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First published online 12 December 2007
doi: 10.1242/dev.014829
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1 Department of Medical and Molecular Genetics, Indiana University School of
Medicine, Indianapolis, IN 46202, USA.
2 Programs in Signal Transduction and Stem Cells and Regeneration, Burnham
Institute for Medical Research, La Jolla, CA 92037, USA.
3 Department of Cellular and Molecular Medicine, Glycobiology Research and
Training Center, University of California San Diego, 9500 Gilman Drive, La
Jolla, CA 92093, USA.
4 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 26 October 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: HSPG, Ndst, FGF, ERK, Branching morphogenesis, Lacrimal gland, Ptpn11, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Makarenkova et al., 2000).
Similarly in humans, autosomal dominant aplasia of lacrimal and salivary
glands, or ALSG syndrome, is associated with FGF10 mutations
(Entesarian et al., 2005).
Conversely, exogenous Fgf10 can induce ectopic lacrimal gland formation in eye
explant cultures, and transgenic expression of Fgf10 leads to
lacrimal gland like structures in the cornea
(Govindarajan et al., 2000;
Makarenkova et al., 2000).
Fgf7, a close homolog of Fgf10, is also expressed in the
periocular mesenchyme. Although a lacrimal gland phenotype has not been
observed in the Fgf7 deletion mutant, Fgf7 can also induce
ectopic lacrimal glands in transgenic animals or explant cultures
(Lovicu et al., 1999;
Makarenkova et al., 2000).
Biochemical studies have shown that both Fgf7 and Fgf10 strongly interact with
Fgfr2b. Indeed, antisense DNA against Fgfr2b in ex vivo
cultures can disrupt the budding outgrowth of the lacrimal gland epithelium,
and the human lacrimo-auriculo-dento-digital (LADD) syndrome, a symptom of
which is lacrimal duct aplasia, is caused by mutations in FGFR2 and
FGFR3 genes (Makarenkova et al.,
2000; Rohmann et al.,
2006). Therefore, FGF signaling is essential for lacrimal gland
development.
The FGF-FGFR interaction is known to require heparan sulfate proteoglycans
at the cell surface. Heparan sulfates are linear glycosaminoglycan molecules
modified by a series of sulfotransferase enzymes, which generate significant
structural diversity among different tissues
(Esko and Selleck, 2002).
However, within each tissue, the composition of heparan sulfate is remarkably
consistent, suggesting that the biosynthesis of heparan sulfate is tightly
regulated during development (Ledin et
al., 2004; Maccarana et al.,
1996; van Kuppevelt et al.,
1998). The sulfation of heparan sulfate is important for its
interaction with FGF and FGFR, as evident in the studies of branching
morphogenesis in the Drosophila trachea and vertebrate lung
development. The Drosophila heparan sulfate
N-deacetylase/N-sulfotransferase (Ndst) gene
sulfateless is required for the FGFR-dependent MAPK activation during
tracheal development, but not for EGFR-dependent MAPK signaling in the
amnioserosa. Moreover, the trachea branching morphogenesis defect in the
sulfateless mutant can be partially rescued by overexpression of FGF
ligand (Lin et al., 1999).
This is consistent with the idea that heparan sulfate is involved in
stabilizing FGF-FGFR interactions. Drosophila Hs2st or Hs6st
mutations abolish 2-O and 6-O-sulfation of heparan sulfate respectively;
however, a compensatory elevation of sulfation levels exists at other
residues. Probably because the overall sulfation level is maintained,
Hs2st or Hs6st single mutants display only partial embryonic
lethality with few tracheal defects
(Kamimura et al., 2006). In
Hs2st; Hs6st double mutants, however, the trachea branching
morphogenesis is severely disrupted because of the loss of FGF signaling. In
the vertebrate lung, it is observed that the branching epithelium tubules
express sulfated heparan sulfate at high levels, and that chemical inhibition
of heparan sulfate sulfation prevents FGF10-induced lung budding
(Izvolsky et al., 2003). In
addition, the Hs6st1 mutant mouse exhibits impaired lung
alveolarization among other defects
(Habuchi et al., 2007).
Therefore, the role of heparan sulfate in FGF dependent branching
morphogenesis is conserved in both fly and mouse.
Inside the cell, FGF signaling is transmitted by multiple pathways,
prominent among which is the Raf/MAP-kinase kinase (MEK)/extracellular
signal-regulated kinase (ERK) pathway. Here, Shp2, a ubiquitously expressed
protein tyrosine phosphatase, positively regulates MAPK activity by modulating
many of its components (Lai et al.,
2004; Neel et al.,
2003). In support of this model, Shp2-null mouse embryos
die at the implantation stage because of a defective Src-Ras-MAPK pathway
downstream of Fgf4 signaling (Yang et al.,
2006). During mouse eye development, both lens and retina
formation depend on FGF signaling mediated by Frs2
(Gotoh et al., 2004). Targeted
mutations in the Shp2-binding site of Frs2 strongly reduce ERK
signaling, thus disrupting lens induction. Recent studies have also suggested
the role of Shp2 in branching morphogenesis. During embryonic lung
development, Shp2 transcripts are predominantly detected at the
epithelial bud cells, and expression of dominant negative Shp2 inhibits
epithelial branching and Fgf10-induced endodermal budding
(Tefft et al., 2005). Further
studies show that the level of phospho-ERK is reduced and that the epithelial
cell proliferation, but not differentiation, is impaired. Interestingly, the
conditional knockout of Shp2 in the mammary gland does not affect
normal branching and terminal budding in virgin females, but pregnancy-induced
lobulo-alveolar outgrowth is disrupted due to a Stat signaling defect
(Ke et al., 2006). Therefore,
further studies are required in order to understand the function of Shp2 in
branching morphogenesis.
We have recently shown that Ndst1 is required for FGF signaling
during lens induction (Pan et al.,
2006). In the present study, we further explore the extracellular
and intracellular regulation of FGF signaling, and demonstrate that the
lacrimal gland bud-specific modification of heparan sulfate by Ndst
controls the selective Fgf10/Fgfr2 interaction at the cell surface. This leads
to Shp2-dependent activation of ERK signaling inside the cell and preferential
outgrowth of the lacrimal gland bud. Therefore, lacrimal gland budding is
controlled by the Ndst-Fgfr2-Shp2 genetic
pathway.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Zhang et al.,
2004). Fgfr2flox mice were kindly provided by
Dr David Ornitz (Washington University Medical School, St Louis, MO)
(Yu et al., 2003).
Ndst2 mice were kindly provided by Dr Lena Kjellén (University
of Uppsala, Uppsala, Sweden) (Forsberg et
al., 1999). Le-Cre mice were kindly provided by Drs
Richard Lang (Children's Hospital Research Foundation, Cincinnati, OH), Peter
Gruss (MPI for Biophysical Chemistry, Göttingen, Germany) and Ruth
Ashery-Padan (Tel Aviv University, Tel Aviv, Israel)
(Ashery-Padan et al., 2000).
R26R mice were obtained from the Jackson Laboratory (Bar Harbor, ME)
(Soriano, 1999). All
experiments were performed in accordance with institutional guidelines.
Carmine staining of lacrimal glands
Lacrimal glands in embryos carrying the Le-Cre transgene were
visualized by their GFP expressions under a Leica MZ16F fluorescent dissecting
microscope. Alternatively, lacrimal glands in newborn animals were revealed by
aceto-carmine stain. Briefly, decapitated heads were dissected to reveal the
lacrimal glands located in the facial subcutaneous tissue between the skin and
cranial bones, then fixed in 4% PFA at 4°C overnight. After dehydration in
70% ethanol overnight, the heads were incubated in 0.5% aceto-carmine [0.5 g
carmine stain (C-1022, Sigma, St Louis, MO) dissolved in 100 ml boiling 45%
acetic acid] for 5-10 minutes, and destained in 70% ethanol for 3 minutes, 1%
acid alcohol (1% HCl in 70% ethanol) for 2 minutes and 5% acid alcohol (5% HCl
in 70% ethanol) for 1 minute. Lacrimal glands were examined under a Leica
MZ16F dissecting microscope and photographed with a Leica DFC320 camera.
BrdU, TUNEL, X-gal staining and immunohistochemistry
BrdU, TUNEL, X-gal staining and heparan sulfate immunohistochemistry were
performed as previously described (Pan et
al., 2006). For regular immunohistochemistry, paraffin sections
were cleared in xylene and rehydrated through a series of decreasing ethanol
solutions. Antigen retrieval was performed by microwave heating for 10 minutes
at sub-boiling condition in citrate buffer (10 mM sodium citrate, pH 6.0).
After cooling, endogenous peroxidase activity was quenched with 3%
H2O2 in 10% methanol/PBS solution for 30 minutes, and
non-specific interaction was blocked by 5% goat serum in PBS at room
temperature for 1 hour. The sections were next incubated with primary antibody
at 4°C overnight in a humid chamber, followed by sequential treatment with
a biotin-conjugated secondary antibody and ABC reagent (Vectastain ABC Kit,
Vector Laboratories, Burlingame, CA). Finally, the sections were incubated
with DAB solution for color reaction.
The antibodies used were: anti-BrdU (G3G4, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), anti-Cre (#69050-3, Novagen, Madison, WI), anti-phospho-ERK1/2 and anti-phospho-Shp2 (#9101 and #3751, Cell Signaling Technology, Beverley, MA), anti-GFP (a gift from Dr Pamela Silver, Harvard Medical School, Boston, MA), anti-Pax6 (PRB-278P, Covance, Berkeley, CA), anti-Shp2 (Sc-280, Santa Cruz Biotechnology, Santa Cruz, CA), and 10E4, 3G10 (Seikagaku, Tokyo, Japan).
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).
Briefly, mouse embryos were fixed in 4% PFA, cryoprotected in 30% sucrose
buffer, and embedded in OCT compound. After sectioning on a Leica CM1900
cryostat, the samples were collected on glass slides and dried. RNA in situ
probes dissolved in hybridization buffer (50% deionized formamide, 10% dextran
sulfate, 1 mg/ml rRNA, 1x Denhardt's solution, 5x SSC, 5 mM EDTA)
were heat denatured at 70°C for 10 minutes and applied to the sections.
The slides were covered with a cover slip to reduce evaporation. After
overnight incubation at 65°C in a humid chamber, the slides were rinsed
three times in wash buffer (1x SSC, 50% formamide, 0.1% Tween-20) at
65°C, and twice in MABT buffer (100 mM maleic acid, 150 mM NaCl, pH 7.5,
0.1% Tween-20) at room temperature. The sections were incubated with blocking
solution (20% heat-inactivated sheep serum/2% blocking reagent (Roche,
Indianapolis, IN) in MABT buffer) for 1 hour at room temperature. To detect
the signal, anti-DIG antibody (Roche, Indianapolis, IN) was diluted 1:1500 in
blocking buffer and added to the slides. After overnight incubation at
4°C, the slides were washed in MABT buffer and stained with BM purple AP
staining solution (Roche, Indianapolis, IN).
The following probes were used: Erm and Pea3 (both from
Dr Bridget Hogan, Duke University Medical Center, Durham, NC), Fgf10
and Fgfr2b (both from Dr Suzanne Mansour, University of Utah, Salt
Lake City, UT), and Ndst1 (Pan et
al., 2006). At least three embryos of each genotype were analyzed
for each probe.
FGF ligand and carbohydrate engagement assay (LACE)
In situ binding of the FGF-FGFR complex with heparan sulfate was carried
out using the LACE assay as previously described
(Allen and Rapraeger, 2003;
Pan et al., 2006). Briefly,
embryos were harvested and fixed in 4% PFA at 4°C overnight prior to
paraffin embedding. Deparaffinized and rehydrated 5 µm sections were
incubated in 0.5 mg/ml NaBH4 for 10 minutes, in 0.1 M
glycine for 30 minutes and 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 15% fetal bovine serum in RPMI-1640 at 4°C
overnight, followed by 2 hours incubation at room temperature with cy3-labeled
anti-human Fc IgG secondary antibody. After washing in PBS, the slides were
mounted with n-propyl gallate (NPG) antifading reagent, and examined using a
Leica DM500 fluorescent microscope.
Explant culture
Lacrimal gland explant cultures were performed according to an established
protocol (Makarenkova et al.,
2000). After washing, 80-120 µm diameter heparin acrylic beads
(Sigma, St Louis, MO) were incubated with 250 µg/ml recombinant FGF
(R&D Systems, Minneapolis, MN) or BSA dissolved in PBS at 4°C
overnight. E13.5-E14.5 embryos carrying the Le-Cre transgene
identified by GFP expression were dissected in PBS to remove the ocular
tissue, which includes the eye and the adjacent mesenchyme and ectoderm. The
FGF- or BSA-soaked beads selected under microscope were nudged into the
periocular mesenchyme using forceps. When placed on a Millipore filter (0.8
µm pore), the eye rudiment floated on top of the culture media [CMRL1066
supplemented with 10% FBS, 4 mM L-Glutamine, 0.1 mM non-essential amino acids
and antibiotics (Gibco, Carlsbad, CA)]. After 2 days' incubation at 37°C
with 5% CO2, the culture was examined for GFP-expressing lacrimal
gland buds.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In the Le-Cre transgenic mouse, GFP
expression was clearly detected in the lens and corneal epithelium at E12.5
prior to lacrimal gland development (data not shown). At E14.5, visible at the
temporal side of the eye was a small protrusion of lacrimal gland primordium,
which continued to extend until E16.5 to form an elongated duct with a rounded
tip at its distal end (Fig.
1A,B, arrowheads). At the time of extensive branching
morphogenesis at P0, the Le-Cre GFP reporter expression illuminated
the fine branches within the exorbital (arrow) and the intraorbital (arrow
head) lobes of the lacrimal gland (Fig.
1C). The GFP expression of the Le-Cre transgene continued
to be present in adult animals, faithfully following the development of the
lacrimal gland (data not shown).
We next examined whether the Le-Cre transgene could also
specifically target Cre expression in the lacrimal gland. For this,
we performed immunohistochemistry on E14.5 embryo sections and observed
co-expression of nuclear Pax6, cytoplasmic GFP and nuclear Cre recombinase in
the lacrimal gland bud cells (Fig.
1D-F). Furthermore, we crossed the Le-Cre with a
Cre reporter mouse, R26R, which expresses lacZ only
after a stop cassette is removed by Cre-mediated recombination
(Soriano, 1999). This
experiment also allowed a genetic fate mapping of the Le-Cre-positive
cells, because even transient expression of Cre under the direction
of Le-Cre permanently marked all the progeny cells with constitutive
lacZ expression. In the Le-Cre;R26R mice, X-gal
staining showed that the lacZ reporter was expressed throughout
lacrimal gland development, closely resembling the GFP expression pattern
described above (data not shown). At birth, both intraorbital and exorbital
lobes of the lacrimal gland were labeled with strong X-gal staining, which
upon sectioning, was found to be present only in the Pax6-positive epithelial
cells but not in any mesenchymal cells
(Fig. 1G-I). This result
demonstrates that the Le-Cre transgene can efficiently delete
loxP flanked DNA in lacrimal gland cells. Furthermore, consistent
with the idea that the lacrimal gland mesenchymal cells are derived from
neural crest cells, our fate-mapping results confirm that the developing
lacrimal bud is restricted to contribute only to the epithelial cells of the
lacrimal gland (Johnston et al.,
1979).
Lacrimal gland bud specific heparan sulfate modification
To investigate the role of heparan sulfate in lacrimal gland morphogenesis,
we examined the distribution of both modified and unmodified heparan sulfate
using the 3G10 antibody, which detects the heparan sulfate stub region exposed
by Heparitinase I digestion (David et al.,
1992). At E15.5, we observed ubiquitous 3G10 staining in many
regions of the eye, including the Pax6-positive conjunctival epithelium and
the protruding lacrimal bud (Fig.
2A, arrow). By contrast, the 10E4 antibody, which recognizes only
the sulfated heparan sulfate, exhibited a similar albeit more restricted
staining pattern (Leteux et al.,
2001; van den Born et al.,
2005). Although 10E4 still labeled both the lacrimal gland bud and
its surrounding mesenchyme, the staining was much weaker in the posterior
stalk region of the lacrimal gland when compared with the very tip of the
lacrimal bud, with the strongest staining in the basolateral membranes of the
tip epithelial cells (Fig.
2D,F, arrow). As a control, no 10E4 staining was observed in
Heparitinase I-treated sections (data not shown). Therefore, the distal tip of
the lacrimal bud expresses the specifically modified heparan sulfate.
This restricted 10E4 staining pattern indicates that heparan sulfate
sulfation is differentially regulated during lacrimal gland development. We
thus performed RNA in situ hybridization to investigate the expression of the
heparan sulfate N-deacetylation and N-sulfation gene Ndst1, which is
essential for generating the N-sulfated 10E4 epitope during embryonic
development (Pallerla et al.,
2007; Pan et al.,
2006). Using an Ndst1 antisense probe, we indeed detected
Ndst1 transcripts in the lacrimal gland buds with strongest
expression in the Pax6-positive tip cells
(Fig. 2G-I, arrowheads). As a
control, the Ndst1 sense probe did not detect any signal (data not
shown). Ndst1 expression thus correlates with the N-sulfation pattern
of heparan sulfate in lacrimal budding.
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;
Pan et al., 2006). At birth,
carmine staining revealed extensively branched lacrimal glands in wild-type
control pups (Fig. 2J, arrow).
Ndst1-/- animals, however, exhibited significant ocular
defects with no detectable lacrimal gland
(Fig. 2K, arrow). To confirm
the absence of the lacrimal gland, we also crossed the Ndst1 null
allele with the Le-Cre transgene. In Ndst1-/-;
Le-Cre pups at P0, again no GFP-positive lacrimal gland was observed
(Fig. 2L, arrow). These results
demonstrate that the heparan sulfate Ndst1 gene is essential for
lacrimal gland development.
Ndst1 is required in lacrimal gland bud epithelium
We next investigated the tissue specific requirement of Ndst1
using a floxed allele of Ndst1
(Grobe et al., 2005;
Wang et al., 2005). For the
mesenchymal-specific knockout of Ndst1, we employed the
Wnt1-Cre transgene, which is specifically activated in the
neural-crest derived mesenchymal cells
(Danielian et al., 1998). This
complemented the Le-Cre transgene mediated Ndst1 knockout in
the lacrimal epithelial cells. In wild-type E14.5 embryos, both the distal
lacrimal gland bud and its surrounding mesenchyme stained for 10E4 antibody
(Fig. 3A). By contrast, 10E4
staining was restricted to the lacrimal gland epithelial cells in the
Ndst1flox/flox; Wnt1-Cre embryos and mesenchymal
cells in the Ndst1flox/flox; Le-Cre embryos,
respectively (Fig. 3B, arrow;
Fig. 3C, arrowhead). In the
Ndst1flox/flox; Wnt1-Cre; Le-Cre and the
Ndst1-/- embryos, 10E4 staining was completely abolished
in the lacrimal gland region, whereas 3G10 staining remained unchanged
(Fig. 3D, arrow; data not
shown). Therefore, complementary abrogations of heparan sulfate sulfation were
achieved with Wnt1-Cre or Le-Cre-mediated Ndst1
knockouts.
To analyze the phenotypes of these tissue-specific Ndst1 deletion
mutants, we next performed carmine staining. At birth, well-formed lacrimal
glands were detected in both control and in the
Ndst1flox/flox; Wnt1-Cre mice without any overt
ocular phenotype (Fig. 3E,F,
arrowheads). By contrast, the Ndst1flox/flox;
Le-Cre pups were frequently micropthalmic without apparent lacrimal
glands (Fig. 3G, arrowhead). To
further characterize the phenotype, we also visualized the lacrimal gland by
GFP expression from the Le-Cre transgene. In contrast to the
extensively branched lacrimal glands in the control Le-Cre embryos,
50% of Ndst1flox/flox; Le-Cre newborn mice showed
much reduced lacrimal glands (Fig.
3H,I, arrows, n=24). In another 46% of the
Ndst1flox/flox; Le-Cre animals, no lacrimal gland
was observed, even though GFP expression was still detectable in the cornea
(Fig. 3J, arrow,
n=22). We suspected that the incompletely penetrant lacrimal gland
defects in Ndst1flox/flox; Le-Cre mice might be
due to genetic redundancy of the Ndst2 gene
(Holmborn et al., 2004).
Indeed, by crossing the Ndst1flox/flox; Le-Cre
with Ndst2 animals, we showed that the double mutants,
Ndst1flox/flox; Ndst2-/-; Le-Cre,
completely abolished lacrimal gland development
(Fig. 3K, arrow,
n=18). Taken together, our results demonstrate that lacrimal gland
development depends on Ndst-mediated heparan sulfate modification in
the epithelial cells.
To determine the specific defect of lacrimal gland development in Ndst1 mutant embryos, we next examined the lacrimal gland budding during embryonic development. At E14.5, a small lacrimal gland bud identified by its GFP fluorescence had grown into the surrounding periocular mesenchyme in the Le-Cre transgenic embryo, but no such outgrowth was observed in the Ndst1flox/flox; Le-Cre embryos under a fluorescent dissecting microscope (Fig. 4A,B, arrow and arrowhead). Fgf10-Fgfr2b and Bmp7 signaling are known to be important for lacrimal gland development, but the expression of Fgf10 and Bmp7 in the periocular mesenchyme, as well as of Fgfr2b in the conjunctival epithelium remained unchanged (Fig. 4C-F, arrows, and data not shown). Similarly, the 3G10 staining of total heparan sulfate in the conjunctival epithelium, which comprises the precursors of the lacrimal gland bud, was indistinguishable between the E12.5 control and Ndst1flox/flox; Le-Cre embryos (Fig. 4G,H, arrows). In the fornix, or the deepest rim of the conjunctival epithelium, however, the Ndst1 mutant cells lacked 10E4 staining in both basal and lateral membranes (Fig. 4I,J, arrows, epithelium outlined with broken lines), although the epithelial cells further away from the eye retained 10E4 staining (Fig. 4I,J, arrowheads). Consistent with this early molecular defect, histological analysis of E14.5 Ndst1 mutant embryos showed either no budding or only very limited outgrowth of the lacrimal gland, probably reflecting the variable severities of the Ndst1 mutant phenotype (Fig. 4L, Fig. 5D-F). The presumptive lacrimal gland budding sites in the E14.5 Ndst1 mutants were also defective in cell proliferation as shown by the lack of BrdU staining, although no abnormal cell apoptosis was observed by TUNEL staining (Fig. 4L, arrow, and data not shown). By contrast, extensive BrdU incorporation was observed in the nuclei of control lacrimal glands (Fig. 4K, arrow). Therefore, the epithelial specific knockout of Ndst1 disrupted heparan sulfate modification and cell proliferation at the lacrimal gland primordium.
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;
Pan et al., 2006). This
Fgfr2b-binding pattern was completely lost in the absence of Fgf10 or on
sections treated with Heparitinase I enzyme, demonstrating an obligatory
complex of Fgf10, Fgfr2b and heparan sulfate in situ (data not shown). As
expected, the immunofluorescence pattern closely resembled the heparan sulfate
10E4 staining pattern, with strongest staining in the cells at the tip of the
lacrimal gland bud (Fig. 5A,
arrow). In the Ndst1flox/flox; Le-Cre embryos,
however, the Fgf10/Fgfr2b staining was abolished in the epithelial cells of
lacrimal gland but not in the surrounding mesenchymal cells
(Fig. 5D, arrow). Similar
bud-specific and Ndst1-dependent staining was also observed for the
Fgf7/Fgfr2b combination, but no binding signal was observed for Fgf10/Fgfr2c,
Fgf7/Fgfr2c or Fgf2/Fgfr2b (Fig.
5B,E, arrow and data not shown). Finally, we have previously shown
that Fgf1/Fgfr2b binding is insensitive to the Ndst1 mutation during
early lens induction. Indeed, we also detected in the lacrimal gland
epithelial and mesenchymal cells almost ubiquitous Fgf1/Fgfr2b binding, which
was unaffected by the Ndst1 mutation
(Fig, 5C,F, arrow). Therefore,
the Ndst1-dependent heparan sulfate modification specifically
regulates Fgf10/Fgfr2b and Fgf7/Fgfr2b binding at the tip of the lacrimal
gland bud.
|
). Finally, we further reduced the extent of heparan sulfate
sulfation by compounding Ndst1 and Ndst2 mutations. In the
Ndst1flox/flox;Ndst2-/-; Le-Cre double
mutants, both endogenous and ectopic lacrimal glands were completely abolished
in the explant culture experiments, demonstrating the essential role of Ndst
genes in Fgf10-induced lacrimal gland budding (P<0.001 for
endogenous budding, P<0.02 for ectopic budding, Fisher's exact
test). Therefore, Fgf10 depends on Ndst function to promote lacrimal
gland development.
Fgfr2 inactivation abolishes lacrimal gland budding
We next sought to provide genetic evidence that Fgfr2, the strong binding
partner of Fgf10 in LACE assay, is also required for lacrimal gland budding.
The Le-Cre transgene was crossed with an
Fgfr2flox allele, which carries two loxP sites
flanking exons 7-10 of Fgfr2 gene
(Yu et al., 2003). As
expected, although the Le-Cre control pups at birth clearly formed
the lacrimal gland as marked by abundant GFP expression, none of the
Fgfr2flox/flox; Le-Cre animals developed lacrimal
glands (Fig. 6A,B). The mutant
phenotype can be traced to lacrimal gland induction, as budding of the
lacrimal gland was observed in Le-Cre controls but not in the
Fgfr2flox/flox; Le-Cre embryos
(Fig. 6C,D). By contrast, no
lacrimal gland defect was observed in the mesenchymal specific Fgfr2
knockout, Fgfr2flox/flox; Wnt1-Cre
(Fig. 6E,F). These results
demonstrate the essential role of Fgfr2 in lacrimal gland epithelium
cells for budding morphogenesis.
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). We thus
examined the activity of Shp2 protein using a phospho-specific antibody. Not
surprisingly, phospho-Shp2 expression was detected in the control lacrimal
gland bud, but not in the Fgfr2 mutant conjunctival epithelium, while
the retinal phospho-Shp2 remained unaffected
(Fig. 6K,L). This suggested
that Fgfr2 inactivation may result in downregulation of MAPK
signaling during lacrimal gland induction.
Ndst1-Fgfr2-Shp2 cascade is required for ERK signaling in lacrimal gland bud
To further explore the FGF downstream signaling, we deleted Shp2 in the
lacrimal gland by crossing the Le-Cre transgene with a
Shp2flox allele that, upon Cre mediated recombination,
creates a Shp2-null allele (Zhang
et al., 2004). Elimination of Shp2 was demonstrated by
immunohistochemistry using an antibody specific to the C-terminal domain of
Shp2. Compared with the ubiquitous expression in control embryos, Shp2 in the
Shp2flox/flox; Le-Cre embryos was specifically
lost in the conjunctival epithelium, as expected with the epithelial-specific
deletion of Shp2 (Fig.
7A,B, broken lines). Similar to the Fgfr2 and
Ndst1 knockouts, the Shp2 mutants never exhibited much cell
proliferation in the fornix of the conjunctival epithelium, as shown by the
BrdU incorporation assay or abnormal cell apoptosis as revealed by TUNEL assay
results (Fig. 7C,D; data not
shown). As a consequence, there was no detectable lacrimal gland budding at
E14.5 or any lacrimal gland structure at birth
(Fig. 7F,H versus control
Fig. 7E,G). Importantly, the
10E4 staining of heparan sulfate was disrupted in the fornix of the
conjunctival epithelium in the Shp2 mutants
(Fig. 7I,J, arrows). Thus, Shp2
function is also essential for heparan sulfate modification during lacrimal
gland development.
We next assayed MAPK signaling activities in Ndst1, Fgfr2 and Shp2 mutants. As a downstream effector of MAPK signaling, phospho-ERK was expressed in the control E12.5 conjunctival fornix, which contained the thickening precursor cells of the lacrimal gland (Fig. 7K, arrow). In all three mutants, no induction of phospho-ERK expression was detected in the same region (Fig. 7L-N, arrowheads). In the control E14.5 lacrimal gland, the strongest phospho-ERK expression was at the very tip of the bud, which correlated with the cell proliferation pattern shown in the BrdU assay as well as heparan sulfate pattern by 10E4 staining (Fig. 7O, arrow). By contrast, the Le-Cre-mediated conditional knockout of Ndst1, Fgfr2 and Shp2 abolished phospho-ERK expression at the conjunctival epithelium, even though retinal expression of phospho-ERK remained unaffected (Fig. 7P-R). The PEA3 family transcription factors, including Pea3 and Erm are well characterized FGF signaling downstream genes. By RNA in situ hybridization, we also observed a complete loss of Pea3 and Erm expressions in the tip of lacrimal gland primordium in all three mutants (Fig. 7S-V, and data not shown). Taken together, our results support that Ndst1, Fgfr2 and Shp2 potentiate FGF-ERK signaling in lacrimal gland development.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Richard et al.,
1995; Sherman et al.,
1998). By genetic manipulation, however, we have recently shown
that heparan sulfate probably functions cell-autonomously in tumor
vasculogenesis (Fuster et al.,
2007). In this study, tissue-specific knockout experiments further
show that both heparan sulfate and Fgfr2 are required in the lacrimal
gland epithelium, and that mesenchymal knockouts of Ndst1 and
Fgfr2 do not perturb lacrimal gland development. Therefore, at least
for lacrimal gland FGF signaling, heparan sulfate appears to function in cis
with Fgfr2 in the lacrimal gland epithelium. We have also provided evidence that the heparan sulfates in the lacrimal gland epithelium are differentially modified. Despite the ubiquitous heparan sulfate expression throughout the length of the lacrimal gland, only the distal tip of the lacrimal gland bud contains uniquely sulfated heparan sulfate as identified by the 10E4 antibody. Notably, this heparan sulfate modification pattern correlates exactly with the in situ binding of Fgf10/Fgfr2b and Fgf7/Fgfr2b at the lacrimal gland bud, shown by LACE assay, and localized activation of FGF signaling, as shown by phospho-ERK staining. By contrast, Fgf1 and Fgf2 exhibit either ubiquitous binding or very weak binding respectively, with Fgfr2b in the developing lacrimal gland. Finally, genetic ablation of Ndst1 and Ndst2 genes disrupted endogenous lacrimal gland development in vivo and Fgf10 induced ectopic lacrimal gland budding in explant culture, demonstrating the essential role of heparan sulfate N-sulfation in Fgf10/Fgfr2b signaling. By its developmentally dynamic expression pattern, heparan sulfate could thus not only potentiate but also restrict FGF signaling within the lacrimal gland tip, providing a mechanism to promote directional outgrowth of the lacrimal gland bud.
How then is the tissue specific heparan sulfate modification generated? It
is known that heparan sulfate biosynthetic genes are differentially expressed
during development, and at least in the case of Ndst genes,
post-transcriptional regulation also plays a role in their expression
(Grobe and Esko, 2002).
However, it remains to be determined what upstream signaling regulates these
heparan sulfate modification genes. In this study, our data show that heparan
sulfate modification itself depends on functional Fgfr2 and
Shp2 in lacrimal gland primordium, suggesting reciprocal regulation
between heparan sulfate and FGF signaling. This feedback model is attractive
because it may help to stabilize the Fgf10 responsive zone to the tip of
lacrimal bud with specific heparan sulfate modification.
In summary, we have provided genetic evidence that specifically modified heparan sulfate at the cell surface plays a functionally important role in lacrimal gland development by spatially regulating Fgf10/Fgfr2b signaling (Fig. 8). This leads to activation of intracellular ERK signaling mediated by Shp2 phosphatase and preferential cell proliferation at the lacrimal gland bud. As restrictive heparan sulfation modification is widely observed in development, the genetic cascade of Ndst-Fgfr2-Shp2 may thus be a general mechanism in the branching morphogenesis process.
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ACKNOWLEDGMENTS |
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|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Allen, B. L. and Rapraeger, A. C. (2003).
Spatial and temporal expression of heparan sulfate in mouse development
regulates FGF and FGF receptor assembly. J. Cell Biol.
163,637
-648.
Ashery-Padan, R., Marquardt, T., Zhou, X. and Gruss, P.
(2000). Pax6 activity in the lens primordium is required for lens
formation and for correct placement of a single retina in the eye.
Genes Dev. 14,2701
-2711.
Dakubo, G. D., Wang, Y. P., Mazerolle, C., Campsall, K.,
McMahon, A. P. and Wallace, V. A. (2003). Retinal ganglion
cell-derived sonic hedgehog signaling is required for optic disc and stalk
neuroepithelial cell development. Development
130,2967
-2980.
Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K. and McMahon, A. P. (1998). Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase. Curr. Biol. 8,1323 -1326.[CrossRef][Medline]
David, G., Bai, X. M., Van der Schueren, B., Cassiman, J. J. and
Van den Berghe, H. (1992). Developmental changes in heparan
sulfate expression: in situ detection with mAbs. J. Cell
Biol. 119,961
-975.
Entesarian, M., Matsson, H., Klar, J., Bergendal, B., Olson, L., Arakaki, R., Hayashi, Y., Ohuchi, H., Falahat, B., Bolstad, A. I. et al. (2005). Mutations in the gene encoding fibroblast growth factor 10 are associated with aplasia of lacrimal and salivary glands. Nat. Genet. 37,125 -127.[CrossRef][Medline]
Esko, J. D. and Selleck, S. B. (2002). Order out of chaos: assembly of ligand binding sites in heparan sulfate. Annu. Rev. Biochem. 71,435 -471.[CrossRef][Medline]
Forsberg, E., Pejler, G., Ringvall, M., Lunderius, C., Tomasini-Johansson, B., Kusche-Gullberg, M., Eriksson, I., Ledin, J., Hellman, L. and Kjellen, L. (1999). Abnormal mast cells in mice deficient in a heparin-synthesizing enzyme. Nature 400,773 -776.[CrossRef][Medline]
Fuster, M. M., Wang, L., Castagnola, J., Sikora, L., Reddi, K.,
Lee, P. H., Radek, K. A., Schuksz, M., Bishop, J. R., Gallo, R. L. et al.
(2007). Genetic alteration of endothelial heparan sulfate
selectively inhibits tumor angiogenesis. J. Cell Biol.
177,539
-549.
Gotoh, N., Ito, M., Yamamoto, S., Yoshino, I., Song, N., Wang,
Y., Lax, I., Schlessinger, J., Shibuya, M. and Lang, R. A.
(2004). Tyrosine phosphorylation sites on FRS2alpha responsible
for Shp2 recruitment are critical for induction of lens and retina.
Proc. Natl. Acad. Sci. USA
101,17144
-17149.
Govindarajan, V., Ito, M., Makarenkova, H. P., Lang, R. A. and Overbeek, P. A. (2000). Endogenous and ectopic gland induction by FGF-10. Dev. Biol. 225,188 -200.[CrossRef][Medline]
Grobe, K. and Esko, J. D. (2002). Regulated
translation of heparan sulfate N-acetylglucosamine
N-deacetylase/n-sulfotransferase isozymes by structured 5'-untranslated
regions and internal ribosome entry sites. J. Biol.
Chem. 277,30699
-30706.
Grobe, K., Inatani, M., Pallerla, S. R., Castagnola, J.,
Yamaguchi, Y. and Esko, J. D. (2005). Cerebral hypoplasia and
craniofacial defects in mice lacking heparan sulfate Ndst1 gene function.
Development 132,3777
-3786.
Habuchi, H., Nagai, N., Sugaya, N., Atsumi, F., Stevens, R. L.
and Kimata, K. (2007). Mice deficient in heparan sulfate
6-o-sulfotransferase-1 exhibit defective heparan sulfate biosynthesis,
abnormal placentation, and late embryonic lethality. J. Biol.
Chem. 282,15578
-15588.
Hadari, Y. R., Kouhara, H., Lax, I. and Schlessinger, J.
(1998). Binding of Shp2 tyrosine phosphatase to FRS2 is essential
for fibroblast growth factor-induced PC12 cell differentiation.
Mol. Cell. Biol. 18,3966
-3973.
Holmborn, K., Ledin, J., Smeds, E., Eriksson, I.,
Kusche-Gullberg, M. and Kjellen, L. (2004). Heparan sulfate
synthesized by mouse embryonic stem cells deficient in NDST1 and NDST2 is
6-O-sulfated but contains no N-sulfate groups. J. Biol.
Chem. 279,42355
-42358.
Izvolsky, K. I., Shoykhet, D., Yang, Y., Yu, Q., Nugent, M. A. and Cardoso, W. V. (2003). Heparan sulfate-FGF10 interactions during lung morphogenesis. Dev. Biol. 258,185 -200.[CrossRef][Medline]
Jakobsson, L., Kreuger, J., Holmborn, K., Lundin, L., Eriksson, I., Kjellen, L. and Claesson-Welsh, L. (2006). Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis. Dev. Cell 10,625 -634.[CrossRef][Medline]
Johnston, M. C., Noden, D. M., Hazelton, R. D., Coulombre, J. L. and Coulombre, A. J. (1979). Origins of avian ocular and periocular tissues. Exp. Eye Res. 29, 27-43.[CrossRef][Medline]
Kamimura, K., Koyama, T., Habuchi, H., Ueda, R., Masu, M.,
Kimata, K. and Nakato, H. (2006). Specific and flexible roles
of heparan sulfate modifications in Drosophila FGF signaling. J.
Cell Biol. 174,773
-778.
Ke, Y., Lesperance, J., Zhang, E. E., Bard-Chapeau, E. A.,
Oshima, R. G., Muller, W. J. and Feng, G. S. (2006).
Conditional deletion of Shp2 in the mammary gland leads to impaired
lobulo-alveolar outgrowth and attenuated Stat5 activation. J. Biol.
Chem. 281,34374
-34380.
Lai, L. A., Zhao, C., Zhang, E. E. and Feng, G. S. (2004). The Shp-2 tyrosine phosphatase. In Protein Phosphatases. Vol. 5 (ed. J. Arino and D. Alexander), pp. 275-299. Berlin, Heidelberg: Springer-Verlag.
Ledin, J., Staatz, W., Li, J. P., Gotte, M., Selleck, S.,
Kjellen, L. and Spillmann, D. (2004). Heparan sulfate
structure in mice with genetically modified heparan sulfate production.
J. Biol. Chem. 279,42732
-42741.
Leteux, C., Chai, W., Nagai, K., Herbert, C. G., Lawson, A. M.
and Feizi, T. (2001). 10E4 antigen of Scrapie lesions
contains an unusual nonsulfated heparan motif. J. Biol.
Chem. 276,12539
-12545.
Lin, X., Buff, E. M., Perrimon, N. and Michelson, A. M. (1999). Heparan sulfate proteoglycans are essential for FGF receptor signaling during Drosophila embryonic development. Development 126,3715 -3723.[Abstract]
Lovicu, F. J., Kao, W. W. and Overbeek, P. A. (1999). Ectopic gland induction by lens-specific expression of keratinocyte growth factor (FGF-7) in transgenic mice. Mech. Dev. 88,43 -53.[CrossRef][Medline]
Maccarana, M., Sakura, Y., Tawada, A., Yoshida, K. and Lindahl,
U. (1996). Domain structure of heparan sulfates from bovine
organs. J. Biol. Chem.
271,17804
-17810.
Makarenkova, H. P., Ito, M., Govindarajan, V., Faber, S. C., Sun, L., McMahon, G., Overbeek, P. A. and Lang, R. A. (2000). FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development. Development 127,2563 -2572.[Abstract]
Neel, B. G., Gu, H. and Pao, L. (2003). The `Shp'ing news: SH2 domain-containing tyrosine phosphatases in cell signaling. Trends Biochem. Sci. 28,284 -293.[CrossRef][Medline]
Pallerla, S. R., Pan, Y., Zhang, X., Esko, J. D. and Grobe, K. (2007). Heparan sulfate Ndst1 gene function variably regulates multiple signaling pathways during mouse development. Dev. Dyn. 236,556 -563.[CrossRef][Medline]
Pan, Y., Woodbury, A., Esko, J. D., Grobe, K. and Zhang, X.
(2006). Heparan sulfate biosynthetic gene Ndst1 is required for
FGF signaling in early lens development. Development
133,4933
-4944.
Richard, C., Liuzzo, J. P. and Moscatelli, D.
(1995). Fibroblast growth factor-2 can mediate cell attachment by
linking receptors and heparan sulfate proteoglycans on neighboring cells.
J. Biol. Chem. 270,24188
-24196.
Rohmann, E., Brunner, H. G., Kayserili, H., Uyguner, O., Nurnberg, G., Lew, E. D., Dobbie, A., Eswarakumar, V. P., Uzumcu, A., Ulubil-Emeroglu, M. et al. (2006). Mutations in different components of FGF signaling in LADD syndrome. Nat. Genet. 38,414 -417.[CrossRef][Medline]
Sherman, L., Wainwright, D., Ponta, H. and Herrlich, P.
(1998). A splice variant of CD44 expressed in the apical
ectodermal ridge presents fibroblast growth factors to limb mesenchyme and is
required for limb outgrowth. Genes Dev.
12,1058
-1071.
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[CrossRef][Medline]
Tefft, D., De Langhe, S. P., Del Moral, P. M., Sala, F., Shi, W., Bellusci, S. and Warburton, D. (2005). A novel function for the protein tyrosine phosphatase Shp2 during lung branching morphogenesis. Dev. Biol. 282,422 -431.[CrossRef][Medline]
van den Born, J., Salmivirta, K., Henttinen, T., Ostman, N.,
Ishimaru, T., Miyaura, S., Yoshida, K. and Salmivirta, M.
(2005). Novel heparan sulfate structures revealed by monoclonal
antibodies. J. Biol. Chem.
280,20516
-2023.
van Kuppevelt, T. H., Dennissen, M. A., van Venrooij, W. J.,
Hoet, R. M. and Veerkamp, J. H. (1998). Generation and
application of type-specific anti-heparan sulfate antibodies using phage
display technology. Further evidence for heparan sulfate heterogeneity in the
kidney. J. Biol. Chem.
273,12960
-12966.
Wang, L., Fuster, M., Sriramarao, P. and Esko, J. D. (2005). Endothelial heparan sulfate deficiency impairs L-selectin- and chemokine-mediated neutrophil trafficking during inflammatory responses. Nat. Immunol. 6, 902-910.[CrossRef][Medline]
Yang, W., Klaman, L. D., Chen, B., Araki, T., Harada, H., Thomas, S. M., George, E. L. and Neel, B. G. (2006). An Shp2/SFK/Ras/Erk signaling pathway controls trophoblast stem cell survival. Dev. Cell 10,317 -327.[CrossRef][Medline]
Yu, K., Xu, J., Liu, Z., Sosic, D., Shao, J., Olson, E. N.,
Towler, D. A. and Ornitz, D. M. (2003). Conditional
inactivation of FGF receptor 2 reveals an essential role for FGF signaling in
the regulation of osteoblast function and bone growth.
Development 130,3063
-3074.
Zhang, E. E., Chapeau, E., Hagihara, K. and Feng, G. S.
(2004). Neuronal Shp2 tyrosine phosphatase controls energy
balance and metabolism. Proc. Natl. Acad. Sci. USA
101,16064
-16069.
Zhang, X., Friedman, A., Heaney, S., Purcell, P. and Maas, R.
L. (2002). Meis homeoproteins directly regulate Pax6 during
vertebrate lens morphogenesis. Genes Dev.
16,2097
-2107.
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