1 Cancer Research UK, Lincoln's Inn Fields, London WC2A 3PX, UK
2 School of Animal and Microbial Sciences, University of Reading, Whiteknights,
Reading RG6 6AJ, UK
3 Department of Zoology and Department of Anatomy and Cell Biology, University
of Florida, P.O. Box 118525, Gainesville, FL 32611, USA
Author for correspondence (e-mail:
cohn{at}zoo.ufl.edu)
Accepted 11 February 2005
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SUMMARY |
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Key words: Fgfr2-IIIb, Tubular morphogenesis, Hypospadias, Urethral plate, Genital tubercle
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Introduction |
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Vertebrate external genital development can be divided into two distinct
phases: the first involves initial outgrowth and patterning of the genital
tubercle, which occurs in both male and female embryos
(George and Wilson, 1994); the
second, hormonally controlled phase, involves either the continued growth and
differentiation of the penis, or the arrest of outgrowth and differentiation
of the clitoris. During the first phase, there are no discernable
morphological differences between male and female external genitalia. In the
mouse, genital tubercle development is initiated at embryonic day (E) 10.25,
when paired buds of lateral plate mesoderm emerge beneath ventral body wall
ectoderm on either side of the cloaca
(Perriton et al., 2002
). An
epithelial extension of cloacal endoderm extends between these swellings to
form the urethral plate, and the left and right swellings merge medially to
form a single genital tubercle (Kurzrock
et al., 1999a
; Perriton et
al., 2002
). The urethral plate epithelium extends to the distal
tip of the genital tubercle where, in the clitoris, it persists as an
epithelial cord or, in the penis, it canalizes to form a urethral tube.
Like the limb, the genital tubercle undergoes proximal to distal outgrowth,
during which cells within the tubercle are patterned along three axes. In the
genitalia, however, these processes must be coordinated with epithelial
tubulogenesis to generate a urethral canal. Epithelial-mesenchymal
interactions play an important role in outgrowth of the genital tubercle.
Surgical removal of the distal epithelium leads to an arrest of outgrowth and
truncation of the phallus (Kurzrock et
al., 1999b; Murakami and
Mizuno, 1986
). This is analogous to the outgrowth of the early
limb bud, which is controlled by the apical ectodermal ridge, a specialized
population of distal epithelial cells. Indeed, both of these structures are
sites of fibroblast growth factor (Fgf) 8 expression, and signaling activity
of these tissues can be replaced with beads soaked in Fgf8 protein
(Crossley et al., 1996
;
Haraguchi et al., 2000
). We
recently identified a new signaling region, the endodermally derived urethral
plate epithelium, which is situated along the ventral side of genital tubercle
and is a site of sonic hedgehog (Shh) expression
(Perriton et al., 2002
). Shh
is required for outgrowth and patterning of the genital tubercle, and mice
with a targeted deletion of Shh have penile and clitoral agenesis
(Haraguchi et al., 2001
;
Perriton et al., 2002
).
Members of the Hox paralogy group 13, Hoxd13 and Hoxa13,
also play an essential role in external genital and limb development
(Kondo et al., 1997
;
Morgan et al., 2003
;
Stadler, 2003
). Loss of
function of both genes results in agenesis of the genital tubercle, and
heterozygosity for either causes patterning defects of the phallus. A recent
study reports that Hoxa13-null mice exhibit hypospadias
(Morgan et al., 2003
), and
mutations in the human HOXA13 gene are responsible for the range of
phenotypes seen in Hand-Foot-Genital Syndrome
(Goodman et al., 2000
;
Mortlock and Innis, 1997
).
Whereas early genital development is controlled by a genetic program that
operates prior to production of steroid hormones, the second phase of penis
development requires exposure to an androgen, either testosterone or
dihydrotestosterone (DHT) (George and
Wilson, 1994). Androgenic steroids, synthesized by the Leydig
cells of the testes, are first seen just prior to the onset of
androgen-induced genital differentiation
(Abney, 1999
). In the
developing external genitalia of mammals, androgen receptors (ARs) are
abundant in the urethral epithelium, with lower concentrations found in the
underlying stromal tissue (Kim et al.,
2002
). Similarly, 5
-reductase Type 2, an enzyme that
converts testosterone to 5
-dihydrotestosterone (DHT), is highly
expressed in the mesenchymal stroma surrounding the urethra
(Kim et al., 2002
;
Tian and Russell, 1997
). Loss
of a functional AR or inability to synthesize DHT causes reduction in penis
growth, hypospadias and, in severe cases, complete loss of phallic development
(George and Wilson, 1994
). By
contrast, exposure of developing females to androgens leads to masculinization
of the external genitalia (George and
Wilson, 1994
).
Normal genital development must involve integration of local and systemic cues; however, neither the interactions between signals that operate in the first and second phases of genital development nor the genetic control of urethral tube closure is well understood. We report that a loss of function mutation in mouse Fgfr2-IIIb causes urethral tube development to arrest, resulting in severe hypospadias. Although Fgfr2-IIIb-/- mice develop a urethral plate that expresses Shh and Fgf8, which mediate its signaling activity, the urothelial progenitor cell population becomes depleted and urethral plate maturation fails prior to the onset of tubulogenesis. We show that transcription of Fgfr2-IIIb and Fgf10 requires AR signaling during the second phase of external genital development, and that the Fgfr2 promoter contains a stereotypic androgen response element (ARE) sequence. Together, these results demonstrate that Fgfr2-IIIb-mediated signaling is essential for development of the urethral tube and suggest that the Fgfr2 gene is a transcriptional target of AR.
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Materials and Methods |
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Histology, in situ hybridization, and immunohistochemistry
Histology and in situ hybridization were carried out using standard
procedures and as previously described
(Perriton et al., 2002;
Revest et al., 2001
). Probes
used were for Fgfr2-IIIb, Shh and Fgf8
(Mailleux et al., 2002
;
Revest et al., 2001
). Wax
sections transverse to the genitalia were dewaxed in xylene, rehydrated
through graded alcohols and used for detection of keratin 14 (K14) (BabCO,
USA), AR (N-20) (Santa Cruz Biotech, USA) or Ki67 (Novocastra, UK). Sections
were boiled twice for 10 minutes in 0.01 M citrate buffer pH 6 then blocked
with swine serum (1:25). All dilutions were in PBS. Anti-K14 (1:1000), anti-AR
(1:100) or anti-Ki67 (1:200) was applied for 2 hours at room temperature. A
biotinylated swine anti-rabbit secondary antibody (DAKO, UK) was applied
(1:500) for 40 minutes followed by streptavidin-peroxidase for 40 minutes
(1:500). A DAB substrate kit (Vector Labs, USA) was used for detection.
Apoptotic cells were either detected by incorporation of terminal
deoxynucleotidyltransferase-mediated dUTP end labeling (TUNEL) or stained
using a commercially available kit (Apoptag ISOL) according to the
manufacturer's protocol (Intergen, UK).
Whole organ culture of genital tubercles
Genital tubercles were dissected in ice-cold medium (MEM supplemented with
10% fetal calf serum, L-glutamine and antibiotic/antimycotic), and transferred
onto Millipore nitrocellulose filters (0.8 µm) supported by wire mesh.
Organs were cultured with the ventral side upwards, immediately below the
media-gas interface, in a humidified incubator at 37°C and 5%
CO2. Cultures were fed with BJGB medium supplemented with 10% fetal
calf serum, L-glutamine and antibiotic/antimycotic. Flutamide (Sigma) was
dissolved in 100% ethanol and added to the cultures at concentrations of
10-5 M, 10-4 M, 5-4 M and 10-3 M.
Control cultures were treated with an identical volume of ethanol.
Dihydrotestosterone (Sigma) was dissolved in ethanol and used at a
concentration of 5-6 M. Cultures were maintained for 48 hours
before being fixed in 4% paraformaldehyde for in situ hybridization or 1%
glutaraldehyde for scanning electron microscopy.
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Results |
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Transcription of Fgfr2-IIIb and Fgf10 requires AR activity
The hypospadias defect of Fgfr2-IIIb-/- mice resembles
the phenotype that results from disruption of androgen signaling. In the
prostate, Fgfr2 can mediate the effects of androgen on tumor progression,
suggesting that Fgfr2 operates as an andromedin
(Nakano et al., 1999). We
therefore investigated the possibility that the Fgfr2 and the AR pathways
interact in genital development. To determine whether AR is required for
Fgfr2-IIIb expression in urethral cells, we cultured both male and
female genital tubercles from E14.5-E15.5 wild-type embryos in the presence of
flutamide, a pharmaceutical AR antagonist that induces hypospadias when
administered to pregnant females
(Imperato-McGinley, 1994
).
Because binding assays have shown that flutamide must be present at 500- to
1000-fold higher concentrations than circulating androgens, in order to
compete for AR binding (Simard et al.,
1986
; Zuo et al.,
2002
), we first carried out a dose-response analysis to identify
physiologically relevant concentrations required to produce a hypospadias
phenotype in vitro. Genital tubercles from male and female embryos were
cultured separately; however, we detected no differences in their responses to
flutamide. Genital tubercles cultured for 48 hours with 10-5 M
flutamide (n=19) developed similar to the controls (see Fig. S1A,B in
the supplementary material). At 10-4 M flutamide, 38% of the
tubercles exhibited hypoplasia of the prepuce and malformation of the glans
(n=13; see Fig. S1C in the supplementary material). In cultures with
5-4 M (n=4) and 10-3 M (n=20)
flutamide, 100% of the genital tubercles had a hypospadias phenotype,
consisting of a prepuce that was either hypoplastic or completely absent, a
superficial urethral plate and a poorly differentiated glans (see Fig. S1D,E
in the supplementary material). Histological analysis of cultured tubercles
revealed that urethral plate differentiation was perturbed by flutamide
treatment. After 48 hours in the presence of 5-4 M flutamide, the
urethral plate still could be detected, but the tissue was severely
disorganized and hypoplastic relative to the neatly stratified, complex
urethral epithelium observed in control cultures (compare Fig. S1G with Fig
S1F in the supplementary material). We next examined Fgfr2-IIIb
expression in a series of genital tubercles cultured under the same
conditions. Organ cultures of male and female genital tubercles treated with
flutamide showed a dose-dependent decrease in Fgfr2-IIIb expression,
which mirrored the dose-dependent induction of hypospadias. Treatment with
10-5 M flutamide resulted in no detectable change in
Fgfr2-IIIb expression relative to controls
(Fig. 6A,B); however, raising
the flutamide dose to 10-4 M resulted in a marked decrease in
Fgfr2-IIIb expression in the urethral plate
(Fig. 6C). When the flutamide
concentration was increased further to 5-4 M or 10-3 M,
Fgfr2-IIIb became undetectable
(Fig. 6D,E). Given that the
urethral plate is present, although disorganized, 48 hours after flutamide
treatment (see Fig. S1G in the supplementary material), we conclude that
absence of Fgfr2-IIIb transcripts after 48 hours in culture is due to
downregulation of the gene, rather than to loss of
Fgfr2-IIIb-expressing cells.
|
The ability of AR to modulate Fgfr2-IIIb transcription raised the
possibility that the Fgfr2 gene may contain an ARE. Direct contact
between AR protein and DNA requires a highly conserved AR recognition site,
the hexanucleotide T-G-T-T-C-T, which functions as common DNA-binding element
for androgen, glucocorticoid and progesterone receptors
(De Vos et al., 1991;
Scheidereit and Beato, 1984
).
We therefore performed an in silico analysis of the Fgfr2 promoter sequence
(GenBank Accession Number, X66455) (Avivi
et al., 1992
) using TRANSFAC
(Wingender et al., 2001
),
which revealed the presence of the T-G-T-T-C-T motif between nucleotides
1193-1198 (Fig. 6G). The
presence of a putative ARE sequence within the Fgfr2 promoter,
together with our finding that antagonism of AR results in loss of
Fgfr2-IIIb transcripts in cultured genital tubercles, suggests that
Fgfr2 could be a direct transcriptional target of the AR.
Fgf10 expression also can be regulated by androgen in the prostate
(Donjacour et al., 2003;
Lu et al., 1999
), although
whether this is direct or indirect is unclear
(Thomson, 2001
;
Thomson and Cunha, 1999
). To
determine whether Fgf10 expression in the genital tubercle may
require androgen signaling, we repeated the flutamide dose-response experiment
describe above and, after 48 hours in culture, genitalia were assayed for
Fgf10 expression. Fgf10 transcripts persisted in control
cultures and in the presence of 10-5 M flutamide; however, as with
Fgfr2-IIIb, doses of flutamide between 10-4 M and
10-3 M resulted in progressive downregulation of Fgf10 in
male and female genital tubercles (Fig.
6H-J; data not shown).
Although Fgfr2-IIIb expression can be regulated by AR, we found no evidence of a positive feedback loop between these two receptors. Immunohistochemical analysis of AR distribution in external genitalia of Fgfr2-IIIb-/- and wild-type embryos at E16.5 revealed no discernable differences (Fig. 7). AR staining was observed in genital mesenchyme and urethral epithelium, with particularly strong staining in the corporal bodies and basal layer of the urethra (Fig. 7A,B). Sections through the pelvic region of the trunk, proximal to the genital tubercle, showed that the proximal urethra remained intact and was positive for AR in Fgfr2-IIIb-/- mutants (Fig. 7A,B, insets). Thus, although AR is a positive regulator of Fgfr2-IIIb in the external genitalia, expression of AR appears to be independent of Fgfr2-IIIb.
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Discussion |
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Fgf10-Fgfr2-IIIb signaling is required for maintenance of urethral progenitor cells
Normal patterning of uroepithelium (urothelium) involves interaction
between stromal (mesenchymal) cells and the adjacent epithelium
(Kurzrock et al., 1999b). We
have shown that, during external genital development, morphogenesis of the
urethral tube is regulated by mesenchymally produced Fgf10 signaling through
Fgfr2IIIb in the urethral epithelium. Although hypospadias is generally
considered to result from defective morphogenesis of the urethral tube, our
findings show that hypospadias in Fgfr2-IIIb-deficient mice results
from a differentiation defect that begins to be manifested prior to the onset
of tubulogenesis. Differentiation of stratified epithelia depends upon
sustained regenerative proliferation, which requires a population of
progenitor stem cells in the basal layer
(Hong et al., 2004
;
Yang et al., 1999
). Our
results suggest that the mechanism by which Fgfr2-IIIb-mediated signaling
regulates urethral tubulogenesis involves the sustained proliferation of basal
cells in the urothelium, where the progenitor cell population resides. The
cessation of cell division in this population causes an abrupt arrest of
urethral differentiation, resulting in development of a thin, disorganized
urethral plate that fails to undergo normal maturation and stratification.
These findings are, to some degree, reminiscent of the bladder defects
observed in Fgf7-null mutant mice, in which bladder urothelium is
abnormally thin and fails to undergo normal stratification
(Tash et al., 2001
). Although
the intermediate cell layer is absent, basal cells are retained in bladder
urothelium of Fgf7-/- mice, suggesting that another factor
may be able to at least partially compensate for Fgf7 in the bladder
urothelium. Fgf10 is expressed in the lamina propria, adjacent to
bladder urothelium and compensation by Fgf10 may be sufficient to maintain the
basal progenitor cell layer in Fgf7-/- mice. The presence
of two Fgfr2-IIIb ligands in the bladder mesenchyme contrasts with the
situation in the genital tubercle, where we detected Fgf10 but not
Fgf7. Thus, our observation that loss of Fgf10 alone results in a
hypospadias phenotype of similar severity to that seen in
Fgfr2-IIIb-/- mice can be explained by a lack of
redundancy in the expression of Fgfr2-IIIb ligands during external genital
morphogenesis.
Regulation of urethral tube closure by androgen is mediated by Fgfr2-IIIb
Disruption of androgen signaling during external genital development has
long been know to result in hypospadias
(Glucksmann et al., 1976).
Here, we have shown that hypospadias can also result from disruption of the
Fgfr2-IIIb pathway. Our discovery that AR activity is required for
Fgfr2-IIIb expression in the urethra suggests that the androgen and
Fgf10 pathways converge at the point of Fgfr2-IIIb to regulate morphogenesis
of the urethral tube. Our observations that both male and female mutants
develop hypospadias, and that the hypospadias phenotype begins to develop in
Fgfr2-IIIb mutants prior to the stages at which androgens act on
genital development suggest that Fgfr2-IIIb also has an early,
androgen-independent role in development of the urethral plate. We favor a
model in which pattern formation of external genitalia at sexually indifferent
stages is controlled by locally regulated expression of Fgfr2-IIIb;
however, as systemic endocrine signals are integrated at later stages,
expression of Fgfr2-IIIb (and perhaps other genes) may be modulated
by this new set of factors. Although the presence of an AR binding site in the
Fgfr2 promoter suggests a possible direct interaction, the data do not rule
out the possibility of intermediate steps. These findings represent, to our
knowledge, the first example of how the developing genitourinary system
integrates cues from systemically circulating steroid hormones (e.g.,
androgen) with signaling by locally expressed growth factors.
Divergent functions of Fgf10-Fgfr2-IIIb signaling in limb and external genital development
Investigation of the molecular genetic control of external genital
development has only recently begun, a striking number of parallels with the
mechanisms of limb development have emerged. Maintenance of limb bud and
genital tubercle outgrowth is controlled by a specialized epithelial signaling
region (the apical ectodermal ridge and the distal urethral epithelium,
respectively), which is a source of Fgf8
(Crossley et al., 1996;
Haraguchi et al., 2000
;
Murakami and Mizuno, 1986
).
Wnt5a, which is expressed in a distal to proximal gradient in both
structures, is required for their distal outgrowth
(Yamaguchi et al., 1999
), as
are Hoxd13 and Hoxa13
(Kondo et al., 1997
;
Morgan et al., 2003
). A
polarizing region that expresses Shh in an asymmetric pattern is also
present in limb buds and genital tubercles, and loss of Shh results in loss of
digits and agenesis of the phallus (Chiang
et al., 1996
; Perriton et al.,
2002
). Curiously, although both Fgf10 and Fgfr2-IIIb are essential
for early outgrowth of the limb buds (De
Moerlooze et al., 2000
; Min et
al., 1998
; Revest et al.,
2001
; Sekine et al.,
1999
), our findings indicate that neither is required for
outgrowth of the genital tubercle. In the limb, Fgfr2-IIIb acts upstream of
Shh (Revest et al.,
2001
). In the genital tubercle, by contrast, Shh
expression does not require Fgf10-Fgfr2-IIIb signaling. These results, taken
together with our previous finding that Shh is an upstream regulator of
Fgf10 expression in the genital tubercle
(Perriton et al., 2002
),
indicate that the relative positions of Fgf10/Fgfr2-IIIb and
Shh are inverted in the pathways that control limb and genital
tubercle outgrowth. Although Shh is essential for maintenance of genital
tubercle outgrowth, it is not required for early induction of the genital
swellings (Perriton et al.,
2002
). Thus, the identity of the gene that initiates the process
of genital tubercle budding remains to be determined.
Implications for the etiology of hypospadias in humans
Humans with Beare-Stevenson cutis gyrata syndrome, which is characterized
in part by external genital defects such as hypospadias and bifid scrotum,
have mis-sense mutations in FGFR2
(Akai et al., 2002;
Przylepa et al., 1996
;
Vargas et al., 2003
;
Wang et al., 2002
). Our data
suggest that a defect in urethral epithelial maturation may underlie this
condition. Malformations of the urogenital tract also occur in Split
Hand/Split Foot syndrome, limb-mammary syndrome, ectrodactyly-ectodermal
dysplasia-cleft lip/palate (EEC) and Hay-Wells syndrome, all of which are
associated with mutations in p63, a homolog of the tumor suppressor gene p53.
Interestingly, it has recently been shown that the p63 sterile-
motif
regulates Fgfr2 splicing and is required for generation of the
IIIb isoform (Fomenkov et al.,
2003
). Although it remains unclear whether these malformations
result from deficiencies in epithelial stem cell proliferation, one possible
explanation for the associated genital malformations is an underlying
Fgfr2-IIIb deficiency. Finally, our findings highlight a mechanism that may
have implications for understanding the global increase in hypospadias
(Paulozzi et al., 1997
).
Fgfr2 and Fgf10 null mutations have widespread effects on
multiple organ systems (De Moerlooze et
al., 2000
; Min et al.,
1998
), making it highly unlikely that the large number of children
presenting with hypospadias carry mutations in any of the genes examined here.
However, our finding that antagonism of AR leads to downregulation of
Fgfr2-IIIb in mouse genitalia raises the possibility that exposure to
anti-androgenic compounds during pregnancy may lead to diminished Fgfr2
activity. This represents a crucial step towards understanding how the genetic
program for urethragenesis is affected by signals from the environment and the
endocrine system during genitourinary development.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/10/2441/DC1
* These authors contributed equally to this work
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