First published online 3 August 2005
doi: 10.1242/dev.01948
Development 132, 3989-4003 (2005)
Published by The Company of Biologists 2005
Maintenance of chondroitin sulfation balance by chondroitin-4-sulfotransferase 1 is required for chondrocyte development and growth factor signaling during cartilage morphogenesis
Michael Klüppel1,
Thomas N. Wight2,
Christina Chan2,
Aleksander Hinek3 and
Jeffrey L. Wrana1,*
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
 |
SUMMARY
|
|---|
Glycosaminoglycans (GAGs) such as heparan sulfate and chondroitin sulfate
are polysaccharide chains that are attached to core proteins to form
proteoglycans. The biosynthesis of GAGs is a multistep process that includes
the attachment of sulfate groups to specific positions of the polysaccharide
chains by sulfotransferases. Heparan-sulfate and heparan
sulfate-sulfotransferases play important roles in growth factor signaling and
animal development. However, the biological importance of chondroitin
sulfation during mammalian development and growth factor signaling is poorly
understood. We show that a gene trap mutation in the BMP-induced
chondroitin-4-sulfotransferase 1 (C4st1) gene (also called
carbohydrate sulfotransferase 11 – Chst11), which encodes an
enzyme specific for the transfer of sulfate groups to the 4-O-position in
chondroitin, causes severe chondrodysplasia characterized by a disorganized
cartilage growth plate as well as specific alterations in the orientation of
chondrocyte columns. This phenotype is associated with a chondroitin sulfation
imbalance, mislocalization of chondroitin sulfate in the growth plate and an
imbalance of apoptotic signals. Analysis of several growth factor signaling
pathways that are important in cartilage growth plate development showed that
the C4st1gt/gt mutation led to strong upregulation of
TGFß signaling with concomitant downregulation of BMP signaling, while
Indian hedgehog (Ihh) signaling was unaffected. These results show that
chondroitin 4-O-sulfation by C4st1 is required for proper chondroitin sulfate
localization, modulation of distinct signaling pathways and cartilage growth
plate morphogenesis. Our study demonstrates an important biological role of
differential chondroitin sulfation in mammalian development.
Key words: Gene trapping, Chondroitin-4-sulfotransferase 1, Bone morphogenesis, Cartilage growth plate, Chondroitin sulfate spatial distribution, Osteoarthritis, Growth factor signaling, Chst11
|
Introduction
|
|---|
Glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin
sulfate (CS) are long chains of repeating disaccharide subunits, which are
covalently linked to core proteins to form proteoglycans
(Sugahara and Kitagawa, 2000
).
During the biosynthesis of GAGs in the Golgi, a number of sulfotransferases
modify the disaccharide subunits and GAG chains through transfer of sulfate
groups to specific positions on the sugar moieties
(Habuchi, 2000;
Kusche-Gullberg and Kjellen,
2003; Selleck,
2000; Sugahara and Kitagawa,
2000). Mature proteoglycans can be cell membrane-bound or are part
of the extracellular matrix (ECM) and are important in a wide range of
biological processes, including cell migration, proliferation and survival, as
well as modulation of growth factor signaling
(Kirn-Safran et al., 2004;
Perrimon and Hacker, 2004;
Selleck, 2000;
Sugahara and Kitagawa,
2000).
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.
|
Materials and methods
|
|---|
Generation of C4st1 mutant mice
ES cells bearing a gene trap mutation in the C4st1 gene
(C4st1gt) were used to generate diploid aggregation
chimeras as previously described (Nagy and
Rossant, 1993). Offspring transmitting the
C4st1gt allele through the germline were used to generate
homozygous C4st1gt animals. Animals were genotyped by
Southern analysis was performed according to the manufacturer's
recommendations (ZetaProbe, BioRad). DNA from tail biopsies were digested with
PstI and blotted onto membranes. The membranes were hybridized to a
4.8 kb en2-lacZ probe fragment containing en2-intronic
sequences as well as the lacZ-coding sequence. The probe fragment was
derived from the PT-1 gene trap vector by EcoRI-PstI digestion and
was 32P-labeled using an oligo-labeling kit (Pharmacia).
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.
|
Results
|
|---|
Severe skeletal abnormalities in mice homozygous for the C4st1gt mutation
The C4st1 gene trap allele represents an integration of the gene
trap vector into the first intron of the C4st1 gene, thus leading to
a fusion transcript containing the first exon of the C4st1 gene
followed by the lacZ-coding sequence
(Fig. 1A). Mice heterozygous
for the C4st1gt mutation were fertile and viable.
Offspring of heterozygous matings were genotyped by measuring the ratio of
lacZ to en2 DNA (Fig.
1B). In order to determine if this gene trap disrupts
C4st1 function, we analyzed C4st1 expression in E10.0
wild-type and homozygous mutant embryos using exon-specific probes and RNA
whole-mount in situ hybridization (Fig.
1C-F). Although exon I-specific probes visualized the previously
reported expression of C4st1 in the branchial arches and the AER of
the developing limb in both wild-type (Fig.
1C) and C4st1gt/gt
(Fig. 1E) embryos, expression
of exons II/III was only apparent in wild-type
(Fig. 1D), but not
C4st1g/gtt embryos
(Fig. 1F). Gene trap
integration in the C4st1 gene thus disrupted expression of exons II
and III of the C4st1 gene, which encode the transmembrane and the
intra-Golgi catalytic domains (Fig.
1A); therefore, C4st1gt probably represents a
null allele of the C4st1 gene.
View larger version (48K):
[in this window]
[in a new window]
|
Fig. 1. Gene trap integration into the C4st1 locus. (A) Schematic
representation of the integration of the PT-1 gene trap vector into intron 1
of the mouse C4st1 locus and the formation of a
C4st1-exonI-lacZ fusion transcript. (B) Embryos from intercrosses of
C4st1gt heterozygous animals were genotyped by Southern
analysis and the ratio of lacZ to en-2 alleles measured. (C-F)
Whole-mount in situ hybridization on E10.0 embryos using the indicated
exon-specific C4st1 probes. In branchial arches (white arrows) and
AER of limb buds (black arrowheads) in C4st1 mutant (gt/gt) embryos,
a C4st1 exon I-specific signal (E) is present, but an exon
II/III-specific signal (F) is absent.
|
|
Genotypes approximately followed Mendelian ratios up to late embryonic
stages (Table 1); however,
C4st1g/gt homozygous mutant animals were not detected 3
weeks after birth (Table 1).
Moreover, the Mendelian ratios indicated that the heterozygous state of the
C4st1 gene trap mutation did not result in lethality
(Table 1). Homozygous
C4st1g/gt mutant animals were born at normal ratios, but
when compared with wild-type animals, displayed severe dwarfism
(Fig. 2A) and died within 6
hours of birth with severe respiratory distress (data not shown).
In order to analyze the apparent dwarfism in more detail, we stained the
skeleton of E19.5 embryos using Alcian Blue/Alizarin Red
(Fig. 2B-H). We observed
multiple skeletal abnormalities, including a small rib cage, a kinked
vertebral column, severely shortened limbs and a dome-shaped skull in mutant
embryos (Fig. 2B, part ii). Cartilage staining by Alcian Blue was reduced in homozygote mutant embryos
(Fig. 2C, part ii), when
compared with wild-type embryos (Fig. 2C,
part). Alcian Blue is known to bind GAGs
(Hronowski and Anastassiades,
1988; Ippolito et al.,
1983), and it has also been reported that undersulfation of
proteoglycans leads to reduced Alcian Blue staining
(Rossi et al., 1996); thus,
the reduction in Alcian Blue staining probably reflects a reduction in GAG
content and C4S levels in the cartilage growth plate. Closer inspection of
mutant skeletons revealed reduced bone length, but increased width in long
bones as well as the iliac bone (Fig. 2C,
parts I and ii), and whereas wild-type embryos had well-developed
vertebrae with prominent dorsal arches
(Fig. 2D, part i), the
vertebrae of mutant embryos were misshapen with poorly formed dorsal arches
(Fig. 2D, part ii). The length
of the mutant scapula was also greatly reduced
(Fig. 2E, part ii). We also
noted that ossification of the talus (Fig.
2F, part ii) and phalanges two and three was absent in both the
fore- and hindlimbs of mutants (Fig.
2F; data not shown), whereas ossification of the calcaneus, the
first phalanges and metatarsal bones occurred
(Fig. 2F). Finally, we examined
skeletal development in mutant heads and found severely shortened facial bones
that included the maxilla, mandible and nasal bones
(Fig. 2G, part ii), but normal
cranial bones (Fig. 2G,H).
View larger version (81K):
[in this window]
[in a new window]
|
Fig. 2. Phenotype of the C4st1gt mutation at E19.5 of
embryogenesis. (A) Gross morphology of wild-type (part i, +/+) and
C4st1gt/gt embryos (ii, part gt/gt). (B-H) Alcian
blue/Alizarin Red skeletal stains. (B) Multiple skeletal abnormalities are
evident in mutant embryos. (C) Higher magnification of hind limbs, showing the
severely shortened and thickened iliac bone (i), femur (f) and tibia and
fibula (ti). Arrowhead indicates Alcian Blue staining of cartilage. (D)
Vertebrae in the mutant display misshapen dorsal arches (arrowhead). (E)
Reduced size of scapula (double-headed while arrow) in mutant embryos. (F)
Phalanges 2 and 3, and the talus bone (arrow) fail to ossify in mutant
hindlimbs (m, metatarsal bones). (G) Lateral view and (H) dorsal view of
skull, showing normal size of frontal (a), parietal (b), interparietal (c) and
supraoccipital (d) bones in mutant embryos, but smaller maxilla (arrowhead),
mandible (arrow) and nasal bones in mutant embryos.
|
|
Altogether, these results demonstrate that C4st1 is required for
morphogenesis of bones formed by endochondral ossification, which includes the
long bones of the limb, vertebrae and facial bones; however, it is not
required for development of cranial bones, which form through intramembraneous
ossification.
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.
View larger version (79K):
[in this window]
[in a new window]
|
Fig. 3. Analysis of cartilage development in C4st1gt/gt embryos
by staining for lacZ. Part i, wild type; part ii, mutant. (A,D)
Identical staining in whole-mount (A) and sectioned (D) forelimb buds of
cartilage aggregations (arrows) in +/gt (i) and gt/gt (ii) E11.5 embryos.
Asterisk indicates AER. (B,E) Whole-mount staining of cartilage primordia (B)
and sectioning of stained tibia primordium (E) at E13.5 in +/gt (i) and gt/gt
(ii) hindlimbs shows no differences in size of cartilage elements or cellular
patterning. Abbreviations in B: d, digits; t, tibia; fi, fibula; f, femur.
Abbreviations in E: d, distal; p, proximal. Arrowhead indicates slight bending
of gt/gt tibia primordium. (C) Whole-mount staining of hindlimbs at E15.5,
showing impaired segmentation of cartilage in digits (arrowhead) and bending
of tibia (arrow) in gt/gt (ii), but not +/gt (i) embryos. d, digits; t, tibia;
fi, fibula. (F) Sectioned proximal tibia of stained +/gt (i) and gt/gt (ii)
E15.5 hindlimbs. Homozygous mutant growth plates are slightly shortened
(double-headed arrow) and show a decrease in the size of the columnar zone
(c). p, proliferative zone; h, hypertrophic zone. Scale bar: 100 µm for
A,D; 300 µm for B; 100 µm for E; 600 µm for C; 200 µm for F.
|
|
The C4st1gt/gt mutation affects growth plate morphogenesis
To investigate the defects in growth plate morphogenesis associated with
the loss of C4st1 in more detail, we focused on E18.5 embryos, at which point
both skeletal abnormalities and growth plate defects were readily apparent.
First, we examined C4st1 mRNA expression in growth plates by RNA in
situ hybridization using the exon 2 and 3 probe
(Fig. 4A). C4st1 was
expressed in the proliferating zone of the wild-type growth plate
(Fig. 4A, part i), whereas in
homozygous mutants, expression of exons 2 and 3 was not observed
(Fig. 4A, part ii), consistent
with a disruption of C4st1 expression by the gene trap integration.
Next, we examined the growth plate of E18.5 tibias using Safranin O, which
stains GAGs in cartilaginous tissue. In mutants, Safranin O staining was
reduced, suggesting reduced GAG content
(Fig. 4B, part ii) and staining
of cartilage islands in wild-type primary bone was absent in the mutants
(Fig. 4C). In wild-type growth
plates, three distinct chondrocyte subpopulations, namely articular
proliferating chondrocytes, columnar proliferating chondrocytes and
hypertrophic chondrocytes were readily apparent
(Fig. 4B, part i). The
morphology of mutant growth plates, however, was disturbed, being severely
shortened, disorganized and hypocellular
(Fig. 4B, part ii), Consistent
with this, the chondrocyte zones and in particular the columnar and
hypertrophic zones were reduced in size
(Fig. 4B, part ii). To explore
the cellular deficits in more detail, we next analyzed Hematoxylin and
Eosin-stained growth plates (Fig.
4D-G). This revealed that in the smaller columnar layer of the
mutant growth plate, the chondrocyte columns, which normally form as flattened
cells oriented along the longitudinal axis of the developing bone
(Fig. 4D, part I; Fig. 4E), were disorganized
(Fig. 4D, part ii;
Fig. 4F). Strikingly, in the
medial region clumps of cells were evident, whereas in the lateral regions,
although columns formed, they were not oriented properly and extended radially
such that some columns were oriented perpendicular to the long axis of the
bone (Fig. 4B, part ii;
Fig. 4D, part ii;
Fig. 4F, part ii).
Interestingly, we also observed an increase in the thickness of the bone
collar in C4st1gt/gt homozygous mutant growth plates
(Fig. 4F). Finally, we examined
the Hematoxylin and Eosin stained sections under dark-field conditions, to
reveal gross features of the extracellular matrix (ECM). This revealed that
wild-type chondrocytes were surrounded by a smooth ECM
(Fig. 4G, part i), whereas in
the mutants there was extensive fibrillation of the ECM
(Fig. 4G, part ii). Of note,
this feature is typically found in the cartilage of osteoarthritic patients.
Altogether, these results indicate that C4st1 is required for proper
chondrocyte development, orientation of chondrocyte stacks and morphogenesis
of the cartilage growth plate.
View larger version (138K):
[in this window]
[in a new window]
|
Fig. 4. Cartilage growth plate defects in the proximal tibia of
C4st1gt mutant embryos at E18.5. (A) RNA in situ
hybridization using a C4st1-exon2+3-specific probe shows
C4st1 expression in proliferating, but not hypertrophic chondrocytes
in wild-type growth plates (i) and the reduction in C4st1 staining in
mutant growth plates (ii). (B,C) Safranin O staining. (B) Safranin O staining
shows a reduction in size of proliferating (p), columnar (c) and hypertrophic
(h) zones, as well as less intense staining in mutant growth plates (ii) when
compared with wild type (i). Brackets indicate areas shown in C. (C) Higher
magnification of the transition zone between hypertrophic cartilage and
primary bone, showing cartilage islands (arrow) in wild-type (i), but not
mutant, bone (ii). (D-G) Hematoxylin and Eosin staining. (D) Mutant growth
plates appear disorganized and contain ECM disruptions (arrowhead) and
misoriented chondrocyte columns (arrow). (E,F) Higher magnification either
wild-type (E) or mutant (F) growth plates (p, c and h are defined in B).
Wild-type chondrocyte columns are oriented parallel to the longitudinal bone
axis, whereas mutant columns are oriented almost perpendicular to it. In
addition there is an increased thickness of the bone collar in the mutants
(arrows). (G) Dark-field images of Hematoxylin and Eosin-stained wild-type (i)
and mutant (ii) proliferating chondrocytes, showing fibrillation of ECM in
mutant growth plates. Scale bar: 400 µm for A; 1 mm for B; 100 µm for
C,D; 30 µm for E,F; 10 µm for G.
|
|
The C4st1gt/gt mutation disturbs chondroitin sulfation balance and chondroitin sulfate spatial distribution in the growth plate
In order to determine how mutation of C4st1 affected CS sulfation
in the growth plate, we quantitated CS species in E18.5 wild-type and mutant
embryonic growth plates using fluorophore-assisted carbohydrate
electrophoresis (FACE) analysis (Fig.
5A). As expected, chondroitin-4-sulfate (C4S), the product of
C4ST, was reduced by more than 90% in homozygous mutant growth plates,
suggesting that C4st1 is the main enzyme for production of C4S in cartilage.
The presence of a small amount of residual C4S might arise from expression of
other C4st1-related enzymes. There was also 
50% drop in
chondroitin-6-sulfate (C6S) and a reduction in unsulfated chondroitin (C0S).
By contrast, hyaluronan (HA) was only modestly affected in the mutants
(Fig. 5A). Therefore, loss of
C4st1 effectively reversed the sulfation balance from predominantly C4S in
wild type to predominantly C6S in mutant cartilage.
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.
View larger version (101K):
[in this window]
[in a new window]
|
Fig. 5. Analysis of growth plate extracellular matrix (ECM) markers of the proximal
tibia at E18.5. (A) Fluorophore-assisted carbohydrate electrophoresis (FACE)
analysis of ECM glycosaminoglycans (GAGs). (B) Chondroitin sulfate (CS)
immunohistochemistry showing ECM staining (black arrow) in all three growth
plate layers (p, proliferating chondrocytes; c, columnar chondrocytes; h,
hypertrophic chondrocytes) of wild-type cartilage (+/+), whereas CS staining
in mutant (gt/gt) proliferating and columnar layers is restricted to the
pericellular space (arrowhead), with very little staining in the ECM (black
arrow). However, in the columnar and hypertrophic layers of mutants, ECM
staining of CS was observed (white arrows). (C) C6S was detected by
immunofluorescence staining and is distributed in the outer-most layers of the
proliferative zone in wild-type cartilage (+/+), whereas in mutant cartilage
(gt/gt), low pericellular C6S staining was observed in both proliferative and
columnar layers. (D) Distribution of aggrecan was analyzed by
immunofluorescence, which revealed localization to the ECM in all layers of
the wild-type cartilage (+/+), as well as in the proliferative layer of mutant
cartilage (gt/gt). However, in the mutant, aggrecan was increasingly
restricted to the pericellular space in columnar and hypertrophic (arrowhead)
layers. (E) Detection of collagen II by immunofluorescence shows strong
staining of the ECM in both wild-type (+/+) and mutant (gt/gt) proliferative
and columnar layers, and decreased staining in the ECM of the hypertrophic
layer. Scale bar: 10 µm.
|
|
C4st1 regulates proliferation and apoptosis, but not the differentiation pattern of chondrocytes
To determine how alteration in chondroitin sulfation affects chondrocyte
differentiation, we first analyzed different chondrocyte subpopulations by
marker analysis using RNA in situ hybridization
(Fig. 6A-C). The expression
patterns of both collagen II (Fig.
6A), a marker for the proliferating zones in the growth plate, and
collagen X (Fig. 6B), a marker
for hypertrophic chondrocytes, were indistinguishable between wild-type and
homozygous mutant growth plates. Furthermore, expression of fibroblast growth
factor receptor type 3 (Fgfr3,
Fig. 6C), a marker for late
columnar proliferating/prehypertrophic chondrocytes, was observed in both
wild-type and homozygous mutant growth plates
(Fig. 6C, part I;
Fig. 6C, part ii). These
results indicate that the pattern of chondrocyte differentiation was not
significantly affected in C4st1gt mutants. Therefore, we
examined whether defects in C4st1gt growth plate
morphology might reflect alterations in the balance of proliferation versus
apoptosis (Fig. 6D-I). In
wild-type growth plates, proliferation, as measured by BrdU-labeled
chondrocytes, was visible throughout the proliferative layers
(Fig. 6D, part i), similar to
mutant growth plates, although the proliferative zone was drastically reduced
in size in mutants (Fig. 6D, part
ii). Interestingly, quantitation of proliferation rates
in the three growth plate zones revealed an approximately twofold increase in
the number of proliferating cells in the proliferative and columnar zones of
homozygous mutants, when compared with wild-type growth plates
(Fig. 6J). When we examined
apoptosis by TUNEL staining (Fig.
6E), we found that wild-type growth plates displayed moderate
apoptosis that was restricted to differentiated chondrocytes in the
hypertrophic zone (Fig. 6E, part
i), whereas mutant growth plates exhibited TUNEL-labeled cells in
all zones of the growth plate (Fig. 6E,
part ii). Quantitation of TUNEL-positive cells in the three growth
plate zones revealed an approximate 3.5-fold increase in TUNEL-positive cells
in the hypertrophic zone of homozygous mutant growth plates
(Fig. 6K). Moreover, a
significant number of TUNEL-positive cells were counted in columnar and
proliferating zones of homozygous mutant growth plates, but not in wild-type
growth plates (Fig. 6K).
View larger version (95K):
[in this window]
[in a new window]
|
Fig. 6. Chondrocyte development and differentiation at E18.5. (A,B,G-I) Proximal
tibia; (C-F) distal tibia. (A-C) Marker analysis by RNA in situ hybridization
in wild-type (+/+) and C4st1gt/gt (gt/gt) growth plates.
Developmental markers for proliferating chondrocytes (collagen II, A),
hypertrophic chondrocytes (collagen X, B) as well as columnar chondrocytes
(Fgfr3, C) were expressed normally in mutant growth plates (ii) when
compared with their wild-type counterparts (i), with the size of their
expression domains reduced in relation to the overall shortening of the growth
plate. (D) BrdU labeling of proliferating chondrocytes was detected by
immunofluorescence (green). In wild-type growth plates (i), the zone of
proliferation (double-headed arrow) excludes the hypertrophic layer. This
proliferative zone is reduced in size in mutant cartilage (ii). (E) TUNEL
staining (green) to identify chondrocytes undergoing apoptosis. In wild-type
cartilage (i), low numbers of only the most differentiated hypertrophic cells
(white bar) showed TUNEL staining, whereas in mutant cartilage, an increased
number of cells in all zones (double-headed arrow) showed TUNEL staining (ii).
(F) Immunofluorescence (red) using an -Bcl2 antibody. Wild-type growth
plates (i) show widespread Bcl2 staining in the columnar zone (double-headed
arrow), whereas staining in gt/gt growth plates (ii) is severely reduced and
present only in lateral areas (arrows). (G) Immunofluorescence (red) using an
-Bax antibody. Wild-type growth plates (i) show strong staining in
prehypertrophic and hypertrophic chondrocytes (double-headed arrow), but not
in columnar and proliferating cells (arrowhead). Homozygous mutant growth
plates (ii), however, display staining in hypertrophic cells (arrow) and
proliferating cells (arrowhead). (H,I) Higher magnification of regions labeled
by white squares in G. Bax expression in wild-type growth plates is restricted
to prehypertrophic and hypertrophic cells (`h' in H, i), and is not present in
columnar cells (`c' in H, i) or proliferating cells (I, i). White line
represents the border between columnar and prehypertrophic and hypertrophic
zones. Bax expression in mutant growth plates is observed in hypertrophic
cells (arrowhead in H, ii), but also in columnar cells (arrow in H, ii) and
proliferating cells (green arrow in H, ii; arrowhead in I, ii). (J)
Quantitation of BrdU-labeled cells in proliferating (prol.), columnar
(column.) and hypertrophic (hypertr.) chondrocytes. Cartilage in mutant
(gt/gt) shows an approximate twofold increase in BrdU-labeled cells in both
proliferating and columnar chondrocytes compared with wild type. (K)
Quantitation of TUNEL-labeled (apoptotic) cells in all three layers of the
growth plate. Mutant growth plates showed a 3.5-fold increase in the number of
cells undergoing apoptosis in the hypertrophic zone and apoptotic cells in
both proliferating and columnar chondrocytes. Scale bar: 150 µm.
|
|
We next wanted to analyze whether this increase in apoptosis correlated
with changes in the balance of pro-versus anti-apoptotic signals. For this, we
analyzed the expression of the anti-apoptotic protein Bcl2 and the
pro-apoptotic protein Bax by immunofluorescence. While wild-type growth plates
exhibited a wide expression domain of Bcl2 in proliferating and columnar
chondrocytes (Fig. 6F, part i), Bcl2 expression in homozygous mutant growth plates was severely reduced and
only present in lateral regions (Fig. 6F,
part ii). Conversely, in wild-type growth plates, high levels of
Bax expression were observed in prehypertrophic and hypertrophic chondrocytes,
but not proliferating or columnar chondrocytes
(Fig. 6G, part i; 6H, part i; 6I, part
i). Homozygous mutant growth plates also exhibited Bax expression
in hypertrophic cells (Fig. 6G, part ii;
6H, part ii), but in contrast to wild-type growth plates, Bax
expression was observed in the proliferating zone as well, thus leading to Bax
expression in most cells of the mutant growth plates
(Fig. 6G, part ii;
Fig. 6H, part ii;
Fig. 6I, part ii). These
results suggest that chondrocytes in mutant cartilage are exposed to
imbalanced apoptotic signaling, with a strong reduction in anti-apoptotic
signals and an increase in pro-apoptotic signals. Based on these data, we
propose that the disturbed morphology of the growth plate reflects both
accelerated maturation of chondrocytes leading to severe shortening of the
proliferative and hypertrophic zones, as well as enhanced apoptosis of
chondrocytes, caused by a disturbed balance of pro-versus anti-apoptotic
signals throughout the growth plate region that cannot be compensated by the
increased proliferative rate.
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.
View larger version (135K):
[in this window]
[in a new window]
|
Fig. 7. Analysis of Ihh, BMP and TGFß signaling in E18.5 cartilage. (A) distal
tibia; (B-H) proximal tibia. (A) Ptch1 RNA in situ hybridization as
output for Ihh signaling reveals expression in columnar chondrocytes in both
wild-type (i) and mutant (ii) cartilage. (B-D) Phosphorylated Smad1
distribution. Wild-type (i) and mutant (ii) growth plates were stained using
an antibody that recognizes Smad1 phosphorylated by activated BMP receptors.
(B) pSmad1 (red) is seen in hypertrophic chondrocytes in wild-type cartilage
(i), and is reduced in mutant cartilage (ii). (C) Higher magnification of
proliferating region (indicated as `C' in B) shows very little pSmad1 staining
in both wild-type (i) and mutant (ii) cartilage. (D) Higher magnification of
early hypertrophic regions (indicated as `D' in B) shows nuclear pSmad1
staining in wild-type chondrocytes (i), which is reduced in mutant
chondrocytes (ii). (E-H) Phosphorylated Smad2 distribution. Wild-type and
mutant growth plates were stained using an antibody that recognizes Smad2
phosphorylated by activated TGFß receptors. (E) Very little nuclear
pSmad2 staining was seen in wild-type growth plates (i) in columnar/early
hypertrophic layers and in lateral aspects of the growth plate (white arrow).
In mutant cartilage (ii), strong pSmad2 staining is seen in all cartilage
layers. (F-H) Higher magnification of regions indicated in E. Arrows indicate
nuclear staining. (F) Higher magnification of proliferative layer. No nuclear
pSmad2 staining was seen in wild-type chondrocytes (i), whereas a high
proportion of mutant chondrocytes (ii) shows moderate nuclear pSmad2 staining.
(G) Higher magnification of late columnar layer. Very little nuclear pSmad2
staining is visible in wild-type cells (i), whereas strong nuclear pSmad2
staining is apparent in all mutant chondrocytes (ii). (H) Higher magnification
of hypertrophic layers. Weak nuclear pSmad2 staining is seen in few wild-type
chondrocytes (i), whereas almost all mutant chondrocytes show strong nuclear
pSmad2 staining (ii). Scale bar: 300 µm for A; 200 µm for B,E; 20 µm
for C,D,F,G,H.
|
|
Treatment of both wild-type and C4st1gt/gt mutant
explants with N-Shh decreased the size of the mineralized part of the explants
and increased the size of the non-mineralized part
(Fig. 8A), albeit to a lesser
degree in the mutants (Fig.
8B). In both wild-type and homozygous mutant explants, N-Shh
induced Ptch1 expression (Fig.
8E,F) and strongly suppressed collagen X expression
(Fig. 8C,D), consistent with
its reported role in blocking hypertrophy. Furthermore, we noted no
significant differences in the response of the cartilage component of
wild-type versus mutant growth plates in any of these assays. However, we did
note in this assay that the perichondrium in C4st1gt/gt
mutant explants underwent neither N-Shh-induced thickening (arrow
Fig. 8C,D), nor N-Shh-induced
Ptch1 expression (Fig. 8E, part
ii; Fig. 8F, part
ii), contrasting wild-type perichondrium (arrow
Fig. 8E, part i;
Fig. 8F, part i). Next, we
examined exogenous TGFß stimulation, which led to a decrease in both
length and width of metatarsal explants
(Fig. 8A,B) and reduction of
collagen X expression (Fig.
8C,D). When we examined pSmad2 levels in untreated wild-type
metatarsals, some nuclear pSmad2 was evident in prehypertrophic and
hypertrophic chondrocytes (Fig.
8G-I; Fig. 8G, part
iii; Fig. 8G, part
iv), and this was strongly increased by TGFß in the
prehypertrophic and hypertrophic zones, which were also reduced in size
(Fig. 8H,I; Fig. 8H, part iii;
Fig. 8H, part iv), in agreement
with TGFß-dependent reduction in collagen X expression. In the
proliferative zone, there was very little discernible pSmad2 staining in
untreated wild-type growth plates (Fig. 8G,
part i; Fig. 8G, part
iii), whereas after TGFß treatment, staining became apparent
in the lateral regions (arrow in Fig. 8H,
part i), with most cells in the center remaining pSmad2-negative
even after TGFß treatment (Fig. 8H,
part iii). These results are in accordance with our in vivo
observations on the activity of the Smad2 pathway in wild-type growth plates.
The C4st1gt/gt explants also recapitulated our in vivo
results, displaying strong nuclear pSmad2 staining in virtually all cells of
the growth plate, including chondrocytes in the proliferative zone, even in
the absence of TGFß stimulation (Fig.
8G, part ii; Fig. 8G, part
v; Fig. 8G, part
vi), although addition of TGFß caused some further
enhancement (Fig. 8H, part ii; Fig. 8H, part v;
Fig. 8H, part vi). This
suggests that the constitutively activated TGFß signaling pathway in
mutant chondrocytes can be further stimulated by exogenous TGFß.
View larger version (75K):
[in this window]
[in a new window]
|
Fig. 8. C4st1gt/gt metatarsal explants are able to respond to
exogenous growth factors. Metatarsals were removed from E18.5 wild-type (+/+)
and mutant (gt/gt) embryos and cultures for 4 days in either the absence
(control) or presence of 2 µg/ml N-Shh, 500 pM TGFß or 10nM BMP2 as
indicated. Metatarsals were photographed (A,B) or processed for RNA in situ
hybridization (C-F) or immunofluorescence (G-J). (A,B) Appearance of explants
after 4 day treatment with no factor (control), N-Shh, BMP2 or TGFß.
(C,D) Effects of growth factor treatment on hypertrophic differentiation as
visualized by collagen X RNA in situ hybridization. Collagen X staining is
reduced in N-Shh and TGFß-treated wild-type (C) and mutant (D) explants
and increased in BMP2-treated wild-type and mutant explants. In addition,
treatment of wild-type, but not mutant explants with N-Shh lead to increased
thickness of the perichondrium (arrows). (E-J) Signaling pathways in
metatarsal explants. (E) Ptch1 staining in untreated wild-type and
mutant explants is restricted to proliferating chondrocytes. No Ptch1
expression is seen in the perichondrium. (F) Treatment of explants with N-Shh
leads to Ptch1 expression throughout the growth plate in both
wild-type and mutant explants. Wild-type explants also showed Ptch1
expression in the perichondrium, which was not observed in mutant explants
(arrows). (G) Nuclear pSmad2 staining in untreated wild-type explants was seen
in some late columnar/early hypertrophic cells (i; see iv for higher
magnification), whereas occasional weak staining in proliferating cells was
also present (i; iii). In untreated mutant explants, strong pSmad2 staining
was apparent in all cells of the growth plate (ii, v, vi). (H) Treatment of
wild-type explants with TGFß lead to an increase in the number of
pSmad2-stained cells and staining intensity in hypertrophic cells (i, iv),
whereas cells in the proliferative layer were still mostly negative for
nuclear pSmad2 (i, iii). Arrow in i indicates increased pSmad2 staining in
lateral regions of the growth plate. Treatment of mutant explants with
TGFß lead to a small increase in pSmad2 staining intensity (ii, v, vi).
(I) Nuclear pSmad1 expression in untreated wild-type explants was apparent in
a subset of proliferating chondrocytes (iii) and in hypertrophic chondrocytes
(iv). Whereas mutant explants also showed nuclear pSmad1 staining in
hypertrophic chondrocytes (ii, vi), staining intensity was lower in
proliferating chondrocytes (ii, v). (J) Both wild-type (i, iii, iv) and mutant
(ii, v, vi) explants treated with BMP2 showed strong nuclear pSmad1 staining
in the expanded region of hypertrophy (iii, iv, v, vi). Scale bar: 5 mm for
A,D; 1 mm for C-F; 500 µm for G-J, parts i, ii; 20 µm for G-J, parts
iii-vi.
|
|
In sharp contrast to TGFß, treatment with BMP2 caused a dramatic
increase in the size of both wild-type and mutant growth plates
(Fig. 8A,B), as well as
inducing collagen X expression (Fig.
8C,D). We next examined pSmad1, which was present mainly in
prehypertrophic and hypertrophic zones, consistent with our in vivo analyses,
although we also observed some pSmad1 in a subset of proliferating
chondrocytes (Fig. 8I, part i;
Fig. 8I, part iii; Fig. 8I, part iv). Treatment
with BMP2 led to an expansion of the hypertrophic domain of pSmad1 staining
(Fig. 8J, part i; Fig. 8J, part iii;
Fig. 8J, part iv). By contrast,
we observed that pSmad1 was reduced in untreated
C4st1gt/gt explants
(Fig. 8I, part ii) and was
restricted to a small region of hypertrophic chondrocytes
(Fig. 8I, part ii;
Fig. 8I, part v;
Fig. 8I, part vi). However,
treatment with BMP2 led to a significant induction in pSmad1
(Fig. 8J, part ii;
Fig. 8J, part v;
Fig. 8J, part vi), corroborating our morphological and gene expression data that
C4st1gt/gt explants have the capability to respond to BMP
signaling.
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.
|
Discussion
|
|---|
We have demonstrated that mice with a mutation in C4st1 die
shortly after birth with severe chondrodysplasia, altered chondrocyte stack
orientation and accelerated chondrocyte differentiation. We show that the
reduction in C4S in C4st1gt/gt mutant mice leads to
changes in the spatial distribution of CS and affects the balance of TGFß
family signaling in the cartilage growth plate
(Fig. 8). Thus, correct
chondroitin sulfation balance is essential for mammalian cartilage
morphogenesis and embryonic development.
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.
View larger version (51K):
[in this window]
[in a new window]
|
Fig. 9. Model of the role of C4st1 during cartilage development. The organized
growth plate of wild-type cartilage contains proliferating (p), columnar (c)
and hypertrophic (h) chondrocytes (grey) surrounded by ECM containing CS
(orange). C4st1gt/gt-mutant growth plates are severely
reduced in length, but display increased width. The chondrocyte layers are
disorganized and chondrocyte columns are not oriented along the longitudinal
axis of the bone. CS (orange) is mostly absent from the ECM and instead is
located in the pericellular space surrounding chondrocytes. There is a
dramatic increase in the thickness of the bone collar (dark grey) in mutant
growth plates and bone. Whereas Ihh signaling to proliferating chondrocytes is
not significantly altered in mutant growth plates (red arrows), BMP signaling
to both proliferating and hypertrophic chondrocytes is reduced (yellow
arrows), and TGFß signaling to proliferating and hypertrophic
chondrocytes (blue arrows) is dramatically increased in mutant growth plates.
Imbalance in apoptotic signals leads to a dramatic increase in the area prone
to undergo apoptosis (indicated by black brackets).
|
|
Altered growth factor signaling in C4st1gt/gt growth plates
A prominent defect in the cartilage of C4st1gt/gt
mutant growth plates is a marked reduction in both the proliferative and
hypertrophic zones. A number of signaling pathways have been shown to be
involved in controlling cartilage morphogenesis
(Karsenty and Wagner, 2002;
Vortkamp, 2001). Of particular
note, BMP family members positively regulate chondrocyte proliferation and
hypertrophy, but negatively regulate terminal chondrocyte differentiation both
in vitro and in vivo (Yoon and Lyons,
2004). This results in BMPs increasing the length of the
proliferative and hypertrophic zones
(Minina et al., 2002).
TGFß1, conversely, negatively regulates chondrocyte proliferation and
hypertrophy (Serra and Chang,
2003). Our analysis of Smad signaling in
C4st1gt/gt mutant growth plates revealed that active
Smad1, which functions in the BMP pathway, was downregulated, whereas Smad2 in
the TGFß pathway was strongly upregulated throughout the growth plate.
Therefore, the combination of reduced BMP signaling and upregulation of
TGFß signaling probably plays a key role in causing the severe reduction
in the size of the proliferative and hypertrophic zones and increased
apoptosis observed in C4st1gt/gt mutant growth plates
(Fig. 9). These results
contrast the relatively normal spatial activation of the hedgehog pathway we
observed, suggesting that the lack of C4S does not have a major impact on the
function of Ihh during growth plate morphogenesis. These results suggest that
the lack of C4S has specific effects on selected growth factor signaling
pathways in the growth plate.
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.
|
Conclusion
|
|---|
In summary, we have shown that the correct balance of chondroitin sulfation
is crucial for endochondral bone formation. Disruption of the C4st1
locus leads to chondrodysplasia, osteoarthritis-like symptoms and affects the
balance of TGFß family signaling in the cartilage growth plate. These
results thus demonstrate a crucial role for a chondroitin sulfotransferase in
mammalian development and disease.
|
ACKNOWLEDGMENTS
|
|---|
We thank Ken Harpal for the Hematoxylin and Eosin staining, and the
histology laboratory in the Department of Pathology in Mount Sinai Hospital
for the Safranin O staining. This work was funded by the Canadian Institutes
of Health Research (CIHR) to J.L.W. M.K. was supported by postdoctoral
fellowships from CIHR and the Canadian Association of Gastroenterology. J.L.W.
is a CIHR Investigator and an International Research Scholar of the Howard
Hughes Medical Institute.
|
Footnotes
|
|---|
4 Department of Molecular and Medical Genetics and Microbiology,
University of Toronto, Toronto M5S 1A8, Canada
|
REFERENCES
|
|---|
Abad, V., Meyers, J. L., Weise, M., Gafni, R. I., Barnes, K. M.,
Nilsson, O., Bacher, J. D. and Baron, J. (2002). The
role of the resting zone in growth plate chondrogenesis.
Endocrinology 143,1851
-1857.[Abstract/Free Full Text]
Attisano, L. and Wrana, J. L. (2002). Signal
transduction by the TGF-beta superfamily. Science
296,1646
-1647.[Abstract/Free Full Text]
Baur, S. T., Mai, J. J. and Dymecki, S. M.
(2000). Combinatorial signaling through BMP receptor IB and GDF5:
shaping of the distal mouse limb and the genetics of distal limb diversity.
Development 127,605
-619.[Abstract]
Calabro, A., Midura, R., Wang, A., West, L., Plaas, A. and
Hascall, V. C. (2001). Fluorophore-assisted carbohydrate
electrophoresis (FACE) of glycosaminoglycans. Osteoarthr.
Cartilage 9,S16
-S22.[CrossRef]
Campos-Xavier, B., Saraiva, J. M., Savarirayan, R., Verloes, A.,
Feingold, J., Faivre, L., Munnich, A., Le Merrer, M. and Cormier-Daire,
V. (2001). Phenotypic variability at the TGF-beta1 locus in
Camurati-Engelmann disease. Hum. Genet.
109,653
-658.[CrossRef][Medline]
Candia, A. F., Watabe, T., Hawley, S. H., Onichtchouk, D.,
Zhang, Y., Derynck, R., Niehrs, C. and Cho, K. W.
(1997). Cellular interpretation of multiple TGF-beta signals:
intracellular antagonism between activin/BVg1 and BMP-2/4 signaling mediated
by Smads. Development
124,4467
-4480.[Abstract]
Deckelbaum, R. A., Chan, G., Miao, D., Goltzman, D. and
Karaplis, A. C. (2002). Ihh enhances differentiation of CFK-2
chondrocytic cells and antagonizes PTHrP-mediated activation of PKA.
J. Cell. Sci. 115,3015
-3025.[Abstract/Free Full Text]
Garcia-Garcia, M. J. and Anderson, K. V.
(2003). Essential role of Glycosaminoglycans in Fgf signaling
during mouse gastrulation. Cell
114,727
-737.[CrossRef][Medline]
Grobe, K., Ledin, J., Ringvall, M., Holmborn, K., Forsberg, E.,
Esko, J. D. and Kjellen, L. (2002). Heparan sulfate
and development: differential roles of the N-acetylglucosamine
N-deacetylase/N-sulfotransferase isozymes. Biochim. Biophys.
Acta 1573,209
-215.[Medline]
Habuchi, O. (2000). Diversity and functions of
glycosaminoglycan sulfotransferases. Biochim. Biophys.
Acta 1474,115
-127.[Medline]
Hildebrand, A., Romaris, M., Rasmussen, L. M., Heinegard, D.,
Twardzik, D. R., Border, W. A. and Ruoslahti, E.
(1994). Interaction of the small interstitial proteoglycans
biglycan, decorin and fibromodulin with transforming growth factor beta.
Bio
TOP
SUMMARY
Introduction
