1 The Neurosciences Institute 10640 John Jay Hopkins Drive, San Diego, CA 92121,
USA
2 Department of Neurobiology, The Scripps Research Institute 10550 North Torrey
Pines Road, La Jolla, CA 92037, USA
* Author for correspondence (e-mail: makarenkova{at}nsi.edu)
Accepted 3 March 2005
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SUMMARY |
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Key words: Barx2, GDF5, Limb development, Adhesion, Chondrogenesis, Joint, BMP, Mouse
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Introduction |
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Several studies indicate that chondrogenesis comprises three main steps:
chondrogenic lineage commitment, mesenchymal cell condensation and
differentiation into cartilage. During the condensation phase, mesenchymal
cells aggregate to form chondrogenic and non-chondrogenic cell populations.
This step involves differential regulation and synthesis of specific adhesion
molecules that change adhesive properties and mediate cell sorting
(Cottrill et al., 1987;
Ide et al., 1994
;
Tavella et al., 1994
). After
mesenchymal condensation is complete, the prechondrogenic cells differentiate
further to produce specific extracellular matrix components, such as collagen
II and aggrecan, both of which are classic markers of chondrogenesis.
Many different signaling molecules are involved in regulation of
chondrogenesis; however, bone morphogenetic proteins (BMPs) and members of the
Sox and homeobox transcription factor families play central roles
(Bi et al., 1999;
Yi et al., 2000
;
Zhao et al., 1997
). In
developing limbs, BMPs and BMP-related molecules, such as the growth and
differentiation factors (GDFs), have been implicated in the establishment of
limb axes, chondrogenesis, osteogenesis and tissue patterning by apoptosis
(Hoffmann and Gross, 2001
;
Macias et al., 1997
;
Niswander, 2002
;
Pizette et al., 2001
;
Tang et al., 2000
;
Yi et al., 2000
). BMPs induce
the expression of specific markers of chondrogenesis
(Enomoto-Iwamoto et al., 1998
;
Shea et al., 2003
;
Tsumaki et al., 2002
) via a
transcriptional cascade that often involves other regulators such as Sox and
homeobox proteins (Baur et al.,
2000
; Boulet and Capecchi,
2004
; Zhang et al.,
2000
).
Sox proteins belong to the high-mobility group (HMG) DNA-binding family of
transcription factors. Members of this family bind to and activate
chondrocyte-specific enhancers in genes encoding the various collagens
(Bi et al., 1999;
Lefebvre et al., 1996
;
Zhou et al., 1998
). In
particular, Sox9, the first transcription factor to specify the chondrogenic
lineage, plays a crucial role in chondrogenesis through activation of the
collagen II (Col2a1) and collagen alpha2(XI) genes (Col11a2)
(Bi et al., 1999
).
The first intron of the rat Col2a1 gene contains a 620 bp
chondrocyte specific regulatory enhancer
(Horton et al., 1987) and two
shorter overlapping fragments of this enhancer direct chondrocyte-specific
expression in transgenic mice (Bell et al.,
1997
; Zhou et al.,
1995
). Moreover, a 48 bp sequence within the region of overlap
between these two fragments that contains three HMG motifs is sufficient to
confer chondrocyte-specific expression in cell lines
(Lefebvre et al., 1996
;
Zhou et al., 1998
). HMG motifs
bind to members of the Sox family and various Sox proteins, including Sox9,
can activate this enhancer (Zhou et al.,
1998
). However, multiple copies of the 48 bp sequence are required
to give the same level and pattern of expression as the larger Col2a1
enhancer fragment in cell lines and transgenic mice
(Lefebvre et al., 1996
),
suggesting that other regions of the Col2a1 enhancer or promoter may
contribute significantly to the level and pattern of Col2a1
expression in vivo.
The Col2a1 gene contains many other potential transcription factor
recognition motifs in addition to those for Sox proteins
(Ala-Kokko et al., 1995;
Ghayor et al., 2000
;
Huang et al., 2002
;
Kamachi et al., 1999
;
Murray et al., 2000
). However,
the identities most of the factors that bind to these motifs and their
possible interactions with Sox proteins have not been elucidated. It is known
that homeobox transcription factors, including members of the Hox, Msx and Dlx
families, coordinate the expression of genes that are essential for
differentiation of skeletal elements
(Ferrari et al., 1994
;
Ferrari et al., 1995
;
Rogina et al., 1992
;
Satokata et al., 2000
).
Several lines of evidence also indicate that homeobox proteins are important
transducers of BMP signaling pathways during chondrogenesis
(Xu et al., 2001
). For
example, activation of the collagen II gene (Col2a1) enhancer in a
chondroblast cell line by BMP2 is eliminated by antisense oligonucleotides
against the mRNA encoding the homeobox factor Dlx2, suggesting that Dlx2 acts
downstream of BMP signals (Xu et al.,
2001
). Whether homeobox transcription factors act cooperatively or
in parallel pathways with Sox proteins remains unknown.
The homeobox transcription factor Barx2 regulates the expression of cell
adhesion molecules (CAMs) including NCAM
(Edelman et al., 2000a;
Meech et al., 1999
;
Meech et al., 2003
) and
cadherin 6 (Sellar et al.,
2001
), suggesting that it can influence processes such as cell
aggregation, formation of intercellular contacts and cell fusion. Our previous
work also indicates that Barx2 is involved in limb development. For example,
we found that Barx2 is required for myotube formation in limb bud cultures and
that overexpression of Barx2 accelerates the fusion of both C2C12 and
embryonic limb myoblasts (Meech et al.,
2003
).
In this study, we report that Barx2 is expressed during limb development in patterns that suggest a role in chondrogenesis. Barx2 is necessary for the formation of primary mesenchymal aggregations and for cartilage differentiation in limb bud cultures, and it regulates the expression of several genes encoding CAMs and extracellular matrix proteins, including NCAM, tenascin C and collagen II. We identify two conserved binding sites for Barx2 within the Col2a1 intronic enhancer and show that addition of Sox9 antibodies, or disruption of an adjacent HMG-box, reduces Barx2 binding, suggesting cooperation between Barx2 and Sox9. In addition, we show that the BMP family members BMP4 and GDF5 regulate Barx2 expression in the developing limb. Overall, these data suggest that Barx2 provides a crucial link between BMP signaling and mesenchymal condensation and differentiation, and that it acts concert with other BMP targets such as Sox9 to directly regulate the expression of chondrogenic genes.
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Materials and methods |
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Cell adhesion assays
D1 cells (ATCC, CRL-12424) were transfected with pcDNA3 or with pcDNA3
containing a mouse full-length Barx2 cDNA and cell aggregation assays were
performed (Kawano et al.,
2002). Briefly, for the Ca+2-dependent assays, cells
were incubated for 20 minutes at 37°C in Ca+2,
Mg+2-free HEPES-buffered saline (HCMF) containing 0.01% trypsin
(type XI, Sigma) and 10 mM CaCl2. Cells were washed and resuspended
at a density of 2x105 cells/ml in DMEM-Hanks solution with 1
mM CaCl2. Cells were transferred to 24-well plates coated with 1%
bovine serum albumin (Sigma) and incubated for 15-30 minutes at 37°C with
constant rotation at 40 rpm. To evaluate cell aggregation, cells were compared
at 0 and 30 minutes incubation. For the Ca+2-independent assay
cells were treated for 20 minutes with 2mM EDTA in HCMF, then dissociated into
a single cell suspension in Hanks solution with 1 mM EDTA and 2% FBS. Cell
aggregation assay was performed as described above. The extent of aggregation
was determined by the measuring the appearance of aggregates larger than 30
µm using a Beckman Coulter Counter (Fullerton, CA).
Antisense inhibition of Barx2 expression in limb bud micromass cultures
To examine the role of Barx2 in mesenchymal condensation and
chondrogenesis, we used morpholino oligodeoxynucleotides (ODNs)
(Heasman, 2002;
Summerton, 1999
;
Summerton and Weller, 1997
).
Antisense and control Barx2 ODNs were synthesized by Gene Tools (Corvalis,
OR). ODNs (2-5 µM) were added to micromass cultures using the osmotic
delivery system recommended by the manufacturer or using Lipofectamine 2000
(Invitrogen). Cultures were maintained for 48-96 hours and ODNs were replaced
every 6-8 hours to increase the efficiency of antisense treatment. After 48
hours, the number of mesenchymal aggregates was assessed and 24-48 hours
later, cultures were fixed in 2% PFA in PBS with 0.05% Triton X-100, and
stained with Alcian Blue to assess the extent of chondrogenesis.
Retroviral construction and packaging
Retroviral vectors were constructed that contained the enhanced green
fluorescent protein (EGFP) gene, full-length mouse Barx2 cDNA, or cDNA
fragments that expressed the following combinations of Barx2 protein domains:
the homeodomain and Barx basic region (HD-BBR); or the homeodomain, Barx basic
region and C-terminal activation domain (HD-BBR-C)
(Edelman et al., 2000a). The
retroviral vector was based on the murine embryonic stem cell (MESV) virus
with modifications (Owens et al.,
2002
). Retroviral particles were packaged in COS1 cells
(Edelman et al., 2000b
).
Supernatant containing retroviral particles was collected after 48 hours,
filtered and used to infect primary cells in suspension.
Retroviral transduction in micromass cultures
Retroviral infection of micromass cultures was carried out as described
(Stott and Chuong, 1997).
Infected cells were cultured for an additional 2-4 days, fixed in 4%
paraformaldehyde (PFA) in PBT (PBS with 0.05% Triton) and processed for
immunostaining or stained with Alcian Blue to quantify levels of
chondrogenesis and to examine nodule formation. At least five micromass
cultures infected with each retroviral construct were analyzed for each
experiment. Each experiment was repeated three or four times, yielding similar
results.
Alcian blue staining and quantitation of chondrogenesis
Micromass cultures were fixed with 2% PFA, washed in PBT and stained with
1% Alcian Blue 8GX (Sigma) in 0.1 N HCl, pH 1 for 5 hours
(Lev and Spicer, 1964).
Cultures were then de-stained with 70% ethanol. Alcian Blue incorporated into
the cell matrix was extracted with 0.5 ml of 4 M guanidine HCl (pH 5.8), and
quantified by measuring absorbance at OD600 nm
(Lev and Spicer, 1964
). The
statistical significance of the difference in Alcian Blue staining between
control and experimental micromass cultures after antisense treatment or
retroviral delivery of Barx2 constructs was assessed using the nonparametric
Wilcoxon signed rank test (Ostle, 1975). A value of P<0.01 was
considered to reflect a statistically significant difference.
Whole limb cultures
Limbs were dissected from E11 and E12.5 embryos, placed on a 0.8 µm
Millipore filter supported by a metal grid, and cultured in Fitton-Jackson
modified BGJb medium (Invitrogen) supplemented with glutamax, human transferin
40 µg/ml, 1x insulin/transferin selenium, and albumax I 50 µg/ml
(Invitrogen). The limbs were incubated at 37°C, in 5% CO2 and
the culture medium was changed daily.
Bead implantation
Heparin acrylic beads (150-200 µm, Sigma) were washed in PBS and soaked
in recombinant human BMP4 (Genetics Institute) or recombinant mouse GDF5
(#853-G5, R&D Systems) at concentrations of either 200 or 500 µg/ml for
3 hours at 4°C. Control beads were soaked in bovine serum albumin (BSA) in
PBS. BMP4-, GDF5- or BSA-loaded beads were implanted in the distal
interdigital mesenchyme of mouse fore- and hindlimbs at E12.5. Limbs with
implanted beads were cultured for 48 hours, fixed in 4% PFA and processed for
in situ hybridization or immunohistochemistry.
Histology and immunohistochemistry
Frozen sections and micromass cultures were stained as described previously
(Makarenkova et al., 1997)
using the following antibodies: mouse monoclonal antibody to collagen II
(Abcam, Clone 5B2.5, AB 3092); rabbit polyclonal antibody to Barx2 (Santa Cruz
Biotechnologies, M-186, sc-9128); and rabbit polyclonal antibody to collagen
type II (Chemicon, ab-2031). Alexa- or rhodamine-conjugated antibodies
(Molecular Probes) were used as secondary antibody. Nuclei were stained with
Oli-Green. ProLong anti-fade reagent was used to reduce sample fading
(Molecular Probes).
In situ hybridization
For in situ hybridization, embryos were fixed overnight in 4% PFA at
4°C. Antisense RNA probes were labeled with digoxigenin and whole-mount in
situ hybridization was performed as described
(Nieto et al., 1996). The
mouse GDF5 probe was generated from a 493 bp RT-PCR product (bases 437-930 of
the mouse GDF5 coding region) and was cloned into pCRTMII-TOPO
(Invitrogen). The mouse Barx2 probe contained a 431 bp
EcoRI-PstI fragment from within the Barx2 coding region
(Jones et al., 1997
).
Limb bud nuclear extract preparation and gel mobility-shift assays
Double-stranded oligonucleotide probes were prepared that correspond to the
HBS and HMG motifs within the Col2a1 intronic enhancer
(Lefebvre et al., 1996).
Probes containing point mutations were also generated (see
Fig. 7A,B). Limb nuclear
extract was prepared from E12.5 embryonic limbs as described previously
(Schreiber et al., 1989
). The
gel mobility-shift assays were performed as described
(Edelman et al., 2000a
).
DNA/protein complexes were then resolved by electrophoresis on an 8% native
polyacrylamide gel at 4°C. Gels were dried and visualized using a
PhosphorImager (Molecular Dynamics).
|
RT-PCR analysis of micromass cultures
Micromass cultures infected with EGFP- and HDBBRC-expressing retroviruses
were harvested after 4 days and total RNA was prepared using Trizol (Gibco).
Each RNA (5 µg) sample was DNAse treated with DNA-free (Ambion), and
reverse transcribed using Superscript reverse transcriptase (Invitrogen) and
random hexamer primers. PCR amplification was performed using a Lightcycler
(Roche) and the Roche HotStart Master SYBR-green kit following the
manufacturer's instructions. Primer sequences are as follows: mouse
Col2a1, forward 5'-GAACCCAGAAACAACACAATCC-3' and reverse
5'-GTTCGGACTTTTCTCCCCTC-3'; mouse cyclophillin, forward
5'-CCAAAGACCACATGCTTGCCATCC-3' and reverse
5'-TGGTCAACCCCACCGTGTTCTTCG-3'.
The relative abundance of the cDNAs representing the Col2a1 and cyclophillin transcripts were determined using a standard curve for each primer set as described (Stevens, 2004). The concentration of Col2a1 cDNA was normalized to the concentration of the housekeeping gene cyclophillin cDNA in each same. To determine the influence of retroviral treatment on the expression of Col2a1, the relative expression of Col2a1 in the HDBBRC-retrovirus treated sample was divided by the relative expression of Col2a1 in the control-retrovirus treated samples. Mean and s.e.m. values were determined from three independent retroviral-transduction experiments. A melting curve analysis was performed for each sample after PCR amplification to ensure that a single amplification product was obtained.
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Results |
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Inhibition of Barx2 expression by morpholino antisense oligonucleotides blocks mesenchymal aggregation and chondrogenic differentiation of limb bud micromass cultures
To determine whether Barx2 functions during primary mesenchymal
condensation and chondrogenic differentiation, its expression was inhibited in
limb bud micromass cultures using morpholino-modified antisense Barx2
oligonucleotides (ODNs). Treatment of micromass cultures with 5 µM Barx2
antisense ODNs (Fig. 3A)
significantly reduced Alcian Blue staining of cartilaginous matrix, whereas
similar treatment with sense and control ODNs showed little or no change. To
quantify the inhibition of chondrogenesis by antisense Barx2 ODNs, the Alcian
Blue that had incorporated into micromass cultures was extracted and
quantified in five separate experiments. As shown in
Fig. 3B, Barx2 antisense ODNs
reduced Alcian Blue staining by 90% relative to control cultures. These
results indicate that expression of Barx2 is required for chondrogenic
differentiation of primary limb bud micromass cultures.
|
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Barx2 does not promote chondrogenesis of mesenchymal cells from proximal regions of the limb bud
During limb development, specification of proximal mesenchyme precedes the
specification of distal mesenchyme, which is in contact with the apical
ectodermal ridge (AER) (Dudley et al.,
2002; Muneoka et al.,
1989
; Vargesson et al.,
1997
). To determine whether Barx2 could influence the
differentiation of proximal limb bud mesenchyme, we compared the effect of
Barx2 expression on chondrogenic differentiation in micromass cultures
prepared from the proximal and distal regions of the limb bud at E11.0. As
shown in Fig. 5D, none of the
Barx2 constructs induced chondrogenesis in micromass cultures prepared from
the proximal region of the limb bud, whereas micromass cultures made from the
distal mesenchyme of the same limb bud showed a fivefold increase in
chondrogenesis after overexpression of Barx2. These results suggest that Barx2
can promote chondrogenesis only in the distal mesenchyme of the limb bud,
which contains mostly uncommitted cells
(Dudley et al., 2002
).
Barx2 regulates expression of Collagen II in limb bud micromass cultures and binds to regulatory sequences of the Col2a1 enhancer
The results described above show that ectopic expression of Barx2 in limb
bud micromass cultures accelerates chondrogenic differentiation. To determine
whether this effect involves regulation of the Col2a1 gene, we first
examined Col2a1 mRNA levels. There was a nearly fivefold upregulation
of Col2a1 mRNA levels (P<0.05; n=4) in Barx2
retrovirus infected cultures, relative to controls
(Fig. 6A), suggesting that
Barx2 can activate the Col2a1 gene. In addition, immunostaining of
limb bud cultures treated with Barx2 retroviruses also showed an increase in
collagen II protein expression compared with control
(Fig. 7B).
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|
The HMG site is important for binding Col2a enhancer by Barx2 and other homeodomain proteins
The consensus binding sequence for Sox proteins is (A/T)(A/T)-CAA(A/T)G
(Lefebvre et al., 2001;
Lefebvre et al., 1998
;
Zhao et al., 1997
). The
Col2a enhancer contains several canonical HMG-box elements that are
conserved in the human, mouse and rat Col2a sequences
(Fig. 7A). The HMG3
(Zhou et al., 1998
) and HMG4
(Bell et al., 1997
) elements
have been shown to interact specifically with Sox9. We identified another
conserved element that matched the HMG-box consensus motif that we refer to as
HMG5 (Fig. 7A). HMG5 is
immediately adjacent to the HBS within the B1 probe, suggesting that it might
influence binding of the probe to Barx2. Mutation of the HMG5 motif within the
B1 probe reduced binding to embryonic limb nuclear extract compared with the
intact B1 probe (Fig. 8C),
suggesting that the HMG motif influences binding of Barx2 to this sequence and
raising the possibility that Barx2 and other homeodomain proteins may bind
cooperatively with Sox proteins to Col2a1 regulatory regions.
To determine whether the DNA-protein complexes formed with the intact B1
probe contain Sox9, we tested the ability of Sox9 antibodies to block the
formation of these complexes. A control probe (S1) was generated containing
the HMG2 and HMG3 motifs that bind to Sox9. Nuclear extracts from embryonic
limb formed several complexes with the S1 probe
(Fig. 8B) in accordance with
previous observations (Lefebvre et al.,
1998). Addition of 2 µg of Sox9 antibody reduced the intensity
of these complexes by
50%, indicating that the antibody can partially
block the binding of Sox9 to the S1 probe
(Fig. 8B). Significantly,
addition of 2 µg of Sox9 antibody also reduced the intensity of the complex
formed with the B1 probe by
50% (Fig.
8B); a reduction similar to that caused by addition of Barx2
antibody (Fig. 8B). By
contrast, the complex formed with the B2 probe was reduced by the addition of
Barx2 antibody, but not by the addition Sox9 antibody
(Fig. 8B). These data suggest
that Sox9 and Barx2 bind cooperatively to adjacent sites in the
Col2a1 enhancer.
Barx2 and Sox9 occupy Col2a1 intronic enhancer during limb chondrogenesis
Our studies indicate that Barx2 can regulate collagen II expression in the
limb mesenchymal cells. We also found that Barx2 can bind to regulatory
element in the Col2a1 enhancer in vitro. To test whether Barx2 might
be actively engaged at the Col2a1 enhancer in vivo, we performed ChIP
assays. Equivalent amount of crosslinked chromatin from limb mesenchymal cells
was immunoprecipitated with Barx2 antibody (Santa Cruz Biotechnology) or with
an irrelevant antibody or rabbit IgG (negative control). The precipitated DNA
then was subjected to PCR amplification using primers that span the region of
Col2a1 enhancer containing putative Barx2-binding site. Antibodies to
Barx2 immunoprecipitated this region of Col2a1 enhancer from limb
mesenchymal cells (Fig. 9A),
whereas the irrelevant antibody or normal rabbit IgG did not
(Fig. 9A, lane 3). This
indicates that Barx2 binds to the enhancer in vivo. In addition, we examined
whether BMPs, well-known regulators of chondrogenesis, can modulate Barx2
binding to the Col2a1 enhancer. Replicate limb micromass cultures
were prepared and treated with BMP4 (200 ng/ml) or BSA and the ChIP assay was
performed. The amount of Col2a1 enhancer DNA precipitated by Barx2
antibodies was greater after BMP treatment
(Fig. 9B). These results
indicate that Barx2 might be an important downstream mediator of BMP signaling
during chondrogenesis. To verify the specificity of Barx2 association with the
Col2a1 enhancer region, we performed a ChIP assay with D1 and C3H10
T1/2 cells transiently transfected with Myc-tagged Barx2 and cultured under
differentiation conditions. The Myc antibody was used to precipitate Barx2. As
shown in Fig. 9A, Barx2 binds
the core regulatory region of the Col2a1 enhancer, while binding was
not observed with rabbit IgG. In addition, we tested whether Sox9 can occupy
the same region of the Col2a enhancer. An equivalent amount of
chromatin from D1 cells co-transfected with Sox9-flag and Barx2-Myc expression
vectors was immunoprecipitated in parallel with Flag, Phospho-Sox9 and Barx2
antibodies. As shown in Fig.
10C, Barx2 binds the regulatory region of Col2a1
enhancer. Both Flag and Phospho-Sox9 antibodies precipitated the
Col2a1 enhancer sequence (Fig.
9D), showing that Sox9 also binds this region of the
Col2a1 gene during chondrogenesis.
|
|
GDF5 and BMP4 application induce ectopic Barx2 expression in embryonic limbs
The implied role of BMP signaling in chondrogenesis and the observation
that Barx2 and GDF5 are co-expressed during limb development prompted us to
explore a functional connection between Barx2 and BMP signaling. Heparin
acrylic beads were soaked in BSA (0.1%), BMP4 or GDF5 (200 and 500 µg/ml),
and implanted into the distal interdigital region of E12.5 mouse fore- and
hindlimbs. Limbs were cultured for an additional 48 hours and were examined
for Barx2 expression by in situ hybridization or immunohistochemistry. Limbs
implanted with BSA-soaked beads showed no changes in the expression of Barx2
mRNA or protein (Fig. 10A,D).
By contrast, application of GDF5 or BMP4 beads induced ectopic expression of
Barx2 mRNA and protein within the distal region of the limb bud
(Fig. 10B,C,E,F). Barx2
expression was not detected in cells immediately adjacent to the GDF5 or
BMP-soaked beads, but was observed only at a defined distance (200 µm)
from the bead, implying that a particular concentration of GDF5 or BMP4 may be
required to induce the expression of Barx2. There was also a difference in the
response of fore- and hindlimbs to GDF5 and BMP4
(Table 1). A higher proportion
of limbs exhibiting induced expression of Barx2 were obtained when GDF5 or
BMP4 beads were implanted in the hindlimb versus the forelimb (89% versus 40%)
(Table 1). These results are
consistent with the idea that the forelimb is more developmentally advanced
than the hindlimb at this stage and thus less responsive to developmental
signals. Taken together, these data suggest that GDF5 and BMP4 regulate Barx2
expression in limb bud mesenchyme in a stage-dependent manner.
|
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Discussion |
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Barx2 regulates mesenchymal condensation and chondrogenic differentiation during limb development
Our experiments indicate that Barx2 is required for adhesion and
aggregation of mesenchymal cells (Fig.
11). Several adhesion molecules, including NCAM and cadherins,
particularly N-cadherin, have been implicated in the formation of mesenchymal
aggregations, initiation of chondrogenesis and limb patterning
(Tavella et al., 1994;
Widelitz et al., 1993
;
Yajima et al., 2002
). Our
previous experiments showed that NCAM, which mediates
Ca+2-independent cell adhesion, is regulated by Barx2
(Edelman et al., 2000a
).
However, although inhibition of Barx2 blocks condensation of mesenchymal
cultures, condensation and chondrogenesis are not disrupted in NCAM knockout
mice (Fang and Hall, 1999
),
suggesting that Barx2 must regulate additional CAMs or cadherins. The finding
that Barx2 is involved in regulation of Ca+2-dependant cell
adhesion suggests that Barx2 can also modulate cadherin expression. This
possibility is supported by a study showing that Barx2 regulates cadherin 6 in
ovarian carcinoma cells (Sellar et al.,
2001
). However, further experiments are required to determine
which cadherins are regulated by Barx2 in developing limb.
|
Barx2 and Sox9 regulate Col2a1 gene expression during chondrogenesis
Overexpression of Barx2 in micromass cultures increased Alcian Blue
staining and nodule formation, suggesting that Barx2 controls chondrogenic
differentiation. It is a formal possibility that Barx2 promotes chondrogenesis
by inducing prechondrogenic condensation alone. However, our results suggest
that Barx2 induces expression of the major cartilage matrix protein collagen
II. We found that Barx2 binds to two different conserved HBS motifs in the
cartilage-specific Col2a1 enhancer region, and that overexpression of
Barx2 in the limb mesenchymal progenitor cells can activate Col2a1
gene expression. Previous studies showed that Sox9 activates this same
enhancer, and identified at least two Sox9-specific HMG motifs
(Bell et al., 1997;
Zhou et al., 1998
). We
identified a fifth conserved element that matches the HMG-box consensus motif
(HMG5), adjacent to one of the Barx2-binding sites (HBS1). We also found that
Barx2 and Sox9 can occupy the same regulatory element of Col2a1 enhancer
during chondrogenic differentiation. This and our other experiments showing
that binding of limb nuclear proteins to the HBS1 element was reduced by
mutation of this adjacent HMG motif and partially disrupted by addition of
Sox9 antibodies, strongly suggests a functional interaction between Barx2 and
Sox9.
GDF5 and BMP signaling regulates Barx2 expression during limb development
Previous and current studies indicate that both Barx2 and the closely
related factor Barx1 are regulated by BMPs. We showed that BMP4 and GDF5 can
induce ectopic expression of Barx2 in developing limbs and promote binding of
Barx2 to Col2a1 enhancer during chondrogenesis. By contrast, BMP4 inhibits
expression of Barx1 and restricts its expression to the proximal, presumptive
molar mesenchyme of mouse embryo (Barlow et
al., 1999; Tucker et al.,
1998
). This difference in responses to BMPs might be due to
activation of various downstream signaling pathways in each cellular context
or to interaction with other developmental signals
(Yoon and Lyons, 2004
). BMP
signals are mainly mediated through ligand binding to receptors followed by
activation of Smad proteins (Chen et al.,
2004
; Nishimura et al.,
2003
; Nohe et al.,
2004
). Recent studies have shown a distinct, structurally related
class of SMADs which inhibits, rather than induces, TGFß family signals
(Christian and Nakayama, 1999
;
Nakayama et al., 1998
).
BMPs are crucial regulators of chondrogenesis that increase condensation of
limb mesenchyme and directly induce chondrogenic genes, including Sox9
(Zehentner et al., 1999).
Moreover, mice carrying different combinations of mutations in the genes
encoding BMPs, GDF5 and their receptors have more severe defects in limb
development than mice carrying single mutations in these genes
(Tsumaki et al., 2002
;
Vortkamp, 1997
;
Yi et al., 2000
). This
suggests that multiple BMPs and GDFs may have redundant or synergistic
functions in the regulation of chondrogenesis and skeletal development.
In our experiments, we found that both BMP4 and GDF5 could induce Barx2
expression in cultured mouse limbs, indicating that various BMPs might
regulate Barx2 expression at different stages of limb development. GDF5 has
been reported to regulate both cellular condensation and chondrogenic
differentiation in cultured limbs and in micromass cultures
(Akiyama et al., 2000;
Buxton et al., 2001
;
Francis-West et al., 1999
;
Hatakeyama et al., 2004
;
Spiro et al., 2001
). BMPs
(BMP2 and BMP4), however, appear to be crucial for later stages of
skeletogenesis involving chondrogenic differentiation and skeletal patterning
(Kameda et al., 2000
;
Tsumaki et al., 2002
). Hence,
GDF5 can induce chondrogenesis in mesenchymal cells that have not yet
condensed, while BMPs induce chondrogenic differentiation only after
condensation (Fig. 11). This
conclusion is reinforced by observations that the effectiveness of BMPs in
inducing chondrogenic differentiation in mesenchymal micromass cultures is
greater in high-density cultures or after induction of cell-cell interactions
(Denker et al., 1999
). Based
on these data and our own observations, we propose that GDF5 and BMPs act in a
sequential manner to regulate Barx2 during mesenchymal condensation and
chondrogenesis (Fig. 11).
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ACKNOWLEDGMENTS |
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REFERENCES |
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