First published online 20 April 2005
doi: 10.1242/dev.01778
Development 132, 2441-2450 (2005)
Published by The Company of Biologists 2005
Development of the mammalian urethra is controlled by Fgfr2-IIIb
Anita Petiot1,*,
Claire L. Perriton2,*,
Clive Dickson1 and
Martin J. Cohn2,3,
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
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Fig. 1. Fgfr2-IIIb is expressed in urethral plate epithelium and prepuce
of the mouse genital tubercle. Using whole-mount in situ hybridization,
Fgfr2-IIIb transcripts were localized in external genitalia of
wild-type mouse embryos at E10.5 (A), E11.5 (B), E12.5 (C), E13.5 (D), E14.5
(E), E15.5 (F) and E16.5 (G). Arrows indicate the position of the urethral
plate epithelium and arrowheads indicate the preputial swellings. Ventral view
of the embryo is shown in A, and ventral views of the genital tubercle are
shown in B-G. Negative control using sense probe is shown in inset of C.
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Fig. 2. Fgfr2-IIIb-/- and Fgf10-/- mutants
develop hypospadias. Scanning electron micrographs of the ventral aspect of
male genital tubercles are shown in A-F and female genital tubercles are shown
in I,J. Scale bar: 100 µm. Arrows indicate position of urethral epithelium.
(A) Wild-type genital tubercle at E13.5. (B) Genital tubercle of
Fgfr2-IIIb-/- mutant at E13.5. Arrowhead indicates
precocious, ectopic opening of urethral plate and white arrow indicates deep
grove in urethral seam. (C) Wild-type genital tubercle at E15.5. Proximal
(arrow) and definitive distal (arrowhead) opening of the urethral plate are
indicated. (D) Genital tubercle of Fgfr2-IIIb-/- mutant at
E15.5 exhibits an open urethral plate along the proximal half of the penis and
agenesis of the prepuce ventrally. (E) Penis of wild-type embryo at E17. The
prepuce (pseudocolored green) has surrounded the glans (pseudocolored red)
ventrally, and the original proximal urethral meatus (arrow in C) has closed.
(F) Penis of Fgfr2-IIIb-/- mutant at E17 exhibits a
completely open urethra. Arrow indicates the dorsal midline (roof) of the
urethral plate, and the walls of the urethra now line the ventral surface of
the glans (red). Prepuce (green) is present laterally but not ventrally. (G,H)
Transverse sections through phallus of E19.5 wild-type (G) and
Fgfr2-IIIb-/- mutant (H) embryos. u, urethral tube; p,
prepuce; cc, corpus cavernosum. Arrows indicate the open urethra in the
Fgfr2-IIIb-/- mutant. (I,J) Hypospadias and agenesis of
ventral prepuce in external genitalia of Fgf10-/- mutants
at E15.5 (I) and E17.5 (J). Arrows indicate ectopic opening of urethra.
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Fig. 3. Expression of Fgf8 and Shh in the urethral epithelium of
wild-type and Fgfr2-IIIb-/- embryos. (A,B) Fgf8
expression in the distal urethral epithelium of male wild-type (A) and
Fgfr2-IIIb-/- (B) genital tubercles at E13.5. (C,D)
Shh expression in the urethral epithelium of male wild-type (C) and
Fgfr2-IIIb-/- (D) genital tubercles. The Shh
domain bifurcates proximally in the Fgfr2-IIIb-/- mutant
(D), where the urethral plate has opened. (E,F) Transverse sections through
the distal genital tubercle of E15.5 wild-type embryo, showing histological
organization (E) and Shh expression (F) in urethral epithelium
(boxed). (G,H) Transverse sections through the distal genital tubercle of
E15.5 Fgfr2-IIIb-/- mutant embryo reveal the lack of a
histologically distinct urethral epithelium (G) and the absence of
Shh expression (H).
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Fig. 4. Disorganization of the urethral plate in Fgfr2-IIIb-/-
mutants. Transverse histological sections through the genital tubercle of
wild-type (A,C) and Fgfr2-IIIb-/- mutant (B,D) genitalia.
Urethral epithelium is shown within the boxes and at higher magnification on
right sides of A and B. (A) At E13.5, cells in the wild-type urethral plate
are stratified and have a complex squamous organization. (B) Urethral plate of
Fgfr2-IIIb-/- mutant is a thin, unstratified plate of
cuboidal cells. (C) Proximal transverse section through wild-type phallus at
E15.5 showing separation of the bilaminar urethral plate to form a urethral
tube. Boxed area indicates urethral epithelium. (D) Proximal transverse
section through phallus of Fgfr2-IIIb-/- mutant showing
that by E15.5, epithelial organization of the urethra has been lost.
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Fig. 5. Loss of urethral progenitors and arrest of cell division in
Fgfr2-IIIb-/- mutant genitalia. Transverse sections
through phalli of wild-type (A,C,E) and Fgfr2-IIIb-/-
mutant (B,D,F) mice. Genitalia of male embryos are shown in A,B,E; genitalia
of female embryos are shown in C,D,F. (A) Immunolocalization of keratin 14
(K14) at E16.5 reveals the presence of urethral epithelial progenitor cells in
the basal layer of the wild-type urethra. (B) K14 staining is absent from the
urethral region of the Fgfr2-IIIb-/- mutant phallus. (C,D)
At E13.5, the proliferation marker Ki67 is seen in the urethral plate of wild
type (C) and Fgfr2-IIIb-/- mutant (D) embryos. (E) Ki67
staining showing sustained proliferation of urethral epithelial cells in
wild-type mouse at E15.5. (F) Ki67 staining is absent from the urethral region
of Fgfr2-IIIb-/- mouse. Boxes indicate position of
urethral epithelium.
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Fig. 6. Expression Fgfr2-IIIb and Fgf10 in urethral epithelium is
regulated by the androgen receptor. (A-F) Ventral views of whole genital
tubercles from male embryos cultured for 48 hours and processed for
whole-mount in situ hybridization with a Fgfr2-IIIb riboprobe. Arrows
indicate the position of urethral plate epithelium. (A) Fgfr2-IIIb
expression in the urethral epithelium of 48 hour control culture. (B-E)
Fgfr2-IIIb expression in genital tubercles cultured with the androgen
receptor antagonist flutamide at concentrations of 10-5 M (B),
10-4 M (C), 5-4 M (D) and 10-3 M (E). There
is a dose-dependent decrease in Fgfr2-IIIb expression, hypoplasia of
the prepuce and malformation of the glans. (F) Fgfr2-IIIb expression
and normal morphology is rescued in urethral plate of genital tubercle treated
with flutamide (5-4 M) by addition of dihydrotestosterone (DHT;
5-6 M) (compare F with D). (G) Partial sequence of Fgfr2
promoter (nucleotides 1041-1610) showing the presence of stereotypical
androgen response element (ARE) in red. (H-J) Ventral views of female genital
tubercles cultured for 48 hours and probed for expression of Fgf10.
(H) Control culture. (I,J) Fgf10 expression persists in mesenchyme of
the glans after treatment with 10-5 M flutamide (I), but is
undetectable after treatment with 10-3 M flutamide (J). Arrowheads
in H,I indicate Fgf10 expression in mesenchyme.
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Fig. 7. Distribution of androgen receptor in Fgfr2-IIIb-/-
genitalia. Immunolocalization of androgen receptor (AR) in wild-type (A) and
Fgfr2-IIIb-/- (B) genitalia of male embryos at E16.5. (A)
AR is detected in the preputial mesenchyme (p), corpus cavernosum (cc) and
urethra (u) of wild-type mouse. (B) Distribution of AR in
Fgfr2-IIIb-/- mutant genitalia is similar to that seen in
wild type (compare A with B). AR is present in urethral region (u) of mutant
phallus, even though epithelial organization has been lost. Insets show
sections taken anterior and proximal to the external genitalia, within the
body wall, and reveal the presence of AR in the anterior urethra of wild-type
(A) and Fgfr2-IIIb-/- (B) mice.
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© The Company of Biologists Ltd 2005
