1 Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive
Cancer Center and Hospital, Keck School of Medicine, University of Southern
California, 1441 Eastlake Avenue, Los Angeles, CA 90089-9176, USA
2 Department of Morphology, Faculty of Medicine, Ben Gurion University of the
Negev, Beer Sheva, Israel
3 Department of Craniofacial Development, King's College, London, UK
4 Institute for Genetic Medicine, Keck School of Medicine, University of
Southern California, 1441 Eastlake Avenue, Los Angeles, CA 90089-9176,
USA
* Author for correspondence (e-mail: maxson{at}hsc.usc.edu)
Accepted 11 August 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Skull vault, Calvarial foramina, Msx2, Twist, Neural crest, Mouse
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The development of the skull vault is of interest not only because it
provides a model of how mesenchymal populations produce patterned structures,
but also because of its relevance to human disease
(Wilkie, 1997;
Cohen and MacLean, 2000
;
Wilkie and Morriss-Kay, 2001
).
Anomalies in skull vault development are common in humans, occurring as
frequently as 1 per 2500 live births (Cohen
and MacLean, 2000
). Among these are craniosynostosis and
persistent calvarial foramina. Craniosynostosis is the premature fusion of the
calvarial bones at the sutures. Persistent calvarial foramina are defects in
the ossification of bones of the skull vault. Several genes responsible for
one or both of these defects have been identified
(Wilkie, 1997
;
Wilkie and Morriss-Kay, 2001
;
Ornitz and Marie, 2002
). These
include FGF receptors 1, 2 and 3 (Jabs et
al., 1994
; Muenke et al.,
1994
; Reardon et al.,
1994
; Meyers et al.,
1995
), the basic HLH gene, Twist
(Wilkie, 1997
), and the
homeobox genes Msx2 and Alx4
(Wilkie and Morriss-Kay, 2001
;
Wilkie et al., 2001
). A
gain-of-function mutation in Msx2 can cause craniosynostosis
(Jabs et al., 1993
).
Heterozygous loss of Msx2 function results in persistant foramina in
the skull vault (Wilkie et al.,
2000
; Wuyts et al.,
2000b
). More recently, haploinsufficiency for Alx4 has
been shown to cause calvarial foramina (Wu
et al., 2000
; Wuyts et al.,
2000a
; Mavrogiannis et al.,
2001
). Intriguingly, heterozygous loss of Twist function
can cause craniosynostosis, and, in some affected individuals, calvarial
foramina (el Ghouzzi et al.,
1997
; Howard et al.,
1997
; Thompson et al.,
1984
; Young and Swift,
1985
). Thus, in humans, Twist is required to prevent
premature suture fusion, and for the normal growth of the calvarial bones.
Similarly, Msx2 is required for calvarial bone growth, and, when
carrying a gain-of-function (P146H) mutation, can cause fusion of calvarial
bones.
Analysis of transgenic mice and targeted mouse mutants has provided results
that parallel findings in humans. Twist mutant mice have synostosis
of the coronal suture (el Ghouzzi et al.,
1997; Howard et al.,
1997
; Carver et al.,
2002
). Overexpression of Msx2 under the control of its
own promoter or heterologous promoters causes overgrowth of the bones of the
skull vault, a phenotype that may mimic the early stages of synostosis
(Liu et al., 1999
). Similarly,
Msx1 and Msx2 mutant mice exhibit calvarial foramina
(Satokata and Maas, 1994
;
Satokata et al., 2000
).
Beyond the identification of these genes and basic descriptions of their mutant phenotypes in humans and mice, there is little information about their roles in the cellular processes underlying normal skull vault development or the pathogenesis of craniosynostosis and calvarial foramina. Neither is it clear whether these genes function in the same or distinct developmental pathways, an issue of significance not only for understanding basic developmental mechanisms but also in the search for modifier genes in humans that can influence the penetrance and expressivity of developmental anomalies. We investigate both of these issues, focusing on the role of Msx2 in skull vault development, and a potential interaction between Msx2 and Twist.
We trace the origin of the calvarial foramen defect in Msx2
mutants to a group of skeletogenic mesenchyme cells that compose the frontal
bone anlagen. We show that this cell population is reduced because of defects
in differentiation and proliferation not because of apoptosis or
deficient migration of neural crest-derived precursor cells. We demonstrate,
in addition, that heterozygous loss of Twist function causes a
foramen in the skull vault resembling that resulting from heterozygous loss of
Msx2. This defect is substantially worse in
Msx2-Twist double heterozygous embryos than in individual
heterozygotes. Underlying this worsening of the skull vault defect are further
deficiencies in both the differentiation and proliferation of frontal bone
skeletogenic mesenchyme. Finally, both genetic and molecular data show that
Msx2 and Twist do not function in a simple, linear pathway
but rather act in parallel. We suggest that Msx2, a target of the BMP
pathway (Vainio et al., 1993),
and Twist, a target of the FGF pathway
(Rice et al., 2000
), integrate
inputs from these two pathways to cooperatively control the differentiation
and proliferation of neural crest-derived skeletogenic mesenchyme and thus the
patterning of the frontal bone.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Histology and immunostaining
To visualize mineralized bone, skullcaps of postnatal day 4 mice were
dissected and stained with a solution of Alizarin Red S (50 mg/l in 0.2% KOH)
for 48 hours. Skulls were then cleared with glycerol.
For whole-mount histochemical staining for alkaline phosphatase, embryos
were fixed in 4% paraformaldehyde in PBS. E12.5 and E14.5 embryo heads were
bisected midsagitally, and the brain and associated dura were removed, leaving
intact the presumptive calvarial bones and epithelium. In the case of E16.5
embryos, the skin was also removed. The specimens were washed with NTMT (0.1 M
NaCl, 0.1 M Tris-HCl pH 9.5, 50 mM MgCl2, 0.1% Tween20), then
stained with NBT and BCIP (Roche). Detection of alkaline phosphatase in tissue
sections was carried out as described previously
(Liu et al., 1999). Apoptotic
cells were detected by means of the TUNEL assay using an In Situ Cell Death
Detection Kit (Roche). FITC signals were visualized by confocal microscopy.
Osteoclast activity was detected by staining for tartrate-resistant acid
phosphatase (TRAP) using the Sigma Acid Phosphatase, Leukocyte Kit
(Rajapurohitam et al.,
1997
).
Analysis of Wnt1-Cre/R26R reporter gene expression was carried out
largely as described previously (Jiang et
al., 2002). ß-galactosidase staining was performed on embryo
whole mounts or 10 µm cryosections. In some experiments, whole
mount-stained embryos were embedded in paraffin wax, sectioned (6 µm) and
counterstained with Nuclear Fast Red.
For immunostaining of frozen sections, embryos were fixed with 4% paraformaldehyde, embedded in HistoPrep (Fisher Scientific) and sectioned in a cryostat (10 µm). Immunohistochemistry was performed using Zymed Histostain-SP anti-rabbit and phosphorylated Histone H3 polyclonal antibody (Upstate, 1:200 dilution) according to the manufacturer's instructions.
In situ hybridization
A Twist 5' cDNA fragment excluding bHLH domain was obtained
by BamHI and PstI digestion of CMV-M-Twist
(Hamamori et al., 1997). This
fragment was subcloned into pBluescriptSKII(+) and used as a template for
synthesis of riboprobes. The Msx2 probe, consisting of the entire
first exon, was amplified from a full-length mouse cDNA by PCR and cloned into
the EcoRI and SalI sites of pBSKII(+). In situ hybridization
probes for Runx2 and Bsp were as described
(Ducy et al., 1997
;
Rice et al., 1999
).
Radioactive in situ hybridization was performed as described
(Kim et al., 1998
;
Rice et al., 2000
).
Whole-mount in situ hybridization was performed as described by Hogan et al.
(Hogan et al., 1994
).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
We next sought to understand why the frontal bone primordia are reduced in size in Msx2 mutants. One possibility is that cells that compose the primordia undergo apoptosis at an increased rate. We carried out TUNEL assays for apoptotic cells to assess survival of skeletogenic mesenchyme cells in Msx2 mutants. Examples of such stains are shown in Fig. 3. Relatively few positive cells were evident in frontal bone rudiments of wild-type embryos at E12.5, and no difference was apparent between Msx2 mutants and wild-type controls. Similar results were obtained E13.5 and postnatal day 4 (data not shown). These data suggest that apoptosis does not contribute significantly to the deficiency of frontal bone osteogenic cells in Msx2 mutants.
|
|
Reduced proliferation of osteogenic cells in the frontal bone
rudiment of Msx2 mutants
Data presented thus far suggest that Msx2 mutants have a defect in
the transition of undifferentiated neural crest-derived mesenchyme to
Runx2-expressing skeletogenic mesenchyme. Previous studies
established that forced expression of Msx genes can promote cell proliferation
(Dodig et al., 1999;
Hu et al., 2001
), and that
inactivation of Msx2 results in reduced proliferation of osteoblastic
cells in metopic sutures of postnatal Msx2 mutant mice
(Satokata et al., 2000
). These
studies raise the possibility that, in addition to defects in the
differentiation of the frontal bone skeletogenic mesenchyme, Msx2
mutants may also have defects in the proliferation of this cell population. To
test this idea, we monitored a phosphorylated form of histone H3, a marker of
M phase. Phosphorylation of histone H3 at serine 10 accompanies and is
required for chromosome condensation during mitosis
(Hendzel et al., 1997
). Using
an antibody specific for serine 10-phosphorylated H3, we assessed
proliferation within the population of ALP-expressing cells composing the
frontal bone rudiment. We focused on an interval spanning the stage at which
the deficiency in the differentiation of skeletogenic mesenchyme was first
evident (Fig. 5). This interval
was prior to the appearance of a morphologically distinct osteogenic front,
defined as a discreet population of proliferative osteogenic cells located at
the edge of the growing bone. At E12.5, we did not detect a difference between
mutant and wild-type mice in the number of 10-phosphorylated H3 positive cells
per unit area of ALP expression (Fig.
5A-E). However, by E14.5, a significant difference was evident
(Fig. 5F-J). These results
suggest (1) that Msx2 mutants have a defect in the proliferation of
the skeletogenic mesenchyme, and (2) that this proliferation defect occurs
after the defect in the differentiation of the frontal bone skeletogenic
mesenchyme. Together, our results show that loss of Msx2 function
results in a decrease in the proportion of proliferative skeletogenic
mesenchyme cells within the frontal bone rudiment between E12.5 and E14.5.
Msx2 therefore is required for the differentiation and subsequent
proliferation of the skeletogenic mesenchyme.
|
We examined Twist mutant mice to determine whether they exhibited
a defect in frontal bone development similar to that of Msx2 mutant
mice and individuals with Saethre-Chotzen syndrome. Mice homozygous for a null
mutation in Twist die at E11 with vascular and neural tube defects
(Chen and Behringer, 1995).
Twist+/ mice are viable and, like individuals with
Saethre-Chotzen syndrome, have craniosynostosis
(el Ghouzzi et al., 1997
;
Carver et al., 2002
). Such mice
also have anterior digit duplications
(Bourgeois et al., 1998
).
Alizarin Red staining of skulls of P4 Twist+/ mice
revealed a subtle but highly penetrant defect in the posterior region of the
frontal bone (Fig. 6A,D).
|
Msx2 and Twist are also co-expressed in developing limb
(Coelho et al., 1991;
Davidson et al., 1991
;
Robert et al., 1991
;
Fuchtbauer, 1995
;
Stoetzel et al., 1995
),
prompting us to ask whether the Msx2 genotype affected the penetrance
of the anterior digit duplication characteristic of
Twist+/ mice
(Bourgeois et al., 1998
). The
incidence of the digit duplication phenotype in
Twist+/ mice (34%, n=58) was identical to
that in Msx2+/; Twist+/
mice (34%, n=109). Thus, Msx2 and Twist did not
interact genetically in the developing limb, despite being co-expressed
there.
Msx2 and Twist cooperatively control the
differentiation and proliferation of the frontal bone skeletogenic
mesenchyme
We knew from our analysis of Msx2 mutant embryos that defects in
the differentiation and proliferation of the skeletogenic mesenchyme were
associated with the frontal foramen. We sought to determine whether a decrease
in Twist dose specifically made these defects more severe. As is
evident in Fig. 7, the
Msx2+/; Twist+/ genotype
resulted in further reductions over Msx2 individual mutants in the
domains of ALP staining and Runx2 expression in the frontal bone
rudiment of E12.5 embryos (Fig.
7A-D). Wnt1-Cre/R26R analysis at E12.5 showed no
difference in neural crest distribution in Msx2-Twist
compound mutants compared with individual Msx2 mutants
(Fig. 7E,F). Nor was there an
increase in numbers of apoptotic cells in Msx2-Twist double
mutants (data not shown).
|
We sought to understand the molecular mechanism by which Msx2 and Twist cooperate in the control of the development of the skeletogenic mesenchyme (Fig. 8). An initial question was whether they function in a simple linear pathway, in which one controls the other's expression. In situ hybridization showed that at E10.5, Msx2 and Twist are co-expressed in presumptive frontal bone mesenchyme (Fig. 8A,G). By E11.5, Twist is expressed broadly in the frontonasal neural crest, while Msx2 is expressed in a band of cells representing the primordia of the frontal and parietal bones (Fig. 8C,I). At E12.5, Msx2 and Twist are co-expressed in the preosteogenic and osteogenic mesenchyme of the frontal bone (Fig. 8E,K). Thus, in principle either gene could regulate the expression of the other. However, in situ hybridization experiments showed that at E10.5, E11.5 and E12.5, the distribution of Msx2 transcripts was not significantly different in Twist+/ mice than in controls (Fig. 8A-F; data not shown). Similarly, analysis of Twist expression in Msx2 mutant embryos showed no apparent change in E10.5, E11.5 and E12.5 embryos (Fig. 8G-L). These data suggest that, within the limits of resolution of in situ hybridization, Msx2 and Twist do not regulate each other's activity at the level of mRNA abundance.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Msx2 is required at or before E12.5 for the development of
the frontal bone skeletogenic mesenchyme from neural crest precursors
Several studies have linked Msx genes to the control of the timing of
cellular differentiation and proliferation
(Woloshin et al., 1995;
Liu et al., 1999
;
Odelberg et al., 2000
;
Hu et al., 2001
). Virally
mediated overexpression of Msx2 in chick primary calvarial
osteoblasts caused an increase in proliferation and slowed differentiation
(Dodig et al., 1999
).
Reciprocally, expression of antisense Msx2 mRNA caused more rapid
differentiation and a reduction of proliferation
(Dodig et al., 1999
).
Abate-Shen and colleagues found that overexpression of Msx1 or
Msx2 in several different cell lines inhibits differentiation and
causes upregulation of cyclin D1 (Hu et
al., 2001
). We showed, in addition, that transgenic overexpression
of Msx2 causes an increase in the number of proliferative osteoblasts
in the osteogenic front of postnatal mice
(Liu et al., 1999
). These
studies have led to the view that a normal function of Msx2 is to
maintain cells in an undifferentiated proliferative state. Accordingly, we
would expect calvarial osteogenic cells of Msx2 mutant mice to
exhibit both premature differentiation and reduced proliferation. Our findings
differ from this expectation in that we did not see any evidence of premature
differentiation at any stage. Furthermore, although we did see an effect on
proliferation at later stages (E14.5 (Fig.
5) and newborn (Satokata et
al., 2000
)), no such effect was evident at E12.5. Thus, the
proliferation of osteogenic cells was sensitive to Msx2 dosage only
at later stages.
That knockdown of Msx2 activity causes premature differentiation of cultured osteoblasts, although targeted inactivation of Msx2 in the mouse has no such effect may be due to a difference in the behavior of osteogenic cells derived from late-stage embryos versus early embryos used in our study. Alternatively, this discrepancy could be a consequence of a selection during the isolation of calvarial osteoblasts. Msx2 may function differently in the subpopulation of osteogenic cells placed in culture than in osteogenic cells in vivo.
In addition to its effect on the proliferation of the skeletogenic mesenchyme, Msx2, in principle, could influence the number of skeletogenic mesenchyme cells through effects on the survival of such cells or their neural crest precursors. However, TUNEL assays at several developmental stages from E12.5 to postnatal day 4 failed to provide evidence for increased apoptosis in Msx2 mutants, arguing against the idea that effect on cell survival contributes significantly to the frontal bone defect.
In Msx2 mutants, a reduction in the expression domain of Runx2 is evident as early as E12.5, the earliest that Runx2 expression could be detected. Changes in ALP activity, another early marker of osteoblasts, are also apparent at E12.5. These differences in the number of cells expressing early osteoblast marker genes could result from a defect in either the specification or differentiation of the skeletogenic mesenchyme. They could also result from defective migration of the subpopulation of neural crest precursor cells that ultimately give rise to the frontal bone anlagen. As markers specific for this precursor cell population are not available, we could not follow the development of cells allocated to the skeletogenic mesenchyme prior to E12.5. However, we were able to examine the influence of Msx2 genotype on neural crest in general by means of the Wnt1-Cre/R26R system.
Analysis of the distribution of neural crest in embryos from E9.5 to newborn led to two important conclusions. First, at E12.5 neural crest cells are present in frontal bone anlagen of Msx2 mutants in normal numbers. Second, by E16.5, prospective frontal bone and frontal suture neural crest lineage cells are distributed normally in Msx2 mutants. These data argue against gross defects in neural crest as a cause of the frontal bone defect. That there is a normal number of neural crest cells in mutants at E12.5, but a reduction in ALP expressing cells at that same stage, is consistent with a defect in the differentiation of osteogenic cells from neural crest precursors. One caveat in this interpretation is that because the Wnt1-Cre/R26R system marks all neural crest cells, our data do not exclude mis-migration or mis-specification events. Such events might result, for example, in a mixed population of neural crest cells in the frontal bone rudiment, including some not competent to differentiate into osteoblasts. Our data also cannot exclude an effect on the proliferation of a small subpopulation of neural crest cells as the cause of the reduction in the number of ALP-positive cells at E12.5.
Despite these caveats, it is interesting that the apparent requirement for
Msx2 in the differentiation of the frontal bone skeletogenic
mesenchyme parallels findings on the function of the msh gene of
Drosophila. Isshiki, Nose and colleagues
(Isshiki et al., 1997;
Nose et al., 1998
) have shown
that msh is required for the development subsets of dorsal
neuroblasts and muscle progenitors. msh does not participate in the
initial specification of these cells, but in the realization of the
differentiated phenotype. Msx2 may play an analogous role in the
development of the frontal bone skeletogenic mesenchyme, controlling its
differentiation into osteogenic cells. We note, however, that
Msx1/Msx2 double mutant embryos have profound defects in neural
crest-derived craniofacial structures, leaving open the possibility that
Msx1 and Msx2 may be required together for earlier events in
neural crest specification or migration
(Satokata et al., 2000
) (M.I.
and R.E.M., unpublished).
Msx2 is likely to have a role in osteogenic cell populations
outside the frontal bone skeletogenic mesenchyme. Satokata et al.
(Satokata et al., 2000)
documented a deficiency in long bone osteoblasts in Msx2 mutants. In
addition, Msx2 mutant mice have a subtle defect in the parietal bone
(Figs 1,
6; data not shown). It is
interesting that calvarial defects in humans with heterozygous loss of
Msx2 function are usually located in the parietal bone
(Wilkie et al., 2000
). This
difference between mice and humans in the relative severity of the parietal
and frontal bone defects may reflect a species difference in the Msx gene
dosage requirements for the development of different calvarial bones.
Msx2 and Twist cooperatively control the
differentiation and proliferation of the frontal bone skeletogenic
mesenchyme
To understand the biological significance of the genetic interaction
between Msx2 and Twist, it was important to know whether the
two genes affect frontal bone patterning through the same development
processes. That reduction of Msx2 or Twist gene dosage
specifically exacerbated the defects in the differentiation and proliferation
of skeletogenic mesenchyme cells composing the frontal bone rudiment is
significant: it narrows the possible cooperative functions of Msx2
and Twist to the same development interval and the same set of
processes. Although our data do not address the molecular pathways through
which Msx2 and Twist exert their effects, they do enable us
to conclude that Msx2 and Twist function cooperatively in
the processes of differentiation and proliferation.
We note that at E12.5, reduced Twist dosage, with or without an
Msx2 mutation, did not affect levels of apoptosis. This is perhaps
surprising in light of findings that Twist can protect against
Myc-induced apoptosis in Rat1 cells
(Maestro et al., 1999), and
that cultured calvarial osteoblasts derived from the fused coronal suture of
an individual with Saethre-Chotzen syndrome with a heterozygous loss of
function in Twist exhibit increased sensitivity to Tnf-induced
apoptosis (Yousfi et al.,
2002
). This difference in the apoptotic behavior of cultured
osteogenic cells compared with frontal bone skeletogenic mesenchyme, in vivo,
may reflect differences between conditions in cell culture versus those in the
whole animal, or the potentially distinct embryological origins of coronal
suture osteoblasts from individuals with Saethre-Chotzen syndrome (mixed
mesoderm and neural crest) versus frontal bone osteogenic cells (exclusively
neural crest).
Tam and colleagues (Soo et al.,
2002) have shown that Twist/
embryos exhibit defects in cranial neural crest migration. Transplantation
experiments demonstrated that Twist is required both in neural crest
cells and in paraxial mesoderm for the guidance of migrating neural crest
cells. In addition, Twist is required for neural crest
differentiation, whereas in wild-type embryos, Sox10 expression is
downregulated progressively in migratory neural crest cells, in mutant embryos
it is maintained at a high level, suggesting that neural crest cells may be
arrested at an early stage of differentiation
(Soo et al., 2002
).
Our analysis shows that in both Twist+/ and in
Msx2-Twist double mutant embryos, cranial neural crest cells
are distributed normally, at least as assessed by the Wnt1-Cre/R26R
marker. That Runx2/ALP-expressing cells are reduced in
Msx2-Twist mutants, while neural crest cells populate the
frontal bone anlagen in normal numbers suggests that Msx2 and
Twist cooperate in the differentiation of the skeletogenic mesenchyme
from neural crest precursors. Whether the differentiation of neural crest
cells is arrested at an early stage in Msx2-Twist double
mutants as it apparently is in Twist/
embryos remains to be established. Our data also demonstrate that presumptive
frontal bone cells of E14.5 Msx2-Twist double heterozygous embryos
proliferate at a lower rate than corresponding cells of
Msx2+/ embryos. This result is consistent with
findings linking forced expression of Twist to uncontrolled
proliferation (Maestro et al.,
1999; Gullaud et al.,
2003
; Pajer et al.,
2003
).
Genetic and molecular data provide some insight into the nature of the
cooperation between Msx2 and Twist. That the foramen was
significantly larger in Msx2/;
Twist+/ mice than in
Msx2/ mice argues against a linear pathway
in which one gene is an obligate downstream effector of the other. This view
is also supported by in situ hybridization data on E12.5 embryos showing no
evidence of crossregulatory interactions between Msx2 and
Twist at the mRNA level (Fig.
8). Msx2 and Twist may thus control
proliferation and differentiation of skeletogenic mesenchyme through parallel
pathways. It remains possible, however, that Msx2 and Twist
interact on a level other than transcription. Consistent with such a
possibility are observations that Msx2 and Twist proteins are capable of
interacting both in vitro and in intact cells (Y. Hamamori and R.E.M.,
unpublished), and that such interactions have been documented for other
homeodomain and bHLH proteins (Knoepfler
et al., 1999; Poulin et al.,
2000
). Although we do not know whether Msx2 and Twist interact in
developing embryos, or, if so, whether this interaction is significant
functionally, the idea of a direct, cooperative protein-protein interaction
does provide a molecular-level hypothesis to explain the cooperativity between
Msx2 and Twist in skull vault development. We note that
Twist has been shown to inhibit acetyltransferase activities of p300 and PCAF
through interactions with their HAT domains
(Hamamori et al., 1999
). In
addition, Msx proteins can serve as transcriptional repressors
(Zhang et al., 1996
;
Newberry et al., 1997
), and,
through interactions with MINT/SHARP, may recruit histone deacetylases
(Newberry et al., 1999
;
Shi et al., 2001
;
Oswald et al., 2002
). It is
thus possible that Msx2 and Twist cooperatively repress one or more genes
whose downregulation is required for the differentiation of frontal bone
skeletogenic mesenchyme.
Msx2 and Twist are both targets of morphogen pathways.
During suture development, Twist integrates inputs from the BMP and
FGF pathways (Rice et al.,
2000). Msx2 is an immediate-early gene in the BMP2/4
pathway (Hollnagel et al.,
1999
). We suggest that in the frontal bone anlagen, Msx2
and Twist act as a nexus for BMP and FGF signaling, and participate
in the control of the identity and/or proliferation of the frontal bone
skeletogenic mesenchyme. We envisage a model similar to a regulatory network
documented in Drosophila, in which msh, together with
ladybird and even skipped, regulate the identity of cardiac
muscle progenitor cells (Jagla et al.,
2002
). Such a combinatorial interaction could maintain stringent
control over the differentiation and proliferation of the skeletogenic
mesenchyme, and thus serve as part of the mechanism that coordinates the
growth of skull with that of the brain. Finally, we our results have
implications for the pathophysiology of familial parietal foramina, and
possibly craniosynostosis. These results predict that in humans,
Twist activity may influence the penetrance of calvarial defects
caused by haploid loss of Msx2 function. Reciprocally, Msx2
activity may influence the penetrance of defects resulting from Twist
mutations. The clinical manifestations in individuals affected with familial
parietal foramina and Saethre-Chotzen syndrome may thus depend on the sum of
the activity of these two genes.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aubin, J. E., Liu, F., Malaval, L. and Gupta, A. K. (1995). Osteoblast and chondroblast differentiation. Bone 17,77S -83S.[CrossRef][Medline]
Bourgeois, P., Bolcato-Bellemin, A. L., Danse, J. M.,
Bloch-Zupan, A., Yoshiba, K., Stoetzel, C. and Perrin-Schmitt, F.
(1998). The variable expressivity and incomplete penetrance of
the twist-null heterozygous mouse phenotype resemble those of human
Saethre-Chotzen syndrome. Hum. Mol. Genet.
7, 945-957.
Carver, E. A., Oram, K. F. and Gridley, T. (2002). Craniosynostosis in Twist heterozygous mice: a model for Saethre-Chotzen syndrome. Anat. Rec. 268, 90-92.[CrossRef][Medline]
Chai, Y., Jiang, X., Ito, Y., Bringas, P., Jr, Han, J., Rowitch,
D. H., Soriano, P., McMahon, A. P. and Sucov, H. M.
(2000). Fate of the mammalian cranial neural crest during tooth
and mandibular morphogenesis. Development
127,1671
-1679.
Chen, Z. F. and Behringer, R. R. (1995). twist is required in head mesenchyme for cranial neural tube morphogenesis. Genes Dev. 9,686 -699.[Abstract]
Coelho, C. N. D., Sumoy, L., Rodgers, B. J., Davidson, D. R., Hill, R. E., Upholt, W. B. and Kosher, R. A. (1991). Expression of the chicken homeobox-containing gene GHox-8 during embryonic chick limb development. Mech. Dev. 34,143 -154.[CrossRef][Medline]
Cohen, M. M., Jr and MacLean, R. E. (2000). Craniosynostosis: Diagnosis, Evaluation, and Management. New York: Oxford University Press.
Couly, G. F., Coltey, P. M. and le Douarin, N. M.
(1993). The triple origin of skull in higher vertebrates: a study
in quail-chick chimeras. Development
117,409
-429.
Davidson, D. R., Crawley, A., Hill, R. E. and Tickle, C. (1991). Position-dependent expression of two related homeobox genes in developing vertebrate limbs. Nature 352,429 -431.[CrossRef][Medline]
Dodig, M., Tadic, T., Kronenberg, M. S., Dacic, S., Liu, Y. H., Maxson, R., Rowe, D. W. and Lichtler, A. C. (1999). Ectopic Msx2 overexpression inhibits and Msx2 antisense stimulates calvarial osteoblast differentiation. Dev. Biol. 209,298 -307.[CrossRef][Medline]
Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L. and Karsenty, G. (1997). Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89,747 -754.[Medline]
el Ghouzzi, V., le Merrer, M., Perrin-Schmitt, F., Lajeunie, E., Benit, P., Renier, D., Bourgeois, P., Bolcato-Bellemin, A. L., Munnich, A. and Bonaventure, J. (1997). Mutations of the TWIST gene in the Saethre-Chotzen syndrome. Nat. Genet. 15, 42-46.[Medline]
Fuchtbauer, E. M. (1995). Expression of M-twist during postimplantation development of the mouse. Dev. Dyn. 204,316 -322.[Medline]
Gullaud, M., Delanouse, R. and Silber, J. (2003). A Drosophila model to study the functions of TWIST orthologs n apoposis and proliferation. Cell Death Differ. 10,641 -651.[CrossRef][Medline]
Hamamori, Y., Wu, H. Y., Sartorelli, V. and Kedes, L. (1997). The basic domain of myogenic basic helix-loop-helix (bHLH) proteins is the novel target for direct inhibition by another bHLH protein, Twist. Mol. Cell. Biol. 17,6563 -6573.[Abstract]
Hamamori, Y., Sartorelli, V., Ogryzko, V., Puri, P. L., Wu, H. Y., Wang, J. Y., Nakatani, Y. and Kedes, L. (1999). Regulation of histone acetyltransferases p300 and PCAF by the bHLH protein twist and adenoviral oncoprotein E1A. Cell 96,405 -413.[Medline]
Hendzel, M. J., Wei, Y., Mancini, M. A., van Hooser, A., Ranalli, T., Brinkley, B. R., Bazett-Jones, D. P. and Allis, C. D. (1997). Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106,348 -360.[CrossRef][Medline]
Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994). Manipulating the Mouse Embryo: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.
Hollnagel, A., Oehlmann, V., Heymer, J., Ruther, U. and
Nordheim, A. (1999). Id genes are direct targets of bone
morphogenetic protein induction in embryonic stem cells. J. Biol.
Chem. 274,19838
-19845.
Howard, T. D., Paznekas, W. A., Green, E. D., Chiang, L. C., Ma, N., Ortiz de Luna, R. I., Garcia Delgado, C., Gonzalez-Ramos, M., Kline, A. D. and Jabs, E. W. (1997). Mutations in TWIST, a basic helix-loop-helix transcription factor, in Saethre-Chotzen syndrome. Nat. Genet. 15,36 -41.[Medline]
Hu, G., Lee, H., Price, S. M., Shen, M. M. and Abate-Shen, C. (2001). Msx homeobox genes inhibit differentiation through upregulation of cyclin D1. Development 128,2373 -2384.[Medline]
Isshiki, T., Takeichi, M. and Nose, A. (1997).
The role of the msh homeobox gene during Drosophila neurogenesis: implication
for the dorsoventral specification of the neuroectoderm.
Development 124,3099
-3109.
Jabs, E. W., Muller, U., Li, X., Ma, L., Luo, W., Haworth, I. S., Klisak, I., Sparkes, R., Warman, M. L., Mulliken, J. B. et al. (1993). A mutation in the homeodomain of the human MSX2 gene in a family affected with autosomal dominant craniosynostosis. Cell 75,443 -450.[Medline]
Jabs, E. W., Li, X., Scott, A. F., Meyers, G., Chen, W., Eccles, M., Mao, J.-I., Charnas, L. R., Jackson, C. E., and Jaye, M. (1994). Jackson-Weiss and Crouzon syndromes are allelic with mutations in fibroblast growth factor receptor 2. Nat. Genet. 8,275 -279.[Medline]
Jagla, T., Bidet, Y., da Ponte, J. P., Dastugue, B. and Jagla, K. (2002). Cross-repressive interactions of identity genes are essential for proper specification of cardiac and muscular fates in Drosophila. Development 129,1037 -1047.[Medline]
Jiang, X., Rowitch, D. H., Soriano, P., McMahon, A. P. and
Sucov, H. M. (2000). Fate of the mammalian cardiac neural
crest. Development 127,1607
-1616.
Jiang, X., Iseki, S., Maxson, R. E., Sucov, H. M. and Morriss-Kay, G. M. (2002). Tissue origins and interactions in the mammalian skull vault. Dev. Biol. 241,106 -116.[CrossRef][Medline]
Karsenty, G. (2001). Minireview:
transcriptional control of osteoblast differentiation.
Endocrinology 142,2731
-2733.
Kim, H. J., Rice, D. P., Kettunen, P. J. and Thesleff, I.
(1998). FGF-, BMP- and Shh-mediated signalling pathways in the
regulation of cranial suture morphogenesis and calvarial bone development.
Development 125,1241
-1251.
Knoepfler, P. S., Bergstrom, D. A., Uetsuki, T., Dac-Korytko,
I., Sun, Y. H., Wright, W. E., Tapscott, S. J. and Kamps, M. P.
(1999). A conserved motif N-terminal to the DNA-binding domains
of myogenic bHLH transcription factors mediates cooperative DNA binding with
pbx-Meis1/Prep1. Nucleic Acids Res.
27,3752
-3761.
Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., Deguchi, K., Shimizu, Y., Bronson, R. T., Gao, Y. H., Inada, M. et al. (1997). Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89,755 -764.[Medline]
Liu, Y. H., Tang, Z., Kundu, R. K., Wu, L., Luo, W., Zhu, D., Sangiorgi, F., Snead, M. L. and Maxson, R. E. (1999). Msx2 gene dosage influences the number of proliferative osteogenic cells in growth centers of the developing murine skull: a possible mechanism for MSX2-mediated craniosynostosis in humans. Dev. Biol. 205,260 -274.[CrossRef][Medline]
Maestro, R., Dei Tos, A. P., Hamamori, Y., Krasnokutsky, S.,
Sartorelli, V., Kedes, L., Doglioni, C., Beach, D. H. and Hannon, G.
J. (1999). Twist is a potential oncogene that inhibits
apoptosis. Genes Dev.
13,2207
-2217.
Mavrogiannis, L. A., Antonopoulou, I., Baxova, A., Kutilek, S., Kim, C. A., Sugayama, S. M., Salamanca, A., Wall, S. A., Morriss-Kay, G. M. and Wilkie, A. O. (2001). Haploinsufficiency of the human homeobox gene ALX4 causes skull ossification defects. Nat. Genet. 27,17 -18.[CrossRef][Medline]
Meyers, G. A., Orlow, S. J., Munro, I. R., Przylepa, K. A. and Jabs, E. W. (1995). Fibroblast growth factor receptor 3 (FGFR3) transmembrane mutation in Crouzon syndrome with acanthosis nigricans. Nat. Genet. 11,462 -464.[Medline]
Muenke, M., Schell, U., Hehr, A., Robin, N. H., Losken, H. W., Schinzel, A., Pulleyn, L. J., Rutland, P., Reardon, W., Malcolm, S. et al. (1994). A common mutation in the fibroblast growth factor receptor 1 gene in Pfeiffer syndrome. Nat. Genet. 8, 269-274.[Medline]
Newberry, E. P., Latifi, T., Battaile, J. T. and Towler, D. A. (1997). Structure-function analysis of Msx2-mediated transcriptional suppression. Biochemistry 36,10451 -10462.[CrossRef][Medline]
Newberry, E. P., Latifi, T. and Towler, D. A. (1999). The RRM domain of MINT, a novel Msx2 binding protein, recognizes and regulates the rat osteocalcin promoter. Biochemistry 38,10678 -10690.[CrossRef][Medline]
Nose, A., Isshiki, T. and Takeichi, M. (1998).
Regional specification of muscle progenitors in Drosophila: the role of the
msh homeobox gene. Development
125,215
-223.
Odelberg, S. J., Kollhoff, A. and Keating, M. T. (2000). Dedifferentiation of mammalian myotubes induced by msx1. Cell 103,1099 -1109.[Medline]
Ornitz, D. M. and Marie, P. J. (2002). FGF
signaling pathways in endochondral and intramembranous bone development and
human genetic disease. Genes Dev.
16,1446
-1465.
Oswald, F., Kostezka, U., Astrahantseff, K., Bourteele, S.,
Dillinger, K., Zechner, U., Ludwig, L., Wilda, M., Hameister, H.,
Knochel, W. et al. (2002). SHARP is a novel component of the
Notch/RBP-Jkappa signalling pathway. EMBO J.
21,5417
-5426.
Otto, F., Thornell, A. P., Crompton, T., Denzel, A., Gilmour, K. C., Rosewell, I. R., Stamp, G. W., Beddington, R. S., Mundlos, S., Olsen, B. R. et al. (1997). Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89,773 -779.[Medline]
Pajer, P., Pecenka, V., Karafiat, V., Kralova, J., Horejsi, Z. and Dvorak, M. (2003). The twist gene is a common target of retroviral integration and transcriptional deregulation in experimental nephroblastoma. Oncogene 22,665 -673.[CrossRef][Medline]
Poulin, G., Lebel, M., Chamberland, M., Paradis, F. W. and
Drouin, J. (2000). Specific protein-protein interaction
between basic helix-loop-helix transcription factors and homeoproteins of the
Pitx family. Mol. Cell. Biol.
20,4826
-4837.
Rajapurohitam, V., Chalhoub, N., Benachenhou, N., Neff, L., Baron, R. and Vacher, J. (1997). The mouse osteopetrotic grey-lethal mutation induces a defect in osteoclast maturation/function. Bone 28,513 -523.[CrossRef]
Reardon, W., Winter, R. M., Rutland, P., Pulleyn, L. J., Jones, B. M. and Malcolm, S. (1994). Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome. Nat. Genet. 8,98 -103.[Medline]
Rice, D. P., Kim, H. J. and Thesleff, I. (1999). Apoptosis in murine calvarial bone and suture development. Eur. J. Oral Sci. 107,265 -275.[Medline]
Rice, D. P., Aberg, T., Chan, Y., Tang, Z., Kettunen, P. J.,
Pakarinen, L., Maxson, R. E. and Thesleff, I. (2000).
Integration of FGF and TWIST in calvarial bone and suture development.
Development 127,1845
-1855.
Robert, B., Lyons, G., Simandl, B. K., Kuroiwa, A. and Buckingham, M. (1991). The apical ectodermal ridge regulates Hox-7 and Hox-8 gene expression in developing chick limb bud. Genes Dev. 5,2363 -2374.[Abstract]
Satokata, I. and Maas, R. (1994). Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat. Genet. 6, 348-356.[Medline]
Satokata, I., Ma, L., Ohshima, H., Bei, M., Woo, I., Nishizawa, K., Maeda, T., Takano, Y., Uchiyama, M., Heaney, S. et al. (2000). Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nat. Genet. 24,391 -395.[CrossRef][Medline]
Schowing, J. (1968). Mise en evidence du role inducteur de l'encephale dans l'osteogenese du crane embryonnaire du poulet. J. Embryol. Exp. Morphol. 19, 83-93.[Medline]
Shi, Y., Downes, M., Xie, W., Kao, H. Y., Ordentlich, P., Tsai,
C. C., Hon, M. and Evans, R. M. (2001). Sharp, an
inducible cofactor that integrates nuclear receptor repression and activation.
Genes Dev. 15,1140
-1151.
Soo, K., O'Rourke, M. P., Khoo, P. L., Steiner, K. A., Wong, N., Behringer, R. R. and Tam, P. P. (2002). Twist function is required for the morphogenesis of the cephalic neural tube and the differentiation of the cranial neural crest cells in the mouse embryo. Dev. Biol. 247,251 -270.[CrossRef][Medline]
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[CrossRef][Medline]
Stoetzel, C., Weber, B., Bourgeois, P., Bolcato-Bellemin, A. L. and Perrin-Schmitt, F. (1995). Dorso-ventral and rostro-caudal sequential expressin of M-twist in the postimplantation murince embry. Mech. Dev. 51,251 -263.[CrossRef][Medline]
Thompson, E. M., Baraitser, M. and Hayward, R. D. (1984). Parietal foramina in Saethre-Chotzen syndrome. J. Med. Genet. 21,369 -372.[Medline]
Vainio, S., Karavanova, I., Jowett, A. and Thesleff, I. (1993). Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissues during early tooth development. Cell 75,45 -58.[Medline]
Wilkie, A. O. (1997). Craniosynostosis: genes
and mechanisms. Hum. Mol. Genet.
6,1647
-1656.
Wilkie, A. O. and Morriss-Kay, G. M. (2001). Genetics of craniofacial development and malformation. Nat. Rev. Genet. 2,458 -468.[CrossRef][Medline]
Wilkie, A. O., Tang, Z., Elanko, N., Walsh, S., Twigg, S. R., Hurst, J. A., Wall, S. A., Chrzanowska, K. H. and Maxson, R. E., Jr (2000). Functional haploinsufficiency of the human homeobox gene MSX2 causes defects in skull ossification. Nat. Genet. 24,387 -390.[CrossRef][Medline]
Wilkie, A. O., Oldridge, M., Tang, Z. and Maxson, R. E., Jr (2001). Craniosynostosis and related limb anomalies. Novartis Found. Symp. 232,122 -133.[Medline]
Woloshin, P., Song, K., Degnin, C., Killary, A. M., Goldhamer, D. J., Sassoon, D. and Thayer, M. J. (1995). MSX1 inhibits myoD expression in fibroblast x 10T1/2 cell hybrids. Cell 82,611 -620.[Medline]
Wu, Y. Q., Badano, J. L., McCaskill, C., Vogel, H., Potocki, L. and Shaffer, L. G. (2000). Haploinsufficiency of ALX4 as a potential cause of parietal foramina in the 11p11.2 contiguous gene-deletion syndrome. Am. J. Hum. Genet. 67,1327 -1332.[Medline]
Wuyts, W., Cleiren, E., Homfray, T., Rasore-Quartino, A.,
Vanhoenacker, F. and van Hul, W. (2000a). The ALX4
homeobox gene is mutated in patients with ossification defects of the skull
(foramina parietalia permagna, OMIM 168500). J. Med.
Genet. 37,916
-920.
Wuyts, W., Reardon, W., Preis, S., Homfray, T., Rasore-Quartino,
A., Christians, H., Willems, P. J. and van Hul, W.
(2000b). Identification of mutations in the MSX2 homeobox gene in
families affected with foramina parietalia permagna. Hum. Mol.
Genet. 9,1251
-1255.
Young, I. D. and Swift, P. G. (1985). Parietal foramina in the Saethre-Chotzen syndrome. J. Med. Genet. 22,413 -414.
Yousfi, M., Lasmoles, F., El Ghouzzi, V. and Marie, P. J. (2002). Twist haploinsufficiency in Saethre-Chotzen syndrome induces calvarial osteoblast apoptosis due to increased TNFalpha expression and caspase-2 activation. Hum. Mol. Genet. 11,359 -369.[CrossRef][Medline]
Zhang, H., Catron, K. M. and Abate-Shen, C. (1996). A role for the Msx-1 homeodomain in transcriptional regulation: residues in the N-terminal arm mediate TATA binding protein interaction and transcriptional repression. Proc. Natl. Acad. Sci. USA 95,1764 -1769.
Related articles in Development: