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First published online 3 January 2007
doi: 10.1242/dev.02741
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Department of Genetics, Cell Biology and Development, University of Minnesota Comprehensive Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.
* Author for correspondence (e-mail: marke032{at}umn.edu)
Accepted 14 November 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Fgfr2, Prostate, Seminal vesicle, Branching morphogenesis, Shh, Gli1, Fgf10, Gli2, Ptch1, Bmp4, Bmp7, svs, Seminal vesicle shape mutation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
altered seminal vesicle morphology results from a complete failure of
branching morphogenesis during seminal vesicle development
(Marker et al., 2003a). In
addition, the svs mutation reduces branching morphogenesis in the prostate
gland by approximately 40% (Marker et al.,
2003a), owing to increased ductal length between the bifurcation
events of the elongating buds, resulting in a reduced number of ductal tips
with no reduction in organ weight (Marker
et al., 2003a).
Branching morphogenesis is a developmental process common to almost all
organisms in the animal kingdom (Davies,
2002). In mammals, the kidney, lung and most glands including the
pancreas, mammary, prostate and seminal vesicles undergo branching
morphogenesis during development. Organogenesis of branched organs has been
described in terms of five steps: organ specification, epithelial bud
initiation, epithelial duct elongation into the mesenchyme, bifurcation of the
ducts leading to complex branching patterns, and cellular differentiation of
the newly branched structure (Affolter et
al., 2003). During prostate and seminal vesicle development, the
svs mutation specifically affects budding/branching morphogenesis because
organ specification, ductal elongation and cellular differentiation are normal
in both organs (Marker et al.,
2003a; Shukri et al.,
1988). This contrasts with many other spontaneous and engineered
mutations that decrease both organ growth and branching simultaneously in the
prostate and/or seminal vesicles. Examples include mutations affecting Ar,
Gdf7, Ghr, Fgf10, Hoxa13, Hoxd13, Igf1 and Srd5a2
(Donjacour et al., 2003;
Marker et al., 2003b;
Settle et al., 2001). In cases
where both growth and branching are affected, it is difficult to determine if
branching morphogenesis defects reflect a direct requirement for the gene in
regulating branching or indirect effects of compromised organ growth. Because
the svs mutation affects branching and not growth, it provides a unique
opportunity to investigate the molecular mechanisms that control branching
morphogenesis in the prostate and seminal vesicles.
Previous work mapped the svs mutation to a 2.7 cM interval on mouse
chromosome 7 that included the Fgfr2 locus
(Marker et al., 2003a).
Fgfr2 was initially considered as a candidate for the gene affected
by the svs mutation, and the Fgfr2 open reading frame was sequenced
but no changes were identified. This was not surprising because there was an
apparent disconnect between phenotypes of known Fgfr2 mutations and
the phenotypes present in svs mutant mice. All previously reported
loss-of-function Fgfr2 mutations in mice caused dysgenesis or
agenesis of organs throughout the body that resulted in embryonic or perinatal
lethality (Arman et al., 1998;
Arman et al., 1999;
De Moerlooze et al., 2000;
Hajihosseini et al., 2001;
Xu et al., 1998), whereas we
had identified only branching defects in the urogenital tract of svs mice.
Additionally, gain-of-function mutations in FGFR2 cause several human
diseases including Crouzon, Jackson-Weiss, Apert and Pfeiffer syndromes
(Hajihosseini et al., 2001;
Hertz et al., 2001;
Robertson et al., 1998),
whereas svs mutant mice do not display any of the phenotypes associated with
these diseases. Furthermore, FGF7 and FGF10 were known to be expressed in the
mesenchyme of the developing prostate and seminal vesicles and to act via
FGFR2(IIIb) expressed in the developing epithelium, and recombinant FGF7 or
FGF10 stimulated both growth and branching of developing prostates and seminal
vesicles in vitro acting at least in part as pro-proliferative signals for the
epithelium (Alarid et al.,
1994; Sugimura et al.,
1996; Thomson and Cunha,
1999). These data suggested that FGFR2 signaling was crucial for
prostate and seminal vesicle growth and therefore did not fit well with the
lack of growth defects in svs mutant prostates and seminal vesicles. The
requirement for FGF10/FGFR2(IIIb) signaling for prostate growth was
subsequently confirmed by analysis of FGF10-knockout mice
(Donjacour et al., 2003).
In order to understand the molecular basis for the branching morphogenesis defects present in svs mutant mice, we took a positional cloning approach to identify the affected gene. The map position of the svs mutation was narrowed to a 410 kb interval predicted to contain eight genes. All eight genes were investigated but no coding region sequence changes were identified. Investigation of non-coding sequences identified a 491 bp insertion in the tenth intron of Fgfr2 as a candidate svs mutation. This insertion was associated with changes in the pattern of Fgfr2 alternative splicing, and an engineered null allele of Fgfr2 failed to complement the svs mutation proving that a partial loss of FGFR2(IIIb) isoforms causes svs phenotypes. In addition, signaling by FGFR2(IIIb) through ERK1/2 (MAPK3/1 - Mouse Genome Informatics) and expression of several genes that regulate branching were found to be defective in svs mutant mice.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). PCR primers to amplify the svs insertion were: outer-svs,
5'-GTGAAGACTGGAGCTGCCCAGT-3' and
5'-AGTTCAATTCCTACCACCTATGCTG-3'; nested-svs,
5'-AAGCTCACGGCTCCTTTGG-3' and
5'-GGCACCTGCACACATGTACTTATAA-3'. Full-length Fgfr2 cDNA was amplified by a dT-primed reverse transcription (RT) followed by a primary and then a nested PCR using High Fidelity Taq according to the manufacturer's guidelines (Invitrogen). Primary primers, 5'-AGCAGGAACAGCAGTAACAACAGC-3' and 5'-ACACACGTGACAATATGCTTCCCAC-3'; nested primers, 5'-CCGCTCGAGCGGATTGGCACTGTGACCATGGTCAGCT-3' and 5'-AAGGAAAAAAGCGGCCGCAAAAGGAAAACACTCATGTTTTAACACTGCCG-3'.
Southern blotting
Genomic DNA from svs mutant mice (Jackson Laboratories) and the two
parental control strains Balb/cBy and C57BL/6By (Jackson Laboratories) was
digested with BamHI, separated on a 0.8% agarose gel, transferred to
Hybond-N+ membrane and then probed with a fragment of Fgfr2
comprising bases 1,024 to 2,254 of GenBank NM_201601.
Complementation test
svs mutant mice were crossed to ß-Actin. CRE transgenic mice (Jackson
Laboratories) as previously described
(Lewandoski et al., 1997) to
generate svs/svs; ß-Actin.CRE mice. This group was crossed with
Fgfr2flox/flox (a generous gift from D. Ornitz, Washington
University, St Louis, MO) to generate cohorts of mice that were
Fgfr2flox/svs or Fgfr2
/svs;ß-Actin.CRE.
Animals were typed by PCR from genomic DNA.
Western blotting
Protein lysates were prepared by sonicating seminal vesicles or ventral
prostates at 30% amplitude for 10 seconds (Ultrasonic Processor) in modified
Ripa's buffer (50 mM Tris-HCl, 1% NP40, 0.25% sodium dodecyl sulfate (NaDOC),
150 mM NaCl, 1 mM EDTA) with protease inhibitor cocktail (Roche). 5 µg of
protein was run on a 4-12% stacking polyacrylamide gel (BioRad) under
denaturing conditions. Protein was transferred to PVDF membrane (Millipore),
blocked with 1% BSA in TBS, then probed with anti-FGFR2 (SantaCruz, sc-122),
anti-FGFR2 (Sigma, F6796), anti-actin (Santa Cruz, sc-1616), anti-P-ERK1/2
(Cell Signaling Technologies, #9101), or anti-ERK1/2 (Cell Signaling
Technologies, #9102) antibodies. Proteins were visualized by enhanced
chemiluminesence exposed to film. Signal on film was quantified using a BioRad
Gel Doc GS700 imaging densitometer.
In-situ hybridization
An Fgfr2 partial cDNA (39,464-39,833 of GenBank AC157606) was used
to synthesize DIG-labeled sense and antisense RNA probes using a DIG RNA
Labeling Kit (Roche) according to the manufacturer's instructions. Fresh
tissues were dissected in PBS at 4°C and embedded into OCT. Tissue
sections (12 µm) were cut, mounted on Superfrost-plus microscope slides
(Fisher), hybridized with the Fgfr2 probe, and visualized as
previously described (Thut et al.,
2001).
Organ cultures
Postnatal day (P) 1 or 5 seminal vesicles were dissected out of CD1 mice
(Charles River Laboratories) into basal medium at 4°C, and cultured in 5%
CO2 at 37°C on Millicell-CM Culture Plate Inserts (30 mm, 0.4
µm pore size; Millipore Corp.) in Nunclon Multidish 4-well plates (Nunc
A/C) at the air/medium interface. Plate inserts were floated upon 0.5 ml of
basal medium consisting of DMEM/F12 50/50 Mix supplemented with 0.37 g/L
L-glutamine, 10 U/ml penicillin, 10 µg/ml streptomycin,
1xITS, and 0.5% DMSO. Additional supplements included 2.5 µg/ml
recombinant FGF10 (Peprotech Inc.), 1x10-8 M testosterone,
and 20 µM UO126 (Cell Signaling Technologies). Culture medium was changed
every other day during the culture period. To monitor branching, pictures were
taken of organs on each day of culture.
Fgfr2 isoform analysis
Full-length Fgfr2 amplicons and pIRES plasmid (Clontech) were
digested with XhoI and NotI (New England BioLabs). pIRES was
treated with shrimp alkaline phosphatase (Promega) and purified.
Fgfr2 amplicons were ligated into linear pIRES and transformed into
Escherichia coli DH5
. Individual clones were characterized by
restriction enzyme digestion and sequencing.
RNA isolation, RT-PCR and real-time PCR
Seminal vesicles were dissected from P5 svs mutant as well as heterozygous
and wild-type littermate mice. Heterozygous and wild-type seminal vesicles
were pooled and are referred to as wild type; svs mutant seminal vesicles were
pooled and are referred to as mutant. Following dissection, pooled seminal
vesicles were crushed in Trizol (Invitrogen) using a pestle and RNA isolated
according to the manufacturer's instructions. Total RNA was amplified using
the SMART RNA Amplification Kit (Clontech Laboratories) following the
manufacturer's instructions.
For RT-PCR, 25 ng of poly(A) RNA was first treated with DNase according to the manufacturer's instructions (Invitrogen). Random primers, MMLV RT (Invitrogen) and other standard reagents were used in the PCR.
Real-time PCR was undertaken using a Lightcycler (Roche). Briefly, the LightCycler FastStart DNA MasterPLUS SYBR Green I Kit (Roche) was used with 5 µl of cDNA from the RT reaction. Primer sequences were as follows:
Shh, 5'-AATGCCTTGGCCATCTCTGT-3' and 5'-GCTCGACCCTCATAGTGTAGAGACT-3';
Ptch1, 5'-CTCTGGAGCAGATTTCCAAGG-3' and 5'-TGCCGCAGTTCTTTTGAATG-3';
Gli1, 5'-GGAAGTCCTATTCACGCCTTGA-3' and 5'-CAACCTTCTTGCTCACACATGTAAG-3';
Gli2, 5'-CCTTCTCCAATGCCTCAGAC-3' and 5'-GGGGTCTGTGTACCTCTTGG-3';
Bmp4, 5'-GGTTACCTCAAGGGAGTCGAGATTG-3' and 5'-TCTTATTCTTCTTCCTGGACCGCTG-3';
Bmp7, 5'-AGTGTGCCTTCCCTCTGAAC-3' and 5'-AGGGCTTGGGTACGGTGT-3'.
Transcript levels were normalized to 18S RNA which was amplified using primer sequences 5'-GCCGCTAGAGGTGAAATTCTTG-3' and 5'-CATTCTTGGCAAATGCTTTCG-3'. An analysis of variance (ANOVA) test was undertaken using StatView 4.1 (Abacus Concepts) to determine statistically significant differences.
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/svs mice were dissected into cold PBS and
embedded in OCT. Tissue sections (12 µm) were cut using a cryostat, mounted
on Superfrost-plus microscope slides, fixed in 100% ethanol at -20°C for 2
minutes, and allowed to dry at room temperature. Slides were stained with
Hematoxylin and Eosin, dehydrated, and mounted using Permount. |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We
have now typed nine additional markers on the cross and localized the svs
mutation to a 410 kb interval between D7Mit134 and D7Mit43
(Fig. 1A). Annotation of the
mouse genome has identified two known and six predicted genes within this
interval (Fig. 1B)
(Birney et al., 2006;
Waterston et al., 2002). We
evaluated the candidate genes present between D7Mit134 and D7Mit43 for
expression in the developing prostate and seminal vesicles by RT-PCR (data not
shown). The predicted open reading frames for all detected candidate
transcripts were sequenced in svs mutant mice and parental control strains
Balb/cBy and C57BL/6By, but no changes were identified (data not shown). We
subsequently initiated an effort to identify structural changes in the
non-coding sequences of the candidate interval using Southern blotting. This
identified a restriction fragment length polymorphism between svs mutant mice
and the parental control strains Balb/cBy and C57BL/6By
(Fig. 2A). The location of the
polymorphism was predicted based on the mouse genome sequence, and
subsequently analyzed by PCR (Waterston et
al., 2002). The amplification of a longer fragment from svs mutant
mice than from the two parental strains Balb/cBy and C57BL/6By suggested the
presence of a novel insertion (Fig.
2B). Sequence analysis identified a 491 bp insertion identical to
a murine leukemia virus long terminal repeat in the tenth intron of
Fgfr2 in svs mutant mice (Fig.
2C). This insertion was absent from all other CXB recombinant
inbred strains of mice (data not shown), consistent with the possibility that
this insertion is responsible for the phenotypes of svs mutant mice.
Partial loss of Fgfr2 causes svs phenotypes
The potential consequences of this insertion for Fgfr2 function
were evaluated in several ways. Initially, we conducted western blot analysis
using a commercially available antibody directed against the carboxy-terminus
of FGFR2 and observed a banding pattern in both wild-type and svs mutant
seminal vesicles similar to that previously reported for this antibody in
experiments on other mouse tissues (Fig.
3A, upper panel). Previous reports indicate that these bands are
specific for FGFR2 because they are eliminated in tissues with an engineered
null mutation in FGFR2 (Xu et al.,
1998), and they appear with the ectopic expression of FGFR2 cDNAs
(Schmahl et al., 2004). In
addition, a qualitatively similar result was obtained with a second antibody
directed against the extracellular domain of FGFR2
(Yan et al., 2005) that
strongly recognizes the lower molecular weight form of FGFR2 also recognized
by the antibody directed against the carboxy terminus
(Fig. 3A, middle panel). These
data suggest that wild-type and svs mutant seminal vesicles express similar
levels of FGFR2 protein. Furthermore, in situ hybridization using a probe
directed against the first coding exon of Fgfr2 showed that
transcripts were correctly localized to the epithelium of the prostate and
seminal vesicles in the control and svs mutant mice
(Fig. 3B-D and data not
shown).
|
). To determine if aberrant alternative splicing of
Fgfr2 was occurring in the svs mutant mice, full-length
Fgfr2 transcripts were amplified by RT-PCR from svs mutant and
control P5 seminal vesicle RNA. A total of 111 control and 96 svs mutant
full-length Fgfr2 transcripts were analyzed by restriction enzyme
digestion and sequencing. There was a surprisingly complex pattern of
alternative splicing present in both control and svs mutant seminal vesicles.
Eleven different isoforms were detected in each group, with only three
isoforms overlapping between the control and svs mutant groups
(Fig. 4A,B). The difference in
isoform distribution between svs and control mice was highly statistically
significantly (Pearson 
2 test, P<0.0001).
Strikingly, all of the Fgfr2 transcripts expressed in control seminal
vesicles included exon 8IIIb, which confers specificity for the FGF ligands
expressed in the mesenchyme of the developing prostate and seminal vesicles
(Thomson and Cunha, 1999). By
contrast, 10 of 11 Fgfr2 transcripts containing exon 8IIIb observed
in wild-type seminal vesicles were reduced in or absent from svs mutant
seminal vesicles.
To determine if partial loss of specific Fgfr2 isoforms caused svs
mutant phenotypes, a genetic complementation test was conducted using a
previously described mutant allele of Fgfr2. This allele has exons 7,
8IIIb, 8IIIc and 9 (7-9) flanked by loxP sites (Fgfr2flox)
and encodes normal Fgfr2 transcripts
(Yu et al., 2003). However, in
the presence of Cre recombinase, exons 7-9 are deleted and a null allele of
Fgfr2 is created (Fgfr2). Crosses were
conducted to create Fgfr2flox/svs and
Fgfr2/svs mice. Seminal vesicles from both
genotypes were evaluated for branching morphology at P5
(Fig. 5A-C) and by histology in
adults (Fig. 5D,E). The
Fgfr2flox allele complemented the svs mutation, but the
Fgfr2 allele did not, proving that svs phenotypes
are due to a partial loss of Fgfr2 function.
Signaling and gene expression change downstream of the svs mutation
FGFR2 is a receptor tyrosine kinase that can signal through multiple
intracellular pathways (Chen et al.,
2000; Kim et al.,
2003; Nakamura et al.,
2001; Newberry et al.,
1997; Sakaguchi et al.,
1999; Xiao et al.,
2004; Yan et al.,
1993). In the Drosophila trachea, the FGFR homolog
breathless controls branching morphogenesis through the MEK-ERK
pathway (Gabay et al., 1997;
Lee et al., 1996;
Sutherland et al., 1996). To
investigate whether this pathway might be important for the phenotypes of svs
mutant mice, the status of ERK1/2 activation was examined in svs mutant and
control seminal vesicles. There was an abundance of phosphorylated ERK1/2 in
control seminal vesicles whereas phosphorylated ERK1/2 could not be detected
in svs mutant seminal vesicles (Fig.
6A). To determine if FGFR2(IIIb) directly activates ERK1/2 in
developing seminal vesicles, wild-type organs were cultured with recombinant
FGF10 protein. FGF10 is expressed by the developing mesenchyme of the prostate
and seminal vesicles and can signal through the IIIb isoform of FGFR2
(Lu et al., 1999). FGF10
activated ERK1/2 in the wild-type seminal vesicles within 20 minutes,
suggesting that this is a direct response of FGFR2(IIIb) activation
(Fig. 6B). To determine if
activated ERK1/2 is a plausible explanation for the loss of branching
morphogenesis in the developing seminal vesicles of svs mutants, organ
cultures of wild-type seminal vesicles were also conducted in the presence of
UO126, a synthetic inhibitor of MEK1/2 (MAP2K1/2 - Mouse Genome Informatics),
the upstream kinases that activate ERK1/2
(Davies et al., 2000).
Following 4 days in culture, seminal vesicles cultured with testosterone
branched significantly, whereas loss of ERK1/2 activation due to UO126 led to
a complete loss of branching morphogenesis
(Fig. 6C,D). These effects were
unlikely to be due to interference with androgen receptor signaling because
the levels of activated ERK1/2 were indistinguishable in organs cultured with
or without testosterone (Fig.
6D). These data demonstrated that activation of the MEK1/2-ERK1/2
signaling pathway downstream of FGFR2(IIIb) is crucial for branching
morphogenesis in the developing seminal vesicles. These data are also
consistent with the possibility that the loss of ERK1/2 activation in svs
mutant seminal vesicles is the proximal mechanism responsible for defects in
branching morphogenesis.
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). We used real time RT-PCR to evaluate the effects of partial
loss of FGFR2(IIIb) on the expression of branching-regulatory genes
(Fig. 7A-G). Although
expression of the upstream ligand Fgf10 was unaffected, mRNA levels
for several other branching regulators, including Shh, Gli1, Gli2, Ptch1,
Bmp4 and Bmp7, were all reduced in svs mutant seminal
vesicles. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10% of spontaneous
mutations in mice are due to transposable elements, including many instances
of de novo murine leukemia virus insertion
(Maksakova et al., 2006). The
main effect of this insertion in the developing seminal vesicles is to change
the pattern of alternative splicing of the Fgfr2 transcript (Figs
3,
4). During seminal vesicle
development, the epithelium expresses Fgfr2 transcripts containing
exon 8IIIb, whereas transcripts containing exon 8IIIc are absent. In other
tissues, mesenchymal cells express transcripts containing exon 8IIIc. The
aberrant alternative splicing in svs mutant mice does not appear to change the
primary localization pattern of Fgfr2 transcripts in the seminal
vesicles; in situ hybridization using a probe containing sequences found in
all isoforms of Fgfr2 exhibited epithelial-specific localization in
both wild-type and svs mutants (Fig.
3). However, the presence of low levels of Fgfr2
transcripts containing exon 8IIIc in the mesenchyme cannot be ruled out. In
situ hybridization was conducted using probes specific to exon 8IIIb and exon
8IIIc of Fgfr2, but these probes were not sensitive enough to detect
Fgfr2 transcripts in svs mutant seminal vesicles (data not
shown).
The change in alternative splicing presumably results from the disruption
of primary sequence, or from secondary structure elements within the
Fgfr2 pre-mRNA that are required for the highly regulated and complex
pattern of Fgfr2 alternative splicing previously described
(Ingersoll et al., 2001).
Alternative usage of exon 8IIIb and 8IIIc is regulated by cis elements present
in the intronic sequences between exon 8IIIb and 8IIIc that are recognized by
transactivating factors such as FOX-2 (RBM9 - Mouse Genome Informatics)
(Baraniak et al., 2006). It is
possible that the svs mutation disrupts these cis-acting regulatory sequences
thereby blocking the recruitment of important transactivating splicing
factors. However, the svs insertion is approximately 2 kb 3' of known
Fgfr2 splicing-regulatory elements, including IAS1 (intronic
activating sequence), ISAR (intronic splicing activator and repressor), IAS1
(intronic activating sequences 1), IAS2, IAS3, ISE1 (intronic silencing
enhancers 1), ISE2 and ISE3 (Baraniak et
al., 2006; Baraniak et al.,
2003; Hovhannisyan and
Carstens, 2005). Thus, the svs mutation may indicate the presence
of one or more previously unrecognized intronic regulatory splicing elements
in the tenth intron of Fgfr2.
During our analysis of Fgfr2 transcripts present during branching
morphogenesis in wild-type organs, we identified 11 distinct splice variants
(Fig. 4B). Although the
importance of alternative splicing has not been well characterized for many of
the alternativelyincluded Fgfr2 exons, the alternative usage of exons
8IIIb and 8IIIc has been extensively studied and shown to function as a key
determinant of FGFR2 ligand specificity
(Ingersoll et al., 2001). All
of the wild-type Fgfr2 transcripts included exon 8IIIb, which is
essential for receptor activation by FGF7 and FGF10, the ligands expressed by
the prostate and seminal vesicle mesenchyme. In svs mutant organs, 10 of the
11 wild-type Fgfr2 splice variants were reduced in abundance or
absent. The dramatic changes observed in alternative splicing, along with the
recessive nature of the svs phenotypes, suggested that partial loss of
FGFR2(IIIb) function was responsible for these svs mutant phenotypes. This was
confirmed by the failure of a known null allele of Fgfr2 to
complement the svs mutation (Fig.
5).
|
;
Shukri et al., 1988). By
contrast, many Fgfr2 loss-of-function mutant alleles result in
lethality due to affects on multiple organ systems. Fgfr2 is known to
be required for early mammalian development because an Fgfr2-null
mutation caused peri-implantation embryonic lethality shortly after embryonic
day (E) 4.5 (Arman et al.,
1998). A strong hypomorphic Fgfr2 allele that deleted the
exons encoding the immunoglobin-like domain III extracellular ligand-binding
domain caused embryonic lethality around E10.5, a failure of limb bud
induction, and placental defects (Xu et
al., 1998). Fgfr2(IIIb), an isoform-specific knockout
allele of Fgfr2 that eliminated receptor isoforms incorporating exon
8IIIb, caused perinatal lethality with multiple organ defects including a
complete failure of developmental growth and branching in the lung
(De Moerlooze et al., 2000).
Subsequent studies revealed that elimination of Fgfr2(IIIb) also
caused growth and branching defects in additional organs including the mammary
glands and pancreas (Mailleux et al.,
2002; Pulkkinen et al.,
2003). Although Fgfr2 has previously been implicated in
branching morphogenesis in several organs outside of the male reproductive
tract, branching morphogenesis in the seminal vesicles and prostate seem
particularly sensitive to the affects of the svs mutation. For example, we
have analyzed whole-mount preparations of svs mammary glands and evaluated the
histology of svs lungs (data not shown). These efforts did not reveal dramatic
branching morphogenesis defects in svs mutants. However, very subtle affects
of the svs mutation on branching in these organs might have been missed.
Initial interest in identifying the gene affected by the svs mutation came
from the previously described svs phenotypes, which include a complete failure
of branching morphogenesis during seminal vesicle development and dramatically
reduced branching morphogenesis in the prostate gland without associated
defects in organ growth or differentiation
(Marker et al., 2003a). Genes
from the fibroblast growth factor family, hepatocyte growth factor family,
epidermal growth factor family, transforming growth factor beta superfamily,
sonic hedgehog pathway and, more recently, notch signaling have all been
implicated as regulators of branching morphogenesis
(Davies, 2002;
Marker et al., 2003b;
Wang et al., 2006). However,
it is often unclear what precise role each gene plays in controlling branching
morphogenesis. Many of the genes are temporally and spatially regulated during
development and are likely to regulate multiple steps during organogenesis.
The multiple roles of key regulatory genes make it difficult to establish a
precise function for each gene during branching morphogenesis.
It has previously been suggested that, in the prostate and seminal
vesicles, signaling by FGF7 and FGF10 through FGFR2(IIIb) is important for
epithelial proliferation and duct elongation during branching morphogenesis
(Thomson, 2001;
Thomson and Cunha, 1999).
Fgf7 and Fgf10 are expressed by the mesenchyme of both the
prostate and seminal vesicles during development, and recombinant FGF7 or
FGF10 stimulated both growth and branching of developing prostates and seminal
vesicles in vitro, acting at least in part as pro-proliferative signals for
the epithelium (Alarid et al.,
1994; Sugimura et al.,
1996; Thomson and Cunha,
1999). The requirement for FGF10 for prostate and seminal vesicle
development was confirmed by experiments showing that Fgf10-null
embryos develop only minimal prostatic organ rudiments and that the caudal
segments of the Wolffian ducts, which are the precursor structures for the
seminal vesicles, degenerate in a majority of Fgf10-null embryos
(Donjacour et al., 2003).
Additionally, grafting of embryonic prostates revealed that minimal prostate
development occurred from Fgf10-null prostates. Similarly, grafting
the caudal Wolffian ducts from the rare Fgf10-null embryos in which
they did not degenerate, revealed that Fgf10-null embryos had a
limited ability to develop rudimentary seminal vesicles, with only one in
eight grafted Wolffian ducts resulting in tissue that resembled immature
seminal vesicle (Donjacour et al.,
2003).
The fact that prostates and seminal vesicles exhibit no size deficit in svs
mutant mice (Marker et al.,
2003a), whereas Fgf10-null embryos exhibit a dramatic
loss of growth for both organs, suggests that FGF10 can partially signal
through the reduced levels of FGFR2(IIIb) still present in svs mutant organs
and that this is sufficient to support organ growth. However, branching
morphogenesis fails completely in svs seminal vesicles and is reduced by
40% in svs prostates (Marker et al.,
2003a). This suggests that peak levels of FGF10 signaling through
FGFR2 are required to induce branching because partial loss of FGFR2(IIIb) in
svs mutant mice blocked branching in the seminal vesicles and reduced
branching in the prostate. FGFR2(IIIb) can activate several downstream
signaling pathways. This study highlights the importance of the MEK1/2-ERK1/2
signaling pathway in branching morphogenesis. The svs mutant seminal vesicles
failed to maintain activation of the MEK1/2-ERK1/2 pathway during branching
morphogenesis despite similar overall levels of FGFR2 protein expression in
wild-type and mutant seminal vesicles. The loss of ERK1/2 activation is likely
to result from the shift in Fgfr2 alternative splicing that decreases
the abundance of exon 8(IIIb)-containing isoforms and results in the ectopic
expression of exon 8(IIIc) isoforms that are normally not expressed during
seminal vesicle development (Fig.
4). FGF7 and FGF10 are thought to be the ligands that activate
FGFR2 during seminal vesicle and prostate development
(Thomson, 2001;
Thomson and Cunha, 1999).
Since these ligands cannot signal via exon 8(IIIc)-containing FGFR2 isoforms,
the partial loss of exon 8(IIIb)-containing isoforms may be sufficient to
explain the loss of ERK1/2 activation in svs mutant seminal vesicles. Previous
studies have also shown that different isoforms of FGFR2 can heterodimerize
(Tanahashi et al., 1996),
suggesting that heterodimers between exon 8(IIIb)-containing and exon
8(IIIc)-containing FGFR2 isoforms in svs organs may further reduce the
availability of functional receptors for FGF7 and FGF10. It is also possible
that the ectopic expression of 8(IIIc)-containing FGFR2 isoforms in svs mutant
mice could cause gain-of-function phenotypes owing to signaling through other
known FGFR2 downstream signaling pathways such as p38 MAPK, AKT or PLC
(Ceridono et al., 2005;
Chen et al., 2000;
Mehta et al., 2001). However,
the failure of an Fgfr2-null mutation to complement svs developmental
phenotypes (Fig. 5) confirms
that the loss-of-function effects of the svs mutation on FGFR2 are responsible
for those phenotypes.
|
|
|
; Lamm et al.,
2001; Podlasek et al.,
1999; Pu et al.,
2004). These data provide genetic support for previous in vitro
studies that have suggested cross-regulation among branching
morphogenesisregulatory genes. Many of the gene expression changes observed in
our experiments could be predicted from previous in vitro studies. For
example, short-term treatment of ventral and lateral prostates with
recombinant FGF10 upregulated expression of Shh, Ptch1, Gli1, Gli2
and Bmp7, and our experiments showed that a partial loss of
FGFR2(IIIb), the FGF10 receptor, resulted in decreased expression of these
genes (Huang et al., 2005).
However, our studies did not always perfectly mirror the results predicted by
short-term in vitro experiments. For example, short-term treatment of ventral
and lateral prostates with recombinant FGF10 downregulated expression of
Bmp4, and our experiments showed that a partial loss of FGFR2(IIIb)
also resulted in decreased expression of Bmp4
(Huang et al., 2005). This
difference in the effects of FGFR2(IIIb) signaling on Bmp4 expression
between the in vitro organ cultures and the in vivo organs is likely to be due
to the different time frames that the organs are exposed to the increased or
decreased receptor activity before examining transcript levels. For the in
vitro organ cultures, the ventral prostate and lateral prostate were treated
with recombinant FGF10 for 24 hours, then RNA was extracted and transcript
levels determined (Huang et al.,
2005). This time frame is short compared with the situation for
the seminal vesicles from the svs mutant mice, which experienced decreased
FGFR2(IIIb) signaling throughout organogenesis. Furthermore, it is likely that
branching morphogenesis regulators participate in feedback loops that are
reiteratively used during ductal elongation and branching, such that long-term
loss of a key gene such as Fgfr2(IIIb) would cause a general
downregulation of the branching process and its associated gene
expression.
Conclusions
The mouse svs mutation causes a complete loss of branching morphogenesis in
the seminal vesicles and a dramatic reduction of branching in the prostate
without changes to organ growth or differentiation. These phenotypes are
caused by a 491 bp insertion in the tenth intron of Fgfr2, which is
associated with aberrant alternative splicing that alters receptor activity
without affecting protein expression levels or transcript localization. The
partial loss of 8IIIb-containing transcripts is responsible for svs phenotypes
because a null allele of Fgfr2 failed to complement the svs mutation.
Furthermore, the reduced FGFR2(IIIb) activity caused a loss of sustained
ERK1/2 activation and a reduction in the transcript levels of Shh, Ptch1,
Gli1, Gli2, Bmp4 and Bmp7, which are important regulators of
branching morphogenesis. Thus, the svs mutation provides a unique model to
study branching morphogenesis of the prostate and seminal vesicles and FGFR2
function during development and in the adult.
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ACKNOWLEDGMENTS |
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