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First published online February 6, 2009
doi: 10.1242/10.1242/dev.028605
Department of Biochemistry and Molecular Biology, Norris Cancer Hospital, University of Southern California Keck School of Medicine, 1441 Eastlake Avenue, Los Angeles, CA 90089-9176, USA.
* Author for correspondence (maxson{at}usc.edu)
Accepted 22 December 2008
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Key words: EphA4, Twist1, Boundary formation, Cell guidance, Craniosynostosis
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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The mammalian skull vault is a composite structure, consisting of membrane
bones with distinct lineage origins (Jiang
et al., 2002
). The frontal bones and the central portion of the
interparietal bone are derived from neural crest, the parietal bones and the
lateral portion of the interparietal bone from paraxial mesoderm. The bones of
the skull vault are separated by sutures, fibrous joints that accommodate the
expanding brain and allow the skull to undergo reshaping during birth.
Craniosynostosis, the premature fusion of calvarial bones at the sutures,
occurs as frequently as 1 in 2500 live births. Affected individuals have
abnormally shaped skulls, and in some instances mental retardation and
impaired vision and hearing (Cohen and
MacLean, 1999; Wilkie and
Morriss-Kay, 2001).
Mutations in a number of genes, collectively functioning in several
signaling pathways, can cause craniosynostosis in humans or mice
(Ornitz and Marie, 2002;
Rawlins and Opperman, 2008;
Wilkie, 1997). Among these
genes are TWIST1, which regulates both BMP and FGF signaling
(Carver et al., 2002;
Connerney et al., 2008;
el Ghouzzi et al., 1997;
Howard et al., 1997;
Rice et al., 2000;
Rice et al., 1999;
Wilkie, 1997), FGFR1,
FGFR2 and FGFR3 (Jabs et
al., 1994; Marie et al.,
2005; Meyers et al.,
1995; Yu et al.,
2003), the Wnt pathway inhibitor, Axin2
(Yu et al., 2005), the Bmp
target, MSX2 (Jabs et al.,
1993), and RAB23, a component of the Shh pathway
(Jenkins et al., 2007). The
cellular mechanisms underlying craniosynostosis have been investigated using
both mouse and tissue culture models
(Maxson and Ishii, 2008;
Rawlins and Opperman, 2008;
Wilkie and Morriss-Kay, 2001).
Mice carrying the S252W or P253R mutation engineered into Fgfr2 mimic features
of Apert syndrome (Wang et al.,
2005; Yin et al.,
2008), and exhibit enhanced RTK signaling
(Shukla et al., 2007).
Activation of Wnt signaling by targeted deletion of Axin2 results in
an expansion of the pool of osteoprogenitors and ultimately to synostosis
(Liu et al., 2007;
Yu et al., 2005). Although
details of underlying developmental mechanisms that lead ultimately to
synostosis are still lacking, one reasonable hypothesis is that it is caused
by changes in the balance of proliferation and differentiation of osteogenic
cells in the developing suture (Bialek et
al., 2004; Chen et al.,
2003; Lee et al.,
1999; Yousfi et al.,
2002; Yousfi et al.,
2001).
Our recent results on the mechanism of Saethre-Chotzen syndrome, caused by
heterozygous loss of function of Twist1, draw attention to the
significance of tissue boundaries in the development of synostosis
(Merrill et al., 2006).
Individuals affected with Saethre-Chotzen have coronal synostosis, fusion of
the frontal and parietal bones at the coronal suture. Twist1 mutant
mice also exhibit coronal synostosis
(Carver et al., 2002;
el Ghouzzi et al., 1997). In
such mice and in cultured osteoblasts, Twist1 can inhibit osteoblast
differentiation by regulating the activity of Runx2
(Bialek et al., 2004;
Guenou et al., 2005;
Yoshida et al., 2005).
Connerney et al. have presented evidence that reduced dosage of Twist1 changes
the proportion of Twist1 homo- and heterodimers within developing sutures and
thereby regulates suture patency (Connerney
et al., 2008; Connerney et al.,
2006). We demonstrated that Twist1 mutant mice have a
deficiency in the neural crest-mesoderm boundary at the coronal suture
(Merrill et al., 2006). The
boundary normally lies between the mesoderm-derived cells of the prospective
suture and the neural-crest-derived osteogenic cells of the prospective
frontal bone (Merrill et al.,
2006; Yoshida et al.,
2008). Thus the boundary not only demarcates neural crest and
mesoderm, but also osteogenic and non-osteogenic sutural cells. In
Twist1 mutants, neural crest cells crossed the boundary into the
mesoderm domain of the suture (Merrill et
al., 2006).
We showed previously that the change in cell behavior at this boundary was
associated with a reduction in the levels of the ephrin ligands, ephrin A2
(Efna2) and ephrin A4 (Efna4), as well as their receptor, EphA4. Moreover, we
identified loss-of-function mutations in EFNA4 in 3/77 patients
(Merrill et al., 2006).
Ephrins are membrane-bound ligands that interact with Eph receptors, a large
family of receptor tyrosine kinases
(Klein, 2004;
Kullander and Klein, 2002;
Wilkinson, 2001). Ephrin-Eph
signaling is bidirectional, through both the receptor and the ligand.
Engagement of Eph receptors by membrane-bound ephrin ligands induces
dimerization and subsequent trans-phosphorylation of the receptors, leading to
changes in the activity of downstream effectors, which include the
mitogen-activated protein kinases ERK, c-Jun N-terminal kinase, Src family
kinases and Ras/Rho family GTPases. Ephrin-Eph signaling regulates a variety
of developmental processes including vascular and neuronal development and the
establishment of developmental boundaries
(Klein, 2004;
Kullander and Klein, 2002;
Martinez and Soriano, 2005;
Palmer and Klein, 2003;
Pasquale, 2005;
Poliakov et al., 2004;
Surawska et al., 2004).
Here we test for a causal connection between Twist1, ephrin A
signaling and craniosynostosis; we also investigate cellular mechanisms
controlled by ephrin A signaling in the developing skull vault. We demonstrate
that loss of function of the Efna4 receptor, EphA4, causes
coronal synostosis in mice, definitively establishing the link between
craniosynostosis and loss of ephrin A signaling suggested by our earlier human
genetic findings (Merrill et al.,
2006). We show further that EphA4 interacts genetically
with Twist1 and acts as a Twist1 effector in the control of
the frontal-parietal boundary and in the regulation of the RTK indicator,
P-Erk1/2 and the BMP pathway indicator, P-Smad1/5/8. Finally we use
1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) labeling
to show that Twist1 and EphA4 control the guidance of
migratory osteogenic cells to the leading edges of the growing frontal and
parietal bones, and that these genes are required to exclude such osteogenic
cells from the coronal suture. Our results suggest that migration of
osteogenic cells is an important element in the patterned growth of calvarial
bones, and that the mis-migration of such cells plays a crucial role in the
development of craniosynostosis in Twist1 and EphA4 mutant
mice.
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),
R26R (Soriano, 1999),
Wnt1-cre (Danielian et al.,
1998) and Mesp1-cre
(Saga et al., 1999) alleles
have been described. We genotyped EphA4, Efna2, Twist1, R26R,
Wnt1-cre and Mesp1-cre alleles by PCR as described
(Chen and Behringer, 1995;
Dottori et al., 1998;
Feldheim et al., 2000;
Jiang et al., 2002;
Saga et al., 1999).
Histology, immunostaining and in situ hybridization
Heads of embryos were embedded in OCT medium (Histoprep, Fisher Scientific)
before sectioning. Frozen sections were cut at 10 µm. Analysis of
β-galactosidase activity of Wnt1-Cre/R26R and
Mesp1-Cre/R26R reporter gene expression was carried out as described
(Ishii et al., 2003).
Immunostaining of frozen sections was largely carried out as previously
reported (Ishii et al., 2003).
Immunohistochemistry was performed using rabbit anti-Runx2 (Sigma), rabbit
P-Erk1/2 (Cell Signaling), rabbit Erk1/2 (Cell Signaling) and rabbit
anti-P-Smad1/5/8 (Cell Signaling) diluted in 1% BSA/PBS and incubated
overnight at 4°C. Detection of primary antibody of anti-Runx2,
anti-P-Erk1/2 and anti-Erk1/2 was performed by incubating goat anti-rabbit-HRP
(Zymed, 1/250) for 1 hour at room temperature and visualizing with DAB
substrate. Detection of anti-P-Smad1/5/8 was performed by incubating
rhodamine-labeled goat anti-rabbit IgG (1:100) for 1 hour at room temperature
followed by DAPI counterstaining and examination by epifluorescence
microscopy. Non-radioactive section in situ hybridization using the tyramide
signal amplification (TSA) method was performed as described
(Adams, 1992;
Paratore et al., 1999;
Yang et al., 1999). Briefly,
to analyze mRNA expression by TSA, DIG-labeled or FL-labeled riboprobes were
allowed to hybridize with the section and were detected with anti-DIG or
anti-FL antibodies conjugated to horseradish peroxidase (POD). Indirect TSA
fluorescence system (TSA-biotin/avidin-FITC) was used to detect the
POD-conjugated antibody (Perkin Elmer). RNA probes were generated as reported:
EphA4 (Nieto et al.,
1992), Twist1 (Rice
et al., 2000).
Whole-mount skull Alizarin Red S staining
Skulls from 21-day-old postnatal mice were stained for bone with 2%
Alizarin Red S in 1% KOH for 1 to 2 days. The specimens were then cleared and
stored in 100% glycerol.
Whole-mount alkaline phosphatase (ALP) staining
Whole-mount staining for alkaline phosphatase was carried out as described
(Ishii et al., 2003).
Embryonic day 13.5 (E13.5) embryo heads were fixed in 4% paraformaldehyde in
PBS, and were bisected midsagitally after fixation. Presumptive calvarial
bones were stained with NBT and BCIP (Roche).
Exo utero DiI labeling of migratory osteogenic precursor cells
Details of the exo utero manipulation have been described
(Muneoka et al., 1986;
Serbedzija et al., 1992).
Briefly, E13.5 embryos with embryonic membranes were carefully exposed by
incising the uterine wall. Two embryos from each side of the uterine horns
were designated as the experimental group, and all others were removed. DiI
(Molecular Probes, 1:10 dilution from 0.5% stock solution) was injected into
the area of the calvarial bone rudiments under a dissecting microscope with a
microelectrode (tip diameter, 20 µm) attached to a mouth pipette
(Yoshida, 2005). After
injection, the embryos were returned to the peritoneal cavity of dams and
allowed to continue development exo utero. After 2-3 days of additional
development, the embryos were removed and examined by epifluorescence
microscopy. The survival rate of the embryos after DiI injection was greater
than 70%.
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), we
assessed compound EphA4-/-; Efna2+/-
mutant mice. Alizarin-Red staining of skulls of EphA4-/-
mice at P21 revealed that the coronal sutures were partially fused, a
phenotype that closely resembles that of Twist1+/- mice
(Fig. 1).
Efna2-/- mutants exhibited normal coronal suture
development. EphA4-/-; Efna2+/- double
mutant skulls had no significant increase in the severity of the synostosis
phenotype over EphA4-/- mutants
(Fig. 1; data not shown),
indicating that EphA4 plays a more prominent role than Efna2
in coronal synostosis, at least in the mouse. We therefore concentrated on
EphA4 mutants in further efforts to understand the relationship
between Twist1, ephrin signaling and craniosynostosis.
We examined the activity of alkaline phosphatase (ALP), an early osteoblast
marker, in EphA4-/- embryos at E14.5 when a loss of
integrity of the boundary between ALP and non ALP-expressing cells is first
apparent in Twist1 mutants
(Merrill et al., 2006)
(Fig. 2). In wild-type embryos,
the prospective coronal suture is evident as a layer of non-ALP-expressing
cells located between the prospective frontal and parietal bones. In
EphA4-/- mutants this layer exhibited a disorganized
appearance and was filled with ALP-expressing cells, as is the case in
Twist1+/- mutants (Fig.
2) (Merrill et al.,
2006). In addition, the expression domain of ALP was substantially
broader in EphA4-/- mutants, similar to a phenotype
observed in Twist1 mutants.
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)
(Fig. 2). We carried out
intercrosses between EphA4 mutants and embryos carrying
Wnt1-Cre and R26R and examined embryos at a series of
developmental stages. In EphA4-/- mutants,
lacZ-positive cells were evident outside the neural crest domain.
This phenotype was first detectable at E14.5 and became more severe at E16.5.
We did not detect a boundary defect in heterozygous mutants. These results
suggest that reduced EphA4 function results in a set of phenotypes in
the coronal suture that resemble those of Twist1 mutants.
EphA4 is a downstream effector of Twist1 in the coronal suture
We sought to determine whether levels of EphA4 transcripts in the
developing calvarial bones and sutures are regulated by Twist1
(Fig. 3). EphA4 mRNA
was localized in the periosteal layers above and below the developing frontal
and parietal bones. It was also expressed in a layer of cells outside
(ectocranial to) the bone layer, and broadly within the suture mesenchyme. In
Twist1+/- mutants, EphA4 expression was reduced
substantially in the ectocranial and periosteal layers, consistent with our
previous finding that Twist1 controls the levels of EphA4 protein
expression in calvarial tissues. Twist1 expression was not changed in
EphA4-/- mutants.
If EphA4 is an effector of Twist1, then combination Twist1+/-; EphA4+/- heterozygotes should exhibit phenotypes of greater severity than individual heterozygous mutants. We crossed Twist1+/- heterozygous mice with EphA4 mutant mice and examined skulls at E13.5, E14.5 and P21 (Fig. 4; Table 1). The penetrance of craniosynostosis as assessed at P21 increased from 73 to 94% in Twist1+/-; EphA4+/- compound mutants (n=33) compared with Twist1+/- mutants (n=26); also, a larger portion of the suture was fused (50 vs 25%; P<0.005) in the compound mutants.
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Twist1 and EphA4 cooperatively control P-Erk1/2 and P-Smad1/5/8 activity in the developing frontal and parietal bones
As part of an effort to understand the molecular basis of the suture
defects, we examined the expression of the RTK effector, P-Erk1/2
(Fig. 5), and the BMP effector,
P-Smad1/5/8 (Fig. 6).
Twist1 and Eph-ephrin are known to function through the RTK pathway
(Guenou et al., 2005;
Pratt and Kinch, 2002;
Vindis et al., 2003). FGF/FGFR
signaling has a well-documented role in craniosynostosis and normal suture
development (Deng et al.,
1996; Johnson et al.,
2000; Marie et al.,
2005; Rice et al.,
2000; Yamaguchi and Rossant,
1995), and Twist1 has been shown to control levels of
FGFR expression and P-Erk1/2 activity in sutures of late embryonic and
postnatal mice (Connerney et al.,
2008; Rice et al.,
2000). Finally, the BMP pathway is known to be involved in the
specification and differentiation of calvarial osteogenic cells
(Kim et al., 1998;
Ryoo et al., 2006); forced
expression of the BMP antagonist noggin can prevent fusion of the
sagittal suture (Warren et al.,
2003).
Immunostaining of sections of E14.5 embryos showed that P-Erk1/2 was expressed in the ectocranial non-osteogenic cell layer as well as in the underlying osteogenic layer (Fig. 5). The number of P-Erk1/2-expressing cells decreased progressively in both layers as the dosages of Twist1 and EphA4 were reduced. Total Erk was unaffected in Twist1+/-; EphA4+/- mutants, demonstrating that Twist1 and EphA4 specifically regulate the distribution of cells expressing phosphorylated Erk1/2.
The distribution of P-Smad1/5/8-expressing cells was also strongly influenced by Twist1 and EphA4 (Fig. 6). Control embryos expressed P-Smad1/5/8 at a high level in the osteogenic fronts of the growing frontal and parietal bones (Fig. 6A,B). Lower levels were evident in more mature osteoblasts, distal to the leading edges. Thus, at E14.5, the highest levels of P-Smad1/5/8 activity were associated with the progenitor cells of the osteogenic fronts and lower levels with the differentiated cells of the developing bone. There was a clear boundary between domains of high and low P-Smad1/5/8 expression in the osteogenic fronts and suture. In both Twist1 and EphA4 mutants, the number of P-Smad1/5/8-expressing cells was reduced significantly in the osteogenic fronts, and the boundary between these cells and the prospective sutural cells was blurred (Fig. 6E-L). Punctate staining of P-Smad1/5/8 was evident throughout the suture. Combination mutants exhibited an even more dramatic reduction in the number of P-Smad1/5/8-positive cells in the suture (Fig. 6M,N). These data suggest that Twist1 and EphA4 together control the number and distribution of P-smad1/5/8-positive cells in the coronal suture. Further, the reduction in the number of P-Smad1/5/8-expressing cells in the suture is consistent with our finding that high levels of P-Smad1/5/8 expression are associated with undifferentiated progenitor cells in the osteogenic fronts, and lower levels with differentiating osteogenic cells within developing bone.
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). In embryos with the Twist1+/-;
EphA4+/- genotype, a substantial number of
lacZ-positive cells were ectopically located in the neural crest
territory. Thus cells derived from mesoderm, as well as neural crest, failed
to respect the neural crest-mesoderm boundary between the frontal and parietal
bones.
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; Yoshida et al.,
2008). This allowed us to examine directly the behavior of
populations of migratory osteogenic cells that contribute to the frontal and
parietal bones. At E12.5, the frontal and parietal bone rudiments consist of
patches of osteogenic precursor cells located in the supraorbital ridge. The
frontal bone rudiment is anterior to the eye, the parietal bone rudiment is
posterior. Between the rudiments is the prospective coronal suture, in later
stages identifiable by the absence of ALP-expressing osteogenic cells. The
prospective frontal and parietal osteogenic cells can be labeled by injecting
DiI into the area of the rudiments (supraorbital ridge) at E13.5
(Yoshida, 2005;
Yoshida et al., 2008) (P.G.R.,
N.L.W. and R.E.M., unpublished observations). During subsequent development of
the injected embryos, exo utero, labeled cells migrate apically, adding to the
leading edge of the frontal and parietal bones. We asked whether such migratory osteogenic cells exhibit abnormal behavior in Twist1-EphA4 mutants. We injected DiI into E13.5 Twist1+/-; EphA4+/- embryos and allowed them to develop exo utero. We then examined the embryos at E16.5 by epifluorescence microscopy. As is evident in Fig. 8, labeled cells were excluded from the area of the prospective suture in wild-type embryos. However, such cells were present in substantial numbers in the sutural space of Twist1+/-; EphA4+/- mutant embryos. In multiple repetitions of this experiment, in which DiI was injected into the frontal bone rudiment as well as the parietal bone rudiment, we obtained closely similar results (Table 2). Thus, Twist1 and EphA4 controlled the distribution of migratory osteogenic precursor cells between the non-osteogenic coronal suture and the prospective frontal and parietal bones. We also examined the distribution of migratory osteogenic precursor cells one day earlier when they are in the process of migration. In control embryos at E15.5, DiI-labeled cells were found largely in the ectocranial, EphA4-expressing layer (Fig. 8O-Q). In EphA4-Twist1 mutants, by striking contrast, labeled cells were located diffusely within and adjacent to the osteogenic layer (Fig. 8R-T). Thus Twist1 and EphA4 determined the partitioning of osteogenic precursor cells between the non-osteogenic ectocranial layer and the ALP-expressing prospective bone. Together these results suggest that Twist1 and EphA4 function in the partitioning of migratory osteogenic cells between osteogenic and non-osteogenic territories in the coronal suture.
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DISCUSSION
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).
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); thus
Efna2 and Efna4 may function redundantly. However, in our
earlier screen of patients with non-syndromic craniosynostosis we did not
detect mutations in Efna2
(Merrill et al., 2006). A
definitive test of the roles of Efna2 and Efna4 in suture
development will have to await an Efna4 knockout mouse.
The expansion of osteogenic marker gene expression into the mesenchyme of
the coronal suture is associated with a reduction in P-Erk1/2 activity in the
non-osteogenic, ephrin A-expressing layer outside the osteogenic layer. Total
Erk activity is not affected, demonstrating that Twist1 and
EphA4 control P-Erk1/2 signaling specifically. It is interesting that
this change in P-Erk1/2 is in the cell layer in which osteogenic precursor
cells migrate, and from which migratory cells are lost in
Twist1-EphA4 combination mutants. Thus the phosphorylation status of
Erk, which is known to be regulated by ephrin-Eph signaling
(Elowe et al., 2001;
Miao et al., 2001;
Pasquale, 2008;
Poliakov et al., 2004;
Pratt and Kinch, 2002;
Schmucker and Zipursky, 2001),
may be related to the migratory properties of osteogenic precursor cells and
to their association with this cell layer.
We note that our results on P-Erk1/2 levels are in apparent contrast with
two recent findings. Yin et al. (Yin et
al., 2008) found that an increase in P-Erk1/2 activity is
associated with craniosynostosis in the Pro253Arg mutant of Fgfr2, which
models Apert craniosynostosis; Connerney et al.
(Connerney et al., 2008) showed
that P-Erk1/2 is upregulated in sutures of Twist1 mutant mice. These results
differ from ours in two important respects. First, both studies analyzed
embryos at E16.5 or older, after the mis-migration/mixing events we document
here have occurred. Second, both examined P-Erk1/2 activity at sites other
than the ectocranial, EphA4-expressing layer. Yin et al.
(Yin et al., 2008) in bone
marrow cells and Connerney et al.
(Connerney et al., 2008) in
osteogenic fronts. These results, taken together with our findings, suggest
that P-Erk signaling functions in two distinct processes, one at E15.5 or
earlier, involving the partitioning of osteogenic cells between the
EphA4-expressing layer and the osteogenic layer, the other at E16.5 or later
involving the differentiation of osteogenic cells in the osteogenic layer or
in the suture. The earlier process is positively regulated by Twist1,
the later process negatively regulated.
Also associated with the expansion of osteogenic marker gene expression
into sutural mesenchyme in individual and combination Twist1 and
EphA4 mutants is a broadening of the distribution of
P-Smad1/5/8-expressing cells and a reduction in their number. That Smad1/5/8
signaling is apparently reduced in craniosynostotic sutures may seem
paradoxical given the general finding that Bmp signaling promotes
osteogenesis. However, we note that in wild-type sutures, high levels of
P-Smad1/5/8 are found in osteogenic fronts, which contain proliferative,
ALP-positive cells, and lower levels are found in differentiating osteoblasts
within the developing bone. Thus while the Bmp pathway has a well-documented
positive role in osteogenesis, the transition from proliferative osteogenic
cells of the osteogenic front to more differentiated osteoblasts in the
mineralizing bone may actually entail a reduction in Bmp signaling. We note
that two studies have reported increases in P-smad1/5/8 levels or Bmp activity
in craniosynostotic sutures (Warren et
al., 2003; Connerney et al.,
2008). However, both focused on late-embryonic or postnatal
stages, and in the case of Warren et al.
(Warren et al., 2003), on a
sagittal suture. Thus, as with P-Erk1/2 signaling, it is likely that these
studies concern processes distinct from the boundary and migration defects we
document here.
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) (A.
Merrill and R.E.M., unpublished), suggesting that ephrin signaling controls
boundary behavior not by regulating cell interactions at the immediate
boundary, but by controlling the guidance or migratory behavior of osteogenic
cells as they move apically from the frontal and parietal bone rudiments in
the supraorbital ridge to the leading edges of the developing bone. We
investigated this hypothesis by means of DiI labeling of embryos in vivo,
followed by exo utero development of injected embryos. Iseki and colleagues
used this technique to demonstrate that migratory cells contribute to the
growing calvarial bones (Yoshida,
2005; Yoshida et al.,
2008). We used this approach because of the lack of a satisfactory
means of labeling and culturing calvarial rudiments in vitro in a way that
mimics normal apical expansion of the frontal and parietal bones. This
labeling technique enables us follow cells injected at E13.5 for periods of up
to five days with no significant dilution of DiI. Moreover, development of
injected embryos is normal.
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),
together with our results (Fig.
8), demonstrates that DiI injected into the frontal or parietal
bone rudiments labels osteogenic precursor cells. Our finding that cells
labeled by injection of DiI into the parietal bone rudiment are present in the
coronal suture of mutant mice strongly suggests that these are cells that
would normally contribute to the parietal bone. Thus, from Wnt1-Cre
and Mesp1-Cre lineage tracing, together with DiI labeling, we
conclude that in Twist1-EphA4 mutant mice, osteogenic cells of neural
crest and mesoderm origin cross a boundary between the osteogenic territories
of the frontal and parietal bones and enter the coronal suture. We conclude
further that the normal function of ephrin-Eph signaling is to target cells to
appropriate sites at the coronal leading edge of the bone and ensure that they
do not enter the coronal suture. How ephrin-Eph signaling achieves this
remains unclear, although such signaling is known to guide cells by means of
repulsive and attractive interactions
(Arvanitis and Davy, 2008;
Egea and Klein, 2007;
Klein, 2004;
Santiago and Erickson,
2002). Twist1 mutant mice exhibit synostosis of the lambdoid suture as well as the coronal (H. Yen and R.E.M., unpublished observations). The lambdoid suture does not coincide with a major lineage boundary like the coronal, raising the question of the extent to which boundary defects are involved in lambdoid synostosis. Our Cre labeling and DiI labeling results suggest that it is not the neural crest-mesoderm boundary per se that is important in the development of coronal synostosis, but rather a defect in a boundary between osteogenic and non-osteogenic compartments. We suggest that such a mechanism may apply generally to the lambdoid and other sutures.
What is the role of mistargeting of osteogenic cells in the development of
synostosis? That reduced dosage of the osteoblast determinant Runx2
can rescue the Twist1 synostosis phenotype
(Bialek et al., 2004) suggests
that inappropriate differentiation of osteogenic cells is part of the
mechanism underlying synostosis in Twist1 mutants. Our present data
show that in control embryos migratory osteogenic cells migrate apically along
the ectocranial layer, ultimately reaching the leading edge of the bone. In
mutant embryos, migratory osteogenic cells are excluded from the ectocranial
layer, moving into the osteogenic layer and the prospective suture.
Consequences of this include the broadening of ALP activity in the osteogenic
layer, the presence of ALP-positive cells in the coronal at E14.5, and the
ultimate formation of bone within the suture. We suggest that two mechanisms -
aberrant migration and a change in osteogenic cell differentiation requiring
Runx2 - work in sequence to produce synostosis. We propose that osteogenic
cells from the frontal and parietal territories invade the coronal suture and
signal normally non-osteogenic sutural cells to assume an osteogenic identity,
thus producing synostosis.
Finally we note that our findings are consistent with the recent results of
Yoshida et al. (Yoshida et al.,
2008) in supporting the view that cell migration is a significant
morphogenetic force in the patterned growth of the skull vault. Lana-Elola et
al. (Lana-Elola et al., 2007)
showed that only a small number of cells of the mesenchyme of the sagittal
suture assume an osteogenic identity and are incorporated into the advancing
parietal bone (Lana-Elola et al.,
2007). However, inhibition of DNA synthesis slowed bone growth
significantly, leading these authors to propose that proliferation of cells of
the osteogenic fronts rather than recruitment of prepositioned mesenchyme is
important for bone growth. Our results, together with those of Yoshida et al.
(Yoshida et al., 2008) suggest
that migration of osteoprogenitor cells from an area at the base of the
growing rudiment also makes a major contribution to the apical expansion of
calvarial bones. More precise identification of these progenitor cell
populations, as well as an understanding of the processes that guide their
migration and differentiation will illuminate the mechanisms that underlie the
patterned growth of the skull as well as the pathophysiology of
craniosynostosis.
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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