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First published online 30 November 2005
doi: 10.1242/dev.02171
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1 Department of Cell Biology, New York University School of Medicine, MSB room
614, 550 1st Avenue, New York, NY 10016, USA.
2 Faculty of Medicine, Centre for Bone and Periodontal Research, McGill
University, Room 2203, 740 Avenue Dr Penfield, Montreal, Québec H3A
1A4, Canada.
3 Departments of Pathology and Dermatology, New York University School of
Medicine, MSB room 614, 550 1st Avenue, New York, NY 10016, USA.
* Corresponding author (e-mail: loomic01{at}med.nyu.edu)
Accepted 19 October 2005
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B ligand (RANKL) in osteoblasts. Thus, during
intramembranous bone formation, EN1 acts both cell autonomously and non-cell
autonomously. In summary, this study identifies EN1 as a novel modulator of
calvarial osteoblast differentiation and proliferation, processes that must be
exquisitely balanced to ensure proper skull vault formation.
Key words: Calvarial bone, En1, Osterix, Osteoblasts, Osteoclasts
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
These populations migrate into defined locations overlying the cerebral
hemispheres, and subsequently differentiate into condensing osteogenic or
chondrogenic mesenchyme (between E7.5-E11.5 in mouse). The osteogenic
mesenchyme is characterized by the expression of Runx2 and
Osterix (Osx; Sp7 - Mouse Genome Informatics), the
earliest molecular determinants of bone formation
(Komori et al., 1997;
Nakashima et al., 2002;
Otto et al., 1997). In
contrast to the endochondral bone formation that ensues from a cartilaginous
template, most elements of the skull vault develop by intramembranous
ossification, characterized by the direct differentiation of osteogenic
mesenchyme into osteoblasts. Osteoblasts are specialized cells that produce
bone extracellular matrix or osteoid, and that subsequently regulate its
mineralization (Aubin,
2002).
During the morphogenetic phases of skull development, a pool of highly
proliferative osteoprogenitors populates the margins (osteogenic fronts) of
each enlarging bone anlagen, thereby maintaining calvarial bone expansion
through osteoblast replenishment (Iseki et
al., 1999; Opperman,
2000). After each bone has acquired its basic form (E15.5), the
individual skeletal elements remain separated by fibrous joints, or sutures,
composed of skeletogenic mesenchyme and fibroblasts
(Opperman, 2000). Continued
production of osteoprogenitors in the sutures ensures that calvarial expansion
is coordinated with growth of the underlying brain. Thus, sutures serve as
major centers for calvarial osteoblast differentiation and new bone formation
postnatally. Finally, in addition to osteoblast-mediated bone formation,
calvarial bones undergo dynamic modeling and remodeling of their
three-dimensional microarchitecture, through the coordinated resorptive
activity of haematopoietically derived osteoclasts
(Takahashi, 2002).
Genetic and molecular evidence has implicated a number of growth and
transcription factors as being important regulators of skull formation. The
fibroblast growth factors (FGFs) are a family of secreted polypeptides that
act through four related tyrosine kinase receptors (FGFR1-FGFR4) to regulate a
plethora of developmental processes, and they are of central significance to
intramembranous ossification (Ornitz and
Marie, 2002). Human diseases that manifest the precocious osseous
obliteration of sutures, known as craniosynostosis, often result from
gain-of-function mutations in FGF receptors 1-3 (FGFR1/2/3)
(Webster and Donoghue, 1996;
Wilkie, 1997). Mouse models of
loss- or gain-of-function mutations in Fgfr1 and Fgfr2 have
provided further evidence that FGF signaling regulates the proliferation and
differentiation of calvarial osteoblasts and osteoprogenitors
(Eswarakumar et al., 2004;
Eswarakumar et al., 2002;
Yu et al., 2003;
Zhou et al., 2000).
Elucidating the precise mechanisms of osteoblastic FGF signaling, however, has
been complicated by the fact that at least four potential ligands (Fgf2,
Fgf4, Fgf9 and Fgf18) for Fgfr1-Fgfr3 are expressed in
the developing mouse calvarium (Kim et
al., 1998; Ohbayashi et al.,
2002; Rice et al.,
2000). In addition, although a growing number of intracellular
antagonists for receptor tyrosine kinases (RTKs) have been identified as being
important modifiers of FGF-responsiveness in many developmental contexts,
information on how these may regulate calvarial bone formation is currently
lacking (Furthauer et al.,
2002; Kawakami et al.,
2003; Wakioka et al.,
2001).
At the transcriptional level, a number of homeodomain proteins have been
shown to participate in regulating calvarial bone development. Perturbed
calvarial ossification is observed in humans and mice harboring
loss-of-function mutations in the homeoproteins Msx2, Dlx5 and
Alx4 (Robledo et al.,
2002; Satokata et al.,
2000; Wilkie et al.,
2000; Wuyts et al.,
2000). Interestingly, regulation of the osteocalcin gene promoter
by Runx2 has been shown to be influenced by FGF signaling and the modifying
activities of Dlx5 and Msx2 (Newberry et
al., 1998). Furthermore, FGF signaling has been shown to directly
induce Msx2 gene expression in calvarial sutures
(Ignelzi et al., 2003). These
findings are indicative of crucial interactions between growth factors and
transciptional regulators of calvarial osteogenesis, and raise the possibility
that other such modifiers remain to be identified.
Engrailed 1 (En1), the homolog of Drosophila en, is a
homeodomain-containing transcription factor that participates in the
regulation of multiple mammalian developmental processes, such as dorsoventral
patterning of the distal limb and mid-hindbrain specification
(Loomis et al., 1996;
Wurst et al., 1994).
Interestingly, several ossification and growth abnormalities observed in the
sternae and phalanges of En1-null mice, as well as the observed
expression of En1 in developing vertebrae, have implicated its
involvement in skeletal development
(Davidson et al., 1988;
Wurst et al., 1994). We show
that En1 is expressed during the early and late phases of calvarial
osteogenesis. Through the characterization of novel phenotypic features of
En1-ablated mice, we demonstrate that EN1 plays a crucial role in
regulating intramembranous ossification during craniofacial bone development.
In addition, evidence is provided to suggest that EN1 regulates calvarial
ossification by influencing FGF responsiveness in osteoblasts. We further show
that EN1 has a non cell-autonomous function in regulating osteoclast
recruitment and activation, thereby affecting calvarial bone resorption and
remodeling.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Matise and Joyner,
1997; Wurst et al.,
1994) were maintained on a wild-type Swiss Webster background.
Mice harboring either the En1hd (homeobox deleted) or
En1lki (lacZ `knock in') null alleles were
intercrossed to generate homozygous mutants lacking En1 function.
Whole-mount skeletal preparations and ß-galactosidase staining
Visualization of the cartilaginous and mineralized skeleton was facilitated
by Alcian Blue and Alizarin Red staining, as previously described
(Loomis et al., 1996). For
detection of ß-galactosidase activity in tissues, specimens were treated
for several hours with a gluteraldehyde fixative, washed in phosphate-buffered
saline (PBS) and stained with standard X-gal solution for 16 hours at 4°C
(Matise and Joyner, 1997).
Quantitative micro-computed tomography (microCT)
Micro-computed tomography (microCT) was performed at the Centre for Bone
and Periodontal Research located on McGill campus (Montreal, Quebec). Data
were acquired on a SkyScan T-1072 microtomograph (Skyscan, Aartselaar,
Belgium). Cross-sections along the specimen axis were reconstructed and images
quantified using the Cone-Beam Reconstruction Software supplied by
SkyScan.
Histological and immunohistochemical analysis of calcified and decalcified tissues
Dissected newborn skulls were fixed overnight at 4°C in 4%
paraformaldehyde (PFA), and decalcified for 4 days in 40 mM EDTA (pH 7.3),
before dehydrating and embedding in paraffin. X-gal-stained tissues were
dehydrated in isopropanol.
Immunolocalization of phosphorylated extracellular signal-regulated kinase (pERK) was performed on 6-µm deparaffinized sections using a rabbit monoclonal antibody specific to phosphorylated ERK1/2, according to the manufacturer's instructions (20G11, Cell Signaling). Signal detection was performed using the ABC-AP Kit (Vector Laboratories) coupled to the BM-Purple substrate (Roche).
In situ hybridization (ISH) and probes
Probes for osteopontin (Opn), osteocalcin (Ocn), bone
sialoprotein (Bsp) and osterix (Osx), were amplified by
reverse transcriptase (RT)-PCR (iScript cDNA Synthesis Kit, BioRad), employing
specific primers to each mRNA and total RNA extracted from primary calvarial
osteoblasts as a template. Amplified cDNA fragments were cloned into the
pGEMT-Easy cloning vector (Promega). Probes for murine Fgfr1 and
Fgfr2 were obtained from Dr G. Morriss-Kay (Oxford). The probe for
Fgf18 was obtained from Dr B. Hogan (Duke). The probes for
Spry1 and Spry2 were obtained from Dr G. Martin (UCSF).
Digoxigenin-UTP labeling of RNA riboprobes was performed with the MEGAscript
transcription kit (Ambion). In situ hybridization was performed on
paraffin-embedded sections or whole-mount calvariae, essentially as described
by Wilkinson (Xu, 1999).
In vivo proliferation analysis
Bromodeoxyuridine (BrdU, Sigma; 100 µg/g body weight) was administered
intraperitoneally into pregnant mice at the indicated gestational stages;
then, 1.5 hours later, mice were sacrificed and embryos collected and fixed
overnight in 4% PFA. Immunodetection of BrdU was performed as previously
described (Ishii et al.,
2003). A comparative analysis of the osteogenic fronts of
En1-null and wild-type littermates was performed, and statistical
significance analyzed by ANOVA one-way assessment of variance (Graph
Prism).
Assessment of alkaline phosphatase (ALP) activity, tartrate-resistant acid phosphatase (TRAP) and mineral content
ALP activity was quantitated as previously described
(Deckelbaum et al., 2002). The
histological detection of TRAP+ osteoclasts in paraffin-embedded
sections was performed as previously described
(Miao et al., 2004).
Whole-mount detection of TRAP+ osteoclasts was performed as
described (Holt et al., 1994).
For the detection of mineralized matrix, cultured cells were formalin fixed,
ethanol dehydrated, stained with 2% AgNO3 under ultraviolet light,
and then treated with 5% sodium thiosulfate. Detection of mineralized bone
matrix in non-decalcified tissue sections was performed as described
previously (Valverde-Franco et al.,
2004).
Culture of murine primary calvarial osteoblasts
Dissected calvarial bones were collected in Hank's balanced salt solution
(HBSS, Gibco). To extract osteoblasts, bone fragments were treated with
collagenase type IA (1 mg/ml in Hank's balanced salt solution; Sigma) for 30
minutes at 37°C, and then with EDTA (4 mM in PBS), and released cells were
plated at a density of 106 cells/35 mm in complete culture media
[cCM: 10% FCS (Gemini), 
-MEM (Gibco), 100 U/ml penicillin, 50 µg/ml
streptomycin]. At confluence, cCM was supplemented with 100 µg/ml ascorbate
and 5 mM ß-glycerophosphate to promote osteogenic differentiation and
mineralization (Bakker,
2003).
Northern blot analysis
Northern blot analysis was performed as previously described using total
RNA (20 µg) extracted from primary osteoblasts, separated on a denaturing
formaldehyde gel, and blotted onto supported nitrocellulose
(Deckelbaum et al., 2002).
Quantitative assessment of autoradiograms was performed using ScionImage
software.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Matise and Joyner, 1997). The
En1-lacZ allele (En1lki), from which the
expression of functional EN1 protein is abrogated, precisely recapitulates the
endogenous En1 expression. Importantly, En1lki/+
heterozygotes display no discernable phenotype, and are used interchangeably
with wild-type mice in this study. Furthermore, the En1 homolog
En2 is not expressed by any calvarial tissues (data not shown),
excluding the possibility that it compensates for En1 function in
these tissues. Calvarial expression of En1, as detected by whole-mount X-gal staining, initiates at embryonic day (E) 11.5 within lateral aspects of the head (Fig. 1A, part a; arrow), overlying the diencephalic-telencephalic border in the forebrain. Between E12.5 and E13.5, En1 expression expands rostrocaudally and anteriorly, encompassing the frontonasal and mandibular prominences (Fig. 1A, parts b,c; arrows). During phases of overt intramembranous ossification [E14.5 to postnatal day (P) 1], En1 is detected in all developing calvarial bones and sutures (Fig. 1A, parts d-f; arrows, asterisk). Coronal sections of E11.5 heads revealed En1 expression within the presumptive calvarial bone mesenchyme, as confirmed by its colocalization with the early osteogenic marker ALP (Fig. 1B, part a; data not shown). By E13.5, this domain had expanded further toward the base and apex of the skull (Fig. 1B, part b). Histological analysis of the neonatal skull showed that En1 is predominantly expressed by ectoperiosteal osteoblasts lining the bone surfaces (Fig. 1B, parts c,d; arrow), as well as by osteoblasts and osteocytes populating the endosteal surfaces and the matrix of the cranial bone trabeculae (Fig. 1B, part d; arrowheads). Curiously, we found En1 expression to differ between the types of calvarial sutures. Within the abutting interfrontal suture, En1 expression remains restricted to the sutural mesenchyme and is excluded from the osteoprogenitors populating the bone margins (Fig. 1B, parts c,e). By comparison, the osteoprogenitors outlining the frontal and parietal bones exhibit En1 expression (Fig. 1B, part f). Taken together, our expression analyses suggest potential roles for En1 in regulating both the primordial and later stages of calvarial ossification.
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), we found no differences between wild-type and
En1-/- mice in this regard.
|
To evaluate the cellular basis for the defects in calvarial bone
mineralization, we evaluated the capacity of En1 mutant osteoblasts
to induce mineralization in culture. Osteoblasts released from newborn
wild-type and En1-/- calvariae were cultured for 21 days
under conditions that promote extracellular matrix mineralization. We observed
that, although wild-type osteoblasts formed extensive mineralized
three-dimensional nodules, En1-/- osteoblasts exhibited a
poor capacity to induce mineralization over the culture period
(Fig. 3D). Moreover, ALP
activity, the osteoblastic expression of which is indispensable for bone
mineralization (Fedde et al.,
1999; Murshed et al.,
2005), was significantly reduced in En1-/-
osteoblasts in comparison to the wild-type population
(Fig. 3E). Collectively, these
results imply that En1 is required for calvarial osteoblast
differentiation and bone mineralization.
Impaired osteogenesis and osteoblast function in calvarial bones of En1-/- mice
We therefore investigated the possibility that the calvarial defects in
En1 mutants result from impaired osteoblast differentiation in vivo.
The expression of osteopontin (Opn), a secreted phosphoglycoprotein
that is normally activated in differentiating calvarial osteoblasts
(Iseki et al., 1997), was
examined by whole-mount ISH. We first detected calvarial expression of
Opn in wild-type mice at E14.5, within the ossification centers of
the presumptive parietal bones (Fig.
4A). By comparison, Opn was nearly absent in
En1-/- skulls. By E16.5, Opn expression extended
into all calvarial membranous bones of wild-type and
En1-/- skulls but its domain was considerably reduced in
the mutants. This deficiency in Opn expression persisted at E18.5 in
En1-/- rudiments, indicating that En1 plays a
role in promoting osteoblast differentiation during the prenatal stages of
calvarial bone development.
To evaluate whether the delay in Opn initiation in En1 mutants arises from impaired commitment and early differentiation of the cranial skeletogenic mesenchyme, we examined the expression of the osteogenic determinant Osx in E13.5 skulls. Interestingly, Osx and En1 expression overlapped in the calvarial skeletogenic mesenchyme of wild-type embryos (Fig. 4B; compare with Fig. 1B, part b). By contrast, virtually no expression of Osx was observed in the calvarial mesenchyme of En1-/- littermates. As comparable levels of Osx occurred within the mutant and wild-type mandibular mesenchyme (not shown), this result suggests that En1 has a selective role in enhancing the osteogenic potential of the skull vault mesenchyme. Furthermore, Osx expression remained reduced in En1-/- calvarial osteoblasts both in vivo and in vitro (Fig. 4B,D). These findings suggest that En1 function is specific to and indispensable for inducing early phases of calvarial osteogenesis, possibly through the activation of Osx.
To gain a more detailed perspective on calvarial osteogenesis, we examined the coronal suture morphology and the expression of osteoblastic genes on sagittal sections of wild-type and En1-/- skulls at P1. We made three unanticipated observations. First, the thin layer of mesenchyme, which typically separates the overlapping parietal and frontal bone fronts in the wild-type coronal suture, was considerably thickened in En1 mutants (Fig. 4C, arrow). Moreover, the margins of the mutant frontal and parietal bones failed to overlap within the suture proper. Second, although En1 mutants at this developmental stage exhibited normal Opn expression in ectoperiosteal and endosteal osteoblasts, terminally differentiated osteocytes continued to express this gene inappropriately (4C, insets, arrowheads). Third, the calvarial expression of osteocalcin (Ocn), a specific marker of late osteoblast differentiation, was almost abolished in En1 mutants. In corroboration, the expression of Ocn and bone sialoprotein (Bsp), an additional marker of the mature osteoblast, was impaired in cultures of differentiating En1-/- calvarial osteoblasts (Fig. 4D). These findings suggest that En1 functions beyond early osteogenesis to regulate the expression of the late osteoblastic genes commonly associated with matrix mineralization. In addition, the morphological changes within the suture suggest that EN1 may regulate osteoprogenitor proliferation and/or differentiation.
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We next evaluated BrdU incorporation within the coronal sutures of wild-type and En1-/- calvariae at E18.5. The wild-type coronal suture characteristically comprised a thin layer of mesenchyme containing relatively few proliferating cells at the osteogenic fronts of the overlapping parietal and frontal bones (Fig. 5C). By contrast, the En1-/- suture was considerably thickened and hypercellular, and contained increased numbers of BrdU-positive cells. These findings indicate that En1 plays differential roles in regulating osteoprogenitor proliferation within distinct suture types.
|
). We found comparable expression of Fgf18 in sutural
osteoprogenitors and mature calvarial periosteal osteoblasts between wild-type
and En1-/- mice. These findings suggest that En1
is crucial for attenuating Fgfr1 and Fgfr2 expression within
the coronal suture mesenchyme, and raises the possibility that En1
regulates osteoprogenitor proliferation through FGFR signaling.
|
). As Spry1 and Spry2 are commonly expressed
in FGF-responsive tissues (Minowada et
al., 1999; Zhang et al.,
2001), we examined their expression in the calvarial bones of
wild-type and En1-/- mice. Although Spry1 was not
detected in wild-type or En1-/- calvariae (data not
shown), Spry2 was expressed by wild-type ectoperiosteal osteoblasts,
osteoprogenitors of the coronal suture, and, to a lesser extent, endosteal
osteoblasts of the frontal bone trabeculae
(Fig. 6C). In contrast to the
expression in wild-type calvariae, we found almost no expression of
Spry2 in parallel sections of En1 mutants. This finding,
together with the FGF ligand/receptor expression data, suggests that EN1 is
required for regulating the signal transduction events downstream of FGFR that
are necessary for activating Spry2 transcription (see
Fig. 6E for proposed
model).
The induction of Spry2 expression is mediated in part by the
ERK-signaling cascade downstream of FGFR
(Ozaki et al., 2001). More
importantly, ERK phosphorylation is crucial for promoting FGFR-mediated
osteogenesis in calvarial cell and organ cultures
(Kim et al., 2003;
Xiao et al., 2002). To
evaluate the role of this signal transduction pathway in vivo, we used an
antibody specific to the phosphorylated form of ERK (pERK) to probe sections
of calvarial bone. As shown in Fig.
6D, we observed weak pERK expression in the osteogenic fronts of
the parietal and frontal bones of wild-type and En1-null calvariae,
whereas no activity was detected in periosteal osteoblasts of either genotype.
By contrast, strong pERK activity was observed in the endosteal osteoblasts of
wild-type frontal bone trabeculae at E16.5 (not shown) and P1. Strikingly, the
number of endosteal osteoblasts exhibiting pERK activity in
En1-/- calvariae was significantly reduced. These in vivo
findings show that Spry2 and pERK lie within distinct spatial domains
of calvarial bone, which strongly suggests that signal transduction pathways
other than the ERK/MAPK cascade mediate osteoblastic induction of
Spry2 downstream of FGFs. In addition, ERK activity correlates with
advanced osteoblast maturation, and its deficiency in
En1-/- calvariae suggests that impairment in FGF signaling
might be a contributing factor to the perturbed osteogenic differentiation
that characterizes En1 mutants.
Increased resorption and aberrant osteoclast activation in En1-/- calvarial bone
In addition to impaired osteogenesis, increases in osteoclast number or
activity could potentially contribute to the calvarial osteopenia in
En1 mutants. Our observations of multiple perforations within the
mature frontal and parietal bones of En1-/- skulls
suggested an osteolytic process (Fig.
6A). This prompted us to evaluate osteoclast activity in wild-type
and En1-/- calvariae by using whole-mount staining for
TRAP activity. TRAPs are produced by both mono- and multi-nuclear activated
osteoclasts, and are localized primarily to the osteoclast ruffled border and
the extracellular resorptive space
(Minkin, 1982). We observed
that TRAP-staining in the parietal bones of wild-type mice at P5 was stronger
near the bone margins, whereas areas distant from the sutures stained less
intensely (Fig. 6B). In
contrast to wild-type bones, the analogous bones of En1-/-
mice showed uniform TRAP activity throughout. Histological analysis of coronal
sections of wild-type calvariae at P1 confirmed that osteoclasts were
restricted to areas of mature trabecularized bone, and predominantly occupied
the endocranial surfaces (Fig.
6C, arrows). By comparison, En1-/- cranial
bones displayed significantly more TRAP+-osteoclasts, which were
often larger and located along trabeculae distant from the endocranial surface
(Fig. 6C, arrowheads).
Substantiating these observations, quantitative histomorphometry performed on
E18.5 and P1 calvariae showed increased osteoclast numbers in En1
mutants, consistent with the hypothesis that dysregulated osteoclastogenesis
underlies the osteolytic phenotype of En1 null calvariae (E18.5: wild
type, 17.11±1.559, n=9; En1 null, 47.11±2.796,
n=9; P1: wild type, 46.83±5.974, n=6; En1
null, 76.50±2.766, n=6). These results suggest that increased
osteoclast numbers may be a contributing factor to the osteopenic phenotype of
En1 mutants.
To determine whether increased osteoclast recruitment in En1
mutants results from alterations in known osteoclastogenic regulators, we
examined the expression of receptor activator of NFB ligand (RANKL;
TNFSF11 - Mouse Genome Informatics), a TNF-related cytokine that promotes
osteoclast differentiation (Takahashi,
2002). Primary osteoblasts were released from wild-type and
En1 mutant calvariae at P2 and cultured for 7 days under conditions
promoting mineralization. Under these conditions, Rankl was strongly
upregulated in En1-/- cells
(Fig. 6E,F). By comparison,
expression of osteoprotegerin (Opg), a decoy receptor and inhibitor
of Rankl, was comparable between wild-type and
En1-/- cells. These findings point toward an additional
and non cell-autonomous role for En1 in regulating osteoclast
differentiation and/or recruitment.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Furthermore, En1 expression initiates in the calvarial skeletogenic
mesenchyme at E11.5, several days following the cessation of CNC migration
(Jiang et al., 2002).
Interestingly, En1 expression temporally precedes that of the
osteogenic determinant Osx, and, in the absence of En1, the
onset of Osx expression is delayed. As Osx is necessary for
potentiating the osteogenic fate of the skeletogenic mesenchyme
(Nakashima et al., 2002), its
perturbed expression provides a mechanistic basis for the delayed calvarial
ossification in En1-/- mice. Furthermore, that
Osx expression remains impaired in En1-null osteoblasts,
suggests that En1 also lies upstream of Osx during later
phases calvarial osteogenesis, and thus mediates distinct functions in
osteoblast differentiation.
Consistent with a later role for En1 in osteoblast differentiation
and function, our quantitative morphometric analysis showing reduced bone
volume in En1 mutants is indicative of generalized calvarial
osteopenia. In correlation with this, En1-/- osteoblasts
were deficient in mediating osteoid mineralization and exhibited reduced ALP
activity, an enzyme that is essential for this process
(Fedde et al., 1999;
Murshed et al., 2005).
Corroborating its role in osteoblast function, En1 is expressed
postnatally by ectoperiosteal and endosteal osteoblasts, as well as by
terminally differentiated osteocytes. Moreover, ablation of En1
results in impaired Ocn and Bsp expression, genes that are
normally associated with advanced osteoblast differentiation
(Aubin, 2002). Ocn
expression has also been shown to be dependent on Osx
(Nakashima et al., 2002).
However, the fact that Opn expression in En1-/-
calvariae is restored to wild-type levels postnatally, suggests that early
phases of osteoblast differentiation can eventually occur in the absence of
En1 (Fig. 4C,D). Taken
together, our results strongly indicate that, in addition to its role in early
osteogenic commitment, En1 is directly required for mediating late
calvarial osteoblast differentiation and bone matrix mineralization.
|
).
Activating mutations in FGFR1 or FGFR2 that cause
craniosynostosis in humans are recapitulated by knock-in mouse models that
exhibit enhanced osteoblast differentiation
(Eswarakumar et al., 2004;
Zhou et al., 2000).
Conversely, selective ablation of Fgfr2IIIc, the mesenchymal splice
variant of the Fgfr2, results in impaired calvarial ossification and
osteoblast differentiation (Eswarakumar et
al., 2002). Mice lacking FGF18 ligand share phenotypic
similarities with En1 mutants: they display defective calvarial
ossification and delayed terminal osteoblast differentiation
(Ohbayashi et al., 2002).
Thus, the associated alterations in osteoblastic differentiation affecting
En1-/- calvariae are consistent with an underlying
perturbation in FGF signaling. Importantly, our data demonstrate that these
effects are not due to changes in the osteoblastic expression of Fgfr1,
Fgfr2 and Fgf18, but are rather attributed to hampered events
downstream of FGF receptor activation.
Two lines of evidence indicate that En1 regulates signaling
mediated by FGFRs. First, the activation ERK, normally restricted to the
mature endosteal osteoblasts of wild-type calvarial bone, is severely impeded
in En1 mutants (Fig.
7D,E). Second, En1 ablation results in loss of the FGF
target gene Spry2 in ectoperiosteal osteoblasts. The significance of
limiting pERK to the most mature osteoblasts in bone is not entirely clear;
however, it correlates with previous studies ascribing inductive functions for
pERK during calvarial bone formation and advanced osteoblast differentiation
(Kim et al., 2003;
Xiao et al., 2002). Therefore,
En1 might mediate the terminal differentiation of endosteal
osteoblasts by potentiating ERK activity
(Fig. 6E). The ERK/MAPK cascade
has been demonstrated as an important intracellular mediator of FGF-signaling
in multiple developmental contexts. Interestingly, recent studies have shown
that ERK activity is frequently limited to the sub-domains of FGF-responsive
regions (Corson et al., 2003).
Reciprocally, a number of FGFR inhibitors (Sprouty, Sef, Spred, Mkp3)
are induced by FGF signaling, and are expressed in patterns consistent with
their role in restricting ERK activity
(Kawakami et al., 2003;
Lin et al., 2002;
Minowada et al., 1999;
Wakioka et al., 2001;
Zhang et al., 2001). Here, we
present novel evidence demonstrating the existence of select domains for ERK
activation in calvarial bone. Accordingly, we found that Spry2, a
biochemical antagonist of the ERK/MAPK pathway
(Hanafusa et al., 2002), is
preferentially expressed by ectocranial osteoblasts and sutural
osteoprogenitors, indicating that it may play a role in spatially modulating
FGF responsiveness. However, the fact that loss of Spry2 expression
in En1-/- calvariae did not result in enhanced ERK
activity in ectoperiosteal osteoblasts suggests that other antagonists may
also modulate ERK. Indeed, in the developing limb bud, Fgf8 signal
responsiveness is attenuated in the mesenchyme by the cooperative activities
of Spry1 and Mkp3, limiting ERK activation to the overlying
ectoderm (Corson et al., 2003;
Kawakami et al., 2003;
Minowada et al., 1999). We
postulate that EN1 regulates the establishment of a negative-feedback loop
within the calvarial skeletal rudiments by inducing the expression of
Spry2 in ectocranial osteoblasts, while potentially repressing the
expression of other FGFR-signaling attenuators (e.g. Mkp3, Sef) in
endosteal osteoblasts (Fig.
6E). Consequently, loss of En1 function would result in
the observed reduction in endosteal pERK
(Fig. 6E). Furthermore,
En1 may regulate alternative FGF-signaling effectors known to affect
osteoblast differentiation, such as p38 MAPK or PKC
(Kozawa et al., 1999;
Lemonnier et al., 2000;
Lomri et al., 2001). A precise
temporal and spatial delineation of these intracellular pathways will enable a
better understanding of how osteoblastic differentiation is coordinated by EN1
and FGFs.
Differential effects of En1-ablation on osteoprogenitor proliferation
In addition to defects in calvarial osteoblast differentiation, altered
osteoprogenitor proliferation is likely to contribute to the frontal foramina
and gaping of the coronal sutures in En1 mutants. The interfrontal
suture forms late in development (E18.5-P1) through the gradual approximation
of the frontal bone margins. Prior to suture closure at the cranial apex, the
bone margins are interposed by extensive mesenchyme that would preclude the
effectiveness of regulatory signals between the opposing osteogenic fronts. By
contrast, the coronal suture is established early (E12-E14) along the
CNC-mesodermal lineage boundary, where a thin layer of mesenchyme maintains a
consistent separation between the closely juxtaposed, but non-fusing frontal
and parietal bones (Jiang et al.,
2002). Together with recent studies showing distinct
FGF-responsiveness between the sutures, it is reasonable to infer that the
interfrontal and coronal sutures represent unique sites for intramembranous
bone formation (Ignelzi et al.,
2003).
Interestingly, En1 is selectively expressed by the interfrontal
sutural mesenchyme, but is excluded from the osteoprogenitors
(Fig. 1). We postulate that the
proliferative defect in the interfrontal suture, which becomes apparent only
by E18.5, stems from deficient commitment of the mesenchyme rather than from a
direct requirement for En1 in promoting osteoprogenitor mitosis. By
comparison, in the coronal suture En1 is expressed by
osteoprogenitors along the opposing bone margins, and its absence results in
increased proliferation and mesenchymal thickening. This suggests that EN1 is
a direct negative regulator of osteoprogenitor proliferation at this location.
Previous studies indicated that Fgfr1 and Fgfr2 elicit a
differential mitogenic response to FGFs in coronal suture osteoprogenitors
(Ignelzi et al., 2003;
Iseki et al., 1997;
Iseki et al., 1999). Moreover,
specific activating mutations in Fgfr2 are known to enhance
proliferation within this population
(Eswarakumar et al., 2004). It
is therefore possible that upregulation of Fgfr2 and Fgfr1
in the coronal sutures of En1-/- calvariae results in the
enhancement of an FGF-mediated mitotic response.
EN1 affects calvarial bone remodeling by regulating osteoclastogenesis
Following the completion of calvarial morphogenesis, prenatal and postnatal
cranial bone expansion is modulated by the resorptive activity of osteoclasts.
Calvarial osteoclast activity has been shown to initiate as early as E16.5 in
the mouse and is important for the modeling and remodeling of the skull vault
during brain growth (Rice et al.,
1997). Here, we show that loss of En1 function results in
focal calvarial osteolytic lesions that correlate with a significant increase
in osteoclast number and activation. Consistent with resorptive bone loss,
En1-ablated mice exhibit reduced calvarial bone volume and increased
marrow space. In agreement with this, osteoclast numbers within the calvarial
bone rudiments of En1-/- mice are significantly
increased.
Osteoclast differentiation and activation occurs in response to specific
cytokines and growth factors secreted by osteoblasts and their progenitors
within the bone marrow microenvironment
(Takahashi, 2002). By binding
to its cellular receptor RANK, RANKL mediates signal transduction pathways
that result in overt osteoclast differentiation. Osteoclastogenesis, in turn,
is balanced by the osteoblast-specific expression and secretion of
osteoprotegrin (OPG), a decoy receptor capable of binding and inhibiting
RANKL. Curiously, osteoprogenitors and less differentiated osteoblasts have
been shown to express higher levels of RANKL and to support osteoclastogenesis
to a greater extent than differentiated osteoblasts
(Atkins et al., 2003;
Gori et al., 2000).
Accordingly, we have demonstrated that En1-null osteoblasts, arrested
in an early stage of differentiation, display a specific increase in RANKL
expression. Furthermore, the aberrant expression of Opn, a known
osteoclastic chemoattractant, by terminally differentiated osteocytes has been
associated with increased bone remodeling and osteoclast recruitment
(Terai et al., 1999;
Yamazaki et al., 1999).
Interestingly, we observed abnormal Opn expression in
En1-/- calvarial osteocytes, suggesting an additional
mechanism for increased osteoclast recruitment in these animals. In addition
to its direct role in regulating calvarial osteogenesis, these findings
demonstrate a novel role for En1 in inhibiting osteoclastogenesis by
osteoblasts. The En1-null mouse thus provides a valuable tool for
studying the interactions between osteogenesis and osteoclastogenesis during
intramembranous ossification.
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ACKNOWLEDGMENTS |
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