Molecular analysis of coordinated bladder and urogenital ... · differentiate into the urogenital and reproductive organs, including the urinary bladder (hereafter described as the

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INTRODUCTIONDuring mouse development, a transient embryonic cavity called thecloaca forms at the caudal end of the hindgut and is subsequentlydivided into the urogenital sinus and rectum (de Santa Barbara andRoberts, 2002; Kimmel et al., 2000; Kondo et al., 1996; Peningtonand Hutson, 2003; Yamada et al., 2003; Yamada et al., 2006). It hasbeen suggested that the cloaca/urogenital sinus and adjacent tissuesdifferentiate into the urogenital and reproductive organs, includingthe urinary bladder (hereafter described as the bladder) in both sexes.This is supported by reports of complex congenital malformationssuch as: exstrophy-epispadias complex (exstrophy of cloaca orbladder and abnormal dorsal external genitalia; hereafter, dorsalexternal genitalia represents upper external genitalia); defectivebody wall, bladder and genitalia formation; and limb body walldefects that are probably caused by abnormalities in earlydevelopment of the cloaca and urogenital sinus (Craven et al., 1997;Manner and Kluth, 2005; Penington and Hutson, 2003). Theconstellation of defects reported in affected individuals suggests thatcoordinated developmental programs regulate bladder and externalgenital morphogenesis. However, the signaling and structuralcontributions of the transient embryonic cloaca to formation ofmultiple urogenital and reproductive organs inside and outside thepelvic cavity remain obscure.

The phenotypes of gene knockout mice have revealed that severalgrowth and signaling factor families play fundamental roles inreproductive and urogenital organ formation (Batourina et al., 2002;Doles et al., 2006; Haraguchi et al., 2001; Haraguchi et al., 2000;Huang et al., 2005; Jamin et al., 2002; Kobayashi and Behringer,2003; Kondo et al., 1996; McMahon et al., 2003; Morgan et al., 2003;Perriton et al., 2002; Suzuki et al., 2003). Shh is expressed in thecloacal epithelia and is vital for the regulation of structures derivedfrom the genital tubercle (GT) and the urogenital sinus, such as theexternal genitalia and the prostate, respectively (de Santa Barbara andRoberts, 2002; Freestone et al., 2003; Pu et al., 2004). Shh null mouseembryos display GT agenesis associated with aberrant cloacalformation, suggesting that Shh signals emanating from the epitheliumof the cloaca may play an essential role in peri-cloacal mesenchyme(PCM) development (Haraguchi et al., 2001; Perriton et al., 2002).

In this manuscript, we report our studies investigating the roleof the hedgehog (HH) signaling pathway and of HH-responsivetissues in urogenital development. We analyzed the expression ofmembers of the HH signaling pathway in structures developingfrom the cloacal region, including the GT, internal urethra (pelvicurethra), lower body wall and bladder. We performed phenotypeanalyses of Shh and Gli mutants, and employed HH-responsivetransgenic marker alleles in mice to examine the lineage of theseHH-responding tissues with regard to the reproductive andurogenital organs. In addition to the cloacal epithelium, Shh isexpressed in the developing urethral plate (UP), internal urethraand epithelia lining the bladder. Shh and Gli mutant micedisplayed hypoplasia and defects of the UP, internal urethra andbladder wall. We discovered that HH-responsive PCM contributesto both the dorsal GT and to mesenchymal precursors of bladdersmooth muscle. These analyses demonstrate that coordinatedformation of the bladder, internal urethra and external genitalia isorchestrated by HH signaling.

Molecular analysis of coordinated bladder and urogenitalorgan formation by Hedgehog signalingRyuma Haraguchi1, Jun Motoyama2, Hiroshi Sasaki3, Yoshihiko Satoh1, Shinichi Miyagawa1, Naomi Nakagata1,Anne Moon4 and Gen Yamada1,*

The urogenital and reproductive organs, including the external genitalia, bladder and urethra, develop as anatomically alignedorgans. Descriptive and experimental embryology suggest that the cloaca, and its derivative, the urogenital sinus, contribute to theformation of these organs. However, it is unknown how the primary tissue lineages in, and adjacent to, the cloaca give rise to theabove organs, nor is bladder formation understood. While it is known that sonic hedgehog (Shh) is expressed by the cloacalepithelia, the developmental programs that regulate and coordinate the formation of the urogenital and reproductive organs havenot been elucidated. Here we report that Shh mutant embryos display hypoplasia of external genitalia, internal urethra (pelvicurethra) and bladder. The importance of Shh signaling in the development of bladder and external genitalia was confirmed byanalyzing a variety of mutant mouse lines with defective hedgehog signaling. By genetically labeling hedgehog-responding tissuelineages adjacent to the cloaca and urogenital sinus, we defined the contribution of these tissues to the bladder and externalgenitalia. We discovered that development of smooth muscle myosin-positive embryonic bladder mesenchyme requires Shhsignaling, and that the bladder mesenchyme and dorsal (upper) external genitalia derive from Shh-responsive peri-cloacalmesenchyme. Thus, the mesenchymal precursors for multiple urogenital structures derive from peri-cloacal mesenchyme and thecoordination of urogenital organ formation from these precursors is orchestrated by Shh signals.

KEY WORDS: Urinary bladder, External genitalia, Cloaca, Internal urethra, Pelvic urethra, Shh (sonic hedgehog), Gli, PCM (peri-cloacalmesenchyme), Smooth muscle, Exstrophy, Mouse

Development 134, 525-533 (2007) doi:10.1242/dev.02736

1Center for Animal Resources and Development (CARD), Graduate School ofMedical and Pharmaceutical Sciences, Kumamoto University, Honjo 2-2-1,Kumamoto 860-0811, Japan. 2Molecular Neuropathology Group, Brain ResearchInstitute, RIKEN, Wako, Saitama, Japan. 3RIKEN Center for Developmental Biology,Chuo-ku, Kobe 650-0047, Japan. 4Department of Pediatrics and Program in HumanMolecular Biology and Genetics, University of Utah, UT 84112, USA.

*Author for correspondence (e-mail: [email protected])

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MATERIALS AND METHODSTransgenic and targeted mouse allelesThe Shh, Gli1 and 2, and Xt (Gli3 – Mouse Genome Informatics) mutantalleles employed here were previously described (Chiang et al., 1996; Ding etal., 1998; Hui and Joyner, 1993; Motoyama et al., 1998; Park et al., 2000), aswas the Gli1-CreERT2 mouse line (Ahn and Joyner, 2004; Ahn and Joyner,2005). The Del5-LacZ reporter transgenic construct included an HH-responding regulatory element (Sasaki et al., 1997) consisting of eightsynthetic ‘del-5’ sequences that each contain a Gli-binding site (TTATGACGGAGG CTAACA AGCAGG GAACAC CCAAGT AGAAGC TGGCTGTC). This element was cloned adjacent to the basal gamma-crystallinepromoter and inserted 5� to the �-galactosidase coding sequence (Sasaki et al.,1997) (see Fig. 5A). This construct (total length 5.2 kb) was microinjected intofertilized C57BL/6 oocytes to generate transgenic animals using standardprocedures (Sasaki and Hogan, 1996). For sampling, pregnant females weresacrificed from embryonic day (E) 10.5 to newborn, and the urogenital andreproductive tissues were examined (Haraguchi et al., 2000).

HistologyMouse embryos were fixed overnight in 4% paraformaldehyde (PFA) orPBS, dehydrated through methanol, embedded in paraffin, and 8 �m serialsections were prepared. Hematoxylin and Eosin (HE) staining wereprocessed by standard procedures (Haraguchi et al., 2000).

Immunohistochemistry and X-Gal stainingImmunohistochemical analysis was performed by standard procedures usingthe following antibodies: anti-AR (Androgen receptor) Ab (1:100; SantaCruz, N-20) or anti-SMM (smooth muscle myosin) Ab (1:200; BiomedicalTechnology BF-562). �-Galactosidase activity was detected with previouslydescribed methods (Suzuki et al., 2003). X-Gal-stained embryos werepostfixed with 4% PFA at 4°C overnight, embedded in paraffin, and 8 mmserial sections were counterstained with Eosin. Newborn animals were fixedin 0.2% PFA in the buffer (1 mmol/l MgCl2, 0.02% NP40 in PBS [pH 7.4])at 4°C overnight, embedded in OCT, and frozen sections were preparedusing a cryostat at a thickness of 10 �m. The sections were then postfixedin 0.2% PFA in the buffer at room temperature for 10 minutes before X-Galstaining.

In situ hybridizations for gene expression analysesSection in situ hybridizations for gene expression analyses were performedas previously described (Suzuki et al., 2000). The antisense riboprobetemplates for Ptc1 (Ptch1 – Mouse Genome Informatics), Gli1 and Shh wereas described by previous authors (Ding et al., 1998; Haraguchi et al., 2001;Motoyama et al., 1998).

Lineage analysis of HH-responding tissuesTissue lineage analyses were conducted by analyzing Gli1-CreERT2;R26Rmice (Ahn and Joyner, 2004; Ahn and Joyner, 2005). The Gli1-CreERT2

mice were crossed with R26R-LacZ indicator mice (Soriano, 1999) to obtainGli1-CreERT2/+;R26R/R26R males, which were subsequently crossed withICR females. Noon of the day of a vaginal plug was designated as E0.5. A20 mg/ml stock solution of tamoxifen (T-5648, Sigma) was prepared in cornoil. Two milligrams of tamoxifen per 40 g body weight was administered tothe pregnant ICR mouse females using oral gavage at E8.75~9.0 to label thePCM region. Mouse embryos and newborn animals were processed forwhole-mount or section X-gal staining. No overt teratologic effects wereobserved after tamoxifen administration at E9.0 of pregnancy under theseconditions (data not shown). We monitored the post-pubescent reproductiveactivity of some animals exposed to tamoxifen as embryos by the currentconditions and found that they had normal reproductive activity (data notshown).

RESULTSExpression analysis of Shh, Ptc1 and Gli1 duringurogenital organ formationWe examined histogenesis during formation of the GT, urethra,lower body wall and bladder and also assayed expression ofmembers of the Shh signaling cascade during this period (Figs 1, 2).

At E10.5 (hereafter E represents embryonic day post coitum), thedeveloping cloaca and caudal hindgut are apparent (Fig. 1A,D). ByE11.5, the GT is observed adjacent to the cloaca as a prominentprotrusion (Fig. 1B,E). Continued growth of the GT is accompaniedby the development of the urethral plate (the primordia of theexternal urethra) and the bladder at E13.5 (Fig. 1C,F). At E10.5-13.5, prominent Shh expression was observed in the epitheliumlining the cloaca, the urethral plate, the internal urethra and theluminal epithelia of the bladder (Fig. 2A-C). By assaying adjacentsections, we observed complementary expression of the HH receptorPtc1, and HH-intracellular effector Gli1 in the PCM (Fig. 2D,G;yellow box). This finding is consistent with an epithelial-mesenchymal interaction between the cloacal epithelium and thePCM in a putative ‘cloacal field’ (Yamada et al., 2006). From E11.5to E13.5, the Shh expression domain expanded further and includedthe developing urethral plate and the epithelia lining the internalurethra and bladder (Fig. 2B,C; note continuity of Shh expression inthese three epithelial structures). Widespread Ptc1 and Gli1expression were detected in the mesenchyme adjacent to theseepithelia (Fig. 2E,F,H,I).

Hypoplasia of urethral plate, internal urethra andbladder wall in Shh and Gli mutant miceTo examine how Shh signaling influences the histogenesis of theaforementioned structures, Shh null mutant embryos were examined(Fig. 3). At E13.5, the GT was hypoplasic and the UP was notvisible, although it is well developed in controls at this stage (Fig.

RESEARCH ARTICLE Development 134 (3)

Fig. 1. Urogenital tissue formation in mice. (A-F) Histogenesis ofthe developing cloaca, GT, UP and bladder at E10.5 (A,D), E11.5 (B,E),E13.5 (C,F). H&E stained mid-sagittal sections of the cloaca, GT andbladder primordia are shown in D-F. b, bladder; bw, body wall; c,cloaca; gt, genital tubercle; pcm, peri-cloacal mesenchyme; t, tail bud;u, internal urethra; uc, umbilical cord; up, urethral plate.

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3A,C). The bladder cavity and surrounding mesenchyme werehypoplasic (Fig. 3D; blue arrow). The normal angle made by theconnection between the internal urethra and the bladder (the internalurethral orifice) was apparent in wild-type embryos but was notobserved in Shh null mutant mice [already prominent at early stages(Fig. 3A,B) and also clear at late stages in wild-type embryos (Fig.3E; data not shown). The curved course of the internal urethra fromthe internal urethral orifice, under the pubic bone, to the GT isapparent in wild-type embryos (Fig. 3E, green arrows). By contrast,in Shh mutants we observed a simple bladder-like lumen attached toa rudimentary tube that extended toward the remnant of the GT (Fig.3G). In humans, the ventral bladder is composed by two inferio-lateral surfaces facing the pubic bones and external genitalia.Although the frontal (ventral) anatomical structures of the mousebladder are not prominently developed, mesenchymal hypoplasiawas very prominent in the frontal regions of the bladder of Shh nullmutants (Fig. 3; data not shown).

HH signaling is mediated in part by the Gli family oftranscription factors (composed of three members, Gli1, Gli2 andGli3 in mammals), and studies in Gli mutants have revealedessential roles for both positive and negative transcriptionalregulation mediated by these factors during normal embryogenesis(Blaess et al., 2006; Ding et al., 1998; Haraguchi et al., 2001;Hardcastle et al., 1998; Hui and Joyner, 1993; Matise and Joyner,1999; Mo et al., 1997; Motoyama et al., 1998). During urogenitaltissue formation (E10.5-13.5), Gli2 and Gli3 expression was

detected in the GT and bladder mesenchyme (Haraguchi et al.,2001) (R.H. and G.Y., unpublished). To confirm a role for HHsignaling in urogenital tissue development, we examined thesestructures in Gli1, Gli2 and Gli1;Gli2 compound mutants (Fig. 4).Gli2–/– mutants have hypoplasia of the external genitalia, internalurethra and bladder (Fig. 4B). Although Gli1–/– mutant mice werephenotypically normal (data not shown), additional loss offunction of a single allele of Gli2 in a Gli1 homozygous mutant(Gli1–/–;Gli2+/–) resulted in similar urogenital malformations tothose observed in Gli2–/– mutants (Fig. 4C). Previous studies of Glicompound mutants demonstrated that Gli family members haveredundant functions in organogenesis (Bai et al., 2004; Hardcastleet al., 1998; Mo et al., 1997; Motoyama et al., 1998; Park et al.,2000). Our findings reveal that such redundancy also occurs duringdevelopment of the urogenital tissues. In summary, mouse mutantslacking either the Shh ligand or HH transcriptional effectorsdisplay similar defects in external genitalia, internal urethra andbladder formation.

Localizing activity of the HH pathway in vivousing reporter mouse strainsNext, we employed a new reporter mouse strain to localize activeHH signaling in vivo (Fig. 5). We developed an HH-responsive,LacZ marker strain called del5-LacZ (see Materials and methods)employing a Gli-responsive binding site that had been previouslyidentified in the 5� flanking upstream sequence of the Foxa2 gene

527RESEARCH ARTICLECoordinated formation of bladder and urogenital organs

Fig. 2. Expression patterns of Shhsignaling genes in developingurogenital tissues. Shh is expressed in thecloacal epithelium, internal urethra,bladder and urethral plate (A-C). Serialtissue sections reveal Ptc1 (D-F) and Gli1(G-I) expression in the adjacentmesenchyme. At E10.5, both Ptc1 (D) andGli1 (G) are expressed in the PCM (yellowbox). At E11.5 and E13.5, both genes areexpressed in the mesenchyme surroundingthe urethra, bladder and urethral plate(E,F,H,I). (J) Schematic depicting urogenitalorgans adjacent to the limb and the tail atE13.5. Shh-expressing epithelia are shownin red. b, bladder; bw, body wall; c, cloaca;cm, cloacal membrane (the ventral side ofthe cloaca composed by inner endodermalepithelia with surface ectoderm); gt,genital tubercle; hg, hindgut; pcm, peri-cloacal mesenchyme; r, rectum; u, internalurethra; uc, umbilical cord; up, urethralplate.

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(Sasaki et al., 1997). Deletion analyses on the upstream sequencesof Foxa2 gene revealed the sequences to detect HH signaling usingin vitro systems (Sasaki et al., 1997) (H.S., unpublished). Based onsuch studies, an HH-responsive transgenic mouse line wasgenerated, employing this Gli-responsive binding site (this study andJ.M. and H.S., unpublished). Here, we assayed the efficacy of thisreporter as a readout of intracellular HH by examining LacZ activityin the E11.5 limb and neural tube (Fig. 6), both embryonic regionsof HH activities (McMahon et al., 2003) (Fig. 6A,C). Furthermore,as would be predicted, del5-LacZ activity was increased in limbbuds lacking the Gli3 repressor (genotype: del5-LacZ/+;Xt/Xt, Fig.6B) and was reduced in the neural tube and notochord of Shh nullmutants (Fig. 6D).

We employed the del5-LacZ reporter mouse to assay for Gli-mediated transcriptional activation in the developing bladder, PCMand urethral plate. The mesenchymal LacZ staining observed in thedel5-LacZ reporter mouse was quite specific and restricted toinclude the PCM adjacent to the cloaca at E10.5 (yellow box in Fig.5B), and the mesenchyme surrounding the bladder at E12.0 andE13.5 (Fig. 5C,D). There was also prominent LacZ staining of theUP at E13.5, but not at earlier stages. The size of these del5-LacZ-positive domains was significantly reduced in Shh null mutants atboth E11.5 and E13.5, (Fig. 7B,D). We attribute the residual signaldetected in these regions to the other HH ligands, e.g. Ihh expressionin the cloaca (data not shown).

We also noted that the intensity of del5-LacZ staining variedwith the embryonic stage and tissue examined: the LacZ signalwas faint in the PCM at E11.5 but quite robust in the bladdermesenchyme by E12-13.5 (Fig. 5C,D, white arrow), whereas nostaining was detected in the GT mesenchyme at these stages(black arrows; see Discussion). Thus, the pattern of del5-LacZexpression is restricted within the broader Ptc1 and Gli1expression domains in urogenital tissues at multiple stages

(compare Fig. 5B with Fig. 2D,G and Fig. 5D with Fig. 2F,I;expression data not shown for E9.5). As our in vitro and in vivodata showed that the expression regulated by the del5 sequencereflects intracellular HH signaling (Sasaki et al., 1997), it is likelythat restricted HH signaling revealed by the del5-LacZ stainingdata reflects the net of positive and negative Gli activity in thisregion. In fact, we detected Gli3 repressor expression duringurogenital tissue development (data not shown).

Analysis of the contribution of HH-respondingurogenital tissues utilizing tamoxifen-inducibletissue labelingAn important feature of urogenital and reproductive organ formationis the dynamic transition of the initial epithelial lined cavity (thecloaca and urogenital sinus) into bladder, GT, urethral plate andurethra. In order to follow the tissue lineages that respond to HHsignals at different time points and locations during development ofthese structures, we used an inducible genetic labeling system.Taking advantage of the regulation of the activity of the Gli1 locusby HH signaling, the Joyner lab generated a unique HH-respondingtissue lineage analysis system by targeting a tamoxifen-inducibleCre recombinase to the Gli1 locus (Gli1-CreERT2; see schematic inFig. 8) (Ahn and Joyner, 2004). This system has been shown to beeffective for analyzing the tissue lineage of HH-responding cellsduring limb formation and central nervous system development(Ahn and Joyner, 2004; Ahn and Joyner, 2005; Harfe et al., 2004).

Based on our data documenting the histogenesis of the cloacaand urogenital sinus and Shh expression by this tissue, we wereinterested in defining the contribution of early HH-responsivecells to the bladder, urethra and external genitalia. We thereforeutilized tamoxifen to induce activity of Cre protein present atE8.75-9.0 (expressed from the Gli1-CreERT2 allele) and thenassayed LacZ activity from the Rosa26 Cre reporter (Ahn and


Fig. 3. Dysgenesis of the body wall, bladder and GT in Shh null mutants. (A-H) H&E-stained mid-sagittal sections of wild-type (A,B,E,F) andShh–/– (C,D,G,H) specimens at E13.5 (A-D) and at newborn stages (E-H). Higher magnifications of the GT and bladder primordia (boxed in A,C,E,G)are shown in B,D,F,H. The blue arrows in D and H indicate mutant bladder primordia; the green arrows in E indicate internal (pelvic) urethra. am,abdominal muscle; b, bladder; bs, bladder smooth muscle; bw, body wall; eg, external genitalia; p, pelvic bone; r, rectum; u, internal urethra; uc,umbilical cord.

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Joyner, 2004; Ahn and Joyner, 2005) at E10-11 and at later stages.Under these temporal conditions, we detected significant LacZstaining in the PCM [Fig. 8A,B; the LacZ signals in the yellowregion correspond to the Ptc1 and Gli1 expression domains in Fig.2D,G; this point is also suggested by the earlier expression of Ptc1and Gli1 (data not shown)]. Remarkably, assaying for LacZactivity at E13.5 revealed that ‘early-onset and long-contributing’lineages in the PCM make a dynamic contribution to the dorsal(upper) GT and the bladder (Fig. 8C,D). This developmental LacZtransition from PCM to GT and bladder suggests a coordinateddevelopmental formation of these organs for the first time (seebelow).

Marker analysis of bladder smooth muscle anddorsal GT mesenchymal differentiationOne of the key characteristics of bladder mesenchymedifferentiation is the expression of smooth muscle markers (Baskinet al., 1996; Yamada et al., 2006). We assayed the LacZ-positivebladder mesenchyme in Gli1-CreERT2 reporter embryos (tamoxifeninduced at E8.75-9.0) for smooth muscle myosin production. Co-staining of the mesenchyme in the bladder wall at E14.5 confirmsthat these smooth muscle myosin-positive tissues derive from earlyHH-responsive lineages (genetically labeled at E9; co-stainingrevealed by blue LacZ signals and brown anti-smooth musclemyosin signals, Fig. 9A,B).

The external genitalia undergo hormone-dependent, dimorphicmorphogenesis after E16.5, which leads to penis and clitorisformation in males and females, respectively (Cobb and Duboule,2005; Dolle et al., 1991; Yamada et al., 2003; Yamada et al.,2006). Androgen receptor (AR) plays a fundamental role indirecting the GT toward penis formation, including tubular urethraformation from the UP. AR gene is prominently expressed in maleGTs later than E16.5 and also its expression is detected in thehormone-independent early phase of GT formation (R.H. andG.Y., unpublished). Thus, we examined mesenchymal ARexpression in the GT mesenchyme of LacZ-stained, tamoxifen-induced HH reporter mice. At E14.5, co-staining for LacZ and ARin the dorsal GT mesenchyme reveals a developmental linkbetween the AR-expressing dorsal GT and early HH-respondingtissues derived from the PCM (Fig. 9C,D). This dorsal GTmesenchyme probably contributes to corporal bodies and penilebones (Yamada et al., 2006) (Fig. 8E). LacZ staining of the dorsalGT and bladder mesenchyme in Gli1-CreERT2 reporter newbornsthat were tamoxifen-induced at E8.75-9.0 also supports thecontribution of early HH-responsive PCM to these structures (Fig.8E-G).

To further confirm these results, we assayed the effects ofreduced Shh gene dosage on the contribution of HH-responsivetissues to the GT and bladder in compound mutants (Shh–/–; Gli1-CreERT; R26R/+, Fig. 9E-J) and noted a significant reduction in


Fig. 4. Urogenital tissue defects in Gli mutant mice. (A-C) Mid-sagittal H&E sections of urogenital organs in newborns with genotypesindicated. Urogenital tissue defects present in Gli2–/– embryos (B) wereexacerbated in Gli1–/–;Gli2+/– embryos (C) as compared with controls(Gli1–/– or Gli2+/– embryos, data not shown). am, abdominal muscle; b,bladder; eg, external genitalia; p, pelvic bone; r, rectum; u, internalurethra.

Fig. 5. The del5-LacZ transgenic reporter allele detects activeHH signaling in vivo. (A) A schematic diagram of the del5-LacZDNA construct to generate transgenic mice. The promoter containseight consensus Gli-binding motifs (red ovals) and a basal gamma-crystalline promoter upstream of the LacZ gene. (B-D) LacZ stainingwas detected in the PCM at E10.5 (A; yellow box and inset) and atsubsequent stages in the PCM, urethral plate and bladder (C,D).Note that at E12.5 and E13.5, there is robust LacZ activity in bladder(white arrow) but not in the GT mesenchyme (black arrow). b,bladder; c, cloaca; gt, genital tubercle; u, internal urethra; uc,umbilical cord; up, urethral plate.

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the contribution of LacZ-positive tissues in Shh null mutants (Fig.9H-J; blue arrow indicates the reduced LacZ activity relative toFig. 9E-G).

DISCUSSIONParticipation of HH signaling in bladder, urethra(external and internal) and external genitaliaformationThe current analyses represent the first demonstration of theessential functions of HH signaling for bladder formation and theimportant structural contributions made by HH-responsive tissuesto the bladder, urethra and dorsal external genitalia (often termed theBWBG region: body wall, bladder and genitalia).

The urogenital and reproductive tissue primordia ultimately formthe organs necessary for uresis, ejaculation and sperm intake andimplantation and urino-retention. How PCM region (partlymentioned as infra-umbilical mesenchyme; basically locating in theupper part of the cloacal field) could contribute to various urogenitaltissues has remained a mystery for decades (Mildenberger et al.,1988). By using a tamoxifen-inducible genetic lineage system tolabel HH-responding tissues, we have been able to define thetemporally distinct contributions of different HH-respondinglineages to the complex urogenital organs (Fig. 9K). Labeling atE8.75-9.0 revealed that early HH-responsive tissues contribute firstto the PCM and later, to the dorsal GT and bladder mesenchyme. Ofthe mesenchyme derived from the PCM, the dorsal GT mesenchymedisplayed sexual dimorphism at late embryonic stages but bladdermesenchyme did not.

The tamoxifen-inducible labeling system described by Ahn et al.is thought to label cells within approximately one day of tamoxifeninjection. In our tamoxifen/Gli1-CreERT2 labeling experiments, theLacZ signal was relatively weak in the PCM compared with theprominent staining and contribution of these cells to bladdermesenchyme and the dorsal GT. We suggest that the initial cellpopulation within the PCM undergoes transient expansion by

proliferation (data not shown). Our data obtained with the HH-responsive marker allele, del5-LacZ, also suggested very dynamicurogenital organ formation, consistent with these findings; in thissystem, LacZ expression is directly regulated by the HH-induciblepromoter in vivo (not from the Rosa locus). In fact, the latter approachrevealed prominent HH signal activation in the bladder mesenchyme,but not in the dorsal GT, at E12-13. Overall, these data illustrate thevarying level of participation of HH-responsive tissues in specificaspects of urogenital organ development at different stages.

Although these data provided compelling evidence for thetemporally dynamic generation of individual urogenital andreproductive tissues from HH-responsive cells, the precise sourceand identity of HH ligands that are required (including Shh and Ihh)and their specific targets will require further investigation, asdetermining the extent of redundancy between Shh and Ihh. We havefound that the PCM adjacent to the cloacal epithelium is positive forSHH protein by immunostaining (data not shown). In the bladder,based on the timing of tamoxifen induction and subsequent timingof contribution of LacZ-positive cells to individual regions, most, ifnot all, of the required HH signaling may arise from the cloaca andurogenital sinus.

Crosstalk between HH and other signaling pathways, such asfibroblast growth factors (Fgfs), bone morphogenetic proteins(Bmps) and Wnts, and integration of these multiple inputs, is likelyto be as crucial for regulating normal development of the thesetissues as has been proposed in other regions of the developingembryo (Lamm et al., 2001; McMahon et al., 2003; Ovchinnikov etal., 2006; Roberts et al., 1998; Yamada et al., 2006; Zuniga et al.,1999).

Genetic cascades for urogenital organogenesisand insights into human birth defectsOrchestration of complex urogenital organ formation is one of thekey events in embryogenesis. How the PCM contributes to multipleurogenital tissues has remained a mystery for decades. Our


Fig. 6. HH-reporter expression in Shh null and Xt/Xt (Gli3) mutantembryos. (A-D) Expression of the del5-LacZ transgene in the limbs andin the neural tube of wild-type (A,C), Xt/Xt (B) and Shh null (D) embryosat E11.5. The expression level of del5-LacZ was reduced in Shh–/– (D)and augmented in Xt/Xt (B) mutant embryos (black arrows).

Fig. 7. Shh null mutants have reduced HH signaling in thedeveloping urogenital organs. A reduction in LacZ activity from thedel5-LacZ reporter of Shh–/– mutant PCM and bladder (B,D) wasobserved when compared with the control specimen (A,C).(C,D) Higher magnifications of the boxed areas show the bladderprimordia. b, bladder; c, cloaca; gt, genital tubercle; pcm, peri-cloacalmesenchyme; u, internal urethra; uc, umbilical cord.

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demonstration of the contribution of early PCM to differenturogenital tissues, including the bladder and external genitalia, is aunique finding that supports previous speculation in medicalembryology text books (Carlson, 1994; Larsen, 1997; Moore, 1998).In human teratology, abnormal human phenotypes reflectingsimultaneous dysgenesis of the bladder and external genitalia havebeen described. Affected human phenotypes often display exstrophy

of the bladder, epispadias, or ‘flattened or hypoplasic externalgenitalia’, which are intriguingly similar to phenotypes in themutants analyzed here. These complex congenital defects promptedthe idea of coordinated urogenital organ development. However,there has previously been little experimental exploration of the tissuelineages or signaling pathways involved that would provide amechanistic basis for such a hypothesis.


Fig. 8. Contribution to HH-responding bladder and externalgenitalia mesenchymes from the PCM. Above is a schematicdepicting the inducible Gli1-CreERT2 allele and the experimentaltimecourse employed to labeling HH-responsive tissues. (A-D,F,G) LacZstaining of urogenital tissues in Gli1-CreERT2;R26R embryos that wereadministered tamoxifen at E8.75~9.0. (A,C) Whole-mount view ofstained embryos at E10.5 and E13.5. (B,D) Mid-sagittal sections showthe contribution of HH-responsive cells initially to the PCM (yellow boxand inset) and to the mesenchyme of the GT and bladder (D, redarrow). (E) Diagram of newborn sagittal specimens; yellow boxesindicate likely primordia of penile bone and corporal bodies and thebladder, labeled and shown in F and G, respectively. b, bladder; c,cloaca; gt, genital tubercle; hg, hindgut; pcm, peri-cloacalmesenchyme; r, rectum; t, tail bud.

Fig. 9. Marker analyses and fate mapping of tissuesresponding to HH signaling in Shh mutants. (A-J) Tamoxifen-induced Cre labeling with the same experimental conditions as thosedescribed in Fig. 8. (A-D) E14.5 specimens were co-stained for LacZactivity (green) and immunohistochemically stained fordifferentiation markers (brown). (A,B) In the bladder (but not in theGT) LacZ labeled mesenchyme co-stained with anti-smooth musclemyosin (SMM). (C,D) The LacZ-positive dorsal GT mesenchymeoverlapped with the tissue expressing AR. (E-J) In Shh mutants,reduced LacZ staining was detected in the embryonic GT and bladdermesenchyme (compare E with H, and F,G with I,J). (K) A schematicdepicting the distribution of HH responding tissues at E9.5-10.5 andE13.5. b, bladder; bw, body wall; c, cloaca; cm, cloacal membrane(the ventral side of the cloaca composed by inner endodermalepithelia with surface ectoderm); gt, genital tubercle; hg, hindgut;pcm, peri-cloacal mesenchyme; up, urethral plate.

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Urethral birth defects are a frequent congenital defect observed inhumans, and some publications suggest that their incidence may beincreasing in certain geographic regions (Baskin, 2004). Moreover,significantly higher frequency of such birth defects in male infantsis reported (Larsen, 1997; Moore, 1998). Defects of the dorsalexternal genitalia include epispadias with urethral defects andvarious dorsal external genitalia anomalies with relatively lowfrequency (Jeffs, 1987). Birth defects affecting the bladder, such asbladder exstrophy with associated malformation of the urethra hasbeen reported in 1 of about 30,000-50,000 births (Cheng et al., 2006;Jeffs, 1987). Affected children often require multiple reconstructivesurgical procedures (Jeffs, 1987; Johnston, 1975), increasing theimpetus for research into the developmental bases of normal andabnormal urogenital organogenesis.

Another point for consideration is the lack of understanding of theembryonic mesenchymal precursors of the bladder walls. Improvingour knowledge in this regard is vital to support medical efforts inbladder reconstruction. In fact, current bladder reconstructionapproaches often utilize other endodermal organs, such as the ileumand the colon as the source of autograft tissue to construct bladder-like reservoirs (Staack et al., 2005). Although the development ofthese endodermally derived tissues was also influenced by Shh, theultimate structure and physiological character of mature gut andbladder are obviously significantly different. Hence, multiplecomplications, including electrolyte imbalance and stone formationwere observed after the use of gastrointestinal mucosa ormesenchyme for bladder repair (Staack et al., 2005). Therefore,elucidation of the developmental mechanisms driving embryonicmesenchyme toward functional bladder tissues is likely to havesignificant impact when translated to the medical arena. Althoughfurther studies are necessary, our work has provided insight into thefunctions of Shh signaling during embryonic bladder formation. Thecurrent findings of the importance of HH signaling and the structuralroles of HH-responsive tissues during urogenital organogenesis maynot only provide clues to understanding the basis of humanurogenital birth defects, but may also stimulate translational researchthat can have an impact on the management of these defects.

We would like to specially thank Drs Alex Joyner and Chi-Chung Hui for theirinvaluable support. We would also like to thank Drs Denis Duboule, ShigeakiKato, Larry Baskin, Jerry Cunha, Tsutomu Ogata, Chisa Shukunami, JohnMcLachlan, Cathy Mendelsohn, Chin Chiang, Melissa Little, MarkLewandoski, David Ornitz, Jeffrey Pollard and Andrew McMahon forencouragement and suggestions. We would also like to express ourappreciation to Shiho Kitagawa for her valuable assistance. This study wassupported by a Grant-in-Aid for Scientific Research on Priority Areas; byMechanisms of Sex Differentiation; by General promotion of Cancer researchin Japan; by the 21st Century COE Research Program; and by a Grant forChild Health and Development (17-2) from the Ministry of Health, Labour andWelfare.

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