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First published online December 20, 2005
doi: 10.1242/10.1242/dev.02200
1 Center for Craniofacial Molecular Biology School of Dentistry University of
Southern California, 2250 Alcazar Street, CSA 103, Los Angeles, CA 90033,
USA.
2 Division of Oral Anatomy, Department of Oral Biological Science, Niigata
University Graduate School of Medical and Dental Sciences, Gakkocho-dori,
Niigata 951-8514, Japan.
* Author for correspondence (e-mail: ychai{at}usc.edu)
Accepted 7 November 2005
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Cranial neural crest (CNC) cell proliferation, Differentiation, Frontal bone development, DLX5, FGFR, TGFß, Twist1, Mouse
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Members of the transforming growth factor ß superfamily mediate a wide
range of biological activities, including cell proliferation, differentiation,
extracellular matrix formation and induction of homeobox genes, suggesting
that TGFß signaling is important in pattern formation during
embryogenesis. TGFß ligands signal through their receptors, which are
members of a transmembrane serine/threonine kinase family
(Massague, 1992;
Massague, 1998;
Eickelberg et al., 2002). The
TGFß ligand binds to the TGFß type II receptor and triggers
heterodimerization with a TGFß type I receptor
(Massague, 1998). Following
heterodimerization, the TGFß IIR transphosphorylates the type I receptor,
resulting in the propagation of a phosphorylation signal to downstream
substrates SMAD2 and SMAD3 (Wrana et al.,
1994; Derynck and Zhang,
1996). To date, overwhelming evidence supports the notion that
both TGFß IR and TGFß IIR are indispensable in eliciting the
biological response of TGFß (Wrana et
al., 1992; Attisano et al.,
1993).
Tgfbr2 is expressed in the CNC-derived ectomesenchyme and cranial
sutures, and has been recently demonstrated to play an important role during
human skull development (Pelton et al.,
1990; Lawler et al.,
1994; Loeys et al.,
2005). We have previously shown that conditional inactivation of
the Tgfbr2 gene in neural crest cells result in cleft palate, small
mandible, calvarial and cardiovascular defects (such as PTA and
aorticopulmonary septum malformations, which are detailed elsewhere) with
complete phenotype penetrance. Furthermore, it is clear that the inductive
signaling within the CNC-derived dura mater is crucial for both the CNC- and
non-CNC-derived calvarial bone development
(Ito et al., 2003). Despite
this information, we still do not have a comprehensive understanding of the
molecular and cellular mechanisms of the TGFß signaling-mediated frontal
bone development.
In this study, we show that TGFß is required for CNC cell
proliferation in both the orbital and the calvarial (lateral) aspects of the
frontal bone primordium. However, osteoblast differentiation is dependent on
TGFß signaling only in the calvarial aspect, not the orbital aspect, of
the developing frontal bone. In searching for members of TGFß signaling
network that are involved in regulating CNC cell proliferation and
differentiation, we show a compromised FGF signaling in the Tgfbr2
mutant. FGF signaling plays a crucial role in regulating calvarial
development. Fgfr2-deficient mice show compromised osteoblast
proliferation (Yu et al.,
2003). In Tgfbr2 mutant mice, Fgf2 and
Fgfr2 expression are significantly reduced. Exogenous FGF2 can rescue
the proliferation defect in the frontal bone primordium of Tgfbr2
mutant, thus demonstrating the biological importance of this signaling cascade
in regulating CNC cell proliferation. Our study provides a useful animal model
towards a comprehensive understanding of normal craniofacial development, as
well as CNC-related congenital malformations.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Soriano, 1999). Mating
Wnt1-Cre and R26R mice generated transgenic mice with
progenies of neural crest cells labeled with indelible ß-galactosidase
(Chai et al., 2000).
Detection of ß-galactosidase (lacZ) activity
Whole embryos (E9.5 and E10.5) were stained for ß-galactosidase
activity according to standard procedures, as previously described
(Chai et al., 2000).
Cryostat sectioning
Mouse embryonic tissue was frozen, sectioned and then stained according to
standard procedures. Specifically, mouse tissue was dissected in PBS and fixed
by immersion in 0.2% glutaraldehyde solution for 30 minutes at room
temperature. Tissue was soaked in 10% sucrose in PBS for 30 minutes at
4°C, incubated in PBS plus 2 mM MgCl2, 30% sucrose and 50% OCT
at 4°C for 2 hours, then frozen in OCT. Sections were cut at 10-20 µm
and mounted on polylysine-coated slides. The mounted tissue sections were
fixed in 0.2% glutaraldehyde for 10 minutes on ice, rinsed briefly in PBS and
rinsed in detergent solution (0.005% NP-40 and 0.01% sodium deoxycholate in
PBS) for 10 minutes at 4°C. Thereafter, the slides were washed in PBS for
10 minutes and stained in X-gal staining solution overnight at room
temperature in the dark. Sections were counterstained with Nuclear Fast Red
and Eosin.
Generation of Tgfbr2fl/fl;Wnt1-Cre mutant mice and histological analysis
Mating Tgfbr2fl/+;Wnt1-Cre with
Tgfbr2fl/fl mice generated
Tgfbr2fl/fl;Wnt1-Cre null alleles that were genotyped
using PCR primers as previously described
(Chytil et al., 2002). All
samples were fixed in 10% buffered formalin and processed into serial paraffin
sections using routine procedures. For general morphology, deparaffinized
sections were stained with Hematoxylin and Eosin.
Analysis of cell proliferation and apoptosis
DNA synthesis activity within the frontal bone primordium was monitored by
intraperitoneal BrdU (5-bromo-2'-deoxy-uridine, Sigma) injection (100
µg/g body weight) at E12.5, 13.5 and 14.5. One hour after the injection,
mice were sacrificed and embryos were fixed in Carnoy's fixative solution and
processed. Serial sections of the specimen were cut at 5 µm intervals.
Detection of BrdU labeled cells was carried out by using a BrdU Labeling and
Detection Kit and following manufacturer's protocol (Boehringer Mannheim).
BrdU-positive and total number of cells within the frontal bone primordium
were counted from five randomly selected sections per sample. Five samples
were evaluated from each experimental group. Student's t-test was
applied for statistical analysis. A P value of less than 0.05 was
considered statistically significant. TUNEL assay was performed using the In
Situ Cell Death Detection (fluorescein) Kit (Roche Molecular Biochemicals) by
following the manufacturer's protocol.
In situ hybridization
In situ hybridization was performed according to the methods of Wilkinson
et al. (Wilkinson et al., 1998). Several negative controls (e.g. sense probe
and no probe) were run in parallel with the experimental reaction. Details of
the experimental procedures are available upon request.
Organ culture of wild type and Tgfbr2fl/fl;Wnt1-Cre mutant frontal bone primordium explants
Timed-pregnant mice were sacrificed on postcoital day 14.5 or 16.5 and
staged according to external developmental characteristics. Frontal bone
primordium explants (six per treatment group) were cultured in serumless,
chemically defined BGJB medium according to standard methods (Chai et al.,
1994).
Preparation and introduction of TGFß or FGF beads
For delivery of TGFß1 or TGFß2, affi-gel blue beads (BioRad),
diameter 50-80 µm, were used. The beads were washed in phosphate-buffered
saline (PBS) and then incubated for 1 hour at room temperature in 10 µg/ml
TGFß1 or TGFß2 (R&D). Heparin-acrylic beads (Sigma, 200 µm
diameter) were soaked in FGF2 (100 µg/ml, R&D) for 1 hour and applied
into the calvaria explant. Control beads were incubated with in 0.1% BSA.
TGFß- or BSA-containing beads were placed adjacent to the margin of
frontal bone primordium. In both wild-type and
Tgfbr2fl/fl;Wnt1-Cre mutant sample, one side of the
frontal bone primordium was treated with BSA bead and the other side treated
with TGFß or FGF2 bead.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Pelton et al., 1990;
Lawler et al., 1994). The
Wnt1 transgene drives Cre expression specifically in the
neural crest lineage and was used to generate Tgfbr2 conditional null
allele (Chai et al., 2000;
Ito et al., 2003). The
anatomical features of a frontal bone include the calvarial aspect, which
covers the frontal lobe of brain, and the orbital aspect, which forms the roof
of bony orbit. At E16.5, all Tgfbr2fl/fl;Wnt1-cre mutants
had identical frontal bone defects, including failed development of the
calvarial aspect of skull and a smaller than normal bony rudiment above the
orbit (Fig. 1B). We performed
histological analyses of the calvaria development at multiple time points in
order to characterize the frontal bone defect in the
Tgfbr2fl/fl;Wnt1-cre mutant sample. Apparent condensation
of the CNC-derived mesenchyme was observed in the frontal bone primordium of
the wild-type embryo at E12.5 (Fig.
1C). There was comparable development of the frontal primordium in
the Tgfbr2fl/fl;Wnt1-cre mutant sample at E12.5
(Fig. 1D). At E13.5, the
frontal primordium of the wild type was more defined and extended superiorly
and laterally (Fig. 1E). In the
Tgfbr2fl/fl;Wnt1-cre mutant, however, the frontal
primordium development was retarded (Fig.
1F). Frontal bone matrix was clearly visible in the wild-type
sample at E14.5 (Fig. 1G). In
the Tgfbr2fl/fl;Wnt1-cre mutant sample, the calvarial
aspect of the frontal bone primordium failed to form bone matrix
(Fig. 1H, arrow), while the
orbital aspect did so (Fig. 1H,
arrowhead). As embryonic development continued, a well-formed frontal bone was
present at both the calvarial and orbital aspects of the frontal bone in the
wild type sample (Fig. 1I,K).
In the Tgfbr2fl/fl;Wnt1-cre mutant sample, only the
orbital aspect of the frontal bone formed, while the calvarial aspect did not
(Fig. 1J,L).
Loss of TGFß IIR signaling does not affect the migration of CNC cells into the frontal primordium
In order to test whether a CNC migration defect might have contributed to
the deficiency of the CNC-derived frontal primordium, and ultimately the
failure of frontal bone development, we crossed the Tgfbr2
conditional allele with R26R transgenic mice and generated embryos
with Tgfbr2fl/fl;R26R;Wnt1-Cre genotype. All of these
embryos had frontal bone hypoplasia identical to that seen in the
Tgfbr2fl/fl;Wnt1-Cre mutant mice. Whole-mount and
sectioned ß-gal staining showed no difference in migration and
distribution of CNC cells within the frontonasal prominence and the first
branchial arch between Tgfbr2fl/fl;R26R;Wnt1-Cre mutant
and the wild-type control embryos from E9.5 to E11.5
(Ito et al., 2003). Following
the beginning of telencephalon development at E10.5, the CNC-derived
mesenchyme was focused above the eye in both the wild-type and the
Tgfbr2fl/fl;R26R;Wnt1-Cre mutant. Histological sections
showed that the CNC-derived mesenchyme began to form the frontal bone
primordium at E11.5 (Fig. 2A).
There was no apparent difference in the contribution of CNC cells within the
frontal primordium between the Tgfbr2fl/fl;R26R;Wnt1-Cre
mutant and wild-type sample (Fig.
2A,B). Our data suggest that there was no CNC migration defect
that might have resulted in a deficient number of CNC-derived mesenchymal
cells within the forming frontal primordium and in failure of frontal bone
development in Tgfbr2fl/fl;Wnt1-Cre mutant mice. At E13.5,
loss of TGFß signaling affected the expansion of the CNC-derived frontal
primordium in the Tgfbr2fl/fl;Wnt1-Cre mutant when
compared with the wild-type control (Fig.
2C,D). Taken together, our data are consistent with a specific
requirement for TGFß signaling in the CNC-derived frontal primordium
during calvarial morphogenesis.
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Disruption of TGFß signaling in the CNC-derived mesenchyme perturbs bone matrix maturation during osteoprogenitor differentiation in the developing frontal bone
To test for an osteoprogenitor differentiation defect that might be
responsible for the failure of frontal bone development in the
Tgfbr2fl/fl;Wnt1-Cre mutant, we performed an osteogenic
marker analysis. As shown in Fig.
1, the frontal bone primordium first appeared to be smaller than
the wild-type control at E13.5. We investigated the differentiation status of
the CNC-derived osteoprogenitors starting at this stage. There was comparable
Runx2, type I collagen, osterix and Ibsp (previously known
as BSP) expression in the developing frontal primordium between the wild-type
and Tgfbr2fl/fl;Wnt1-Cre mutant samples
(Fig. 5A-H), suggesting normal
osteoblast differentiation in Tgfbr2fl/fl;Wnt1-Cre mutant
at E13.5. At E14.5, the number of cells expressing Runx2 reduced in
the calvarial aspect of the frontal bone primordium of the
Tgfbr2fl/fl;Wnt1-Cre mutant when compared with the
wild-type control (Fig. 5I,J).
These data suggest that there was a defect in osteoprogenitor cell
differentiation within this cell population in the absence of TGFß
signaling. Interestingly, analysis of two of the early osteogenic lineage
markers revealed comparable expression of type I collagen and osteonectin in
the frontal primordium of both the wild-type control and the
Tgfbr2fl/fl;Wnt1-Cre mutant
(Fig. 5K-N), suggesting the
presence of early osteoprogenitor cells in the frontal primordium of the
Tgfbr2fl/fl;Wnt1-Cre mutant mice.
Osterix is a zinc-finger-containing transcription factor that is required
for osteoblast differentiation and acts downstream of RUNX2 during osteoblast
differentiation (Nakashima et al.,
2002). The expression of osterix was detected in the calvarial
aspect of the frontal primordium in the wild-type sample
(Fig. 5O, arrow). No osterix
expression was detected within the calvarial aspect of the frontal primordium
in the Tgfbr2fl/fl;Wnt1-Cre mutant sample, suggesting a
defect in osteogenic progenitor differentiation
(Fig. 5P, arrow). Although
reduced, osterix expression was evident in the orbital aspect of the frontal
bone (Fig. 5P, arrowhead),
suggesting that the osteoprogenitor differentiation within this portion of the
frontal bone was not dependent on TGFß signaling. To evaluate
osteoprogenitor terminal differentiation and bone matrix maturation, we
examined the expression of late osteogenic markers. There was virtually no
detectable expression of osteopontin, Ibsp, osteocalcin and ALP in
the calvarial aspect of the frontal primordium (arrow) in the
Tgfbr2fl/fl;Wnt1-Cre mutant, suggesting a bone matrix
maturation defect in the mutant mice (Fig.
5Q-X). Overall, our osteogenic marker analysis indicated that the
CNC-derived osteogenic progenitor cells were unable to complete the
differentiation process (e.g. matrix maturation and mineralization) in the
calvarial aspect of the frontal primordium in the
Tgfbr2fl/fl;Wnt1-Cre mutant.
FGFR2 functions as a downstream mediator of TGFß signaling to control cell proliferation activity within the frontal bone primordium
As shown in this study, loss of Tgfbr2 affects osteoprogenitor
cell proliferation and differentiation in the
Tgfbr2fl/fl;Wnt1-Cre mutant. The identity remains unclear
of the molecular signaling cascade involved in regulating osteoprogenitor cell
proliferation and differentiation during frontal bone development. To
investigate this molecular mechanism, we examined the expression of genes
known to be crucial in regulating skull development. Recent studies suggest
that FGFR2 is crucial for osteoprogenitor cell proliferation during skull
development and conditional inactivation of Fgfr2 results in a
decrease in osteoblast proliferation and bone formation (Iseki et al., 1999;
Yu et al., 2003). To test
whether TGFß and FGF signaling cascade may work together to regulate cell
proliferation, we compared the expression of Fgfr2 between the
wild-type and the Tgfbr2fl/fl;Wnt1-Cre mutant samples. At
E13.5, the expression of Fgfr2 in the frontal primordium was reduced
in the Tgfbr2fl/fl;Wnt1-Cre mutant when compared with the
wild type (Fig. 6A,B). At
E14.5, the defect of the frontal primordium was more evident and there was no
detectable Fgfr2 expression in the calvarial aspect of the frontal
primordium in the Tgfbr2fl/fl;Wnt1-Cre mutant sample
(Fig. 6D). In the wild-type
sample, however, active Fgfr2 expression was detected within the
frontal primordium (Fig. 6C).
Interestingly, the expression of Fgfr2 was also reduced in the
orbital aspect of the frontal bone in the
Tgfbr2fl/fl;Wnt1-Cre mutant, supporting the argument that
TGFß mediated Fgfr2 expression was crucial for osteoprogenitor
proliferation (Fig. 6D,
arrowhead). Because there was no change in apoptotic activity within the
frontal primordium of the Tgfbr2fl/fl;Wnt1-Cre mutant, our
data suggest that TGFß-mediated FGF signaling is involved in controlling
osteoprogenitor proliferation during calvarial development.
TGFß signaling mediates Dlx5 and Twist1 expression during frontal bone primordium development
Dlx5 is known as a crucial mediator of calvarial osteoblast
differentiation. Loss of Dlx5 results in multiple craniofacial
defects including agenesis of frontal bones
(Depew et al., 1999;
Tadic et al., 2002;
Holleville et al., 2003). The
similarity in frontal bone development defect between the
Tgfbr2fl/fl;Wnt1-Cre and Dlx5 mutant mice
suggests that TGFß and DLX signaling network may function together to
regulate osteoblast differentiation during calvarial morphogenesis. To test
this hypothesis, we examined the expression of Dlx5 in the frontal
primordium of the Tgfbr2fl/fl;Wnt1-Cre mutant samples. At
E13.5, well-defined expression of Dlx5 was detected within the
CNC-derived frontal primordium of the control sample
(Fig. 6E). Dlx5
expression was also detected in the frontal bone primordium of the
Tgfbr2fl/fl;Wnt1-Cre mutant
(Fig. 6F). As bone matrix began
to form, Dlx5 was continuously expressed in the frontal primordium of
the wild-type sample at E14.5 (Fig.
6G). In the Tgfbr2fl/fl;Wnt1-Cre mutant,
however, Dlx5 expression was not detectable on the calvarial aspect
of the frontal primordium (Fig.
6H). Interestingly, the osteogenic differentiation defect became
apparent in the Tgfbr2fl/fl;Wnt1-Cre mutant sample at this
stage (Fig. 5), suggesting that
TGFß-mediated Dlx5 expression may play a crucial role in
controlling osteoprogenitor cell differentiation during calvarial
morphogenesis. However, Dlx5 expression was unaffected in the orbital
aspect of the frontal bone in the Tgfbr2fl/fl;Wnt1-Cre
mutant (Fig. 6H, arrowhead).
The fact that osteoblast differentiation occurred successfully in this region
(Fig. 5) suggests that
TGFß signaling is dispensable for osteogenesis at the orbital aspect of
the frontal bone.
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). Loss of
Twist1 function results in defects in the head mesenchyme, failure of
neural tube closure in the cranial region, and other abnormalities
(Chen and Behringer, 1995). An
important function of TWIST1 is to suppress Fgfr gene expression, thereby
inhibiting osteogenic differentiation of the cranial suture mesenchyme, thus
maintaining the potency of cranial sutures during craniofacial development.
During frontal bone development, Twist1 is expressed in the
undifferentiated CNC-derived mesenchyme prior to osteoblast differentiation
(Rice et al., 2000). In the
Tgfbr2fl/fl;Wnt1-Cre mutant, elevated Twist1
expression was detected in the frontal bone primordium where bone matrix
failed to form at E14.5 (Fig.
6J, arrow). In the wild-type sample, bone matrix was formed in the
frontal bone primordium and there was no detectable Twist1 expression
at E14.5 (Fig. 6I), suggesting
downregulation of the Twist1 gene following osteoblast
differentiation. In the orbital aspect of the frontal bone where bone matrix
was detected in both the Tgfbr2fl/fl;Wnt1-Cre mutant and
the wild type, there was no detectable Twist1 expression
(Fig. 6I,J, arrowhead). These
data suggests that TGFß signaling is required to suppress Twist1
expression in order to achieve normal CNC proliferation and differentiation
during frontal bone development.
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We hypothesized that TGFß is crucial for regulating the expression of FGF ligand that itself induces Fgfr2 expression. E14.5 frontal bone primordium explant treated with BSA beads (n=6) showed normal levels of Fgfr2 and Fgf2 expression (Fig. 7G, data not shown). TGFß1 beads treated explants (n=6) showed a dramatic increase in Fgfr2 expression, suggesting that TGFß signaling can induce FGF signaling in the developing calvaria (Fig. 7H). Finally, in order to test whether FGF signals downstream of TGFß in regulating CNC proliferation in the frontal primordium, we performed experiments using calvarial explants from the Tgfbr2fl/fl;Wnt1-Cre mutant sample. BSA beads (n=4) were unable to restore cell proliferation when compared with the wild-type control (Fig. 7I). FGF2 beads treated explants (n=6) showed dramatic increase of Fgfr2 expression, restored cell proliferative activity back to normal, and greatly expanded the cell mass in the Tgfbr2fl/fl;Wnt1-Cre mutant frontal bone primordium explant (Fig. 7J). These results demonstrate that FGF signaling is a crucial downstream component of the TGFß pathway/network in regulating CNC proliferation during frontal bone development.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Francis-West
et al., 1998; Wilkie and
Morris-Kay, 2001). The important findings of this study include
that TGFß signaling is specifically required in the CNC-derived frontal
bone primordium during skull development. TGFß IIR appears to be a
crucial regulator for proliferation and differentiation of osteoblasts, but is
not required for the survival of osteoblast progenitors. Most importantly,
TGFß-mediated FGF signaling controls CNC proliferation in the skull. This
discovery provides a link between two important families of signaling
molecules and begins to provide a cellular mechanism for how different growth
factors may work together to exert their specific regulatory function during
frontal bone development.
Role of Tgfbr2 in frontal bone development
Tgfbr2 is specifically expressed during the condensation,
proliferation and differentiation stages of the frontal bone development,
suggesting that it has a crucial role in regulating osteogenesis
(Lawler et al., 1994;
Iseki et al., 1995). Previous
studies showed that TGFß signaling plays a pivotal role in regulating
postnatal calvarial development and repair. In particular, TGFß signaling
stimulates osteogenic progenitor cell proliferation and can induce premature
suture obliteration in cultured fetal rat calvaria
(Opperman et al., 2000). In
addition, TGFß signaling in the immature dura mater (in newborn and
immature animals) can induce calvarial bone repair, while diminished TGFß
signaling in the mature dura mater fails to repair calvarial defects,
suggesting that TGFß signaling is a crucial regulator for calvarial
ossification (Greenwald et al.,
2000).
Deletion of any one Tgfb isoform is probably compensated for by the
presence of the other, as the three TGFß isoforms are expressed in
overlapping patterns (Pelton et al.,
1991; Dickson et al.,
1995; Sanford et al.,
1997). Inactivation of both Tgfb2 and Tgfb3
results in severe developmental defects that include craniofacial
malformations and early embryonic lethality, suggesting that the concerted
function of TGFß isoforms in regulating craniofacial development
(Dunker and Krieglstein,
2002). More recently, an animal model with a compound mutation of
SMAD2 and SMAD3 has demonstrated a critical function of TGFß signaling in
regulating craniofacial development (Liu
et al., 2004). As there is no CNC migration defect in the
Tgfbr2fl/fl;Wnt1-Cre mutant, our observations are
consistent with a general model in which local signals regulate the fate and
function of neural crest cells as they migrate to their final destinations.
TGFß is one such signal and is required for the proper development of the
CNC-derived frontal primordium during calvarial morphogenesis.
TGFß regulates FGF signaling to control osteogenic progenitor cell proliferation within the CNC-derived frontal bone primordium
TGFß is known to regulate the fate of multipotential progenitor cells
instructively by regulating the expression or function of tissue-specific
growth and transcription factors (Moses
and Serra, 1996). We provide evidence that TGFß signaling is
specifically required in the CNC-derived frontal bone primordium during
initial calvarial morphogenesis. Loss of Tgfbr2 affects
osteoprogenitor cell proliferation. Although osteoprogenitor cells produce
bone matrix proteins, their main function is to maintain the progenitor pool
through cell proliferation (Yu et al.,
2003). It is important to point out that, although both the
calvarial and orbital aspects of the frontal primordium show a significant
defect in cell proliferation, only the calvarial aspect of the frontal bone
primordium fails to form, while the orbital aspect is developed (although
smaller than the one of the wild type) in the
Tgfbr2fl/fl;Wnt1-Cre mutant sample. This discrepancy may
reflect differential regulatory mechanisms for osteoprogenitor differentiation
between different regions of the frontal bone.
Fgfr2 gene expression is clearly downregulated in the frontal bone
primordium of the Tgfbr2fl/fl;Wnt1-Cre mutant.
Fgfr2 is one of the earliest genes expressed in the condensing
mesenchyme and its expression can be induced by FGF2
(Delezoide et al., 1998;
Orr-Urtreger et al., 1991;
Ornitz and Marie, 2002;
Yu et al., 2003). At the
functional level, FGFR2 signaling regulates proliferation of osteoprogenitor
cells and the anabolic function of mature osteoblasts, which apparently has
also been compromised in the Tgfbr2fl/fl;Wnt1-Cre mutant
sample. In addition, the expression of Fgfr1 is also dramatically
reduced in the frontal bone primordium (data not shown). As both Fgf2
and Fgfr2 expression are dramatically reduced and exogenous FGF2
restores Fgfr2 expression as well as rescues CNC proliferation in the
cultured frontal bone primordium in Tgfbr2fl/fl;Wnt1-Cre
mutant, our study suggests that TGFß-regulated FGF signaling is crucial
for CNC proliferation and provides the first in vivo model to demonstrate the
crucial role of this signaling cascade in regulating the fate of CNC cells
during calvarial morphogenesis. Besides using FGF2, we have also attempted to
rescue cell proliferation defect by introducing other morphogens (such as
BMP4) into the frontal primordium of Tgfbr2fl/fl;Wnt1-Cre
mutant sample. To date, only exogenous FGF signaling was able to restore cell
proliferation activity in the mutant sample, demonstrating the functional
specificity of TGFß-mediated FGF signaling in regulating osteoprogenitor
proliferation.
TGFß signaling mediates Twist1 and Dlx5 expression and the differentiation of osteoprogenitor cells during calvarial morphogenesis
TGFß is deposited at high level in the forming skeletal tissue and has
important effects on bone cell function during development and repair
(Filvaroff et al., 1999). Loss
of Tgfbr2 in neural crest derivatives affects the differentiation of
osteoblast cells during frontal bone development. The presence of early
osteogenic differentiation markers (such as type I collagen and osteonectin)
in the frontal primordium suggests that the CNC-derived frontal mesenchyme is
present but fails to differentiate into an osteogenic lineage in the
Tgfbr2fl/fl;Wnt1-Cre mutant at E14.5
(Fig. 5). The failure to
express mature osteogenic differentiation makers (such as osterix, osteopontin
or osteocalcin) in the CNC-derived frontal bone primordium suggests that
TGFß signaling is required for osteoprogenitor cell differentiation.
Conversely, the orbital aspect of the frontal bone anlagen expresses all
osteogenic differentiation markers and can form bone in the
Tgfbr2fl/fl;Wnt1-Cre mutant, demonstrating a differential
regulatory mechanism during osteogenesis.
Of the possible downstream transcription factors that may be controlled by
TGFß signaling and are crucial for osteoblast differentiation,
Dlx5 and Twist1 expression are clearly affected in the
frontal primordium of the Tgfbr2fl/fl;Wnt1-Cre mutant.
Dlx5 plays a crucial role in skull formation
(Depew et al., 1999;
Acampora et al., 1999).
Targeted inactivation of Dlx5 results in multiple craniofacial
defects, including frontal bone malformations
(Depew et al., 1999). Several
lines of evidence suggest that Dlx genes are crucial for regulating osteoblast
differentiation. First, Dlx single (e.g. Dlx5) or double
(Dlx5/Dlx6) null mutation results in abnormal skull development
(Depew et al., 1999;
Depew et al., 2002). Second,
Dlx5 is crucial for regulating osteocalcin expression and promoting
the maturation of bone cell phenotypes
(Ryoo et al., 1997;
Newberry et al., 1998;
Depew et al., 1999). Third, a
recent study suggests that Dlx5 binds directly to the alkaline
phosphatase promoter to stimulate Alp expression in response to BMP2
treatment. Msx2 counteracts the osteogenic-inducing property of
Dlx5 during osteoblast differentiation
(Kim et al., 2004). Our study
shows that Dlx5 expression is significantly suppressed in the frontal
bone primordium of the Tgfbr2fl/fl;Wnt1-Cre mutant. Taken
together, we propose that TGFß-mediated Dlx5 expression plays a
crucial role in regulating the terminal differentiation of osteoblast during
calvarial morphogenesis.
TWIST proteins are basic helix-loop-helix (bHLH)-containing transcription
factors. Haploinsufficiency of Twist1 results in craniosynostosis and
suggests that Twist1 generally functions as an osteoblast
differentiation inhibitor (el Ghouzzi et
al., 1997; Howard et al.,
1997). The increased Twist1 expression in the frontal
bone primordium in the Tgfbr2fl/fl;Wnt1-Cre mutant has
significant implications. In terms of osteoblast progenitor proliferation,
Fgfr2 and Twist1 have overlapping expression patterns and
FGFRs may mediate a signal downstream of Twist1 because murine Twist1
expression precedes that of Fgfr genes during skull development
(Jabs, 2001;
Holleville et al., 2003). It
is conceivable that elevated Twist1 expression inhibits Fgfr gene
expression in the frontal primordium, which results in a compromised
osteoprogenitor cell proliferation in the
Tgfbr2fl/fl;Wnt1-Cre mutant. In parallel, TWIST1 protein
has recently been shown to inhibit RUNX2 function during skeletogenesis
(Bialek et al., 2004). Elevated
Twist1 expression may inhibit Dlx5 expression to inhibit the
terminal differentiation of osteoprogenitor cells within the CNC-derived
frontal bone primordium in the Tgfbr2fl/fl;Wnt1-Cre mutant
sample and to prevent normal bone formation.
This study clearly demonstrates the important inductive function of dura
mater in regulating calvarial bone development as conditional inactivation of
TGFß signaling in the CNC-derived dura mater resulted in a defect in the
mesoderm-derived parietal bone primordium (T.S., Y.I., P.B. and Y.C.,
unpublished). The successful induction of Fgfr2 expression by
implanted TGFß beads in the parietal bone primordium of the
Tgfbr2fl/fl;Wnt1-Cre mutant sample shows that the
TGFß signaling cascade is intact in this mesoderm derived tissue and the
parietal bone defect may be the result of a defect in another CNC-derived
tissue. This finding is consistent with previously published results in which
the ossification of parietal bone requires interaction with the underlying
neural crest derived meninges (Jiang et
al., 2002). However, it is not clear whether the TGFß
signaling mediated ossification of the CNC-derived frontal bone is cell
autonomous or is completely dependent on the inductive signals from dura
mater, which can only be tested by a specific inactivation of TGFß
signaling in the meninges.
In summary, our study shows that loss of Tgfbr2 in the neural crest does not affect the migration of CNC cells during frontal bone development. TGFß IIR is specifically required for proliferation and terminal differentiation of the osteoprogenitor cells. TGFß-mediated FGF signaling is crucial for the CNC-derived osteoprogenitor proliferation. As exogenous FGF signaling can rescue only the defect in cell proliferation but is unable to promote differentiation of osteoprogenitors in the frontal bone primordium of Tgfbr2fl/fl;Wnt1-Cre mutant sample, it is apparent that there are additional signaling pathways that rely on TGFß signaling to induce osteoblast differentiation in the calvarial aspect of the frontal bone primordium. Specifically, TGFß-mediated Twist1 and Dlx5 expression may play a crucial role for osteoblast differentiation and osteoid mineralization. In the orbital aspect of the frontal bone primordium, however, TGFß signaling is apparently dispensable for differentiation of osteoprogenitor cells. It is tempting to speculate that signals from the orbit may compensate the loss of TGFß signaling in the orbital aspect of frontal bone and allow the successful differentiation of osteoprogenitor cells in the Tgfbr2fl/fl;Wnt1-Cre mutant sample. Future studies using this animal model will provide useful information on the mechanism of TGFß signaling in regulating human skull development.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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