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Regular Article |
From the Laboratory of Developmental Biology and
Repair,*
The Institute of Reconstructive Plastic Surgery,
and The Department of Surgery, New York University Medical Center, New
York, New York; the Department of
Cardiology,
Molecular Cardiology Unit, Kyushu
University School of Medicine, Fukuoka, Japan; and the Department of
Surgery,
Stanford University School of
Medicine, Stanford, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To understand the events that lead to
premature cranial suture fusion, numerous investigators have relied on
animal models of cranial suture fusion. Murine suture development, in
which the posterior frontal (PF) suture undergoes programmed sutural
fusion shortly after birth, provides an ideal model to study the
biomolecular mechanisms that occur before, during, and after cranial
suture fusion.7,8
In these models, the PF cranial suture
fuses between postnatal days 12 to 22 in the rat and days 25 to 45 in
the mouse whereas all other sutures, including the coronal (COR) and
sagittal (SAG), remain patent (Figure 1)
.
Using these models, our group and others have demonstrated that the
dura mater directly underlying a cranial suture regulates sutural fate
(ie, fusion or patency).8-11
In addition, we have shown
that FGF-2 mRNA and protein expression in the fusing PF suture is
up-regulated in the suture-associated dura mater just before and during
active sutural fusion.12,13
These findings implicate FGF
biological activity in the regulation of cranial suture fusion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All experiments were approved by the Institutional Use and Care Committee at New York University Medical Center. Time-dated pregnant Sprague-Dawley rats were purchased from Taconic Laboratories (Germantown, NY). CD-1 mice (24 days old) were purchased from Charles River Laboratories (Wilmington, MA). For all animals, the first day of life was considered the first day after birth.
Preparation of Adenoviral Vectors
A replication-deficient recombinant adenovirus encoding a truncated form of the chicken FGF-R1 gene (AdCAFGF-TR) was constructed as previously described.14 The truncated FGF-R1 was a 1.3-kb cDNA fragment lacking the sequence coding its cytoplasmic tyrosine kinase domain and tagged with an influenza virus hemagglutinin epitope at its C-terminus. This fragment was subcloned into an adenoviral expression vector (type 5 genome with deletions of E1 and E5 regions) containing a chicken {beta}-actin promoter with a cytomegalovirus enhancer unit. This construct has been shown to inhibit signal transduction by FGF-R1, FGF-R2, and FGF-R3 in vitro.15
AdCAsFGF-2 was prepared as previously described.16 Briefly, a recombinant cDNA for the secreted form of human FGF-2 was constructed by adding the signal sequence of FGF-4 to the 5' end of cDNA encoding the full-length human FGF-2. The control adenovirus encoding the Escherichia coli lacZ ({beta}-galactosidase cDNA) was constructed in a similar manner.14 All adenoviruses were propagated in human 293 cells and purified by centrifugation in cesium chloride step gradients before dialysis against 10% glycerol in phosphate-buffered saline (PBS) as previously described.17
Tissue Harvest for PF-Dural Cell and Osteoblast-Enriched Cell Cultures
Dural cell cultures derived from PF-associated dural tissues (PFDCs) from postnatal day 6 rats were established as previously reported.18 Osteoblast-enriched cell cultures (NRCs) derived from neonatal rat calvaria were established as previously described by Frick and colleagues.19 First passage cells were used for all experiments.
Western Blot Analysis for FGF-TR, Phosphorylated ERK-1 and -2, and FGF-2
Western blot analyses were performed to assess transgene expression and phosphorylation of proteins involved in FGF signal transduction. One million first passage NRCs and PFDCs were plated on tissue culture plates. Once confluent, cells were infected with vehicle, AdCALacZ (100 pfu/cell), or AdCAFGF-TR (50 and 100 pfu/cell). Protein was collected 48 hours after infection and an immunoblot analysis of hemagglutinin protein expression (ie, marker on the FGF-TR transgene) was performed using a monoclonal hemagglutinin antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Twenty-four hours after infection, an additional group of infected cells was placed in serum-free media. After 24 hours (ie, 48 hours after infection), 10 ng/ml of recombinant human basic FGF (rhFGF-2; R&D Systems, Minneapolis, MN) was added to the serum-free media. After 30 minutes, total cellular protein was collected for Western blot analysis. Immunoblot analysis was performed using an antibody to phosphoERK-1 and -2 (ERK = extracellular regulated protein kinase; New England BioLabs, Beverly, MA) and reprobing the same membrane with a nonphosphorylation-specific ERK-2 antibody that recognizes ERK-1 to a lesser extent (Santa Cruz).
To verify overexpression of FGF-2 by the AdCAsFGF-2 construct, cells were infected with vehicle, AdLacZ, and AdCAsFGF-2 as described above. Protein was collected 48 hours after infection. Western blot analysis for FGF-2 was performed using a monoclonal human FGF-2 antibody (clone no. 354F1; Texas Biotechnologies, Houston, Texas). Immunoblot analyses of phosphoERK-1 and -2 and total ERK were performed as described above without rhFGF-2 stimulation of vehicle and AdCALacZ-infected cells.
Cranial Suture Organ-Culture System
To analyze the effects of alterations in FGF biological activity,
a cranial suture organ-culture system was established as previously
described (see Figure 3A
).7,8
In this model, the mouse PF
suture fuses after 30 days in culture. Mouse calvarial explants are
ideal for these studies because their small size and thin bone cortices
allow prolonged survival in culture whereas the thicker, larger bone in
rat calvaria precludes similar analysis in vitro. Briefly,
after euthanizing 30 CD-1 mice (24 days old), a portion of the calvaria
inclusive of the PF, SAG, and COR sutures and their underlying dural
tissues (suture complex) was isolated, and washed copiously with PBS.
The suture complexes were then divided into three groups
(n = 10 per group). Dural tissues underlying the
PF suture were infected with AdCAFGF-TR (1 x
109
pfu), AdCALacZ (1 x
109
pfu), or vehicle (PBS; 20 µl). Infected
suture complexes were then placed into organ culture wells consisting
of an insert with a 3.0-µm pore size (Millipore, Bedford, MA) After
placement of the suture complex into the inserts, 500 µl of
serum-free suture media was placed (BGJb medium supplemented with 500
µ/ml penicillin, 500 µg/ml streptomycin, 0.25 µg amphotericin,
100 µg/ml ascorbic acid, and 0.3 mmol/L sodium phosphate, pH 7.4)
into each well and changed every other day.
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Targeted, in Utero Delivery of AdCAFGF-TR and AdCAsFGF-2
Pregnant Sprague-Dawley rats (embryonic day 18; E18) were purchased from Taconic Laboratories. Modulation of FGF biological activity at E18 was chosen as FGF-2 mRNA is highly expressed by the calvarial dural tissues at this time point.20 After attainment of adequate anesthesia, a midline laparotomy incision was made. The fetal rat heads were exposed by making a hysterotomy. For experiments examining the effect of FGF biological activity abrogation, the fetal PF suture was identified, and 1 x 109 pfu of AdCALacZ, AdCAFGF-TR, or 20 µl of PBS (ie, vehicle) was injected into the region of the PF dura after piercing the calvaria adjacent to the PF suture with a tuberculin syringe. For experiments in which FGF biological activity was increased, the fetal COR suture was identified, and 1 x 109 pfu of AdCALacZ, AdCAsFGF-2, or 20 µl of PBS (ie, vehicle) was injected into the dural tissues underlying the left COR suture. After injection, the fetal head was returned to the uterus and the hysterotomy was repaired with fine suture material. Four of the 11 to 12 fetuses per pregnant rat were treated. The remaining fetuses received a 40-µl injection of India Ink in the dorsal soft tissues of the thorax to facilitate their postnatal identification. After delivery, untreated neonatal rats injected with India Ink were sacrificed. Two pregnant rats (four treated fetuses/rat times two pregnant rats equals eight treated fetuses/group) were used for AdCAFGF-TR and AdCAsFGF-2 transfections. Four pregnant rats each were used for AdCALacZ and vehicle transfections (two in each group received injections targeting the PF dura; two in each group received injections targeting the COR suture).
Treated animals were sacrificed on postnatal day 30. Calvaria were harvested and immediately fixed in formalin. X-gal staining of AdCALacZ-treated animals was performed. After decalcification, 5-µm sections were prepared from the midpoint of the PF and COR sutures. A total of 40 sections was analyzed from each PF and COR sutures and assessed histologically for fusion or patency. To assess the presence of the truncated FGF-R1 construct, immunohistochemistry was performed using a rabbit anti-hemagglutinin antibody. Immunohistochemical techniques were performed as previously described.13
RNA Isolation from NRCs and PFDCs
RNA isolation was performed as previously described.17 Briefly, one million first-passage NRCs or PFDCs were plated on 100-mm culture dishes. After reaching confluence, cells were infected with AdCALacZ (100 pfu/cell), AdCAFGF-TR (50 and 100 pfu/cell), or vehicle. Twenty-four hours after infection, cells were placed in serum-free media for 24 hours and 10 ng/ml of rhFGF-2 (R&D Systems) was added to the media. RNA was isolated after 6 and 48 hours of rhFGF-2 (R&D Systems) stimulation and analyzed for expression of TGF-{beta}1 and collagen type I mRNA, respectively. RNA isolated from infected cells in serum-free media and not exposed to rhFGF-2 served as controls for all groups. RNA was isolated from an additional group of infected cells 48 hours after infection and probed for expression of the transgene encoded by AdCAFGF-TR (see below).
RNA isolation from cells infected with AdCAsFGF-2, vehicle and AdCALacZ was performed 48 hours after infection (serum-free media x 24 hours) and analyzed for alterations in baseline TGF-{beta}1 mRNA expression.
Probe Preparation
Rat probes for TGF-{beta}1 and collagen I
I were 535- and 727-bp
polymerase chain reaction-amplified fragments, respectively, as
previously described.21
The murine FGF-R1 cDNA probe was a
1.2-kb EcoRI-BamHI fragment from the full-length
FGF-R1 cDNA.22
Glycerol aldehyde phosphate dehydrogenase
(GAPDH) was a 1-kb fragment complementary to the human GAPDH-coding
region with strong cross-reactivity against rat GAPDH (Clontech, Palo
Alto, CA). Approximately 100 ng of each probe were labeled with 50
µCi of [-32P]-dCTP (New England Biotech,
Piscataway, NJ). Northern blot analysis was performed as previously
described.23
All experiments were performed in duplicate.
Cellular Proliferation: BrdU Incorporation and Nonradioactive Proliferation Assay
To assess the effects of AdCAFGF-TR infection on cellular proliferation, NRCs and PFDCs were infected with AdCALacZ (100 pfu/cell), AdCAFGF-TR (50 pfu and 100 pfu/cell), or vehicle. On the following day, 20,000 infected (vehicle, 100 pfu/cell AdCALacZ, 50 and 100 pfu/cell AdCAFGF-TR) NRCs were grown in 16 wells of a 24-well tissue-culture plate into which glass coverslips had been placed. After 24 hours, serum-free media was added to each well. On the following day, 10 ng/ml of rhFGF-2 was added to the media of all wells. All cells were labeled with BrdU (10 µmol/L final concentration) for 1 hour before fixation with 10% formalin. BrdU-positive cells were identified using a monoclonal BrdU antibody (Amersham Pharmacia Biotech, Uppsala, Sweden) and a fluorescein-conjugated anti-mouse immunoglobulin (Jackson Immunoresearch Laboratories, West Grove, Penn) followed by incubation with Hoechst stain (1 µg/ml in deionized water) (Sigma, St. Louis, MO).24,25
Proliferation as assessed by BrdU incorporation was corroborated by performing a nonradioactive proliferation assay. Proliferation of infected cells was assessed using the WST-1 cell proliferation reagent assay (Boehringer Mannheim, Indianapolis, IN) after 1, 3, and 5 days in culture. All experiments were performed in triplicate for each time point.
Similar proliferation analysis was performed on cells infected with AdCAsFGF-2, vehicle, and AdCALacZ. In these studies, vehicle and AdCALacZ cells were not treated with rhFGF-2. All experiments were performed on similarly treated PFDCs.
Statistical Analysis
Statistical analyses of BrdU incorporation and the nonradioactive
proliferation assay were performed using two-way ANOVA with
*P 
0.05 considered significant. Post hoc tests were
performed using the Tukey-Kramer multiple comparison test. Statistical
analysis of Northern blot and Western blot analyses were not performed
because these studies represent semiquantitative measures. All Northern
and Western blot analyses were performed in duplicate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To assess the efficacy of the adenoviral constructs to alter FGF
biological activity in infected cells, osteoblast-enriched
cultures (NRCs) were infected with AdCAFGF-TR or AdCAsFGF-2.
High-level, dose-dependent, transgene expression was detected by
Northern and Western blot analysis (Figure 2A)
. Furthermore, transgene expression
resulted in a biologically active product resulting in abrogation of
ERK-1 and -2 phosphorylation in AdCAFGF-TR-infected NRCs stimulated
with rhFGF-2 (Figure 2B)
. As expected, NRCs infected with vehicle and
AdCALacZ demonstrated ERK-1 and -2 phosphorylation in response to
rhFGF-2 stimulation.26
Similarly, Western blot analysis of
FGF-2 demonstrated that AdCAsFGF-2-infected cells express greater
amounts of FGF-2 than control or AdCALacZ-infected cells resulting in
increased FGF biological activity as assessed by ERK-1 and -2
phosphorylation (Figure 2, C and D)
. Similar results were obtained on
identically treated cells derived from PF-associated dural tissues
(PFDCs) (data not shown).
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To assess sutural development in response to alterations in FGF
biological activity we chose first to use an in vitro organ
culture system in which mouse cranial sutures, together with the
underlying dura mater, are placed in serum-free culture conditions
(Figure 3A)
. We chose to analyze cranial
suture fusion in vitro as this system enables isolation of
the dura mater-cranial suture complex from humoral or mechanical forces
that may contribute to sutural fusion/patency. To examine the effects
of abrogating FGF biological activity, the dural tissues of mouse
calvarial explants inclusive of the PF and SAG sutures were infected
with vehicle, AdCALacZ, or AdCAFGF-TR, and cultured for 30 days as
previously described.7,8
X-gal staining of
AdCALacZ-infected suture complexes demonstrated significant transgene
expression within cells localized to the underlying dura mater of the
suture at the end of the culture period (Figure 3, B and C)
. In
addition, examination of PF sutures under x25 magnification
demonstrated complete fusion of all vehicle or AdCALacZ-infected PF
sutures (n = 10 in each group) after the 30-day
culture period (Figure 3D)
. In contrast, all PF sutures infected with
the AdCAFGF-TR (n = 10) were widely patent at
the end of the culture period (Figure 3E)
. Histological examination
corroborated these findings (Figure 3, F and G)
.
Abrogation of FGF Biological Activity in Utero Prevents Programmed PF Suture Fusion
Based on our in vitro organ culture findings, we
evaluated the fate of PF suture complexes treated with the
truncated FGF-R1 adenovirus in utero. Rat embryos (E18)
in which the PF-associated dural tissues were infected with
AdCAFGF-TR demonstrated no gross phenotypic abnormalities. Similar
to our in vitro findings, transgene expression as assessed
by X-gal or immunohistochemical staining of sutural complexes harvested
on postnatal day 30 demonstrated high levels of transgene expression in
the dura mater underlying the PF suture (Figure 4
; A, B, D, G, and H). In addition, as
expected, all PF sutures infected with vehicle or AdCALacZ demonstrated
complete sutural obliteration on postnatal day 30 (Figure 4, B and C)
.
In contrast, all PF sutures infected with AdCAFGF-TR were widely patent
(Figure 4, E and F)
. Thus, targeted in utero delivery of the
truncated FGF-R1 virus prevented programmed postnatal PF sutural
fusion.
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To evaluate the effects of increased FGF biological activity on
normally patent cranial sutures, the left COR suture complexes of
embryonic rats (E18) were infected with control (vehicle or LacZ) or
the FGF-2 overexpression adenovirus. This experiment was designed to
mimic the presumed effects of FGF-R mutations in syndromal
craniosynostoses (ie, increased FGF biological activity). X-gal
staining of AdCALacZ-infected COR sutures localized high-level
expression of the LacZ transgene along the dural aspect of the left COR
(Figure 5B
; and Figure 6, A and B
). In addition, all COR sutures
infected with vehicle or AdCALacZ demonstrated complete histological
patency (Figure 6, A–C)
. In contrast, all COR sutures infected with
AdCAsFGF-2 demonstrated marked thickening of both the parietal and
frontal bones bordering the COR suture with fusion of the suture
(Figure 6, D–F)
. In addition, animals with fused left COR sutures
demonstrated the characteristic craniofacial morphology associated with
unilateral coronal synostosis (ie, frontal plagiocephaly; Figure 5
).
Compensatory or continued growth perpendicular to the patent right COR
suture secondary to left COR suture fusion resulted in a left-sided
deviation of the normally linear plane made by the PF and SAG sutures
(Figure 5, C and D)
.
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To investigate possible cellular mechanisms underlying our
in vitro and in vivo cranial suture findings,
effects of cellular infection with AdCAFGF-TR and AdCAsFGF-2 on
TGF-{beta}1 gene expression were explored. Similar to FGF-2, TGF-{beta}s have
been implicated in the regulation of sutural fusion.12,27
Infected cells were stimulated with rhFGF-2 (10 ng/ml) for 6 hours and
TGF-{beta}1 mRNA expression was assessed. Both vehicle and
AdCALacZ-infected cells demonstrated increased TGF-{beta}1 mRNA expression
in response to rhFGF-2 stimulation. In contrast, NRCs infected with
AdCAFGF-TR (50 and 100 pfu/cell) failed to increase expression of
TGF-{beta}1 mRNA in response to rhFGF-2 (untreated and {beta}-gal-infected
NRCs demonstrated a greater than fourfold increase in TGF-{beta}1 mRNA
expression; Figure 7A
).
|
). These data demonstrate that
changes in FGF biological activity modulate TGF-{beta}1 expression and may
contribute to the regulation of sutural fate. Northern blot analysis
performed on similarly treated PFDCs demonstrated identical changes in
TGF-{beta}1 gene expression (data not shown). Alterations of FGF Biological Activity Result in Changes in Cellular Expression of Collagen I by Cranial Suture-Derived Dural Cell Cultures
Opperman and colleagues28
have demonstrated increased
collagen synthesis in fusing sutures as compared to nonfusing cranial
sutures. In addition, FGF-2 has been shown to inhibit collagen type I
mRNA synthesis in fibroblasts and osteoblasts and may thus represent a
potential mechanism by which alterations in FGF biological activity may
mediate sutural fate in our system.29
Interestingly,
abrogation or stimulation of FGF biological activity in NRCs failed to
significantly alter the expression of collagen I mRNA (data not shown).
In contrast, rhFGF-2 stimulation of PF suture-derived dural cell
cultures was associated with an increase in collagen type I mRNA
expression, whereas abrogation of FGF biological activity with the
truncated FGF-R1 virus attenuated this response (2.3-fold increase in
collagen I mRNA expression in untreated, FGF-stimulated PFDCs; Figure 7B
). Thus, in our system, dural cell populations respond differently to
FGF stimulation than osteoblast-enriched cultures to FGF stimulation.
Alterations of FGF Biological Activity Affect Osteoblast and Dural Cell Proliferation
FGF-2 is a potent mitogen for many cell types including
osteoblasts.30
To assess the ability of
AdCAFGF-TR-infected cells to resist the mitogenic effects of rhFGF-2
stimulation, cellular proliferation of infected cells was assessed
using BrdU incorporation and a nonradioactive proliferation assay.
Results of cellular BrdU incorporation are depicted in Figure 7C
. NRCs
infected with AdCAFGF-TR (14 ± 7% BrdU-positive cells/HPF; 100
pfu/cell) demonstrated significantly less BrdU incorporation than both
NRCs infected with AdCALacZ (36.2 ± 4.2%) and vehicle (33.6
± 6.7%). A nonradioactive proliferation assay demonstrated similar
results with AdCAFGF-TR-infected cells proliferating significantly
more slowly in response to rhFGF-2 compared to vehicle and
AdCALacZ-infected cells (Figure 7D)
. In contrast, cells infected with
AdCAsFGF-2 proliferated significantly more rapidly than vehicle or
AdCALacZ-infected cells (Figure 7, F and G)
. Similar results were
obtained on identically treated PFDCs (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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There are four known FGF-Rs and mutations in three of them (FGF-R1, FGF-R2, and FGF-R3) have been identified in patients with syndromic craniosynostosis. The gain-of-function associated with the mutated FGF-Rs has been attributed to ligand-independent dimerization/activation, and more recently to increased affinity of the mutated FGF-Rs for ligand, specifically FGF-2.2-6 The mechanism for ligand-independent FGF-R dimerization and subsequent activation has been attributed to disruption of intramolecular disulfide bonds in the third immunoglobulin loop of the FGF-R as a result of a point mutation and loss of an IgIII-associated cysteine residue.2-5 Similarly, Robertson and colleagues36 demonstrated that FGF-R mutations not involving cysteine substitutions may disrupt intramolecular disulfide bonds by altering the conformation of the IgIII domain. Recently, in vitro analysis of the most common Apert mutations (FGF-R2 mutations; Ser252Trp and Pro253Arg) demonstrated increased affinity for FGF-2 ligand as compared to wild-type FGF-R2.6
Our findings demonstrate that AdCAFGF-TR infection of the dural tissues underlying the PF cranial suture inhibited physiological cranial suture fusion, whereas AdCAsFGF-2 infection of the dural tissues underlying the COR suture resulted in fusion of this normally patent suture. In addition, we show that alterations in FGF biological activity is associated with significant changes in cellular proliferation and TGF-{beta}1 expression by NRCs and PFDCs. Moreover, stimulation of PFDCs with rhFGF-2 results in significant up-regulation of collagen I mRNA. Thus, sutural patency or fusion secondary to alterations in FGF biological activity is likely because of changes in cellular proliferation, TGF-{beta}1 expression, and collagen I expression by the underlying dura mater, and sutural mesenchyme. This conclusion is supported by the fact that the osteogenic actions of TGF-{beta}1 and FGF-2 are closely related because: 1) each cytokine directly enhances the expression of the other; 2) TGF-{beta}1 potentiates the mitogenic effects of FGF-2 on osteoblast-like cells; and 3) TGF-{beta}1 potentiates the expression of type I collagen, the major component of the bone extracellular matrix.30,37-40 Moreover, we have shown that increased expression of TGF-{beta}1, similar to FGF-2, is temporally and spatially related to programmed sutural fusion and patency.12,27
It is interesting to note that Mehrara and colleagues18 demonstrated increased type I collagen production by cells derived from PF-associated dural tissues as compared to cells derived from the dural tissues underlying a patent cranial suture. Additionally, Opperman et al28 demonstrated increased collagen synthesis in fusing cranial sutures as compared to patent sutures in vitro. Finally, osteoblasts derived from human synostotic sutures (ie, isolated and syndromic sutures) demonstrate greater basal levels of type I collagen and noncollagenous matrix molecules than control osteoblasts.41-43 Taken together, these studies suggest that alterations in the expression of osteogenic cytokines (ie, FGF-2 and TGF-{beta}1) and extracellular matrix molecules likely determine the fate of the overlying cranial sutures. These conclusions are supported by our current study.
Manipulations of FGF-dependent signaling in the cranial sutures have been previously described. After placement of FGF-4-soaked beads along the osteogenic fronts of embryonic day 15.5 mouse SAG suture explants, Kim and colleagues44 demonstrated close apposition of the parietal bones with evidence of SAG sutural fusion. These same authors demonstrated increased proliferation in the cells of the osteogenic front in the region adjacent to the FGF-4 beads, suggesting that increased proliferation of osteoblasts lining the osteogenic front contributes to osseous fusion of the SAG suture. Placement of FGF-2-soaked beads on E15 mouse COR sutures induced osteopontin expression in the sutural mesenchyme (ie, normally not mineralizing) in the area of the bead.45 These studies suggest that increased availability of FGF ligand leads to alterations in mineralization and proliferation, and ultimately may alter the histological fate (ie, fusion or patency) of the cranial sutures. It is likely that similar alterations in dural cell and osteoblast proliferation and subsequent changes in gene expression secondary to modulation of FGF biological activity contributed to the alterations of cranial suture fate observed in our study.
Taken together with the identification of gain-of-function FGF-R mutations in syndromic craniosynostosis, these studies suggest that FGF biological activity critically regulates suture fusion and patency and that this regulation occurs, at least in part, via alterations in cellular proliferation, TGF-{beta}1 expression, and collagen expression. We are currently investigating alterations in the temporal and spatial expression of numerous osteoinductive and osteogenic cytokines as well as their inhibitors in adenovirally treated PF and COR sutures to further elucidate the biomolecular mechanisms that resulted in the phenotypes reported in this study. Using targeted, in utero delivery of transgenes, we successfully altered postnatal cranial suture fate. As prenatal diagnosis and fetal manipulation become a reality, our data may facilitate the development of less invasive treatment options for craniosynostosis.
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Footnotes |
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Supported by National Institute of Dental and Craniofacial Research grant RO1-DE13194.
Accepted for publication October 13, 2000.
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