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First published online 15 November 2006
doi: 10.1242/dev.02679
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1 Department of Medical and Molecular Genetics, Indiana University of Medicine,
Indianapolis, IN 46202, USA.
2 Department of Cellular and Molecular Medicine, Glycobiology Research and
Training Center, University of California, San Diego, 9500 Gilman Drive, La
Jolla, CA 92093, USA.
3 Department of General Zoology and Genetics, Westfälische
Wilhelms-Universität Münster, Schlossplatz 5, 48149 Münster,
Germany.
* Author for correspondence (e-mail: xz4{at}iupui.edu)
Accepted 4 October 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Ndst, HSPG, BMP, Wnt, FGF, Erk, Signaling, Lens, Induction, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Wawersik et al.,
1999). Similarly, suppression of FGF signaling via a
pharmacological inhibitor or dominant-negative FGF receptor 1 (FGFR1) resulted
in the downregulation of Pax6, Sox2 and Foxe3 expression,
and in defects in lens formation (Faber et
al., 2001). In further support of a role of FGF signaling in early
lens development, a mutation in the FGFR signaling mediator Frs2
led to anophthalmia or microphthalmia
(Gotoh et al., 2004). Hh
signaling is necessary for early eye-field specification and lens regeneration
(Macdonald et al., 1995;
Tsonis et al., 2004). However,
hyperactive Hh signaling from the embryonic midline may also result in lens
degeneration, as demonstrated in cavefish development
(Yamamoto et al., 2004). Wnt
signaling is also known to be involved in the development of the lens.
Misexpression of a Wnt receptor, frizzled 3, led to ectopic
Pax6 expression and eye formation in Xenopus
(Rasmussen et al., 2001). By
contrast, a mouse mutant deficient for the Wnt signaling co-receptor
Lrp6 exhibited abnormal cell death in lens epithelium, whereas the
conditional knockout of ß-catenin in periocular ectoderm resulted in
ectopic lentoid formation (Smith et al.,
2005; Stump et al.,
2003).
Many of the signaling pathways described above are known to be dependent on
the presence of heparan sulfate proteoglycans (HSPGs) on the cell surface
(reviewed in Lin, 2004). HSPGs
are glycoproteins containing covalently linked heparan sulfate
glycosaminoglycan chains. These linear polysaccharides exhibit enormous
structural heterogeneity because of variable N-deacetylation of
N-acetylglucosamine residues, N- and O-sulfation, and epimerization
of uronic acid residues (Esko and Selleck,
2002). Previous studies have demonstrated that cell-surface
proteoglycans can serve as co-receptors for FGF
(Rapraeger et al., 1991;
Yayon et al., 1991). This is
supported by crystallographic structures of heparan sulfate associated with an
FGF-FGFR complex (Pellegrini et al.,
2000; Schlessinger et al.,
2000). Recently, the role of HSPGs in morphogen diffusion was
illuminated by genetic studies of Drosophila proteoglycan core
proteins and glycosaminoglycan biosynthetic enzymes. It was demonstrated that
loss of HSPGs prevented the transport of Dpp, wingless (Wnt) and Hh molecules,
resulting in the disruption of morphogen gradients
(Belenkaya et al., 2004;
Han et al., 2004;
Kirkpatrick et al., 2004). In
vertebrate development, the proteoglycan core-protein gene glypican 3
(Gpc3) genetically interacts with Bmp4 during limb
development, and loss of Wnt signaling is also observed in mouse Gpc3
mutants (Paine-Saunders et al.,
2000; Song et al.,
2005). By contrast, a mutation in the glycosaminoglycan
biosynthetic gene UDP-glucose dehydrogenase (Ugdh) inhibited
the signaling of FGF, but not of Nodal or Wnt3, in mesoderm- and
endoderm-migration during gastrulation
(Garcia-Garcia and Anderson,
2003). Another glycosaminoglycan biosynthetic gene, Ext1,
is required for FGF8 signaling in CNS development, whereas a hypomorphic
mutation in Ext1 expanded the range of Indian hedgehog (Ihh)
signaling during chondrocyte maturation
(Inatani et al., 2003;
Koziel et al., 2004).
Interestingly, recent studies have shown that zebrafish ext2 and
extl3 regulate Fgf10, but not Fgf4, signaling during limb development
(Norton et al., 2005). These
findings demonstrate the potential of HSPGs in regulating specific signaling
pathways in a contextdependent manner.
The enzyme N-acetylglucosamine
N-deacetylase-N-sulfotransferase (Ndst) catalyzes the first
sulfation step during the synthesis of heparan sulfate. Consistent with its
crucial role in HSPG modification, a Drosophila Ndst mutant,
sulfateless, exhibited a segment-polarity phenotype as a result of
impaired Wnt signaling (Lin and Perrimon,
1999). Furthermore, FGFR-dependent MAPK activity was also reduced
in the sulfateless mutants during mesoderm and trachea development,
and genetic interactions were demonstrated between sulfateless and
the FGF-receptor gene (Lin et al.,
1999). Finally, mosaic analysis showed that the loss of
sulfateless prevented the diffusion of Dpp- and Hh-molecules in wing
imaginal discs (Belenkaya et al.,
2004; Han et al.,
2004). These results established that Ndst genes are essential for
the transport of morphogenic molecules and for their subsequent signaling.
There are four known Ndst enzymes in mammals, and biochemical experiments
suggest that they might have different substrate specificities
(Aikawa et al., 2001). Targeted
deletion of Ndst1 in mice resulted in embryonic lethality as a result
of lung defects, whereas brain and ocular defects had also been noted
(Fan et al., 2000;
Grobe et al., 2005;
Grobe et al., 2002;
Ringvall et al., 2000).
Ndst2 mutants had impaired mast-cell development
(Forsberg et al., 1999;
Humphries et al., 1999),
whereas the Ndst3 mutant did not exhibit an obvious phenotype
(Grobe et al., 2002).
Importantly, Ndst1 and Ndst2 double-homozygous mutants
exhibited early embryonic lethality, similar to that observed in
Ext1- and Ext2-null mutants
(Forsberg et al., 1999;
Grobe et al., 2002;
Lin et al., 2000;
Stickens et al., 2005). These
results demonstrate both the functional specificity and redundancy among the
Ndst-family enzymes.
We have previously characterized cranial facial-developmental defects in
Ndst1 mutants and showed that Ndst1 genetically interacted
with Shh. In addition, we found that fibroblast cells derived from
Ndst1-mutant embryos failed to respond to FGF stimulation in vitro,
suggesting a role of Ndst1 in FGF signaling
(Grobe et al., 2005). In this
study, we further examined lens development in Ndst1 mutants, and
demonstrated that loss of Ndst1 function disrupted lens-vesicle
invagination and lens cell differentiation. Importantly, we showed that BMP-
and Wnt-signaling were not affected in Ndst1-mutant lenses. Instead,
Ndst1 loss of function led to a reduced binding of FGF ligand or
FGF-FGFR complex on the cell surface. Consistent with this, MAPK signaling was
downregulated during lens development. Therefore, Ndst1 was important
for lens-specific FGF signaling during development.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Bmp4 mice were kindly provided by Simon Conway (Indiana University
School of Medicine, Indianapolis, IN, USA) and Bridget Hogan
(Lawson et al., 1999). TOPGAL
mice are obtained from Jackson Laboratory
(DasGupta and Fuchs,
1999).
RT-PCR
Lens tissue was dissected in ice-cold PBS and immediately placed in liquid
nitrogen. RNA was isolated from tissue extracts using a RNA-isolation kit
(Qiagen, Valencia, CA), and reverse transcription was carried out according to
the manufacturer's instructions (Invitrogen, Carlsbad, CA). The primers used
for PCR were: Ndst1 (forward:
5'-ACCACAGCCAGCCAGAACGCTTGTG-3'; reverse:
5'-AGCTGCGCTCTTCCCCTTTACTGTC-3'), Ndst2 (forward:
5'-CCTTGCAGAACCGTTGTC-3'; reverse:
5'-CAGCCATTCCAATCCTG-3'), Ndst3 (forward:
5'-TGTGTTTCCTGTGAGTCCAGATGTGTG-3'; reverse:
5'-ATTGTCCTCCTCACTTCCATCAGCCTG-3') and Ndst4 (forward:
5'-AACAGGAAATGACACTTATTGAAACG-3'; reverse:
5'-ACTTTGGGGCCTTTGGTAATATG-3').
BrdU and TUNEL analysis
Pregnant mice were injected 2 hours prior to dissection with BrdU dissolved
in PBS at 0.1 mg BrdU per 1 g body weight. The embryos were fixed in 4% PFA at
4°C overnight, incubated in 30% sucrose/PBS at 4°C overnight and
embedded in OCT compound. Antigen retrieval was performed on 10 µm
cryosections by microwave heating for 10 minutes at sub-boiling condition in
citrate buffer at pH 6.0, and treated with 1 N HCl for 90 minutes at room
temperature. Next, the sections were blocked with 10% normal goat serum in PBS
at room temperature for 2 hours prior to the addition of an anti-BrdU antibody
(Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA,
USA). After overnight incubation at 4°C, the sections were treated with
secondary antibody and with the nuclear stain Hoechst for 2 hours at room
temperature, and then examined under a Leica DM500 fluorescent microscope. The
cell-proliferation rate was calculated as the ratio of BrdU-positive cells
versus Hoechst-positives cells, and analyzed by the Student's
t-test.
TUNEL staining was performed with an in situ cell-death detection kit (Roche, Indianapolis, IN, USA). Briefly, cryosections were processed for antigen retrieval as described above, incubated with blocking buffer (0.1 M Tris-HCl, pH 7.5, 3% BSA, 20% serum) for 30 minutes at room temperature and then with TUNEL reaction mixture for 2 hours at 37°C. After rinsing with PBS, the sections were blocked again with 0.05% blocking reagent (supplied in the TSA Indirect Tyramide Signal Amplification Kit, Perkin Elmer Life Science, Boston, MA, USA) for 30 minutes and then incubated with TUNEL-POD for 30 minutes at 37°C. Finally, the signal was developed with DAB substrate and detected under a Leica DM500 microscope.
RNA in situ hybridization
RNA whole-mount in situ hybridization was performed as previously described
(Zhang et al., 2002). RNA in
situ hybridization on sections was carried out according to a standard
protocol (Dakubo et al., 2003).
The following probes were used: BF2, ERM (from Bridget Hogan, Duke
University Medical Center, Durham, NC, USA), Hes1 (from Naoki
Takahashi, Nara Institute of Science and Technology, Nara, Japan),
Math5 (also known as Atoh7 - Mouse Genome Informatics; from Tom
Glaser, University of Michigan, Ann Arbor, MI, USA), Ndst1, Pax6,
Six3 (from Guillermo Oliver, St Jude Children's Research Hospital,
Memphis, TN, USA) and Sox2. At least three embryos of each genotype
were analyzed for each probe.
Erk-P and Smad1-P immunohistochemistry
X-gal staining, in situ hybridization and regular immunohistochemistry were
performed as previously described (Zhang
et al., 2003). Immunohistochemistry of phospho-Smad
(Smad-P) and phospho-Erk (Erk-P) was carried out according
to published procedures (Ahn et al.,
2001). Briefly, mutant and control embryos were matched by somite
numbers, and processed for coronal section on a Leica cryostat. For antigen
retrieval, the sample slides were incubated in citrate buffer (10 mM sodium
citrate, pH 9.0) at 80°C for 30 minutes, followed by treatment with 2%
H2O2 to quench the endogenous peroxidase activity. After
1 hour of blocking at room temperature with 5% goat serum in PBS, the slides
were incubated with primary antibody diluted in the blocking solution
overnight at 4°C. Next, the slides were blocked for 30 minutes with 0.05%
blocking reagent (TSA Indirect Tyramide Signal Amplification Kit, Perkin Elmer
Life Science, Boston, MA, USA) and sequentially incubated with a biotin
conjugated anti-rabbit antibody and ABC reagent (Vectastain ABC Kit, Vector
Labs, Burlingame, CA, USA). To amplify the immunoperoxidase signal, the
specimens were incubated with biotinyl tyramide diluted 1:50 in tyramide
diluent for 10 minutes and then in 1:250 streptavidin-HRP for 30 minutes.
Finally, the sections were incubated with DAB solution for color reaction.
As a control, we also performed phospho-Erk1/2 immunostaining on embryos
treated with the FGFR1 inhibitor PD-173074 (a gift from Pfizer, New Jersey,
NJ, USA) or the MAPK kinase inhibitor U0126 (Cell Signaling Technology,
Beverly, MA). Prior to immunohistochemistry, the control embryos were
incubated in RPMI containing 1% BSA; 50 µM U0126 or 40 µM PD-173074 at
37°C; and 5% CO2 for 30 minutes. This effectively abolished the
phospho-Erk1/2 expression in the embryos, thus validating the specificity of
the phospho-Erk1/2 staining in our experiment
(Corson et al., 2003).
The antibodies we used were: anti-phospho-Erk1/2, anti-phospho-Smad1/5/8,
anti-phospho-Smad2 (all from Cell Signaling Technology, Beverly, MA, USA),
anti-phospho-Smad1 (PS1) antibody [kindly provided by Peter ten Dijke (Leiden
University Medical Center, Leiden, The Netherlands) and Carl-Henrik Heldin
(Ludwig Institute for Cancer Research, Uppsala, Sweden)], anti-Pax6 (the
Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA),
anti-AP2 (Santa Cruz biotechnology, Santa Cruz, CA, USA), anti-Pax2,
anti-Prox1 (both from Covance, Berkeley, CA, USA), anti-A crystallin
(kindly provided by Samuel Zigler, National Institute of Health, Bethesda,
Maryland, USA), anti-Six3 (kindly provided by Guillermo Oliver, St Jude
Children's Research Hospital), and 10E4, HepSS-1 and 3G10 (all from Seikagaku,
Tokyo, Japan).
|
) and
incubated with the sections at 4°C overnight. The bound FGF2 was detected
using a Vectastain ABC kit (Vector Labs, Burlingame, CA, USA) and stained in
DAB solution (Sigma, St Louis, MO, USA). As a negative control, heparan
sulfate on sections was degraded with 10 units of heparitinase I (Seikagaku,
Tokyo, Japan) in 50 mM Tris-HCl pH 7.4, 50 mM sodium acetate, 1 mM calcium
chloride and 1 mg/ml BSA at 37°C for 2 hours prior to the assay.
For in situ binding of the FGF-FGFR complex with heparan sulfate, we
carried out the ligand and carbohydrate engagement (LACE) assay, as described
(Allen and Rapraeger, 2003).
Briefly, the frozen sections were incubated in 0.5 mg/ml NaBH4 for
10 minutes, in 0.1 M glycine for 30 minutes and then blocked with 2% BSA.
Next, the slides were incubated with 20 µM FGF, 20 µM human FGFR-Fc
chimera (both from R&D Systems, Minneapolis, MN) and 10% fetal-calf serum
in RPMI-1640 at 4°C overnight. After washing in PBS, the bound FGFR-Fc was
detected using a cy3-labeled anti-human Fc IgG secondary antibody, and the
fluorescence signal was examined using a Leica DM500 fluorescent
microscope.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Next, we sought to confirm the in situ hybridization results by RT-PCR
analysis. Total RNA was isolated from the lens and retina of E12.5 mice, and
subjected to RT-PCR with specific primers for Ndst1-4 and for the
mitochondria ribosomal subunit L19
(Fig. 1B). In the lenses, we
detected the expression of Ndst1 and, to a lesser extent,
Ndst2 only. By contrast, transcripts of all four Ndst genes were
present in the retinae. As a control, similar levels of ribosomal subunit
L19 signals were observed in the retina and in the lens. Furthermore,
no signal was detected in a RT-PCR reaction without reverse transcriptase.
Previous studies have shown that Ndst2-null mice are normal, with the
exception of defects in connective-tissuetype mast cells
(Forsberg et al., 1999;
Humphries et al., 1999).
Together, these results suggest that, of the Ndst genes, Ndst1 most
probably plays the dominant role during lens development.
Inactivation of Ndst1 disrupts eye development
We next examined homozygous Ndst1-null mice from E12.5 to E17.5
and observed ocular phenotypes in all embryos collected (n=18,
Fig. 2A). Among them, four out
of 18 embryos (22%) exhibited microphthalmia with reduced retinae and lenses,
eight out of 18 embryos (44%) retained the retina but lacked lens, and the
remaining six out of 18 embryos (34%) had no retina or lens. The size of
embryos without lenses was indistinguishable from that of wild-type litter
mates, whereas the embryos without eye structures were sometimes smaller and
showed additional brain defects.
To investigate the origin of the ocular defects, we next analyzed homozygous Ndst1 mutants at E13.5. The range of lens phenotypes was apparent in the Ndst1-mutant embryos and, even in the least-affected embryos, lenses were smaller and pinched at the anterior region (Fig. 2D,E). Unlike the wild-type littermates, the lens lumen of which was mostly filled with lens fibers, much of the anterior-lens vesicle was still empty in mutant lenses. To uncover the molecular changes in Ndst1 mutants, we also performed RNA in situ hybridization on frozen sections to stain for Pax6 gene expression (Fig. 2B-I). Pax6 expression should be restricted to the epithelial cells of the lens at this stage; however, in mutants, Pax6 transcripts were detected throughout the lens (Fig. 2E). In more-severely affected mutants, the entire lens was reduced to a small cluster of cells connected to the surface ectoderm with a residual lens stalk (Fig. 2G, arrow) and the retinae were mis-shaped. Finally, some Ndst1 mutants lacked any apparent retina or lens structure (Fig. 2H). There were sometimes pigmented cells left at the presumptive eye region (Fig. 2I, arrowhead). Interestingly, Pax6 expression could still be detected in the surface ectoderm (Fig. 2I). The severity of the lens defects observed in these E13.5 Ndst1 embryos suggested that failure of lens development probably originated even earlier during development.
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;
Darnell et al., 1999;
Furukawa et al., 2000;
Wang et al., 2001;
Yuasa et al., 1996;
Zhu et al., 2002). RNA in situ
hybridization showed that transcripts of these genes were present in both
wild-type and Ndst1-mutant retinae. Therefore, retinal patterning and
differentiation appeared to have at least been initiated in the absence of the
lens in Ndst1 mutants.
Early lens defects in Ndst1-knockout embryos
In search of the mechanism for lens defects, we next studied lens induction
in Ndst1 mutants. At the 24-somite stage, the mutant lens placode was
morphologically indistinguishable from wild-type controls
(Fig. 3A). However, at the
30-somite stage, wild-type embryos had formed the lens pit, whereas
Ndst1-mutant embryos exhibited less-advanced indentation in the lens
placode (Fig. 3A). At the
35-somite stage (E11.0), lens vesicles were either entirely absent (data not
shown) or reduced in size in Ndst1 mutants
(Fig. 3A). Throughout
lens-vesicle development, the rates of BrdU incorporation in mutant embryos
were consistently reduced in comparison to wild-type controls
(Fig. 3B). By contrast, few
apoptotic cells were observed in either wild-type or Ndst1-mutant
lens vesicles (Fig. 3C, arrow),
even though there was a significant increase in TUNEL staining in periocular
mesenchyme in Ndst1-mutants (Fig.
3C, arrowhead). These results suggest that Ndst1 mutants
were defective in lens cell proliferation.
|
AP2, a transcription factor required for lens development, was found
to be expressed in both wild-type and Ndst1-mutant lens primordia at
the 32-somite stage (data not shown). In 35-somite embryos, however, some of
the Ndst1 mutants expressed AP2 in overlying head ectoderm
only, and not in lens vesicles (Fig.
4I, five out of eight embryos). Therefore, AP2 expression
was specifically lost between the 32- and 35-somite stages. Normally, A
crystallin is expressed in the lens pit at E10.5 and Prox1 expression
initiates in the lens placode at E9.5
(Robinson and Overbeek, 1996;
Wigle et al., 1999). None of
these molecules were observed in the more severely affected homozygous
Ndst1-null mutants (Fig.
4L, four out of six mutants for Prox1;
Fig. 4O, two out of three
mutants for A crystallin). Notice that some Ndst1 mutants
exhibited relatively mild lens-vesicle defects and that these embryos also
preserved Pax6, AP2, Prox1 and A crystallin expressions. This is
consistent with the observation that Ndst1 mutants displayed a range
of phenotypes, including some that developed both lens and retina. Taken
together, these molecular defects show that Ndst1 inactivation
results in a delay or even failure of lens-vesicle development.
Ndst1 knockout did not affect canonical BMP and Wnt signaling in the lens
BMP/TGFß signaling are known to play important roles in lens
development. We thus examined the intracellular mediators of BMP/TGFß
signaling - the phospho-Smad1 (Smad1-P) and phospho-Smad2
(Smad2-P) proteins - in Ndst1-mutant lenses. To validate the
Smad1-P antibody, we first compared its staining pattern in wild-type
and Bmp4-mutant embryos. Consistent with previous reports, we
observed specific Smad1-P expression in the first branchial arch
(Fig. 5A, arrowhead) and
olfactory placode (Fig. 5A,
arrow) at E9.5 (Ahn et al.,
2001; Faber et al.,
2002). Not surprisingly, this coincides with strong Bmp4
expression in these locations (Dudley and
Robertson, 1997). In Bmp4-knockout embryos, however, this
Smad1-P staining pattern was abolished. Therefore, Smad1-P
immunohistochemistry reliably detected active Bmp4 signaling during embryonic
development.
In wild-type embryos, Smad1-P was present in the presumptive lens
ectoderm and optic vesicle as early as the 20-somite stage (E9.5), forming an
anterior-posterior gradient (Fig.
5B). This expression pattern persisted in 30 somite-stage embryos
(E10.5) as lens placodes invaginated. In homozygous Ndst1-knockout
embryos (KO/KO), similar Smad1-P staining was detected, even as the
lens-vesicle formation was disrupted (Fig.
5B, arrowhead). Furthermore, both wild-type and
Ndst1-mutant lens vesicles strongly expressed the Smad2-P
protein at E11.0 (Fig. 5B,
arrow). Therefore, the BMP/TGFß signaling mediated by phospho-Smad
proteins was unaffected in Ndst1 mutants. To further test the genetic
interaction between Ndst1 and Bmp4, we crossed
Ndst1 mice with a Bmp4-mutant strain carrying a
LacZ knock-in allele (Bmp4LacZ)
(Lawson et al., 1999). As
shown in Fig. 5C, the loss of
one copy of the Bmp4 gene did not enhance the lens phenotype in
either heterozygous or homozygous Ndst1-mutant embryos (n=24
for Ndst1KO/+ Bmp4LacZ/+, n=10 for
Ndst1KO/KO Bmp4LacZ/+), and the Bmp4
expression reported by the ß-galatosidase activity was also unchanged in
the Ndst1-mutant background. Taken together, these results suggest
that BMP/TGFß signaling was not affected by Ndst1 inactivation
in the lens.
|
), we
also assayed the canonical Wnt signaling activity in Ndst1-mutant
lenses (Fig. 5D). In both
wild-type and Ndst1-mutant embryos, similar TOPGAL transgene
expressions were observed in periocular tissue, whereas no ß-galatosidase
activity was detected in the lens. Therefore, the lens defects in
Ndst1 mutants are unlikely to be caused by abnormal canonical Wnt
signaling.
Ndst1 mutants are defective in heparan sulfate synthesis and FGF-FGFR binding
We next analyzed the expression pattern of heparan sulfate during lens
development. The monoclonal antibody 10E4 recognizes an epitope unique to
heparan sulfate, whereas the HepSS-1 antibody binds to N-sulfated
heparan sulfate domains (Leteux et al.,
2001; van den Born et al.,
2005). In wild-type embryos, both antibodies stained the basal
membranes of the optic vesicle and the lens vesicle
(Fig. 6). We further
demonstrated that this staining pattern was specific to heparan sulfate
because sections treated with heparitinase I completely lost the staining
(Fig. 6). Heparitinase I
digestion also generated a heparan sulfate `stub' motif, which was the epitope
of the 3G10 antibody (David et al.,
1992). In heparitinase I-treated sections, we observed specific
3G10 staining in the developing eye (Fig.
6). Therefore, heparan sulfate was abundantly expressed during
lens formation.
Ndst1 catalyzes the N-deacetylation and N-sulfation of heparan sulfate. Interestingly, we observed a complete loss of 10E4 and HepSS-1 staining in KO/KO embryos, but 3G10 staining after the Heparitinase I treatment remained intact (Fig. 6). Because the 3G10 antibody detects heparan sulfate stubs that remain after heparitinase digestion, these findings indicate that heparan sulfate chains were still being made in the Ndst1-mutant embryos, but that these were undersulfated.
The sulfation pattern of heparan sulfate is important for its interaction
with FGF ligands and receptors. We thus performed LACE assays and asked
whether Ndst1 mutants defective in heparan sulfate modification also
exhibited reduced FGF binding (Allen and
Rapraeger, 2003). Embryo sections were incubated with FGF2 tagged
with biotin and the binding of FGF2 to eye tissue was detected by biotin
histochemistry. In wild-type eyes, biotinylated FGF2 was specifically
localized at the basal membrane of lens- and retinal-cells, and the
FGF2-binding pattern closely resembled the distribution of endogenous heparan
sulfate during eye development (Fig.
7A). As a control, no staining was observed in the absence of
biotinylated FGF2 (data not shown). More importantly, prior treatment of
embryo sections with heparitinase I completely abolished the staining
(Fig. 7A). This demonstrates
that the binding of biotinylated FGF2 on these tissue sections crucially
depends on intact heparan sulfate. In Ndst1-mutant embryos,
incubation with the same concentration of biotinylated FGF2 resulted in
much-weaker staining as compared with the wild-type controls, and significant
binding of FGF2 to lens cells was observed only after a 10-fold increase in
FGF2-ligand concentration (Fig.
7A). Therefore, the Ndst1 mutation resulted in a reduced
affinity of FGF2 to the lens cell basement membrane.
|
;
McWhirter et al., 1997;
Vogel-Hopker et al., 2000).
Eye sections from E10.5 embryos were incubated with purified FGF and with FGFR
fused with the human IgG Fc domain (FGFR-Fc), and bound FGFR-Fc was probed
with an anti-IgG antibody. In wild-type-embryo sections, we observed specific
binding of FGFR2c and FGFR3c to lens- and retina-cells in the presence of
FGF8b (Fig. 7B). As a control,
no signal was detected without FGF8b or after the treatment of tissue sections
with heparitinase I (data not shown). This demonstrated that the observed FGFR
binding in situ was mediated by FGF and cell-surface heparan sulfate. In E10.5
Ndst1-mutant sections, binding of FGFR3c-FGF8b was reduced throughout
the eye region (Fig. 7B). In
comparison, FGFR2c-FGF8b binding was weaker in the retina
(Fig. 7B, arrow) and became
almost undetectable in the Ndst1-mutant lens
(Fig. 7B, arrowhead). The
more-pronounced loss of FGFR2c-FGF8b binding to the lens was especially
interesting considering the significant lens phenotype in Ndst1
mutants. Finally, FGFR4-FGF19 exhibited weak but unchanged binding to both
wild-type and Ndst1-mutant eyes, suggesting that Ndst1
inactivation disrupted the assembly of some, but not all, FGF-FGFR pairs in
the eye tissues.
|
; de Iongh and
McAvoy, 1993; Kitaoka et al.,
1994; Lovicu and Overbeek,
1998; Martinez-Morales et al.,
2005; McWhirter et al.,
1997; Nguyen and Arnheiter,
2000; Vogel-Hopker et al.,
2000; Zhao et al.,
2001). This includes all the FGF and FGFR variants known to be
present during early eye development. It also represents all six subfamilies
of FGFs, except the FGF11-14 subfamily, which does not interact with FGF
receptors. In wild-type embryos, the binding of each FGF-FGFR pair largely
confirmed previous results obtained in cell culture or in biochemical studies
(Fig. 7C). In Ndst1
mutants, however, FGF-FGFR interactions were altered. Except for FGF19, the
interaction of each FGF with at least one FGFR variant was disrupted by
Ndst1 deletion. Different FGF-FGFR pairs were affected differentially
by Ndst1 inactivation, and each pair exhibited distinct binding
activities in different tissues (Fig.
7C and data not shown). Overall, 18 FGF-FGFR pairs required Ndst1
function in the lens, suggesting that Ndst1-modified heparan sulfate
potentially regulates a large number of FGF-FGFR interactions during lens
development.
FGF-signaling targets were downregulated in Ndst1 mutants
The significant loss of in situ FGF-FGFR binding to Ndst1-mutant
tissue raised the possibility that FGF signaling was compromised during eye
development. To test this idea, we first examined the expression of the
phospho-Erk1/2 (Erk-P) proteins - the downstream effectors of the
FGF-MAPK pathway. Using a phospho-specific antibody against Erk1/2, we
detected Erk-P expression in the developing optic cup and lens
vesicle in E10.5 embryos (Fig.
8A). The specificity of the immunohistochemistry assay was
demonstrated in embryos cultured in the presence of the MEK inhibitor U0126,
which acts upstream of Erk1/2 (Favata et
al., 1998). After treatment, Erk-P expression was
completely lost throughout the embryos, including the eye tissues
(Fig. 8A). Furthermore, we
cultured wild-type embryos with PD173074, a potent FGFR inhibitor
(Skaper et al., 2000). This
treatment also abolished Erk-P staining in the developing optic cup
and lens vesicle (Fig. 8A).
Together, these results confirm previous reports that Erk-P
expression directly correlates with FGFR-MAPK signaling activity during eye
development (Corson et al.,
2003; Govindarajan and
Overbeek, 2001; Lovicu and
McAvoy, 2001).
In wild-type embryos, strong Erk-P immunostaining was observed in lens placodes at the 27-somite stage, whereas little Erk phosphorylation was detected in the Ndst1-mutant lens ectoderm (Fig. 8B, arrowhead). Similarly, wild-type embryos exhibited strong Erk-P expression in the invaginating lens vesicle at the 30- and 32-somite stages. In mutant embryos, where lens development failed to progress beyond initial lens-placode invagination, the lens tissues expressed Pax6 but not the Erk-P proteins (Fig. 8B, arrow). Interestingly, strong expression of Erk-P remained in the mutant optic vesicle throughout development. These results show that the MAPK pathway was specifically disrupted in the Ndst1-mutant lens.
|
). In
particular, previous experiments have demonstrated that the expression of the
Pea3 (also known as Etv4 - Mouse Genome Informatics) group of ETS-domain
transcription factors [ER81 (also known as Etv1 - Mouse Genome Informatics),
ERM (also known as Etv5 - Mouse Genome Informatics) and Pea3] closely mimic
FGF-signaling activities (Munchberg and
Steinbeisser, 1999; Raible and
Brand, 2001; Roehl and
Nusslein-Volhard, 2001). Using RNA in situ hybridization, we
observed expression of ERM in developing wild-type lens vesicles at
E10.5. By contrast, ERM expression was significantly downregulated in
Ndst1-mutant lenses. Consistent with lens-specific loss of
Erk-P, the reduction of ERM was also confined to the
developing lens, because midbrain- and branchial-arches still exhibited strong
ERM expression. Together, these results show that FGF-MAPK signaling
was disrupted in the Ndst1-mutant lens. |
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). Thus, in Drosophila tissue where Dpp is
abundantly expressed, heparan sulfate mutation has no obvious effect on
Dpp-controlled patterning (Haerry et al.,
1997; Lin and Perrimon,
1999). By contrast, cell-surface heparan sulfate acts as a
co-receptor for FGF signaling, forming a trimeric complex with FGF and the FGF
receptor. Therefore, all mutant cells deficient for heparan sulfate fail to
respond to FGF signaling. In vertebrate lens development, the lens vesicle
develops from a single layer of placodal cells, which is directly exposed to
strongly expressed inductive signals, such as BMP4, from the optic vesicle. It
is therefore expected that the Ndst1-mutant lens vesicle will
maintain its BMP response. By contrast, heparan sulfate is required for
FGF-signal binding on the cell surface, and Ndst1 inactivation will
thus abrogate the FGF-signaling activity during lens-vesicle development.
|
).
Consistent with this model, we observed reduced FGF2 binding to lens cells in
Ndst1 mutants. In addition, Ndst1 inactivation significantly
reduced binding of lens cell heparan sulfate to FGF8b-FGFR2c and FGF8b-FGFR3c
complexes. Surprisingly, the binding affinity of FGF19-FGFR4 to lens cell
heparan sulfate remained unchanged in Ndst1 mutants. This suggested
that Ndst1-mediated structural remodeling of heparan sulfate was necessary to
allow for the assembly of some, but not all, FGF-FGFR complexes on the cell
surface. Such distinct requirement for heparan sulfate motifs in FGF signaling
has also been observed in earlier studies
(Ford-Perriss et al., 2002).
In particular, a mutation in the heparan sulfate 2-O-sulfotransferase
(HS2ST) gene was shown to disrupt binding of FGF8b-FGFR2c, but not
FGF8b-FGFR3c, on E10.5 heart sections
(Allen and Rapraeger, 2003). In
zebrafish limb development, ext2 and extl3 mutants
specifically affected FGF10, but not FGF4, signaling
(Norton et al., 2005). Taken
together, these results suggest that heparan sulfate-synthesizing enzymes can
play an active role in regulating different FGF-signaling pathways.
In this study, we have systematically analyzed in situ FGF-FGFR binding on
the cell surface of the lens. Our results are mostly consistent with
mitogenesis studies performed in cell culture and with binding studies by
surface plasmon resonance, although a few differences were noted
(Mohammadi et al., 2005;
Ornitz et al., 1996;
Zhang et al., 2006). These
differences probably result from the fact that our assay involved endogenous
heparan sulfate on developing lenses, whereas the other systems depend on
exogenous heparin. Nevertheless, our data confirm that many of the FGF-FGFR
interactions require N-sulfated glucosamine residues in heparan
sulfate, suggesting that Ndst1 inactivation could potentially disrupt
multiple FGF-FGFR-signaling pathways during eye development. Recent studies
demonstrating modest. or even no, lens defects in FGFR1, FGFR2 and FGFR3
single mutants support this idea (Garcia
et al., 2005; Huang et al.,
2003; Zhao et al.,
2006). Therefore, our study of the Ndst1 gene provides an
attractive model to unravel the complexity of FGF signaling in eye
development.
|
ACKNOWLEDGMENTS |
|---|
|
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|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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