1 Programme in Molecular Biology and Cancer, Samuel Lunenfeld Research
Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G
1X5, Canada
2 The Hope Heart Program, Benaroya Research Institute at Virginia Mason, 1124
Columbia Street, Seattle, WA 98104-2046, USA
3 Division of Cardiovascular Research, The Hospital for Sick Children, Toronto,
Ontario M5G 1X8, Canada
* Author for correspondence (e-mail: wrana{at}mshri.on.ca)
Accepted 20 June 2005
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SUMMARY |
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Key words: Gene trapping, Chondroitin-4-sulfotransferase 1, Bone morphogenesis, Cartilage growth plate, Chondroitin sulfate spatial distribution, Osteoarthritis, Growth factor signaling, Chst11
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Introduction |
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Although the role of heparan sulfation in development and growth factor
signaling has been extensively studied
(Garcia-Garcia and Anderson,
2003; Grobe et al.,
2002
; Kirn-Safran et al.,
2004
; Koziel et al.,
2004
; Merry and Wilson,
2002
; Nybakken and Perrimon,
2002
; Perrimon and Hacker,
2004
; Shworak et al.,
2002
; Wilson et al.,
2002
), the biological function of chondroitin sulfation is less
well understood. During development and in disease, chondroitin sulfation on
carbon positions 4 (C4S) and 6 (C6S) is tightly controlled both spatially and
temporally (Kitagawa et al.,
1997
; Theocharis et al.,
2003
; Tsara et al.,
2002
). Furthermore, although deletion of mouse
chondroitin-6-sulfotransferase 1 (C6st-1; Chst3
Mouse Genome Informatics) does not affect skeletal development, in humans,
mutations in C6ST1 (CHST1; Human Gene Nomenclature Database)
are associated with chondrodysplasia
(Thiele et al., 2004
).
Most skeletal structures are formed by endochondral ossification, in which
a transient cartilage template is replaced by bone. During cartilage
morphogenesis, chondrocytes in the growth plate undergo a complex and highly
regulated program of proliferation and differentiation
(Karsenty and Wagner, 2002;
Shum et al., 2003
). The
periarticular region of the growth plate contains a reservoir of immature
resting as well as non-directionally proliferating chondrocytes. Subsequently,
chondrocytes form a columnar layer by assuming a flattened cell shape and
proliferate in stacks along the longitudinal axis of the developing bone. In
the hypertrophic zone, chondrocytes terminally differentiate and elaborate a
mineralized vascularized matrix that is then replaced by osteoblasts to
generate primary bone (Karsenty and
Wagner, 2002
; Shum et al.,
2003
). By contrast, many cranial bones as well as the midshaft of
long bones are formed directly by intra-membraneous ossification without a
cartilage intermediate (Karsenty and
Wagner, 2002
).
Several signaling pathways control the morphogenesis of the cartilage
growth plate, including Ihh, parathyroid hormone-like peptide (Pthlp),
fibroblast growth factor (FGF) and others. For example, the Ihh-Pthlp
negative-feedback loop regulates the size of proliferative zone and the onset
of hypertrophy (Vortkamp,
2001) and chondrocyte maturation is a complex, tightly regulated
developmental process (Karsenty and
Wagner, 2002
; Shum et al.,
2003
; Vortkamp,
2001
). Members of the transforming growth factor (TGFß)
family also play important roles during cartilage morphogenesis
(Karsenty and Wagner, 2002
;
Klüppel et al., 2000
;
Serra and Chang, 2003
). In
particular, TGFß1 promotes chondrogenesis in cultures of early
undifferentiated mesenchyme, but inhibits both chondrocyte proliferation and
hypertrophy in long bone organ cultures
(Serra and Chang, 2003
).
Activating mutations in TGFß1 have been identified in Camurati-Engelmann
disease, which is characterized by a thickening of the bone collar of long
bones (Campos-Xavier et al.,
2001
; Janssens et al.,
2000
; Janssens et al.,
2003
; Saito et al.,
2001
). Targeted deletion of the TGFß2 gene results in
alterations in size and shape of limb rudiments and bifurcation of the sternum
(Sanford et al., 1997
). In
contrast, the TGFß-related bone morphogenetic proteins (BMPs) positively
regulate both chondrocyte proliferation and hypertrophy
(Horiki et al., 2004
;
Yoon and Lyons, 2004
). For
example, mice with mutations in the BMP receptor type 1B develop brachydactyly
(Baur et al., 2000
;
Yi et al., 2000
), and mice
overexpressing the negative regulators of BMP signaling, Smad6 and Smurf1,
display delayed chondrocyte hypertrophy and dwarfism
(Horiki et al., 2004
). These
studies establish that skeletal patterning and development are major targets
for this morphogen superfamily
(Klüppel et al., 2000
;
Rountree et al., 2004
;
Yoon and Lyons, 2004
).
During an induction gene trap screen in ES cells and embryoid bodies for
target genes of TGFß and BMP signaling, we identified the chondroitin
4-sulfotransferase 1 (C4st1; Chst11 Mouse Genome
Informatics) gene as a target of TGFß and BMP signaling
(Klüppel et al., 2002).
The C4st1 gene encodes a Golgi enzyme that catalyzes the transfer of
sulfate groups to the 4-O position of chondroitin and dermatan sulfate
(Hiraoka et al., 2000
;
Okuda et al., 2000
;
Yamauchi et al., 2000
). Here,
we report on the consequences of inactivation of C4st1 on cartilage
development by using the C4st1 gene trap ES cell line
(C4st1gt) to generate mice deficient in C4st1.
Homozygous mutant mice die within hours of birth and display a severe
chondrodysplasia that is restricted to bones formed through endochondral
ossification. Detailed analysis of the developing skeleton and the cartilage
growth plate showed that loss of C4st1 disturbs the balance of
chondroitin sulfation, causes abnormal chondroitin sulfate localization and
leads to strong upregulation of TGFß signaling with concomitant
downregulation of BMP signaling. These defects result in abnormal chondrocyte
differentiation and orientation within the growth plate that cause severe
disturbances in growth plate morphogenesis.
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Materials and methods |
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Embryo processing, histology and staining
For Hematoxylin and Eosin, and Safranin O staining, embryos were dissected
in PBS and fixed in formalin for several days. Subsequently, embryos were
embedded in paraffin, sectioned and stained as previously described. For RNA
in situ hybridization and immunofluorescence, embryos were dissected in PBS
and fixed in cold 4% PFA/PBS overnight. Tissues were rinsed in cold PBS and
cryo-protected by shaking the tissues in cold 0.5 M sucrose/PBS for 12-24
hours. Tissues were embedded in OCT compound (Tissue Tek), snap-frozen in a
dry-ice/ethanol bath and subsequently stored at 70°C. Bones and
cartilage of E19.5 mouse embryos were stained with Alizarin Red/Alcian Blue as
previously described (McLeod,
1980).
Fluorophore-assisted carbohydrate electrophoresis (FACE)
FACE analysis was performed as previously described
(Calabro et al., 2001).
Briefly, E18.5 growth plates were separated from the mineralized parts of the
bone. Glycosaminoglycans were extracted and enzymatically cleaved to create
disaccharides, which were then fluorotagged by reductive amination with
2-aminoacridone. The tagged products are then displayed by electrophoresis,
identified by their characteristic migration and chemistry, and quantitated by
their molar fluorescence.
RNA in situ hybridization
RNA in situ hybridization on whole-mount E10.0 embryos was performed as
previously described (Kluppel et al.,
2002). Section RNA in situ hybridization was essentially performed
as previously described (Klüppel et
al., 1997
). Some experiments employed the TSA-Plus DNP (AP) signal
amplification kit (Perkin Elmer Life Sciences, Boston).
Immunofluorescence and antibodies
Embryo cryostat sections (7 µm) were air-dried for 2 hours, post-fixed
for 10 minutes in 4% paraformaldehyde at room temperature and washed three
times with PBS. For the mouse monoclonal 1C6 -aggrecan antibody
[developed by Dr Bruce Caterson and obtained from the Developmental Studies
Hybridoma Bank at University of Iowa (DSHB) under the auspices of the NICHD],
sections were then digested with 0.1 U of Chondroitinase ABC (Seikagaku,
Japan) for 45 minutes at 37°C, followed by three washes with PBS. For the
mouse monoclonal CIIC1
-collagen II antibody (developed by Drs Rikard
Holmdahl/Kristofer Rubin, obtained from DSHB), sections were pre-treated with
2.5% hyaluronidase/PBS for 45 minutes at room temperature, followed by three
washes in PBS. Subsequently, sections were blocked, incubated with primary and
secondary antibodies and mounted. Primary antibodies and dilutions used were:
mouse
-aggrecan 1C6 (DSHB, 1:100), mouse
-collagen II 8A4 (DSHB,
1:100), mouse
-chondroitin-6-sulfate (Seikagaku, 1:100), mouse
-chondroitin-sulfate (Sigma, 1:100), rabbit
-pSmad1 (Cell
Signaling, 1:50), rabbit
-pSmad2 (Cell Signaling, 1:50) and rabbit
-Bcl2 (Santa Cruz, 1:200), rabbit
-Bax (Santa Cruz, 1:200).
Secondary antibodies were either Cy2 (green) or Cy3 (red) conjugated. For the
TUNEL stain, an in situ Cell Death Detection Kit (Roche) was used according to
the manufacturer's instructions. For BrdU labeling, pregnant mice were
injected twice with 600 µl of 10 mM BrdU (Roche), 5 hours and 2 hours
before sacrificing. After sectioning, pictures were taken on a Leica DMR
fluorescence microscope and processed with Metamorph software.
Metatarsal explant cultures
The three medial metatarsal bones were removed from the hindlimbs of E18.5
embryos and incubated overnight in explant medium as previously described
(Serra et al., 1999). After 24
hours, explants were incubated in medium containing growth factors N-Shh
(R&D Systems, 2 µg/ml final concentration), TGFß (R&D Systems,
500 pM final concentration) or BMP2 (Genetics Institute, 10 nM final
concentration) for 4 days, with daily change of medium and growth factors.
Subsequently, explants were fixed and processed as described for embryos
above.
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Results |
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|
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The C4st1gt/gt mutation does not affect early cartilage development
Endochondral ossification is a multi-step process that initiates with the
aggregation of mesenchymal cells, the subsequent differentiation of these
cells into chondrocytes and lastly the coordinated proliferation and
differentiation of chondrocytes to form a scaffold for developing bones
(Karsenti and Wagner, 2002). We have shown previously that embryos
heterozygous for the gene trap mutation in the C4st1 gene display
prominent lacZ staining in the developing embryonic cartilage
(Klüppel et al.,
2002).
To determine which steps of endochondral ossification are affected in C4st1gt/gt embryos, we compared the lacZ expression pattern in embryos heterozygous and homozygous for this gene trap mutation by both whole-mount staining and sectioning (Fig. 3). At E11.5, we observed identical lacZ staining pattern in early cartilage aggregations in the forelimbs of both heterozygous (Fig. 3A, part I; Fig. 3D, part i) and homozygous embryos (Fig. 3A, part ii; Fig. 3D, part ii). At E13.5, staining of cartilage primordia of digits, tibia, fibula and femur were also identical in whole mount preparations of both heterozygous (Fig. 3B, part i) and homozygous embryos (Fig. 3B, part ii), and sectioning of stained limbs revealed no difference in size or cellular structure of these elements (Fig. 3E), although we did note a slight bending of the tibial primordium in homozygous embryos (Fig. 3E, part ii). At E15.5, the overall length of limbs was similar in heterozygous and homozygous embryos (Fig. 3C). By whole-mount lacZ staining, limbs from heterozygous embryos revealed staining in all cartilage elements, and a reduction of staining in developing joints and hypertrophic areas (Fig. 3C, part i). Limbs from E15.5 homozygous embryos, however, displayed an impaired segmentation of cartilage in digits that corresponds to the defects in phalange formation we observed at E19.5 and bending of the tibial cartilage was still apparent (Fig. 3C, part ii). Notably, sectioning of the cartilage elements revealed a slightly shortened cartilage growth plate in homozygous embryos (Fig. 3F, part ii), that was accompanied by a reduction in the size of the columnar, but not proliferative or hypertrophic regions (compare Fig. 3F, parts ii and i). Together, these results suggest that mesenchymal aggregation and cartilage primordium formation are not affected in homozygous C4st1gt/gt embryos. However, the C4st1gt/gt mutation affects chondrocyte differentiation during cartilage growth plate morphogenesis. Furthermore, the severe reduction in bone length observed in E19.5 homozygous embryos was not yet apparent at E15.5, indicating that the effects of the loss of C4st1 on skeletal development readily apparent at E18.5-E19.5 reflect defects in cartilage growth plate function.
|
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Next, we analyzed the spatial distribution of chondrotin sulfate in E18.5
mutant and wild-type growth plates, using antibodies specific to CS and C6S
(Fig. 5B-E). CS
antibody, which recognizes chondroitin irrespective of sulfation status,
stained the ECM in all three zones of the wild-type growth plate
(Fig. 5B), albeit levels in the
hypertrophic zone were reduced when compared with other areas. In the
periarticular zone of mutant growth plates, CS displayed pericellular and
intracellular localization, and was not observed in the ECM
(Fig. 5B). However, in the late
columnar as well as the hypertrophic zones of homozygous mutant growth plates,
CS accumulated in the ECM, and continued to display pericellular and
intracellular localization (Fig.
5B). Interestingly, CS staining in the hypertrophic zone again
visualized the fibrillation of the mutant ECM. Next, we used an antibody
specific to C6S, which revealed that in wild-type growth plates, C6S was
restricted to the ECM of the outermost layer of the periarticular zone
(Fig. 5C). By contrast, C6S in
mutant growth plates was predominantly localized to the pericellular space and
extended throughout the periarticular zone and into the columnar layer of the
growth plate (Fig. 5C).
To determine if the C4st1gt mutation affected the spatial distribution of major cartilage ECM proteins, we analyzed the CS-proteoglycan aggrecan (Fig. 5D) as well as collagen II (Fig. 5E). In all three zones of wild-type growth plates, we observed strong ECM staining for aggrecan (Fig. 5E), whereas in homozygous mutants, aggrecan staining was similar to wild type in the proliferative zone, but displayed a more pericellular deposition in both the columnar and hypertrophic layers (Fig. 5D). Collagen II expression in wild-type growth plates again marked the ECM, with strong labeling in the periarticular and columnar regions and a reduction of staining in the hypertrophic zone (Fig. 5E). Homozygous mutant growth plates showed similar collagen II staining with a typical ECM pattern in all three layers. This indicates that loss of C4st1gt does not lead to a general deficiency in ECM. Altogether, these results demonstrate that loss of C4st1gt leads to specific defects in chondroitin sulfation balance, a reduction in chondroitin and disturbances in the distribution of chondroitin sulfate.
|
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The C4st1gt mutation exhibits differential effects on growth factor signaling in the embryonic growth plate
Signaling pathways such as Ihh, BMP and TGFß and have been shown to be
involved in the regulation of chondrocyte development
(Karsenty and Wagner, 2002;
Minina et al., 2002
;
Vortkamp, 2001
). Therefore, we
wanted to determine if the deficiency in chondroitin sulfonation in
C4st1gt/gt growth plates affected these signaling
pathways. For this, we first assessed the expression of target genes or the
activity of signaling mediators of these pathways in E18.5 growth plates.
Ihh signaling upregulates expression of Ptch1, a negative
regulator of hedgehog signaling (Vortkamp,
2001). RNA in situ hybridization showed expression of
Ptch1 in columnar proliferating chondrocytes in both wild-type and
mutant growth plates in a similar pattern
(Fig. 7A, part i; Fig. 7A, part ii), although the
size of the expression domain in mutants was reduced compared with the overall
size of the growth plate.
BMP and TGFß signaling have been shown to lead to the phosphorylation
and nuclear translocation of Smad1 and Smad2, respectively, which subsequently
control transcriptional responses (Attisano
and Wrana, 2002; Massague,
2000
; Miyazono et al.,
2004
; ten Dijke and Hill,
2004
). Therefore, to examine BMP signal transduction, we first
examined nuclear pSmad1 using a phosphospecific Smad1 antibody. In both
wild-type and homozygous mutant growth plates, we observed low background
levels of staining in the periarticular and columnar layers
(Fig. 7B, part i;
Fig. 7B, part ii; Fig. 7C, part I;
Fig. 7C, part ii), but
prominent nuclear pSmad1 staining was observed in wild-type prehypertrophic
and hypertrophic chondrocytes (Fig. 7B,
part i; Fig. 7D, part
i). However, in the hypertrophic zone of homozygous mutant growth
plates, pSmad1 was strongly reduced, with only an occasional cell in the
hypertrophic zone displaying nuclear p-Smad1
(Fig. 7B, part ii; Fig. 7D, part ii). When we
examined pSmad2, we observed low background levels of staining throughout
wild-type growth plates (Fig. 7E, part
i; Fig. 7F, part i;
Fig. 7G, part i;
Fig. 7H, part i), although in
the most lateral regions of the proliferative zone and in some hypertrophic
cells, we could detect some p-Smad2 (Fig.
7E, part i). In stark contrast, when we examined
C4st1gt/gt mutant growth plates, we observed a dramatic
upregulation of pSmad2 levels (Fig. 7E,
part ii), with virtually all cells exhibiting strong nuclear
pSmad2 staining (Fig. 7F, part
ii; Fig. 7G, part
ii; Fig. 7H, part
ii). These data demonstrate that whereas the
C4st1gt mutation has minimal effects on Ihh signaling, it
dramatically affects the balance of TGFß family signaling by strongly
downregulating BMP signaling, while potently upregulating TGFß
signaling.
Metatarsal explant cultures: C4st1gt/gt growth plates retain the capacity to respond to exogenous growth factors
To determine whether growth plates in C4st1gt mutant
animals have an inherent alteration in their ability to respond to distinct
extracellular cues, we analyzed the potential of wild-type and mutant
metatarsal bone explants to respond to exogenously added growth factors.
Metatarsal bones from E18.5 wild-type and mutant embryos were dissected and
cultured for 4 days in the absence or presence of TGFß1, BMP2 and N-Shh,
which has been shown to mimic the effects of Ihh
(Deckelbaum et al., 2002). The
explants were then analyzed for gross morphological changes as well as
sectioned to examine growth plate hypertrophy, as measured by Collagen
X mRNA expression and the activity of Ihh, TGFß and BMP
signaling.
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Altogether, these results demonstrate that while the balance of TGFß and BMP signaling are disturbed in vivo, mutant growth plates retain the inherent capacity to respond to exogenous growth factors. C4st1gt mutant metatarsal explants were able to respond to exogenous BMP2 and also to TGFß, despite the constitutive activation of this pathway. Thus, interfering with chondroitin-4-sulfation causes spatial pathway-specific defects in the elaboration of morphogen signaling in the cartilage growth plate.
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Discussion |
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Cartilage growth plate morphogenesis requires functional C4st1
Disruption of expression of C4st1 did not interfere with early steps in
endochondral ossification, including mesenchymal aggregation and cartilage
primordia formation. However, cartilage growth plate morphogenesis was
disturbed. This was not due to defective growth plate patterning, but to a
severe reduction in the size of the proliferating, columnar and hypertrophic
zones. Further, cartilage islands that are evident in the region of primary
bone in wild-type long bones were absent in the mutants. In addition, we
observed a strong increase in the rate of apoptosis in the mutant growth
plates, caused by an imbalance of pro-versus anti-apoptotic signals. All of
these results are consistent with a model
(Fig. 9) in which chondrocytes
in mutant growth plates undergo accelerated differentiation and apoptosis that
leads to reduced growth plate size and subsequent bone length.
We also demonstrated an altered orientation of mutant chondrocyte stacks,
leading to stacks that are oriented perpendicular to the longitudinal axis of
the bone. This has not, to the best of our knowledge, been reported
previously. This phenotype might be related to physical changes in the growth
plate. Alternatively, there is evidence for an as yet unidentified
periarticular factor that provides an instructive signal for chondrocyte stack
orientation (Abad et al.,
2002). It is tempting to speculate that chondroitin sulfation may
control the transmission or reception of such a signal thereby controlling the
polarity of chondrocyte stacks. Future analysis of
C4st1gt/gt growth plates will give more insight into this
phenotype. The orientation of chondrocyte stacks along the longitudinal axis
of the bone is considered a key determinant of bone longitudinal growth and
morphogenesis (Karsenty and Wagner,
2002
; Shum et al.,
2003
). Therefore, the altered orientation of stacks in mutant
growth plates might be an important contribution to the reduced longitudinal
growth of the mutant embryonic long bones and their enhanced thickness.
|
It is unclear what molecular mechanism underlies altered TGFß family
signaling in C4st1gt/gt mutant growth plates. However,
mutant metatarsal explants treated with exogenous BMP2 responded with an
increase in explant size, collagen X expression and Smad1 activation.
Therefore, the loss of BMP signaling in the mutants is not due to an intrinsic
inability of the cells to respond to BMP. Therefore, expression of endogenous
BMP ligands may be affected, or the ability of BMPs to diffuse or access all
surface signaling receptors may be compromised. Alternatively, TGFß
ligand has been shown to interact with the small leucine-rich CS-containing
proteoglycans, Decorin and Biglycan, which modulate TGFß activity. Of
particular relevance, Decorin negatively regulates TGFß signaling
(Hildebrand et al., 1994;
Kresse and Schonherr, 2001
).
As the distribution of CS and the CS-proteoglycan aggrecan were shifted to the
pericellular environment of the proliferating and hypertrophic zones in mutant
growth plates, altering the balance of CS sulfation may interfere with the
proper sequestration of TGFß in the ECM and allow for constitutive
TGFß signaling. Finally, studies in Xenopus and mammalian cell
culture models have highlighted dose-dependent antagonistic crosstalk between
TGFß and BMP signaling pathways
(Candia et al., 1997
). Thus,
strong upregulation of TGFß signaling in the mutants may indirectly
antagonize endogenous BMP signals.
The absence of C4st1 leads to osteoarthritis-like symptoms
C4st1gt/gt mutant growth plates are disorganized,
hypocellular, display accelerated chondrocyte maturation and have a
fibrillated ECM. Furthermore, there is a reduction in GAG content and CS and
in particular aggrecan content in mutant growth plates. Many of these
cartilage growth plate deficiencies are characteristic of the degenerative
changes that occur in the cartilage in osteoarthritis (OA)
(Martel-Pelletier, 2004).
Treatment with C4S and CS can prevent cartilage degradation, partially by
inhibiting the catabolism of proteoglycans and collagens
(Uebelhart et al., 1998
).
Thus, C4st1gt/gt mice with their reduction in cartilage
C4S and CS levels have features of an osteoarthritic phenotype. TGFß
signaling has been shown to have a dual role during OA: while it can
counteract GAG loss, it also promotes the development of osteophytes, the
occurrence of which is strongly associated with OA
(Scharstuhl et al., 2002
).
Moreover, BMP signaling has also been implicated in a protective role during
OA development (Rountree et al.,
2004
; Scharstuhl et al.,
2003
). Thus, the combined reduction in BMP signaling and
upregulation in TGFß signaling might be functionally involved in the
development of OA-like symptoms in C4st1-mutant mice.
Mutations in the latency-associated peptide (LAP) domain of TGFß1
leads to secretion of constitutively active TGFß1 ligand are associated
with Camurati-Engelmann disease in humans
(Campos-Xavier et al., 2001;
Janssens et al., 2000
;
Janssens et al., 2003
;
Saito et al., 2001
). This
condition is characterized by a thickening of the bone collar of the long
bones. In C4st1gt/gt long bones, we also observed a strong
increase in the thickness of the bone collar, suggesting that the observed
upregulation of TGFß signaling may have a similar effect as that observed
in individuals with Camurati-Engelmann disease.
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Conclusion |
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ACKNOWLEDGMENTS |
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Footnotes |
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