Coordinated activity of Spry1 and Spry2 is required for normal
development of the external genitalia
Saunders T. Chinga,b, Gerald R. Cunhab, Laurence S. Baskinb,
M. Albert Bassonc, Ophir D. Kleina,d,e,f,n
aDepartment of Orofacial Sciences, University of California, San Francisco, United States
bDepartment of Urology, University of California, San Francisco, United States
cDepartment of Craniofacial Development and Stem Cell Biology, King's College, London, UK
dProgram in Craniofacial and Mesenchymal Biology, University of California, San Francisco, United States
eInstitute for Human Genetics, University of California, San Francisco, United States
fDepartment of Pediatrics, University of California, San Francisco, United States
a r t i c l e i n f o
Received 13 August 2013
Received in revised form
5 December 2013
Accepted 10 December 2013
Available online 18 December 2013
a b s t r a c t
Development of the mammalian external genitalia is controlled by a network of signaling molecules and
transcription factors. Because FGF signaling plays a central role in this complicated morphogenetic
process, we investigated the role of Sprouty genes, which are important intracellular modulators of FGF
signaling, during embryonic development of the external genitalia in mice. We found that Sprouty genes
are expressed by the urethral epithelium during embryogenesis, and that they have a critical function
during urethral canalization and fusion. Development of the genital tubercle (GT), the anlage of the
prepuce and glans penis in males and glans clitoris in females, was severely affected in male embryos
carrying null alleles of both Spry1 and Spry2. In Spry1?/?;Spry2?/?embryos, the internal tubular urethra
was absent, and urothelial morphology and organization was abnormal. These effects were due, in part,
to elevated levels of epithelial cell proliferation in Spry1?/?;Spry2?/?embryos. Despite changes in
overall organization, terminal differentiation of the urothelium was not significantly affected. Character-
ization of the molecular pathways that regulate normal GT development confirmed that deletion of
Sprouty genes leads to elevated FGF signaling, whereas levels of signaling in other cascades were largely
preserved. Together, these results show that levels of FGF signaling must be tightly regulated during
embryonic development of the external genitalia in mice, and that this regulation is mediated in part
through the activity of Sprouty gene products.
& 2013 Elsevier Inc. All rights reserved.
Abnormalities of the external genitalia are among the most
common birth defects in humans. Hypospadias is a condition in
which the urethral meatus is abnormally placed along the ventral
side of the penis rather than at the distal tip. It affects approxi-
mately 1 in every 250–300 live male births, and in spite of the best
efforts at surgical reconstruction, it can result in severe psycho-
sexual problems and voiding abnormalities (Paulozzi et al., 1997).
Studies of patients with hypospadias have identified rare muta-
tions in several genes (Chen et al., 2007; Fukami et al., 2006; Silver
and Russell, 1999), but the majority of cases remain idiopathic.
Despite the high incidence of congenital anomalies affecting the
external genitalia, the molecular mechanisms that control its
development are not completely understood.
Development of the external genitalia in humans begins
around 4 weeks of gestation, when mesenchymal cells migrate
to and cluster at the border of the cloacal membrane. These cells
form the genital tubercle (GT), which is the anlage of the male
glans penis and female glans clitoridis. Appearance of the GT is
accompanied by formation of paired genital swellings flanking the
cloacal membrane. Shortly thereafter, urogenital folds are formed
along the lateral edges of the cloacal membrane. Up until week 12,
the male and female external genitalia are indistinguishable.
Subsequent differentiation of male genitalia is hormone-driven.
Androgens act on the genital tissues to induce outgrowth of the
GT, remodeling of the urogenital folds into a penis with an
internalized urethra, and fusion of the genital swellings to form
the scrotum (Baskin, 2004; Cunha and Baskin, 2004).
GT morphogenesis in mice closely parallels that in humans
(Fig. 1). Initiation of GT formation occurs around embryonic day
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nCorresponding author at: Department of Orofacial Sciences, University of
California, San Francisco, United States. Tel: +1 415 476 4719.
E-mail address: firstname.lastname@example.org (O.D. Klein).
Developmental Biology 386 (2014) 1–11
(E) 10.5 with the emergence of bilaterally paired genital swellings
composed of mesenchyme and overlying ectoderm encompassing
an endoderm-derived epithelium. The genital swellings fuse into a
single GT by E11.75. An endoderm-derived urethral plate epithe-
lium spans the proximo-distal axis of the GT along the midline.
Preputial swellings, which give rise to the prepuce, appear lateral
to the GT at E13.5. By E16.5, the preputial swellings have fused at
the ventral midline of the tubercle, enclosing the urethral plate
epithelium and forming the internalized tubular urethra (Perriton
et al., 2002). At this stage, the GT is ambisexual and the male organ
is indistinguishable from the female.
A network of signaling molecules and transcription factors
mediates the epithelial–mesenchymal interactions that pattern
the GT. The distal urethral epithelium (DUE) directs outgrowth
and patterning of the GT by acting as a primary signaling center,
similar to the apical ectodermal ridge of the developing limb.
Removal of the DUE results in reduced outgrowth of the embryo-
nic GT and downregulation of mesenchymal Fgf10, an important
morphogen during GT development (Haraguchi et al., 2000; Ogino
et al., 2001; Satoh et al., 2004). Previous studies have identified
several genes expressed in the urethral epithelium (urothelium)
that promote growth and differentiation of the GT. For example,
Sonic hedgehog (Shh) regulates expression of several signaling
molecules and transcription factors in the surrounding mesench-
yme, including Fgf10, Bmp4, Wnt5a, Nog, Msx2, Hoxa13 and Hoxd13
(Lin et al., 2009). Altered expression of any of these mesenchymal
factors disrupts crucial processes such as cell proliferation, apop-
tosis, and responsiveness to extracellular molecular signals, ulti-
mately leading to abnormal formation of the GT (Haraguchi et al.,
2000; Lin et al., 2009; Morgan et al., 2003; Satoh et al., 2004;
Suzuki et al., 2003; Warot et al., 1997; Yamaguchi et al., 1999).
The Fibroblast Growth Factor (FGF) family of secreted ligands
plays a particularly important role in epithelial–mesenchymal
interactions during GT morphogenesis. Deletion of either Fgf10
from the GT mesenchyme or its cognate receptor Fgfr2-IIIb from
the urothelium results in an ectopic opening along the ventral
surface of the GT at a time when the preputial folds should
normally have fused to enclose the urethra (Haraguchi et al.,
2000; Satoh et al., 2004). Decreased FGF signaling impairs cell
proliferation in the GT, affects organization of the urethral plate
epithelium, and reduces the expression of Shh in the epithelium
(Petiot et al., 2005).
Based on its expression in the DUE, it was initially thought that
Fgf8 was responsible for GT outgrowth and patterning. However,
while Fgf8 is strongly expressed in the DUE at the earliest stages of
GT outgrowth, deletion of Fgf8 from the DUE did not appear to
have any adverse effects on the genitalia (Haraguchi et al., 2000;
Seifert et al., 2009b). Interestingly, ectopic expression of Fgf8 led to
overproliferation of cells in the GT and aberrant morphology
during embryogenesis, suggesting that excessive amounts of FGF
can also cause genital defects (Lin et al., 2013). Despite its strong
expression during GT outgrowth, the lack of phenotypic defects in
Fgf8-null mice suggests that redundancy of FGF signals in the GT
may be important. This has recently been shown to be the case for
FGF receptors present in the developing GT. Deletion of both Fgfr1
and Fgfr2 from the urethral epithelium resulted in abnormal
maturation of the urethral epithelium, while ablation of FGF
signaling in the GT mesenchyme led to significantly decreased
outgrowth. These results show that FGF signals control different
developmental processes in the urethral epithelium as opposed to
the surrounding GT mesenchyme (Lin et al., 2013).
Sprouty genes are intracellular inhibitors of FGF-activated
receptor tyrosine kinase (RTK) signaling. First discovered in a
screen for mutations affecting Drosophila tracheal branching,
Sprouty genes have since been shown to be involved in the
development of several organs via modulation of RTK signaling
induced by FGF, EGF, or GDNF (Basson et al., 2005; Chi et al., 2006,
2004; Hacohen et al., 1998; Klein et al., 2006; Mahoney Rogers
et al., 2011; Mailleux et al., 2001; Minowada et al., 1999; Shim
et al., 2005). The function of Sprouty genes as inhibitors of FGF
signaling led us to hypothesize that they function in GT develop-
ment. We found that the combined deletion of Spry1 and Spry2 in
the male mouse embryo profoundly affected genital morphogen-
esis. Our results show that Sprouty genes are critical regulators of
FGF signaling during embryonic patterning of the male GT and are
necessary for the normal development of the urethral epithelium
and formation of the tubular urethra.
Materials and methods
Mouse maintenance and treatment
All mouse studies were carried out under an approved protocol
in strict accordance with the policies and procedures established
by the University of California, San Francisco (UCSF) Institutional
Animal Care and Use Committee (protocol AN084146). Mice were
maintained in a temperature-controlled facility with access to food
and water ad libitum. Spry1?/?, Spry2?/?, and Fgf10?/?mutant
mouse alleles have been described previously (Basson et al., 2005;
Min et al., 1998; Shim et al., 2005). Spry1?/?;Spry2?/?double
knockout embryos were generated by breeding male mice homo-
zygous for the β-Actin-Cre transgene and heterozygous for both
Spry1 and Spry2 (β-Actin-CreTg/Tg;Spry1þ/?;Spry2þ/?) to female
mice homozygous for floxed alleles of both Spry1 and Spry2
(Spry1fl/fl;Spry2fl/fl) (Petersen et al., 2011). To generate embryos at
specific timepoints, adult mice were mated overnight and females
were checked for a vaginal plug in the morning. The presence of a
vaginal plug was designated E0.5. For cell proliferation assays,
pregnant female mice were administered a 100 μL dose of BrdU
(10 mg/mL) via intraperitoneal injection. Mice were then sacrificed
2 h after injection.
Quantitative real-time PCR
Total RNA was isolated from E14.5 male lungs, kidneys, and GTs
using Trizol, and 500 ng from each sample was used to synthesize
cDNA (M-MLV RT, Promega, Madison, WI). Quantification of Spry1,
Spry2, Spry4, Etv4, and Etv5 was performed using SsoAdvanced
SYBR Green Supermix (Bio-rad, Hercules, CA) carried out in a Bio-
rad C1000 Touch thermal cycler attached to a CFX96 Realtime
optical detection module. Rpl19 (60S ribosomal protein L19) was
used to normalize gene expression levels. Three to four biological
samples were used to quantify transcript levels for each gene, and
each reaction was run in triplicate. The Student's t-test was used to
determine whether changes in Etv4 and Etv5 gene expression
between control and Sprouty mutants were significant.
Fig. 1. Timeline of mouse external genital development. Cartoons represent a view
from the ventral side of the mouse external genitalia during embryonic develop-
ment. Beginning at E10.5, paired genital swellings (GS) appear. By E12.5, genital
swellings have fused to form the genital tubercle (GT). Outgrowth of the GT
continues through E14.5 and bilateral preputial swellings (PS) have also appeared.
By E16.5, the preputial swellings have fused at the ventral midline to form a
continuous prepuce (P) that surrounds the GT.
S.T. Ching et al. / Developmental Biology 386 (2014) 1–11
Histology and 3D reconstruction
GT samples were fixed in 4% paraformaldehyde overnight at
4 1C and processed for paraffin sections. 7 μm paraffin sections
were cut and stained with hematoxylin and eosin for histological
analysis. For 3-dimensional reconstruction of control and mutant
GTs, brightfield images of stained serial GT sections were collected
using a Leica DM5000B upright microscope connected to a Leica
DFC500 digital camera. Images were then analyzed, annotated,
and rendered 3-dimensionally using BioVis3D software (Montevi-
Scanning electron microscopy
Tissue was fixed in 0.1 M sodium cacodylate buffer, 1% osmium
tetroxide in 0.1 M sodium cacodylate, and then dehydrated for
scanning electron microscopy (SEM). Specimens were dried in a
Tousimis AutoSamdri 815 Critical Point Dryer and scanning elec-
tron micrographs were obtained using a Hitachi TM-1000 scan-
ning electron microscope. All SEM was performed at the University
of California, Berkeley Electron Microscope Lab.
In situ hybridization and immunohistochemistry
Samples were collected and fixed in 4% paraformaldehyde
overnight at 4 1C, immersed in 30% sucrose/PBS overnight at
4 1C, and embedded in O.C.T. compound (Sakura Finetek, Torrance,
CA). 10 μm frozen sections were cut using a Microm 550 cryostat
and hybridized to DIG-labeled RNA probes for in situ detection of
RNA transcripts. Sections were treated with 10 μg/mL of protei-
nase K and acetylated prior to hybridization with probe. DIG-
labeled RNA probes were synthesized from plasmids containing
full-length cDNA or fragments of Spry1, Spry2, Spry4, Fgf10, Fgfr1,
Fgfr2, Etv4, Etv5, Aldh1a2, Shh, Bmp4, and Wnt5a.
Cell proliferation was assessed using immunohistochemical detec-
tion of BrdU on paraffin sections using a rat monoclonal antibody
specific for BrdU (Abcam, Cambridge, MA; 1:1000 dilution). Slides
were treated with 0.2 N HCl in water prior to applying antibody, and
positive cells were visualized by diaminobenzidine (DAB) staining
after incubation with an HRP-conjugated secondary antibody. Cell
proliferation was quantified in coronal sections of control and mutant
GTs by calculating the ratio of BrdU-positive cells to the total number
of nuclei in the urethral epithelium as determined by counterstaining
with hematoxylin. Proliferation was analyzed in two male embryos
Fig. 2. Sprouty and FGF pathway genes are expressed in the embryonic GT. In situ hybridization was used to determine expression patterns of Sprouty and FGF pathway
genes in sagittal sections of E14.5 male mouse GTs. Spry1, Spry2, and Spry4 mRNA transcripts primarily aggregate in the urethral epithelium, with higher levels of Spry1 and
Spry2 expression relative to Spry4 (A–C0). Spry1, but not Spry2, is detected in a small mesenchymal region on the dorsal region of the distal GT (A and B), while low levels of
Spry4 are diffusely distributed throughout the GT mesenchyme (C). Quantitative PCR detection of Spry1, Spry2, and Spry4 transcripts in the male GT at E14.5 show higher
expression levels for Spry1 and Spry2 in comparison to Spry4 (D). Both Fgf10 and Fgfr1 are expressed in the GT mesenchyme adjacent to the urethral epithelium, while Fgfr2
expression overlaps with Sprouty in the urothelium (E–G0). Etv4 and Etv5 expression is also restricted to the epithelium (H–I0). Black boxes in low-magnification images (A–C,
E–I) represent regions shown under high magnification (A0-C0, E0-I0). Urethral epithelium is outlined with black dots in high-magnification panels (A0–C0, E0–I0). Scale bars,
400 μm (A–C, E–I), 50 μm (A0–C0, E0–I0). D: dorsal; V: ventral; epi: epithelium; and mes: mesenchyme.
S.T. Ching et al. / Developmental Biology 386 (2014) 1–11
of each genotype, and a minimum of five representative BrdU-stained
sections from each GT was counted to attain cell counts.
Tissue was prepared for paraffin embedding by ethanol dehy-
dration and xylene treatment, 7 μm paraffin sections were cut on a
Microm HM325 microtome. Uroplakin III (Nichirei Bioscience Inc.,
Tokyo, Japan; 1:1000 dilution) and phospho-MEK1/2 (Cell Signal-
ing Technology, Danvers, MA; 1:200 dilution) were detected by
immunohistochemistry using standard protocols.
Sprouty genes are expressed in the developing GT and reflect active
FGF signaling in the urethral epithelium
There are four mammalian Sprouty gene family members in the
mouse genome, and these have differing patterns of expression in
adult and embryonic tissues. Spry1, Spry2, and Spry4 are expressed
in a number of developing tissues, including heart, lung, brain, and
skeletal muscle; whereas Spry3 is primarily expressed in the adult
testis and brain (Minowada et al., 1999). We first assayed Sprouty
gene expression in the embryonic GT using in situ hybridization to
detect transcripts of Spry1, Spry2, and Spry4. Spry1 and Spry2
expression was primarily localized to the urethral epithelium at
E14.5 (Fig. 2A–B0), a stage at which the GT and urethral plate
epithelium undergo significant morphogenetic changes. Faint
expression of Spry1 was also detected in the distal mesenchyme
on the dorsal surface of the GT (Fig. 2A0). Expression of Spry4 in the
GT mesenchyme and urethral epithelium was low (Fig. 2C and C0),
suggesting that Spry1 and Spry2 are the primary Sprouty genes
acting in embryonic GT development.
Quantitative real-time PCR was also used to measure levels of
Sprouty gene expression in the embryonic male GT. By qPCR, we
confirmed that Spry1 and Spry2 were expressed at higher levels in
the GT compared to Spry4, and Spry2 transcripts were the most
abundant, with an approximately 2-fold increase relative to Spry1
(Fig. 2D). Other known Sprouty-expressing tissues were also
analyzed by qPCR to determine expression levels relative to the
GT. Expression of Spry1 in the GT was higher than Spry1 levels in
the lung, but less than the kidney. By comparison, Spry2 expres-
sion was higher in the GT relative to both kidney and lung. Finally,
Spry4 expression was low in the lung, kidney, and GT, although
levels were comparable between kidney and GT (Supplemental
Sprouty gene products are intracellular inhibitors of FGF
signaling that act in the cells in which they are expressed. To
determine whether Sprouty expression domains overlap with
regions of FGF signaling in the GT, we assayed the expression
patterns of Fgf10, Fgfr1, and Fgfr2, all of which are critical for
normal urogenital development (Haraguchi et al., 2000; Petiot
et al., 2005; Satoh et al., 2004; Lin et al., 2013). In accordance with
previous findings, Fgf10 transcripts were localized to the GT
mesenchyme at E14.5, with levels appearing to be highest in the
mesenchyme adjacent to the urothelium (Fig. 2E and E0). Expres-
sion of Fgfr1 was also largely confined to the mesenchymal
compartment of the GT, but the regions of highest expression
appeared to be in the distal mesenchyme along the dorsal and
ventral GT (Fig. 2F and F0). By contrast, expression of Fgfr2, the
primary receptor for FGF10, overlapped with Spry1 and Spry2
expression in the urethral epithelium (Fig. 2G and G0). The
expression of Fgfr2 suggests that the urethral epithelium is the
primary target for FGF10 protein produced in the surrounding
Fig. 3. GT development is abnormal in Spry1?/?;Spry2?/?and Fgf10?/?embryos. Scanning electron micrographs and histological sections of Spry1?/?;Spry2?/?and
Fgf10?/?GTs at different stages of development reveal abnormal organogenesis. Gross morphology of the male GT in Spry1?/?;Spry2?/?embryos is similar to controls at
E14.5 (A and B). In male E14.5 Fgf10?/?embryos, preputial swellings are less prominent (C, blue asterisks) and the urethral meatus appears significantly larger than controls
(yellow arrowhead). Fusion of the preputial and labioscrotal folds along the ventral surface of the GT is disrupted in E16.5 male Spry1?/?;Spry2?/?embryos, resulting in the
absence of an internalized urethra in the proximal GT (E, red arrow). Fgf10?/?GTs are hypoplastic with an ectopic urethral opening at the base of the GT (F, green arrow), but
fusion of the labioscrotal folds is not extensively affected. Scale bar, 500 μm. Coronal histological sections of E18.5 control and mutant GTs show that many features of the
embryonic GT are preserved in both Spry1?/?;Spry2?/?and Fgf10?/?embryos, despite the absence of an internal tubular urethra (G–I, asterisks). Mesenchymal
condensations, preputial glands, preputial stroma, and glandular epithelium are not visibly altered (H and I). GT: genital tubercle; PS: preputial swelling; Pre: prepuce; LS:
labioscrotal fold; UO: urethral opening; and Ur: urethra. Scale bar, 400 μm.
S.T. Ching et al. / Developmental Biology 386 (2014) 1–11
mesenchyme. To identify the regions of active FGF signaling in the
GT, we probed for Etv4 and Etv5, two members of the ETS family of
transcription factors that are critical downstream effectors of FGF
signaling. Expression of both Etv4 and Etv5 was essentially
restricted to the urethral epithelium, indicating that this is the
primary tissue target for FGF ligands in the developing GT
Deletion of both Spry1 and Spry2 in the urethral epithelium leads to
abnormal development of the male mouse GT
To understand the role of Sprouty genes in the development of
the GT, we analyzed mice carrying mutations in one or more
Sprouty genes. Based on our findings that Spry1 and Spry2 were
the two predominant Sprouty family members expressed in the
urethral epithelium, we examined GT development in embryos
lacking Spry1 (Spry1?/?), Spry2 (Spry2?/?), or both Spry1 and
Spry2 (Spry1?/?;Spry2?/?). No defects of GT development were
apparent in embryonic or adult mice lacking a single Sprouty gene
(data not shown), but embryos in which both Spry1 and Spry2
were deleted showed gross morphological abnormalities in male
GT development. At E16.5 in control male embryos, the preputial
swellings have encircled the GT by growing ventrally and fusing
along the ventral midline, while the more proximal labioscrotal
swellings have also fused along the midline to form a continuous
scrotum (Fig. 3D). In Spry1?/?;Spry2?/?male embryos, fusion of
both the preputial and labioscrotal folds was interrupted, resulting
in a bifid scrotum and an ectopic opening along the proximal-
ventral surface of the GT (red arrow, Fig. 3E).
Histological examination of E18.5 Sprouty mutant GTs revealed
the complete absence of an internal urethra and preputial space
(Fig. 3H; asterisk); whereas control GTs contained an internal
tubular urethra (Fig. 3G). Other features of GT development such
as mesenchymal condensation, formation of preputial glands and
stroma, and appearance of the epithelium covering the GT
mesenchyme remained largely intact. Interestingly, we only
observed GT defects in male Spry1?/?;Spry2?/?embryos, whereas
GT development in female Spry1?/?;Spry2?/?embryos appeared
normal (Supplemental Fig. 2). To determine if abnormal GT
morphology was present at earlier stages of development, we also
analyzed E12.5 and E14.5 Spry1?/?;Spry2?/?embryos and found
no observable defects (data not shown, Fig. 3A and B). Overall size
of the GT in Sprouty mutants also did not appear significantly
affected, indicating that Sprouty genes do not play a major role in
Absence of an internal urethra has been described in mice
carrying mutations in Bmp7 or EphB2 as a consequence of
incomplete urorectal septation (Dravis et al., 2004; Wu et al.,
2009). We therefore investigated whether this was the cause of
the ectopic opening in Spry1?/?;Spry2?/?GTs. Histological ana-
lysis of sagittal E16.5 embryo sections showed two distinct
epithelial-lined sinuses separated by a urorectal septum extending
to the base of the GT, indicating that extension of the urorectal
septum to the ectoderm was complete (Supplemental Fig. 3).
To compare the effects of elevated FGF signaling in Sprouty
mutants with mutants in which FGF signaling is decreased, we
examined GT development in Fgf10?/?embryos. GT defects that
resembled those in the Sprouty mutants have previously been
characterized in these Fgf10?/?embryos (Haraguchi et al., 2000;
Satoh et al., 2004), although some notable differences were found.
At E14.5, the preputial swellings in Fgf10?/?embryos were
reduced in size relative to the GT; whereas the proximal opening
of the urethra was significantly larger compared to both control
and Spry1?/?;Spry2?/?embryos (Fig. 3C; yellow arrowhead). The
E16.5 GT in Fgf10?/?embryos was also considerably smaller than
either control or Sprouty mutant GTs, whereas the ectopic opening
at the base of the GT did not extend into the scrotal region,
indicating that the effects of Fgf10 deletion were restricted to GT
growth and urethral development. Despite these gross morpholo-
gical differences between Fgf10?/?and Spry1?/?;Spry2?/?GTs at
earlier stages of embryonic development, organization of Fgf10?/?
GTs was similar to Spry1?/?;Spry2?/?GTs. Histological analysis of
Fgf10?/?GTs at E18.5 showed that, while the overall size of the GT
was small in comparison to the Spry1?/?;Spry2?/?GT, both
mutants lacked an internal urethra. Formation of the surrounding
mesenchyme, preputial stroma, and preputial glands was unper-
turbed (Fig. 3H and I). In contrast to the male-specific defect seen
in Spry1?/?;Spry2?/?embryos, GT defects were found in both
male and female Fgf10?/?embryos (data not shown).
Because Spry1 was also expressed at low levels in the GT
mesenchyme, we performed tissue-specific deletion of Spry1 and
Spry2 to determine whether they regulate GT development
through the urethral epithelium, GT mesenchyme, or both. We
generated ShhCreEGFP;Spry1?/?;Spry2?/?embryos to remove Spro-
uty genes in the endoderm-derived urethral epithelium and
Tbx4Cre;Spry1?/?;Spry2?/?embryos to delete Sprouty genes in
the genital mesenchyme (Harfe et al., 2004; Luria et al., 2008).
GT development in Tbx4Cre;Spry1?/?;Spry2?/?embryos was indis-
tinguishable from control embryos; whereas GT morphology in
male ShhCreEGFP;Spry1?/?;Spry2?/?embryos phenocopied the glo-
bal Sprouty knockout embryos, confirming that Spry1 and Spry2
are critically required in the urethral epithelium, but not in the GT
(Supplemental Fig. 4).
morphogenesisof the GT
Deletion of Spry1 and Spry2 affects cell proliferation in the urethral
epithelium, but expression of urothelial markers is not perturbed
To construct a more detailed picture of how Sprouty gene
deletion affected the development of the GT, we generated 3-
dimensional renderings of the GT, urethra, and urethral lumen
from serial histological sections at E14.5. In control embryos, the
urethra runs along the ventral midline of the GTand forms a three-
horned tube at the proximal end. The lumen forms in a proximal-
to-distal direction, and it has not reached the distal end of the GT
at this stage (Fig. 4A and A0). The urethra in Sprouty mutant
embryos was significantly enlarged at the proximal end and did
not form recognizable horns. Moreover, the lumen failed to extend
as far into the distal GT as in controls (Fig. 4B and B0). By contrast,
the urethra in Fgf10?/?GTs was hypoplastic, with a less defined
proximal urethra and an ectopic opening at the base of the GT
(Fig. 4C and C0; black arrow). Although the gross morphology of
Spry1?/?;Spry2?/?GTs at E14.5 was not significantly affected,
histological analysis revealed abnormal organization of the
urothelium. In normal GTs, the urothelium is a 2–3 cell-layered
transitional epithelium, with cuboidal or columnar-shaped cells
lining the basal lamina and flattened, squamous-like cells lining
the urethral lumen (Fig. 4D). Compared to controls, the urothelium
in Spry1?/?;Spry2?/?embryos was thicker, the cells lining the
urethral lumen did not form a smooth, flattened surface, and basal
cells appeared slightly elongated (Fig. 4E). Interestingly, deletion of
Fgf10 had the opposite effect on the urothelium. In Fgf10?/?
embryos at E14.5, the urothelium was thin in comparison to
controls and was composed of only 1–2 layers of tightly packed
cells (Fig. 4F).
We examined the fate of the urothelium in late-stage embryos
to determine whether deletion of Sprouty genes led to abnormal
differentiation or degradation of the epithelium normally lining
the urethra. Detection of keratin 14 (K14), an epithelial marker,
showed that in control GTs, the urothelium is composed of a basal
layer of cuboidal, K14-positive cells and an apical layer of squa-
mous, K14-negative cells (Fig. 4G and G0; red and black
S.T. Ching et al. / Developmental Biology 386 (2014) 1–11
Fig. 4. Altered levels of FGF signaling lead to changes in urothelial morphology and structure, but not cell identity. Serial histological sections were used to create
3-dimensional renderings of the GT in control and mutant embryos (A–C0). Coronal view of the urethra (red) in control embryos shows a three-horned structure at the
proximal end of the GT (A). A sagittal view shows that the urethral lumen (white) has extended approximately halfway to the distal end of the GT (A0). Both the Spry1?/?;
Spry2?/?urethra and urethral lumen are dramatically expanded at the proximal end (B and B0); whereas the urethral epithelium in Fgf10?/?GTs is hypoplastic and the
proximal urethral opening fails to close (C, black arrow). Black dotted lines in A0–C0indicate where sections were collected for (D–F). Coronal sections of the urethra in E14.5
control and mutant male GTs were stained with hematoxylin and eosin (D–F). In control GTs, the urethral epithelium is composed of a semi-stratified transitional epithelium
with columnar cells lining the basal membrane and flattened cells lining the lumen (D). In Spry1?/?;Spry2?/?GTs the urothelium is significantly thicker with few luminal
cells exhibiting a squamous morphology (E). The urethral epithelium in Fgf10?/?GTs consists of only a thin layer of cuboidal cells (F). Urethral epithelium is outlined in black
dotted lines. Scale bar, 30 μm. Characterization of epithelium was done by immunohistochemical staining for K14 (G–H0) and uroplakin III (I and J) on coronal sections of
E18.5 male control and Spry1?/?;Spry2?/?GTs. Urothelium consists of basal K14-positive cuboidal cells (G0; black arrowheads) and luminal K14-negative squamous cells (G0;
red arrowheads). High magnification of K14-stained epithelium in control and Spry1?/?;Spry2?/?GTs (G0and H0), dotted lines separate poorly stratified ventral-medial
epithelium from lateral epithelium resembling ectoderm-derived epidermis (H0). Scale bars, 200 μm (G and H), 30 μm (G0and H0), 50 μm (I and J).
S.T. Ching et al. / Developmental Biology 386 (2014) 1–11
arrowheads). In Spry1?/?;Spry2?/?embryos, the epithelium cov-
ering the ventral-medial aspect of the GT appeared less differ-
entiated based on its rounded morphology and poor stratification.
Nevertheless, positive staining of K14 in the basal cells indicated
that the early stages of epithelial differentiation had occurred
(Fig. 4H and H0). To determine whether terminal differentiation of
the urothelium was ultimately affected in Sprouty mutants, we
also stained for uroplakin III, a specific marker for differentiated
urothelial cells. In control embryos, uroplakin III was expressed on
the apical side of cells lining the urethral lumen (Fig. 4I). Uroplakin
III staining was similarly detected on the apical surface of
epithelial cells lining the ventral surface of the Spry1?/?;Spry2?/
?GTs, indicating that the epithelium retained its urothelial
identity even in the absence of both Sprouty genes (Fig. 4J).
One important function of FGF signaling is to regulate cell
proliferation (reviewed in (Turner and Grose, 2010)). In particular,
FGF10 is known to induce proliferation in urothelial cells and
prostate epithelium (Bagai et al., 2002; Thomson and Cunha,
1999); whereas a decrease in cell proliferation was observed in
the urothelium of Fgfr2-IIIb?/?embryos (Petiot et al., 2005). This
led us to ask whether the hyperplasia of the urethral epithelium at
E14.5 in Spry1?/?;Spry2?/?embryos was due to elevated levels of
cell proliferation. We measured the percentage of proliferating
urothelial cells by counting the number of BrdU-positive cells in
the urethral epithelium and comparing it to the total number of
urothelial cells in multiple coronal sections of the GT at E14.5. Cell
proliferation was elevated by approximately 30% in Spry1?/?;
Spry2?/?embryos relative to littermate controls (Fig. 5A, B, and
D). Although previous studies have shown that injecting mice with
recombinant FGF10 caused an increase in the number of dividing
cells in the urothelium, an in vivo analysis of proliferation had not
been performed in embryos lacking Fgf10. Therefore, we quantified
the number of BrdU-positive cells in the urethra of Fgf10?/?
embryos and found that the percentage of cells undergoing cell
division was decreased by approximately one-third (Fig. 5C and D).
Levels of cell death were also measured by TUNEL staining in
control and mutant GTs to determine whether changes in apop-
tosis were responsible for the abnormal urethral development
observed in Sprouty and FGF mutants, but no significant differ-
ences were found (data not shown). Taken together, these results
demonstrate that while the effects of increased or decreased FGF
signaling may have opposing effects at the cellular level, they can
result in similar morphological defects at the organ level.
Activation of MAPK signaling is elevated in Spry1?/?;Spry2?/?GTs,
but other signaling pathways appear unperturbed
Because Sprouty genes are important inhibitors of FGF signal-
ing, we hypothesized that abnormal GT morphology in Sprouty
mutant embryos may be a result of aberrant regulation of the FGF
pathway. To test this, we examined whether activation of signaling
components downstream of the FGF receptor was greater in
Spry1?/?;Spry2?/?GTs. We first investigated whether levels of
phosphorylated MEK1/2, the activated form of two key MAPK
protein kinases, was elevated in Sprouty mutants. In control GTs,
throughout the urethral epithelium but was absent in the sur-
rounding genital mesenchyme (Fig. 6A). In Spry1?/?;Spry2?/?
GTs, phospho-MEK1/2 intensity was markedly increased through-
out the epithelium, suggesting that activation of MAPK signaling
was increased in the absence of Sprouty genes (Fig. 6B).
We next asked if transcriptional regulation of gene targets down-
stream of FGF signaling was also elevated in Sprouty mutant GTs. Etv4
and Etv5, two members of the ETS family of transcription factors, are
downstream targets of FGF signaling whose expression reflects the
levels of FGF activity (Kobberup et al., 2007; Liu et al., 2003).
Fig. 5. Levels of cell proliferation are affected in Spry1?/?;Spry2?/?and Fgf10?/?GTs. Representative coronal sections of E14.5 male GTs from control, Spry1?/?;Spry2?/?,
and Fgf10?/?embryos stained for BrdU (brown) and counterstained with hematoxylin (blue) to visualize cells undergoing proliferation (A–C). Epithelium is outlined in black
dotted lines. Scale bar, 50 μm. The proportion of urothelial cells undergoing proliferation was calculated by quantifying the number of BrdU-positive cells and dividing by the
total number of epithelial cells. A higher percentage of cells in the urethral epithelium of Spry1?/?;Spry2?/?GTs is undergoing cell division compared to controls, whereas a
lower percentage of proliferating cells is seen in the Fgf10?/?urethra (D).npo0.05.
S.T. Ching et al. / Developmental Biology 386 (2014) 1–11
Expression of both genes appeared to be greater in the urothelium of
Spry1?/?;Spry2?/?E14.5 male GTs compared to controls (Fig. 6C–F).
Quantification of Etv4 and Etv5 RNA transcript levels by qPCR in
mutant and control GTs at this stage demonstrated that expression of
Etv4 was significantly increased in Spry1?/?;Spry2?/?male GTs.
Although Etv5 expression also showed a slight increase, our data did
not reach statistical significance (Fig. 6G). Collectively, these data
support the hypothesis that deletion of both Spry1 and Spry2 in the
GT leads to reduced inhibition of the FGF pathway and abnormal
upregulation of MAPK signaling and transcriptional activation of
Initiation, outgrowth, and patterning of the mammalian GT
require a number of important signaling molecules and transcrip-
tion factors, and disruption of these signaling pathways may lead
to abnormal formation of the GT. Therefore, we sought to establish
whether expression of genes involved in GT development was
altered in Spry1?/?;Spry2?/?embryos. One of the central players
in genital morphogenesis is the Hedgehog pathway. Shh is
expressed in the urethral plate epithelium and urothelium
throughout embryonic development, and controls the expression
of numerous genes in the GT mesenchyme including Ptc1, Bmp4,
Fgf10, Hoxd13, Hoxa13, Nog, Fgf8, Wnt5a, and Msx1 (Haraguchi
et al., 2007; Lin et al., 2009; Perriton et al., 2002). Deletion of Shh
early in embryogenesis results in genital agenesis (Haraguchi et al.,
2001), and a separate role for Shh in urethral formation later in
development was found with conditional inactivation of Shh after
the initiation stage of GTorganogenesis (Haraguchi et al., 2007; Lin
et al., 2009). Because cell proliferation was reduced in Shh?/?GTs
(Haraguchi et al., 2001), we tested whether the elevated levels of
proliferation in Spry1?/?;Spry2?/?
through increased Shh expression. We examined Shh expression
at E14.5, the first stage at which changes in the urethral epithe-
lium were detected in Sprouty mutant GTs. We found strong
expression of Shh throughout the urethral epithelium of both
control and Spry1?/?;Spry2?/?GTs, although levels of Shh in
Sprouty mutant GTs did not appear significantly elevated com-
pared to controls (Fig. 6H and I). These findings were confirmed by
quantitative PCR (data not shown). This suggests that deletion of
Sprouty genes in the urothelium does not significantly affect levels
of Shh expression.
Fgf10 expression in the GT is dependent on a feedback loop with
Fgfr2-IIIb and is also a target of Shh (Haraguchi et al., 2001; Petiot
et al., 2005), but Fgf10 expression in the GT mesenchyme of Spry1?/?;
Spry2?/?embryos was comparable to controls (Fig. 6J, K). Wnt5a and
embryos were mediated
Fig. 6. Activation of FGF signaling is increased in Spry1?/?;Spry2?/?GTs, but initiation and maintenance of other signaling pathways is preserved. Levels of MAPK activation
in control and Sprouty mutants were assessed by detection of phospho-MEK1/2 in sagittal sections of E14.5 male GTs (A and B). Scale bar, 50 μm; epi, epithelium; mes,
mesenchyme. In situ hybridization was used to examine expression of Etv4 and Etv5 in sagittal sections of E14.5 control and Spry1?/?;Spry2?/?GTs. Insets show higher
magnification images of the urothelium in control and Spry1?/?;Spry2?/?embryos (C–F). Scale bar, 400 μm. Quantitative real-time PCR detection of Etv4 and Etv5 transcript
levels showed elevated Etv4 expression in Spry1?/?;Spry2?/?GTs (G;*po0.05). In situ hybridization was also used to evaluate expression patterns of Shh, Fgf10, Wnt5a,
Bmp4, and Aldh1a2 in E14.5 male control and Spry1?/?;Spry2?/?embryos (H–Q).
S.T. Ching et al. / Developmental Biology 386 (2014) 1–11
Bmp4 are two mesenchymal factors that regulate GT outgrowth and
proliferation (Suzuki et al., 2003; Yamaguchi et al., 1999). We
investigated whether expression of either of these genes was reduced
in Spry1?/?;Spry2?/?GTs. In both control and Sprouty mutant GTs,
Wnt5a transcripts accumulated in the distal mesenchyme of the GT;
whereas Bmp4 expression was evident in the dorsal mesenchyme
at the distal tip and along the ventral mesenchyme (Fig. 6L–O).
These results suggest that FGF signaling is not a major regulator of
either Wnt or Bmp signaling in the GT at E14.5. Finally, we asked if
Sprouty deletion could lead to changes in retinoic acid signaling,
which plays an important role in patterning of the external genitalia
(Liu et al., 2011; Ogino et al., 2001). We measured expression
of Aldh1a2, a gene encoding the enzyme that controls the rate-
limiting step in retinoic acid synthesis. Although the region of
Aldh1a2 was somewhat expanded, differences in its expression levels
were not obvious between control and mutant embryos (Fig. 6P
In mammals, the evolution of external genitalia is a prerequi-
site for internal fertilization, and abnormal development of the
external genitalia presumably lowers reproductive fitness. It is
therefore somewhat surprising that in humans, congenital defects
of the external genitalia are common. Hypospadias, the most
frequent genital anomaly in humans, affects approximately 1 in
250–300 live male births, and reports suggest that its incidence
has increased over the last two decades in some populations (Lund
et al., 2009; Paulozzi et al., 1997). To date, causal mutations in
human patients with hypospadias have only been identified in a
few genes, including SRD5A2, CXorf6, FGF8, and FGFR2 (Beleza-
Meireles et al., 2007; Kalfa et al., 2008; Ogata et al., 2008; Samtani
et al., 2011; Silver and Russell, 1999). However, the majority of
these are rare mutations in isolated cases of hypospadias, implying
that a considerable number of mutations have yet to be discov-
ered. A better understanding of the molecular mechanisms that
control genital development will improve our ability to identify
candidate genes involved in genital birth defects.
Here, we present evidence that Sprouty genes are critical mod-
ulators of FGF signaling in the embryonic mouse GT and are required
for normal development of the external genitalia during embryogen-
esis. In the absence of Spry1 and Spry2, male embryos exhibit an
ectopic opening along the ventral side of the GT, reflecting an
essential component of human hypospadias. Later in gestation, these
embryos completely lack an internal tubular urethra, suggesting that
the urethral closure defects seen at earlier stages are not simply due
to delays in development. Expression of Sprouty genes in the
embryonic GT was primarily restricted to the urothelium, which is
also the site of Fgfr2-IIIb expression. Deletion of Fgf10, Fgfr2-IIIb,
or Fgfr1 and Fgfr2 in combination in the mouse also causes a
hypospadias-like phenotype, highlighting the need for functional
FGF signaling during GT development (Haraguchi et al., 2000; Petiot
et al., 2005; Lin et al., 2013). Expression of Sprouty genes in the
urothelium also overlapped with Etv4 and Etv5, two known effectors
of FGF signaling, suggesting that urethral defects in Spry1?/?;
Spry2?/?embryos may be mediated through these two factors.
Interestingly, loss of Sprouty function did not appear to affect
terminal differentiation of the urothelium, as shown by expression
of both K14 and uroplakin III, indicating that failure to form the
urethra may not be a result of aberrant epithelial differentiation.
We found that deletion of Spry1 and Spry2 led to hyperplasia of
the urethral epithelium and increased cell proliferation. Sprouty
proteins antagonize FGF-induced RTK signaling by interacting with
several downstream components of the Ras/MAPK cascade (Casci
et al., 1999; Edwin et al., 2009). One of the primary cellular
processes regulated by FGF signaling is proliferation, and changes
in the number of cells undergoing division have been reported in
many studies in which GT development has been affected (Morgan
et al., 2003; Petiot et al., 2005; Seifert et al., 2009a; Suzuki et al.,
2003). A major question that has not been resolved from these
studies is how changes in cell proliferation translate into the
hypospadias phenotype. We found that both increased and
decreased levels of FGF signaling affected the organization and
thickness of the urothelium. In Spry1?/?;Spry2?/?embryos, the
urothelium was considerably thicker than controls; whereas in
Fgf10-null embryos, the urothelium was composed of 1–2 layers of
seemingly less mature epithelial cells. Recent studies have linked
cell proliferation and cell polarity (Bilder, 2004). The urothelium is
a polarized epithelium composed of several cell layers that differ
in morphology and function. Whether the changes in proliferation
in both Spry1?/?;Spry2?/?and Fgf10?/?GTs disturb epithelial cell
polarity, and ultimately, urethral fusion will need to be explored.
Alternatively, changes in cell proliferation may be a secondary
effect of disrupted cell polarity caused by aberrant FGF signaling.
It is striking that, in contrast to other mutants displaying
similar GT anomalies, the defect in Spry1?/?;Spry2?/?embryos
only occurs in males and not in females. Hormone-dependent
sexual differentiation of the GT is not apparent until E16.5 (Suzuki
et al., 2002), suggesting that the sexually dimorphic effects of
Sprouty deletion are not a result of differences in androgen or
estrogen activity. It is particularly interesting that despite some
evidence of being under transcriptional regulation by androgens,
mutations in either Fgf10 or Fgfr2-IIIb do not lead to sex differences
in GT morphogenesis (Petiot et al., 2005). Therefore, our findings
raise the possibility that Sprouty genes represent one of the
earliest targets of sexually dimorphic signaling in the developing
GT. We were unable to detect any differences in Sprouty gene
expression between male and female GTs by in situ hybridization
(data not shown), but a more detailed exploration of androgen
effects on FGF signaling will be required.
The role of Sprouty proteins as inhibitors of FGF signaling was
highlighted by our findings that deletion of both Spry1 and Spry2 in
the embryonic GT led to increased activity of MAPK signaling, as well
as increased expression of downstream targets such as Etv4 and Etv5.
However, characterization of signaling pathways such as Hedgehog,
Bmp, and Wnt in the Spry1?/?;Spry2?/?male GT revealed a
surprising lack of altered gene expression levels between controls
and mutants. In published reports on GT development in Fgf10?/?
and Fgfr2-IIIb?/?embryos, marked differences in Shh, K14, and Bmp4
expressions were described (Petiot et al., 2005; Satoh et al., 2004).
Therefore, there are likely additional genes that have yet to be
identified which are involved in mediating the developmental events
studied here. Despite the fact that E12.5 Sprouty mutant male GTs do
not appear abnormal, changes in expression of critical genes at early
stages of embryogenesis may establish aberrant morphogenetic
patterns that later lead to defective development. It is interesting
to note that in spite of the large variety of genetic deletions that lead
to abnormal GT morphogenesis, many of them produce very similar
defects in epithelial fusion along the ventral surface of the GT. This
would suggest that internalization of the urethra, as well as forma-
tion of a continuous prepuce and scrotum, is highly susceptible to
morphological disruption during development. This may also explain
why genital defects in humans are such a common occurrence.
Taken as a whole, our findings show that Sprouty genes are
necessary to regulate FGF activity in the developing mouse GT.
Spry1?/?;Spry2?/?male mice represent a good model for gaining
insight into the molecular mechanisms underlying embryonic
development of mammalian external genitalia. They also offer a
potential entry point for improved understanding of the etiology
of congenital defects that affect the external genitalia such
as hypospadias. Finally, given the paucity of causal mutations
S.T. Ching et al. / Developmental Biology 386 (2014) 1–11
known to induce hypospadias in humans, Sprouty genes are
strong candidates for mutation screening in patients with this
This work was supported by the National Institutes of
Health (R01DK095002 and RO1 DK0581050), T32 training grant
(5T32DK007790), National Science Foundation (IOS-0920793).
This work was also supported in part by a grant from the Urology
Care Foundation Research Scholars Program and Amgen, Inc.
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