Molecular genetic cascades for external genitalia formation: An emerging organogenesis program

DEVELOPMENTAL DYNAMICS 235:1738 –1752, 2006

REVIEWS–A PEER REVIEWED FORUM

Molecular Genetic Cascades for External Genitalia Formation: An Emerging Organogenesis Program G. Yamada,1* K. Suzuki,1 R. Haraguchi,1 S. Miyagawa,1 Y. Satoh,1 M. Kamimura,1 N. Nakagata,1 H. Kataoka,2 A. Kuroiwa,3 and Y. Chen4

External genitalia are anatomical structures located at the posterior embryonic region as part of several urogenital/reproductive organs. The embryonic anlage of the external genitalia, the genital tubercle (GT) develops as a bud-shaped structure with an initial urethral plate and later urethra. Embryonic external genitalia are considered to be one of the appendages. Recent experiments suggest that essential regulatory genes possess similar functions for the outgrowth regulation of the GT and limb appendages. The transient embryonic epithelia located in the distal GT are called the distal urethral epithelium (DUE) regulating, at least in part, the (distal) GT development. This review covers the available data about early patterning of GT and discusses the molecular developmental similarities and points of divergence between the different appendages. Development of the male and female external genitalia is also reviewed. Developmental Dynamics 235:1738 –1752, 2006. © 2006 Wiley-Liss, Inc. Key words: reproduction; urogenital organ; sex differentiation; growth factors; androgen; hormone; genitalia; appendage development; penis; clitoris; genital tubercle; cloaca; tail; anus Accepted 23 February 2006

INTRODUCTION The urogenital/reproductive organs are those necessary for various aspects of the reproduction of a species. They include external genitalia and

internal reproductive organs. The latter includes the vagina, uterus, and oviduct in females and the prostate, seminal vesicles, and vas deferens in males. The gonads, namely the ovaries and testes, are organs that gener-

ate and supply germ cells for reproduction. These organs have developed to enable efficient internal or external fertilization. Anatomically, male external genitalia include the penis, urethra, and scrotum. The female exter-

ABBREVIATIONS ARM anorectal malformation AER apical ectodermal ridge AP anterior-posterior AR androgen receptor BMP bone morphogenetic protein CM cloacal membrane DUE distal urethral epithelium FGF fibroblast growth factor GT genital tubercle KO knockout PCM peri-cloacal mesenchyme PD proximo-distally SEM scanning electron microscope SHH sonic hedgehog UP urethral plate URS urorectal septum VER ventral ectodermal ridge

1 Center for Animal Resources and Development (CARD), Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan 2 Second Department of Pathology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan 3 Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan 4 Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana Grant sponsor: Scientific Research on Priority Areas, Mechanisms of Sex Differentiation; Grant sponsor: General Promotion of Cancer Research in Japan; Grant sponsor: 21st Century COE Research Program; Grant sponsor: Grant for Child Health and Development (17-2) from Ministry of Health, Labour and Welfare. *Correspondence to: G. Yamada, Center for Animal Resources and Development (CARD), Graduate School of Medical and Pharmaceutical Sciences, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan. E-mail: [email protected]

DOI 10.1002/dvdy.20807 Published online 5 April 2006 in Wiley InterScience (www.interscience.wiley.com).

© 2006 Wiley-Liss, Inc.

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nal genitalia cover regions inside the labia majora including labia minora, clitoris, and vestibule of the vagina. Great progress has recently been achieved in molecular embryology that has provided clues regarding how embryonic organ development is coordinated by regulatory genes and growth factors. In addition, advances in mouse molecular genetics have now made it possible to utilize conditional gene knockout (KO) mouse models to analyze questions concerning various organs. Such experimental strategies have established mouse models as one of the suitable tools to analyze the genetic cascades for organogenesis. The application of mouse molecular genetics to analyze another embryonic developmental context, i.e., urogenital/ reproductive organ formation has also been initiated (Cobb and Duboule, 2005; Kobayashi and Behringer, 2003). Due to the “limited” number of developmental regulatory genes in the genome, some of the regulatory genes involved with other organs have been suggested to regulate urogenital/reproductive organogenesis. However, the molecular mechanism of reproductive organ formation, with the exception of some pioneering research into gonadal development, still represents an unexplored research area. This might be a reflection that (1) mouse genetic studies have only recently been utilized for it, and (2) the unisexual to sexually dimorphic developmental processes have so far been insufficiently analyzed. During development, limbs, fins, and external genitalia protrude from the body wall and are termed appendages by embryologists (Ogino et al., 2004). The ways in which these structures develop can differ in many ways. In this review, a unique developmental viewpoint is presented and discussed in relation to the molecular developmental similarity and the divergence for external genitalia formation as one of the appendages. Hence, emphasis is placed on the early embryonic GT and the limb. Our descriptions mainly cover embryonic external genital patterning and not much about hormone-dependent tissue differentiation. For their general developmental processes including female external genitalia development, previous reviews and texts are also

referred to (Yamada et al., 2003; Larsen, 2001). Limb outgrowth is positioned in the body trunk in a way that is linked with the main body skeleton, enabling efficient motion control for the species. Arms/legs develop proximo-distal structures called stylopod, zeugopod, and autopod. External genitalia also display proximo-distally differentiated structures that develop in cooperation with urinary/reproductive organs. The male external genitalia have the following characteristics: 1. They are equipped with a physiological architecture enabling an erection for copulation and folding/ retracting for time other than copulation. 2. The external genital appendages usually develop tubular or groovelike epithelia necessary for uresis or ejaculation. As the genital tubercle (GT) develops in association with urinary/reproductive systems, its outgrowth initiation follows vital endodermal morphogenesis in the posterior of embryos such as in the cloacal region formation. However, how all the above morphogenetic requirements are fulfilled by sets of developmental programs has not been clarified until recently. These findings are described in this review. Regarding later development, external genitalia develop as male or female specific organs in their adult form. This means that hormone-dependent development, i.e., the sexually dimorphic development of the external genitalia, is observed. Male anatomical structures have evolved in insects, fishes, amphibians, birds, and mammals. Masculine development of the external genitalia is generally androgen dependent (Wilson et al., 2002; Zhou et al., 2002). The actions of androgens during external genital morphogenesis generally occur late in embryogenesis, namely after the initial unisexual patterning of the external genital anlagen. The research field dealing with external genitalia formation is interdisciplinary, and it is categorized variously as molecular developmental biology, reproductive biology (McLachlan, 2001). Integrated studies are required to explore the differ-

ent developmental and physiological questions related to the basic sciences involved, along with the associated clinical aspects, in this research field. Reports dealing with the molecular developmental mechanisms of mammalian external genitalia development are increasing. The integrated knowledge covering the various topics touched upon here will be beneficial for the fields of human medical genetics, teratology, and molecular developmental studies. In this review, a general overview of external genital morphology and recent molecular analyses on their development will be presented.

EMBRYONIC EXTERNAL GENITALIA DEVELOPMENT: GENITAL TUBERCLE (GT) FORMATION IN MOUSE EMBRYOS During mouse development, a protrusion is observed in the caudal region of the embryo. The genital tubercle (GT), an embryonic anlage of the external genitalia, later differentiates into a penis in males and a clitoris in females. Previous studies have suggested GT developmental processes show some similarities with limb development. Both structures exhibit, at least superficially, similar developmental processes such as displaying prominent outgrowth as an embryonic bud structure before their differentiation (Dolle et al., 1991; Yamaguchi et al., 1999). A scanning electron microscope (SEM) analysis of GT development shows that the cloacal membrane (CM) is located at the ventral midline in the caudal regions of the embryo before the protrusion of the GT (Fig. 1A). Embryonic regions bilaterally adjacent to the CM form genital folds with the CM, visible as a shallow “groove”-like region. Virtually no tissue lineage analyses have been performed regarding the question of the origin of various GT regions at late stages back to the peri-cloacal region. Because mouse embryos are experimentally useful species to analyze tissue lineage contribution in mammalian urogenital/reproductive organs, genetic lineage analyses using transgenic embryos should be developed.

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It is speculated that growth factors expressed in the CM may be involved in the epithelial–mesenchymal interactions between the CM peri-cloacal mesenchyme (PCM). These events contribute to regions adjacent to GT as tissue lineages (see below). Research focusing on the PCM is very limited to date. Such a region has only been referred to as the posterior ventral tail mesenchyme in studies dealing with epithelial– (VER; ventral ectodermal ridge) mesenchymal interactions (Goldman et al., 2000). Apart from such interactions, virtually no studies have so far been reported on cloaca–PCM interactions. Parts of the cloaca are caudally connected to a blind-ended tail gut. After E10.5 in the mouse (hereafter E represents the murine embryonic days of development), the GT starts to develop rostrally to the CM. The GT appears as a prominent structure at E11.5, with bilaterally located ventral swellings (Fig. 1B). It then further grows out proximo-distally (PD) at E12.5 (Fig. 1C). The urethral plate (UP) is formed at the ventral (lower) median plane, which is continuous with the CM on its caudal side. The cloaca is subdivided into the urogenital sinus and anus, concomitant with the growth of the urorectal septum (URS). This separation, accompanied by the CM “breakdown,” has been considered to be the result of URS development in some previous studies (Nievelstein et al., 1998). The regulation of the breakdown including apoptotic regulation requires further analysis. The mechanisms of URS growth approaching the CM involves several combinatorial events including the growth and positioning of different embryonic regions including the genito-urinary sinus, the hindgut, the GT, and the mesenchyme close to the umbilical cord (infra-umbilical mesenchyme), and CM (Penington and Hutson, 2003; Mo et al., 2001). The urogenital folds surround the orifice of the urogenital sinus. Various types of anorectal malformations (ARM) have been reported with different degrees of separation of the anus. An abnormal development of the CM or dorsal part of the cloaca has often been discussed in relation with such malformations (e.g., Nievelstein et al., 1998). When part of the CM develops

Fig. 1. Development of the genital tubercle (GT) by SEM. All photographs are arranged with the tail bud oriented toward the right of the photo. The cloacal region at E10.5 (A), E11.5 (B), and E12.5 (C) in mouse embryos White arrow indicates cloaca in A. The GT (genital tubercle) develops anterior to the CM (cloacal membrane) and grows out proximo-distally (arrow in B). Part of the hindlimb apical ectodermal ridge (AER) is indicated by an arrowhead. The distal-most protrusion at the tip of the GT is shown by the white arrowhead in C. The developing urethral plate is visible (arrow in C). D,E: Sexually dimorphic development of the GT at E18.5 in mouse embryos. Transverse sections of the GT show internally developing urethra in the case of male GT (D). Transverse sections of the female GT are shown in E.

abnormally, an abnormal hindgut opening or future anal opening (orifice) occurs (Nievelstein et al., 1998). Several signaling pathways have been implicated in the formation of the anorectal region in mouse, including Sonic hedgehog (Shh) and fibroblast growth factor (Fgf) 10 (Mo et al., 2001; Fairbanks et al., 2004). Some morphological features related to the distal GT region, and tail development deserve comment. Recently, the molecular mechanisms of GT formation have been investigated and it has been proposed that the distal urethral epithelium (DUE), the distal-most epithelial region, regulates several aspects of the outgrowth of the murine GT. It has been shown that the DUE fulfills an essential role in the initial outgrowth control of the GT (Haraguchi et al., 2000, 2001; Ogino et al., 2001; Morgan et al., 2003). The roles of DUE during GT morphogenesis are, therefore, considered to be particularly intriguing when viewed from a molecular developmental perspective (see below). Of note is the presence of the distalmost region located at the tip of the GT, which is related to the distal morphology (marked by white arrowheads at E12.5; Fig. 1C). This distal morphology has been prominently observed in human embryonic GT by a SEM analysis (Glenister, 1954; Suzuki et al., 2004a). The developmental significance of such an anatomically

prominent distal tip region of the human GT remains to be investigated. Human embryos generally show morphological similarities with the mouse, albeit with slight morphological variations such as the tail length and distal GT regions (Fallon and Simandl, 1978). Several histological differences have also been observed between human and mouse external genitalia development. The CM is observed in human embryos at Carnegie stage 14 (Suzuki et al., 2004a). Human tail development is well known to initially be prominent while later becoming hypoplastic in comparison to mouse tail development. Apoptotic regulation has been implicated in this process (Fallon and Simandl, 1978). Human caudal dysgenesis syndromes such as sacral/caudal agenesis have been known but mechanisms underlying such symptoms in relation to apoptotic regulation have been not known. Regarding tail development, the tail gut, namely the endodermal organ connected to the cloaca, is prominently observed during mouse development (Suzuki et al., 2004a). Apart from such early urogenital developmental divergences, the mature human penis develops no penile bones in contrast to the murine penis (Murakami and Mizuno, 1986; Murakami, 1987; Yamada et al., 2003; Kamikawa-Miyado et al., 2005). In the mouse, the first morphological differences between the male and

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Fig. 2. Expression patterns of several genes (Fgf8, Shh, Wnt5a, Bmp4, Bmp7) during mouse genital tubercle (GT; B–F) and limb development (G–K). A: SEM photo of an embryonic GT and hindlimbs at E12.0. Arrow: Distal urethral epithelium (DUE) and urethral plate; Arrowheads: apical ectodermal ridge (AER) in hindlimb. The embryonic tail region was removed. Whole-mount in situ hybridization with digoxigenin-labeled Fgf8, Shh, Wnt5a, Bmp4, and Bmp7 probes. B–F: Murine embryonic GT at E12.5. Lower (ventral) side of GT is shown. G–J: Embryonic limb at E11.5. K: Murine embryonic limb at E10.5. Note the “similar” expression of Fgf8 and Wnt5a in the distal epithelia and distal mesenchyme of limbs and GT. Shh is expressed in the urethral plate (UP) (C) and in ZPA (H). Bmp4 is expressed in ventral-distal mesenchyme and ventral bilateral mesenchyme close to the urethral plate (E). Bmp4 is expressed in limb mesenchyme (J). Bmp7 is expressed in the DUE (F) and also in AER of limbs (K).

after E16.5. The canalization of the urethral plate proceeds proximo-distally in the male GT (Fig. 1D). During female external genitalia development, the GT develops less prominently, thus forming the clitoris. The urogenital folds and areas of genital swelling are unfused to form the labia minora and majora. The urethra and vagina are connected to the vestibule of the vagina. Inside the female GTs, although they develop the common UP in earlier stages, the urethral folds are not completely fused and the urethral groove thus remains in the ventral midline (Fig. 1E; see also below).

Fig. 3. Signaling cascades for genital tubercle (GT) formation (A) and for limb formation (B). DUE, the signaling epithelia of the GT, expresses Fgf8 and Bmp7. Adjacent to DUE, the distal mesenchyme of the GT expresses Bmp4 and Wnt5a. In the GT, the urethral plate expresses Shh in the ventral midline (red region in A). Bilateral to the urethral plate, Fgf10 is expressed (C). AER is located in the distal tip of the developing limb (B) and it expresses Fgf8, Fgf9, and Fgf4. Shh is expressed in ZPA of the posterior limb and Wnt5a is expressed in the distal mesenchyme of limb (B). In the GT, midline Shh is expressed adjacent to the bilateral mesenchyme, which expresses Fgf10 (D, E). Shh (D) and Fgf10 (E) gene expression in the E13.5 embryonic mouse GT.

female GT are evident from E16.5. The fusion of the urethral folds in the ventral midline leads to the formation

of the urethral groove, and subsequently the mature urethra. This event is prominent in the male GT

FORMATION OF DISTAL SIGNALING EPITHELIUM (DUE) IN AN EMBRYONIC GT: APICAL ECTODERMAL RIDGE (AER) AND DUE AS COMPARISONS Recently, the molecular mechanisms of GT formation have been investigated. It has been proposed that the DUE, the region marked by Fgf8 expression, regulates the initial outgrowth of the murine GT (Fig. 2B).

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TABLE 1. Developmental Regulators for Embryonic External Genitalia (GT) Developmenta Hoxa13, d13

Model for hand-foot-genital syndrome

Dolle et al. (1991) Mortlock and Innis (1997) Warot et al. (1997)

Wnt5a, Ror2

Proximo-distal hypoplasia of several appendages

Yamaguchi et al. (1999) Oishi et al. (2003)

Fgf8

Demonstration of distal signaling epithelia (DUE) in the GT appendage

Haraguchi et al. (2000) Ogino et al. (2001) Morgan et al. (2003) Yucel et al. (2004)

Fgf10

Regulation of urethral plate (UP) to urethral-formation

Haraguchi et al. (2000) Ogino et al. (2001) Yucel et al. (2004)

RAR

Regulation of urethral plate (UP) formation

Ogino et al. (2001)

Shh, Gli2

Regulation for GT initial outgrowth and urethral plate (UP) formation

Haraguchi et al. (2001) Perriton et al. (2002) Podlasek et al. (2003a)

Hoxa13, Bmp7

Hoxa13 and distal morphogensis, corporal body formation and cloaca formation

Morgan et al. (2003) de Santa Barbara and Roberta (2002)

Hoxd13

Anal sphincter formation

Kondo et al. (1996)

BmprIa, Bmp7, (noggin)

Regulation of proximo-distal outgrowth; GT hyperplasia

Suzuki et al. (2003a) Ptikus et al. (2004)

Fgfr2IIIb

Fgf10-Fgfr2IIIb regulation for urethral development

Satoh et al. (2004) Petiol et al. (2005)

Ephrin-B2, Eph-B2

Ephrin-Eph signaling for urethra formation

Dravis et al. (2004)

Recent histological and molecular analyses have suggested Fgf8-expressing DUE to be adjacent to the outer ectodermal epithelia (Suzuki et al., 2003a). Based on the findings of several functional assays, the DUE of the developing GT functions as signaling epithelia, thereby orchestrating at least the early patterning and the development of the external genitalia (Table 1; Haraguchi et al., 2000; Ogino et al., 2001; Morgan et al., 2003). As for external genitalia formation, the DUE controls both mesenchymal gene expression and GT outgrowth (Haraguchi et al., 2000, 2001). FGF8-soaked beads regulate the GT mesenchymal gene expression such as Fgf10, bone morphogenetic protein (Bmp) 4, and Msx1 (Haraguchi et al., 2000). The surgical removal of DUE leads to hypoplastic GT development in vitro. The AER (apical ectodermal ridge), another transient specialized distal epithelium, is essential for vertebrate embryonic limb outgrowth along the PD axis and it also expresses Fgf8 and other Fgfs (Figs. 2G, 3B). Coordinated

limb development of the AP and PD axis has been shown. In addition to Fgf8, other growth factors also play a role in limb development. The example of Shh, which is expressed in the ZPA (zone of polarizing activity), is well known. Several growth factors have been implicated for anterior-posterior (AP) and PD regulated limb development. The SHH/FGF feedback signaling loop that operates between the polarizing region and the AER may coordinate the growth and patterning of the limb (Fig. 3B; Niswander, 2003). In addition to several Fgfs, the expression of multiple regulatory genes implicated in the formation of AER has also been detected in the developing GT (Suzuki et al., 2003a; Ogino et al., 2001). Besides Fgf8 and Shh expression, several BMP signaling molecules are expressed in the DUE, and in the distal-ventral mesenchyme adjacent to the DUE. The developmental cascade related to DUE formation is relevant for reviewing GT formation. In Drosophila, Decapentaplegic (DPP) is a

downstream target gene of HH signaling. There is also evidence that BMP4 may be a downstream target of SHH (Ingham and McMahon, 2001; McMahon et al., 2003) or it may be located upstream of SHH by regulating its expression in some developmental contexts, e.g., in the mouse dental epithelium (Zhang et al., 2000). Members of the Bmp and Fgf gene families have been suggested to regulate various epithelial–mesenchymal interactions during limb development (Hogan, 1996; Martin, 1998; Dahn and Fallon, 2000; Mariani and Martin, 2003) including possible opposite roles during limb outgrowth regulation depending on the context of the PD development or during digit formation (Dahn and Fallon, 1999; Niswander and Martin, 1993; Ganan et al., 1996; Macias et al., 1997; Pizette and Niswander, 1999). Current analyses of DUE revealed dynamic and complex gene expressions including those of Fgf8 and Bmp7 (or osteogenic protein-1; Fig. 2B,F). Several BMP signaling mole-

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cules are expressed in and adjacent to DUE. In addition, both Bmp7 expression (Table 1; Morgan et al., 2003; Suzuki et al., 2003a) and Fgf9 expression are detected in DUE (Satoh et al., 2004). Bmp7 has also been recognized to be involved in epithelial-to-mesenchymal transition (EMT) during kidney development, and other developmental processes (Dudley et al., 1995, 1999; Zeisberg et al., 2003). Bmp7 has also been reported to play an important role in the hindbrain, tooth, eye development, and skeletal patterning (Luo et al., 1995; Arkell et al., 1997; Godin et al., 1998; Zakin et al., 2005). In urogenital/reproductive systems, it functions in apoptotic regulation and the tissue remodeling of the oviduct (Monroe et al., 2000). Although antagonistic interactions between several BMPs-FGFs have been reported, the role of Bmp7 expression in the transient urogenital epithelia remains to be analyzed (Morgan et al., 2003; Suzuki et al., 2003a). Conditional BmprIa mutant mice GT shows hyperplasia and over-expression of noggin also leads to GT hyperplasia (Suzuki et al., 2003a; Plikus et al., 2004). GT hyperplasia at late embryonic stages or at neonatal stages are reported in both cases, which leaves a question that additional late-staged DUE independent abnormalities of the outgrowth induced by Bmp-BmprIa crosstalks may exist (Suzuki et al., 2003a). A redundant mode of several growth factor functions has been reported in the case of developing limbs (Lewandoski et al., 2000; Moon and Capecchi, 2000; Sun et al., 2000). Some similarities in redundancy for urogenital development have been suggested based on several conditional gene knockout mouse analyses. Of note are the similarity and the degree of divergences between fore- and hindlimb appendage development in terms of a redundant Fgf expression (Sun et al., 2002). In addition to the differences in the timing of gene expression between the forelimb and the hindlimb appendages, genes giving positional cues for the AP body axis are differentially expressed between the forelimb and the hindlimbs such as in the case of Tbx genes (Rodriguez-Esteban et al., 1999; Agarwal et al., 2003; Naiche and Papaioannou, 2003; Rallis et al., 2003; Takeuchi et al., 2003).

Between the limbs and external genitalia, the onset timing of Fgf8 expression in AER and DUE also differs, and the FGF8 expression in DUE is generally observed later than that in AER. Hence, it is expected that there exists some conceivably intriguing developmental similarities and divergences between the genital and limb appendages in terms of the timing and the functions of developmental regulatory genes. These developmental similarities may suggest the existence of a “shared” molecular developmental program for vertebrate appendages. As for late-staged GT formation, Fgf8 is not detected after about E14.5, while GT outgrowth continues further later. Late-staged GT outgrowth irrespective of the presence of DUE and definition of DUE detection per se should be further examined (Suzuki et al., 2003a). The assessment of AER for the regulation of limb outgrowth and for various limb differentiation programs has been a research topic for decades. The AER function for outgrowth and/or differentiation has been suggested for AER initiation, establishment, and maintenance phases during limb formation. Molecular and genetic assessment of DUE for regulating initial GT outgrowth and/or concomitant epithelial-mesenchymal regulation during each stage of E10.5–E14.5 should be further defined and evaluated by several strategies.

LIMB DEVELOPMENT: A SIMILAR DEVELOPMENTAL PROGRAM? A limb appendage developmental program requires a precise set of genetic cascades; first regulating the positioning of the limbs in the body, the initiation processes, and the subsequent outgrowth control of limb appendages. In addition to the intriguing similarities in the outgrowing processes of the limbs and GTs, as described above, the developmental “field” formation of the limbs and GTs may represent an interesting topic of future research for comparison purposes. Limb initiation develops as a result of developmental crosstalk by the genes expressed in lateral plate mesoderm leading to the formation of signaling epithelia (AER) for its subsequent out-

growth. Complex genetic interplay between several transcriptional regulators and growth factors has been reported during the initiation processes of limb bud formation. These include interactions between Tbx(s) and dHand, Gli3, Wnt genes possibly lying upstream of the Shh gene in the developmental cascade, which eventually regulates the PD and AP limb pattern (Ros et al., 1996; Logan and Tabin, 1999; Rodriguez-Esteban et al., 1999; Takeuchi et al., 1999; Wang et al., 2000; Chiang et al., 2001; te Welscher et al., 2002a,b; Naiche and Papaioannou, 2003). An embryonic GT bud is formed adjacent to the developing tail at the midline in between the hindlimb region. In comparison to the cascade for limb initiation, very little information has been presented for the developmental field (cloacal field) formation before GT outgrowth. A developmental gene expression cascade has been suggested for the initiation and subsequent development of GT formation as part of caudal embryonic development. The proper regulation of growth factor– crosstalk between the cloaca and PCM has been shown to be essential for subsequent GT formation (Haraguchi et al., 2001). It is conceivable that one of the fundamental characters of the GT appendage is to develop endodermal epithelia along with GT appendage formation, initially as CM and later forming UP subsequently as tubular urethra in the male GT. In this way, the epithelia to mesenchymal influences in the “cloacal field,” namely the effect of Shh expressing CM to PCM, may be one of the characteristic events for subsequent GT formation. A gene KO mouse analysis has indicated that Shh expression in the CM to be one of the vital developmental events for regulating the cloacal field before GT outgrowth (Table 1; Haraguchi et al., 2001). A phenotypic analysis of Shh mutant mice revealed GT development to be arrested at the initial outgrowth stage. The modulation of the gene functions expressed in CM leads to the alteration of the gene expression in PCM. Bmp4 is normally expressed in the PCM adjacent to the cloaca. Upregulation of Bmp4 accompanied by augmented apoptosis in Shh mutants has been shown (Table

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1; Haraguchi et al., 2001; Perriton et al., 2002). Given the significant degree of the conservation of genetic programs between appendage formation, we have examined the expression and function of various early regulators in the mesenchyme of the PCM, such as Fgf10, Tbx(s), dHand, and Gli3 during external genitalia formation (Haraguchi et al., unpublished data). The Fgf10 gene, for example, does not display drastic GT outgrowth phenotypes by gene KO mice in initiation processes, in contrast to the case of the limb where Fgf10 KO mice show limb agenesis (Min et al., 1998; Sekine et al., 1999). Many gene KO mice for the above genes have been reported as early lethal before the organogenesis stages. Cases of Tbx(s) and dHand KO mice have been reported to be lethal before E10.0 (Agarwal et al., 2003; Davenport et al., 2003; Naiche and Papaioannou, 2003; Rallis et al., 2003). Hence, conditional KO analyses are required to clarify the functions of these genes in urogenital/reproductive tissues. Many Creexpressing mouse lines have been generated to analyze various organ development such as limb formation. However, only limited conditional gene KO analyses have been performed to study GT development, partly due to the lack of appropriate Cre-expressing mouse lines (Suzuki et al., 2003a). Some Cre-expressing mouse lines such as Hox-Cre mice may be useful for the mutation of the genes expressed in posterior embryonic regions. However, precise Cre gene regulation is necessary to analyze particular urogenital/reproductive organs before utilizing them. One may learn such examples by the case of precise AER-Cre line characterization in order to accurately mutate the AER-specific genes during its initiation or maturation. Hence, the characterization of the promoters for GTs or for CM and PCM should be performed. Wnt-family proteins have also been implicated in a variety of developmental processes (Wodarz and Nusse, 1998). Previous studies indicate that Wnt-family proteins can be classified into at least two subfamilies (Kuhl et al., 2000); one is the Wnt1 class (e.g., Wnt1) that activates the canonical Wnt/␤-catenin pathway (Cadigan and

Nusse, 1997; Sokol, 1999), while the other is the Wnt5a class that activates the non-canonical Wnt pathway to regulate cell polarity in Drosophila and convergent extension movements (Moon et al., 1993; Heisenberg et al., 2000; Sokol, 2000; Tada and Smith, 2000; Oishi et al., 2003). Among the Wnt-family genes in mammals, the expression of Wnt5a is detected in the embryonic face, limbs and tail, lungs, and GTs (Yamaguchi et al., 1999). In appendages, Wnt5a is expressed both in the distal mesenchyme of limbs and GTs (Fig. 2D, I). Wnt5a KO mice display facial abnormalities, short limbs and tail, and an absence of a GT, along with abnormalities in lung morphogenesis (Yamaguchi et al., 1999; Li et al., 2002; Suzuki et al., 2003a). Hence, Wnt5a may offer a unique research subject for its similarity in the appendage phenotypes as the mesenchymal regulator.

APPENDAGE PHENOTYPE ELICITED BY ABERRANT GROWTH FACTOR GENE EXPRESSION: THE FORMATION OF THE “CLOACAL FIELD” IN CAUDAL EMBRYONIC DEVELOPMENT Because of the adaptation of the partially conserved developmental pathway, it has often been observed that the manipulation of some key upstream genes leads to drastic appendage phenotypes such as neogenesis of an “additional” limb by the overexpression of such factors as Fgf (Ohuchi et al., 1994; Cohn et al., 1995). In the case of limb formation, developmental cascades composed of several developmental genes, such as Wnt8, Fgf10, and Tbx(s) have been suggested to be early regulators before the onset of regulatory genes for outgrowth. Although more analyses are required, these cascades may lie in one of the upstream programs of the limb bud outgrowing processes regulating the induction/establishment of key developmental events before limb bud emergence. Two research strategies have thus been adopted to analyze limb field formation, i.e., the manipulation of early regulators in the chick embryonic

trunk region and the genetic manipulation of the genes utilizing several gene knockout mouse models. Little is still known about the existence of a developmental field orchestrated by developmental genes before GT bud outgrowth in the tail region. Analyzing the developmental cloacal field is, therefore, essential in order to obtain a better understanding of naturally occurring birth anomalies such as external genitalia malformation including duplicated genitalia or penile agenesis. Molecular developmental analyses of the causative factors for hindlimb fusion with GT defects and penile agenesis have yet to be undertaken. Although the molecular mechanisms remain completely unexplored, penile agenesis has been reported as a human urogenital birth defect with a low frequency (Perovic, 1999). Another caudal phenotype elicited by the manipulation of early key developmental regulators could arise from the possible “reduction” of the cloacal field as the Sirenomelia phenotype displays hindlimb fusion and the ventral posterior embryonic dysgenesis known also as Mermaid Syndrome (Zakin et al., 2005). Sirenomelia is a human developmental cloacal malformation characterized by the fusion of both hindlimbs (Kampmeier, 1927; Hoornbeek, 1970). Although Sirenomelia has variable phenotypes with multiple causes, it is intriguing to consider the mechanisms of dysgenesis possibly related to the cloacal field in between the hindlimbs. Various experimental systems are necessary to analyze the developmental mechanisms of the cloacal field and tail formation. Studies on the chick embryonic cloacal field in addition to a lineage analysis for tail ventral mesenchyme should, therefore, contribute to the understanding of this field (Ohta et al., unpublished results). A coordinated model of the development of the caudal embryonic region including the cloaca, tail gut, and VER has been reported (Miller and Briglin, 1996; Knezevic et al., 1998; Kurzrock et al., 1999b; Liu et al., 2004). Using a tail explant culture system, the importance of VER–tail bud interaction and also in the regulation of tail elongation has been suggested (Goldman et al., 2000). It is noteworthy that such

EXTERNAL GENITALIA FORMATION 1745

pioneering studies focus on the functions of VER or the tail bud in the context of tail morphogenesis with little attentions to the cloacal field. There are also other lines of studies on tail development but focusing on caudal neural tube formation (Schoenwolf, 1984). Hence, more investigations are necessary to clarify the regulatory genes and the developmental significance of the cloacal region as part of the study of caudal embryonic morphogenesis. Posterior to the GT bud, the tail region develops, as does the lower body wall region anterior to the GT underneath the infra-umbilical region (Penington and Hutson, 2002a,b). It has thus been speculated that GT development requires correct adjacent tissue differentiation, such as lower body wall formation. Abnormal lower body wall formation associated with bladder/cloaca extrophy often simultaneously displays genital abnormalities such as the upper GT defects, and epispadias (Zhang et al., 1996; Brewer and Williams, 2004; Ogi et al., 2005; Haraguchi et al., unpublished results). In relation with such phenotypes, the presence or absence of cell migration between the umbilical region (later the lower body wall) and the (upper) GT bud requires further analyses. It has recently been shown that muscle precursor migration exists between the hindlimb and cloacal regions as judged by the origin of cloacal muscles (Valasek et al., 2005).

REITERATED INVOLVEMENT OF REGULATORY GENES FOR APPENDAGE DEVELOPMENT When one looks at the roles of developmental regulators for early stages to late GT formation, transcription factors and growth factors function at each developmental context. Generally, essential regulatory transcription factor or growth factor genes may often be involved at various times during organ formation, e.g., during appendage positioning, initiation, and subsequent outgrowth. In the case of limb development, essential regulatory genes may be sequentially in-

volved in the initiation of budding from lateral plate mesoderm and subsequent outgrowth including epithelial-mesenchymal interactions for limb differentiation, thus resulting in normal pattern formation. Recent work has revealed the reiterated functions of essential regulatory genes, such as Hox genes in appendage development (Table 1; Zakany et al., 2004; Kmita et al., 2005). Furthermore, genetic interactions between such genes may differ depending on the developmental context in early limb bud and late stage limb formation (Zakany et al., 2004). They have been reported to play major roles in the patterning of the limbs and external genitalia (Duboule, 1994; Innis, 1997; Kondo et al., 1997; Hashimoto et al., 1999; Innis et al., 2002; Morgan et al., 2003; Cobb and Duboule, 2005). Nested Hox gene expression along with PD limb axis and embryonic GT axis have been reported in a pioneering study (Dolle et al., 1991). Moreover, the gene regulatory mechanisms of Hox gene expression in the limbs and other tissues including GT, suggest the presence of conserved gene regulation (Kondo et al., 1997; Shou et al., 2005; Scott et al., 2005). In addition, there have been reports of human malformations of the limbs and external genitalia associated with Hox gene mutations (Goff and Tabin, 1996; Mortlock and Innis, 1997). Both Hoxa-13 and Hoxd-13 are strongly expressed in the mesenchyme of the GT (Warot et al., 1997). One interesting recent finding is the “similar” hypoplasia or agenesis phenotypes of the limb and external genitalia in such affected patients of Hand-Foot-Genital Syndrome (Table 2; Goodman et al., 2000). Some Hox gene mutations affect the development of the distal (cloaca) portion of the hindgut and the GT (Tables 1, 2; Kondo et al., 1996; de Santa Barbara and Roberts, 2002). Hoxa-13/Hoxd-13 double null mice fail to develop a GT or cloaca (Kondo et al., 1996; Warot et al., 1997). Compound Hoxa-13 and Hoxd-13 null mice have defects in the anal sphincter and the absence of an anal opening (Warot et al., 1997). Although it is becoming clear that the Hox genes play an important role in the development of the lower gastrointestinal, urogenital/reproductive system and the GT, further

molecular and genetic studies are necessary to elucidate the genetic cascade in relation to other regulatory genes or their downstream genes (Stadler et al., 2001, Morgan et al., 2003; Suzuki et al., 2003b; Knosp et al., 2004; Yin and Ma, 2005). Searching for the Hox downstream genes utilizing target chromosome isolation is in progress (Morgan et al., 2003). These strategies include Hox interaction partner assays during limb formation (Suzuki et al., 2003b; Knosp et al., 2004; Chen et al., 2004). The prominent aplastic GT phenotype, such as that seen in Hand-FootGenital Syndrome may reflect the Hox gene functions both in GT formation per se and in the initial cloaca formation. The proper formation of the “cloacal field” is a unique aspect of endodermal regulation before a bud emergence compared with limb field formation (see below). One can see another example of such reiterated functions for the case of Tbx genes, which have been recognized to be key regulators in limb, heart, and other organ development (Rodriguez-Esteban et al., 1999; Agarwal et al., 2003; Davenport et al., 2003; Naiche and Papaioannou, 2003; Rallis et al., 2003; Takeuchi et al., 2003; Suzuki et al., 2004b). Multiple developmental functions for both early and late stage limb formation, e.g., forelimb initiation and digit formation, have been reported (Suzuki et al., 2004b). Concomitantly, the developmental regulation of the AP and PD axes occur during limb appendage development. Cell migration from the trunk, including myogenic precursors, generates developing musculature in coordination with limb outgrowth. Such developmental sequences are necessary to form anatomically and functionally mature arms/legs. Studies that analyze GT differentiation, e.g., the characterization of UP, penile bone formation, cavernous body formation, will be necessary to address the possible reiteration of these gene functions. As a case of reiterated developmental functions of growth factor genes, Shh will be an example to possess several functions in GT appendage initiation and subsequent UP development as below (Haraguchi et al., 2001).

1746 YAMADA ET AL.

TABLE 2. Representative Human Syndromes Affecting External Genitalia Formationa

Syndrome

OMIM

Candidate gene

Phenotype on external genitalia

References

Opitz syndrome

300000

MID1

Hypospadias

Opitz et al. (1969) Wilson and Oliver (1988)

Robinow syndrome

180700

Ror2

Micro or absent penis

Oishi et al. (2003) Turken et al. (1996) Wadington et al. (1973) Vera-Roman (1973)

Pallister-Hall syndrome

148510

Gli3

Micropenis, hypospadias

Stoll et al. (2001) Topf et al. (1993) Hall et al. (1980)

Ulnar-mammary syndrome

181450

Tbx3

Micropenis

Sasaki et al. (2002)

Hand-foot-gential syndrome

140000

Hoxa13, d13

Hypospadias

Halal (1988) Stern et al. (1970)

Rieger syndrome, type 1

180500

Pitx2

Hypospadias

Chisholm and Chudley (1983) Jorgenson et al. (1978)

X-linked lissencephaly with ambiguous genitalia

300215

Arx

Micropenis, ambiguous genitalia

Kitamura et al. (2002) Ogata et al. (2000) Dobyns et al. (1999)

a

Syndromes affected by hormone receptors (AR, ER) and hormonal regulators have been omitted from the table. Basically, human syndromes affected by developmental regulators are listed.

THE DIFFERENTIATION OF THE GT: URETHRA, PENIS, AND CLITORIS FORMATION What is the primary character of a GT appendage in comparison with a limb appendage? As a copulatory appendage, external genitalia are required to develop a tubular or groove-like epithelial structure for uresis and sperm ejaculation/intake. Hence, the male GT appendage undergoes tissue-specific morphogenesis, e.g., the development of a UP leading to tubular urethra formation. At earlier embryonic stages (before E16), the external genitalia of male and female fetuses are indistinguishable and consist of a common GT (Fig. 1). Such undifferentiated GTs corresponds to the stages of 8 –12 weeks of gestation in humans and E11–16 of gestation in mouse. An epithelial groove structure (the urethral groove) appears on the ventral aspect of a GT while it shows outgrowth. In the distal region of a GT, such a urethral groove forms a solid plate of epithelial cells (also termed as urethral plate [the UP]). Such a solid UP canalizes and extends the urethral groove distally

into the male glans region. Later, a urethral fold develops further toward the midline and fuses in the midline to generate the tubular penile urethra (Fig. 1D). The initial urethral fold fusion site is marked by the lower (ventral) midline seam (Baskin et al., 2001; Yamada et al., 2003). Later, the edges of the urethral folds fuse again adjacent to the initial site and form the definitive urethral seam inside the mouse GT (Baskin et al., 2001; Yamada et al., 2003). The progressive formation of the urethral groove leading to midline fusion occurs from the proximal to distal GT. The conversion from a urethral groove to tubular male penile urethra is a unique aspect of male external genital morphogenesis (Yamada et al., 2003; Kurzrock et al., 1999a). Recent reports on endodermal urogenital sinus formation, using a tissue recombination assay, indicate that the distal glandular urethra (urethra in the glans) is composed of a stratified squamous epithelium (Kurzrock et al., 1999a,b). The involvement of Shh signaling in the formation of the cloacal field, epithelia–mesenchyme interaction has

been recently reported (Table 1; Haraguchi et al., 2001; Perriton et al., 2002; Yamada et al., 2003; Satoh et al., 2004). Several other studies have been performed concerning the role of Shh signaling in later stages of GT development. Shh is expressed in the developing UP (Figs. 2C, 3C). Gain-of-function and loss-of-function experiments were performed using SHH-beads application and anti-SHH antibody in addition to explant cultures (Haraguchi et al., 2001). Both experiments suggested the presence of epithelial– mesenchymal interactions during UP formation. The reduction of mesenchymal Fgf10 gene expression by antiSHH antibody together with the finding that Fgf10 gene mutation induces severe ventral GT dysmorphogenesis and urethral defects suggest the importance of Shh-Fgf10 interactions for urethra formation (Haraguchi et al., 2000; Yucel et al., 2004). Hence, Shh is required not only for the initiation of GT outgrowth but also for subsequent tissue differentiation, e.g., the ventral side of GT differentiation (Fig. 3C,D). Such a dual mode of the Shh function in organogenesis would be a

EXTERNAL GENITALIA FORMATION 1747

unique aspect of Shh signaling in comparison with other organs. Furthermore, the possibility of the expression of a set of genes and growth factors, such as Shh, for the late stage male urethra or the cavernous body formation has been proposed and awaits further analysis (Podlasek et al., 2003a). It has been speculated that defects in maternal glucose or other metabolic abnormalities including cholesterol alterations may lead to embryonic defects with hedgehog pathways (Podlasek et al., 2003b). Megalourethra or other birth defects, e.g., holoprosencephaly has been reported in relation to maternal diabetes (Vaux et al., 2005). The roles of Shh in embryonic urethra formation related with the downstream signaling pathway have been studied. The Gli family of transcription factors are targets of the Shh signal pathway. Mice null for Gli2 and/or Gli3 develop a variety of foregut defects (Motoyama et al., 1998). Gli transcription factors are also required for the normal development of the GT. In Gli2 mutant mice, embryonic urethral formation is defective (Haraguchi et al., 2001), while the Gli3 mutation in humans is responsible for the Greig cephalopolysyndactyly (GCPS) syndrome characterized by post-axial polysyndactyly in the hands, and various other abnormalities (Tables 1, 2). Compound mutant analysis for several Gli genes should be performed. Apart from the Shh signaling pathway, Fgf ligands and their receptors have also recently been implicated in epithelial–mesenchymal interactions for embryonic urethral morphogenesis before sexual differentiation (Satoh et al., 2004; Petiot et al., 2005). FGFR2IIIb has also been reported to be able to bind and transduce signals by FGF1, FGF3, FGF7, and FGF10 and it is expressed predominantly in many epithelia; while FGFR2IIIc binds FGF1, FGF2, FGF4, FGF6, and FGF9, and is mainly expressed in the embryonic mesenchyme (Orr-Urtreger et al., 1993; Ornitz et al., 1996). Fgfr2IIIb is expressed in the distal urethral plate epithelium and in the UP between E10.5 and E13.5 in mouse development. Following GT mesenchymal differentiation, Fgf10 is expressed in the bilateral mesenchyme adjacent to the midline UP

(Fig. 3E) and Fgfr2IIIb is expressed in the UP epithelium when there is no gross sexual dimorphism (Fig. 1; Satoh et al., 2004; Petiot et al., 2005). FGF10 mutant mice exhibit the complete absence of both fore- and hindlimbs but do not display prominent GT outgrowth defects (Min et al., 1998; Sekine et al., 1999). The phenotype of Fgf10 mutant mice closely resembles that of Fgfr2IIIb mutant mice. Both Fgf10 and Fgfr2IIIb mutant mice are viable until birth, but are lethal due to aberrant lung formation (Arman et al., 1998; Min et al., 1998; Sekine et al., 1999; Ohuchi et al., 2000). These results suggest that FGF10/FGFR2IIIb signals play important roles in the developmental processes of prepuce fusion and urethra formation, but they are not essential for the early phase of GT outgrowth. Whether the FGF10Fgfr2IIIb interaction also plays a role in sexually dimorphic late-staged urethra morphogenesis requires more investigation (Petiot et al., 2005). FGF10 and Fgfr2IIIb are also detected in such late stages by RT-PCR (Satoh et al., unpublished results). Other roles of the ligand-receptor systems have been elucidated. The Eph family of receptor tyrosine kinases and their membrane-anchored ephrin ligands are a large group of highly conserved molecules functioning in various cell– cell recognitions. Such recognition includes axon pathfinding and the formation of the cardiovascular system (Wilkinson, 2001; Cowan and Henkemeyer, 2002). It has recently been reported that such an Ephrin–Eph system is involved in midline urethral fusion and scrotum formation (Cowan and Henkemeyer, 2002). One of the important features of GT differentiation is sex-dependent differentiation in females and males. Very little is known about the involvement of regulatory gene functions for late staged male or female urethra formation. Regarding hormone-dependent male external genitalia formation, a high frequency of birth defects has been observed (Baskin, 2004). A frequent example is hypospadias, which results from the failure of the formation or fusion of the urethral folds. The site of the failure of urethral fold fusion corresponds to the position

of the abnormal opening of the urethra. Impaired signaling through the androgen receptor (AR) is one of the causes for this defect in at least a minority of patients (Ogata et al., 2001; Sasagawa et al., 2002). There have been some reports indicating the possibility of an increase in the frequency of hypospadias in human newborn births (Paulozzi et al., 1997; Paulozzi, 1999). Hypospadias is currently repaired by pediatric surgery (Baskin, 2001). As for estrogen-dependent processes of differentiation, its effect on male urogenital organ formation also still remains poorly described (Hess, 2003; Pu et al., 2004; Huang et al., 2005; Goyal et al., 2004). Penis and clitoris development is influenced by androgens (Matsumoto et al., 2005). Masculine phenotypes are regulated by androgens such as testosterone and 5a-dihydrotestosterone (DHT) in mammals. Evidence supporting the crucial role of androgen is based on pharmacological and genetic data. Androgen antagonists (Silversides et al., 1995; Suzuki et al., 2002) or inhibitors of its synthesis (Imperato-McGinley et al., 1985; Clark et al., 1990) interfere with testicular function and external genitalia development. Mutations in androgen receptor gene (Murakami, 1987; Yeh et al., 2002; Sato et al., 2003) or for androgen synthetic enzymes (Andersson et al., 1991; Wilson, 1992) result in abnormalities in male sexual differentiation and development. Clitoromegaly is also known to be induced by androgen, e.g., by prenatal exposure to androgens by congenital adrenal hyperplasia (CAH). In the case of female urethra formation, mature tubular urethra is not formed though the initial staged midline UP and the open ventral urethra in late stages is formed. The lack of the fusion of urethral folds underlies such events and it is considered as under hormonal influences (Yamada et al., 2003; Baskin, 2004). The epithelial edges fuse proximally but they do not form the midline mesenchymal seam as seen in male urethral formation. The mechanisms of such hormonal effects including the urethral seam formation are not well understood (Baskin, 2004). In addition to sexually dimorphic urethra formation, the molecular

1748 YAMADA ET AL.

mechanisms of female external genitalia formation in the late stages remain unexplored. Although some natural mutants with abnormal female external genitalia are known, virtually no developmental gene functions are known. The perineum is prominently marked by the rostral urogenital region (urogenital triangle) and anal region in humans. The urogenital folds develop the urogenital ostium and the genital swellings laterally. The genital swellings remain separated and form the labia in females. In contrast, the genital swellings fuse in the midline to form the scrotum in males (Shapiro et al., 2004; Larsen, 2001). The urogenital folds remain separate and form the labia minora of the vestibule of the vagina minora in females. The labia minora is well developed in humans and not prominent in other species. The proper apoptotic regulation of cell death is required for the regulation of the vaginal opening (Rodriguez et al., 1997). The presence of the vaginal septum often has been found in some specimens of breeding mouse colonies.

PERSPECTIVES As a research topic, embryonic external genitalia formation is an interdisciplinary science using approaches in developmental biology, reproductive biology, and human genetics. In molecular developmental biology, researchers will continue to analyze the extent of developmental similarities and divergence among different cascades of appendage formation in mammals. For example, the initiation, outgrowth, and subsequent differentiation of external genital appendage (bud) will offer useful research material for a better understanding of embryonic appendage formation. Like the physiological/anatomical organization of arms/legs with body skeleton and musculature, external genitalia should establish anatomic structural connections with urinary/reproductive organs in the posterior body. External structures develop in coordination with such “pelvic organs” underneath the peritoneal cavity. How this is achieved is in need of investigation. Another important unexplored area is the molecular mechanisms of male

and female external genitalia formation. Control of developmental regulation in the context of hormonal actions is still not understood. Further identification of hormonal target genes will extend from developmental regulatory genes to general regulators for cell growth/differentiation, e.g., cell cycle regulators, growth factors (Shiina et al., 2006; Matsumoto et al., 2005). Studies on the mechanisms of hormone development urogenital tumor growth or nuclear hormone receptor gene KO mouse studies will continue to provide information about this area. Later in development, almost no developmental gene functions are known for the female external genitalia, labia and clitoris, formation. Complex and coordinated development of female perineal regions must be studied by conditional mouse mutant models and not by organ culture studies. Genes expressed during such processes need to be identified and developmental mechanisms understood. Urinary tract abnormalities in newborn humans, such as abnormal ureter– bladder or ureter– kidney connections, have been shown to have a high frequency as birth defects in comparison with other organ abnormalities (Batourina et al., 2005). Similarly, hypospadias is a relatively common urinary-external genital birth defect (Baskin, 2004). The growing understanding of the molecular mechanisms that allow normal urogenital development must now be put to use to work out the mechanisms of other urogenital birth defects.

ACKNOWLEDGMENTS We thank Drs. Denis Duboule, Shigeaki Kato, Alex Joyner, Tsutomu Ogata, Rolf Zeller, Chi-Chung Hui, Gail Martin, Cheng-Ming Chuong, Uli Ruether, Virginia E. Papaioannou, John McLachlan, Benoit Bruneau, Jacques Drouin, Lee Niswander, Anne M. Moon, David Ornitz, Mark Lewandoski, Yukiko Ogino, Saverio Belluci, Giovanni Levi, Irma Thesleff, and John Fallon for their encouragement and suggestions. We also appreciate the assistance of Shiho Kitagawa. This research was supported by a Grant-in-Aid for Scientific Research on Priority Areas; General promotion of Cancer research in Japan, by a

Grant-in-Aid for Scientific Research on Priority Areas; Mechanisms of Sex Differentiation, by the 21st Century COE Research Program; and by a Grant for Child Health and Development (17-2) from the Ministry of Health, Labour and Welfare.

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