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First published online 14 January 2009
doi: 10.1242/dev.029355
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1 Department of Molecular Biology, and Faculty of Dentistry, McGill University,
Montreal, Quebec H3A 2B2, Canada.
2 Department of Anatomy and Cell Biology, and Faculty of Dentistry, McGill
University, Montreal, Quebec H3A 2B2, Canada.
3 Department of Molecular Genetics, University of Texas Southwestern Medical
Center, Dallas, TX 75235, USA.
4 Department of Internal Medicine, University of Texas Southwestern Medical
Center, Dallas, TX 75235, USA.
5 Department of Pathology, University of Texas Southwestern Medical Center,
Dallas, TX 75235, USA.
* Author for correspondence (e-mail: hiromi.yanagisawa{at}utsouthwestern.edu)
Accepted 11 December 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Branchial arch, Intramembranous ossification, Neural crest, Craniofacial, Osteoblast, Mandible, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Shigetani et al., 2002).
Primitive neural crest-like populations are present in cyclostomes, including
hagfish (jawless, vertebraeless) and lamprey (jawless vertebrate), which are
considered to be a sister group to the vertebrates
(Ota et al., 2007). The
identification of factors involved in the differentiation and regulation of
cranial neural crest derivatives therefore provides insight into vertebrate
evolution. Improper development of neural crest cells accounts for at least
one-third of all birth defects in humans
(Trainor, 2005). Syndromic
mandibular dysmorphogenesis is common among craniofacial malformations, often
accompanying clinical features of agnathia, micrognathia and macrognathia.
Hand1 and Hand2 are highly conserved basic helix-loop-helix (bHLH) proteins
that are implicated in the development of the neural crest, heart, placenta
and limb (Cross et al., 1995;
Cserjesi et al., 1995;
Riley et al., 1998;
Srivastava et al., 1995).
Gain-of-function studies have shown that Hand1 supports the proliferation of
cardiac myocytes and prevents differentiation associated with cell-cycle exit
(Risebro et al., 2006).
Conversely, Hand1 induces differentiation of the trophoblast lineage by
interacting with multiple bHLH proteins and Sox15
(Riley et al., 1998;
Scott et al., 2000;
Yamada et al., 2006). Neural
crest-specific knockouts for Hand2 demonstrated that Hand2 is
necessary for the differentiation of catecholaminergic neurons in the
sympathetic nervous system and of enteric neurons
(D'Autreaux et al., 2007;
Hendershot et al., 2008;
Morikawa et al., 2007). The
coordinated roles of Hand1 and Hand2 in heart and branchial arch development
were demonstrated by generation of an allelic series of Hand mutant mice
(Barbosa et al., 2007;
McFadden et al., 2005;
Yanagisawa et al., 2003).
Furthermore, the regulation and mechanism of Hand functions have been
investigated at a molecular level. Phosphorylation-dependent subnuclear
localization of Hand1 is essential for cell fate determination of the
trophoblast lineage (Firulli et al.,
2003; Martindill et al.,
2007). Gene dosage-dependent antagonistic interaction of
Hand2 and Twist1, as well as phosphorylation-regulated dimer
choice between Hand2, Twist1 and E-proteins, have profound effects on limb and
craniofacial development (Firulli et al.,
2005; Firulli et al.,
2007). Although Hand proteins act predictably according to bHLH
models in vitro (Hollenberg et al.,
1995; McFadden et al.,
2002; Scott et al.,
2000), biological functions of Hand proteins are largely dependent
on cell type, and the precise molecular mechanisms of action of Hand proteins
in the development of branchial arches are not fully understood.
Hand1 is expressed in the distal (ventral) zone of the first and
second branchial arches, and the Hand2 expression domain includes
that of Hand1 and extends laterally to occupy two-thirds of the
branchial arches (Clouthier et al.,
2000; Srivastava et al.,
1997). An endothelin-1-dependent ventrolateral Hand2
enhancer has been extensively characterized in vivo
(Charité et al., 2001;
Yanagisawa et al., 2003). Mice
lacking this enhancer (termed Hand2BA/BA) develop a
spectrum of craniofacial defects, including hypoplastic mandible, cleft palate
and abnormal middle ear ossicles, confirming the importance of Hand2
expression driven by this enhancer in the development of subsets of cranial
neural crest cells (Yanagisawa et al.,
2003).
Craniofacial skeletons, which are derivatives of cranial neural crest
cells, are formed preferentially by intramembranous ossification. Membranous
bones are formed directly by osteoblast differentiation of the condensed
mesenchyme, whereas endochondral bones use cartilaginous templates that
develop through endochondral ossification to form bones
(Erlebacher et al., 1995).
Runx2, one of three mammalian orthologs of the Runt-related transcription
factor in Drosophila, is a crucial early determinant of the
osteoblast lineage. Upregulation of Runx2 is both necessary and
sufficient to induce osteoblast differentiation by regulating expression of
numerous osteoblast-specific genes, including Runx2 itself
(Ducy et al., 1997;
Komori et al., 1997).
Mutations in the RUNX2 gene result in autosomal-dominant
cleidocranial dysplasia in humans, which is characterized by
hypoplasia/aplasia of clavicles, patent fontanelles, supernumerary teeth,
short stature and other defects in skeletal patterning and growth
(Mundlos et al., 1997;
Otto et al., 1997). Here, we
show that branchial arch-specific deletion of Hand2 in mice results
in a previously uncharacterized phenotype involving accelerated osteoblast
differentiation. We provide molecular evidence of the function of Hand
proteins, which involves direct interaction with, and inhibition of, Runx2
activity. These results reveal a novel role for Hand proteins as negative
regulators of bone development, specifically in controlling the
differentiation of osteoblasts derived from neural crest cells.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Yanagisawa et al.,
2003).
Cartilage and bone staining
Skeletal preparations were stained using Alcian Blue for cartilage and
Alizarin Red for bone as described
(McLeod, 1980).
Histology and in situ hybridizations
Paraffin-embedded sections were stained with Hematoxylin and Eosin
(H&E) for routine histology or with von Kossa for detection of
mineralization. In situ hybridization of sectioned tissues was performed as
previously described (Shelton et al.,
2000). Images were captured using Openlab 4.0.1 acquisition and
analysis software (Improvision) and processed with Photoshop CS2 (Adobe).
β-galactosidase staining
β-galactosidase staining of transgenic embryos was performed as
described (Yanagisawa et al.,
1998).
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed as described
(Barbosa et al., 2007). For
whole-mount staining for alkaline phosphatase, embryos were stained with BM
Purple (Roche) according to the manufacturer's instructions.
TUNEL and TRAP staining
TdT-mediated dUTP nick end labeling (TUNEL) staining was performed using
the In Situ Cell Death Detection Kit (Roche) according to the manufacturer's
protocol. Osteoclast activity was detected by staining for tartrate-resistant
acid phosphatase (TRAP) using the Acid Phosphatase Leukocyte Kit
(Sigma-Aldrich).
Immunostaining
Immunocytochemistry was performed as described
(Funato et al., 2001).
Phospho-histone H3 antibody (Upstate Biotechnology) was used according to the
manufacturer's instructions to evaluate cell proliferation on
paraffin-embedded sections.
Micro-computed tomography (Micro-CT)
An X-ray microtomograph (SkyScan1072, Kontich) was used to scan entire
hemimandibles for 30 minutes at a rate of 1.9 seconds per scan (at 80 kV, 100
µA) using a rotational angle of 180° and a rotational step of 0.9°.
For each sample, 
1000 X-ray slices, with increments of 15 µm covering
the entire specimen, were reconstructed from the acquired scan.
Three-dimensional reconstruction to visualize calcified tissue was performed
based on triangular surface rendering using the manufacturer's software
(3D-Creator). Measured parameters recorded for statistical analysis by the
instrument and software included total mineralized tissue volume within each
hemimandible (n=4).
Real-time quantitative PCR (qPCR)
Total RNA was isolated from mandibles (n=6-7 per genotype) using
Trizol (Invitrogen) without pooling the samples. cDNA was synthesized using
the Transcriptor First-Strand cDNA Synthesis Kit (Roche). qPCR was performed
with an ABI Prism 7000 system using SYBR Green PCR Master Mix (Applied
Biosystems) according to the manufacturer's instructions. Amplification of
single products was confirmed by monitoring the dissociation curve.
Gapdh mRNA level was used for normalization. Primer sequences are
available upon request.
Cell culture and transfection assays
Expression vectors for Hand2, Hand1 and Runx2 deletions were generated by
PCR. p6OSE2-luc and the expression vector for Twist1 were described previously
(Ducy and Karsenty, 1995;
Sosic et al., 2003). Reporter
plasmid (100 or 150 ng) and indicated amounts of expression plasmids were
transfected into COS cells using Fugene6 (Roche) with 50 ng of an
RSV-β-galactosidase expression plasmid to monitor transfection
efficiency. Cell extracts were prepared 48 hours after transfection and
assayed for luciferase activity (Promega). To inhibit histone deacetylase
(HDAC) activity, trichostatin A (TSA) (Sigma-Aldrich) at a final concentration
of 100 nM, or an identical amount of ethanol (control), was added 24 hours
before collecting cell lysates. For stable transfections, cells were selected
using 400 µg/ml G418 and individual clones were harvested and amplified
prior to analysis. Western blot analysis and immunostaining verified the
presence of transgene in each clone analyzed, and more than four positive
clones were combined for each stable cell line. For osteoblast differentiation
assays, ROS 17/2.8 cells were grown to confluence and maintained in
β-minimal essential medium (β-MEM) containing 10% FBS, with or
without 10 mM β-glycerophosphate and 50 mg/ml ascorbic acid for the
indicated number of days.
Co-immunoprecipitation (Co-IP) and western blotting
Cell lysates of COS cells transfected with the indicated expression
constructs were harvested in lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM
NaCl, 1 mM EDTA, 1% Triton X-100, 1x Roche complete protease inhibitor
cocktail]. Lysates were incubated with specific-antibody-conjugated Protein
G-Sepharose beads (Zymed) in lysis buffer with 0.1% BSA. Co-IP and western
blotting were performed using rabbit anti-Myc (A14), mouse anti-Myc (9E10)
(Santa Cruz Biotechnology) or mouse anti-Flag (M2; Sigma-Aldrich).
In vitro binding assays
Glutathione S-transferase (GST)-fusion proteins and maltose-binding protein
(MBP)-tagged proteins were isolated from bacterial lysates using glutathione
beads (Amersham) and amylose resin (NEB), respectively, according to
manufacturers' directions. Glutathione beads conjugated with 2 µg of
GST-fusion protein were incubated with 1 µg of MBP-tagged protein in NET
buffer [50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM EDTA, 0.5% NP40, 0.5% BSA].
The beads were then washed and eluted in 10 mM glutathione buffer. Elutes were
subjected to SDS-PAGE, transferred to nitrocellulose membrane and
immunoblotted using anti-Myc antibody.
Chromatin immunoprecipitation (ChIP) assays
ChIP assay was performed as described
(Funato et al., 2003) with
minor modifications. In brief, cells were formaldehyde cross-linked and lysed.
Sheared chromatin was immunoprecipitated with anti-Runx2 antibody (M-70; Santa
Cruz Biotechnology), and DNA was isolated and analyzed by PCR with osteocalcin
gene primers. PCR primer sequences are available upon request.
Statistical analysis
The experimental data were analyzed by two-tailed Student's t-test
and expressed as the mean ± s.e.m. A P-value less than 0.05
was considered significant.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Bone and cartilage staining with Alizarin Red and Alcian Blue, respectively,
revealed hypoplastic mandibles with abnormal ossification in
Hand2BA/BA mice at postnatal day (P) 1
(Fig. 1A). Membranous bones of
the mandible appeared porous and ectopic bony processes were observed on the
ventral surface of the mandible (Fig.
1Ad, arrowheads and arrows). The angular process was severely
reduced in the mutants (Fig.
1Af, agp). The porotic bone phenotype was further confirmed by
micro-CT, which indicated a 52% reduction of mineralized tissue volume in the
Hand2BA/BA mice (Fig.
1C,D). The abnormal membranous bone phenotype provides strong
support for the involvement of Hand proteins in the control of osteoblast
differentiation.
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During intramembranous ossification in the skull and ribs, the release of
negative regulation is thought to be a prerequisite for
Runx2-positive mesenchymal progenitors to initiate osteoblast
differentiation (Bialek et al.,
2004). The abnormal membranous bone in
Hand2BA/BA mice suggested that the primary defect might
involve control of osteoblast differentiation. To precisely define the
molecular defects resulting from the absence of Hand2 in the
branchial arches, we assessed the activity and expression of known osteoblast
differentiation markers: alkaline phosphatase (ALP; Alp1 - Mouse Genome
Informatics), an early osteoblast differentiation marker expressed in
preosteoblasts and post-proliferative osteoblasts; bone sialoprotein
(Bsp; Ibsp - Mouse Genome Informatics), which is expressed
predominantly in osteoblasts; and osteocalcin (Bglap1/2 - Mouse
Genome Informatics), an indicator of mature osteoblasts and osteocytes
(Aubin et al., 1995). At E12.0,
when the mandibular primordium is formed, ALP activity was barely detectable
in wild-type embryos (Fig.
2Aa). By contrast, we detected a larger population of cells
positive for ALP activity in Hand2BA/BA mutants
(Fig. 2Ab). Although no
significant upregulation of Alp or Bsp expression was
detected by qPCR using whole-mandible RNA preparations
(Fig. 2B), these results
suggested that osteoblast differentiation had initiated in mutant embryos at
E12.0. At E13.5, ALP activity was increased in the mandible of
Hand2BA/BA as compared with wild-type embryos
(Fig. 2Ca,d), specifically in
the developing mandibular bone (Fig.
2Ac,d). Bsp and osteocalcin were also expanded in the
mandible of Hand2BA/BA as compared with wild-type
littermates (Fig. 2C), and the
transcripts of all three markers were dramatically increased
(Fig. 2D). Section in situ
hybridization of E15.5 embryos also showed that the zone of Bsp- or
osteocalcin-expressing cells extended further toward the midline in
Hand2BA/BA than in wild-type mandible (see Fig. S1b,c,f,g
in the supplementary material). Since TRAP staining showed no difference in
osteoclast activity in the mutant mandible at E15.5 (data not shown), it is
unlikely that a local increase in bone resorption was solely responsible for
the porotic change. Furthermore, no difference was observed in the
proliferation or apoptosis of mesenchymal cells in the mandibular arch between
wild-type and Hand2BA/BA embryos at E11.5 and E13.5 (data
not shown). Taken together, these findings indicate that Hand2
insufficiency results in precocious and accelerated osteoblast differentiation
in the mandible, which in turn leads to insufficient mineralization of the
mandible.
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;
Garg et al., 2005;
Komori et al., 1997). Runx2
also binds its own promoter, establishing a positive autoregulatory loop
(Ducy et al., 1999). Since
ectopic bone formation was observed in Hand2BA/BA embryos
(Fig. 1Ad), we speculated that
the absence of Hand2 affected the expression of Runx2. Consistent
with this hypothesis, the Runx2 expression domain was expanded in the
mandible of the mutant at E13.5 (Fig.
2Ea,c) and E15.5 (see Fig. S1d,h in the supplementary material),
and the Runx2 transcript level was markedly increased at E13.5
(Fig. 2F). These findings
suggested that misregulation of Runx2 expression/activity is an
underlying molecular defect of abnormal intramembranous ossification in
Hand2BA/BA mice.
Hand2 and Runx2 expression during osteoblast differentiation
We performed in situ analysis to determine whether Hand2 is
expressed in a pattern compatible with a regulator of osteoblast
differentiation. First, we analyzed Hand1, Hand2 and Runx2
expression in developing wild-type mandibles at E12.5 using whole-mount in
situ hybridization. All three genes are expressed in the mandibular arch
(Fig. 3A) and lacZ
expression driven by the 11 kb Hand2 promoter recapitulates
endogenous expression (Fig.
3Ad) (Charité et al.,
2001).
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), and no skeletal abnormalities are observed in
Hand1loxP/loxP; Wnt1-Cre mice
(Barbosa et al., 2007), we
focused our attention on Hand2. We examined, by in situ hybridization, the
localization of Hand2 and Runx2 in the mandibular component
of the first branchial arch prior to initiation of mandibular ossification. At
E11.5, Runx2 was expressed in the mandibular primordium
(Fig. 3Ba) and Hand2
expression modestly overlapped with the medial aspect of the Runx2
expression domain in wild-type embryos
(Fig. 3Bb). In
Hand2BA/BA embryos, the Runx2 expression domain
was slightly expanded (Fig.
3Bc; Fig. 2E) and
Hand2 was absent from the corresponding region of the mandibular
arch, although Hand2 expression in the tongue and surrounding
connective tissue was maintained (Fig.
3Bd). At E12.5, Hand2 expression was decreased, and only
overlapped with Runx2 in the most medial component of the mandibular
primordium (Fig. 3Be,f).
Although we could not detect broad overlap between Hand2 and
Runx2 in the mandibular primordium, we could not formally exclude the
possibility that Hand2 expression was below the detection limit of in
situ hybridization. Expression analysis using Hand2-lacZ embryos,
which recapitulate endogenous Hand2 expression
(Ruest et al., 2003), showed
that the mandibular bone and surrounding connective tissues were positive for
lacZ, exhibiting considerable overlap with Runx2 expression
at E12.5 (compare Fig. 3Be with
Fig. S2a in the supplementary material). At E14.5, when osteoblast
differentiation and intramembranous ossification occurs, lacZ
expression was not detected in the mandible (see Fig. S2b, arrow, in the
supplementary material). These data demonstrate that Hand2 is
transiently expressed in the primordium of mandibular bone and that
Runx2 induces intramembranous ossification after the decline in
Hand2 expression.
Hand2 is sufficient to inhibit osteoblast differentiation in vitro
Next, we asked whether Hand2 directly affects osteoblast differentiation.
We stably transfected rat osteosarcoma ROS 17/2.8 osteoblastic cells with
Hand2 or empty expression vectors. ROS 17/2.8 cells express markers for
osteoblast progenitors, such as type I collagen
(Bialek et al., 2004), and
markers for differentiated osteoblasts, such as osteocalcin
(Ducy et al., 1997), both of
which are Runx2 target genes, but do not express Hand1 or
Hand2 (data not shown). Upon induction of osteoblast differentiation
in β-MEM medium with or without β-glycerophosphate and ascorbic
acid, control ROS 17/2.8 cells sufficiently differentiated by day 5
(Fig. 3C). By contrast, in
Hand2-overexpressing cells, differentiation was delayed. This was
accompanied by marked reduction of Alp, osteocalcin and
Runx2 transcripts (Fig.
3D). These experiments indicate that Hand2 inhibits the initiation
of osteoblast differentiation in vitro.
Hand proteins are Runx2 inhibitors
Hand2BA/BA mice exhibit ectopic bone formation and
accelerated osteoblast differentiation in the first branchial arch. This
phenotype, coupled with our observation that Hand2 is expressed in
mandibular primordium, led us to speculate that the initiation and/or
progression of osteoblast differentiation might require release from
Hand-mediated repression of Runx2. To address this possibility, we tested the
effect of Hand proteins on the transcriptional activity of Runx2 in COS cells.
Hand1 and Hand2 inhibited Runx2-dependent activation of luciferase activity
through tandem copies of the Runx2 binding site (p6OSE2-luc) in a
dose-dependent manner (Fig.
4A). To examine homo- or heterodimerization of Hand1 and Hand2 as
a possible mechanism for repression of Runx2 activity, we used Hand2 with a
phenylalanine-to-proline mutation in the first helix (mutant F119P), which is
unable to dimerize with ubiquitous bHLH E-protein E12 (Tcfe2a - Mouse Genome
Informatics) (McFadden et al.,
2002). Hand2 (F119P) was unable to dimerize with Hand1 or Hand2
(Fig. 4C); however, it still
inhibited Runx2 transactivation with Hand1 or Hand2
(Fig. 4B). Furthermore, Hand2
(
HLH), which lacks the domain necessary for dimerization with other
bHLH proteins, showed similar results (Fig.
4B), confirming that neither a functional HLH motif nor
dimerization with other bHLH proteins was required for Hand2 inhibitory
function.
To determine the specificity of Hand proteins in Runx2 inhibition, we
tested the Hand-related bHLH proteins paraxis (Tcf15)
(Burgess et al., 1995) and
capsulin (Tcf21) (Funato et al.,
2003) in this assay. Paraxis is 62% identical and 78% similar to
Hand2 within the HLH domain (Burgess et
al., 1995). Neither of these proteins was able to inhibit Runx2
transactivation function in this assay. Finally, we examined Twist1, which is
abundantly expressed in developing branchial arches and was previously shown
to dimerize with Hand2 upon phosphorylation and to function antagonistically
to Hand2 (Firulli et al.,
2005; Fuchtbauer,
1995). Although the expression of Twist1, Hand1 and Hand2 was
comparable (data not shown), Twist1 itself only marginally inhibited
Runx2-dependent transcription activity
(Fig. 4B), and did not
antagonize the Hand1 or Hand2 effect on Runx2 activity (data not shown). Taken
together, these results indicate that Hand proteins possess a unique property
that is not shared by the related bHLH protein paraxis, and that the
inhibitory effects of Hand proteins are independent of dimerization with bHLH
proteins.
|
The transcriptional co-factors CREB-binding protein (CBP)/p300 (Crebbp -
Mouse Genome Informatics) and four and a half LIM domains 2 (Fhl2), a member
of the LIM-only subclass of the LIM protein superfamily, interact with Runx2
and increase its transcriptional activity
(Gunther et al., 2005;
Sierra et al., 2003). CBP/p300
physically interacts with Hand2 (Dai and
Cserjesi, 2002) and Fhl2 is known to bind Hand1 and inhibit
Hand1/E12 coactivation of a target gene reporter
(Hill and Riley, 2004).
However, neither CBP/p300 nor Fhl2 affected the ability of Hand2 to repress
activation of Runx2-responsive reporters
(Fig. 5D). Histone deacetylases
(HDACs) are also known to directly interact with Runx2 and inhibit its
transactivation activity (Vega et al.,
2004); however, Hand-mediated repression was unaffected by the
HDAC inhibitor TSA, suggesting that the repression was independent of HDAC
catalytic activity (Fig.
5E).
The N-terminal domain of Hand2 inhibits Runx2 by binding to its Runt DNA-binding domain
We next searched for Runx2 domains that interact with Hand2. All Runx2
deletion mutants used in immunoprecipitation experiments contained an
endogenous nuclear localization sequence (NLS)
(Fig. 6A) and were expressed in
nuclei (data not shown). Hand2 was immunoprecipitated by Runx2 and all
deletion mutants except Runt, which lacked the Runt DNA-binding domain
(Fig. 6A,B). Conversely, the
Runt domain alone was sufficient to bind Hand2
(Fig. 6A,B). These experiments
establish that the Runt DNA-binding domain is necessary and sufficient for
interaction with Hand2.
To identify the regions of Hand2 responsible for the inhibition of Runx2
function, we generated a series of Hand2 deletion mutants
(Fig. 6C). Although some were
also expressed in the cytoplasm, all deletion mutants were detectable and
predominantly localized in the nucleus
(Fig. 6E). Runx2 was
immunoprecipitated by Hand2 deletion mutants 155-217, HLH and
basic, but not 1-90 (Fig.
6D). Interestingly, although the C-terminal amino acids of Hand2
are highly conserved among all orthologs of Hand proteins, deletion of this
region (155-217) still resulted in a marked reduction in the transcriptional
activity of Runx2 (Fig. 6F). By
contrast, 1-90 failed to inhibit Runx2 transactivation function, and
further analysis of the N-terminus showed that the Runx2 inhibitory domain of
Hand2 was localized to amino acids 32-59
(Fig. 6C,F). Indeed, deletion
of this region (Hand2 32-59) completely abolished the binding and
inhibition of Runx2 (Fig.
6D,F).
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32-59) was unable to interact with
Runx2 (see Fig. S3B in the supplementary material). Although Hand1
(32-59) and Hand1 (1-87) mutants localized to the nucleus (see
Fig. S3C in the supplementary material), they failed to inhibit Runx2-mediated
transcriptional activity (see Fig. S3D in the supplementary material). We
conclude that Hand protein binding to Runx2 is responsible for repression of
Runx2 activity (Fig. 6C).
Finally, to determine whether direct DNA binding is required for Hand2
activity in our transactivation assay, we used a DNA-binding mutant of Hand2
(RRR109-111EDE) (McFadden et al.,
2002) in which three conserved arginine residues in the basic
region were mutated (Fig. 6C).
As Fig. 6F shows, the
RRR109-111EDE mutant was a potent repressor of Runx2, indicating that Hand2
does not require direct DNA binding to inhibit Runx2 transactivation
function.
Detection of Hand2-Runx2 interaction by ChIP
The observation that Hand2 interacts with the Runx2 DNA-binding domain
suggested that Hand2 might also inhibit the DNA-binding activity of Runx2. To
address this, we examined the effect of Hand2 on Runx2 association with the
osteocalcin promoter by ChIP of extracts from ROS 17/2.8 cells stably
transfected with full-length Hand2, Hand2 (1-90) or Hand2
(32-59) expression vectors. As shown in
Fig. 6Ga,b, the association of
Runx2 with the osteocalcin promoter was readily detectable by ChIP assay in
the presence of Hand2 (1-90) or Hand2 (32-59), and this
interaction was abolished in the presence of wild-type Hand2. We conclude that
the association of Hand2 with the Runt domain of Runx2 interferes with DNA
binding by Runx2 and, consequently, inhibits activation of Runx2 target genes
(Fig. 7A).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Regulation of osteoblast differentiation by Hand proteins
Our current findings, coupled with recently published work, suggest that
Hand proteins have multiple roles in craniofacial development, as depicted in
Fig. 7B. Hand proteins are
involved in the early development of postmigratory neural crest cells by
maintaining and/or patterning subsets of neural crest cells. Failure to
maintain the distal midline mesenchyme in Hand1/2 compound mutant
embryos (Barbosa et al., 2007),
and the interrupted Meckel's cartilage and hypoplastic angular process in
Hand2BA/BA mice, may be attributable to the loss of Hand
functions in patterning or maintenance of neural crest cells at the early
post-migratory stage.
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). However,
we cannot formally exclude the possibility that abnormal osteoblasts in
Hand2BA/BA embryos affected the function of osteoclasts,
which are linked to osteoblast differentiation
(Engin et al., 2008). Taken
together, these data suggest a model in which Hand2 controls proper temporal
and spatial expression of osteoblast differentiation genes by regulating
Runx2.
Mechanism of Runx2 repression by Hand proteins
The expression and activity of Runx2 must be regulated to prevent aberrant
ossification or delayed bone development. Our current study establishes that
Hand proteins tightly regulate Runx2 activity in the branchial arches. We
propose two mutually compatible mechanisms for the regulation of Runx2 by Hand
proteins. First, Runx2 is a primary target of Hand2 repression in vivo. The
absence of Hand2 might accelerate the positive autoregulatory loop of Runx2.
Second, Hand2 physically interacts with Runx2 and inhibits its ability to
transactivate promoters of osteoblast-specific Runx2 target genes. This
repression does not require recruitment of HDAC proteins or HDAC activity and
no interaction was detected between Hand2 and Hdac5, a class II HDAC expressed
in the developing craniofacial skeleton (data not shown). Rather, the
inhibitory function is through direct interaction between Hand2 and Runx2, as
we observed by ChIP assays. The repressive influence of Hand proteins must
then be released in a timely manner for Runx2 to bind osteoblast-specific gene
promoters and execute osteoblast differentiation in the branchial arch.
Interestingly, this type of repression that causes `sequestration' of
DNA-binding factors in the nucleus is similar to previously reported
inhibitory functions of Hdac4 and Twist1 on Runx2
(Bialek et al., 2004;
Vega et al., 2004). However,
the biological impact of Hdac4 and Twist1 inhibition is distinct from that of
Hand proteins in several aspects. Hdac4 expression is restricted to
chondrocytes and Hdac4-null mice show acceleration of chondrocyte
hypertrophy and, consequently, precocious endochondral mineralization
(Vega et al., 2004). By
contrast, the phenotype of Hand mutant mice reflects a defect in osteoblast
differentiation and maturation in the branchial arch. The possibility exists
that Hand2 affects chondrocyte maturation by regulating the antagonistic
function of Runx2 on Fgf18 at the perichondrium
(Hinoi et al., 2006) and/or on
Indian hedgehog, which is a target gene of Runx2 that is expressed in
pre-hypertrophic chondrocytes (Yoshida et
al., 2004), as Hand2 is also expressed in these regions
of the developing ulna (McFadden et al.,
2002). Whereas both Hdac4-null mice and Hand2
mutant mice show ectopic bone formation with upregulation of Runx2
expression in vivo, Twist1 inhibits osteoblast differentiation in the skull
and ribs without affecting Runx2 expression and without the
development of ectopic bones or insufficient mineralization
(Bialek et al., 2004).
Hand2 function is independent of DNA binding and dimerization
The bHLH domain of Hand proteins has been considered to be essential for
their function. However, our data show that the repressive function of Hand2
on Runx2 does not require the basic region, suggesting that the underlying
mechanism is independent of E-box-mediated transcriptional regulation. In
addition, Hand2 mutants lacking DNA-binding activity (RRR109-111EDE) or HLH
protein-binding activity (F119P) are as potent as the wild-type protein in
inhibiting Runx2-dependent transcriptional activation, indicating that Hand2
uses a mechanism independent of direct DNA binding or dimerization with other
bHLH proteins. Antagonistic interaction between Hand2 and Twist1 has been
implicated in Saethre-Chotzen syndrome
(Firulli et al., 2005).
However, Twist1 did not antagonize the effect of Hand1 or Hand2 on Runx2
transcriptional activity. Our results clearly show that Hand proteins can use
a mechanism that is different from the conventional functional model of bHLH
proteins to exert their functions according to cell type and biological
process.
Function of the N-terminal domain of Hand2
Molecular evidence establishes that the anti-osteogenic function of Hand
proteins is mediated by an N-terminal domain that is distinct from its bHLH
domains. We showed that Hand2 (32-59) and Hand1 (32-59) could
not inhibit Runx2 transactivation function. hand2 zebrafish mutant
alleles were identified in a screen for mutations affecting heart, jaw and
pectoral fin development, including a null allele completely deleting the
hand2 locus (hans6) and a truncated allele
lacking the N-terminal domain (hanc99)
(Yelon et al., 2000).
Interestingly, the hanc99 mutant exhibited continuous
upper jaw joints, and the deletion is equivalent to amino acids 1-53 of mouse
Hand2 (Miller et al.,
2003). Although the N-terminal domain of Hand2 is conserved among
jawed vertebrates, including stickleback, pufferfish and zebrafish, it is not
conserved in fruit fly or sea squirt (our unpublished observations),
suggesting that this N-terminal region might exert a crucial biological
function required for jaw formation. One may speculate that gene elongation at
the N-terminus of Hand might have occurred upon evolution of vertebrates from
lower chordates. Sequence analysis of Hand genes in agnathans might provide a
link between the N-terminal domains of Hand genes and the appearance of neural
crest and/or jaw development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/4/615/DC1
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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