Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea, Seoul, Korea
* Author for correspondence (e-mail: ckjoo{at}catholic.ac.kr)
Accepted 31 December 2003
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SUMMARY |
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Key words: Lens, Wnt signaling, Fibroblast growth factors, Lens fiber cell differentiation, Rat, ß-crystallin, ß-catenin
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Introduction |
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Wnt proteins are known to control morphogenetic events during embryonic and
postembryonic development (Moon et al.,
1997). Wnts have been divided into functional classes, namely
transforming and non-transforming Wnts, based on assays performed in mammalian
cell lines (Wong et al.,
1994
). Ectopic expression of Wnt1, Wnt3a, Wnt7a and Wnt8 induces
morphological transformation, whereas Wnt4 and Wnt5a lack transforming
activity. Based on studies in various developmental systems, a mechanistic
model of Wnt action has been proposed. Association of Wnt and its seven pass
transmembrane receptor protein, Frizzled, leads to activation of Disheveled,
resulting in the inhibition of GSK-3ß, and in the subsequent accumulation
and nuclear translocation of ß-catenin. In the nucleus, ß-catenin
activates transcription factors of the T-cell factor (Tcf)/lymphoid enhancer
factor (LEF) family, thereby affecting cell fate
(Nusse and Varmus, 1992
;
Cadigan and Nusse, 1997
). Wnts
can also have a direct effect on the shape and fate of the cell through
cytoskeletal reorganization (Shibamoto et
al., 1998
). For example, in vitro and in vivo studies show that
the small GTPase, RhoA and Rac are directly activated by Frizzled-Disheveled
signaling (Habas et al.,
2003
).
Wnts and proteins in the Wnt signaling pathway are expressed in the lens
throughout its development. The Wnt receptors Frizzled 1, Frizzled 2 and
Frizzled 7 were detected in the lens placode during lens induction
(Stark et al., 2000).
Transcripts encoding Wnt3, Wnt5a, Wnt5b, Wnt7a, Wnt7b, Wnt8a, Wnt8b and Wnt13,
and Frizzled receptors were identified in the postnatal lens epithelium and
transitional zone at the lens equator
(Jasoni et al., 1999
;
Stump et al., 2003
;
Liu et al., 2003
). In
addition, expression of Dishevelleds and Dickkopfs, which are involved in Wnt
signaling, were detected in the lens
(Stump et al., 2003
). Although
several Wnts and Frizzleds are expressed in lens cells and deletion of the Wnt
co-receptor LRP5 leads to the death of central lens epithelial cells
(Stump et al., 2003
), Wnts
have not been shown to direct lens formation or the differentiation of lens
fiber cells.
In this study, we used three independent approaches, to show that Wnt signaling is involved in lens fiber cell differentiation: First, GSK-3ß activity was decreased and nuclear ß-catenin increased in the elongating fiber cells at the equatorial zone of the lens. Second, after FGF priming, Wnt induced cell elongation and the accumulation of ß-crystallin. Finally, ß-catenin activated the transcription of ßB2-crystallin. These results suggest that Wnt acts as a regulatory factor of lens differentiation by regulating morphological change and the accumulation of lens proteins.
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Materials and methods |
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To activate the Wnt signal pathway, Wnt 3a-conditioned medium was prepared
as described previously (Lyu et al.,
2003), and the conditioned medium was diluted with normal culture
medium (final concentration from 5x to 10x). Control treatment
corresponds to the use of conditioned medium from the lacZ expressing
L cells.
Bovine aqueous and vitreous humor were prepared from the eyes of freshly
slaughtered animals as reported previously
(Schulz et al., 1993), and
then used to treat
TN4, B3 or HEK 293 cells at a final concentration of
20% aqueous- or vitreous humor.
For blocking experiments with sFRP-1, sFRP-1 CM was prepared from Myc-tagged sFRP1-expressing HEK 293 cells. Mouse vitreous humor was incubated with control medium and sFRP-1 CM for 30 minutes and then added to cells.
Immunochemical staining
For immunofluorescent labeling, explants were fixed with 4%
paraformaldehyde and washed in phosphate-buffered saline (PBS), then
permeabilized with 0.05% Triton X-100 in PBS. Fixed explants were incubated
with blocking solution containing 1% normal goat serum (Jackson ImmunoResearch
Laboratories, West Grove, PA, USA) and 2% BSA (Sigma) for 1 hour and incubated
overnight at 4°C with rabbit polyclonal anti-Ser9 phospho-GSK-3 ß
(Cell Signaling Technology, Beverly, MA, USA), mouse monoclonal
anti-GSK-3ß (Transduction Laboratories, Lexington, KY, USA), rabbit
polyclonal anti-Aquaporin-0 (Calbiochem, La Jolla, CA, USA), mouse monoclonal
anti-ß-catenin (Transduction Laboratories), or rabbit polyclonal
anti-ß-crystallin (kindly provided by John W. McAvoy, Sydney University).
After a rinse with PBS, the explants were incubated with rhodamine, or
FITC-conjugated (fluoresceinisothiocyanate; Jackson ImmunoResearch
Laboratories) secondary antibodies at room temperature for 1 hour, and
counterstained with Hoechst dye (Molecular Probes, Eugene, OR, USA). Labeled
explants were rinsed with PBS, mounted and examined using a fluorescence
microscope (Zeiss).
For immunofluorescent labeling of eye sections, dissected eyes from a neonatal (P4-P5) C57 mouse were immersed in Tissue Tek OCT compound and frozen in liquid nitrogen. Cryosections of eyes were fixed in 4% paraformaldehyde in PBS, and then incubated for 30 minutes in blocking solution. Antibodies to Ser9 phospho-GSK-3ß and ß-catenin were used on frozen sections to detect expression in the lens.
RNA isolation and RT-PCR analysis
Total RNA was isolated from the central epithelium (containing
undifferentiated cells) and equatorial epithelium (containing both the
proliferating epithelial cells and the elongating epithelial cells) of
neonatal mouse lens, whole lenses, or explants using TRIzol Reagent (Life
Technologies), according to the manufacturer's recommendations. Total RNA (2
µg) was reverse transcribed using the Superscript II kit (Invitrogen) and
random hexamers. PCR amplification was performed using the following
individual primer sets: for mWnt1, 5'-CAGTAGTGGCCGATGGTG-3' and
5'-ATCGATGTTGTCACTGC-3'; for mWnt2b,
5'-GCCAAAGAGAAGAGGCTTAA-3' and
5'-TCAGTCCGGGTGGCGTGGCG-3'; for mWnt3,
5'-GCCGACTTCGGGGTGCTGGT-3' and
5'-CTTGAAGAGCGCGTACTTAG-3'; for mWnt3a,
5'-TAGTGCTCTGCAGCCTGAA-3' and
5'-CCACAGATAGCAGCTGAT-3'; for mWnt4,
5'-ACAGTCCTTTGTGGACGT-3' and 5'-CGTCAATGGCTTTAGATG-3';
for mWnt5a, 5'-ATTGGAATATTAAGCCCG-3' and
5'-GTGACCATAGTCGATGTT-3'; for mWnt5b,
5'-AGCTCTCATGAACCTACA-3' and 5'-TGCAGTGGCAGCGTTCCA-3';
for mWnt7a, 5'-AACATGAAGCTGGAGTGT-3' and
5'-TTACACTTGACGTAGCAGCA-3'; for mWnt7b, TCTGAGCAATTGTGGCTG; for
rßB2-crystallin, 5'-CAGGCTCCGTCCTGGTGCAG-3' and
5'-CGACGCACAGATTGCACCTG-3'; for ß-actin,
5'-AGGCCAACCGCGAAGATGACC-3' and
5'-GAAGTCCAGGGCGACGTAGCAC-3'; for rGAPDH,
5'-CCATGGAGAAGGCTGGGG-3' and
5'-CAAAGTTGTCATGGATGACC-3'. PCR was performed at 94°C for 30
seconds, 55°C (for mWnt1, mWnt4, mWnt5a, mWnt5b, mWnt7a, mWnt7b,
rßB2-crystallin) or 58°C (for mWnt2b, mWnt3, mWnt3a, rGAPDH) or
60°C (for beta-actin) for 40 seconds, and 72°C for 1 minute for 30
cycles. The sequences of all PCR products were confirmed by automated
sequencing.
Western blot analysis
Lenses of neonatal Sprague Dawley rats were dissected and the lens
epithelium and the differentiating fiber cells of the zone of early fiber
elongation at the equator were isolated under a dissecting microscope. Each
tissue sample and explants was homogenized in lysis buffer containing 20 mM
Tris-Cl (pH 7.5), 1% Triton X-100, 0.1% SDS, 0.5% deoxycholic acid, 10%
glycerol, 100 mM sodium orthovanadate and protease inhibitor cocktail (1 mM
EDTA, 1 mM PMSF, 5 µg/ml Aprotinin, 1 µg/ml leupeptin, 1 µg/ml
pepstatin). Homogenates were clarified by centrifugation at 14,000
g for 15 minutes at 4°C and supernatant was collected as
the total extract. Protein concentration was measured using Micro BCA Reagent
kit (Pierce, IL, USA). Lysate was separated by SDS-PAGE (8-10%) and
transferred onto nitrocellulose membranes. Blots were blocked with buffer
containing 5% non-fat dry milk (Santa Cruz) and then reacted sequentially with
the following antibodies: Mouse monoclonal antibodies for E-cadherin
(Transduction Laboratories), N-cadherin (Transduction Laboratories), p27Kip1,
-SMA (Sigma), Actin (Sigma),
-tubulin (Sigma), and a rabbit
polyclonal antibody for p57Kip2 (Santa Cruz Biotechnology) were used to detect
the corresponding proteins. Peroxidase-conjugated goat anti-mouse and
anti-rabbit secondary antibodies (Sigma) were then applied, and the proteins
were detected by using an enhanced chemiluminescence (ECL) reagent (Santa Cruz
Biotechnology).
For western blotting assays of Ser9 phospho-GSK-3ß, tissue samples were homogenized in lysis buffer containing 20 mM Tris-HCl, pH 7.5, 270 mM sucrose, 1 mM EDTA, 1 mM EGTA, 0.5% Triton X-100, 100 nM NaF, 1 mM sodium orthovanadate, 100 nM okadaic acid with protease inhibitors. Extracts were cleared by centrifugation at 13,000 g for 15 minutes at 4°C, and then the protein concentration was determined. Lysates were subjected to western blotting analysis with anti-Ser9 phospho-GSK-3ß or a mouse monoclonal antibody specific to GSK-3ß for normalization of GSK-3ß. Band intensities were quantified using ImageMaster VDS (Pharmacia).
Nuclear extracts were prepared from lens epithelial cells and
differentiating fiber cells in the zone of early fiber elongation at the
equator, or from explants by the method described previously
(Dignam et al., 1983). Protein
concentration was determined using Bradford reagent (Bio-Rad). Western
blotting was performed with anti-ß-catenin antibody or a monoclonal
anti-lamin A/C (Cell Signaling Technologies), for normalizing of nuclear
protein loading.
-tubulin was not detected when blots were probed with
anti-
-tubulin, suggesting that is was not due a contamination of
cytoplasmic proteins.
In vitro GSK-3ß kinase assay
Protein lysates were prepared from lens tissues or explants using lysis
buffer. Total lysates were immunoprecipitated with antibody to GSK-3ß.
The immune complexes were washed three times in lysis buffer and three times
in kinase assay buffer, and then were incubated for 20 minutes at 30°C
with 62.5 µM glycogen synthase peptide-2 (Upstate Biotechnologies, Waltham,
MA, USA), 100 µM [32P]ATP (0.25 µCi/ml), 10 mM
MgCl2. 32P-labeled reactions were spotted onto P81 phosphocellulose
papers, and then were washed and then subjected to liquid scintillation
counting. Kinase activity was normalized to background levels when 40 mM LiCl
was included in the reaction.
Bromodeoxyuridine proliferation assay
Bromodeoxyuridine (BrdU) incorporation was determined by a BrdU labeling
kit, according to the manufacturer's recommendations (Roche, Indianapolis, IN,
USA). Nuclei were counterstained with Hoechst 33258. BrdU-reactive cells were
captured by using an Axio camera and AxioVision 2.05 image analysis software
(Zeiss) under a fluorescence microscope, and the number of BrdU-positive cells
was determined.
Transient transfections and reporter gene assay
HEK 293 cells, B3 or TN4 were plated into 5x105 per
35 mm tissue culture dish and transfected by Lipofectamine (Life Technologies)
with reporter plasmid (TOPFLASH, FOPFLASH, pßB2-crystallin luciferase)
and the internal control pRL-TK. Luciferase assays were performed 24 hours
after transfection using the dual luciferase assay system (Promega).
Construction of plasmids
To construct reporter plasmids, a 624 bp ßB2-crystallin promoter
(614/+10) was amplified from mouse genomic DNA using primers
(Chen et al., 2002); for
ßB2-crystallin promoter, 5'-ATAGAACCCAGGACCACCAG-3' and
5'-GAGTGCCGTGAAGCCAGGCT-3'. The PCR products were inserted in pGL2
basic-vector (Promega). For the pcDNA3-Myc/sFRP-1 and pcDNA3-Myc/ICAT
constructs, a cDNA fragment for mouse sFRP-1 and ICAT was amplified by RT-PCR
from mouse mRNA, as a template. Cloned cDNA was inserted into the pCS2-MT
vector for Myc tagging of N-terminal, and subcloned into pcDNA3 vector. All
constructs were confirmed by sequencing.
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Results |
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It is generally considered that canonical Wnt signaling has mitogenic activity. To determine the effects of Wnt on cellular proliferation in lens epithelial cells, we performed BrdU-incorporation as a marker of DNA synthesis. Medium harvested from confluent cultures of L cells expressing lacZ (control medium) or Wnt3a was applied to rat lens epithelial explants for 2 days. BrdU was administered 6 hours before collection of the explants. Significantly more BrdU-labeled cells were present in explants cultured in Wnt CM than in those cultured in control medium (Fig. 2).
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Induction of ß-crystallin expression by the Wnt/ß-catenin-dependent pathway
Lithium inhibits GSK-ß and mimics Wnt signaling through ß-catenin
(Stambolic et al., 1996). We
therefore determined whether lithium could induce fiber differentiation in
explants. Cell elongation was not observed when epithelial explants were
treated with FGF for 1 hour, followed by continuous exposure to 10 mM LiCl
(Fig. 10A). To test whether
GSK-3ß responds to lithium in lens epithelial explants, extracts were
analyzed by Western blot assay using antibodies against Ser9
phospho-GSK-3ß. Treatment with FGF2/LiCl resulted in an increase in Ser9
phospho-GSK-3ß, suggesting that GSK-3ß was inactivated
(Fig. 10B). We next examined
the accumulation of ß-crystallin in explants. Explants cultured with
FGF2/LiCl or LiCl accumulated ß-crystallin without cell elongation,
whereas explants cultured with FGF/NaCl or NaCl did not accumulate
ß-crystallin (Fig. 10C).
As shown earlier, explants cultured with FGF2/Wnt CM accumulated
ß-crystallin and elongated. We noted that cell elongation was inhibited
by U0126, and explants cultured with Wnt CM accumulated ß-crystallin
without cell elongation (Fig.
10C). These results confirm that ß-crystallin expression and
cell elongation can be uncoupled, as shown previously
(Lovicu and McAvoy, 2001
).
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Discussion |
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In lens development, diffusible factors in the ocular environment are an
important part of cellular development. The aqueous humor from the anterior
chamber (adjacent to the lens epithelium) favors proliferation, and the
vitreous body (adjacent to the fiber cells) promotes epithelial-to-fiber
differentiation (Schulz et al.,
1993). For example, the BMPs FGFs that are required for lens fiber
cell differentiation are produced by the retina during development and are
secreted into the vitreous humor
(Belecky-Adams et al., 2002
;
Nakashima et al., 1999
). We
showed that vitreous humor induced the activation of the TOP promoter and that
the activity in vitreous humor was inhibited by the Wnt antagonist sFRP-1
(Fig. 1). In other experiments,
CyclinD1 and fla-1 levels were significantly increased by treatment of HEK293
cells with vitreous humor (data not shown), consistent with induction by
ß-catenin (Shtutman et al.,
1999
; Mann et al.,
1999
). These results suggest that vitreous humor, the fluid that
bathes differentiating fiber cells, contains Wnt proteins or factors that can
activate the Wnt/ß-catenin pathway.
Recent studies reported that several different Wnt genes are expressed in
lens cells. Wnt5a, 5b, 7a, 7b, 8a and 8b are expressed in the lens epithelium
of postnatal mice, including the transitional zone, and they are reduced
outside the zone of early fiber elongation at the equator
(Stump et al., 2003). In the
embryonic mouse lens, Wnt3 is expressed in the lens epithelium, Wnt5b is
expressed at the lens equator, and Wnt7a is expressed in the lens fibers
(Liu et al., 2003
). Frizzled
receptors, which have at least 10 orthologs in mammals, were detected in the
lens. Frizzled 1, 2 and 7 are expressed in the chick lens placode
(Stark et al., 2000
). Also,
Frizzled 1, 2, 3, 4, 6 and 7 are expressed in the embryonic and postnatal lens
(Stump et al., 2003
;
Liu et al., 2003
). Consistent
with these studies, we also showed the expression of Wnt2b, Wnt3, Wnt3a,
Wnt5a, Wnt5b, Wnt7a and Wnt7b in lens. Levels of Wnt3a and Wnt7a at the
equator of the neonatal rat lens, where the epithelial cells undergo early
fiber differentiation. Previous studies have shown that Wnt3a and 7a lead to
ß-catenin nuclear translocation
(Shimizu et al., 1997
).
Consistent with this, we found that ß-catenin accumulated in the nuclei
of differentiating cells at the lens equator. These findings, together with
the pattern of Wnt expression, indicate that Wnt signaling in the transitional
zone at the lens equator may have a role in promoting aspects of fiber
differentiation, including changes in cell shape and the expression of genes
that are involved in fiber cell differentiation.
In the rat lens epithelial explants system that we used, treatment with Wnt
alone lead to the accumulation of ß-crystallin without cell elongation.
Although lens fiber differentiation usually involves cell elongation and
crystallin expression, these events are not necessarily linked
(Lovicu and McAvoy, 2001). We
also showed nuclear translocation of ß-catenin, along with decreased
GSK-3ß kinase activity, in Wnt-treated explants. ß-catenin has been
implicated as a transcriptional component in Wnt signal transduction. We
therefore examined the ability of ß-catenin to activate the
ß-crystallin promoter; as expected, ß-catenin increased the activity
of the ß-crystallin promoter. This effect of ß-catenin is further
supported by the demonstration that overexpression of ICAT and GSK-3ß,
negative regulators of Wnt/ß-catenin signaling, inhibited the increase in
activity induced by ß-catenin. In the canonical mechanism of Wnt
signaling, ß-catenin activates transcription via its interactions with
the Tcf/LEF family of transcription factors, which bind to the sequence
5'-A/T A/T CAAAG-3' (van de et
al., 1997
). However, the promoter of ß-crystallin does not
contain the core Tcf/LEF binding site. It is possible that ß-catenin
activates ß-crystallin expression indirectly, through the activation of
another factor that binds to this promoter.
Cell elongation is a well-recognized component of lens fiber
differentiation (Fromm and Overbeek,
1996). FGF induces cell elongation in rat lens explants. This
event requires a continuous signal from FGF
(Lovicu and McAvoy, 2001
). We
showed that cell elongation is induced by a longer exposure to FGF alone, but
not by exposure to Wnt alone. Interestingly, after priming by FGF, Wnt induced
cell elongation accompanied with ß-crystallin expression. Previous
studies have shown that although a brief FGF signal is not sufficient to
induce cell elongation, the process of lens fiber differentiation can be
initiated by a short pulse of high-dose FGF if that pulse is followed by the
longer-term application of a second factor, such as insulin or IGF-1
(Leenders et al., 1997
;
Klok et al., 1998
). Thus,
these findings indicate the necessity of the initial signal of FGF for
morphological differentiation. What mechanism might account for the
Wnt-induced cell elongation after FGF priming? FGF is essential to lens
differentiation but the molecular mechanism is not clear
(Schulz et al., 1993
). FGF2
induces Lef/Tcf-dependent transcription of cyclin D1, a known target gene of
the ß-catenin/Lef pathway, and this growth factor enhances the nuclear
translocation of ß-catenin and reduces GSK-3 activity in human umbilical
vein endothelial cells (Holnthoner et al.,
2002
). In primary rat neuronal cells, FGF1 promotes GSK-3ß
inactivation through ERK-independent Akt phosphorylation and stimulates the
translocation of ß-catenin into the nucleus
(Hashimoto et al., 2002
). This
suggests the possibility that an FGF/GSK-3ß-mediated increase of nuclear
levels of ß-catenin is enhanced further by Wnt, thereby promoting the
expression of genes required for morphological differentiation, such as those
involved in cell adhesion and exit from the cell cycle. However, we did not
observe any inactivation of GSK-3ß by FGF2 in lens explants (data not
shown), and showed that cell elongation is dependent on ERK activity
(Fig. 5H). In addition, TOP
promoter activity was not increased significantly by FGF2 in a lens epithelial
cell line (Fig. 1C). Therefore,
FGF signaling is unlikely to cause the inhibition of GSK-3ß kinase
activity. Another possibility is that ß-catenin-independent Wnt pathways
cooperate with an initial FGF signal to induce cytoskeletal reorganization, an
event shown previously to be critical for the requisite change in cell shape
that occurs during morphogenetic differentiation
(Ferreira-Cornwell et al.,
2000
). Wnt3a is able to induce cytoskeletal reorganization
(Shibamoto et al., 1998
).
Recent evidence has shown that Wnt signaling can activate both the
ß-catenin pathway and the small GTPases Rho and Rac separately or
together (Pandur and Kuhl,
2000
; Ziemer et al.,
2001
; Habas et al.,
2003
). Interestingly, recent studies in lens cells showed that Rho
and Rac GTPase are activated by FGF signaling within 1 hour and that
cytoskeletal reorganization in lens cells may require the function of Rho and
Rac GTPase (Maddala et al.,
2003
). Thus, FGF-induced Rho or Rac activation may be enhanced by
a ß-catenin-independent Wnt pathway to induce cell elongation. In this
context, further study will be needed to analyze direct cross-talk between the
initial FGF signal and the ß-catenin-independent Wnt pathway.
It is generally considered that Wnt/ß-catenin signaling promotes cell
proliferation, rather than triggering differentiation. In our studies, Wnt
induced mitogenic activity and fiber cell differentiation in lens epithelial
cells. These results raise the question of how the same Wnt protein directs
two opposite functions, namely mitogenic activity and fiber cell
differentiation. Previous studies have shown that lower doses of FGFs
stimulate the proliferation of lens epithelial cells, whereas higher doses
promote fiber differentiation (Chamberlain
and McAvoy, 1987). It is possible that the effects of Wnt
signaling are also dependent on dose or that different Wnt proteins
preferentially mediate different responses in lens cells. These issues will
have to be addressed by studies that define the function of the Wnt signaling
pathway in the lens and the roles of each of the different Wnts in lens cell
proliferation and differentiation.
In conclusion, our results strongly suggest that Wnt signaling has the capacity to induce fiber differentiation in cultured rat lens epithelial explants via the regulation of ß-crystallin expression and cell elongation. Our findings also imply that at least two different Wnt pathways are involved in lens fiber differentiation: one is a ß-catenin-dependent pathway that promotes ß-crystallin expression, and the other is a ß-catenin-independent pathway that promotes morphological differentiation. Furthermore, given the fact that lithium mimics some but not all of the activities of Wnt in explants pre-stimulated by FGF, Wnt signaling will probably function in a second yet-to-be-defined pathway, cooperating with the initial signal of FGF to induce cell elongation, but not involving GSK-3ß/ß-catenin. Our continuing studies will focus on the direct contribution of the Wnt/Frizzled pathway to FGF-triggered fiber differentiation and the molecules involved in the convergence of the FGF and Wnt pathway that mediate the morphological aspects of fiber cell formation.
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ACKNOWLEDGMENTS |
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