Yoshiyuki Shiroyanagi . Benchun Liu . Mei Cao .
Koray Agras . Jiang Li . Michael H. Hsieh . Emily J.
Willingham . Laurence S. Baskin
Urothelial sonic hedgehog signaling plays an important role in bladder
smooth muscle formation
Received March 15, 2007; accepted in revised form March 23, 2007
senchyme differentiates into smooth muscle (SM) under
the influence of urothelium. The gene(s) responsible for
this process have not been elucidated. We propose that
the Sonic hedgehog (Shh) signaling pathway is critical
in bladder SM formation. Herein, we examine the role
of the Shh-signaling pathway during SM differentiation
in the embryonic mouse bladder. Genes in the Shh
pathway and SM expression in mouse embryonic (E)
bladders (E12.5, 13.5, and 14.5) were examined by
immunohistochemistry (IHC), in situ hybridization, and
reverse transcription polymerase chain reaction (RT-
PCR). To examine the effects of disrupting Shh signal-
ing, bladder tissues were isolated at E12.5 and E14.5,
that is, before and after bladder SM induction. The
embryonic bladders were cultured on membranes float-
ing on medium with and without 10mM of cyclopamine,
an Shh inhibitor. After 3 days, SM expression was
examined by assessing the following: SM a-actin
(SMAA), SM g-actin (SMGA), SM-myosin heavy chain
(SM-MHC), Patched, GLI1, bone morphogenic protein
4 (BMP4), and proliferating cell nuclear antigen
(PCNA) by IHC and RT-PCR. SM-related genes and
proteins were not expressed in E12.5 mouse embryonic
bladder before SM differentiation, but were expressed
by E13.5 when SM differentiation was initiated. Shh
During bladder development, primitive me-
was expressed in the urothelium in E12.5 bladders. Shh-
related gene expression at E12.5 was significantly higher
than at E14.5. In cyclopamine-exposed cultures of
E12.5 tissue, SMAA, SMGA, GLI1, and BMP4 gene
expression was significantly decreased compared with
controls, but PCNA gene expression did not change. In
cyclopamine-exposed E14.5 cultures, SMGA and SM-
MHC gene expression did not change compared with
controls. Using an in vitro embryonic bladder culture
model, we were able to define the kinetics of SM- and
Shh-related gene expression. Cyclopamine inhibited
detrusor SM actin induction, but did not inhibit SM-
MHC induction. SMAA and SMGA genes appear to be
induced by Shh-signaling pathways, but the SM-MHC
gene is not. Based on Shh expression by urothelium and
the effects of Shh inhibition on bladder SM induction,
we hypothesize that urothelial-derived Shh orchestrates
induction of SM in the fetal mouse bladder.
hedgehog ? organ culture ? development
bladder ? smooth muscle ? sonic
The bladder develops from the urogenital sinus (UGS),
which is an endodermal tube, derived from the hindgut.
The hindgut terminates as a caudal expansion called the
cloaca. The urorectal septum subdivides the cloaca into
the UGS ventrally and the rectum and the anal canal
dorsally. We have previously shown that an epithelial
signal is necessary for the induction of bladder smooth
muscle (SM) (Baskin et al., 1996). We have also pro-
posed that the SM-inducing signal is a diffusible factor
originating from the urothelium (Liu et al., 2000). In-
terestingly, the epithelial signal is not specific for age in
that embryonic, newborn, and adult urothelium all have
Yoshiyuki Shiroyanagi ? Benchun Liu ? Mei Cao ? Koray
Agras ? Jiang Li ? Michael H. Hsieh ? Emily J. Willingham ?
Laurence S. Baskin (.*)
Fax: 11 415/476 8849
Department of Urology
UCSF Children’s Hospital
University of California San Francisco
P. O. Box 0738, 400 Parnassus A640
San Francisco, CA 94143-0738
Tel: 11 415/476 1611
Differentiation (2007) 75:968–977 DOI: 10.1111/j.1432-0436.2007.00187.x
r 2007, Copyright the Authors
Journal compilation r 2007, International Society of Differentiation
the capacity to induce bladder SM differentiation
(DiSandro et al., 1998). Furthermore, the bladder
SM-inducing signal is not specific to urothelium in
that epithelial cells from organs such as the uterus, gut,
and stomach, all associated with a thick SM wall, can
also induce bladder SM formation (DiSandro et al.,
1998). This report focuses on the signals or gene prod-
ucts from the urothelium that control bladder SM for-
Sonic hedgehog (Shh) is a secreted signaling factor
that regulates cell function and fate in development and
adulthood (Ericson et al., 1995a, 1995b). Bitgood and
McMahon (1995) examined Shh expression in detail in
mouse embryos. In the mouse bladder, Shh is detected
in the epithelium of the bladder during the establish-
ment of the bladder precursor at E11.5 (Bitgood and
McMahon, 1995). The Shh pathway functions when the
secreted Shh peptide binds to its membrane-bound re-
ceptor, Patched (Ptc) (Ho and Scott, 2002; Ruiz i Alt-
aba et al., 2003). Shh binds Ptc, which represses Smo,
initiating an intracellular signal transduction cascade
activating three highly related Gli transcription factors:
Gli 1, Gli 2, and Gli 3. Gli 1 and Gli 2 are transcrip-
tional activators targeting Shh genes such as the Wnt
family and the bone morphogenic proteins (BMPs) that
are essential for normal embryonic development and
differentiation of many tissues.
Little is known about Shh signaling in urinary tract
development. The mesenchyme on the ureter, like the
gut and bladder, express two developmental fates (SM
versus fibroblasts of the lamina propria) dependent up-
on their locations relative to the Shh-expressing ureteral
epithelium (Yu et al., 2002). However, the role of Shh
signaling in bladder SM development is unclear.
The Shh-null mice die before birth because of a severe
phenotype that includes holoprosencephaly, distal
anteroposterior limb deficiencies, and absence of the
genital tubercle (Chiang et al., 1996, 1999, 2001).
Ramalho-Santos et al. (2000) noted that both Shh and
Indian hedgehog mutant mice show reduced SM, gut
malrotation, and an annular pancreas. Mo et al. (2001)
reported that Shh signaling is essential for normal
development of the distal hindgut in mice, and that
mutations affecting Shh signaling produce a spectrum
of anorectal malformations. In patients with anorectal
malformations, it is thought that the Shh/BMP4 path-
way is disrupted, thus perturbing normal hindgut for-
mation (Sasaki et al., 2004). Shh is clearly critical for
bladder development as inactivation of Shh in mice has
a severe embryonic lethal phenotype with cloacal exs-
trophy, penile agenesis, and deficiency of SM in pelvic
organs (Yucel et al., 2004). Such a phenotype is con-
sistent with the concept that Shh plays a major role in
bladder SM development.
In this study, we have investigated expression of Shh
mRNA and other downstream genes in the Shh-signaling
pathway during detrusor SM development. We have
also assessed changes in detrusor SM gene expression
by blocking Shh signaling with cyclopamine using an
in vitro bladder organ-culture system.
Materials and methods
Mouse bladder development in vivo
We studied normal, timed mated outbred CD-1 mice (Charles Riv-
er, Wilmington, MA). Noon on the day of the vaginal plug was
designated as embryonic day 0.5. Macroscopic results confirmed
that the UGS is separated from the hindgut by the urorectal septum
on embryonic day 12, with the bladder becoming a distinct entity
on embryonic day 12.5. Hence, the stages examined were embryonic
days 12.5, 13.5, 14.5, and 15.5. For histological analysis, whole
embryos were fixed with 4% formalin. For mRNA expression
analysis, embryonic bladders were isolated, and the umbilical ar-
teries were removed by dissection. Total RNA was isolated using
the RNeasy micro kit (Qiagen, Valencia, CA). All procedures were
performed in Hank’s balanced salt solution on ice. All animal pro-
cedures were performed with the approval of the laboratory animal
resource center at University of California, San Francisco.
Embryonic urinary bladder immunohistochemistry (IHC)
For immunohistochemical studies of embryonic bladders, whole
embryos were fixed in 4% formalin solution and embedded in par-
affin; sections were cut at 5mm. After dewaxing and hydration,
endogenous peroxidase was quenched with 0.3% hydrogen perox-
ide in methanol for 20min at room temperature. A mouse on
mouse kit (Vector Laboratories, Burlingame, CA) was used ac-
cording to supplied protocols to detect primary antibody with the
ABC kit (Vector Laboratories) and 3,30-diaminobenzidine (DAB)
(Vector Laboratories). Mouse monoclonal antibody 1A4 specific
for SM a-actin (SMAA) (Sigma, St. Louis, MI) was used at 1:2,000
dilution, and mouse monoclonal antibody B4 specific for SM
g-actin (SMGA) (MP Biomedicals, Aurora, OH) was used at
Embryonic urinary bladder in situ hybridization
In situ hybridization was performed on 10mm cryosections accord-
ing to standard protocols. Both Shh and Ptc probes were kindly
provided by Dr. Grieshammer (UC, San Francisco). Digoxygenin-
labeled RNA of Shh (Echelard et al., 1993) and Ptc (Goodrich
et al., 1996) were used as probes.
mRNA expression of embryonic bladder in vivo
Total RNA from each embryonic bladder was extracted using the
RNeasy micro kit (Qiagen). The yield and the quality of RNA were
assessed by using a Bioanalyzer (Agilent, Palo Alto, CA). RNA was
incubated with DNase (DNA-free; Ambion, Austin, TX) to remove
contaminating host and viral DNA. The DNase was inactivated
and removed according to the manufacturer’s specifications. ‘‘No
Reverse transcriptase’’ controls were performed on all pools to
confirm that genomic DNA was not present. Relative mRNA ex-
pression was measured quantitatively using a 50-fluorogenic nuc-
lease assay in quantitative polymerase chain reaction (Q-PCR) on
the ABI PRISMs7900 (Applied Biosystems, Foster City, CA).
Quantitative detection of specific nucleotide sequences was based
on the fluorogenic 50-nuclease assay, as described previously (Gin-
zinger, 2002). Briefly, RNA was reverse transcribed into cDNA
with iScript (BioRad, Hercules, CA). The primer and probe for
the assays were obtained from Applied Biosystems ‘‘assays on
demand’’ Taqman expression kits (Catalogue# Mm00725412_s1
[SMAA], Mm00656102_m1 [SMGA], Mm00432087_m1 [BMP4],
Mm00494645_m1 [GLI1], and Mm00448100_g1 [PCNA]; Applied
Biosystems]. Taqman reaction used 5ng of cDNA per well. Relative
expression in comparison with control gene ‘‘GAPDH’’ was cal-
culated using the methods described (Livak and Schmittgen, 2001).
Each experiment was repeated three times, and the mean value was
calculated as expression value for each stage of embryonic bladder
Embryonic bladder organ culture
Based on our preliminary analysis of embryonic bladder develop-
ment in vivo (see Fig. 1), we chose E12.5 bladders before SM for-
mation and E14.5 bladders following SM formation for blocking
experiments in vitro. Isolated E12.5 and E14.5 embryonic bladders
were cultured on 0.4mm pore Millicell insert membranes (Cat# 3460
Clear Coning, Corning, NY) on DMEM/F12 50:50 supplemented
with insulin-transferrin (10mg/l) (Sigma) and antibiotics. Ten blad-
ders were placed in each well. Cultures were incubated in 95% air/
5% CO2at 371C. At culture day 3, tissues were harvested and fixed
for histology, and total RNA was isolated using the RNeasy micro
kit for real-time RT-PCR processing.
Undifferentiated bladders cultured with specific signal pathway
To study the role of the Shh-signaling pathway in bladder SM
induction, the steroidal alkaloid cyclopamine (Biomol inter-
national GR-334, Plymouth Meeting, PA), known to interrupt
Shh signaling, was added to the culture medium at 10mM. E12.5
bladders before SM induction and E14.5 bladders after the expres-
sion of SM genes were treated with cyclopamine for 3 days.
Cyclopamine was prepared in 100% ethanol at 10mM, stored at
?151C. Before the experiments, cyclopamine was diluted with the
culture medium to a final concentration of 10mM as described
previously (Sukegawa et al., 2000; Mistretta et al., 2003; Nanba et
al., 2003; McKinnell et al., 2004). Control cultures were supple-
mented with an equivalent volume of ethanol. We also cultured
bladder tissues from E14.5 embryos after SM induction with 10mM
cyclopamine. Eighty bladders per group were used for Shh pathway
After 3 days in vitro cultivation, each embryonic bladder was
observed under the phase contrast microscope, and the size of tissue
was measured based on pixel area using Adobe PhotoShop.
For immunohistochemical studies, cultured tissues were fixed
with formalin and embedded in paraffin. Sections were cut at 5mm.
The staining procedure was the same as for embryonic bladders.
Relative gene expression was analyzed by real-time RT-PCR de-
Fig.1 Macroscopic findings: whole embryo (upper panels), genital
track (middle panels), and bladder with umbilical arteries (lower
panels). The white arrows indicate the urinary bladder. The black
arrows indicate the umbilical arteries. Ruler51mm. Bars in the
middle panels are 1mm. Bars in the lower panels are 100mm.
Laser capture microdissection of embryonic bladder mesenchyme
To compare the expression level of Ptch1 mRNA at different loca-
tions within the bladder mesenchyme with respect to location relative
to the urothelium, embryonic mouse bladders were collected at E12.5,
13.5, 15, and 16, respectively. Mesenchymal locations were chosen
either next to the urothelium or next to the serosal layer in the E12.5
and E13.5 bladders. In the E15 and E16 bladders, an additional lo-
cation was captured half-way between the serosal and urothelial lay-
ers. The embryonic bladders were cut into 6mm sections and stained
using a Histogenet solution (Arcturus Engineering Inc., Mountain
View, CA). For mesenchyme microdissection, slides were immediately
transferred to a PixCellsII laser capture microscope (Arcturus En-
gineering Inc.). Pictures of samples were taken using a Hitachi KP-
D580 high-sensitivity autogain thermoelectrically cooled digital signal
processor color CCD camera (Hitachi Kokusai Electric, Woodbury,
NY). Cell clusters were transferred to caps with a thermoplastic
polymer film by laser hits. Each cap was placed in an ExtracSuret
device, and RNA extraction was performed with the PicoPuret RNA
Isolation Kit (Arcturus Engineering Inc.). Two rounds of linear RNA
amplifications were performed with the RiboAmpt RNA Amplifi-
cation Kit (Arcturus Engineering Inc.) according to the manufactur-
er’s protocol. The expression level of Ptch1 mRNA was measured
using the SYBR Green qRT-PCR technique on the Prisms7300
Real-Time PCR System (Applied Biosystems, Foster City, CA).
An unpaired t-test was used for statistical analysis.
In vivo bladder development
Figure 1 shows whole embryos, genital tracks, and the
bladders with umbilical arteries attached. The stage of
urinary tract development was defined as shown in
Figure 1. If embryos in each stage did not develop nor-
mally, they were excluded. Note that the bladder de-
velops between the umbilical arteries, and bladder size
increases rapidly between E13.5 and E14.5.
IHC for both SMAA and SMGA revealed that these
proteins were not expressed in the E12.5 bladder
detrusor wall, but did localize to the umbilical arteries
(Figs. 2A,2E). At E13.5, the detrusor wall was lightly
stained with the SMAA antibody, but not with anti-
SMGA (Figs. 2B,2F). In E14.5 and E15.5 embryos, SM
markers were expressed in the outer layer of the bladder
wall. During development, the bladder lumen became
larger and the bladder wall became thicker (Figs.
Shh mRNA expression was detected in the epitheli-
um of the urinary bladder at E12.5 and E13.5 embryos
(Figs. 3A,3B). Similar expression was seen in the uro-
thelium of E14.5 and E15.5 bladders (data not shown).
Ptc expression was observed in the adjacent bladder
mesenchyme at E12.5 (Fig. 3C). In E13.5 bladder, Ptc
was detected in the submucosal layer, but not in the
outer prospective SM layer (Fig. 3D).
mRNA gene expression normalized to GAPDH
mRNA by real-time RT-PCR in embryonic bladder
tissue from E12.5 to E15.5 is shown in Figure 4.
SMGA, SM-myosin heavy chain (SM-MHC), and Cal-
ponin1 were not detected in E12.5 bladder tissue. They
were first expressed in E13.5 bladders with increasing
expression at E14.5 and E15.5. E12.5 bladder tissue had
Fig.2 Smooth muscle (SM) actin staining of the embryonic blad-
der. (A–D) Smooth muscle a-actin (SMAA), (E–H) smooth muscle
g-actin (SMGA). E12.5 (A and E), E13.5 (B and F), E14.5 (C and
G), and E15.5 (D and H). Note the positive staining of the um-
bilical arteries. Original magnification ?10. Same magnification
the highest mRNA expressions of Shh, Ptc, GLI1, and
BMP4 decreasing in a linear fashion to E15.
Bladder organ culture
When E12.5 bladders were cultured in control medium,
SMAA and SMGA mRNA induction was observed on
culture day 3. Shh gene expression did not change (data
not shown). A phase contrast microscope revealed that
there were no gross differences between control and
cyclopamine treatment bladders (Figs. 5A,5B). IHC re-
vealed SMAA protein expression in the outer layer of
cultured bladders on culture day 3 (Figs. 5C,5D). The
amount of SMAA was qualitatively reduced in the Shh-
treated bladder (Fig. 5D) compared with untreated
bladders (Fig. 5C). The size of control and 10mM
cyclopamine-treated bladders after 3 days of cultivation
under the phase contrast microscope was not signifi-
cantly different (Fig. 5E).
Hedgehog signaling pathway disruption in vitro with
In cyclopamine-exposed cultures of E12.5 bladder
tissue, real-time RT-PCR data showed that SMAA
(p50.03), SMGA (p50.00), GLI1 (p50.00), and
BMP4 (p50.00) gene expression significantly de-
(PCNA) (p50.88) gene expression did not change
(Fig. 6). In the cyclopamine-exposed E14.5 (post-SM-
related gene and protein induction) cultures, SMAA
gene expression significantly decreased compared with
control (p50.00), but SMGA (p50.07) and SM-MHC
(p50.43) gene expression did not change compared
with control. GLI1 (p50.00) and BMP4 (p50.00)
gene expression significantly decreased compared with
controls in cultures of E14.5 bladders. PCNA (p50.00)
gene expression significantly increased compared with
control (Fig. 7).
Laser capture microdissection analysis for the Ptch1
In E12.5 and E13.5 embryonic bladders, mesenchymal
cells were captured from two different locations relative
to the urothelium: (a) adjacent to the serosa and (b)
adjacent to the urothelium (Fig. 8A). In the larger E15
and E16 bladders, three areas were captured: (a1) ad-
jacent to the serosa, (a2) in between the serosa and
urothelium, and (b) adjacent to the urothelium (Fig.
8B). Approximately 150 bladder mesenchymal cells of
same capture site at each embryonic stage were pooled
from four fetuses from two dams for RNA extraction.
All samples contained intact RNA and were suitable for
analysis based upon assessment using an Agilent 2100t
Biolanalyser (data not shown).
Ptc1 mRNA was consistently elevated in the me-
senchymal layer adjacent to the urothelium (zone b)
compared with that in all peripheral locations (zones a,
a1, and a2) at each embryonic stage (Fig. 8C, po0.05).
Areas containing the serosa (zones a1and a2) consis-
Fig.3 In situ hybridization of the embry-
(A and B), Patched (Ptc) (C and D).
E12.5 bladder (A and C), E13.5 bladder
(B and D).
tently had the lowest expression of Ptc1 mRNA. Ex-
pression of Ptc1 mRNA in the subepithelial bladder
mesenchymal cells (zone b) was significantly higher in
the E12.5 bladders just before bladder SM formation
compared with that of the later stage bladders (Fig. 8C,
Detrusor SM development in vivo
The mouse bladder has proven to be an excellent model
for the study of visceral SM formation. From a tech-
nical perspective, we have confirmed that the bladder
can be isolated using a dissection microscope at gesta-
tion day 12 in CD-1 mice (Staack et al., 2005). Exper-
imental isolation of the embryonic bladder following
removal of the umbilical arteries allowed quantitative
gene analysis before and after SM differentiation.
Detrusor SM is first identified in the mouse embryonic
bladder at 13.5 days based on protein expression of
SMAA, followed by SMGA (Fig. 2). The location of
SM differentiation is in the periphery of the bladder
mesenchyme in a concentric pattern away from the in-
ducing urothelium. At E12.5 before SM differentiation,
SMAA and SMGA are expressed in the umbilical ves-
sels. Visceral SM differentiation in the mouse has been
identified in the aorta as early as E9, followed by the
differentiation of visceral SM in the foregut (McHugh,
1995). Our findings are consistent with Smeulders et al.
(2002) who demonstrated that SMAA is first expressed
in the E14 mouse bladder, and McHugh (1995), who
documented that the expression of the SMAA preceded
that of the SMGA. These results indicate that the
SMAA is the earliest known marker for detrusor SM
Shh and Ptc expression in embryonic bladder
Shh mRNA is expressed in the urothelial layer in
E12.5–E15.5 bladders (data shown for E12.5 and E13.5,
Figs. 3A,3B). During the same period, the receptor for
Shh, Ptc is expressed in mesenchyme surrounding the
epithelium. The expression patterns of Shh and Ptc
mimics the expression pattern in the developing kidney
Fig.4 Percent RNA gene expression normalized to GAPDH
mRNA by real-time reverse transcription polymerase chain reac-
tion (RT-PCR) in embryonic bladders from E12.5 to E15.5 mice.
(Y-axis: % mRNA gene expression against GAPDH by real-time
Fig.5 Phase contrast findings of
E12.5 cultured bladder for 3 days
in vitro (A, control; B, with cyclopa-
mine). a-smooth muscle staining of
E12.5 cultured bladder (C, control;
D, with cyclopamine). (E) Pixel
numbers (?1000 as a measure of ar-
ea) calculated by Adobe Photoshop
mine-treated bladder (0, control;
10, cyclopamine treated).
and gut, where Shh has been suggested to regulate SM
development (Sukegawa et al., 2000; Yu et al., 2002).
We propose that Shh signals from the urothelium to
the surrounding mesenchyme, inducing SM differenti-
ation. This is consistent with Shh acting as a paracrine
signal from the urothelium, inhibiting SM differentia-
tion at a high concentration of Shh (close to the uro-
thelium in the bladder submucosa), and inducing
mesenchymal cell differentiation into SM as the con-
Fig.6 Percent mRNA gene expression against GAPDH by real-
time reverse transcription polymerase chain reaction in E12.5-
cultured embryonic bladders after 3 days of organ culture. (X-axis:
0, control; 10, 10mM cyclopamine treated;?po0.05).
Fig.7 Percent mRNA gene expression against GAPDH by real-
time RT-PCR in E14.5 cultured embryonic bladders. (X-axis: 0,
control; 10, 10mM cyclopamine treated;?po0.05).
Fig.8 Relative expression of Ptc1 mRNA in the bladder me-
senchyme as a function of distance from the urothelium. (A) Laser-
assisted capture microdissection (LCM) of E12.5 embryonic mouse
bladder mesenchyme. HE, hematoxylin and eosin staining; HS,
Histogenet staining; w/o LCM, before laser capture; w/ LCM,
after microdissection of consecutive sections; E, epithelium. (a)
Mesenchymal layer adjacent to the serosa. (b) Mesenchymal layer
adjacent to the epithelium, scale bar, 50mm. (B) Microdissection of
E15 mice embryonic bladder mesenchyme. (a1) Mesenchymal layer
adjacent to the serosa. (a2) Mesenchymal layer between the serosa
and urothelium. (b) Mesenchyme adjacent to the epithelium, scale
bar, 50mm. (C) Relative expression of Ptc1 mRNA in microdis-
sected mesenchymal compartments at each embryonic stage;
?po0.05; #po0.01. (a), (a1), (a2), and (b) correspond to the loca-
tion in the mesenchyme as described above at each embryonic age.
centration gradient decreases (bladder periphery). Shh
is known to pattern tissue during development by form-
ing a concentration gradient. Cells sense their position
within the gradient and differentiate into distinct cell
types as a function of Shh concentration as described in
the developing neural tube and spinal cord (Ericson
et al., 1995a, 1995b). The Shh network is thought to
function as a genetic switch with the ability to switch
cell fate choices based on threshold concentrations of
Shh (Lai et al., 2003). The network is composed of a
positive transcriptional feedback loop embedded within
a negative signaling feedback loop. Positive feedback by
the transcriptional factor Gli up-regulates its own ex-
pression. Gli also up-regulates the signaling repressor
Ptc, which acts as a negative feedback on the Gli
autoregulatory loop (Lai et al., 2003). Thus, Shh is a
prime candidate to act as both an inducer as well as an
inhibitor of bladder SM formation as a function of
The location of the receptor for Shh, Ptc in the blad-
der mesenchyme as well as downstream-inducing mol-
ecules may also play a role in the patterning of bladder
SM differentiation. Figure 3 shows the location of the
Ptc receptor adjacent to the urothelium in the submu-
cosa. Our in situ hybridization data show decreasing
expression of Ptc outward to the peripheral bladder
mesenchyme, the location where bladder SM differen-
tiation occurs. These results were corroborated using
microdissection laser capture (Fig. 8). These findings
are consistent with Shh signaling from the urothelium
through the Ptc located in the submucosa of the bladder
mesenchyme to inhibit SM differentiation in the sub-
mucosa. In turn, we propose that Ptc induces SM dif-
ferentiation via up-regulation of downstream molecules
such as Serum Response Factor (SRF) in peripheral
bladder mesenchyme where Ptc levels are exceedingly
low (Li et al., 2006). SRF is a transcription factor for
the SMAA and for the vascular SM protein, SM22-a
protein (Miano et al., 1994); both SMAA and SM22-a
promoters have binding sites for SRF. We previously
found that SRF protein is present globally in E12 blad-
der mesenchyme. Subsequently, during bladder devel-
opment, SRF localizes to the peripheral bladder
mesenchyme in the same pattern as SM proteins. This
gene is also a strong candidate for having a role in
SMAA induction secondary to its expression at the time
of SM differentiation (Li et al., 2006).
Quantitative analysis of SM and Shh-signaling pathway
mRNA expression in bladder development in vivo
According to our real-time RT-PCR data from isolated
embryonic bladder, SM-related genes are first expressed
between E12.5 and E13.5 (Fig. 4). SMAA, SMGA,
SM-MHC, and Calponin 1 mRNA expressions are
undetectable in E12.5 bladder. In E13.5 bladder, the
expression pattern of the above genes is much higher
than at E12.5. SM maturation in the embryonic bladder
begins in this stage. mRNA detection by real-time RT-
PCR is about a day earlier than corresponding protein
expression. Our work is consistent with the work of
McHugh and Smeulders, who demonstrated that SM
development in mouse bladder occurs at the E14
(Smeulders et al., 2002). SMAA and desmin protein
expression in the developing bladder increased progres-
sively after E14, consistent with the increased expres-
sion of SMAA, SMGA, SM-MHC, and Calponin 1 that
we have also documented.
Shh-signaling pathway-related mRNA expression in
the embryonic bladder was noted to be decreasing dur-
ing development in inverse proportion to SM develop-
ment. Shh mRNA expression is the highest in E12.5
bladder. Other genes in the Shh-signaling pathway (Ptc,
Gli1, BMP4) were also highly expressed in E12.5 blad-
der. Meanwhile, SM-related genes were not expressed in
E12.5 bladder, but their expression increased from
E13.5 to E15.5. We propose that a higher concentration
of Shh is necessary for the initiation of SM formation
than for SM proliferation during bladder development
accordingly. Therefore, at the initiation of SM forma-
tion, we documented the highest concentration of Shh,
which subsequently decreased along with other genes in
the Shh-signaling pathway. It is possible that Shh
expression is initiated before E12, a stage when the blad-
der is not formed and therefore impossible to isolate.
Bladder organ culture model for cyclopamine treatment
To assess the role of the Shh-signaling pathway in
bladder development, an in vitro embryonic bladder
organ culture system was developed. The SMAA and
SMGA mRNA expression levels after 3 days of culti-
vation were almost at the same level as E15.5 bladders
in vivo. The SM-MHC level was almost the same level of
E14.5 (compare mRNA expression levels in Figs. 2,5).
This in vitro culture model system simulated bladder
development in vivo in that SM-related genes were ex-
pressed and SM differentiation took place during the
culture period. However, in vitro the SM developed
closer to the urothelium, and the SM layer was qual-
itatively thinner than E15.5 embryonic bladders in vivo.
The relative expression of SMAA and SMGA is similar
between the in vitro 3-day culture model and E15 blad-
der in vivo.
Other investigators have used organ culture systems
to confirm the role of the Shh-signaling pathway in the
development of the gut (Sukegawa et al., 2000), tongue
(Mistretta et al., 2003), skin (Nanba et al., 2003), feath-
er buds (McKinnell et al., 2004), and prostate (Free-
stone et al., 2003; Doles et al., 2005). The cyclopamine
concentration in these experiments was 0.1–50mM. We
chose a midrange dose of 10mM cyclopamine in the
culture medium to disrupt the Shh-signaling pathway.
The expression of Gli 1, a downstream transcription
factor in the Shh pathway, in the cultured bladder tissue
was completely inhibited by 10mM cyclopamine. The
BMP4, which is a Shh-target gene, was also inhibited
significantly by 10mM cyclopamine. However, the size
of cultured bladder was not significantly different be-
tween the control and cyclopamine treatment group.
Accordingly, PCNA expression levels were not signif-
icantly altered, suggesting that 10mM cyclopamine in
this culture model did not affect the bladder tissue
growth and proliferation.
Disruption of Shh signaling by cyclopamine signifi-
cantly reduced SMAA and SMGA mRNA expression
in E12.5 bladder tissue cultured for 3 days. SM-MHC
expression did not change significantly, but tended to
decrease somewhat. However, when E14.5 bladders
(SM differentiation has already occurred) were cultured
in the presence of cyclopamine, SMGA expression was
not significantly decreased. Interestingly, SM-MHC ex-
pression increased as a result of Shh signal disruption.
The expression of SMAA serves as an indication of
commitment to a SM cell lineage. However, as this actin
isoform is also expressed in developing cardiac and
skeletal muscle as well as in myofibroblasts, it cannot be
used to identify SM unequivocally (Desmouliere et al.,
1993). On the other hand, SMGA and SM-MHC have
been shown to be markers of mature or more fully
differentiated SM cells (Sawtell and Lessard, 1989;
McHugh et al., 1991; Miano et al., 1994).
Cyclopamine inhibited the expression of SM-specific
markers at the timing of SM induction, but cyclopa-
mine did not inhibit these markers, except for SMAA,
after SM is already induced. At the E14.5 stage, Shh-
signaling disruption increased the expression of smooth-
muscle-related genes. Interestingly, the E14.5 bladders
treated with cyclopamine expressed PCNA mRNA to a
significantly greater degree than non-treated bladders.
This finding also suggested that the Shh-signaling path-
way may inhibit proliferation following bladder SM in-
duction when SM differentiation is taking place. Yu
et al. (2002) demonstrated that the addition of Shh in-
hibits SM differentiation-related genes in a dose–
dependent manner. This finding supports our hypoth-
esis that Shh may inhibit SM differentiation after SM
induction. We propose that Shh acts as both an inducer
of detrusor SM in the early stage of development as well
as an inhibitor of bladder SM formation as has been
described in the development of the gut (Sukegawa
et al., 2000). The proposed dual function of Shh in SM
patterning (inhibition of SM differentiation near the
epithelium and induction of SM differentiation in the
subserosal zone) could be due to a Shh gradient or a
gradient in the expression of Ptc1 as suggested by our
laser microdissection studies. Given the facts that de-
velopment of bladder SM is inhibited by removal of the
Shh-positive epithelium or cyclopamine inhibition of
Shh, it is evident that Shh is a key player in visceral SM
differentiation even though molecules downstream of
Shh are surely critical in SM differentiation as well.
These findings may contribute to clarify the pathogenic
mechanism of detrusor SM deficiency.
In conclusion, our results support the hypothesis that
Shh acts as both an inducer and an inhibitor of bladder
SM formation through the Shh-signaling pathway.
with in situ hybridization. This work was supported by NIH grant
RO1 DK073449 (L. S. B.), the Toyobo Biotechnology Foundation
(Y. S.), and the California Urology Foundation (L. S. B. and Y. S.).
We thank Dr. Uta Grieshammer for assistance
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