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 27 September 2002
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SUMMARY |
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Key words: Microarray, Fibroblast growth factor receptors, FGF3, FGF7, FGF10, BMP4, BMP7, Submandibular gland, Organ culture, SU5402, Mouse
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INTRODUCTION |
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The initiation of mouse SMG development occurs at embryonic day 11 (E11),
and by E12, a single epithelial bud forms within a condensed mesenchyme. By
E13, clefts form in the epithelial bud, which continues to proliferate and
cleft in successive rounds of branching, giving rise to multiple cords and
buds by E14 (Jaskoll et al.,
2001) (Fig. 1). By
E17, morpho-differentiation and lumenization of ducts and terminal buds has
occurred. Functional differentiation of the gland begins just before birth and
continues on day 1 (D1) after birth when the gland starts to secrete saliva.
Most acinar and ductal cell differentiation occurs by D5. The
androgen-dependent differentiation of the granular convoluted tubules (GCT)
occurs at puberty (Gresik,
1975
). The developmental stages are not absolute, and there is a
range of developmental variation occurring within an individual gland and
between glands from animals in the same litter.
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Branching morphogenesis of SMGs in vitro involves EGF signaling
(Kashimata and Gresik, 1997;
Kashimata et al., 2000
;
Morita and Nogawa, 1999
),
integrin
6, laminin-1 (Hosokawa et
al., 1999
; Kadoya et al.,
1995
), the TNF/TNFR1/IL6 pathways
(Melnick et al., 2001b
;
Melnick et al., 2001c
), and
FGF7 (Morita and Nogawa,
1999
). Fibroblast growth factors (FGFs) and their receptors
(FGFRs) are important in many developmental events, including branching
morphogenesis in other organs (Martin,
1998
; Metzger and Krasnow,
1999
; Ornitz,
2000
; Spencer-Dene et al.,
2001
). FGFs are a family of intercellular signaling molecules that
contains at least 22 members (Ornitz and
Itoh, 2001
). The role of BMPs as agonists or antagonists to FGFs
has been studied in other developmental systems including tooth formation
(Neubuser et al., 1997
), lung
organ culture (Weaver et al.,
2000
; Weaver et al.,
1999
), limb growth (Niswander
and Martin, 1993
) and kidney development
(Piscione et al., 2001
).
There are four known FGF receptors, and receptors 1, 2 and 3 each have two
isoforms due to alternate splicing in their extracellular domains
(Ornitz et al., 1996).
Recently, a fifth FGFR has been identified with two splice isoforms but no
intracellular kinase domain (Kim et al.,
2001
; Sleeman et al.,
2001
). The roles of FGFRs, particularly FGFR1, and FGFs in SMG
branching morphogenesis and differentiation are not well defined. Although
FGF7 promotes stalk elongation of SMG epithelial explants cultured in the
absence of mesenchyme (Morita and Nogawa,
1999
), branching morphogenesis in Fgf7 knockout mice
occurs normally (Guo et al.,
1996
). Abnormal salivary gland phenotypes were reported in mice
with a heterozygotic abrogation of Fgfr2c-exon 9(IIIc),
Bmp7-null and Pax6-null mice
(Jaskoll et al., 2002
).
Further, mice engineered to lack either the Fgfr2b isoform or
Fgf10 do not develop SMGs (De
Moerlooze et al., 2000
; Ohuchi
et al., 2000
; Sekine et al.,
1999
). These data show that FGFR2b and FGF10 are necessary for SMG
development, but do not provide information on the mechanisms of FGF and FGFR
action during branching morphogenesis and gland differentiation.
Complementary DNA (cDNA) arrays generate profiles of gene expression and
provide insight into the genetic regulation of complex biological systems.
Array analysis has been used to compare gene expression profiles in cells
(Rolli-Derkinderen and Gaestel,
2000), tissues (Lemkin et al.,
2000
), organisms (Galitski et
al., 1999
), disease states
(Selaru et al., 2002
) and
developmental stages (Hilsenbeck et al.,
1999
; White et al.,
1999
), as well as to predict disease prognosis
(van 't Veer et al., 2002
) and
to define the patterns of gene expression during kidney development and
maturation (Stuart et al.,
2001
). We used cDNA arrays to profile gene expression in
developing mouse SMGs to investigate the genetic mechanisms involved in gland
development. We found that Fgfr1 is expressed most highly early in
gland development. Using organ culture of embryonic submandibular glands, we
investigated the biological function of FGFR1 by decreasing Fgfr1
gene expression and by inhibiting FGFR1 signaling. Analysis of Fgfr,
Fgf and Bmp expression downstream of FGFR signaling suggests
that FGFR1 signaling regulates the expression of growth factors important for
early SMG branching morphogenesis. Our studies begin to define the FGF- and
BMP-dependent mechanisms involved in branching morphogenesis in SMG
development. FGFs and BMPs play reciprocal roles in regulating branching
morphogenesis and FGFR1 signaling plays a central role by regulating both FGF
and BMP expression.
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MATERIALS AND METHODS |
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RT-PCR
Array results were confirmed using RT-PCR. cDNA was generated using
Advantage RT for PCR Kit, and PCR was performed with either Advantaq Plus or
Titanium Taq PCR kits using Atlas Primers (all reagents from Clontech
Laboratories, Inc., Palo Alto, CA). PCR was performed with a Robo-Cycler
(Stratagene, La Jolla, CA). 20 ng of each cDNA was amplified with an initial
denaturation at 95°C for 3 minutes, then 95°C for 1 minute, and
68°C for 2.5 minutes for 18, 23, 28, and 33 cycles and a final elongation
step of 68°C for 5 minutes. Aliquots (5 µl) were removed after 18, 23,
28, 33, and 38 cycles and separated on 2% agarose gels. The band intensity was
measured with a Stratagene Eagle Eye II (Stratagene, La Jolla, CA). The
optimum number of PCR cycles for each primer pair was determined when linear
amplification of the product was still occurring as estimated by band
intensity. Therefore, the difference in the intensity of the PCR product
reflects the difference in the message level at each stage of gland
development. The amount of starting cDNA was adjusted so that the band
intensity of Gadp and Actb were equal. The PCR results
presented in Figs 3 and
4 were repeated at least twice
from a particular cDNA sample, and from at least 2 independent cDNA
preparations.
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SMG organ culture
SMGs dissected from either E12 or E13 ICR mice were cultured on Whatman
Nucleopore Track-etch filters (13 mm, 0.1 µm pore size, VWR, Buffalo Grove,
IL) at the air/medium interface. The filters were floated on 200 µl of
DMEM/F12 in 50 mm glass-bottom microwell dishes (MatTek, Ashland, MA). The
medium was supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin,
150 µg/ml vitamin C, and 50 µg/ml transferrin. Six SMGs were cultured on
each filter at 37°C in a humidified 5% CO2/95% air atmosphere.
Glands were photographed after approximately 2, 20, and 44 hours and the
number of end buds was counted at each timepoint. Each experiment was repeated
at least three times.
Antisense oligonucleotides and controls directed to FGFR1 were designed and manufactured by Biognostik, Gottingen, Germany. FITC-labeled oligonucleotide uptake into the mesenchyme and epithelial buds was detected with confocal microscopy analysis at 20 and 44 hours. The oligonucleotides (2 µM) were added at the beginning of each experiment, and the media and oligonucleotides (1 µM) were replaced after 44 hours of culture. RT-PCR was used to determine the decrease in gene expression.
SU5402 (Calbiochem, La Jolla, CA) was a gift from Dr F. Unda, Universidad
del Pais Vasco, Spain. SU5402, a FGFR1 tyrosine kinase inhibitor that does not
inhibit EGF, PDGF, or the insulin receptor
(Mohammadi et al., 1997), was
added to the culture media at the beginning of the experiment. Control glands
were cultured with an equal volume of the vehicle DMSO.
Detection of cell proliferation and apoptosis
After 20 hours of culture, the glands were incubated with 10 µM BrdU
(BrdU Labeling and Detection Kit 1, Roche Molecular Biochemicals,
Indianapolis, IN) for 90 minutes at 37°C. Then the medium was replaced
with PBS, and after 20 minutes at 37°C, the glands were fixed in 50 mM
glycine in 70% ethanol, pH 2.0, for 20 minutes at -20°C. The anti-BrdU
antibody was incubated for 2 hours at 37°C, and the secondary antibody was
incubated for 1 hour at 37°C. Immunofluorescence was examined using a
Zeiss LSM 510 microscope.
Apoptosis was detected by TUNEL staining using an In Situ Cell Death Detection Kit, TMR-red (Roche, Indianapolis, IN) as described in the manufacturer's instructions, except that glands were fixed for 60 minutes and permeabilized for 10 minutes. Apoptosis was also detected using the Vybrant Apoptosis Assay Kit (Molecular Probes, Eugene, OR) that uses YO-PRO-1 dye and propidium iodide to distinguish apoptotic and necrotic cells. Glands were also stained with peanut lectin conjugated to either FITC or Rhodamine (Vector Laboratories Inc., Burlingame, CA) to stain epithelial cells, and an anti-perlecan antibody (Chemicon, Temecula, CA) to stain the basement membrane and mesenchyme cells. The fluorescent BrdU and apoptosis staining were quantitated using the MetaMorph image analysis program (Universal Imaging Corp, Dowingtown, PA). The fluorescent pixels from all optical sections of each gland were measured and expressed as a ratio of the total pixel area of the gland. At least five glands per condition were used for quantitation and the experiments were repeated three times.
Localization of gene expression in E13 glands
E13 SMGs with sublingual glands removed were treated with 1.6 U/ml Dispase
1 (Roche, Indianapolis, IN) at 37°C for 20 minutes. The mesenchyme and
epithelium were separated with fine forceps and pools of RNA enriched in
mesenchyme or epithelium were prepared using the RNAqueousTM-4PCR kit
with DNase treatment (Ambion, Inc., Austin, TX). All purified RNA was checked
for DNA contamination by PCR with Gapd primers. RT-PCR was performed
as described above, using 10 ng of cDNA per reaction.
Real time PCR
For analysis of gene expression, at least six E13 SMGs were cultured with
either 5 µM SU5402 or an equal volume of DMSO for 2, 6 or 20 hours. RNA was
prepared as described above and TaqManTM reverse transcription reagents
(Applied Biosystems, Foster City, CA) were used to make cDNA. Real time PCR
was performed using Clontech primers and SYBR Green PCR Master Mix and a
TaqManTM 7700 thermocycler (both from Applied Biosystems, Foster City,
CA). The PCR conditions were the same as described above, and aliquots of the
PCR reactions were analyzed by gel electrophoresis to confirm that a single
product of the expected size was amplified. The reactions were run in
triplicate, the experiment repeated three times, and the results were combined
to generate the graphs.
Addition of exogenous growth factors and rescue of SU5402-mediated
inhibition of branching morphogenesis with FGFs
A range of concentrations of exogenous FGF1 (1-500 ng/ml), FGF2 (1-500
ng/ml), FGF7 (1-500 ng/ml), FGF10 (10-2500 ng/ml), BMP7 (1-1000 ng/ml), and
BMP4 (50-1000 ng/ml), were added to glands and cultured for 20 hours.
E13 gland rudiments were cultured with 1.5 µM SU5402, the concentration
required to give 50% inhibition of branching. The rudiments were
incubated with the inhibitor for 30 minutes and then FGF1 (1, 20, 100, or 200
ng/ml), FGF7 (50, 100, 200, 500, 1000 ng/ml), FGF10 (100, 500, 1000, 2500
ng/ml), or BMP7 (20, 50 100, 200, 500 ng/ml), alone or in combination, were
added to the cultures (all growth factors were purchased from R&D Systems,
Minneapolis, MN).
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RESULTS |
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Cluster Group 1 contains 91 genes that are expressed highly at early stages of gland development. We are interested in this group because they are highly expressed during branching morphogenesis before cell differentiation occurs. Group 2 contains 198 genes that are highly expressed at postnatal day 5. Some Group 2 genes have a biphasic profile with another peak in expression at E17. These may play a role in postnatal morphogenesis and secretory cell differentiation, and be involved in late embryonic gland development. Similar patterns of gene expression may occur during embryonic and postnatal gland morphogenesis. Group 3 contains 49 genes that are expressed highly after birth and increase in expression after D1. This group may be involved in postnatal development and secretory function of the gland. Group 4 contains 21 genes that are expressed highly at D1. These genes may be involved in the initiation of secretory cell function or be involved in ductal morphogenesis and differentiation. Group 5 contains 34 genes with increasing expression during development. Group 6 contains 21 genes that are highly expressed in adult glands. These genes may be involved in either the exocrine or endocrine secretory functions of the adult gland, although, the major known secreted salivary proteins are not on the array filter. Group 7 contains 27 genes with increased expression between postnatal day 1 and 5 and which levels off or decreases, in the adult gland. These genes may be involved in development and homeostasis of the adult gland. Group 8 contains 27 genes with a level profile of gene expression, and both Gapd and Actb fall within this group.
Confirmation of array results by RT-PCR
We used RT-PCR to confirm the array results for 13 genes including
Gapd and Actb as housekeeping genes
(Fig. 3). The array results are
presented as individual graphs of the expression profile
(Fig. 3A) together with the
RT-PCR result (Fig. 3B). In
general, the array results are confirmed by the RT-PCR. In our experience with
Atlas arrays, if a gene is barely detectable, but is reproducible, it will be
detected by RT-PCR. Since we are interested in Group 1 genes, with high
expression early in development, we also confirmed the expression profiles of
six other Group 1 genes (not included in
Fig. 2B), including Fgfr1,
Bmb7, Igf2, Igf24, Pref1 and necdin (Ndn)
(Fig. 3B). In addition, the
expression profiles of Bmp1, a gene most highly expressed on
postnatal D5, FZ6, a member of the Frizzled family of WNT receptors that
increases gradually during development, Ngfa and Wnt4, both
highly expressed in adult glands, and Tmsb4x (thymosin B4), a gene
whose expression gradually increases then levels off in the adult gland, were
all confirmed by RT-PCR (Fig.
3).
Fgfr and Fgf expression are developmentally
regulated during SMG development
We focused on the Fgfrs and Fgfs, which are important in
many developmental events, and extended the array results by including cDNAs
from E12 and E13 in our RT-PCR analysis. We also extended our RT-PCR analysis
to include the Fgfr1 and Fgfr2b and c splicing
isoforms, and Fgfr3, Fgfr4 (Fig.
4A), and additional FGFs (Fig.
4B) that were not present on the arrays. RT-PCR analysis of the
Fgfr isoforms reveals distinct developmental regulation of the
different receptors (Fig. 4A).
The highest expression occurs from E13-E14 with another increase in expression
at postnatal D5 for all isoforms except Fgfr3. At E13, the gland is
undergoing the first round of clefting and then through D1 it undergoes
multiple rounds of branching morphogenesis. At E12, when the gland is a single
epithelial bud, it begins to undergo cell proliferation, and Fgfr1b,
Fgfr2b, Fgfrc and Fgfr4 are all expressed. At D5, where
postnatal growth and secretory cell differentiation are occurring, there is
also increased expression of Fgfr1c, Fgfr2b and Fgfr4. In
the adult, the most detectable isoforms expressed are Fgfr1c and
Fgfr2b. Fgfr3 is barely detected by RT-PCR compared with the other
Fgfr isoforms and requires more PCR cycles.
The expression patterns of Fgfs reveal significant developmental regulation of the different isoforms. Analysis of Fgf1, 2, 3, 7, 8, 10 and 13 expression at different stages of SMG development is shown (Fig. 4B). Fgf1 is expressed at all developmental stages but is most highly expressed at postnatal D5 and adult. Fgf2 has a biphasic pattern of expression being most highly expressed at E13 and then again at postnatal D5. Fgf3 expression is present highest at E12, E13, and E14 with another peak at D5. Fgf7 and Fgf10 show increased expression at E13, when branching morphogenesis begins to occur. Fgf8 is expressed at E12, E13, and E14. Fgf13 is expressed highest at E13, but is expressed at a similar level in all other stages except adult.
Antisense oligonucleotides to Fgfr1 decrease branching
morphogenesis of E12 SMGs
We next focused on Fgfr1 and tested the functional importance of
this receptor in an organ culture assay. Antisense oligonucleotides to
Fgfr1 decrease branching morphogenesis of cultured E12 glands by
50% compared to an oligonucleotide control
(Fig. 5A,B). FITC-labeled
oligonucleotides were used to monitor antisense uptake after 20 (data not
shown) and 44 hours (Fig. 5C).
Confocal microscopy sections show uptake in both mesenchyme and epithelium,
with the mesenchyme showing greater uptake. The decrease in Fgfr1
gene expression with antisense treatment was measured by RT-PCR after 20
hours. The antisense oligonucleotides recognize a sequence in both
Fgfr1 splicing isoforms, however, RT-PCR analysis with
isoform-specific primers shows a
75% reduction in message level of
Fgfr1c (Fig. 5D) and
25% reduction in message level of Fgfr1b, as compared to control
glands. The greater decrease in Fgfr1c expression may be due to the
greater uptake of oligonucleotide by the mesenchyme. These data demonstrate
Fgfr1 expression levels are important during branching morphogenesis
of SMGs in vitro.
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Inhibition of FGFR1 signaling decreases SMG branching
morphogenesis
SU5402 inhibits FGFR1 signaling and decreases branching morphogenesis of
E12 (data not shown) and E13 SMGs in a dose-dependent manner
(Fig. 6A). After 20 hours of
treatment, the epithelial buds remain small and do not enlarge and cleft.
After 44 hours, the gland forms epithelial finger-like projections, although
the duct of the gland lengthens and undergoes lumen formation
(Fig. 6A, arrowhead). A range
of concentrations (2.5-25 µM) inhibits branching
(Fig. 6B), at 1 µM a partial
inhibition is seen, and at lower concentrations, 0.5, 0.1 and 0.02 µM, no
effect on branching is apparent. The effect of SU5402 is reversible, as the
glands resume branching if it is washed out after 20 hours (data not shown).
These data demonstrate that FGFR1 signaling is required for epithelial buds to
enlarge and undergo further clefting during SMG branching morphogenesis.
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SU5402 inhibits cell proliferation and does not cause epithelial
apoptosis
BrdU incorporation into SU5402-treated E13 glands reveals a decrease in
epithelial cell proliferation after 20 and 44 hours
(Fig. 7A-D,I). Epithelial cell
proliferation in the control gland is concentrated in the terminal buds
(Fig. 7B), with less labeling
detected in the mesenchyme. SU5402 treatment dramatically decreases epithelial
cell proliferation and has less effect on the mesenchyme. Total BrdU labeling
was quantitated at 20 and 44 hours (Fig.
7I). These data demonstrate that FGFR1 signaling affects
epithelial cell proliferation during SMG branching morphogenesis.
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Apoptosis was measured to determine if the decrease in branching with SU5402 treatment was mediated by an increase in apoptosis. Apoptosis of mesenchyme cells at the edges and on the surface of the glands in culture (Fig. 7E-H) was detected by both TUNEL staining (Fig. 7E-H) and the Vybrant apoptosis kit (data not shown). SU5402 treatment does not cause a significant increase in apoptosis at 20 or 44 hours, although there is a slight increase in apoptosis after 44 hours.
Fgfr, Fgf and Bmp gene expression was localized to
either epithelium or mesenchyme
E13 submandibular glands were micro-dissected into epithelium and
mesenchyme and analyzed by RT-PCR (Fig.
8A). The enzymatic separation is not absolute and the pools of RNA
are enriched for each cell type, however, our findings are in good agreement
with previously published data on Fgfr and Fgf expression.
At E13, Fgfr1b and 2b are expressed in the epithelium and
Fgfr1c, 2c, 3 and 4 are in the mesenchyme. Fgf2, 3,
7 and 10 are all expressed in the mesenchyme while Fgf1,
8 and 13 are expressed in both epithelium and mesenchyme.
Bmp1, 3 and 7 are all expressed in both mesenchyme and
epithelium, although Bmp7 is more abundant in the epithelium.
Bmp2 and Bmp4 are expressed in the mesenchyme. These data
provide information on the localization of gene expression at E13 and help
interpret our organ culture experiments.
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SU5402 treatment modulates gene expression of Fgfrs,
Fgfs and Bmps
Expression of Fgfs, Fgfrs and Bmps was analyzed
by real time PCR at 2, 6 and 20 hours of SU5402 treatment, to investigate the
molecular mechanism by which SU5402 inhibits branching. Glands treated with
SU5402 for 2 and 6 hours look identical to control glands and the first round
of branching in culture has not begun (Fig.
6A), but by 20 hours, morphological changes are apparent
(Fig. 6A). After 2 hours of
SU5402 treatment there was an increase in the transcription of Fgfr1b
and 1c (Fig. 8B), suggesting that inhibition of FGFR1 signaling results in up-regulation of
receptor expression via an autocrine feedback mechanism. However, by 6 hours
the expression of Fgfr1b and 1c drop below control levels.
After 6 hours of SU5402 treatment, there was also decreased expression of
Fgf1, Fgf2, Fgf3 and Bmp7, and after 20 hours of SU5402
treatment, there is still decreased expression of Fgfr1b, Fgf1, Fgf3
and Bmp7 (Fig. 8B),
suggesting that these are important regulators of branching morphogenesis.
There is also increased expression of Fgf2, Fgf7, Fgf10 and
Bmp4 at 20 hours, suggesting these are downstream and may be indirect
targets of FGFR1 signaling. Some of the genes had only minor (<1.5 fold) or
no changes in gene expression at all time points, including, FgfrR2b,
Fgf8, Fgf13, Bmp1 (all in Fig.
8B) Fgfr3, Fgfr4, Bmp2 and Bmp3 (data not
shown). Fgfr2c expression increased at all times with SU5402
treatment, which may suggest FGFR1 signaling regulates Fgfr2c
transcription. These data suggest that Fgf and Bmp
expression, downstream of FGFR1 signaling, regulate branching
morphogenesis.
Exogenous FGFs and BMPs have different morphological effects on
submandibular gland epithelium
Exogenous growth factors were added in different doses for 20 hours
(Fig. 9A). FGF1 had the least
effect and there was no increase in the number of buds
(Fig. 9A). In contrast, FGF2
treatment decreased the size and number of the buds, and FGF7 and FGF10 both
increase the size of the buds and the width of the duct adjacent to the
terminal bud, resulting in the appearance of deep clefts in the epithelium.
FGF7 also stimulates elongation of the main ducts
(Fig. 9A). Exogenous BMP4 and
BMP7 have opposite effects on the glands: BMP7 increases the number of buds
and BMP4 inhibits the number of buds and further branching. Glands treated
with BMP4 for 44 hours have similar morphology to SU5402-treated glands
(Fig. 9A). Taken together with
the increase in Bmp4 expression after 20 hours of SU5402 treatment
(Fig. 8B), these data suggest
increased expression of BMP4 may be responsible for the morphology of
SU5402-treated glands. Thus, BMP4 and FGF2 decrease the size and number of
buds, while FGF7 and FGF10 stimulate bud size and BMP7 increases the number of
buds. These data suggest that FGFs and BMPs are involved in reciprocal
networks that regulate branching morphogenesis.
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FGF7, FGF10 and BMP7 rescue the SU5402-mediated inhibition of
branching morphogenesis
Based on the previous experiments we rescued SU5402-treated glands by
adding downstream targets of FGFR1 signaling. Different doses of commercially
available FGF1, FGF2, FGF7, FGF10, BMP4 and BMP7, were added alone and in
combination, to glands treated with 1.5 µM (IC50 for number of buds) of
SU5402 (Fig. 9B,C). Active
recombinant FGF3 is not available. FGF7 (500 ng/ml), FGF10 (500 ng/ml), and
BMP7 (100 ng/ml), but not FGF1, FGF2 and BMP4, were able to rescue the glands
(Fig. 9B), further suggesting
that FGF7, FGF10 and BMP7 are important regulators of branching. These growth
factors did not have an additive effect of stimulating branching when combined
at their maximal dose. If FGF1, FGF2 or BMP4 were added in combination with
BMP7, FGF7 or FGF10, they decreased the rescue of branching (data not shown).
Taken together with the results of adding exogenous growth factors, these data
suggest that both BMP7 and FGFR2b signaling (via FGF7 and FGF10) are
downstream of FGFR1 signaling.
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DISCUSSION |
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We extended our array data by including E12 and E13 glands in the RT-PCR analysis of the developmental expression of Fgfrs and Fgfs. At E12, the SMG is an epithelial stalk with a single bud, and the sublingual gland is a small epithelial bud, both within a condensed mesenchyme. At E13, the SMG begins to undergo the first round of clefting, and with epithelial cell proliferation, the first round of branching, while the sublingual gland forms an epithelial stalk with a single bud. The major feature of the developmental regulation of the Fgfrs and Fgfs is the increased expression at E13, when branching morphogenesis begins. A similar increase in expression appears again at D5 when acinar cell differentiation is occurring. Similar patterns of gene expression may be conserved and used at different stages of development.
Data from knockout mice provides information about the role of FGFs in SMG
development. The Fgf10-null
(Ohuchi et al., 2000;
Sekine et al., 1999
) and the
Fgfr2b-null mice have multi-organ agenesis, including absence of the
salivary glands (De Moerlooze et al.,
2000
). The role of FGF10 and FGFR2b during branching morphogenesis
cannot be determined in the knockout models where agenesis of the salivary
glands occurs. The known ligands for FGFR2b are FGF1, FGF3, FGF7 and FGF10.
Abnormal SMG phenotypes were recently reported in mice with a heterozygotic
abrogation of Fgfr2c, Bmp7-null, and Pax6-null mice
(Jaskoll et al., 2002
).
Fgfr2c-hemizygotic SMGs (at E16.5) have decreased branching and lumen
formation; the mice also have lobulation defects of liver and lungs and fewer
nephrons in their kidneys (Hajihosseini et
al., 2001
). The Bmp7-null (at E17.5) and
Pax6-null (at E18.5) SMGs also have decreased epithelial branching
and disorganized mesenchyme (Jaskoll et
al., 2002
).
We focused on FGFR1 because of its increased expression at early stages of
SMG development and because little is known about its role in branching
morphogenesis. We used antisense oligonucleotides to decrease Fgfr1
levels, and a chemical inhibitor of FGFR1 signaling to identify an important
role for FGFR1 in regulating branching morphogenesis. Multiple downstream
signaling pathways are activated after FGFR stimulation in different
experimental systems, including the p42/p44 MAP kinase
(Chikazu et al., 2000), p38 MAP
kinase (Mehta et al., 2001
),
PI3 kinase (Chen et al., 2000
),
and Src family kinases (Landgren et al.,
1995
; Yayon et al.,
1997
). The most well-characterized signaling pathway involved in
branching morphogenesis of embryonic SMGs involves EGF receptor
phosphorylation, which is inhibited by PD98059, an inhibitor of MEK1
phosphorylation (Kashimata et al.,
2000
). PD98059-mediated inhibition of branching is not as dramatic
as SU5402 (data not shown), suggesting that MAP kinase-independent pathways
are downstream of FGFRs. Herbimycin, a broad tyrosine kinase inhibitor, also
inhibits branching morphogenesis (data not shown). Intracellular signaling
through FGFR1, which is inhibited by SU5402, is different from FGFR2-mediated
signaling (Rosenthal et al.,
2001
). In these studies, a FGF2-stimulated calcium channel influx
was blocked by the tyrosine kinase inhibitor lavendusdin, but was not blocked
by either SU5402 or herbimycin A.
SU5402 inhibits branching morphogenesis by decreasing epithelial cell
proliferation. SMG branching morphogenesis involves a clefting event that is
independent of epithelial cell proliferation
(Nakanishi et al., 1987;
Spooner et al., 1989
).
However, for continued rounds of branching to occur, proliferation-mediated
expansion of the bud is required for complete formation of new clefts and
lobules (Bernfield and Banerjee,
1982
). Therefore, SU5402 inhibits epithelial proliferation but
clefting still occurs in the bud. Without further epithelial proliferation and
bud expansion no further branching occurs. However, SU5402 did not induce
apoptosis of the epithelial bud or in ducts undergoing lumen formation. We did
not detect epithelial cell apoptosis by either TUNEL staining or with YO-PRO-1
dye/propidium iodide staining, which stains apoptotic/necrotic cells in
unfixed glands. However, we detected apoptosis in mesenchymal cells. We
presume the mesenchyme apoptosis is a result of the culture conditions at the
air-medium interface because apoptosis was not detected in E13 glands
immediately after dissection. Others have detected extensive epithelial cell
apoptosis in E15 submandibular glands cultured for 2 days with SN50, a peptide
that inhibits NF-
B nuclear translocation. Apoptosis was increased 10
fold over control peptide-treated glands and almost 50% of the cells underwent
apoptosis (Melnick et al.,
2001a
).
We localized the expression of Fgfrs, Fgfs and
Bmps, to either the epithelium or the mesenchyme by RT-PCR. Recently,
the stage-specific localization of FGFR, FGF and BMP proteins in the
developing submandibular gland was reported
(Jaskoll et al., 2002). These
immunohistochemical results are generally in agreement with our PCR data in
terms of which FGFRs, FGFs and BMPs are present in the glands. Since
antibodies of FGFRs do not distinguish the receptor isoforms, our PCR data
provide additional information about receptor isoform localization at E13. The
localization of the Fgfr1 and r2 splicing isoforms are
consistent with in situ data, showing at early stages the `b'
isoforms are in the epithelium and the `c' isoforms are in the
mesenchyme (Orr-Urtreger et al.,
1993
; Peters et al.,
1992
).
We analyzed gene expression after SU5402 treatment to identify downstream targets. Our results show that FGFR1 signaling directly regulates multiple receptors and ligands, and begins to define the sequence of transcriptional regulation and the molecular mechanisms involved in SMG branching morphogenesis. A surprising finding was that SU5402 treatment for 2 hours results in an increase in Fgfr1b and 1c transcription, presumably via autocrine feedback. Therefore, an initial response to blocking FGFR1 receptor phosphorylation is to increase the receptor transcription. SU5402 also increased Fgfr2c transcription. It is not known if this is a direct effect of FGFR1 signaling or a direct effect on FGFR2c signaling.
The first round of SMG branching in culture starts within 6-9 hours, and after 6 hours of SU5402 treatment there is decreased gene expression of Fgfr1b, (in the epithelium), Fgf1, Fgf2, Fgf3 (all in the mesenchyme), and Bmp7 (mainly in epithelium) suggesting these genes may be important mediators of branching. FGF1 binds to all FGFRs and exogenous FGF1 does not rescue SU5402-treated glands or stimulate branching in vitro. FGF2 binds to FGFR1 and 2c isoforms, and exogenous FGF2 decreases the size of epithelial buds. Therefore FGF2 expression may be localized at sites where bud expansion does not occur. FGF3 binds to FGFR1b and 2b, and has the greatest decrease in expression with SU5402 treatment. Also, Fgfr1b expression decreases with SU5402 treatment, suggesting that the FGF3/FGFR1b interaction may be important for branching. Interestingly, the SU5402-treated glands could be rescued with FGF7 and FGF10, which bind similar receptors as FGF3 (FGFR1b and 2b), and may functionally compensate for the decrease in FGF3 expression. Also, after 20 hours of SU5402 treatment there is increased expression of the Fgf7 and Fgf10 genes (both in the mesenchyme). Taken together, these data suggest the endogenous increase of FGF7 and FGF10 expression after 20 hours of SU5402 treatment is too late, or not high enough, to rescue the glands.
The role of BMPs as agonists or antagonists to FGFs has been studied in
other developmental systems. During neural cell development, SU5402 increases
Bmp4 and Bmp7 and suppresses Fgf3 gene expression
(Wilson et al., 2000). FGF and
BMP signaling pathways have antagonistic interactions during tooth formation
(Neubuser et al., 1997
). In
embryonic lung organ culture, BMP4 from the endoderm and FGF10 from the
mesenchyme play opposing roles in mediating branching morphogenesis of the
lung (Weaver et al., 2000
;
Weaver et al., 1999
). FGF4 and
BMP2 were shown to have opposite effects on limb growth
(Niswander and Martin, 1993
).
BMP7 increases epithelial cell proliferation during kidney development,
although at high doses it had the opposite effect
(Piscione et al., 2001
).
Bmp7 has a similar expression profile to Fgfr1 in our array
analysis. Furthermore, BMP7 increases the number of SMG buds when added
exogenously, its expression is decreased 6 hours after SU5402 treatment, and
most importantly it was able to rescue SU5402-treated glands
(Fig. 9B). Our data suggest
that Bmp7 expression downstream of FGFR1 signaling is important for
branching morphogenesis and provides a mechanism by which crosstalk between
FGF and BMP signaling occurs. Our studies show opposite roles for BMP4 and
BMP7 during submandibular gland development. Exogenous BMP4 inhibits branching
and the glands appear similar to SU5402-treated glands. Interestingly, there
is increased Bmp4 expression after 20 hours of SU5402 treatment.
Taken together, these data suggest that Bmp4 expression is indirectly
regulated by FGFR1 signaling and that it plays a negative regulatory role on
branching. Reciprocal patterns of Fgf and Bmp expression may
define and regulate areas of epithelial bud expansion, cleft initiation,
and/or progression. Our data suggest that FGF7, FGF10, and BMP7 have positive
regulatory roles on the number and size of epithelial buds, whereas FGF2 and
BMP4 play negative regulatory roles, and potentially decrease epithelial cell
proliferation and may define the sites of cleft formation.
We conclude that FGFR1c signaling in the mesenchyme and FGFR1b expression
levels and signaling in the epithelium are important for branching. Taken
together, with the results using Fgfr2b-null mice
(De Moerlooze et al., 2000), it
is likely that both FGFR1 isoforms and 2b are involved in SMG branching
morphogenesis. FGFR1 signaling may be upstream of FGFR2 by directly regulating
Fgf1 and Fgf3 expression, and indirectly regulating FGF7 and
FGF10 expression. Branching morphogenesis of SMGs involves proliferation,
migration, cleft formation, duct elongation, and differentiation; therefore,
FGFR isoforms, FGFs, and BMPs may have different functions in these processes
in both the mesenchyme and the epithelium.
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ACKNOWLEDGMENTS |
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Footnotes |
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