Research Institute of Molecular Pathology (I.M.P.), Dr Bohr-Gasse 7, 1030 Vienna, Austria
* Author for correspondence (e-mail: wagner{at}imp.univie.ac.at)
Accepted 20 August 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: AP1, AP-1, Fra2, Fra-2, Growth plate, Cartilage, Type X collagen, Mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The AP1 (activator protein 1) transcription factor consists of dimers of
the Fos (Fos, Fra1, Fra2 and FosB) and Jun (Jun, JunB and JunD) families of
basic leucine zipper domain proteins. AP1 is involved in several biological
processes, including differentiation, proliferation, apoptosis and oncogenic
transformation (Jochum et al.,
2001). Jun proteins seem to play important roles during
development as the absence of Jun (Hilberg
et al., 1993
; Johnson et al.,
1993
) and JunB
(Schorpp-Kistner et al., 1999
)
results in embryonic lethality. A bone phenotype was described only recently
in embryo-specific Junb knockout mice, which develop osteopenia and
suffer from a chronic myeloid leukemia (CML)-like disease
(Hess et al., 2003
;
Kenner et al., 2004
), as well
as in cartilage-specific Jun knockout mice, which display scoliosis
(Behrens et al., 2003
). All
four members of the Fos family seem to be involved in bone development.
Fos knockout mice lack osteoclasts because of a complete block in
osteoclast differentiation, resulting in an osteopetrotic phenotype
(Wang et al., 1992
;
Grigoriadis et al., 1994
),
whereas ubiquitous expression of Fos in transgenic mice leads to the formation
of osteosarcomas (Ruther et al.,
1989
). When Fra1 (encoded by Fosl1) is expressed from the
Fos locus, the osteopetrotic Fos knockout phenotype is
partly rescued (Fleischmann et al.,
2000
). Overexpression of
FosB, a splice variant of FosB,
leads to an osteosclerotic phenotype because of increased numbers of mature
osteoblasts (Sabatakos et al.,
2000
). Similarly, transgenic mice overexpressing Fra1 develop
osteosclerosis because of a cell-autonomous increase in the number of mature
osteoblasts (Jochum et al.,
2000
), whereas embryo-specific Fosl1 knockout mice
display osteopenia (Eferl et al.,
2004
). Little is known about the role of Fos proteins, in
particular Fra2 (encoded by Fosl2), in cartilage development.
Overexpression of Fos in embryonic stem (ES) cell chimeras leads to the
development of chondrosarcomas (Wang et
al., 1991
). By contrast, overexpression of Fos in the chondrogenic
cell line ATDC5 inhibits chondrocyte differentiation
(Thomas et al., 2000
). Fra2 is
expressed at high levels in ovary, stomach, intestine, brain, lung and heart
(Foletta et al., 1994
), and in
differentiating epithelia, the central nervous system and developing cartilage
(Carrasco and Bravo, 1995
). In
addition, expression of Fra2 has been found to be distinct from other Fos
members, suggesting that it has unique functions during embryonic development
and adulthood.
As Fra2 is expressed during bone development, in particular in
differentiating chondrocytes (Carrasco and
Bravo, 1995), we determined the role of Fra2 in cartilage biology
and bone growth. Initially, we investigated its function in embryos and
newborns lacking Fra2. The zones of hypertrophic chondrocytes were narrower
throughout embryonic and early postnatal development and less calcified matrix
was deposited in Fosl2/ growth plates. This
is probably due to impaired chondrocyte differentiation in vivo and in vitro.
Moreover, endochondral ossification was delayed in developing vertebral
columns of Fosl2/ embryos. As
Fosl2/ pups die shortly after birth, we
generated floxed Fosl2 mice and crossed them to coll2a1-Cre
transgenic mice. The conditional Fosl2 knockout mice have a rather
broad spectrum of Fosl2 deletion; however, they display a similar
defect in chondrocyte differentiation and suffer from a kyphosis-like
phenotype.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Skeletal staining
Animals were skinned, eviscerated and dehydrated in 95% ethanol overnight
and in acetone again overnight. Skeletons were stained with 0.015% Alcian
Blue, 0.05% Alizarin Red and 5% acetic acid in 70% ethanol for several days.
Next, the skeletons were cleared in 1% KOH for an age dependent period, passed
through a decreasing KOH series and stored in glycerol.
In situ hybridization
Digoxigenin-labeled riboprobes were synthesized according to the
manufacturer's instructions (DIG RNA labeling kit, Boehringer-Mannheim). For
in situ hybridization analyses, sections were deparaffinized and hybridization
was performed according to standard procedures
(Murtaugh et al., 1999). The
signal was detected according to the manufacturer's (Boehringer-Mannheim)
instructions using BM-purple AP-substrate solution. Sections were then washed,
fixed in 4% PFA and mounted.
BrdU labeling
For in vivo labeling, 100 µg/g body weight BrdU were injected
intraperitoneally in pregnant females at E17.5 and E18.5, and embryos were
isolated by Caesarian section 32 hours and 2 hours later, respectively.
Sections were deparaffinized, unmasked by boiling in citrate buffer (0.1 mM
citrate acid, 0.8 mM sodium citrate), blocked in 20% horse serum and incubated
with a FITC-labeled -BrdU antibody (Becton Dickinson) for 30 minutes in
the dark. Cells were counterstained with 4',6-Diamidino-2-phenylindole
(DAPI). At least 200 cells were counted in the zone of proliferating
chondrocytes.
Primary rib cage chondrocytes were incubated with 4 µM/ml BrdU for 2
hours. Cells were fixed in 70% ethanol and permeabilized with 0.07 N NaOH for
2 minutes at room temperature. Cells were incubated with FITC-labeled
-BrdU antibody for 30 minutes in the dark, counterstained with DAPI and
mounted.
Von Kossa staining
Paraffin sections were deparaffinized and incubated in 2% silver nitrate in
a coplin jar placed directly in front of a 60 W lamp for 1 hour to detect
matrix-bound Ca2+. After the staining, sections were fixed with
2.5% sodium thiosulfate for 5 minutes. The sections were washed, dehydrated
and mounted.
Ki67 staining
Paraffin embedded sections were deparaffinized and unmasked as described
above. To block endogenous peroxidase activity, sections were incubated in 3%
H2O2 for 30 minutes at room temperature. Unspecific
binding sites were blocked using 20% horse serum for 20 minutes at room
temperature, followed by incubation with an -Ki67 antibody (Novo
Castra) for 1 hour at room temperature. Secondary
-rabbit antibody and
Vectastain solution (Vectastain ABC kit, Vector Laboratories) were used
according to the manufacturer's recommendations. After washing, the sections
were incubated for 2-10 minutes with DAB substrate solution (DAB Peroxidase
Substrate kit, Vector Laboratories), washed and mounted.
Cell culture
Rib cage chondrocytes were isolated from
Fosl2/ and Fosl2 wild-type neonatal
mice. Rib cages were sequentially digested twice for 30 minutes and once for 4
hours at 37°C in a 0.2% collagenase solution in serum-free Dulbecco's
Modified Eagle Medium (DMEM) with antibiotics (100 µg/ml Streptomycin, 100
U/ml Penicillin). Single cells were cultured overnight over 1.5% agarose in
DMEM, 10% fetal calf serum (FCS), 100 µg/ml Streptomycin, 100 U/ml
Penicillin and 5 µM L-Glutamate (P/S/G) to obtain fibroblast-free
cultures.
Cumulative cell number assay
Primary rib cage chondrocytes were seeded in six-well plates at a number of
1x105 cells/well. After 2 days in culture, cells were counted
and replated at 1x105 cells per well. Counting was repeated
for at least four passages.
Differentiation assay
Primary chondrocytes were seeded in 24-well plates
(1-2x105 cells/well) and cultured in DMEM, 10% FCS, P/S/G, 5
mM ß-glycerophosphate and 100 µg/ml ascorbic acid for 12 days. Half of
the media was exchanged every other day. Differentiated cells were stained in
0.1% Alcian Blue in 0.1 N HCl. The dye was extracted with 4M
guanidine-hydrochloride and absorbance measured at 595 nm.
Semi-quantitative RT-PCR and real time PCR
Total RNA was isolated from primary rib cage chondrocytes or knee joints
from newborn mice (P2) using TRIzol reagent (Invitrogen), according to the
manufacturer's recommendations. RNA (2-4 µg) was used for cDNA synthesis
using Ready-To-Go You-Prime First-Strand Beads (Amersham Biosciences) and 1
µl of Random Primers (Invitrogen) according to the manufacturer's
instructions. After an initial denaturation at 95°C for 2 minutes, the PCR
reactions were carried out as follows: denaturing for 30 seconds at 95°C,
annealing for 45 seconds at 55°C and extension for 90 seconds at 65°C.
The reaction was completed by a 7 minute extension step at 65°C. For
realtime PCR, light cycler Fast start DNA Master SYBR Green (Roche
Diagnostics) was used. The following primers for were used: aggrecan (forward)
5'-tcgcccaggctccaccagatact-3' and (reverse)
5'-ccagccagccagcatagcacttgt-3'; type II collagen (forward)
5'-gcgagaggggactgaagggacacc-3' and (reverse)
5'-cggggctgcggatgctctcaat-3'; type X collagen (forward)
5'-gaccccctggcccctctgga-3' and (reverse)
5'-atctcacctttagcgcctggaatg-3'; Ihh (forward)
5'-caagcagttcagccccaacg-3' and (reverse)
5'-acgtgggccttggactcgta-3'; Fosl2 (forward)
5'-ttatcccgggaactttgacacctc-3' and (reverse)
5'-cggcgttcctcggggctgatt-3'; tubulin (forward)
5'-gacagagccaaactgagcacc-3' and (reverse)
5'-caacgtcaagacggccgtgtg-3'. The expression levels of RNA
transcripts were calculated with the comparative CT method. The individual RNA
levels were normalized for tubulin and depicted as relative expression levels
with the corresponding controls set to 1.
RNase protection assay (RPA)
Total RNA was isolated with the TRIzol protocol (Sigma) and 10 µg were
used for each RPA reaction. RPA was performed using the RiboQuant multiprobe
RNase protection assay system mJun/Fos (PharMingen) according to the
manufacturer's instructions.
Statistical analysis
All experiments were repeated at least three times and carried out in
triplicate. Statistical analysis was performed using Student's
t-test, P<0.05 was accepted as significant. Data are
shown as mean and the error bars represent the standard deviation.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Defective chondrocyte differentiation and extracellular matrix production
To further investigate the role of Fra2 on chondrocyte proliferation and
differentiation, the fate of BrdU-labeled cells was examined in the growth
plate. It has been shown that impaired chondrocyte differentiation leads, over
time, to an accumulation of BrdU-labeled cells in the proliferating zone
(Naski et al., 1998).
Interestingly, the percentage of labeled cells in the zone of proliferating
chondrocytes in Fosl2/ growth plates 32
hours after BrdU injection was sixfold higher than in littermate controls,
whereas no difference was observed 2 hours after BrdU injection
(Fig. 2A). Numbers of
Ki67-positive cells were unchanged during embryonic development
(Fig. 2B), and no premature or
increased apoptosis was observed in growth plates of
Fosl2/ embryos (data not shown). These
findings suggest that impaired chondrocyte differentiation might lead to the
reduced zones of hypertrophic chondrocytes in
Fosl2/ embryos and newborns. Impaired
chondrocyte differentiation into mature hypertrophic chondrocytes may cause
reduced ECM production in the epiphysis. At E15.5, we detected slightly
reduced calcified matrix using von Kossa staining in
Fosl2/ long bones and the difference became
more pronounced at E17.5, suggesting reduced extracellular matrix (ECM)
deposition in long bones of Fosl2/ embryos
compared with control littermates (Fig.
2C).
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Fra2 deficiency led to narrower hypertrophic zones in femoral and tibial
growth plates and this correlated with reduced expression of the chondrocyte
markers type X collagen and aggrecan in vivo. Impaired proliferation or
increased apoptosis of growth plate chondrocytes did not contribute to the
shortened hypertrophic zones in Fosl2/
embryos. As BrdU-labeled cells accumulated in proliferation zones of
Fosl2/ embryos in a time-dependent manner,
impaired differentiation into hypertrophic chondrocytes most probably caused
the shortened hypertrophic zones. Besides Fra2 expression in chondrocytes,
Fra2 has been shown to be expressed in dividing and apoptotic rat calvarial
osteoblasts, but is even higher expressed in differentiating osteoblasts
(McCabe et al., 1995) and
becomes the principal Fos protein in fully differentiated osteoblasts in vitro
(McCabe et al., 1996
). This
suggests a role for Fra2 in differentiation of mesenchyme-derived cells. We
could not observe any differences in expression of typical cartilage markers,
such as type II collagen, Runx2 and Ihh. Moreover, resting and proliferating
zones had unchanged sizes, indicating that Fra2 very specifically affects
chondrocyte differentiation. As Fosl2/
chondrocytes fail to differentiate properly, their matrix deposition was
reduced. This might impair the bone-forming activity, as less scaffold is
provided, leading to decreased bone mass. The function of Fra2 in the
interplay between cartilage and osteoblasts and osteoclasts remains to be
determined. Interestingly, the related Fos family member Fra1 has recently
been shown to also regulate bone mass through bone matrix production by
osteoblasts and chondrocytes (Eferl et al.,
2004
).
Primary cultures of Fosl2/ rib cage
chondrocytes displayed impaired differentiation in vitro, consistent with the
in vivo finding. In addition, Fosl2/
chondrocytes continued to proliferate and gained a large, flattened morphology
later when compared with wild-type cells. The in vitro differentiation defect
was accompanied by markedly reduced expression of the hypertrophic
chondrocyte-specific marker type X collagen and to a lesser extent aggrecan.
However, Fra2 does not bind to the AP1 site in the type X collagen promoter
(Harada et al., 1997) in EMSAs
(data not shown), suggesting that Fra2 might not directly regulate ECM protein
expression. Direct regulation of ECM proteins might be accomplished by Fos, as
it has been described to mediate PTH/PTHrP regulated type X collagen
expression in chicken (Ionescu et al.,
2001
; Riemer et al.,
2002
). Expression of other markers that affect chondrogenesis,
such as FGFR3, FGF18, FGF9, bone morphogenetic protein 4 (BMP4), PTHrP,
PTH/PTHrP receptor (PPR), Ihh, Runx2, alkaline phosphatase and TGFß was
found unchanged in vitro (data not shown). Fra2 seems to be an inducer of
hypertrophic differentiation and its absence might keep chondrocytes in a
proliferative state, which might contribute to reduced chondrocyte
differentiation in vivo and in vitro. Presumably, Fra2 can mediate signals
elicited by Ihh/PTHrP or FGF, which control cartilage differentiation
(Kronenberg and Chung, 2001
;
Coumoul and Deng, 2003
),
although the pathway and the target genes of Fra2 remain to be determined.
We also employed a conditional approach to investigate postnatal cartilage
development. Unfortunately, we found Fra2 deleted to some extent in most
organs of coll2a1-Cre, Fosl2f/f mice, indicating that this
Cre mouse strain is not chondrocyte-specific in postnatal tissues.
Nevertheless, we found deletion of Fra2 in the cartilage of the sternum and we
were able to describe a cartilage phenotype. Similar to
Fosl2/ embryos, coll2a1-Cre,
Fosl2f/f mice showed a reduced zone of hypertrophic
chondrocytes, but at later postnatal stages suffered from an abnormal
dorsoventral bending of the vertebral column (kyphosis). Interestingly, both
Fosl2/ and coll2a1-Cre,
Fosl2f/f mice are growth retarded after P1 but show no
growth defect during embryogenesis. The reduced hypertrophic differentiation
in Fra2-deficient cartilage is likely to account for the observed growth
retardation, as it has been described in other studies with defective
hypertrophic chondrocyte differentiation
(Horiki et al., 2004).
Chondrocyte proliferation appears to be insensitive to the effects of Fra2
deficiency during embryonic and early postnatal development, as we observed no
differences in Fosl2/, coll2a1-Cre,
Fosl2f/f and wild-type mice until P10. However, only 1-5%
of coll2a1-Cre, Fosl2f/f mice survive until weaning, and
we found reduced chondrocyte proliferation and a reduction in the size of the
zone of proliferating chondrocytes in femoral growth plates of these mice at
P23. Fra2 might therefore have no effect on chondrocyte proliferation during
early (pre-weaning), but a role in proliferation at later (post-weaning)
stages of bone development. This has also been reported for other factors
involved in bone development, e.g. FGFR3
(Naski et al., 1998) and
insulin-like growth factor 1 (IGF1) (Baker
et al., 1993
; Liu et al.,
1993
). However, the broad spectrum of Fra2 deletion in
coll2a1-Cre, Fosl2f/f mice suggests that the proliferation
defect in post-weaning mice might simply be secondary to the severe growth
retardation and to profound defects in various Fra2 deficient organs, which
most probably also accounts for the lethality.
In developing vertebral bodies of Fosl2/
embryos, endochondral ossification appears to be delayed for 1-2 days. At
E14.5, differentiation into hypertrophic chondrocytes, and at E16.5,
osteoblast invasion and matrix production are markedly reduced. Moreover, Fra2
deficiency in the spine of coll2a1-Cre, Fosl2f/f mice
leads to kyphosis, a skeletal malformation. An abnormal bending of the spine
was also observed when Jun was conditionally deleted using
coll2a1-Cre transgenic mice (Behrens et
al., 2003), suggesting that Fra2 and Jun might form the
predominant AP1 dimer in vertebral development. In contrast to
Fosl2/ spines, endochondral ossification is
not delayed in long bones and no malformations were observed in the
appendicular skeletons of Fosl2/ mice,
indicating different functions of Fra2 in the development of the appendicular
and axial skeleton.
In conclusion, mice lacking the transcription factor Fra2 display reduced chondrocyte differentiation throughout development, which leads to growth retardation postnatally, and delayed endochondral ossification of the spine. The identification of Fra2 target genes during chondrocyte differentiation may lead to a better understanding of pathways that coordinate cartilage development.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baker, J., Liu, J. P., Robertson, E. J. and Efstratiadis, A. (1993). Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75, 73-82.[Medline]
Behrens, A., Haigh, J., Mechta-Grigoriou, F., Nagy, A., Yaniv,
M. and Wagner, E. F. (2003). Impaired intervertebral disc
formation in the absence of Jun. Development
130,103
-109.
Carrasco, D. and Bravo, R. (1995). Tissue-specific expression of the fos-related transcription factor fra-2 during mouse development. Oncogene 16,1069 -1079.
Cohen, M. M., Jr (2002). Some chondrodysplasias with short limbs: molecular perspectives. Am. J. Med. Genet. 112,304 -313.[CrossRef][Medline]
Coumoul, X. and Deng, C. X. (2003). Roles of FGF receptors in mammalian development and congenital diseases. Birth Defects Res. Part C Embryo Today 69,286 -304.[CrossRef][Medline]
Deng, C., Wynshaw-Boris, A., Zhou, F., Kuo, A. and Leder, P. (1996). Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell 84,911 -921.[Medline]
Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L. and Karsenty, G. (1997). Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89,747 -754.[Medline]
Eferl, R., Hoebertz, A., Schilling, A. F., Rath, M., Karreth,
F., Kenner, L., Amling, M. and Wagner, E. F. (2004).
The Fos-related antigen Fra-1 is an activator of bone matrix formation.
EMBO J. 23,2789
-2799.
Fleischmann, A., Hafezi, F., Elliott, C., Reme, C. E., Ruther,
U. and Wagner, E. F. (2000). Fra-1 replaces
c-Fos-dependent functions in mice. Genes Dev.
14,2695
-2700.
Foletta, V. C., Sonobe, M. H., Suzuki, T., Endo, T., Iba, H. and Cohen, D. R. (1994). Cloning and characterisation of the mouse fra-2 gene. Oncogene 9,3305 -3311.[Medline]
Grigoriadis, A. E., Wang, Z. Q., Cecchini, M. G., Hofstetter, W., Felix, R., Fleisch, H. A. and Wagner, E. F. (1994). c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science 266,443 -448.[Medline]
Haigh, J. J., Gerber, H. P., Ferrara, N. and Wagner, E. F.
(2000). Conditional inactivation of VEGF-A in areas of
collagen2a1 expression results in embryonic lethality in the heterozygous
state. Development 127,1445
-1453.
Harada, S., Sampath, T. K., Aubin, J. E. and Rodan, G. A.
(1997). Osteogenic protein-1 up-regulation of the collagen X
promoter activity is mediated by a MEF-2-like sequence and requires an
adjacent AP-1 sequence. Mol. Endocrinol.
11,1832
-1845.
Hess, J., Hartenstein, B., Teurich, S., Schmidt, D.,
Schorpp-Kistner, M. and Angel, P. (2003). Defective
endochondral ossification in mice with strongly compromised expression of
JunB. J. Cell Sci. 116,4587
-4596.
Hilberg, F., Aguzzi, A., Howells, N. and Wagner, E. F. (1993). c-jun is essential for normal mouse development and hepatogenesis. Nature 365,179 -181.[CrossRef][Medline]
Horiki, M., Imamura, T., Okamoto, M., Hayashi, M., Murai, J.,
Myoui, A., Ochi, T., Miyazono, K., Yoshikawa, H. and Tsumaki, N.
(2004). Smad6/Smurf1 overexpression in cartilage delays
chondrocyte hypertrophy and causes dwarfism with osteopenia. J.
Cell Biol. 165,433
-445.
Ionescu, A. M., Schwarz, E. M., Vinson, C., Puzas, J. E.,
Rosier, R., Reynolds, P. R. and O'Keefe, R. J. (2001).
PTHrP modulates chondrocyte differentiation through AP-1 and CREB signaling.
J. Biol. Chem. 276,11639
-11647.
Jochum, W., David, J. P., Elliott, C., Wutz, A., Plenk, H., Jr, Matsuo, K. and Wagner, E. F. (2000). Increased bone formation and osteosclerosis in mice overexpressing the transcription factor Fra-1. Nat. Med. 6,980 -984.[CrossRef][Medline]
Jochum, W., Passegue, E. and Wagner, E. F. (2001). AP-1 in mouse development and tumorigenesis. Oncogene 20,2401 -2412.[CrossRef][Medline]
Johnson, R. S., van Lingen, B., Papaioannou, V. E. and Spiegelman, B. M. (1993). A null mutation at the c-jun locus causes embryonic lethality and retarded cell growth in culture. Genes Dev. 7,1309 -1317.[Abstract]
Karsenty, G. (1999). The genetic transformation
of bone biology. Genes Dev.
13,3037
-3051.
Karsenty, G. and Wagner, E. F. (2002). Reaching a genetic and molecular understanding of skeletal development. Dev. Cell 2,389 -406.[Medline]
Kenner, L., Hoebertz, A., Beil, T., Keon, N., Karreth, F.,
Eferl, R., Scheuch, H., Szremska, A., Amling, M., Schorpp-Kistner, M.
et al. (2004). Mice lacking JunB are osteopenic due to
cell-autonomous osteoblast and osteoclast defects. J. Cell
Biol. 164,613
-623.
Kobayashi, T., Chung, U. I., Schipani, E., Starbuck, M., Karsenty, G., Katagiri, T., Goad, D. L., Lanske, B. and Kronenberg, H. M. (2002). PTHrP and Indian hedgehog control differentiation of growth plate chondrocytes at multiple steps. Development 129,2977 -2986.[Medline]
Komori, T. (2002). Runx2, a multifunctional transcription factor in skeletal development. J. Cell Biochem. 87,1 -8.[Medline]
Kronenberg, H. M. and Chung, U. (2001). The parathyroid hormone-related protein and Indian hedgehog feedback loop in the growth plate. Novartis Found. Symp. 232,144 -152.[Medline]
Li, Y. and Olsen, B. R. (1997). Murine models of human genetic skeletal disorders. Matrix Biol. 16, 49-52.[CrossRef][Medline]
Liu, J. P., Baker, J., Perkins, A. S., Robertson, E. J. and Efstratiadis, A. (1993). Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75,59 -72.[Medline]
Liu, Z., Xu, J., Colvin, J. S. and Ornitz, D. M.
(2002). Coordination of chondrogenesis and osteogenesis by
fibroblast growth factor 18. Genes Dev.
16,859
-869.
McCabe, L. R., Kockx, M., Lian, J., Stein, J. and Stein, G. (1995). Selective expression of fos- and jun-related genes during osteoblast proliferation and differentiation. Exp. Cell Res. 218,255 -262.[CrossRef][Medline]
McCabe, L. R., Banerjee, C., Kundu, R., Harrison, R. J., Dobner, P. R., Stein, J. L., Lian, J. B. and Stein, G. S. (1996). Developmental expression and activities of specific fos and jun proteins are functionally related to osteoblast maturation: role of Fra-2 and Jun D during differentiation. Endocrinology 137,4398 -4408.[Abstract]
Murtaugh, L. C., Chyung, J. H. and Lassar, A. B.
(1999). Sonic hedgehog promotes somitic chondrogenesis by
altering the cellular response to BMP signaling. Genes
Dev. 13,225
-237.
Naski, M. C., Colvin, J. S., Coffin, J. D. and Ornitz, D. M.
(1998). Repression of hedgehog signaling and BMP4 expression in
growth plate cartilage by fibroblast growth factor receptor 3.
Development 125,4977
-4988.
Ohbayashi, N., Shibayama, M., Kurotaki, Y., Imanishi, M.,
Fujimori, T., Itoh, N. and Takada, S. (2002). FGF18 is
required for normal cell proliferation and differentiation during osteogenesis
and chondrogenesis. Genes Dev.
16,870
-879.
Pavlov, M. I., Sautier, J. M., Oboeuf, M., Asselin, A. and Berdal, A. (2003). Chondrogenic differentiation during midfacial development in the mouse: in vivo and in vitro studies. Biol. Cell. 95,75 -86.[CrossRef][Medline]
Riemer, S., Gebhard, S., Beier, F., Poschl, E. and von der Mark, K. (2002). Role of c-fos in the regulation of type X collagen gene expression by PTH and PTHrP: localization of a PTH/PTHrP-responsive region in the human COL10A1 enhancer. J. Cell Biochem. 86,688 -699.[CrossRef][Medline]
Ruther, U., Komitowski, D., Schubert, F. R. and Wagner, E. F. (1989). c-fos expression induces bone tumors in transgenic mice. Oncogene 4,861 -865.[Medline]
Sabatakos, G., Sims, N. A., Chen, J., Aoki, K., Kelz, M. B., Amling, M., Bouali, Y., Mukhopadhyay, K., Ford, K., Nestler, E. J. et al. (2000). Overexpression of DeltaFosB transcription factor(s) increases bone formation and inhibits adipogenesis. Nat. Med. 6,985 -990.[CrossRef][Medline]
Schorpp-Kistner, M., Wang, Z. Q., Angel, P. and Wagner, E.
F. (1999). JunB is essential for mammalian placentation.
EMBO J. 18,934
-948.
St-Jacques, B., Hammerschmidt, M. and McMahon, A. P.
(1999). Indian hedgehog signaling regulates proliferation and
differentiation of chondrocytes and is essential for bone formation.
Genes Dev. 13,2072
-2086.
Thomas, D. P., Sunters, A., Gentry, A. and Grigoriadis, A.
E. (2000). Inhibition of chondrocyte differentiation in vitro
by constitutive and inducible overexpression of the c-fos proto-oncogene.
J. Cell Sci. 113,439
-450.
Wang, Z. Q., Grigoriadis, A. E., Mohle-Steinlein, U. and Wagner, E. F. (1991). A novel target cell for c-fos-induced oncogenesis: development of chondrogenic tumours in embryonic stem cell chimeras. EMBO J. 10,2437 -2450.[Abstract]
Wang, Z. Q., Ovitt, C., Grigoriadis, A. E., Mohle-Steinlein, U., Ruther, U. and Wagner, E. F. (1992). Bone and haematopoietic defects in mice lacking c-fos. Nature 360,741 -745.[CrossRef][Medline]