Molecular Analysis of Genital Tubercle Formation 2473

Development 127, 2471-2479 (2000) 2471 Printed in Great Britain © The Company of Biologists Limited 2000 DEV2522

Molecular analysis of external genitalia formation: the role of ﬁbroblast growth factor (Fgf) genes during genital tubercle formation

R. Haraguchi1, K. Suzuki1,2, R. Murakami3, M. Sakai2, M. Kamikawa1, M. Kengaku4, K. Sekine5, H. Kawano5, S. Kato5, N. Ueno6 and G. Yamada1,2,* 1Center for Animal Resources and Development (CARD), Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan 2Research Center for Innovative Cancer Therapy, Kurume University, Kurume, Japan 3Department of Physics, Biology and informatics, Yamaguchi University, Yamaguchi 753-8512, Japan 4Department of Biophysics, Faculty of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan 5Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0033, Japan 6Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan *Author for correspondence (e-mail: [email protected])

Accepted 27 February; published on WWW 10 May 2000

SUMMARY

The molecular mechanisms underlying the development of also promoted the outgrowth of the GT. Experiments the external genitalia in mammals have been very little utilizing anti-FGF neutralizing antibody suggested a examined. Recent gene knockout studies have suggested growth-promoting role for FGF protein(s) in GT that the developmental processes of its anlage, the genital outgrowth. In contrast, despite its vital role during limb- tubercle (GT), have much in common with those of limb bud formation, Fgf10 appears not to be primarily essential buds. The Fgf genes have been postulated as regulating for initial outgrowth of GT, as extrapolated from Fgf10−/− several downstream genes during organogenesis. Fgf8 was GTs. However, the abnormal external genitalia expressed in the distal urethral plate epithelium of the development of Fgf10−/− perinatal mice suggested the genital tubercle (GT) together with other markers such as importance of Fgf10 in the development of the glans penis the Msx1, Fgf10, Hoxd13 and Bmp4 expressed in the and the glans clitoridis. These results suggest that the FGF mesenchyme. To analyze the role of the FGF system during system is a key element in orchestrating GT development. GT formation, an in vitro organ culture system was utilized. It is suggested that the distal urethral plate epithelium of GT, the Fgf8-expressing region, regulates the Key words: FGF8, FGF10, Msx1, Hox, Bmp4, Genital tubercle, outgrowth of GT. Ectopic application of FGF8 beads to the Clitoris, Penis, Limb, AER, Epithelial-mesenchymal interaction, murine GT induced mesenchymal gene expression, and Gene knockout mouse

INTRODUCTION developmental analysis. Pioneering study has suggested that GT development may have some similarities to limb Recent molecular cloning of many of the genes controlling development, with both structures exhibiting organ outgrowth development, including growth factors and homeobox genes, (Dolle et al., 1991; Yamaguchi et al., 1999). Vertebrate limb has led to the formulation of basic plans of organogenesis development depends on the establishment and maintenance of (Kessel and Gruss, 1990; Chisaka et al., 1992; Gehring et al., the apical ectodermal ridge (AER), a specialized ectoderm 1994; Johnson and Tabin, 1997; Hogan, 1996, 1999; Gehring, region at the distal tip of the limb bud (Duboule, 1993; Tickle 1999). During fetal and neonatal development of mammals, the and Eichele, 1994; Johnson and Tabin, 1997). It has been last structures to develop along the embryonic anteroposterior speculated that the GT epithelium and mesenchyme develop (AP) axis are the external genitalia, organs displaying through presumptive epithelial-mesenchymal interactions, characteristic proximodistal-oriented outgrowth adjacent to the although no discrete structures resembling an AER have been cloacal membrane (Fig. 1). The mammalian external genitalia described, nor have any candidate molecules for those “AER are highly developed structures to permit efficient internal functions“ been suggested in studies of external genitalia fertilization. formation (Murakami and Mizuno, 1986; Dolle et al., 1991). The external genital anlage, the genital tubercle (GT), Epithelial-mesenchymal interactions play an essential role in differentiates into a penis in males and a clitoris in females regulating a wide variety of developmental processes (Le (Murakami and Mizuno, 1986; Hildebrand, 1995). Because of Douarin and Jotereau, 1975; Hall 1981; Slavkin et al., 1984; the lack of a suitable experimental system, the mechanisms of Thesleff et al., 1991, 1995; Tickle and Eichele 1994; Johnson GT formation have been very little studied by molecular and Tabin, 1997; Hogan, 1999). Signaling between the 2472 R. Haraguchi and others

with animals were done in accordance with the experimental animal rights guidelines of Kumamoto University. Embryos were aseptically dissected and genital tubercles were microsurgically dissected. In some cultures, the distal urethral plate epithelium of the GT was removed by a tungsten needle to eliminate the Fgf8-expressing region. Dissected genital tubercles were oriented with the dorsal surface upward on a 5% gelatin-coated Millipore type HTTP filter in tissue culture dishes. Cultures were fed with BGJb Medium (Gibco BRL) containing 0.1 mg/ml L-ascorbic acid and 10 mM Hepes (pH 7.4). Genital tubercles were maintained at the interface between the gas Fig. 1. The developing external genitalis. The genital tubercle shows (5% CO2) and liquid phases of the culture, and the level of the medium characteristic proximodistal-oriented outgrowth adjacent to the was adjusted so that the tissue was immersed but not covered by the cloacal membrane. medium. After 1-4 days in culture, the genital tubercles were processed for histological analysis. For antibody inoculation, anti- FGF8 antibodies (MAB323 or AF-423-NA, R&D systems Inc, CA, epithelium and the mesenchyme governs many aspects of USA) or control Ig class-matched Ab (PM-01020D, anti-CD90 organogenesis, from the initiation of organ development to antibody, Pharmingen) were added to the culture medium at a concentration of 50 µg/ml. Both anti-FGF8 antibodies gave same differentiation (MacKenzie et al., 1992; Dassule and results. McMahon, 1998; Tucker et al., 1999). The developing limb has long served as a good model system for studying such Preparation of FGF8 protein beads mechanisms during organ development (Tabin, 1991, 1995; Recombinant mouse FGF8b protein and human FGF10 protein (423- Duboule, 1993, 1994). The limb bud is initiated through the F8 and 345-FG, R&D systems Inc, CA, USA) were used at a continued proliferation of cells of the lateral plate mesoderm. concentration of 1.0 mg/ml in phosphate-buffered saline (PBS). Signals emanating from the rapidly proliferating mesodermal Heparin acrylic beads (Sigma H-5263) were washed with PBS and cells induce the limb-bud ectoderm to form the AER. Once subsequently soaked overnight with the above proteins at 4°C. Control induced, the AER becomes essential for sustained outgrowth beads were treated with PBS containing 0.1% bovine albumin (Sigma, and the patterning of a limb by interacting with the underlying USA). mesenchyme through molecules, including fibroblast growth In situ hybridization for gene expression factors (FGFs) (Niswander and Martin, 1992; Niswander et al., Whole-mount in situ hybridization was performed with digoxigenin- 1993, 1994; Laufer et al., 1994; Tickle and Eichele 1994; labeled probes by standard procedures (Wilkinson, 1992). The Niswander, 1999; Pizette and Niswander, 1999). following probes were used for in situ hybridization: Fgf8 (kindly FGFs are essential signaling molecules for embryogenesis provided by B. L. Hogan), Fgf10 (kindly provided by H. Ohuchi and (Cohn et al., 1995; Wall and Hogan, 1995; Yamasaki et al., N. Itoh), Fgfr2 (Riken Inst.), Hoxd13 (Dolle et al., 1991), Bmp4 1996; Bellusci et al., 1997; Martin, 1998; Ornitz, 2000). One (Jones et al., 1991), Shh (kindly provided by C. Shukunami) and Msx1 of the Fgf family genes, Fgf8, is expressed and functions in a (Satokata and Maas, 1994). Histological sectioning was performed as variety of developmental events, including gastrulation and the described previously (Belo et al., 1998). development of limbs, the central nervous system (CNS), the Histological analysis mandible and teeth (Ohuchi et al., 1994; Crossley and Martin, Newborn tissues were fixed in Bouin’s fixative or paraformaldehyde. 1995; Crossley et al., 1996a,b; Johnson and Tabin, 1997; Lee They were dehydrated in graded ethanol, embedded in paraffin and et al., 1997; Neubuser et al., 1997; Bei and Maas, 1998). Fgf8- sectioned. The sections were stained with Hematoxylin and Eosin. deficient mice show a variety of malformations, lacking all embryonic mesoderm-derived structures, including the heart Fgf10-deficient mice and somites (Lewandoski et al., 1997; Meyers et al., 1998). Fgf10−/− newborns were identified as described previously and Previous studies have shown that Fgf8 is important for the external genitalia were analyzed using standard histological growth and patterning of embryonic limbs (Crossley et al., procedures (Sekine et al., 1999). 1996b; Ohuchi et al., 1997), and an elegant recent conditional gene knockout study indicated its importance in development of the jaw (Trumpp et al., 1999). It has been suggested that RESULTS Fgf10 functions in brain, lung and limb development based on its spatiotemporal expression (Yamasaki et al., 1996; Ohuchi Expression of Fgf8, Fgf10, Fgfr2 and other epithelial et al., 1997), and, in support, Fgf10 knockout mice displayed or mesenchymal genes during murine genital drastic outgrowth defects in the limbs and died at birth due to tubercle development the lack of lung development (Min et al., 1998; Sekine et al., Before the onset of GT outgrowth, Fgf8 and Fgf10 were both 1999). Roles of the FGF system during the outgrowth of GT expressed in the epithelium of the outermost part of the and in the subsequent development of the penis and the clitoris urogenital sinus at 10.5 d.p.c. (Fig. 2A,B,D,E). Following are examined in this paper. initial expression, Fgf8 was expressed at the distal region of urethral plate epithelium of the GT in embryos from 11.5 d.p.c. (Fig. 2F) and, subsequently, Fgf8 mRNA levels decreased MATERIALS AND METHODS gradually up to 14.5 d.p.c. (data not shown). Yamaguchi et al. (1999) has also partly reported expression of Fgf8 in GT. Mouse genital tubercle organ culture In contrast, Fgf10 was expressed in the mesenchyme ICR mice (6 week old; Japan SLC Inc) were utilized. All experiments abutting the distal region of the urethral plate epithelium during Molecular analysis of genital tubercle formation 2473

Fig. 2. Whole-mount in situ gene expression analysis for Fgf10 (A-C) and Fgf8 (D-F). Initially, Fgf8 and Fgf10 were both expressed in the epithelium of the outermost part of the urogenital sinus at 10.5 d.p.c. (A,B,D,E) B,E show transverse sections performed from A,D, respectively. Expression in the urogenital sinus epithelium and limb is indicated by an arrow and an arrowhead, respectively (A,D). At 11.5 d.p.c., Fgf10 expression is mainly localized to the distal regions of the mesenchyme adjacent to the distal urethral plate epithelium and is also detected as a low-level expression in the subectodermal mesenchyme as shown in a transverse section (C). Fgf8 expression is detected in a transverse section (F) at the distal region of the urethral plate epithelium at 11.5 d.p.c. Fgfr2 is expressed in the GT epithelium (black arrow) and mesenchyme (white arrows) at 11.5 d.p.c. (G). Hoxd13 (H), Msx1 (I) and Bmp4 (J) were expressed in the mesenchyme of the developing GT at 11.5 d.p.c. Expression of the Sonic hedgehog (Shh) gene was found to be expressed in the distal region of the urethral plate epithelium where Fgf8 was also expressed (K). Bars: (A,D,G-K) 0.5 mm; (B,C,E,F) 0.125 mm. the outgrowth phase of GT (Fig. 2C). It was also expressed at BGJb medium to examine the effects of growth factor activities a low level in the broad subectodermal mesenchyme (Fig. 2C). (see Materials and Methods). Genital tubercles were Hence, both Fgf8 expression in the distal urethral plate maintained at the interface between the gas and liquid phases epithelium and mesenchymal Fgf10 expression continued of the culture so that the tissue was immersed but not covered through the outgrowth period of the GT from 11.5 to 14.5 d.p.c. with the medium. It is known that multiple Fgf genes are expressed in an GTs at several embryonic stages (from 11.5 d.p.c. up to 13.5 overlapping manner during organ formation. Fgf4 expression d.p.c.) could be cultured to a stage approximately equivalent was not evident during GT development (data not shown). FGF to embryonic 14 d.p.c. Fig. 3 demonstrates the outgrowth ligand activities are mediated by FGF receptors, which contain tyrosine kinase domains (reviewed in Johnson and Williams, 1993; Arman et al., 1998; Partanen et al., 1998). Fgfr2 was found to be expressed in the GT epithelium (shown by a black arrow in Fig. 2G) and mesenchyme (shown by white arrows). During limb development, the epithelial-mesenchymal interaction is considered essential in the regulation of the expression of mesenchymal genes. Therefore, expression of several makers, such as the Hoxd13, Msx1 and Bmp4 genes, was examined. Intriguingly, they were expressed in the mesenchyme of the outgrowing GT from 10.5 to 14.5 d.p.c. (Fig. 2H-J). Expression of Bmp2 was not detected (data not shown). The other key signaling gene for limb patterning, sonic hedgehog (Shh), was found to be expressed in the distal region of the urethral plate epithelium where Fgf8 was also expressed (Fig. 2K). Urogenital expression of Shh and Bmp4 has been previously reported (Bitgood and McMahon, 1995). The expression pattern of these genes led us to investigate whether similar gene expression regulatory mechanisms occur during Fig. 3. Kinetics of cultured GT formation. GT outgrowth in vitro. The cultured GT tissues display Induction of Fgf10, Msx1, Bmp4 and Hoxd13 gene equivalent outgrowth in expression by FGF8 protein; GT outgrowth is vitro (1-4 days) compared promoted by the distal urethral plate epithelium and with corresponding GTs FGF8 protein from embryonic stages (12.5-14.5 d.p.c.). To analyze GT development, a GT organ culture system was (E,I) Expression patterns of established. Genital tubercles were microsurgically removed Fgf8 for both control and in from embryos and were oriented with the dorsal surface vitro GTs indicate similar upward on gelatin-coated membrane filters in tissue culture development. Bars: (A-I) dishes. Cultures were performed without serum in a modified 0.5 mm. 2474 R. Haraguchi and others kinetics of the cultured GT in vitro, as compared with corresponding GTs in vivo (12.5-14.5 d.p.c.). Analysis of gene expression patterns after culture also showed GT development to be equivalent to that observed in vivo (an example of Fgf8 expression is shown in Fig. 3E,I). Thus, this organ culture system was helpful in analyzing murine GT formation. During GT development, the role of the distal region of the urethral plate epithelium, the Fgf8-expressing region, in the regulation of the expression of several genes was first examined by cultivating GTs after microsurgically removing the region. Mesenchymal (endogenous) Fgf10 expression was markedly reduced 24 hours after the removal (Fig. 4B). In support of the above results, when FGF8 beads were transplanted to the GT mesenchyme, expression of the mesenchymal genes, Fgf10, Msx1, Bmp4 and Hoxd13, were augmented (Fig. 4C-F; compare with the endogenous signal by control beads in the opposite side of the same specimen). In loss-of-function experiments, administrating anti-FGF8 neutralizing antibody reduced mesenchymal (endogenous) Bmp4 expression 24 hours after addition (Fig. 4G,H). It is not known why the expression of other mesenchymal genes, Fgf10 Fig. 4. The distal region of urethral plate epithelium of GT, the and Msx1, was not inhibited under the same conditions (data Fgf8-expressing region, could regulate mesenchymal gene not shown). Augmentation of mesenchymal gene expression expression during GT development. Endogenous mesenchymal (Fgf10, Msx1, Hoxd13 and Bmp4) was observed 24 hours after, Fgf10 expression was significantly reduced 24 hours after removing but not 12 hours after, application of FGF8 beads (Fig. 4I; the distal tip of the urethral plate epithelium (B) in contrast to the control (A). Augmentation of Fgf10 (C), Msx1 (D), Bmp4 (E) and Bmp4 expression is the same for FGF8 and control beads). Hoxd13 (F) expression in the cultured murine GTs by FGF8 beads. Thus, it appears that induction may be due to several sequential The GT explants from 11.5 d.p.c. embryos were cultured for 24 responses as elicited by FGF. An elegant recent experiment, hours after applying the FGF8 protein beads and control beads in using another FGF, suggested that these proteins may diffuse the same specimen (f, FGF8 beads; c, control BSA beads). To detect from beads 4 hours after being implanted (Iseki et al., 1999). augmentation of gene expression, color staining reaction for in situ More studies are required to analyze the regulation of these expression analysis was performed for 6 hours (E,F), or for 14 hours genes in GT mesenchyme. (C,D). In C,F, induction of gene expression by the beads was To check the competence of mesenchymal responsiveness, relatively strong, resulting in the detection of weak endogenous several stages of GT explants were tested for gene expression signals. Anti-FGF8 neutralizing antibody reduced mesenchymal in response to FGF8 bead transplantation. The inducibility (endogenous) Bmp4 expression 24 hours after addition (Fig. 4G,H; 16 hours in situ color staining). Bmp4 expression was not decreased significantly in mesenchymes at 12.5 d.p.c. and 13.5 augmented 12 hours after FGF8 beads addition (I). Bars: (A-I) d.p.c. (data not shown). We next examined the growth- 0.5 mm. promoting role of the corresponding region and of FGF proteins in GT organ culture. The GT from 11.5 d.p.c. embryos

Fig. 5. GT outgrowth is dependent on the presence of the distal region of the urethral plate epithelium. Absence of Fgf8 expression was confirmed after removing the urethral plate epithelium (A) in contrast to the control (B). The absence of the Fgf8-expressing region is indicated by an arrowhead (A). After removing the tip of the urethral epithelium (the Fgf8- expressing region), GTs from 11.5 d.p.c. embryos were cultured for 48 hours with exogenously applied FGF8 beads (D) or with control BSA beads (C). The GT outgrowth was restored by the exogenous FGF8 bead application but was not with control beads. Anti-FGF8 neutralizing antibody addition (F) inhibited GT outgrowth in contrast to the controls (E). (G) Summary of the growth inhibition or promotion rate by anti-FGF8 antibody administration or by FGF8 or FGF10 beads transplantation. The length of the outgrowth in between the position of prepuce and the distal tip of the GT was measured (length in control taken as 100%). Bars: (A-F) 0.5 mm. Molecular analysis of genital tubercle formation 2475 were cultured for 48 hours after removing the distal urethral plate epithelium (the Fgf8-expressing region). In order to confirm the extent of the removal, absence of Fgf8 expression was checked (Fig. 5A,B). GT outgrowth was found to be dependent on the presence of the Fgf8-expressing region (Fig. 5C). In contrast, the GT with exogenously applied FGF8 beads had outgrowth restored (Fig. 5D). Experiments utilizing anti- FGF8 neutralizing antibody confirmed the growth-promoting role of FGF in GT outgrowth (Fig. 5E,F; see Discussion). The growth-promoting activities of FGF10 beads were found to be not as marked as those of FGF8 beads (Fig. 5G). Fgf10 gene mutation did not affect initial GT outgrowth Since Fgf10−/− embryos display drastic outgrowth defects in limbs (shown by an arrow in Fig. 6A; Min et al., 1998; Sekine et al., 1999), we next examined the external genitalia of Fgf10 knockout mice. Mutant embryos did not show marked GT outgrowth defects, as judged by the size of each GT and the normal Fgf8 expression patterns at early stages (compare Fig. 6B and E (at 10.5 d.p.c.), Fig. 6C and F (at 11.5 d.p.c.)). This suggested that early expression of Fgf10 may not be essential during GT development from 10.5 to 11.5 d.p.c. Because Fgf10 knockout Fig. 6. (A) Fgf10−/− displayed prominent outgrowth defects in the abolished Fgf8 gene expression in the limb bud, cross- limb (arrow) but not in the GT (arrowhead) at 12.5 d.p.c. Early stage regulatory gene expression mechanisms have been suggested Fgf10 mutant embryos show no abnormalities in outgrowth or previously (Min et al., 1998; Sekine et al., 1999). patterning of the urethra, as determined by comparing size and Fgf8 During the outgrowth phase of the GT from 11.5 d.p.c. to expression pattern at 10.5 d.p.c. (B, E) or 11.5 d.p.c. (C, F). At 13.5 d.p.c., Fgf8 expression was altered and the several morphological 14.5 d.p.c., the mutant mice GTs were comparable to the wild- −/− type GTs in proximal-distal outgrowth, however, significant changes were observed in the Fgf10 GT (G) as compared with the growth retardation was observed from 12.5 d.p.c. (Fig. 7C,D). wild type (D). (B-D, wild type; E-G, mutant). Bars: (A) 1.25 mm; (B,C,E,F) 0.5 mm; (D,G) 0.25 mm. Following this retardation, the development of the urethral plate was also consequently affected (shown by Fgf8 expression pattern at 13.5 d.p.c. in Fig. 6D,G). These results knockout newborn mouse was histologically examined. suggested that Fgf10 mutation leads to GT growth retardation, Characteristic morphological defects were found in the ventral although the phenotype is much less prominent than that (lower) side of the glans penis and the clitoridis, especially in observed in the limbs. Abnormal glans penis and glans clitoridis formation in newborn Fgf10 knockout mice The penis and the clitoris of the Fgf10

Fig. 7. (A) Urethral tube formation in the glans penis. In normal development of the glans penis, urethral plate becomes separated from the surface epithelium of the glans penis with the prepuce growing (indicated by white arrow), being fused at the ventral (lower) midline of the GT. Morphogenesis of the urethra and prepuce in the glans clitoridis is largely similar to that of penis, though the incorporation of the tubular urethral epithelium is incomplete. (B) The mesenchymal expression of Fgf10 adjacent to the midline urethral plate (groove) at 13.5 d.p.c. (C,D) External morphology of 13.5 d.p.c. GT (C, wild type; D, Fgf10−/−). (E-H) Fgf10−/− newborn mice show drastic abnormalities in the urethral tube formation and the prepuce fusion defects. P, prepuce; pg, preputial gland; u, urethral tube or urethral groove. Bars: (B,E-H) 0.25 mm; (C,D) 0.5 mm. 2476 R. Haraguchi and others the urethral epithelium and prepuce (Fig. 7F,H). In normal type organogenesis (De Molerlooze et al., 2000). Analysis of such female newborn mouse, morphogenesis of the urethra and receptor functions during GT formation awaits further analysis. prepuce in the glans clitoridis is largely similar to that of penis, An in vitro system was used to culture GT from 11.5-13.5 though the incorporation of the tubular urethral epithelium is d.p.c. mouse embryos up to the stage closely corresponding to incomplete (Fig. 7G). In mutants, tubular urethra was not 14.5 d.p.c. in vivo. The success of the culture seems to depend formed at the glans penis and the clitoridis, and fusion of the largely on keeping organs at the interface of the medium. prepuce at the ventral (lower) midline of the glans region was Currently it is not feasible to cultivate GTs at later stages, completely suppressed (Fig. 7F,H). It is suggested that the which presumably requires additional improvements, e.g., urethral plate epithelium remains as an epithelium on the higher doses of oxygen supply. ventral surface of the glans (Fig. 7F,H). Rudiments of corpus Other experimental methods could potentially be applicable cavernosum glandis were found in both sexes of Fgf10 to study GT development; however, the whole embryo culture mutants. system can only be utilized for embryos up to about 10.5 d.p.c. Following mesenchymal differentiation in the genital (Tam, 1989), and, in the case of exo utero techniques, it is tubercle, Fgf10 was expressed in the mesenchyme adjacent to technically difficult to manipulate murine GTs through the the midline urethral plate (groove) at 13.5 d.p.c., when there yolk sac. Chick embryos have served as excellent tools for was no gross sexual dimorphism (Fig. 7B). These results experimental manipulations, especially in the area of limb suggested that Fgf10 might play an important role in the development (Cohn and Tickle, 1996). However, chicks, which morphogenesis of the most distal structure of the glans penis, copulate by pressing their cloacas together, might exhibit and of the glans clitoridis, becoming involved in the moderately diverged GT morphogenesis compared with developmental process of prepuce fusion and urethral tube mammals (Bakst, 1986; Hildebrand, 1995). formation (see Discussion). Utilizing our serum-free organ culture system, it is suggested that the distal region of the genital urethral plate epithelium, Fgf8-expressing region, has a role in controlling DISCUSSION mesenchymal gene expression and outgrowth of the GT anlage, presumably acting as a signaling center for GT development. Organogenesis has been assumed to include several Ectopic application of FGF8 beads induced expression of fundamental processes: firstly, the initiation of organ formation mesenchymal markers such as Fgf10, Msx1, Bmp4 and by inducing organ outgrowth, and, secondly, subsequent tissue Hoxd13, and induced GT outgrowth. Experiments with differentiation through various mechanisms e.g. epithelial- neutralizing antibodies also suggested a role for FGF8 or mesenchymal interactions (Hogan, 1999). The external related FGF(s) in GT outgrowth. genitalia show marked morphological variations among It was previously shown that the anti-FGF8 antibody used vertebrates but little research has been undertaken into their in this study has a specific neutralizing activity for FGF8- development. mediated cell proliferation (Dorkin et al., 1999). This Hox gene expression in developing GT (Dolle et al., 1991) specificity was also shown in embryonic tissues (Jung et al., and Hoxd13 and Shh expression in urogenital sinus (Oefelein 1999). Moreover, a different anti-FGF8 antibody showed et al., 1996; Podlasek et al., 1999) have been reported. The similar results (see Materials and Methods). However, it is posteriormost Hoxd and Hoxa genes are required for proper possible that other related molecule(s), e.g., other FGF(s), will growth and patterning of GT (Kondo et al., 1997; Warot et al., show similar activities in this system. 1997). Gene regulatory mechanisms of Hox gene expression in Transplanted FGF10 beads displayed growth-promoting limbs and other tissues, including GT, suggest the presence of activities, but less prominent compared with those of FGF8 shared aspects of gene regulation (Kondo et al., 1998). In beads. In this assay, FGF10 beads were applied to GT after addition, there have been reports of human malformations of removing the Fgf8-expressing region, leading to limbs and external genitalia associated with Hox gene downregulation of endogenous Fgf10 in the treated mutations (Goff and Tabin, 1996; Kondo et al., 1997; Mortlock mesenchyme. One might speculate that FGF8 could induce and Innis, 1997). Another line of recent molecular analysis has growth-promoting factor(s) other than FGF10, but it remains demonstrated that Wnt5a can regulate the outgrowth of the to be analyzed further. embryonic axis and facial, genital, ear and limb primordia; Fgf4 expression was not evident during GT development indicating that Wnt5a might be located downstream of such (data not shown) although it is expressed in posterior AER and transcriptional regulatory genes (Yamaguchi et al., 1999). is postulated to function in the control of limb mesenchyme proliferation. This may, therefore, represent a different mode Expression and function of the FGF system during of gene expression profile during GT formation compared with GT formation that of limbs. Several other Fgf genes are expressed in the Fgf8 was expressed in urogenital sinus and its expression urethral plate epithelium or in GT mesenchyme (R. H. and gradually confined to the distal region of the urethral plate G. Y., unpublished results). Combinations of such Fgf gene epithelium. Fgf10 was expressed in outgrowing GT expressions may constitute a redundant network and play mesenchyme while urethral expression of Fgf8 remained. various roles during GT development. Such redundant mode of A null mutation of Fgfr2 results in peri-implantation FGF system was also shown by the absence of limb phenotypes lethality at 4.5-5.5 d.p.c. (Arman et al., 1998, 1999), while in Fgf4 conditional mutants (Moon et al., 2000). embryos with homozygous hypomorphic alleles die around To our knowledge, neither such Fgf8-expressing regions nor 10.5 d.p.c. with no limb buds (Xu et al., 1998). Recent elegant candidate molecules for GT growth promotion have been Fgfr2 knockout experiments also suggest its importance during previously identified. Furthermore, the mesenchymal Fgf10 Molecular analysis of genital tubercle formation 2477 gene was shown to be important during GT formation based limb formation. This difference might also be due to gene on the analysis of the gene knockout phenotypes (see below). compensatory mechanisms by other functionally and/or Thus, the FGF system appears to be one of the key signals structurally related genes during GT development as generally involved in controlling outgrowth and in regulating various shown in other recent gene knockout studies (Rudnicki et al., developmental processes of mammalian GT. 1993; Kondo et al., 1997; Belo et al., 1998; Sun et al., 1999). The inductive effects of epithelia on the development of Redundant gene networks may also be indicated by the various tissues through epithelial-mesenchymal interactions absence of obvious phenotypes during GT formation of have been reported previously (Cunha et al., 1983; Roberts et Msx1−/− mice (unpublished results). The results from Msx1 and al., 1995). Such interactions have been also reported in Msx2 double knockout mice, may be of future interest as the developing urogenital systems by groups such as the Cunha Msx2 gene is also expressed during GT development (R. H. et laboratory (Cunha and Chung, 1981; Hayward et al., 1998; al., unpublished results). Kurzrock et al., 1999b). Fgf genes have frequently been Fgf8 expression in later stage mutant GTs was altered, described as part of gene regulatory loops (Thomson and possibly as a result of the several morphological changes of the Cunha, 1999; Lebeche et al., 1999). It has also been suggested lower (ventral) part of the urethral plate. that several surface epithelia of genital tubercles are involved in epithelial-mesenchymal interactions in relation to the Abnormal glans penis and glans clitoridis formation androgen system (Cunha and Lung, 1978; Murakami and in newborn Fgf10 knockout mice Mizuno, 1986; Murakami, 1987). Following initial outgrowth, distal portions of GTs During limb-bud development, Fgf8 transcripts have been differentiate into penes or clitorides with the prepuce glands, detected in the prelimb field ectoderm prior to the formation urethrae, prepuces and the corpus cavernosum glandis. In of AER (Tickle and Eichele, 1994; Johnson and Tabin, 1997; normal development of the penis, urethral plate becomes Ohuchi et al., 1997). It is known that beads releasing different separated from the surface epithelium of the glans penis in FGFs are able to induce the development of additional limbs proximodistal sequence and forms an epithelial tube that runs when applied to the flank of chick embryos (Cohn et al., 1995; through the glans penis (Grenister, 1954; Murakami, 1987). Crossley et al., 1996b; Ohuchi et al., 1997). It should be noted The prepuce grows to cover the glans penis, being fused at that, in GT development, Fgf8 expression resides in the the ventral (lower) midline of the GT (Fig. 7A). urethral plate epithelium, which is endodermal in origin. Morphogenesis of the urethra and prepuce in the glans Extensive recent experiments confirmed the distal end of the clitoridis is largely similar to that of penis. Extensive urethra as endodermal in origin (Kurzrock et al., 1999a). One anatomical evaluations and related references about the mode might still envisage that part of the initial GT outgrowth before of urethral tube formation are found in recent papers urethral tube formation might be achieved by applying FGF (Kurzrock et al., 1999a,b). proteins. However, it would be necessary to exploit another Histological analysis of newborn Fgf10 mutant external experimental system that would allow the application of genitalia revealed striking defects, including defects in urethral protein beads to murine embryos at earlier stages, because our tube formation of the glans regions. Mesenchymal-derived organ culture can only be efficiently performed during the elements in the glans region, such as the rudiment of the corpus outgrowth phase of GTs. cavernosum glandis and the rudiment of os penis/clitoridis, In this paper, alterations of GT mesenchymal competence on were found to be formed as mesenchymal condensations in the application of FGF8 beads are suggested (data not shown). glans penis/clitoridis of the knockout mice. This responsiveness correlates well with the endogenous Fgf8 It was shown that Fgf10 is expressed in mesenchyme gene expression profile, with strong expression at 11.5 d.p.c. adjacent to the midline at later stages of GT development, when and a gradual decrease later. the differentiation of urethral tube formation occurs. These It has been suggested that the BMP system could exert phenotypes, together with the above mesenchymal expression negative activities against growth-promoting signals emanating of Fgf10, may suggest an essential role of Fgf10 in the from the AER in limbs (Niswander, 1999). The effect of BMP4 morphogenesis of the distalmost structures of GTs, especially beads application on the regulation of GT outgrowth was not those in the lower (ventral) side of GTs formation. Mutant male as prominent under similar culture conditions (data not shown). newborns did not show drastic phenotypes in the testis (data More detailed analysis would be necessary as negative not shown). activities may be balanced by positive (growth-promoting) Multiple morphogenic events would be involved during signals (Niswander, 1999). formation of the urethral plate and the subsequent urethral tube formation. Previous histological studies have suggested a Fgf10 knockout mice did not show drastic outgrowth dynamic expression profile of cell-matrix proteins, e.g. the defects in the GT tenascin gene, in these processes (Murakami et al., 1990). We have shown that Fgf10 may not be essential for the early Analysis of these candidate molecules is in progress (R. H. and phase of GT development, but Fgf10 knockout drastically G. Y., unpublished results). Further molecular analysis is affects morphogenesis of the glans penis and the glans required to clarify whether Fgf10 might regulate such clitoridis, the most distal structures of GTs. molecules or not. It is of interest that Fgf8 gene expression remained normal In humans, one of the frequent hereditary genital in Fgf10−/− GT at 10.5 and 11.5 d.p.c., suggesting that Fgf10 abnormalities, hypospadias, displays an abnormal positioning is not required for the induction of Fgf8 expression in GTs. of the urethral opening (Sutherland et al., 1996). Multiple One might speculate that the gene regulatory loop between causative genetic backgrounds have been suggested as Fgf8 in AER and the mesenchymal Fgf10 might only apply to potential candidate genes for hypospadias, including androgen 2478 R. Haraguchi and others systems (Anderson and Clark, 1990; Nordenskjold et al., Dassule, H. R. and McMahon, A. P. (1998). Analysis of epithelial- 1999). Because Fgf10 knockout mice display multiple agenesis mesenchymal interactions in the initial morphogenesis of the mammalian of vital organs such as the lung, it might be implied that Fgf10 tooth. Dev. Biol. 202, 215-227. De Moerlooze, L., Spencer-Dene, B., Revest, J., Hajihosseini, M., Rosewell, gene mutation in humans would result in more severe I. and Dickson, C. (2000). An important role for the IIIb isoform of phenotypes than those in hypospadias. fibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelial signalling during mouse organogenesis. Development 127, 483-492. We would like to thank Dr Eisuke Mekada, Dr Gail R. Martin, Dr Dolle, P., Izpisua-Belmonte, J. C., Brown, J. M., Tickle, C. and Duboule, Juan-Carlos Izpisua-Belmonte, Dr Brigid L. Hogan, Dr Yuji D. (1991). HOX-4 genes and the morphogenesis of mammalian genitalia. Yokouchi, Dr Atsushi Kuroiwa, Dr Sumihare Noji, Dr Juha Partanen, Genes Dev. 5, 1767-1767. Dr Janet Rossant, Dr Peter Lonai, Dr Akira Tanaka, Dr Mizuyo Dorkin, T. J., Robinson, M. C., Marsh, C., Bjartell, A., Neal, D. E. and Leung, H. Y. (1999). FGF8 over-expression in prostate cancer is associated Tsugane, Dr Paul T. Sharpe, Dr Nobuyuki Itoh, Dr Hideyo Ohuchi, with decreased patient survival and persists in androgen independent Dr Chisa Shukunami, Dr Toru Imamura, Mr Seiji Nakamura and Ms disease. Oncogene 18, 2755-2761. Aki Kuboyama for their suggestions and help. This work was Duboule, D. (1993). The function of Hox genes in the morphogenesis of the supported by a grant-in-aid from The SAGAWA Foundation for vertebrate limb. Ann. Genet. 36, 24-29. Promotion of Frontier Science and in part by the Research for The Duboule, D. (1994). How to make a limb? Science 266, 575-576. Future Program by JSPS. Gehring, W. J., Affolter, M. and Burglin, T. (1994). Homeodomain proteins. Annu. Rev. Biochem. 63, 487-526. Gehring, W. J. (1999). 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