1 Matrix and Morphogenesis Unit, Craniofacial Developmental Biology and
Regeneration Branch, National Institute of Dental and Craniofacial Research,
National Institutes of Health, 30 Convent Drive, MSC 4370, Bethesda, MD
20892-4370, USA
2 Developmental Mechanisms Section, Craniofacial Developmental Biology and
Regeneration Branch, National Institute of Dental and Craniofacial Research,
National Institutes of Health, 30 Convent Drive, MSC 4370, Bethesda, MD
20892-4370, USA
* Author for correspondence (e-mail: mhoffman{at}mail.nih.gov)
Accepted 22 December 2004
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SUMMARY |
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Key words: Fibroblast growth factor receptors, FGF7, FGF10, Signaling, Salivary gland development, Organ culture
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Introduction |
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Branching morphogenesis of ex vivo SMGs involves both FGFR signaling
(Hoffman et al., 2002;
Jaskoll et al., 2002
;
Morita and Nogawa, 1999
) and
EGFR signaling (Kashimata and Gresik,
1997
; Kashimata et al.,
2000
). Previously, we identified a role for FGFR1 and
phosphatidylinositol 3-kinase (PI3K) in SMG branching morphogenesis
(Larsen et al., 2003
). Here,
we investigate the role of FGF7 and FGF10 signaling through FGFR2b. The known
ligands for FGFR2b are FGF1, FGF3, FGF7 and FGF10. FGF7 and FGF10 both bind
with high affinity to FGFR2b (Yeh et al.,
2003
), although FGF10 also binds FGFR1b
(Igarashi et al., 1998
).
Matrix metalloproteinases (MMPs) regulate branching morphogenesis in many
organ systems. During ureteric bud branching, growth factors and extracellular
matrix (ECM) components modulate MMP expression
(Pohl et al., 2000). In
mammary epithelium, interplay between MMPs, growth factors and morphogens is
necessary for branching, although the MMPs are not required for proliferation
(Simian et al., 2001
). In SMG
organ culture, exogenous collagenase decreased cleft formation, and TIMP1
increased it, by regulating collagen III fibril formation at the cleft site
(Hayakawa et al., 1992
;
Nakanishi et al., 1986
).
However, the role of MMPs in SMG morphogenesis may not only be to cleave the
collagen matrix, but also to release FGF-binding heparan sulfates from
proteoglycans, or to directly cleave FGFRs to regulate FGFR function
(Powers et al., 2000
). MMP2
cleaves the extracellular domain of FGFR1, resulting in a cleavage product
that can still bind FGFs and may regulate FGF signaling
(Levi et al., 1996
).
A fundamental but complex question is how FGFs interact with multiple FGFRs and result in distinct downstream signaling that coordinates proliferation, migration, differentiation and, ultimately, morphogenesis. Here, we investigate the role of FGFR2b and its ligands in serum-free ex vivo submandibular gland organ culture. We used salivary epithelium, separated from mesenchyme, to define distinct morphogenetic roles for FGF7 and FGF10. The downstream signaling and gene expression is ERK1/2-dependent and involves a regulatory network of FGFR1b/FGF1/MMP2 expression. FGF7 and FGF10 cause localized cell proliferation and they require both FGF1 and MMP activity to regulate epithelial budding and duct elongation during branching morphogenesis.
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Materials and methods |
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Mesenchyme-free epithelia were cultured as previously described
(Morita and Nogawa, 1999),
with some modifications. E13 glands were incubated in 1.6 U/ml of Dispase in
Hanks' balanced salt solution (Roche Molecular Biochemicals, Indianapolis, IN)
at 37°C for 20 minutes. Epithelia with up to five buds were separated from
the mesenchyme with fine forceps in Hanks' solution containing 10% BSA. Most
importantly, horse serum was omitted from the media. The epithelial rudiments
were placed on a Nuclepore filter, covered with 15 µl of laminin-1 (1
mg/ml; Trevigen, Gaithersburg, MD) or growth factor-reduced Matrigel, diluted
1:1 in medium (5 mg/ml; BD Biosciences, San Jose, CA), and the filter was
floated on top of 200 µl of medium as described above. FGFs were added
either alone or in combination to the media, and the glands were cultured for
up to 44 hours. FGF1 (1, 20, 100, 500 ng/ml), FGF2 (1,10, 100, 200 ng/ml),
FGF3 (1, 10, 100, 1000 ng/ml), FGF7 (50, 100, 500, 1000 ng/ml), or FGF10 (200,
500, 1000, 2000 ng/ml), alone or in combination, was added to the cultures
(all growth factors were purchased from R&D Systems, Minneapolis, MN).
Antisense experiments
Antisense and sense oligonucleotides were minimally phosphorothioated as
previously described (Uhlmann et al.,
2000). Residues that were phosphorothioate-modified are shown in
bold: KGFR sense (1372-1390),
3'-caggccaaccagtctgcct-5'; KGFR
antisense (1390-1372),
3'-aggcagactggttggcctg-5'
(Post et al., 1996
); Bek
antisense (first two residues modified for mouse),
3'-tggcagaactgtcaac-5'; Bek
sense, 3'-gttgacagttctgcca-5';
antisense to both KGFR/Bek,
5'-gctgaccatggtcac-3'; sense to both
KGFR/Bek, 5'gtgaccatggtcagc-3'
(Post et al., 1996
).
Oligonucleotides (2 µM) were added to the media and replaced after 44
hours. RT-PCR was used to determine the decrease in gene expression.
FITC-labeled oligonucleotide uptake was detected with confocal microscopy
(data not shown) [similar to Hoffman et al.
(Hoffman et al., 2002
)].
Addition of recombinant FGFRs and FGF antibodies
E12 or E13 SMGs were cultured for 48 hours as described above with
recombinant (r) FGFRs or neutralizing antibodies to FGFs. The human rFGFR1b
and rFGFR1c, and mouse rFGFR2b, rFGFR2c and rFGFR3c, chimeras were all added
alone (1-20 µg/ml) and in various combinations (R&D Systems,
Minneapolis, MN). Neutralizing antibodies to FGF1 (AF232), FGF2 (AF233), FGF7
(MAB251) and Mouse IgG (MAB002) (R&D Systems), an FGF10 antibody
previously used for neutralization of FGF10 activity (sc7375-L)
(Harada et al., 2002), and a
goat IgG control (sc2028-L; Santa Cruz Biotechnology) were added to the
culture medium at concentrations of 10, 25 and 50 µg/ml. Combinations of
either two or three anti-FGF antibodies (25 µg/ml each) were also tested.
Glands were cultured for 44 hours, and branching morphogenesis was quantitated
as described above.
Time-lapse microscopy
SMGs or epithelial rudiments in laminin-1 were prepared as described above
and incubated in an environmental chamber fitted on a Zeiss SV25 inverted
microscope. Images were acquired once every 20 minutes for 36 hours using
MetaMorph Software (Universal Imaging, Dowingtown, PA) and assembled into
movies.
Whole-mount immunofluorescence
Proliferation and apoptosis were detected as previously described
(Hoffman et al., 2002), using
a BrdU Labeling and Detection Kit and an In Situ Cell Death Detection Kit,
TMR-red (Roche Molecular Biochemicals, Indianapolis, IN), as described in the
manufacturer's instructions. Nuclei were stained with SYBR-green, and for
immunostaining, mouse-on-mouse reagents were used (M.O.M. reagents; Dako,
Carpenteria, CA). With the apoptosis kit, the epithelium was stained with
FITC-peanut lectin (Vector Laboratories, Burlingame, CA). The BrdU and
apoptosis staining were quantitated using MetaMorph Software. The fluorescent
pixels from all optical sections were measured and expressed as a ratio of the
total pixel area of the gland or BrdU:SYBR-green. Five glands/condition were
used, and the experiments were repeated three times.
Epithelial glands or rudiments cultured in laminin were also fixed on the filters in 4% paraformaldehyde (PFA) for 1 hour, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked overnight with 10% donkey serum, M.O.M. blocking reagent, and 1% BSA. Primary antibodies were added in M.O.M. protein reagent for 3 hours at room temperature and secondary reagents were added in PBS-Tween 20 (0.1%) for 2 hours. Primary antibodies included Flg (sc-121), bek (sc-122) (Santa Cruz), E-cadherin Mab 36 (BD Biosciences, Pharmingen), MMP2-hinge and MMP9-hinge (Triple Point Diagnostics), alpha-6 integrin (GoH3, Chemicon), and perlecan (MAB1948, Chemicon). Secondary antibodies were all donkey F(ab)2 fragments labeled with Cy2, Cy3 and Cy5 (Jackson Immunoresearch Laboratories, West Grove, PA). All images were obtained with a Zeiss LSM 510 confocal microscope.
Rescue of rFGFR2b treatment with exogenous FGFs
E13 salivary gland rudiments were cultured with 1.6 µg/ml rFGFR2b, the
concentration required to give 50% inhibition of branching (data not
shown). All FGFs tested were added at the same time as the rFGFR2b at the
beginning of the experiment. A range of doses was tested, but only a single
dose is shown. FGF1 (20, 100, 500, 1000 ng/ml), FGF3 (100, 500, 1000 ng/ml),
FGF7 (50, 100, 500, 1000 ng/ml), FGF10 (100, 500, 1000, 2500 ng/ml), FGF2 (10,
100, 500, 1000 ng/ml), FGF4 (10, 100, 500, 1000 ng/ml) and BMP7 (10, 100, 500,
1000 ng/ml) were added to the cultures.
Inhibition of FGF-induced epithelial morphogenesis
Epithelial rudiments were dissected free of mesenchyme and cultured in
laminin-1 gels. The growth factors, inhibitors and rFGFRs were added at the
same time. DMSO or PBS with 0.1% BSA was used as a carrier control. Su5402 (1,
2.5, 5, 10 µM) was a gift from Dr F. Unda (Universidad del Pais Vasco,
Spain). LY294002 (5 and 10 µM), UO126 (5 and 10 µM) and GO6983 (1.5
µM) were all from Calbiochem (La Jolla, CA). The rudiments were cultured
for 44 hours and photographed, and the bud number and duct length were
measured using MetaMorph Software.
Gelatin zymography of epithelium-conditioned media
The media were concentrated in centrifugal filters (Millipore Biomax 10K
NMWL Membrane, 0.5 ml volume). Glands were lysed, and total protein
quantitated (BCA Assay, Pierce). The volume of media concentrate was
normalized to protein in the gland lysates and analyzed on a 10% gelatin
zymogram gel developed using Invitrogen Zymogram Buffers (Invitrogen,
Carlsbad, CA). HT1080-conditioned medium (Chemicon, Temecula, CA) was included
as a positive control.
Real-time PCR
At least eight epithelial rudiments were cultured in 15 µl of laminin-1
as described above. DNase-free RNA was prepared using an RNAqueous-4 PCR kit
and a DNA-free DNase removal reagent (Ambion, Austin, TX). TaqManTM reverse
transcription reagents (Applied Biosystems, Foster City, CA) were used to make
cDNA. Real-time PCR was performed using primers designed with similar
properties using Beacon Designer Software (Biorad, Hercules, CA), SYBR-green
PCR Master Mix (Applied Biosystems), and a Biorad MyIQ real-time PCR
thermocycler. Each cDNA (5-10 ng) was amplified with an initial denaturation
at 95°C for 10 minutes, then 95°C for 15 seconds and 68°C for 30
seconds, for 40 cycles. Gene expression was normalized to the housekeeping
gene S29. Melt-curve analysis was routinely run, and the PCR
reactions were also analyzed by gel electrophoresis to confirm that a single
product of the expected size was amplified. The reactions were run in
triplicate, repeated three times, and the results combined to generate the
graphs.
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Results |
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FGF7 and FGF10 rescue rFGFR2b-treatment, and neutralizing antibodies to FGF1, FGF7 and FGF10 are required to inhibit branching morphogenesis
Exogenous FGF7 and FGF10 specifically rescued SMGs inhibited with rFGFR2b
(Fig. 4A), whereas FGF1, FGF2,
FGF3 and BMP7 did not. Soluble rFGFR2b binds to multiple FGFs, therefore we
added neutralizing FGF antibodies alone and in combinations to identify the
FGFs produced by the endogenous mesenchyme that were required for branching
(Fig. 4B). None of the
antibodies tested alone could inhibit branching; however, combinations of
neutralizing antibodies to FGF1, FGF7 and FGF10 did significantly inhibit
branching. These data suggest that multiple FGFs are involved, and that FGF1,
FGF7 and FGF10 are important during ex vivo branching morphogenesis.
|
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FGF-induced morphogenesis is dependent on localized epithelial cell proliferation
Proliferation was measured at 8 hours, prior to morphogenesis, and the
ratio of BrdU:SYBR-green was quantitated
(Fig. 6A). The FGFs all
increased proliferation, BMP4 decreased proliferation, and proliferation in
BMP7-treated samples was similar to control
(Fig. 6A,B). Therefore,
multiple FGFs can stimulate epithelial proliferation, which is independent of
later morphogenesis. However, by 44 hours
(Fig. 6B) there were distinct
patterns of epithelial cell proliferation with different FGFs. Proliferation
was concentrated at the tips of the ducts with FGF10 treatment, and occurred
throughout the buds and along the ducts with FGF7 treatment. The data suggest
that downstream gene expression may regulate factors that specify where
localized proliferation occurs. Either the localization of the FGFRs or the
expression of downstream mediators of morphogenesis could specify the sites of
proliferation.
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We analyzed MMP activity in the conditioned media by gelatin zymography, after 24 and 44 hours of FGF treatment (Fig. 9C). FGF7 increased both the pro and active forms of MMP2 when compared with FGF10. FGF10 increased MMP9 levels when compared with FGF7, although MMP9 is barely detectable by zymograms, as a few remaining mesenchyme cells produced it. Therefore, the changes in MMP gene expression are consistent with the changes in MMP activity. Epithelial morphogenesis is dependent on MMP activity, since GM6001, a broad MMP inhibitor, inhibited the enlargement of the epithelial buds and elongation of the ducts (Fig. 9D). In addition, neutralizing antibodies to FGF1 were required to inhibit morphogenesis in the intact SMG, so we added them to FGF7- and FGF10-treated epithelium. Anti-FGF1 inhibited growth, suggesting the epithelial production of FGF1 was required for FGF7- and FGF10-mediated morphogenesis. These data also support the hypothesis that FGF1 signaling (which may be through FGFR1b) and FGFR2b are required for branching morphogenesis. Taken together, our data suggest that MMP and FGF1 are mediators of morphogenesis, and are required for both FGF7- and FGF10-dependent morphogenesis.
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Discussion |
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Multiple FGFs and FGFRs are present in SMGs
(Hoffman et al., 2002), and
defining which FGFs are important in SMG development is hampered by the fact
that many FGFs have overlapping functions. FGF10-knockout mice have no
salivary glands (Ohuchi et al.,
2000
), suggesting that gland initiation is dependent on FGF10, but
our ex vivo data suggest that, once the gland has formed, inhibition of FGF10
with neutralizing antibodies or decreasing FGF10 expression by
antisense treatment (M.P.H., unpublished) does not inhibit branching. The lack
of a SMG phenotype in single FGF-null mice, such as the FGF2- and FGF7-null
mice (Sun et al., 2002
;
Zhou et al., 1998
), could be
interpreted as meaning they are not important for SMG development; however,
overlapping FGF function is likely to compensate during development. Our
experiments requiring combinations of neutralizing FGF1, FGF7 and FGF10
antibodies to inhibit branching when the endogenous mesenchyme is present
support this hypothesis.
Previous reports show that FGF7 induces SMG epithelial proliferation but
that epithelial budding also requires EGFR ligation
(Morita and Nogawa, 1999).
However, media with 10% horse serum was used in these experiments, which
contains mitogenic growth factors and makes comparison difficult with our
serum-free conditions. The EGFR-null mouse
(Jaskoll and Melnick, 1999
)
has macroscopically normal glands with a reduction of buds/area, suggesting
that EGFR regulates SMG branch number in vivo but is not essential for SMG
initiation or early branching. An exogenous EGFR ligand is not required for
epithelial branching; however, the ECM may contain EGFR ligands or the
epithelium may produce an endogenous EGFR ligand. MMP degradation of laminin-5
is reported to release an EGF-like fragment during mammary involution (Schenk
et al., 2003).
We have identified striking morphological differences between FGF7- and
FGF10-treated SMG epithelium (Fig.
5). The function of FGFs in SMG epithelium is different from other
branching organs. In the lung, FGF10 plays an important role during
morphogenesis and induces primary and secondary bud formation, FGF1 induces
robust growth with elongated buds, and FGF7 induces a cyst-like structure
(Bellusci et al., 1997;
Cardoso et al., 1997
;
Izvolsky et al., 2003a
;
Weaver et al., 2000
). During
uteric bud outgrowth, FGF1 and FGF7 are at opposite ends of a morphogenic
spectrum, with FGF10 and FGF2 in between
(Qiao et al., 2001
). FGF1
induces elongated uteric buds with proliferating tips, whereas FGF7 induces
proliferation with an amorphous morphology.
FGF10 is involved in Harderian and lacrimal gland development, where it
induces epithelial proliferation but not branching morphogenesis
(Govindarajan et al., 2000;
Makarenkova et al., 2000
).
FGF10 is important during tooth development
(Kettunen et al., 2000
), where
it is a survival factor for stem cell populations
(Harada et al., 2002
). In SMG
epithelium, FGF10 promotes duct elongation by localized proliferation at the
tip of the duct through ERK1/2-dependent pathways. Nuclear localization of
FGFRs has been reported (Wells and Marti,
2002
), and FGFR2 was recently found in the nucleus during
FGF9-induced proliferation (Schmahl et
al., 2004
). FGFR1b and FGFR2b are also localized throughout the
epithelium; therefore, a co-receptor or cofactor is likely to be required to
allow FGF10 to specifically stimulate just the tip cells to proliferate. The
most likely candidates are heparan sulfate-containing cofactors or receptors,
on the cell surface or in the ECM. The mitogenic activity of FGF10 is
stimulated by heparin, whereas FGF7 is inhibited
(Igarashi et al., 1998
),
suggesting the effects of FGF7 and FGF10 may be regulated by heparan sulfate.
In the lung bud, the localization of heparan sulfate isoforms regulates FGF
function at a particular stage or localization during development
(Izvolsky et al., 2003a
;
Izvolsky et al., 2003b
),
although other transmembrane regulators of FGF signaling such as sef
and XFLRT3 could be involved
(Tsang and Dawid, 2004
). This
hypothesis remains to be tested in the context of salivary gland
morphogenesis.
Both FGF7 and FGF10 signal through FGFR2b in an ERK1/2-dependent manner,
but FGF7-mediated morphogenesis is also PI3K-dependant. In corneal epithelial
cells, FGF7 also stimulates PI3K activity but is not inhibited by MAPK
inhibitors (Chandrasekher et al.,
2001). In our studies, FGF7 also increased FGFR1b and
FGF1 expression compared with FGF10. The PI3K-dependent pathway
(Fig. 8) and FGF1 activity
(Fig. 9D) are both necessary
for epithelial budding. PI3K may be downstream from either FGF10/FGFR1b, or
from FGF1 binding either FGFR1b or FGFR2b. Therefore the downstream signaling
from FGF7 may act in concert with FGF1 signaling to increase widespread
proliferation resulting in epithelial budding. FGF-mediated increases in FGF
gene expression have been identified in other systems, such as the lung, where
FGF7 also increases expression of FGF1
(Lebeche et al., 1999
). Many
studies on FGFR signaling focus on the roles of FGF1 and FGF2 in either
migration or proliferation, and suggest that cells use Src and p38MAPK for
migration and ERKs for proliferation
(Boilly et al., 2000
). Our data
show that ERKs are necessary for proliferation; however, the role of cell
migration is clearly important in SMG morphogenesis (see Movies 1 and 2 in
supplementary material), and identifying the mechanisms of cell migration will
be important for understanding gland
development.
|
In conclusion, branching morphogenesis of mouse submandibular glands ex vivo is regulated by multiple FGFs and FGFRs. Downstream of FGF7 or FGF10 binding to FGFR2b there is a regulatory network of FGFR1b/FGF1/MMP2 expression that influences morphogenesis. Localized cell proliferation results in either epithelial budding or duct elongation. Increased MMP2 activity may increase ECM remodeling and FGFR cleavage, allowing localized expansion of the epithelium into the ECM at sites where proliferation occurs. The increased expression of FGFR1 and FGF1 with FGF7 could reinforce epithelial proliferation in an autocrine manner, resulting in widespread proliferation and budding. On the contrary, FGF10 treatment results in elongation of the duct behind the proliferating tip, where co-receptors or cofactors define the site of FGF10/FGFR2b signaling. The difference between FGF7- and FGF10-mediated morphogenesis is likely to be due to the factors that localize FGF binding to specific cells expressing FGFRs, resulting in different amounts of FGFR2b signaling, proliferation, and downstream expression of autocrine FGF signaling loops and proteases. Therefore, identifying the co-receptors or cofactors that regulate localized FGF binding and FGFR activation in the developing submandibular gland is an important future goal.
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Supplementary material |
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ACKNOWLEDGMENTS |
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