1 GSF-National Research Center for Environment and Health, Institute of
Developmental Genetics, Ingolstädter Landstrasse 1, 85764 Neuherberg,
Germany
2 MRC Human Genetics Unit, Western General Hospital, Crewe Rd, Edinburgh EH4
2XU, UK
3 German Research Centre for Biotechnology, Mascheroder Weg 1, 38124
Braunschweig, Germany
4 University of East Anglia, School of Biological Sciences, Norwich NR4 7TJ,
UK
* Present address: Instituto Cajal, CSIC, Dr Arce 37, 28002 Madrid, Spain
Author for correspondence (e-mail:
imai{at}gsf.de)
Accepted 31 October 2002
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SUMMARY |
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Key words: Pax1, Pax9, Bapx1, Shh, Sclerotome, Chondrogenesis
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INTRODUCTION |
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Pax1 and Pax9 play a critical role in the formation of the axial skeleton.
They belong to the Pax family of transcription factors, characterized by the
presence of a conserved DNA binding domain: the paired box. Pax genes were
originally identified in Drosophila, but later they have been found
in numerous organisms where they play important roles in embryonic patterning
and organogenesis. Among them, Pax1 and Pax9 constitute a paralogous group
characterized by the presence of the paired-box and the octapeptide domain,
and the absence of a homeobox (Strachan
and Read, 1994).
Pax1 and Pax9 show a similar but not identical expression
pattern during mouse development. They are expressed in the developing
sclerotome, where no other Pax genes are expressed. Pax1 is first
expressed in nascent somites shortly before de-epithelialization, by cells
located in the ventromedial part, which marks the emerging sclerotome
population. Pax1 is initially expressed in all sclerotomal cells, but
later its expression becomes stronger in the posterior ventromedial
compartment. Subsequently, Pax9 is expressed in the posterior
ventrolateral compartment of the sclerotome. Thus, Pax1 is
predominantly expressed in the region of the future vertebral bodies and
intervertebral discs, whereas the Pax9 expression domain extends more
laterally in the region of the future neural arches and the proximal part of
the ribs (Deutsch et al.,
1988; Neubüser et al.,
1995
).
Pax1 is required for the proper formation of ventral structures of
the vertebral column (Balling et al.,
1988; Wallin et al.,
1994
; Wilm et al.,
1998
). Although Pax9-deficient mutant mice have no
apparent malformations in the axial skeleton
(Peters et al., 1998
),
Pax1;Pax9-double mutants show a dramatic increase in the severity of
the vertebral defects when compared with Pax1-deficient mutants. In
the absence of both Pax1 and Pax9, the ventromedial
structures of the vertebral column are not formed
(Peters et al., 1999
). Thus,
there is a functional redundancy between the two Pax genes during vertebral
column development. The sclerotomal cells of embryos deficient for both
Pax1 and Pax9 do not properly condense around the notochord
and cannot initiate chondrogenesis. There is a decrease in the proliferation
rate of the sclerotomal population in Pax1;Pax9-double mutants,
followed by an increase in apoptosis
(Peters et al., 1999
).
The secreted molecule Sonic hedgehog (Shh), which is produced by the
notochord and the floor plate, plays pivotal roles in the dorsoventral
patterning of somites and in the survival of sclerotomal cells
(Fan and Tessier-Lavigne,
1994; Johnson et al.,
1994
; Chiang et al.,
1996
). Sclerotomal expression of several genes including
Pax1 and Pax9 is known to be dependent on Shh
(Brand-Saberi et al., 1993
;
Dietrich et al., 1993
;
Koseki et al., 1993
;
Neubüser et al.,
1995
).
Bapx1 (also known as Nkx3-2) and Nkx3-1 genes
are the mouse homologs of Drosophila Nk-3/bagpipe (bap), a mesodermal
gene essential for the formation of the visceral musculature
(Azpiazu and Frasch, 1993).
They are expressed in the sclerotome
(Tribioli et al., 1997
;
Tanaka et al., 1999
;
Kos et al., 1998
) in a
Shh-dependent manner (Murtaugh et al.,
2001
; Kos et al.,
1998
). Nkx3-1-deficient mice show no sclerotomal defects
(Bhatia-Gaur et al., 1999
;
Schneider et al., 2000
).
Interestingly, mouse mutants lacking Bapx1 show a vertebral phenotype
strikingly similar to that of mice mutant for both Pax1 and
Pax9 (Lettice et al.,
1999
; Tribioli and Lufkin,
1999
; Akazawa et al.,
2000
). Overexpression experiments in the chick have recently
demonstrated the role of Bapx1 in chondrogenic differentiation of
sclerotomal cells in response to Shh
(Murtaugh et al., 2001
). As
neither Pax1 nor Pax9 expression is altered in
Bapx1 mutants (Lettice et al.,
1999
; Tribioli and Lufkin,
1999
; Akazawa et al.,
2000
), the possibility exists that Bapx1 might be a
downstream target of Pax1 and Pax9.
In this study, this possibility was tested by a combination of genetic and molecular approaches. We show that Bapx1 expression in the sclerotome is dependent on the two Pax genes. We also show that Pax1 and Pax9 are not only necessary, but also sufficient for Bapx1 expression and for the induction of sclerotome chondrogenesis. Furthermore, we have obtained strong evidence that Bapx1 is a direct target of Pax1 and Pax9 in the sclerotome. Taken together, we conclude that Pax1 and Pax9, as main mediators of Shh signaling, are essential and sufficient for the induction of sclerotome chondrogenesis.
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MATERIALS AND METHODS |
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Retroviral vectors
pRCAS(A)-Pax1
The coding region of mouse Pax1 was excised from pPax1 (see below)
with NcoI/EcoRI and subcloned in
NcoI/EcoRI-digested pSlax13 shuttle vector (a gift from C.
Tabin) (Logan and Tabin, 1998)
to create pSlax-Pax1. To engineer an HA N-terminal epitope, the following two
oligonucleotides were annealed to create an HA linker with NcoI
cohesive ends: 5'-CATGTACCCATACGATGTTCCAGATTACGCT-GG-3' and
5'-CATGCCAGCGTAATCTGGAACATCGTATGGGTA-3'. This HA linker was
ligated to NcoI linearized pSlax-Pax1 to generate a HA-Pax1 fragment.
This fragment was excised from pSlax13 with ClaI and subcloned into
ClaI-digested pRCASBP(A) (Hughes
et al., 1987
). The identity of the final product was confirmed by
sequencing. Western blot on RCAS-A-Pax1 infected chick embryo fibroblasts
(CEF) with HA11 antibody (BAbCO) against the HA epitope confirmed expression
of HA-Pax1 protein (not shown). Immunocytochemistry analysis on
RCAS-Pax1-infected CEF showed nuclear localization of HA-Pax1 (not shown), as
expected for its role as transcription factor. The control vector pRCAS(A)-AP
contains a human placental alkaline phosphatase cDNA (a gift from C. Tabin)
(Fekete and Cepko, 1993
).
Explant cultures, viral infection and RT-PCR
Fertile chicken White Leghorn eggs were obtained from Needle farm,
Hertfordshire, UK. Chick presomitic mesoderm from HH10 stage embryos were
isolated and embedded in collagen gels as described
(Münsterberg et al.,
1995). We used the semi-defined serum-free culture medium as
described (Murtaugh et al.,
1999
) with or without Shh (Ontogeny) supplement at 500 ng/ml.
Retroviral infection was performed as described
(Maroto et al., 1997
) with
4x104 cfu of RCAS-Pax1 and 2x105 cfu of
RCAS-AP. Explants were harvested on the fifth day and processed for
RT-PCR.
RT-PCR was carried out essentially as described
(Münsterberg et al.,
1995), except that 10% DMSO was included in the RT reaction
mixture. Amplification of chick Bapx1 was performed in the presence
of 5% formamide at annealing temperature 65°C with primers
5'-GCTCCCGCGCCGCCTTCTCC-3' and
5'-GGCGGCCGCGGCA-CAGGACAG-3'. The specificity of the amplified
product was confirmed by subcloning and sequencing. Aggrecan was amplified as
described (Murtaugh et al.,
1999
) at annealing temperature 50°C. Mouse Pax1 was
amplified at annealing temperature 60°C with primers
5'-GCTGCCTACTCCCCCAAGA-3' and
5'-CGCTGTA-TACTCCGTGCTG-3'.
Plasmid construction for transactivation assays
Expression plasmids
pPax1 and pPax9 contain mouse Pax1- and Pax9-coding
sequences, respectively, under control of the CMV promoter cloned in pcDNA3
(Invitrogen). To generate pPax1, the coding region of mouse Pax1 was amplified
by RT-PCR from RNA isolated from E11.5 mouse embryos, with primers
5'-CGTTCCATGGAGCAGACGTACGGC-3' and
5'-GTAGAATTCCTCTGAACCGGGCTGTGGCTC-3', which contain NcoI
and EcoRI sites, respectively. The PCR product was cloned in
pCR2.1-TOPO vector. The insert was excised with EcoRI, subcloned in
EcoRI-digested pcDNA3 vector, checked for correct orientation and
sequenced. pPax9 was a generous gift from J. Gerber.
Reporter plasmids
p5.3Bp-luc, p2.8Bp-luc, p1.9Nk-luc, p0.9Bp-luc, p0.7Bp-luc and p0.3Bp-luc
contain genomic sequences of the mouse Bapx1 gene [5' end
positions at -5285, -2762, -1947, -880, -748 and -270, respectively; and
3' end +109 (+1 is the first nucleotide in the published cDNA sequence
with GenBank Accession Number U87957)], cloned into pGL3-Basic vector
(Promega) with the firefly luciferase gene as reporter. To generate
p5.3Bp-luc, a 7 kb Bapx1 genomic clone based on pBluescriptII SK
(Lettice et al., 1999) was
partially digested with KpnI/BamHI, and the corresponding
fragment was subcloned into KpnI/BglII digested pGL3-Basic.
The 3' end of the insert in the final construct was confirmed to
correspond to position +109 by sequencing. p2.8Bp-luc was obtained by excising
KpnI/BglII fragment from p5.3Bp-luc. p1.9Bp-luc, p0.9Bp-luc,
p0.7Bp-luc and p0.3Bp-luc were generated by controlled treatment with
exonuclease III after KpnI/PmlI digestion of p5.3Bp-luc
(Erase-a-Base, Promega). To generate intron constructs pBpIA-luc and
pBpIB-luc, which contain the intron segment in forward and reverse
orientation, respectively, a 1.3 kb Bapx1 intron segment obtained as
a RsrII/MluI fragment from the 7 kb Bapx1 genome
clone was subcloned into MluI digested pGL3-Promoter vector (Promega)
with the SV40 promoter to direct firefly luciferase expression. Integrity of
all plasmids was confirmed by sequencing. pRL-SV40 vector (Promega) contains
the reporter Renilla luciferase gene upstream of SV40 early
enhancer/promoter, and was used to normalize the transfection efficiency among
different experiments.
Transient transfection assays
Mouse embryonic fibroblasts NIH3T3 or monkey kidney COS-7 cells were plated
into six-well plates in DMEM medium (GibcoBRL) supplemented with 10% fetal
calf serum (GibcoBRL). When cells reached about 35% confluency, the DNA was
transfected with Lipofectamine Plus reagent (Invitrogen). In each experiment,
0.5 µg of firefly reporter plasmid was co-transfected with different
amounts of pPax1, pPax9 or pcDNA3 control vector. The total amount of
transfected DNA was made equal in each experiment by completing with pcDNA3.
In addition, 20 ng of pRL-SV40 vector was always cotransfected for
normalization. Forty-two hours after transfection, at about 100% confluence,
cell extracts were collected and firefly and Renilla luciferase
activities were measured (Dual-Luciferase Reporter Assay, Promega). Firefly
luciferase activity in each sample was normalized to Renilla
luciferase to correct for variations in transfection efficiency. For each
assay, two to nine experiments were performed in duplicate.
Sequence analysis
Mouse and human genome sequences including Bapx1 used for
comparison are taken from NW_000229 and AF009802, respectively. Percent
Identity Plot (PIP), TFSEARCH and MatInspector analyses were carried out with
programs available at the websites of The Pennsylvania State University
(http://bio.cse.psu.edu),
Computational Biology Research Center
(http://www.cbrc.jp)
and Genomatix
(http://www.genomatix.de),
respectively.
Electrophoretic mobility shift assay (EMSA)
Pax1, Pax9 and luciferase (control) proteins were synthesized by
TNT-coupled wheatgerm extract system (Promega). EMSA was performed basically
as previously described (Hennighausen and
Lubon, 1987). Protein translation extracts (2 µl) were
incubated at room temperature for 15 minutes with 5x104 cpm
of each corresponding 32P-labeled double stranded oligonucleotide
and 1 µg of poly(dI-dC) in 15 µl of 20 mM HEPES pH 7.9, 60 mM KCl, 10%
glycerol, 0.1 mM EDTA, 2 mM MgCl2, 1 mM dithiothreitol and 0.1%
bovine serum albumin. The reaction mixture was analyzed by electrophoresis and
visualized by autoradiography. In competition assays, a 250 or 500-fold excess
of cold double-stranded oligonucleotide was pre-incubated at room temperature
for 15 minutes before the addition of the labeled oligonucleotide. In
reactions including antibodies, 1 µl (2 µg) of anti-Pax1 goat polyclonal
antibody (M-19, Santa Cruz Biotechnology) or 3 µl (1.2 µg) of goat
anti-mouse antibody as control (115-035-068, Jackson Immuno Research
Laboratories) was added and incubated for 20 minutes at room temperature
before the addition of the probe. The oligonucleotides B4, B5, S4, S1 and S2
from the Bapx1 promoter region are as shown in
Fig. 7B. The
Drosophila-derived oligonucleotides e5-5 and e5-3 correspond to the
oligonucleotides 5 and 3, respectively, described elsewhere
(Chalepakis et al., 1991
).
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RESULTS |
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Meox1 and Meox2 (also known as Mox1 and
Mox2, respectively) constitute a subfamily of homeobox-containing
genes that are expressed in the sclerotome
(Candia et al., 1992).
Meox1 plays a critical role in axial skeleton development
(Stamataki et al., 2001
),
while Meox2 is dispensable for the formation of the vertebral column
(Mankoo et al., 1999
). As
shown in Fig. 1, somitic
expression of neither Meox1 (Fig.
1A,B) nor Meox2 (Fig.
1C,D) is altered in the absence of both Pax1 and
Pax9. In single Pax1 or Pax9 homozygous mutant
embryos, expression of Meox1 and Meox2 is also not affected
(not shown).
|
We then examined expression of Nkx3-1 and Bapx1 in Pax1;Pax9 mutants. Nkx3-1 expression in the sclerotome of Pax1;Pax9-double homozygous embryos remains unchanged (Fig. 1E,F). Normal expression pattern is observed in single Pax1 or Pax9 mutant embryos, as well as in Pax1;Pax9 mutant embryos with other genotype combinations (not shown). Interestingly, Bapx1 expression in the sclerotome is undetectable in the absence of both Pax1 and Pax9 (Fig. 1G,H), whereas Bapx1 expression in other domains like the mandibular region of the first branchial arch, mesenchyme of the limb buds (see Fig. 1H) or splanchnic mesoderm (not shown) is not affected. We did not detect Bapx1 transcripts even in the youngest somites of the double mutants. The lack of Bapx1 expression in the Pax1;Pax9-deficient sclerotome was confirmed by in situ hybridization on sagittal and transverse sections (Fig. 2). It is noteworthy that sclerotomal cells that express Meox1, Meox2 and Nkx3-1 are present in the double mutants. This indicates that the loss of Bapx1 expression is not a secondary consequence of lack of sclerotomal cells, but it is rather a very early molecular defect caused by the absence of the sclerotomal Pax genes.
|
When we analyzed Pax1-single homozygous mutant embryos, we invariably observed a decrease in the intensity of Bapx1 staining in the sclerotome, when compared with wildtype littermates (Fig. 3A,B,E,F). However, Bapx1 expression in the sclerotome of Pax9-single deficient embryos did not show significant changes (Fig. 3C,D). When only one functional copy of Pax1 is present (Pax1+/-, Pax9-/-), the level of Bapx1 expression is considerably reduced (compare Fig. 1G with Fig. 1E). Bapx1 expression is even weaker, when only one functional Pax9 gene copy is present (Pax1-/-, Pax9+/-; Fig. 1H). These results indicate that somitic Bapx1 expression is dependent on the presence of Pax1 and Pax9 in a dose-dependent manner, with Pax1 having a stronger role than Pax9.
|
In summary, these data indicate that Meox1, Meox2 and Nkx3-1 expression in the sclerotome is not dependent on the Pax1/Pax9 activity. They may act either upstream, or in different regulatory pathways. However, somitic expression of Bapx1 requires Pax1 and Pax9.
Pax1 can substitute for Shh in the induction of Bapx1
expression and sclerotome chondrogenesis
In order to assess the potential of Pax1 and Pax9 to activate
Bapx1, we have employed a retroviral system to overexpress
Pax1 in explants of chick presomitic mesoderm (PSM). When the
explants were grown in the absence of Shh, we did not detect Bapx1
expression (Fig. 4A, lane 1).
However, we observed Bapx1 induction when the explants were exposed
to Shh (Fig. 4A, lane 3), as
previously reported (Murtaugh et al.,
2001). Because one of the early effects of Shh is the induction of
Pax1 (Fan and Tessier-Lavigne,
1994
; Johnson et al.,
1994
; Münsterberg et al.,
1995
; Murtaugh et al.,
1999
), there is the possibility that Shh acts through
Pax1 to activate Bapx1 expression. Interestingly, we
observed that overexpression of mouse Pax1 in chick PSM explants cultured in
medium with Shh led to a significant increase of the Bapx1 expression
levels (Fig. 4A, lane 4,
compare with lane 3), indicating that a high dose of Pax1 can enhance the
effect of Shh on the induction of Bapx1. Furthermore, exogenous
expression of Pax1 was sufficient to induce Bapx1, even in the
absence of Shh (Fig. 4A, lane
2).
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To further explore the role of Pax1 in sclerotome differentiation, we have
analyzed expression of the early chondrocyte marker aggrecan in this
in vitro system. It has previously been reported that aggrecan is
induced by Shh treatment (Murtaugh et al.,
1999), or by overexpression of Bapx1
(Murtaugh et al., 2001
) in
chick PSM explants. Consistent with these published observations, as well as
our finding that Pax1 alone can induce Bapx1, we have observed that
overexpression of Pax1, in the absence of Shh, promotes aggrecan expression
(Fig. 4B, lane 2).
Pax1 and Pax9 can transactivate Bapx1 promoter
Our data suggest that Pax1 and Pax9 may directly activate Bapx1
expression. To test this hypothesis, we analyzed putative Bapx1
regulatory sequences for their response to transactivation by Pax1 and Pax9 in
transient transfection assays. The genomic structure of Bapx1, like
other NK family members, is simple, consisting of two exons and a
single intron of 1.3 kb. Thus, as first approximation we have analyzed the
intron as well as a 5.4 kb upstream segment
(Fig. 5A). We first tested if
the 1.3 kb intron can act as an enhancer regulated by Pax1/Pax9. No specific
effect is observed when Pax1 and/or Pax9 expression plasmids are
co-transfected with a reporter plasmid containing the 1.3 kb intron in forward
or reverse orientation upstream of the SV40 promoter (constructs pBpIA-luc and
pBpIB-luc, see Materials and Methods), in NIH3T3 and COS7 (data not
shown).
|
We next tested the 5' region of Bapx1 with the plasmid p5.3Bp-luc that contains the 5.4 kb fragment, including a part of 5'UTR and the putative promoter region of Bapx1 (positions -5285 to +109, with position +1 being the published 5' end of Bapx1 exon 1) (Fig. 5A). This fragment possesses a basal promoter activity, as it can drive luciferase expression in NIH3T3 cells (22% of SV40 promoter activity, data not shown). Interestingly, when p5.3Bp-luc is co-transfected with the Pax1-expression plasmid, there is a dose-dependent increase in the promoter activity, from 2.9 times induction (0.5 µg of the Pax1-expression plasmid) to 3.8 times induction (1.5 µg) (Fig. 5B). Co-transfection with the Pax9 expression plasmid also induces a significant increase in the Bapx1 promoter activity up to 3.9 times (Fig. 5B). The maximum transactivation effect is reached when both Pax1 and Pax9 plasmids are co-transfected. This transactivation effect is specific for the 5.4 kb fragment, as neither Pax1 nor Pax9, or both in combination, can activate the control vector pGL3-Basic (see Fig. 6B and data not shown). Similar transactivation properties are also observed in COS7 cells (not shown).
|
To further narrow down the region(s) responsible for the transactivation activity of Pax1/Pax9, we generated a series of 5' deletion constructs, as schematized in Fig. 6A, and analyzed their response to transactivation by Pax1 and/or Pax9. Co-transfection of p2.8Bp-luc (-2762 to +109) with the Pax1 and/or Pax9 expression plasmids leads to a significant increase in the promoter activity of up to about eight times, when both expression plasmids are co-transfected (Fig. 6B). Similar activities are observed for p1.9Bp-luc (-1947 to +109) and p0.9Bp-luc (-880 to +109) (Fig. 6B). Interestingly, when additional 5' 132-bp sequences in p0.9Bp-luc are deleted (p0.7Bp-luc; -748 to +109), the activation properties of Pax1/Pax9 significantly drops by more than half, but still with 3.0- to 3.4-fold transactivation capacity. The shortest segment tested in p0.3Bp-luc (-270 to +109) is similarly activated by Pax1/Pax9 (Fig. 6B). These data indicate that the region between -880 and +109 contains cis-regulatory elements responsible for Pax1/Pax9-induced transactivation.
Comparative sequence analysis of the Bapx1 promoter
region
Sequence comparison between 8550 bp mouse and 8600 bp human genomic regions
encompassing the Bapx1 gene shows a significant degree of
similarities even outside the coding sequences
(Fig. 7A). In the region
between -880 and +109, overall sequence conservation is as high as 72% between
the two species. Most of conserved noncoding sequences (CNS) as revealed by
PIP analysis (Schwartz et al.,
2000) are located in this interval
(Fig. 7A). Two prominent CNS
segments are found in the regions between -543 and -408 (136 nucleotides, 78%)
and between +92 and +198 (107 nucleotides, 82%). The interval between -880 and
-748 (boxed in green in Fig.
7A, and marked in green in Fig.
7B), which is important for Pax1/Pax9-responsiveness, also
includes two short CNS segments (69 nucleotides, 80% and 69 nucleotides, 78%)
that are separated by a single-base gap. These data suggest that most of
important cis-regulatory elements of Bapx1 reside in the
region between -880 and +109.
Pax1 and Pax9 directly interact with Bapx1 promoter
sequences
In order to ascertain whether Pax1 and Pax9 directly activate
Bapx1 via binding to its cis-regulatory sequences, we
performed electrophoretic mobility shift assays (EMSA) with in vitro
translated Pax1 and Pax9 proteins and a series of labeled oligonucleotides
from the region between -880 and -748. When EMSA was performed with the
labeled oligonucleotides B5 or S4 (Fig.
7B) and Pax1 protein, we did not detect any retarded band (not
shown). Interestingly, when we incubated the labeled oligonucleotide B4
(Fig. 7B) with Pax1 protein, we
detected two retarded protein-DNA complexes
(Fig. 7C, lane 1). These bands
represent the complex of Pax1 protein with B4, as they did not appear when an
in vitro synthesized control protein was employed
(Fig. 7C, lane 6), and as they
were specifically competed by an excess of the cold e5-5 oligonucleotide that
has previously been shown to bind Pax1
(Fig. 7C, lanes 2, 3); but not
by e5-3, a mutated sequence that does not bind Pax1
(Fig. 7C, lanes 4, 5). The
lower band could be due to partial Pax1 synthesis or to a Pax1 degradation
product. The binding of the larger form of Pax1 to B4 was specifically blocked
when an anti-Pax1 antibody was included in the binding reaction
(Fig. 7D, lane 4), confirming
that the bound protein is Pax1.
By competition experiments with the oligonucleotides S1 and S2, located in the 5' or 3' part of B4, respectively (see Fig. 7B), we could narrow down the binding region of Pax1 to positions -880 to -844, as an excess of the cold S1 specifically competed Pax1 binding to B4, whereas S2 did not (Fig. 7E, lanes 4, 5 and lanes 6, 7). Furthermore, by labeling S1 and S2, we confirmed that Pax1 binds to S1, but not to S2 (Fig. 7F, lanes 2, 5). Similarly, we did not detect any retarded band when we incubated Pax9 protein with the labeled B5, S4 and S2 oligonucleotides (Fig. 7F, lane 6, and data not shown), but we detected a weak, but specific, band when Pax9 was incubated with B4 and S1 (Fig. 7F, lane 3, and data not shown).
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DISCUSSION |
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Despite the similarity in the expression patterns between
Pax1/Pax9 and Bapx1 in the early phase of sclerotome
development, their expression profiles significantly differ at the later
stages. Once chondrogenesis has started, both Pax1 and Pax9
are rapidly downregulated, while Bapx1 expression is maintained in
chondrocytes even after Pax1/Pax9 expression diminishes
(Tribioli et al., 1997;
Murtaugh et al., 2001
). This
observation suggests that Pax1/Pax9 may be required only for the
initiation of Bapx1 expression, but not for its maintenance.
Accordingly, it has recently been proposed that an autoregulatory loop between
Bapx1 and Sox9 maintains expression of both in sclerotome derivatives
(Zeng et al., 2002
). As
expression of Sox9 is initiated in young somites of
Pax1;Pax9-deficient embryos
(Peters et al., 1999
),
induction of Sox9 expression in sclerotome cells does not appear to
be dependent on Pax1 and Pax9, and probably not on Bapx1. How sclerotomal
Sox9 expression is initiated remains to be elucidated.
A conserved regulatory pathway
The Drosophila homolog of Bapx1, bagpipe (bap),
is expressed in a subset of dorsal mesodermal cells and in the absence of the
bap function, the development of the visceral musculature is
disrupted (Azpiazu and Frasch,
1993). As Bapx1 mutant mice show no defects in the
formation of the gut musculature, it has been proposed that there is no
equivalent function for the mouse and Drosophila genes
(Lettice et al., 1999
).
Nevertheless, there could be still common regulatory mechanisms. In
Drosophila embryos, the dorsal mesoderm homeobox gene tinman
(tin), in combination with decapentaplegic (dpp),
activates bap expression (Azpiazu
and Frasch, 1993
;
Staehling-Hampton et al.,
1994
). The zinc-finger transcription factor schnurri
(shn) has been proposed to mediate dpp-mediated bap
activation (Staehling-Hampton et al.,
1995
). In addition, the ectodermal segmental regulators
hedgehog (hh), wingless (wg) and
sloppy paired (slp) restrict bap to segmental
clusters of cells within the dorsal mesoderm, with hh having a
positive and wg and slp a negative effect on bap
regulation (Azpiazu et al.,
1996
; Riechmann et al.,
1997
; Lee and Frasch,
2000
). However, wg and hh signals are not
sufficient to mediate normal mesoderm segmentation and bap activation
(Azpiazu et al., 1996
). It is
postulated that as yet unidentified genes, expressed in stripped pattern in
the early mesoderm, are responsible for early bap segmental
expression, and that pox meso (poxm) might fulfill those
conditions (Azpiazu et al.,
1996
). poxm is the Drosophila paired gene that
is most related to Pax1/Pax9, as it is expressed in mesoderm and
lacks a homeodomain (Bopp et al.,
1989
). Thus, the regulatory pathway involving positive regulation
of Bapx1 by Pax1/Pax9 in mesodermal tissues could be
conserved through evolution.
Pax1 and Pax9 in sclerotome chondrogenesis
It is proposed that Shh alters the competence of somitic cells to activate
the chondrogenic differentiation program in response to subsequent BMP
signals, and that Shh induces the expression of still unknown chondrogenic
`competence factors' (Murtaugh et al.,
1999). Recently, it has been shown that Bapx1 is one of
such competence factors, as it is induced by Shh, and that overexpression of
Bapx1 in chick PSM mimics the chondrogenic effects of Shh
(Murtaugh et al., 2001
).
Misexpression of Bapx1 in vivo through retroviral infection in the chick
embryo leads to an expansion of the axial skeleton with thickened and often
fused neural arches, and an ectopic eighth `riblet'
(Murtaugh et al., 2001
). In
the present study, we show that Pax1 also possesses chondrogenic properties,
as it can induce aggrecan expression in cultures of chick PSM
(Fig. 4B). Therefore, Pax1 can
also be regarded as one of the competence factors.
We also tried to overexpress Pax9 in chick PSM using a retroviral construct
RCAS(B) (Hughes et al., 1987)
engineered to express mouse Pax9 (RCAS-B-Pax9). We observed neither
Bapx1 nor aggrecan upregulation when chick PSM explants were
exposed to RCAS-B-Pax9 viral supernatant. The lack of positive results with
the Pax9 retroviral system could be due to technical problems, such as the low
viral titer obtained (<107 cfu/ml), or the use of a different
viral vector (RCAS-B) that might infect sclerotomal cells with lower
efficiency. We have occasionally observed that injection of RCAS-B-Pax9 viral
supernatant in ovo into the PSM of the chick embryo induces fusion of the
proximal part of the ribs, thickening of the neural arches and an ectopic
eighth rib (I. R., A. M. and K. I., unpublished). This phenotype is strikingly
similar to the one observed by overexpressing Bapx1
(Murtaugh et al., 2001
).
Therefore, together with the observed synergistic function between Pax1 and
Pax9 in sclerotome development in the mouse and the capacity of Pax9 to
transactivate the Bapx1 promoter, it is conceivable that Pax9 also
can activate Bapx1 and initiate chondrogenesis in vivo.
Activation of Bapx1 expression by Pax1 and Pax9
The results from our transactivation study and EMSA assay strongly suggest
that Pax1 and Pax9 directly activate Bapx1. From the transient
transfection experiments, we assumed that there are at least two regions
responsible for positive regulation by Pax1 and Pax9 in the Bapx1
promoter region (Fig. 6): the
intervals from -880 to -748 and from -270 to +109. We have found that
Pax1/Pax9 directly bind to a motif located in the segment between -880 and
-844 (Fig. 7). Our analysis
failed to find Pax1/Pax9-binding sequences in the interval between -270 and
+109. It is notable that transactivation by Pax1 and Pax9 significantly
enhanced when the interval between -5285 and -2762 was removed (compare
p5.3Bp-luc with p2.8Bp-luc in Fig.
6B). This observation suggests the presence of
cis-elements that negatively influence on the Pax1/Pax9
transactivation.
Pax proteins bind to specific DNA sequences through the 128 amino acid long
paired domain. The paired domains in Pax1 and Pax9 are almost identical (98%),
suggesting that they could bind the same DNA sequences. Indeed, DNA-binding
studies have shown that Pax1 and Pax9 can bind the modified e5 motif derived
from the paired binding sequence in the Drosophila
even-skipped promoter (Chalepakis et
al., 1991; Neubüser et
al., 1995
). Pax1 also binds in vitro with high affinity to CD19-2
(A-ins) and H2A-17C, two modified sequences identified originally as
recognition sites for Pax5 (Adams et al.,
1992
; Czerny et al.,
1993
). Similarly, the paired box of zebrafish Pax9 recognizes in
vitro with high affinity the original CD19-1 and modified CD19-2 (A-ins)
sequences as well as a modified sequence of e5
(Nornes et al., 1996
).
However, most of these motifs are artificial consensus binding sequences, and
none of the genes from which these motifs originate is related to normal
expression or function of Pax1 or Pax9. It has been reported
that Pax1 can transactivate PDGFR
promoter in transient transfection
experiments in some cell lines, but the maximum effect was observed with a
mutated Pax1 protein, and the effects were dependent on the cellular context
(Joosten et al., 1998
).
Therefore, Bapx1 may be the first example of direct targets of Pax1
and Pax9 with a physiological relevance. Furthermore, the sequences contained
in the oligonucleotide S1 can be considered as a novel DNA motif for binding
of Pax1 and Pax9. In silico analysis by TFSEARCH
(Heinemeyer et al., 1998
) and
MatInspector (Quandt et al.,
1995
) predicts a potential Pax6 binding site within the S1 region
(positions -873 to -853, nucleotides marked in red in
Fig. 7B). This site might turn
out to be a Pax1/Pax9-binding site. Future study will define the nucleotide
motif in the S1 region that is specifically bound by Pax1 and Pax9.
Pax1/Pax9 as main mediators of Shh signaling in
sclerotome differentiation
Sclerotome differentiation is controlled by a number of molecules and
signaling pathways, the hierarchy of which we are beginning to understand.
Based on the data from published studies, together with the evidence reported
here, we propose a model for sclerotome differentiation as schematized in
Fig. 8. Paraxial mesoderm cells
experience sequential changes in their responsiveness to specific signals, as
they progress through developmental stages in the PSM, in the nascent somites
and in the sclerotome. During the transition from PSM to somites (somite
formation), paraxial mesoderm cells become competent to respond to Shh. It is
proposed that Wnt signaling plays a key role in the establishment of this
competence, via differential regulation on Gli genes in paraxial mesoderm
cells (Borycki et al., 2000).
After somites are formed, Shh plays an essential role in the induction,
differentiation and survival of sclerotomal cells, by activating several
transcription factors, including Pax1, Pax9, Nkx3-1 and
Bapx1 (thin arrows in Fig.
8). Once sclerotomal cells are specified, some of these
transcription factors (circled in Fig.
8; i.e. Meox1, Pax1 and Pax9) play a role in
further sclerotome differentiation and in chondrogenesis (green arrows in
Fig. 8), by activating their
downstream targets, including Bapx1. Although Nkx3-1 and
Meox2 are shown to be dispensable for proper axial skeleton
formation, their potential roles in the sclerotome still cannot be ruled out.
They might have a functional redundancy with their closely related genes
Bapx1 and Meox1, respectively. Therefore, whether mice
double mutant in Nkx3-1;Bapx1 or in
Meox1;Meox2 show synergistic defects is of great interest.
Finally, whether the Shh signaling pathway directly activates Pax1
and Pax9 is also not known. Shh signaling might indirectly activate
Pax1 and Pax9, by activating other transcription factors
like Meox1 and Meox2. Analysis of Pax1 and
Pax9 expression in mice deficient in both Meox1 and
Meox2 will clarify this point.
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ACKNOWLEDGMENTS |
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