulator of hypertrophic chondrocyte differentiation downstream of Ihh, we next analyzed the expression of PTHrP. In wild-type mice PTHrP is expressed in the most distal chondrocytes at E14.5 whereas at E16.5 its expression domain has shifted towards the center of the cartilage anlagen. At this stage the most distally located chondrocytes have ceased to express PTHrP (Fig. 5D). In Gli3/ mice we did not detect an obvious downregulation of the level of PTHrP expression. Instead we found that the shift of the expression domain has not occurred at E16.5 (Fig. 5E).
As the PTHrP expression domain is restricted to the most distal cells in Gli3/ mice we reinvestigated the onset of hypertrophic differentiation by analyzing the distance between the central end of the PTHrP expression domain and the distal end of the Ihh expression domain. We found a similar distance in Gli3/ and wild-type mice, indicating that the onset of hypertrophic differentiation is not accelerated if compared with the source of its regulator, PTHrP (Fig. 5M, n=6, P=0.3; Table S1A in supplementary material). By contrast, the distance between the central side of the PTHrP expression domain and the distal end of the skeletal elements is significantly reduced in Gli3/ mutants compared to wild-type mice (Fig. 5M, 71%, n=6, P=0.0001; Table S1A in supplementary material).
|
In summary, the reduced expression domain of Fgfr1 identifies Gli3 as a negative regulator of the differentiation of distal chondrocytes into columnar chondrocytes. Furthermore, as the level of PTHrP expression is not altered in Gli3 mutants this regulation occurs independently of PTHrP. In contrast, the differentiation of columnar chondrocytes into hypertrophic chondrocytes seems to be regulated by the level of PTHrP. To support such a model, we analyzed which types of chondrocytes are present in Ihh-deficient mice. As Fgfr3 is expressed in proliferating and in early hypertrophic cells it cannot be used to determine the size of the zone of columnar chondrocytes. We therefore analyzed the expression of Fgfr1 in distal chondrocytes in relation to that of parathryroid hormone receptor 1 (Pthr1), which is expressed in prehypertrophic chondrocytes overlapping with the expression of Ihh. In wild-type limbs the expression domains of Fgfr1 and Pthr1 are clearly separated by columnar chondrocytes. Remarkably, in Ihh/ limbs, which do not express PTHrP, a small stripe of Fgfr1-expressing distal chondrocytes is directly flanked by Pthr1-expressing prehypertrophic chondrocytes (Fig. 6A-D). We can, thus, conclude that the domain of columnar chondrocytes is significantly reduced or lost in Ihh/ mutants. Furthermore, the remaining proliferating cells in these mutants represent distal chondrocytes. Correspondingly, no columnar chondrocytes could be detected by morphological analysis (Fig. 2H,J).
We next analyzed Fgfr1 expression in Ihh/;Gli3/ double mutants, in which PTHrP expression is restored, but Gli3 function is lost. In these mutants Fgfr1 expression is restricted to the most distal chondrocytes, as it is in Gli3/ mutants. In contrast, the region of columnar chondrocytes between the Fgfr1 and Pthr1 expression domains is significantly expanded (Fig. 6E-H). In summary, these results strongly support a dual role for Gli3 in regulating distal chondrocyte differentiation and the onset of hypertrophic differentiation, by two independent mechanisms.
|
|
We next analyzed the expression of PTHrP, which is upregulated in Col2a1-Ihh mutants, leading to the delayed differentiation of columnar chondrocytes into hypertrophic chondrocytes. Similar to the differentiation of distal chondrocytes, this upregulation of PTHrP expression could be attributed to the Ihh-dependent inactivation of the Gli3 repressor function. However, Gli3/ mice, which do not express any Gli3 repressor, do not display upregulated levels of PTHrP expression. To investigate the activating potential of Gli3 we analyzed PTHrP expression in Col2a1-Ihh;Gli3/ mice. Loss of Gli3 in Col2a1-Ihh mice reduces the domain of PTHrP expression (Fig. 8T) as in Gli3/ mutants. Furthermore, the expression level of PTHrP, which is upregulated in Col2a1-Ihh mice, seems to be reduced to a level similar to that in wild-type or Gli3/ mice, implicating an activating role of Gli3 in regulating the expression of PTHrP. The reduced size of the expression domain in combination with reduced levels of PTHrP expression seems, thus, to be responsible for the accelerated onset of hypertrophic differentiation.
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Discussion |
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Loss of Gli3 partially rescues the Ihh/ phenotype
During endochondral ossification, the three vertebrate homologues,
Gli1, Gli2 and Gli3, are expressed in overlapping domains in
proliferating chondrocytes and in the developing bone (tissues that react to
Ihh). Neither their expression pattern nor the deletion of single Gli
genes identifies one of them as a main tranducer of the Ihh signal
(Mo et al., 1997).
To obtain deeper insight into the interaction of Gli genes and Ihh we started to analyze the role of Gli3 in regulating Ihh target genes, focusing on the role of Ihh in controlling chondrocyte differentiation. Despite the fact that Gli3-deficient mice display only a mild bone phenotype our investigations demonstrated that loss of Gli3 in Ihh mutants rescues the Ihh/ phenotype to a significant degree: the region of hypertrophic cells, which represents most of the chondrocytes in Ihh/ mutants, is clearly restricted to a central region, flanked by non-hypertrophic, proliferating cells, on either end. Interestingly, in these phenotypically rescued limbs, chondrocyte proliferation is upregulated, the expression of Ptch and PTHrP is restored and hypertrophic differentiation of chondrocytes is delayed. It can thus be concluded that in the absence of Ihh signaling, Gli3 acts as a strong repressor of chondrocyte proliferation and of at least the expression of Ptch and PTHrP. Furthermore, similar to Shh signaling, the activating role of Ihh in these processes seems to be mainly mediated by inhibiting the repressor function of Gli3.
|
Despite the dramatic rescue of many aspects of the Ihh/ phenotype, loss of Gli3 does not fully convert the Ihh/ phenotype: the perichondrium seems to ossify only in isolated, restricted areas and PTHrP expression is not restored to normal levels in the distal chondrocytes. Whether these processes require an activating function of Gli3 or are regulated by another member of the Gli family will be the subject of future studies. In the proliferating chondrocytes, Gli2 expression overlaps with that of Gli3 and both genes are expressed more strongly in the distal regions. Remaining Gli2 repressor activity might thus downregulate the expression of PTHrP in Ihh/;Gli3/ double mutants. Alternatively, activating functions of either Gli2 or Gli3 might be required for full activation of PTHrP expression.
Gli3 regulates chondrocyte differentiation in PTHrP dependent and independent mechanisms
Although endochondral long bones of Gli3 mutant mice develop
similarly to those of wild-type mice, our analysis of chondrocyte
differentiation revealed a reduced domain of proliferating chondrocytes,
indicating an accelerated onset of hypertrophic differentiation. Surprisingly,
the expression levels of PTHrP, the main regulator of differentiation
from proliferating into hypertrophic chondrocytes, and its receptor
Pthr1 (data not shown) seem to be normal in
Gli3/ mutants, indicating that the
accelerated differentiation is induced by a PTHrP-independent mechanism.
Instead loss of Gli3 leads to a shift in the expression domain of
PTHrP towards the distal ends of the cartilage elements at E16.5.
Furthermore the region of Fgfr1-expressing distal chondrocytes is
reduced in size in these mice. Thus, Gli3 seems to act as a negative regulator
of an early step of chondrocyte differentiation, i.e. the transition from
distal into columnar cells, thereby positioning the PTHrP expression
domain. The reduced zone of proliferating chondrocytes in
Gli3/ mice can therefore be attributed to an
accelerated differentiation of distal into columnar cells. In summary, we can
conclude that Gli3 regulates two steps of chondrocyte differentiation: the
transition from distal into columnar cells in a PTHrP-independent mechanism,
and the transition of columnar into hypertrophic cells by regulating
PTHrP expression.
Successive roles for Gli3 and PTHrP in determining the switch from distal into columnar chondrocytes and from columnar into hypertrophic chondrocytes, respectively, are supported by the investigation of various combinations of Ihh and Gli3 alleles. In Ihh/ mice, the remaining non-hypertrophic cells are distal, Fgfr1-positive chondrocytes, which are presumably maintained by the strong Gli3 repressor function. As PTHrP expression is absent, these distal cells differentiate directly into Pthr1-expressing hypertrophic chondrocytes. Because of the lack of a positive marker we cannot completely exclude the possibility of a short columnar phase. However, no chondrocyte columns can be detected morphologically. Loss of Gli3 in Ihh/ mutants restores the expression of PTHrP and subsequently delays the onset of hypertrophic differentiation. As Gli3 function is lost the differentiation of distal into columnar chondrocytes is accelerated and the majority of proliferating chondrocytes are of the columnar type. In Col2a1-Ihh mice the repressor function of Gli3 is antagonized by overexpression of Ihh. Accordingly, the differentiation of distal into columnar chondrocytes is accelerated, leading to a shortened zone of distal chondrocytes. In parallel, upregulated PTHrP expression leads to a delayed onset of hypertrophic differentiation. Complete loss of Gli3 in these mutants (Col2a1-Ihh;Gli3/) results in a further acceleration of distal chondrocyte differentiation. Consequently the reduced domain of PTHrP expression, in combination with a possibly reduced PTHrP expression level, accelerates the transition from columnar to hypertrophic cells.
Is activation by Gli3 required for PTHrP expression?
Whereas the differentiation of distal into columnar chondrocytes seems to
be regulated by Gli3 repressor activity, the regulation of PTHrP
expression by Gli3 might be more complex. Loss of Gli3 in
Ihh/ mice restores the expression of
PTHrP, clearly demonstrating that Gli3 negatively regulates
PTHrP expression. However, in Gli3/
mice, PTHrP expression is not upregulated compared to wild-type mice
whereas Col2a1-Ihh mice express significantly upregulated levels of
PTHrP. Therefore, high expression of PTHrP in chondrocytes
seems to require the release of a potential repression by Gli2 or an
activation by either Gli3 or Gli2. Interestingly, loss of Gli3 in
Col2a1-Ihh mutants not only leads to a reduction in the size of the
PTHrP expression domain but, in addition, seems to downregulate the
level of PTHrP expression. As the function of Gli2, acting as a
repressor or an activator, should not be affected by loss of Gli3,
this experiment suggests that upregulation of PTHrP expression in
these limbs requires activation by Gli3. We can not, however, determine by
this experiment whether Gli3 directly activates the PTHrP promotor or
if it acts as a repressor of other inhibitory transcription factors.
It is difficult to determine if PTHrP expression requires activation by Gli3 in wild-type limbs. The activating function of Gli3 might be minimal in wild-type limbs and might only be induced by ectopic overexpression of Ihh. However, the normal level of PTHrP expression in Gli3/ mice is at least in agreement with a simultaneous loss of a Gli3 activator and repressor function. Furthermore, the role of Gli2 in this process remains obscure, as overexpression of Ihh should convert Gli2 into a strong activator. Loss of repression by Gli3 should thus allow Gli2 to activate PTHrP expression, again supporting an activating role for Gli3. Further studies addressing the role of Gli2 are obviously required to fully understand the regulation of PTHrP.
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Conclusions |
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PTHrP-dependent and -independent regulation of chondrocyte differentiation
has been proposed previously from the analysis of mice carrying hypomorphic
and null alleles of Pthr1. Based on cell morphology, Kobayashi et al.
predicted that PTHrP regulates the switch from columnar into hypertrophic
cells, whereas Ihh accelerates the differentiation from distal into columnar
cells (Kobayashi et al., 2002;
Kobayashi et al., 2005
). Our
analysis has, for the first time, used Fgfr1 and Fgfr3 as
markers to define the domains of distal and columnar chondrocytes. In
addition, we identified Gli3 as a regulator of both differentiation steps.
Combining the investigations of both groups we would thus propose a model in
which Gli3 repressor function delays distal chondrocyte differentiation.
Inactivation of the Gli3 repressor function by Ihh from the prehypertrophic
region would induce the differentiation from distal into columnar
chondrocytes. The distance from the Ihh expression domain at which
the differentiation occurs would be determined by the level of Ihh signaling:
more distal if the Ihh signal is high and more central if the signal is low.
In addition Ihh-dependent inactivation of the Gli3 repressor function would
determine the level of PTHrP expression and thus the transition from
columnar into hypertrophic chondrocytes
(Fig. 9).
|
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material available online at http://dev.biologists.org/cgi/content/full/132/23/5249/DC1
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