1 Shriners Hospital for Children, Research Division, Portland, Oregon 97239,
USA
2 Department of Molecular and Medical Genetics, Oregon Health and Science
University, Portland, Oregon 97239, USA
3 Department of Biochemistry and Molecular Biology, Oregon Health and Science
University, Portland, Oregon 97239, USA
Author for correspondence (e-mail:
hss{at}shcc.org)
Accepted 24 June 2004
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SUMMARY |
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Key words: HOXA13, BMP2, BMP7, Limb, Apoptosis, Gene regulation
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Introduction |
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Although many of the genes required for normal limb development have been
identified (for reviews, see Niswander,
2003; Mariani and Martin,
2003
; Logan, 2003
;
Gurrieri et al., 2002
;
Tickle, 2003
), surprisingly
little is known about how these genes are transcriptionally regulated. Among
the factors likely to regulate the expression of limb patterning genes are the
5' HOX transcription factors, whose loss-of-function phenotypes
demonstrate their capacity to regulate key developmental processes such as
cell adhesion, apoptosis, proliferation and migration (Dollé et al.,
1989; Davis and Capecchi, 1994
;
Davis et al., 1995
;
Fromental-Ramain et al.,
1996a
; Fromental-Ramain et
al., 1996b
; Stadler et al.,
2001
; Wellik and Capecchi,
2003
; Boulet and Capecchi,
2004
; Spitz et al.,
2003
; Kmita et al.,
2002
). Although it is clear that HOX proteins mediate many of the
cellular events during limb morphogenesis, the mechanistic links between these
transcription factors, their target genes, and their effects on specific
cellular processes are still largely unknown
(Stadler et al., 2001
;
Morgan et al., 2003
;
Wellik and Capecchi, 2003
;
Boulet and Capecchi, 2004
). One
reason for this is that HOX proteins may not regulate the global expression of
any particular target gene, but instead control the development of specific
tissues through direct interactions with tissue-specific gene regulatory
elements (Hombría and Lovegrove,
2003
; Grenier and Carroll,
2000
; Weatherbee et al.,
1998
).
In the developing limb, HOXA13 localizes to discrete domains within the
interdigital and interarticular regions, where its function is required for
interdigital programmed cell death (IPCD), digit outgrowth and chondrogenesis
(Stadler et al., 2001;
Fromental-Ramain et al.,
1996a
). This observation led us to hypothesize that HOXA13
directly regulates genes whose products are necessary for IPCD and joint
formation. Testing this hypothesis, we detected changes in the expression of
the genes encoding bone morphogenetic proteins 2 and 7 (Bmp2, Bmp7)
in the interdigital and distal joint tissues, suggesting that HOXA13 may
directly regulate their expression in these discrete regions. Scanning the DNA
sequences upstream of Bmp2 and Bmp7, we identified a series
of nucleotide sequences preferentially bound by the HOXA13 DNA-binding domain
(A13-DBD). In vitro characterization of these DNA sequences revealed they
function as enhancer elements that, in the presence of HOXA13, activate the
expression of reporter constructs, independent of sequence orientation.
Furthermore, in the developing autopod, endogenous HOXA13 binds these same
enhancer elements, which can be immunoprecipitated with a HOXA13-specific
antibody. In Hoxa13 mutant limbs, both IPCD and Msx2
expression are partially restored with exogenous BMP2 or BMP7 treatment,
suggesting that HOXA13 is required for sufficient levels of BMP2 and BMP7 to
be expressed in the interdigital tissues, as well as for these tissues to
fully respond to BMP signaling. Together, these results provide the first
molecular evidence that HOXA13 controls distal limb morphogenesis through the
direct regulation of Bmp2 and Bmp7 expression, whose
combined functions are necessary for normal digit morphogenesis and IPCD.
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Materials and methods |
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RNA in situ hybridization and immunohistochemistry
Antisense riboprobes specific for Bmp2, Bmp4 and Bmp7
were generated using plasmids kindly provided by B. Hogan (Bmp2 and
Bmp4; Duke University, NC) and R. Beddington (Bmp7; NIMR,
London), whereas Hoxa13, Msx2, and BMP-receptor IA, IB and
II riboprobes were produced as described
(Morgan et al., 2003). Whole
mount in situ hybridization, immunohistochemistry and confocal imaging of limb
frozen sections were performed as described by Manley and Capecchi, and by
Morgan et al. (Manley and Capecchi,
1995
; Morgan et al.,
2003
). Antibodies to BMP2/4 (AF355) and BMP7 (AF354) were
purchased from R&D Systems, and were used at 0.02 µg/ml on frozen limb
sections.
Analysis of programmed cell death
TUNEL analysis was performed as described by Stadler et al.
(Stadler et al., 2001). Limbs
from E13.5 Hoxa13 and Bmp7 wild-type, heterozygous- and
homozygous-mutant embryos were examined using a Bio-Rad MRC 1024 confocal
laser imaging system fitted with a Leica DMRB microscope. Images were produced
by compiling z-series scans of the intact limb buds, typically averaging 40-50
sections at 1 µm per section. Kalman Digital Noise reduction was used for
all samples.
Limb organ culture and BMP supplementation
Affi-gel blue beads (Bio-Rad) washed five times in sterile PBS were
incubated at 4°C overnight in solutions containing 0.1 mg/ml of rhBMP2
(R&D Systems) or rhBMP7 (R&D Systems), or in sterile PBS. Beads were
inserted into the interdigital mesenchyme of E13.5 wild-type, heterozygous-
and homozygous-mutant limbs between digits II and III, and III and IV. Limb
explants containing the beads were placed on 0.4 µm HTTP Isopore membrane
filters (Millipore), coated with gelatin in 60 mm organ culture dishes
(Falcon), and grown in BGJb media (Invitrogen), supplemented with 50% rat
whole-embryo culture serum (Harlan Bioproducts), 50 U/ml penicillin and 50
µg/ml streptomycin, for 8 hours in an incubator at 37°C, 10%
CO2. The limb explants were examined for induced Msx2
expression or for changes in programmed cell death, using TUNEL analysis of
frozen sections as described (Morgan et
al., 2003).
Synthesis and structural characterization of the HOXA13 DNA-binding domain
Protein synthesis and purification
The 67 amino acid HOXA13 DNA-binding domain (A13-DBD)
(GRKKRVPYTKVQLKELEREYATNKFITKDKRRRISATTNLSER QVTIWFQNRRVKEKKVINKLKTTS) was
synthesized on an ABI 443A peptide synthesizer (Applied Biosystems, Foster
City, CA), using Fmoc-Ser(tBu)-PEG-PS resin (PerSeptive Biosystems, 0.16
mmol/g) and Fmoc amino acids (Anaspec). After synthesis, the peptide was
purified to greater than 95% purity, using a Gilson Model 321 High Performance
Liquid Chromatography (HPLC) system fitted with a semi-preparative
reversed-phase peptide column (Grace Vydac, C18, 15 µm, 300 Å,
250x25 mm). The mass and purity of the A13-DBD peptide was verified
using a Micromass time-of-flight mass spectrometer (Q-tof micro, Waters,
Manchester, UK), peptide sequencing and amino acid analysis.
Circular dichroism spectroscopy
The thermal stability and folding of the A13-DBD peptide into a
helix-turn-helix DNA-binding motif was verified by circular dichroism (CD).
The A13-DBD peptide was reconstituted to a final concentration of 100 µM in
50 mM NaF (buffered to pH 7.2 with phosphate buffer). CD spectra were obtained
using an Aviv 202 spectropolarimeter and a 0.1 mm path length rectangular cell
(Hellma, Müllheim, Germany). The wavelength spectra represent at least an
average of 10 scans with 0.1 nm wavelength steps. Thermal transitions were
recorded at a heating rate of 10°C/hour in a 1 mm cell.
DNA-binding site identification with the HOXA13 DNA-binding domain
The A13-DBD peptide was solubilized to a concentration of 500 nM in PBS,
and incubated with radioactively labeled Bmp2 and Bmp7
upstream regions (200-300 base pair fragments) at 4°C in gel shift buffer
(Promega). 5' upstream sequences for Bmp2 and Bmp7,
representing nucleotides 600 to +1 for Bmp2 (Ensembl ref:
ENSMUSG00000027358), and 3113 to +1 for Bmp7 (Ensembl ref:
ENSMUSG00000008999), were examined for binding by the A13-DBD peptide, using
electrophoretic mobility shift assays (EMSA) on a 4% non-denaturing
polyacrylamide gel buffered with 0.5xTBE. PCR fragments exhibiting
reduced electrophoretic mobility in the presence of the A13-DBD peptide were
subdivided into 30-50 base-pair (bp) regions and synthesized as self-annealing
oligonucleotides (Oligos Etc, Wilsonville, OR) to narrow the regions bound by
HOXA13. Double-stranded oligonucleotides were prepared by heating 1 µM
oligonucleotide stock solutions in 50 mM Tris-HCl (pH 7.2), 50 mM NaCl to
95°C for 5 minutes and allowing them to cool to room temperature. Annealed
oligonucleotides (12 nM) were radiolabeled using terminal transferase and
32P-ddATP (5000 Ci/mmol) (Amersham), as described by the
manufacturer (Roche). Peptide concentrations and EMSA assay conditions were
the same as described, with the exception that 6% polyacrylamide 0.5xTBE
gels were used to resolve the protein-DNA complexes.
Sequences of the self-annealing oligonucleotides
Cell culture and luciferase assays
Luciferase plasmid constructs
DNA regions bound by the A13-DBD peptide were amplified from mouse genomic
DNA by using PCR and the following primers:
PCR conditions used a 2 minute soak at 94°C, followed by 35 cycles of 94°C, 54°C and 72°C for 30 seconds each step. Amplified PCR products were cloned in both orientations into the SmaI site of the pGL3 luciferase vector (Promega) to produce the pGL3BMP2FLuc, pGL3BMP2RLuc, pGL3BMP7FLuc and pGL3BMP7RLuc plasmids. The sequences and orientation of the cloned fragments were verified using di-deoxy terminator fluorescent sequencing.
Hoxa13-HA expression plasmid
The Hoxa13-HA expression plasmid (pCMV-A13) used in the luciferase
and immunoprecipitation assays was produced using a 2-kb genomic region
containing the murine Hoxa13 locus. An HA epitope tag was added to
the 3' end of the Hoxa13 coding region, using a unique
SpeI restriction site to add the following annealed
oligonucleotides:
Cell culture and transfection
NG108-15 cells were grown as recommended by the supplier (ATCC). At 90%
confluence, the cells were passaged into 12-well dishes (Costar), grown at
37°C, 10% CO2 for 24 hours, and then transfected with 2 µg
of pGL3BMP2 or pGL3BMP7, 2 µg of pCMV-A13, or 2 µg of empty pCMV vector,
and 0.5 µg of the pRL-CMV Renilla plasmid (Promega) to normalize for
transfection efficiency. All transfections used the Superfect Reagent, as
recommended by the manufacturer (Qiagen). Forty-eight hours after
transfection, cells were rinsed with PBS, lyzed with M-Per lysis reagent
(Pierce), and processed to detect luciferase activity using the Dual-Glo
Luciferase Assay System (Promega). Luciferase activity was determined as the
average of three separate readings of each well (1 second/read), using a
Packard Fusion Universal Microplate Analyzer (Perkin Elmer). For each
experimental condition four separate transfections were performed. Results
were normalized for transfection efficiency using relative Renilla luciferase
expression levels, as described by the manufacturer (Promega), and plotted
using SigmaPlot 8.0 (SPSS).
Hoxa13 antibody production
A HOXA13 peptide, corresponding to amino acids 1-43 of the murine HOXA13
(MTASVLLHPRWIEPTVMFLYDNGGGLVADELNKNMEGAAAAAA)
(Mortlock and Innis, 1997) was
used to immunize New Zealand White rabbits. Samples exhibiting high
Hoxa13 antibody titers by ELISA were assessed for specificity to the
full-length HOXA13, using western blot and immunohistochemistry of cultured
limb mesenchyme derived from mice heterozygous for a temperature-sensitive
T-antigen (Immorto® mice, Charles River Laboratories) and homozygous for
the HOXA13-GFP mutant allele. A Cytological Nuclear Stain Kit (Molecular
Probes) was used to label nuclei with DAPI.
Chromatin immunoprecipitation (ChIP)
ChIP assays were performed using a chromatin immunoprecipitation assay, as
described (Upstate Biotechnology), with the following modifications: limb buds
from E12.5 Hoxa13GFP wild-type and homozygous-mutant
embryos were dissected in PBS containing 15 µl/ml protease inhibitor
cocktail (PIC) (Sigma). Lysates were sonicated for four periods of 10 seconds
at 4°C using a Microson (Misonix) sonicater at 15% output. To avoid sample
heating, tubes were placed in an ice/EtOH bath for 5 seconds before and after
each sonication. Cell lysates were pre-cleared with 80 µl Gammabind
(Amersham) containing 40 µg/ml tRNA (Roche) in 10 mM Tris-HCl (pH 8), 1 mM
EDTA (GBS). After pre-clearing, all centrifugation steps were performed at 100
g for 2 minutes. Fifteen microliters of Hoxa13
antibody or control solution (PBS) were used at the immunoprecipitation step.
For collection of Hoxa13 antibody/HOXA13/DNA complexes, 60 µl of
GBS was added to the samples. The Gammabind/Hoxa13
antibody/HOXA13/DNA complexes were washed twice for 5 minutes at 4°C for
each wash step. DNA was eluted from the immune complexes by digesting
overnight at 45°C in 50 mM Tris-HCl (pH 8.4), 1 mM EDTA, 0.5% Tween-20
containing 20 µg/ml Proteinase K (Invitrogen). Eluted DNA from the antibody
or no antibody control samples were assessed for the presence of the Bmp2,
Bmp7s1 or Bmp7s2 DNA regions using PCR and the primers described
above. For Bmp7s2, the following primers were used to PCR amplify
immunoprecipitated chromatin: 5'-GCCTCTGTTCTTGCTGCGCT-3' and
5'-ACATGAACATGGGCGCCG-3'.
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Results |
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Reduced Bmp2 and Bmp7 expression in Hoxa13 mutant autopods
Recognizing that limbs lacking Hoxa13 exhibit severe defects in
the initiation of IPCD and joint formation
(Stadler et al., 2001)
(Fig. 1), and that BMP2 and
BMP7 function as key regulators of IPCD and digit chondrogenesis
(Zuzarte-Luís and Hurlé,
2002
; Merino et al.,
1998
; Macias et al.,
1997
; Zou et al.,
1997
; Yokouchi et al.,
1996
; Zou and Niswander,
1996
), we examined their expression in Hoxa13 wild-type
and mutant forelimbs. Analysis of Bmp7 expression in E12.5-13.5
wild-type embryos revealed high levels of expression in the interdigital
tissues (Fig. 1G,I). By
contrast, age-matched homozygous mutants exhibited a marked reduction in
Bmp7 expression in the interdigital and peridigital regions,
specifically in domains that co-express Hoxa13
(Fig. 1H,J,S-V).
Bmp2 expression was also reduced in the interdigital tissues, the developing nail beds, and the distal joints of E12.5 and E14.5 homozygous mutants, which exhibited a diffuse distribution of Bmp2 transcripts when compared with wild-type controls (Fig. 1A,C,K-N). Next, to determine whether the loss of BMP signaling in Hoxa13 mutant limbs affects the expression of BMP-regulated genes, we examined Msx2 expression in the distal interdigital tissues. In Hoxa13 mutant limbs, Msx2 expression was noticeably reduced in the interdigital tissues when compared with wild-type controls at E12.5 and E13.5 (Fig. 1O-R).
To verify that BMP7 and HOXA13 proteins co-localize to the same cells in developing interdigital tissues, we examined BMP7 and HOXA13 protein distribution using a BMP7 antibody and the HOXA13-GFP fusion protein. In E12.5 autopods, HOXA13-GFP and BMP7 proteins co-localized to the same cells in the distal interdigital tissues of both heterozygous and homozygous mutants (Fig. 1S-V). However, the interdigital tissues of Hoxa13 homozygous mutants consistently exhibited fewer BMP7-positive cells, as predicted by the decreased levels of Bmp7 transcripts in this region (Fig. 1H,J). Co-localization of HOXA13-GFP and BMP2 proteins could not be determined by immunohistochemistry because commercially available BMP2 antibodies failed to detect BMP2 protein in autopod frozen sections at E12.5 or E13.5 (data not shown).
HOXA13 directly binds DNA regions upstream of Bmp2 and Bmp7
Because Bmp2, Bmp7 and Hoxa13 are co-expressed in the
developing limb, and their expression is reduced in Hoxa13 mutants,
we hypothesized that HOXA13 may directly regulate Bmp2 and
Bmp7 expression in the distal autopod. To address this possibility, a
HOXA13 DNA-binding domain peptide (A13-DBD) was used to screen DNA regions
upstream of Bmp2 and Bmp7 for direct HOXA13 binding. Prior
to this analysis, it was necessary to confirm that the putative DNA-binding
domain present in the C terminus of HOXA13 actually folds into a DNA-binding
structure and to determine the biophysical conditions that maintain proper
folding for subsequent DNA-binding studies.
Characterization of the A13-DBD secondary structure by circular dichroic
spectroscopy (CD) revealed the peptide stably folds into an -helical
structure, consistent with the predicted helix-turn-helix DNA-binding motif
encoded by the A13-DBD peptide (Fig.
2A, left panel). The folded A13-DBD peptide also exhibited
reasonable thermal stability, maintaining its
-helical structure
between 4 and 25°C (Fig.
3A, right panel). At temperatures higher than 25°C, the
A13-DBD peptide showed a cooperative transition to a denatured conformation
(Fig. 2A, right panel),
confirming that the majority of the A13-DBD peptide folds into the detected
-helical motif. Based on this analysis, we performed our DNA-binding
experiments at temperatures between 4 and 25°C in order to maintain
A13-DBD secondary structure stability. By contrast, full-length HOXA13 could
not be examined for proper folding and function, as the molecule prepared by
in vitro translation was only soluble in a denatured state, preventing CD
analysis, as well as its use as a DNA-binding molecule (data not shown).
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Analysis of the Bmp7C1 binding site revealed the highest A13-DBD affinity for any of the A13-DBD bound sequences, requiring competitor DNA concentrations of greater than 750 nM to completely displace the radiolabeled BMP7C1 oligonucleotide (Fig. 2B,E). The DNA-binding specificity of A13-DBD was confirmed using a randomized self-annealing oligonucleotide that exhibited no changes in electrophoretic mobility in the presence of 4-fold higher concentrations of A13-DBD peptide (Fig. 2D). Similarly, concentrations of control oligonucleotide greater than 750 nM could not displace the A13-DBD-bound Bmp7C1 oligonucleotide, confirming high affinity and specificity for the Bmp7C1 binding site (Fig. 2E).
Analysis of the DNA sequences bound by the A13-DBD peptide revealed a
HOX-specific TAAT nucleotide core sequence
(Fig. 2F). However the
nucleotides flanking the TAAT site varied greatly from sequences bound by
other HOX proteins, suggesting that HOXA13 may recognize unique DNA sequences
to facilitate its tissue-specific regulation of gene expression
(Catron et al., 1993;
Pellerin et al., 1994
).
Interestingly, a second TAAT-containing region (Bmp7s2) was
identified in the sequences upstream of Bmp7; however, in the
presence of the A13-DBD peptide the Bmp7s2 sequence did not exhibit
any change in electrophoretic mobility (data not shown), suggesting that
nucleotides flanking the TAAT core sequence in Bmp7s1 confer binding
specificity.
The Bmp2 and Bmp7 sequences bound by A13-DBD function as enhancers of gene expression in vitro
A 384 bp fragment containing the four Bmp2 binding sites
(BMP2C1-C4) was cloned into a luciferase reporter plasmid and tested for in
vitro transcriptional regulation by HOXA13. Cells transfected with the
Bmp2 luciferase vector and the Hoxa13 expression plasmid
(pCMV-A13) exhibited a consistent increase in relative luciferase activity
(RLA) when compared with control transfections lacking pCMV-A13
(Fig. 3A). Comparisons of
forward and reverse orientations of the 384 bp upstream region revealed a 2.5-
and 1.8-fold increase in RLA, suggesting that the 384 bp Bmp2 region
functions as a HOXA13-regulated enhancer of gene expression
(Fig. 3A,C). Similarly, a 216
bp fragment containing the BMP7C1-binding site also caused an increase in
luciferase reporter expression, resulting in a 1.8- and 2.0-fold increase in
RLA for the forward and reverse orientations, respectively
(Fig. 3B,C). Our detection of
direct interactions between A13-DBD and these enhancer elements, as well as
the ability of these enhancer elements to drive reporter gene expression only
in the presence of HOXA13 strongly supports our hypothesis that HOXA13
directly regulates the expression of Bmp2 and Bmp7 in the
developing autopod.
HOXA13 associates with the Bmp2 and Bmp7 enhancer elements in the developing autopod
To verify that HOXA13 also binds the Bmp2 and Bmp7
enhancer regions in vivo, a Hoxa13 antibody was developed to
immunoprecipitate endogenous HOXA13-DNA complexes from limb bud chromatin. To
identify both wild-type and mutant HOXA13 proteins, the Hoxa13
antibody (Hoxa13) was raised against the N-terminal region of HOXA13,
which is conserved in both wild-type and mutant protein isoforms. Analysis of
the proteins recognized by
Hoxa13 revealed two predominant bands by
western blot hybridization whose sizes matched the predicted molecular weights
of HOXA13 wild-type (43 kDa) and mutant proteins (64 kDa)
(Fig. 3D). The ability of
Hoxa13 to immunoprecipitate native HOXA13 was confirmed by western blot
analysis of precipitated cell lysates expressing full-length HOXA13 tagged
with an HA epitope (Fig. 3E).
In cultured limb mesenchyme, the Hoxa13 antibody co-localizes with
endogenous HOXA13-GFP, confirming the specificity of the Hoxa13
antibody (Fig. 3F-I).
Next, to verify that endogenous HOXA13 binds the same Bmp2 and
Bmp7 enhancer elements in the developing limb, Hoxa13 was used
to immunoprecipitate chromatin from wild-type and homozygous mutant limbs. In
wild-type limbs, chromatin immunoprecipitated with
Hoxa13 consistently
contained the enhancer elements bearing the Bmp2C1, C3 and
C4, and Bmp7s1 nucleotide sequences
(Fig. 3J,K). By contrast,
chromatin immunoprecipitations from Hoxa13 homozygous mutant limbs
did not contain these same enhancer regions, which is consistent with the
ablation of the DNA-binding domain in the Hoxa13GFP mutant
allele (Stadler et al., 2001
)
(Fig. 3J,K).
To confirm the in vivo specificity of HOXA13 for the TAAT-containing
sequences in the Bmp2 and Bmp7 enhancers, wild-type
Hoxa13 chromatin immunoprecipitates were also examined for the presence
of the Bmp7s2 sequence, which contains a TAAT core sequence but is
not bound by the A13-DBD peptide (data not shown). In all cases examined, the
Bmp7s2 sequence could not be detected in wild-type chromatin
immunoprecipitates, confirming the in vivo specificity of HOXA13 for the
Bmp2 and Bmp7s1 sequences
(Fig. 3L). These results
strongly suggest that endogenous HOXA13 directly binds the Bmp2 and
Bmp7 enhancer sequences, and that it is through these interactions
that HOXA13 controls the expression of Bmp2 and Bmp7 in the
distal limb.
Reduced Bmp2 and Bmp7 expression underlies the loss of IPCD in the Hoxa13 homozygous mutant limb
Recognizing that HOXA13 can regulate gene expression through the
Bmp2 and Bmp7 enhancer elements in vitro
(Fig. 3A-C), and that it
associates with these enhancer sites in the developing limb
(Fig. 3J,K), we hypothesized
that the limb phenotypes exhibited by Hoxa13 homozygous mutants must
be due, in part, to insufficient levels of BMP2 and BMP7. Testing this
hypothesis, we examined whether increasing the levels of BMP2 or BMP7 in
Hoxa13 mutant autopods could rescue some aspects of the interdigital
or interarticular joint phenotypes. For the interarticular malformations,
supplementation of the autopod tissues with BMP2- or BMP7-treated beads could
not restore the normal formation of the joint regions
(Fig. 1E,F; data not shown). By
contrast, supplementation of the interdigital tissues with either BMP2- or
BMP7-treated beads restored distal IPCD in Hoxa13 homozygous mutants,
whereas control experiments using PBS-treated beads did not affect the levels
of IPCD in either homozygous mutant or heterozygous control limbs
(Fig. 4A-F). These results
suggest that BMP insufficiency may underlie the loss of IPCD in
Hoxa13 mutant limbs.
|
IPCD is delayed in Bmp7 mutant mice
TUNEL analysis of Bmp7 mutant limbs at E13.5 revealed a
significant delay in IPCD between digits II and III
(Fig. 5A-D), which is the same
interdigital region lacking IPCD in Hoxa13 homozygous mutants
(Stadler et al., 2001). This
result suggests that reductions in BMP7 in Hoxa13 mutant limbs can
affect IPCD initiation, particularly in tissues where responsiveness to BMP
signals is also reduced. For the region between digits III and IV,
TUNEL-positive cells were detected at slightly elevated levels in
Bmp7 mutants, suggesting that BMP7 may regulate IPCD in a
differential manner between digits II and III, versus digits III and IV.
|
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Discussion |
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Hoxa13 coordinates BMP signaling during distal limb development
In the present study one of our major conclusions is that HOXA13 directly
regulates the expression of both Bmp2 and Bmp7 in the distal
autopod. In the absence of HOXA13 function, the interdigital tissues also
exhibit reduced responsiveness to BMP-induced programmed cell death. Together
these results suggest that HOXA13 functions at multiple levels of the
BMP-signaling pathway to direct IPCD and distal digit development. The
function of HOX proteins to regulate multiple steps in a developmental pathway
is well described in Drosophila where Ubx function is required to
regulate multiple genes during the specification of wing and haltere
structures (Weatherbee et al.,
1998). Studies of BMP-receptor function support the conclusion
that HOXA13 regulates BMP-signaling to control digit chondrogenesis, as mice
lacking BMPR-IB in the distal limb exhibit defects in chondrogenesis
remarkably similar to Hoxa13 mutants
(Stadler et al., 2001
;
Baur et al., 2000
;
Yokouchi et al., 1995
).
Similarly, the expression of dominant-negative isoforms of BMPR-IB in chick
also affect IPCD, as well as the chondrogenic capacity of the distal limb
mesenchyme, phenotypes both described in Hoxa13 mutant limb
mesenchyme (Zou and Niswander,
1996
; Zou et al.,
1997
; Stadler et al.,
2001
).
In mice, the function of BMP2 during distal limb development is unknown as
Bmp2 homozygous mutants fail to develop to the limb bud stage
(Zhang and Bradley, 1996). In
Bmp7 homozygous mutants, the predominant limb phenotype is the
addition of an extra anterior digit, whereas the interdigital tissues are
completely resolved (Dudley et al.,
1995
; Luo et al.,
1995
; Hofmann et al.,
1996
). The resolution of the interdigital tissues in Bmp7
mutant mice has been explained by compensatory BMPs co-expressed in these same
tissues (Francis-West et al.,
1999
; Dudley and Robertson,
1997
; Dudley et al.,
1995
; Luo et al.,
1995
; Lyons et al.,
1995
; Francis et al.,
1994
; Hogan et al.,
1994
; Kingsley,
1994
; Lyons et al.,
1989
). In the limb, IPCD can be stimulated by BMP2, BMP4 and BMP7
(Guha et al., 2002
;
Merino et al., 1999a
;
Gañan et al., 1996
;
Yokouchi et al., 1996
).
However, in Bmp7 mutants, the delay in IPCD at E13.5 provides strong
evidence that compensatory factors, such as BMP2 and BMP4, do not use the same
BMP-signaling components as BMP7, suggesting that additional factors like
bioavailability, oligomerization or receptor utilization may also regulate the
timing of IPCD (Keller et al.,
2004
; Groppe et al.,
2002
; Nohe et al.,
2002
; Bosukonda et al.,
2000
; Capdevila and Johnson,
1998
; Zou et al.,
1997
; Zou and Niswander,
1996
; Lyons et al.,
1995
). Thus, in Hoxa13 homozygous mutants, two
compounding events contribute to the loss of IPCD. First, BMP2 and BMP7 are
reduced in the mutant interdigital tissues to levels below the threshold
necessary for IPCD initiation. Second, the competency of the interdigital
tissues to respond to BMP signals is affected by loss of Hoxa13
function.
Surprisingly, mice heterozygous for both Bmp2 and Bmp7
(Bmp2/7) do not exhibit any defects in limb development
(Katagiri et al., 1998). This
result is most likely explained by compensatory levels of additional BMPs or
the upregulation of BMP2, BMP7, or their receptors in Bmp2/7
compound heterozygotes, which to date have not been determined in these mice.
Clearly, conditional mutants producing combinatorial losses of redundant BMP
proteins will be required to define the functions of these growth factors
during distal limb development, particularly in regions where BMPs regulate
both digit chondrogenesis and IPCD. Interestingly, the differential use of
BMPs to direct digit chondrogenesis may also affect IPCD, as apoptosis in limb
mesenchyme is directly linked to the level of chondrogenesis
(Omi et al., 2000
;
Merino et al., 1999b
). This
finding is consistent with the differential IPCD in Hoxa13 mutant
limbs, where digits II and III exhibit the least amount of chondrogenesis and
IPCD (Fig. 1D-F).
Isolation of HOXA13-Bmp2 and -Bmp7 enhancer complexes in the developing autopod defines these genes as direct targets of HOXA13 regulation
Using a biochemical approach, we confirmed the C-terminal region of HOXA13
folds into a functional DNA-binding motif and binds DNA in a sequence-specific
manner. In vitro, the A13-DBD peptide facilitated the identification of
HOXA13-binding sites upstream of Bmp2 and Bmp7. Quantitation
of A13-DBD affinity for these enhancer elements revealed a novel series of
nucleotide sequences that are preferentially bound with affinities almost
2-fold greater than the DNA regions bound by other homeodomain peptides
(Catron et al., 1993).
In vivo, the interactions between HOXA13 and the Bmp2 and
Bmp7 enhancer regions are conserved, as both enhancer elements were
present in immunoprecipitated complexes of HOXA13 bound to DNA. The
association of HOXA13 with the Bmp2 and Bmp7 enhancer
regions in the developing limb strongly suggests that HOXA13 directly
regulates the expression of these genes during autopod formation. It is
important to note that because chromatin immunoprecipitations do not separate
protein complexes prior to immunoprecipitation, we cannot exclude the
possibility that HOXA13 interacts with the Bmp2 and Bmp7
enhancer regions as part of a protein complex. However, this possibility seems
unlikely because of the absence of the Bmp2 and Bmp7
enhancer regions in immunoprecipitations using Hoxa13 mutant limbs
that lack the HOXA13 DNA-binding domain, and because of the utilization of
these enhancers by full-length HOXA13 to direct gene expression in vitro. In
addition, the specificity of HOXA13 DNA-binding is confirmed by the presence
of an additional TAAT-containing sequence upstream of Bmp7 that is
not bound by either the A13-DBD peptide (data not shown) or the full-length
endogenous HOXA13, as determined by chromatin immunoprecipitation from
wild-type autopods (Fig. 3L).
This finding confirms that the nucleotides flanking the core TAAT sequence are
crucial for HOXA13 binding, supporting our hypothesis that HOXA13 interacts
with specific DNA sequences to facilitate its tissue-specific regulation of
gene expression (Catron et al.,
1993; Pellerin et al.,
1994
).
Previous investigations of HOXA13 function indicate that Bmp4
expression may also be regulated by HOXA13 DNA-binding
(Suzuki et al., 2003).
However, by our analysis, the expression of Bmp4 appears to be normal
in Hoxa13 mutant limbs (data not shown), suggesting that the
cooperative regulation of Bmp4 by SP1 may compensate for the loss of
HOXA13 DNA-binding function. This finding is consistent with the absence of
the Bmp4 sequence in the wild-type limb chromatin immunoprecipitated
with a Hoxa13 antibody (data not shown). In the N terminus of HOXA13,
protein-protein interactions may also facilitate some aspects of its function,
as mutations expanding the number of N-terminal polyalanine residues cause
defects in limb and genitourinary development that are similar to those
resulting from mutations ablating the HOXA13 DNA-binding domain
(Goodman et al., 2000
;
Mortlock and Innis, 1997
).
In this paper, we present the first evidence that during limb development HOXA13 directly interacts with gene-regulatory elements upstream of Bmp2 and Bmp7. In vivo, the loss of HOXA13 DNA-binding function abolishes interactions with these enhancer elements and reduces the expression of Bmp2 and Bmp7 in the developing autopod, causing defects that phenocopy malformations associated with perturbations in BMP signaling. Together, these findings strongly suggest that HOXA13 controls the morphogenesis of discrete autopod tissues by regulating Bmp2 and Bmp7 expression through direct interactions with Bmp2 and Bmp7 enhancer elements, confirming our hypothesis that the phenotypes exhibited by Hoxa13 mutant mice reflect a loss in the tissue-specific regulation of direct target genes.
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ACKNOWLEDGMENTS |
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