Mesonephric FGF signaling is associated with the development ... - Core

Developmental Biology 280 (2005) 150 – 161 www.elsevier.com/locate/ydbio

Mesonephric FGF signaling is associated with the development of sexually indifferent gonadal primordium in chick embryos Hidefumi Yoshiokaa,c, Yoshiyasu Ishimarua,c, Noriyuki Sugiyamab,c, Naoki Tsunekawad, Toshiaki Noced, Megumi Kasaharaa, Ken-ichirou Morohashib,c,T a

Department of Natural Sciences, Hyogo University of Teacher Education, 942-1, Yashiro-cho, Kato Gun, Hyogo 673-1494, Japan b Department of Developmental Biology, National Institute for Basic Biology, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan c Core Research for Evolutional Science and Technology, Japan Science and Technology, Kawaguchi, Saitama 332-0012, Japan d Mitsubishi-Kasei Institute of Life Sciences, 11-Minami-Ooya, Machida, Tokyo 194-8511, Japan Received for publication 27 October 2004, revised 11 January 2005, accepted 12 January 2005

Abstract The gonad as well as the reproductive tracts, kidney, and adrenal cortex are derived from the intermediate mesoderm. In addition, the intermediate mesoderm forms the mesonephros. Although the mesonephros is the source of certain testicular cell types, its contribution to gonad formation through expression of growth factors is largely unknown. Here, we examined the expression profiles of FGF9 in the developing mesonephros of chick embryos at sexually indifferent stages, and found that the expression domain is adjacent to the gonadal primordium. Moreover, FGFR3 (FGF receptor 3) showed a strong expression in the gonadal primordium. Next, we examined the functions of FGF signal during gonadal development with misexpressed FGF9. Interestingly, misexpression of FGF9 led to gonadal expansion through stimulation of cell proliferation. In contrast, treatment with a chemical inhibitor for FGFR decreased cell proliferation and resulted in reduction of the gonadal size. Simultaneously, the treatment resulted in reduction of gonadal marker gene expression. Our study demonstrated that FGF expressed in the developing mesonephros is involved in the development of the gonad at the sexually indifferent stages through stimulation of gonadal cell proliferation and gonadal marker gene expression. D 2005 Elsevier Inc. All rights reserved. Keywords: FGF9; Ad4BPSF-1; DMRT-1; Gonadal primordium; Mesonephros; Chick development

Introduction Basically, the developmental process of the reproductive tissues seems conserved among vertebrates. In fact, several transcription factors such as WT-1, SOX9, DAX-1 (NR0), Ad4BP/SF-1 (NR5A1) and DMRT-1 (doublesex- and mab3-related transcription factor) implicated in mammalian gonadal development are conserved in other vertebrate animal species (Capel, 2000; Morohashi, 1997). Moreover, some of them are known to be involved in gonadal T Corresponding author. Department of Developmental Biology, National Institute for Basic Biology, Myodaiji-cho, Okazaki, Aichi 444-8787, Japan. Fax: +81 564 59 5866. E-mail address: [email protected] (K. Morohashi). 0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.01.011

development even in non-vertebrate animals (Asahina et al., 2000; Zarkower, 2001). Therefore, a similar set of transcription factors and thereby similar mechanisms are likely to be involved in gonadal differentiation. Developmentally, the reproductive tissues are derived from the intermediate mesoderm, which initially appears between the paraxial and lateral plate mesoderms (Kaufman, 1992). As an early event occurring in the intermediate mesoderm, the nephric duct (feature Wolffian duct) progresses rostrocaudally towards the cloaca. From the rostrocaudal aspect, this structure is divided into three distinct regions; the pronephros, mesonephros, and metanephros. In the three nephric regions, the nephrogenous mesenchyme medial to the nephric duct undergoes extensive epithelial transformation to form nephric tubules, while

H. Yoshioka et al. / Developmental Biology 280 (2005) 150–161

the nephric duct develops into the male reproductive tract (Saxen, 1987). In the female, the Mqllerian duct develops later into the mesonephros along with the nephric duct. In addition to these reproductive tracts, the urogenital ridge, the primitive stage of the gonad, starts to develop at the ventral surface of the mesonephros. Studies of the contribution of the mesonephros in this early process of gonadal development demonstrated that mesonephric cells migrate into the primitive gonad and eventually differentiate into peritubular myoid and vascular endothelial cells (Buehr et al., 1993; Martineau et al., 1997). However, the tissue contribution has not been studied yet with respect to the source of growth factor signals. Patterning of multicellular tissue is regulated by extracellular signals derived from neighboring cells. The roles of various members of TGFh/BMP, WNT, FGF and Hedgehog families have been highlighted from many aspects of developmental processes in a variety of animals (Huelsken and Birchmeier, 2001; Ingham and McMahon, 2001; Massague and Chen, 2000; Ornitz and Itoh, 2001). With respect to the urogenital development, the functions of these growth factors have been elucidated by gene disruption studies in mice. For example, Wnt4 is required for tubulogenesis in the kidney (Stark et al., 1994) and differentiation of the female gonad and reproductive tract (Vainio et al., 1999). Fgf9 is required for male gonadal differentiation (Clark et al., 2000; Colvin et al., 2001), while Dhh expressed in Sertoli cells is implicated in Leydig cell differentiation (Schmahl and Capel, 2003; Yao et al., 2002). Thus, all these growth factors produced in the developing gonad seem to be essential for gonadal differentiation in mammals. Nevertheless, the contribution of the growth factors expressed in the mesonephros is largely unknown. In the present study, we defined the expression of FGF9/Fgf9 in the chick and mouse mesonephroi at sexually indifferent stages. To elucidate the functions of the growth factors in the process of gonadal differentiation, we adapted misexpression and chemical inhibition studies in chick embryos. Based on their effects on the expressions of the marker genes and morphological changes, the present study revealed that the FGF signal plays a crucial role at the sexually indifferent stages of gonadal development.

Materials and methods In situ hybridization and immunohistochemistry Section and whole-mount in situ hybridization were performed essentially as described previously (Wilkinson, 1992) with minor modifications (Kawabe et al., 1999; Yoshioka et al., 1998). The chick and mouse cDNA probes used in this study were as follows. Chick Ad4BP/SF-1, AMH, and SOX9 were provided by Kudo and Sutou (1997),

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Eusebe et al. (1996), and Kamachi et al. (1999), respectively. FGFR1 (FGF receptor 1), FGFR2 (FGF receptor 2), and FGFR3 (FGF receptor 3) were provided by Dr. Nohno (Noji et al., 1993). FGFR4 (Clone ID; ChEST205p12) was supplied by MRC Geneservice (Cambridge, UK). Chick cDNA for DMRT-1 was obtained as described previously (Raymond et al., 1999). To isolate mouse Fgf9 cDNA probe (NM 013518), total RNA prepared from 12.5 dpc (days postcoitum) fetuses was amplified by reverse transcriptionpolymerase chain reaction (RT-PCR) with primers, 5VTGAAGTTGGGAGCTATTTCGGTGTGC-3V, and 5VGTTCAGGTACTTTGTCAGGGTCCACT-3V. To isolate chick FGF9 cDNA, RT-PCR was performed using total chick embryonic RNA at stage 30. Degenerate primers were designed to target the amino acid sequences, MAPLGEVG (5V primer) and KVPELYKD (3V primer). RT-PCR with the primers produced a 609-bp fragment encoding FGF9 cDNA lacking carboxy terminal. To obtain cDNA encoding the carboxy terminal region, 3V-rapid amplification of cDNA end was performed according to the instructions provided by the manufacturer (Stratagene, La Jolla, CA). The fulllength cDNA for the chick FGF9 was obtained from the above two cDNA fragments. The nucleotide sequence of the chick FGF9 cDNA was deposited in the GenBank database (AB108842). Two-color whole-mount in situ hybridization with digoxigenin-labeled probes (Roche Molecular Biochemicals, Inc., NJ) was performed as described previously (Dietrich et al., 1997), except that Fast Red and BCIP/NBT (Roche Molecular Biochemicals) were used for the first and second staining, respectively. After colorimetric reaction, the embryos were fixed with 4% paraformaldehyde/phosphate-buffered saline (PBS), and embedded in 2% agarose or paraffin followed by sectioning using a vibratome or microtome, respectively. For immunohistochemical analysis of chick VASA homologue (CVH), the tissues were fixed with 4% paraformaldehyde/PBS and embedded in paraffin. Paraffin sections were dewaxed and hydrated by passing through a xylene-ethanol series. To inactivate endogenous alkaline phosphatase (AP) activity, the sections were treated at 658C for 60 min. After three 20-min wash in PBS containing 3% bovine serum albumin, the sections were incubated overnight at 48C with a rabbit anti-CVH (1:10,000) (Tsunekawa et al., 2000). AP-conjugated antirabbit IgG (Vector Laboratories, Burlingame, CA) was used as the secondary antibody. Section in situ hybridization was performed according to the method described previously (Xu and Wilkinson, 1998). Manipulation of chick embryos with recombinant retroviruses The chick FGF9 cDNA was cloned into an avian retrovirus vector RCASBP(A) (Hughes et al., 1987) and transfected into chick embryonic fibroblasts (Fekete and Cepko, 1993). The fibroblasts were trypsinized and packed into a block by centrifugation to use for implantation

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(Riddle et al., 1993). The embryos were staged according to the Hamburger and Hamilton’s staging table (Hamburger and Hamilton, 1951). With embryos at stages 12–13, the FGF9-expressing cells were implanted into the nephrogenous mesenchyme at the presumptive level of 23–24 somites since part of the nephrogenous mesenchyme at this level forms the future gonads (Rodemer et al., 1986). The embryos at appropriate stages were fixed with 4% paraformaldehyde in PBS, and thereafter processed for wholemount in situ hybridization, section in situ hybridization or immunohistochemistry.

amplification of W-specific XhoI repeat was performed with a genomic DNA as a template (Kent et al., 1996). The sex of mouse embryo was determined by PCR amplification of the Sry (Gubbay et al., 1992) and Zfy (Hogan et al., 1994).

Implantation of FGF9 and SU5402-soaked beads

Previous studies reported that several genes corresponding to those involved in mammalian gonadal development are also expressed in the embryonic chick gonads (Smith and Sinclair, 2001). We determined in detail the expression profiles of Ad4BP/SF-1 (gonadal and adrenal marker) and DMRT-1 (gonadal marker) at sexually indifferent stages of the chick embryos. Gonadal sex differentiation is known to start at around stages 28–30. Whole-mount in situ hybridization revealed that the expression of Ad4BP/ SF-1 in the developing mesonephric mesenchyme starts by stage 19 and continues in the following stages (Figs. 1A, D, J). The expression of DMRT-1, which was undetectable at stage 19 (data not shown), appeared by stage 21 (Fig. 1G). At the following stage 24, the expression was detected in two separate domains at the lateral sides of each mesonephros (arrowheads in Fig. 1M). Although the expression patterns of DMRT-1 in the two sexes were quite similar, the expression in the male was higher than in the female (data not shown), presumably because of its localization on the Z sex chromosome (Raymond et al., 1999). Cross-sections revealed that Ad4BP/SF-1 was expressed as a single cell group at the medial side of the mesonephric mesenchyme at stages 19 and 21 (Figs. 1B, C, E, F). By stage 24, the cell group divided into two main subgroups, one was located proximal to the coelomic epithelia (ventral domain) and the other proximal to the dorsal aorta (dorsal domain) (Fig. 1L). Based on our previous observation in rat fetuses (Hatano et al., 1994, 1996), the ventral and dorsal domains seem to correspond to the gonadal (gp in Fig. 1) and adrenal (adp in Fig. 1) primordia, respectively, and the single cell group observed at stage 19 is probably the adreno-gonadal primordium (agp in Fig. 1). The expression of DMRT-1 was localized in the ventral aspect of the mesonephric mesenchyme, which positionally corresponds to the gonadal primordium (Figs. 1I, O). In addition, the expression was clearly observed at the lateral aspect to the mesonephros (Figs. 1N, O). Considering that the expression of DMRT-1 at stage 21 overlaps with that of Ad4BP/SF-1 at the ventral but not the dorsal part, the cells expressing Ad4BP/SF-1 seem to have the potential to develop into the gonad or adrenal cortex before they separate into the two subgroups (Fig. 1F).

AG1-X2 ion exchange resin beads (150–200 Am diameter; Bio-Rad, Richmond, CA) were prepared (Eichele et al., 1984). They were soaked for 1 h in 10 mM solution of SU5402 (Calbiochem, La Jolla, CA; Mohammadi et al., 1997) in dimethyl sulfoxide (DMSO) or otherwise in DMSO alone as control. The beads were washed with PBS and thereafter implanted into the coelomic epithelium corresponding to the presumptive FGF9 expressing-region at stage 18–19. The implanted embryos were incubated for 36 h at 388C. Heparin acrylic beads (Sigma, St Louis, MO) were soaked in 0.5 mg/ml of human recombinant FGF9 (Pepro Tech EC, London, UK) or bovine serum albumin (BSA) for 1 h at 48C. The beads were implanted into the coelomic epithelium corresponding to the presumptive gonadal region at stage 18–19. The implanted embryos were incubated for 24 h at 388C. BrdU labeling and detection To examine cell proliferation, 1 Al bromodeoxyuridine (BrdU) solution (10 Ag/Al in H2O with 0.01% Fast Green) was injected into embryos implanted with the FGF9expressing cells, FGF9-soaked, or SU5402-soaked beads through the vitelline vein. After 60-min incubation at 388C, the embryos were fixed with 4% paraformaldehyde/ PBS and embedded in paraffin. The paraffin sections were dewaxed, hydrated and then treated with 10 mM trisodium citrate (pH 6.0) for 20 min to activate nuclear antigens (Nomura et al., 1998). After two 5-min washes with PBS, the sections were incubated with a mouse antiBrdU monoclonal antibody (Roche, 1:100) for 1 h at 378C. The sections were washed five times with PBS, each for 5 min, and further incubated with Cy3-labeled anti-mouse antibody (IgG, Jackson Immunoresearch Laboratories, West Grove, PA, 1:500) for 1 h at 378C. After washing with PBS again, the sections were examined for immunofluorescence. Sexing of tissue samples The sex of the chick embryo was determined by the presence or absence of the W chromosome. PCR

Results Expression of Ad4BP/SF-1 and DMRT-1 in the developing gonadal and adrenal primordia


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Fig. 1. Expression of Ad4BP/SF-1 and DMRT-1 in the developing nephrogenous mesenchyme. Expression of Ad4BP/SF-1 and DMRT-1 in the developing nephrogenous mesenchyme of the chick embryos was examined. Whole mount in situ hybridization was performed with the probes for Ad4BP/SF-1 (A–F, J–L) and DMRT-1 (G–I, M–O). Ventral views of the examined embryos are shown (A, D, G, J, M). These samples were cross-sectioned (30 Am) at the indicated levels (levels B and C in A, levels E and F in D, levels H and I in G, levels K and L in J, and levels N and O in M). Chick embryos were used at stage 19 (A, B, C), stage 21 (D–I), and stage 24 (J–O) as indicated in the panels. Arrowheads in M indicate two separate DMRT-1-expressing domains. The expressions of male embryos are shown. agp; adreno-gonadal primordium, gp; gonadal primordium, adp; adrenal primordium. Scale bars in A, D, G, J, and M = 200 Am, while in B, C, E, F, H, I, K, L, N, and O = 100 Am.

Expression of FGF9/Fgf9 in the sexually indifferent stages of chick and mouse mesonephroi The intermediate mesoderm is initially defined as a discrete cell aggregate between the paraxial and lateral plate mesoderms at stage 9 of chick embryos, and subsequently develops into the nephric duct and nephrogenous mesenchyme. The nephrogenous mesenchyme forms condensed cell aggregates, where mesenchymal to epithelial transformation occurs extensively, followed by development of the mesonephric tubules (Friebova-Zemanova, 1981). The expression of FGF9 started at the anterior side of the mesonephros as early as stage 18 and thereafter expanded posteriorly (data not shown). At stage 21 when the mesonephric tubules had developed extensively, FGF9 was expressed in the developing mesonephros of male chick embryos (Fig. 2A). Likewise, the expression was observed in female embryos (data not shown). It is well known that the development of Bowman’s capsule and thus its antecedent structure, a comma-shaped body, is essential to establish the excretory apparatus in the kidney, and therefore these structures are characteristic primarily of the metanephros in mammals. However, in chick embryos, the particular structures are well organized even in the mesonephros (Friebova-Zemanova, 1981). The comma-shaped bodies start to appear at stage 18 at the mesenchyme adjacent to the lateral tip of the mesonephric

tubules, and subsequently develop into Bowman’s capsule as illustrated in Fig. 2B. At stage 21, the expression of FGF9 was clearly observed in this particular structure, the comma-shaped body (Fig. 2C). At the later stage 30, when sexually indifferent gonads start to develop sex-dependently, the expression of FGF9 was decreased in the mesonephros, whereas the expression appeared in the epithelial cells of the gonads of both sexes. The FGF9 expression in the male was higher than that in the female (Figs. 2D, E, F, G). Interestingly, the expression in the left gonad of the female was stronger than that in the right gonad (Fig. 2G). It is well known that the right ovary degenerates gradually soon after the sex of indifferent gonads is determined into the ovary. The downregulation of FGF9 expression in the female right gonad may lead to the following ovarian degeneration. Although it was shown that Fgf9 is expressed in the sexually differentiating mouse gonads (Colvin et al., 2001; Schmahl et al., 2004), its expression at the developing mesonephros has not yet been defined. Sexually indifferent mouse fetuses at 9.5 and 10.5 dpc (days postcoitum) were subjected to whole mount in situ hybridization. Similar to the chick embryos, Fgf9 was expressed in the developing mouse mesonephric mesenchyme at 9.5 dpc (Fig. 2H). Cross-sections clearly showed that certain part of the mesonephric mesenchyme adjacent to the mesonephric duct was the expression domain of Fgf9 (Fig. 2I). Any clear

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Fig. 2. Expression of chick and mouse FGF9/Fgf 9 in developing urogenital regions. The expression of FGF9 was examined in the developing urogenital region of the sexually indifferent stage (stage 21) of male chick embryos (A). The developing nephrogenous mesenchyme is schematically represented (B). cb; comma-shaped body, nd; nephric duct, nt; nephric tubule, gp; gonadal primordium, adp; adrenal primordium. A cross-section (30 Am) (C) was prepared at the level indicated in A. The expression of FGF9 was examined in the mesonephros and sexually differentiating gonads of male (D) and female (F) at stage 30. Cross-sections (30 Am) (E and G) were prepared at the levels indicated in D and F. R; right gonad, L; left gonad. The Fgf 9 expression in the developing mouse urogenital region was examined with sexually indifferent stages, 9.5 (H) and 10.5 dpc (J), of male fetuses. Cross-sections (30 Am) (I and K) were prepared at the levels indicated in H and J. The sex of each chick and mouse embryo is indicated. Scale bars in A, D, and F = 300 Am, in C, H, and J, = 100 Am, and in E and G = 50 Am, in I and K = 20 Am.

difference in the mesonephric expression was not observed between the two sexes (data not shown). The mesonephric expression decreased at 10.5 dpc (Figs. 2J, K) and disappeared eventually (data not shown). Spatially correlated expression of gonadal marker genes with FGF9 and FGFRs in the developing mesonephros Based on the expression patterns of FGF9, Ad4BP/SF-1, and DMRT-1 described above, the expression of these genes seemed spatially related. To define the relationship, the expression of FGF9 was compared directly with those of Ad4BP/SF-1 and DMRT-1 by two-color whole mount in situ hybridization. At stage 21, FGF9 was expressed along with Ad4BP/SF-1 and DMRT-1 in the mesonephric mesenchyme (Figs. 3A, C). Cross-sections revealed that the expression domain of FGF9 was adjacent to the ventral but not dorsal half of the expression domain of Ad4BP/SF-1 (Fig. 3B). Likewise, the expression domain of FGF9 was adjacent to that of DMRT-1 (Fig. 3D), indicating that FGF9 is expressed adjacent to or somewhat overlapped with the gonadal primordium. The expression patterns of FGF receptors (FGFR1, FGFR2, FGFR3, and FGFR4) were examined in chick embryos. It has been established that these receptors exhibit different expression patterns in various developing tissues.

Likewise, their expressions in the mesonephric region were specific to each receptor. At stage 19, FGFR1 was expressed in the whole part of the mesonephric region (Figs. 3E, I). A strong expression of FGFR2 was observed in the nephric duct while a modest expression was observed in the mesonephros near the mesentery (Figs. 3F, J). Showing an expression pattern nearly complementary to FGFR2, FGFR3 was expressed in the mesonephros except the nephric duct (Figs. 3G, K). No clear expression of FGFR4 was detected in the mesonephric region (Figs. 3H, L). At the following stage 21, the expression of FGFR1 was retained in the mesonephric mesenchyme proximal to the nephric duct and in the ventral epithelial cell layer covering the mesonephros (Fig. 3M). The expression of FGFR2 was basically identical to that at stage 19 (Fig. 3N). Interestingly, the expression of FGFR3 appeared to be confined to the gonadal primordium at this stage (Fig. 3O). Finally, FGFR4 was weakly expressed in the nephric duct but not in other parts of the mesonephric region (Fig. 3P). Expansion of chick gonad by misexpressed FGF9 The expression profiles described above strongly suggested the involvement of FGF9 in the sexually indifferent stages of gonadal development. To examine this possibility, FGF9 was misexpressed in the developing


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Fig. 3. Spatial correlation of the expression domain of FGF9 with those of Ad4BP/SF-1, DMRT-1, and FGF receptors in the developing mesonephros. With chick embryos at stage 21, two-color whole mount in situ hybridization was performed with probes for FGF9 (blue signal) and Ad4BP/SF-1 (red signal) (A) or FGF9 (blue signal) and DMRT-1 (red signal) (C). Cross-sections (B, D) (30 Am) were prepared as indicated in A and C. Whole-mount in situ hybridization probed with FGF receptors, FGFR1 (E, I, M), FGFR2 (F, J, N), FGFR3 (G, K, O), and FGFR4 (H, L, P) was performed in male chick embryos at stages 19 (E–L) and 21 (M–P). Cross-sections (30 Am) prepared from the examined embryos are shown. Insets in E, F, G, and H correspond to I, J, K, and L, respectively. Arrowheads in B, D, and M–P indicate the gonadal primordium, while arrows with cb indicate comma-shaped body. nd; nephric duct. Scale bars in A and C = 350 Am, and in B, D, and E–P = 100 Am.

right mesonephric region by using a replication competent retrovirus vector. The viruses expressing FGF9 were spread throughout the operated right mesonephros (data not shown). The chick embryos implanted with the FGF9expressing cells were incubated until the gonads underwent sex differentiation (stage 30–31). Interestingly, the whole view of the gonad highlighted by the expression of Ad4BP/SF-1 showed that the misexpression of FGF9 affected gonadal development (Fig. 4A). In fact, crosssections clearly revealed irregular expansion of the gonads (Fig. 4B). Likewise, the expression of DMRT-1 was also detected in the particular cells, indicating that the cells acquired specificity as gonadal cells (Figs. 4C, D). Unexpectedly, however, all embryos were dead by day five after operation possibly because of the toxic effects of the misexpressed FGF9. Thus, it was unclear whether the expanded gonadal region would potentially develop into a functional tissue. In addition to examining the effects of the misexpressed FGF9 on the gonad, we also checked its effects on the mesonephros. However, we could not detect any obvious expansion of the mesonephroi in the embryos

implanted with the FGF9-expressing cells. Therefore, it is unlikely that FGF9 is implicated in the mesonephric development. A null mutation of Fgf9 in the mouse was reported to result in a decrease in the marker gene expression for Sertoli and Leydig cells of the fetal testis, and thus, it was concluded that the XY gonads of the gene disrupted mice were somewhat sexually reversed to female (Colvin et al., 2001). To investigate whether misexpression of FGF9 affects the chick gonadal sex, we examined the expressions of SOX9 and Aromatase genes, which are representative marker genes for the testis and ovary, respectively. Although the expression of SOX9 was detected in the expanded region of the male gonad (Figs. 4E, F), the expression was neither detected in the expanded female gonad nor in the authentic female gonad (Figs. 4G, H). Conversely, the expression of Aromatase was detected in the expanded region of the female gonad (Figs. 4I, J), but not in that of the male (Figs. 4K, L). Implantation of the AP-expressing cells did not affect the expressions of these marker genes in both sexes (Figs. 4M–Q, T).

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Fig. 4. Gonadal expansion by misexpression of FGF9. FGF9-expressing cells (A–L, R, S) or AP-expressing cells (M–Q, T, U) were implanted into the nephrogenous mesenchyme at stage 12–13. After incubation for 5 days (stage 30), the embryos were subjected to in situ hybridization probed with Ad4BP/SF-1 (A, B), DMRT-1 (C, D), SOX9 (E–H), and Aromatase (I–L). The FGF9-expressing cells were implanted into 421 embryos, and 186 embryos that developed up to stage 30 were studied; analysis of Ad4BP/SF-1 expression was conducted in 18 male and 19 female embryos, analysis of SOX9 expression was conducted in 16 male and 18 female embryos, analysis of DMRT-1 expression was conducted in 19 male and 20 female embryos, and analysis of Aromatase expression was conducted in 15 male and 15 female embryos. The expanded gonads indicated by arrowheads were observed in 78% of the embryos examined. The APexpressing cells were implanted into 247 embryos and 113 embryos that developed up to stage 30 were studied; analysis of Ad4BP/SF-1 expression was conducted in 13 male and 11 female embryos, analysis of SOX9 expression was conducted in 10 male and 10 female embryos, analysis of DMRT-1 expression was conducted in 13 male and 14 female embryos, and analysis of Aromatase expression was conducted in 11 male and 10 female embryos. The tissues were cross-sectioned (B, D, F, H, J, L) at the levels indicated in A, C, E, G, I, and K. Likewise, the embryos implanted with the AP-expressing cells were used for in situ hybridization probed with Ad4BP/SF-1 (M, N), DMRT-1 (O), SOX9 (P), and Aromatase (Q). A–F and K–P represent studies in male embryos, while G– J and Q were conducted in female embryos. Arrows in A and M indicate developing adrenal cortex. For analyses of AMH and CVH expressions, the embryos implanted with the FGF9 (R, S) or AP-expressing cells (T, U) were incubated for 5.5 days (stage 31). The FGF9-expressing cells were implanted into 75 embryos and 31 (16 male and 15 female) embryos that developed up to stage 31 were used in this study (R, S). The AP-expressing cells were implanted into 51 embryos and 22 (12 male and 10 female) embryos that developed up to stage 31 were used for this study (T, U). Tissue sections prepared from the male embryos were subjected to in situ hybridization for AMH (R, T) and immunohistochemical analysis for CVH (S, U). The asterisk in S indicates the location corresponding to the expanded gonad in R. Arrows show PGCs distributed in the expanded gonad (S). Scale bars in A, C, E, G, I, K, and M = 300 Am, and those in B, D, F, H, J, L, N, O, P, Q and R–U = 50 Am.

We then examined whether the primordial germ cells (PGCs) migrate toward and settle even at the gonad of the expanded region. In order to visualize PGCs, immunohistochemical studies were performed with an antibody to a chick vasa homologue (CVH) (Tsunekawa et al., 2000), which is a marker for PGCs (Olsen et al., 1997; Yoon et al., 1997). To localize the expanded region of the gonad, the section was subjected to in situ hybridization probed with AMH (Fig. 4R), and the adjacent section was used for CVH staining. As shown in Fig. 4S, PGCs mostly migrated into the gonad while a number of PGCs remained at or around the expanded gonadal region. Expectedly, all PGCs were localized in the gonad when treated with the AP-expressing cells (Figs. 4T, U).

Effects of FGF signal on gonadal cell proliferation To elucidate the mechanisms of gonadal expansion by the misexpressed FGF9, we asked whether FGF9 stimulates gonadal cell proliferation. In order to confine the effects of FGF9 within the gonadal region, FGF9-soaked (Figs. 5A–F) and BSA-soaked beads (Figs. 5G–L) were implanted into the developing right mesonephric mesenchyme at stage 18–19. Since the expression profiles of FGF9 and FGFRs did not show any difference between the two sexes and a similar gonadal expansion was observed in both sexes, male embryos were used in this study. After incubation for 24 h, the primitive but structurally distinguishable gonad was developed. BrdU was injected into the operated chick embryos and BrdU-


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Effects of FGF signal inhibitor on gonadal marker gene expression and gonadal cell proliferation

Fig. 5. Effects of FGF9 on gonadal cell proliferation. FGF9-soaked beads were implanted into the coelomic epithelium at stage 18–19 of male embryos (A–F). As a control, BSA-soaked beads were used (G–L). Asterisks indicate the location of beads (A, B, G, and H). BrdU was injected into the operated embryos 1 h prior to the end of 24-h incubation after operation (stage 24). Six embryos were used for each beadsimplantation study and three of them were subjected to immunohistochemical analysis with anti-BrdU antibody (B, D, H, J), while the nuclei were stained with DAPI (A, C, G, I). Insets in C, D, I, and J are enlarged in E, F, K, and L, respectively. The gonadal region was determined by in situ hybridization probed with Ad4BP/SF-1 using consecutive sections (data not shown). Five tissue sections were selected randomly, and numbers of the immunoreactive and total cells in the areas encircled by dotted lines in E, F, K, and L corresponding to the gonads were counted and are plotted in M. Data shown in M are mean F SD numbers of immunoreactive cells relative to the total number of cells when the relative cell number in the right gonad of the embryo implanted with the BSA-soaked bead was considered 1 unit. BSA R and BSA L; the right and left gonad of the embryo implanted with the BSA-soaked bead, respectively, FGF9 R and FGF9 L; the right and left gonad of the embryo implanted with the FGF9-soaked bead, respectively. The immunoreactive cell numbers per total cell numbers are as follow. Experiment 1; BSA R = 24/114, BSA L = 32/186, FGF9 R = 30/161, and FGF9 L = 20/147, Experiment 2; BSA R = 14/95, BSA L = 19/114, FGF9 R = 17/62, and FGF9 L = 11/101, Experiment 3; BSA R = 14/62, BSA L = 26/134, FGF9 R = 20/71, and FGF9 L = 13/95. Scale bars in A–D and G–J = 100 Am, while in E, F, K, and L = 25 Am.

labeled cells in the gonadal region were detected immunohistochemically. As indicated in Fig. 5M, the relative number of the proliferating cells in the operated right gonad (Fig. 5E) was approximately 1.3-fold higher than that in the unoperated left gonad (Fig. 5F). No such enhanced cell proliferation was noted when the BSAsoaked beads were implanted (Figs. 5K, L).

To characterize the effects of FGF signal on gonadal marker gene expression, beads soaked with SU5402, an inhibitor for FGF receptor, were implanted. After the embryos were incubated for 36 h, they were cross-sectioned at the level of the implanted bead (Figs. 6A, C) and both at anterior (Figs. 6B, D) and posterior (data not shown) levels relative to that of the implanted bead. In situ hybridization with these sections revealed marked reduction of the gonadal expression of Ad4BP/SF-1 when compared with sections of the fetuses implanted with control solvent (DMSO)-soaked bead (Figs. 6I, J). The effect of SU5402 was detected even in the unoperated left gonad although the effect seemed weaker than that in the right gonad. Similar to the effects on the expression of Ad4BP/SF-1, the expression of DMRT-1 decreased (Figs. 6C, D). The beads soaked with control solvent (DMSO) had no effect on the expressions of DMRT-1 (Figs. 6K, L). Interestingly, the expression of Ad4BP/SF-1 at the adrenal primordium seemed unaffected (indicated by arrows with ad in Figs. 6A, B). In addition to the effects on the marker gene expression, the gonads of the embryos implanted with the SU5402soaked beads were smaller than those with the DMSOsoaked beads. Therefore, we examined whether SU5402 inhibits gonadal cell proliferation. The bead-implanted embryos were labeled with BrdU and subjected to immunohistochemical analysis (Figs. 6E–H, M–P). Implantation of the SU5402-soaked beads resulted in 37% reduction in the number of proliferating cells at the operated right gonad compared with the embryos implanted with the DMSO-soaked beads (Figs. 6G, O, Q). Approximately 10% reduction of cell proliferation was observed even at the left gonad of the SU5402 bead implanted embryo (Figs. 6H, P, Q). Since the chemical reagent can potentially inhibit signals from all types of FGFs, the inhibition study does not necessarily lead to the conclusion that FGF9 is essential for gonadal cell proliferation. In fact, it was reported that FGF2 is expressed in the developing mesonephric mesenchyme of the chick embryo (Savage and Fallon, 1995), and the receptor specificity of FGF2 overlaps with that of FGF9 (Ornitz et al., 1996). Therefore, although the exact FGF molecule responsible for gonadal cell proliferation remains elusive, it is reasonable to conclude that the FGF signal is essential for early gonadal cell proliferation.

Discussion A novel expression domain of FGF9/Fgf9 during gonadal development In mammals, Bowman’s capsule appears in the metanephric kidney and functions as a filter for renal

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Fig. 6. Effects of FGF signal inhibitor, SU5402, on gonadal marker gene expression and gonadal cell proliferation. SU5402-soaked beads were implanted into the coelomic epithelium at stage 18–19 of male embryos (A–H). As a negative control, DMSO-soaked beads were used (I–P). BrdU was injected into the operated embryos 1 h prior to the end of 36-h incubation after operation (stage 25). After fixation, they were subjected to in situ hybridization probed with Ad4BP/SF-1 (A, B, I, J) and DMRT-1(C, D, K, L) or used for immunohistochemical analysis for BrdU incorporation (F, N). The sections used for detection of BrdU were stained with DAPI (E, M). The insets in E, F, M, and N are enlarged and indicated as merged images (G, H, O, P). Five tissue sections were selected randomly, and the numbers of the immunoreactive cells and total cells encircled the dotted lines corresponding to the gonads were counted. The gonadal region was determined by in situ hybridization probed with Ad4BP/SF-1 using consecutive section (O, P) or predicted structurally (G, H). The immunoreactive cell numbers relative to the total cell numbers are presented when the relative cell number in the right gonad of the embryo implanted with the DMSO-soaked bead was considered 1 unit (Q). Six embryos were used for each beads-implantation study. Three of the SU5402-beads and DMSO-beads implanted embryos were subjected to immunohistochemical analysis using anti-BrdU antibody. DMSO R and DMSO L; the right and left gonad of the embryo implanted with the DMSOsoaked bead, respectively, SU5402 R and SU5402 L; the right and left gonad of the embryo implanted with the SU5402-soaked bead, respectively. Data shown in Q are mean F SD of three embryos. The immunoreactive cell numbers per total cell numbers are as follow. Experiment 1; DMSO R = 65/260, DMSO L = 64/ 280, SU5402 R = 24/113, and SU5402 L = 60/300, Experiment 2; DMSO R = 112/490, DMSO L = 139/600, SU5402 24/203, and SU5402 L = 52/259, Experiment 3; DMSO R = 79/334, DMSO L = 90/390, SU5402 R = 21/138, and SU5402 L = 51/267. Arrowheads marked R and L indicate the right and left gonad, respectively, while arrows marked ad indicate the adrenal primordia. Asterisks indicate the implanted beads. Scale bars in A–F and I–N = 100 Am, while in G, H, O, and P = 25 Am.

blood flow. In the chick embryo, the structure develops in the mesonephros in addition to the metanephros (Friebova-Zemanova, 1981), and functions in a manner similar to that in the metanephric kidney (Zemanova et al., 2002). Interestingly, we found that FGF9 was expressed in the primitive stage of the developing Bowman’s capsule (corresponding to the comma-shaped body), which is localized adjacent to the tip of the growing mesonephric tubule. Moreover, the expression profile of FGF9 was comparable with those of gonadal markers, DMRT-1 and Ad4BP/SF-1. Based on the expression domains of the gonadal marker genes, the expression domain of FGF9 was found to be adjacent to the gonadal primordium.

Similar to the chick FGF9, the expression of Fgf9 was confirmed in the mesonephros of mouse fetuses. Although it was difficult to identify structurally the developing commashaped body in the mouse mesonephros, Fgf9 seems to be expressed adjacent to the gonadal primordium and function as the signal for the sexually indifferent stage of gonadal development. In addition to this mesonephric expression, Fgf9 has been reported to be expressed in sexually differentiating gonads of the mouse fetuses (Colvin et al., 2001). Similarly, the present study revealed that FGF9 is expressed in epithelial cells of the chick embryonic gonads. As described below, FGF9/Fgf9 expressed in these two distinct domains probably have distinct functions during gonadal development.


Functions of FGF signal at the sexually indifferent stage of urogenital development The FGF signal is mediated by four receptor molecules, FGFR1 to FGFR4. We found in the present study that FGFRs are expressed in the developing mesonephric mesenchyme in a manner specific for each receptor, some of which are consistent with the observation by Walshe and Mason (2000). Noticeably, FGFR3 is clearly expressed in the gonadal primordium. Considering that FGF9 potentially binds to FGFR3 with high specificity (Ornitz et al., 1996), it is reasonable to assume that FGF9 expressed in the developing comma-shaped body stimulates the neighboring gonadal primordium. Based on these observations, we investigated whether FGF9 acts as a signal for gonadal differentiation by implantation of FGF9-expressing cells and FGF9-soaked beads. Surprisingly, misexpression of FGF9 resulted in gonadal expansion as was seen in the limb bud (Niswander, 2003) and genital tubercle (Haraguchi et al., 2000) when they were stimulated by particular types of FGF molecules. Because of increased BrdU incorporation at the gonadal region, this effect of gonadal expansion by FGF9 misexpression is potentially caused by increased cell proliferation. Consistent with this observation, studies using a chemical inhibitor of FGFR clearly indicated that inhibition of FGF signal resulted in decreased cell proliferation and eventually reduction of the gonadal size. Our results are in agreement with those of recent in vitro tissue (Schmahl et al., 2004) and cell (Willerton et al., 2004) culture studies of mouse fetal gonads, which showed stimulation of gonadal cell proliferation by human recombinant FGF9. Taken together, this function of FGF9 in the developing gonad seems to be common action in the two animals. When discussing the FGF9 function, the phenotype of the Fgf9 gene disrupted mouse (Colvin et al., 2001) should be considered. Although the mouse showed male-to-female sex reversal, the gonad was obviously present. Since the mesonephric Fgf9 should disappear from the gene disrupted mouse, the presence of the gonad indicated that mesonephric Fgf9 is not essential for gonad development. However, our present study in chick indicates that FGF9 alone can induce gonad formation. A proper interpretation of these two results is that another unknown FGF is expressed in the mesonephros and compensates for Fgf9 deficiency. Considering that SU5402 markedly decreased the gonadal size and the expression levels of the gonad marker genes, it can be assumed that both FGF9 and the presumptive FGF potentially stimulate gonadal cell proliferation. In fact, the expression of FGF2 was described in the chick mesonephric mesenchyme (Savage and Fallon, 1995). Moreover, since FGF2 potentially binds to FGFR3 as FGF9 does (Ornitz et al., 1996), this FGF is a potential candidate to compensate the FGF9 function during the early gonad development. We also noted the expression of FGFR2 in the developing gonadal primordium although the signal was weaker than

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that of FGFR3. Similar to the possible contribution of multiple FGFs, it is possible to assume that multiple FGFRs are involved in the process of the sexually indifferent gonadal development. With respect to the molecular mechanism underlying gonadal expansion, we should consider the possibility that the expanded gonadal cells originated from the cells once committed to mesonephric cell lineage. In the misexpression study, however, it was confirmed that the ectopic FGF9 expression spread throughout the mesonephric region. Nevertheless, gonadal expansion occurred at restricted areas neighboring the authentic gonad, suggesting that not all mesonephric cells necessarily differentiate into the gonadal cells upon FGF9 stimulation. Concerning the distinct effects of FGF9 on the gonad and mesonephric cells, it is noteworthy that FGF18 has dual function to stimulate the differentiation and proliferation of osteogenic cells and simultaneously suppress those of chondrogenic cells, and importantly these distinct functions are mediated by distinct types of FGFRs (Liu et al., 2002; Ohbayashi et al., 2002). The distinct response to FGF9 stimulation might be due to different types of FGFR expressed in the gonadal primordium and other mesonephric mesenchyme. Function of FGF signal at the stage of gonadal sex differentiation It was reported that Fgf9 gene disruption results in male-to-female sex reversal in mice, displaying marked reduction of both Sertoli (SOX9 and AMH) and Leydig (cholesterol side chain cleavage P450) cell markers accompanied by structural alterations in the testis (Colvin et al., 2001). In contrast, the present FGF9 misexpression study failed to activate SOX9 expression in the chick ovary. In agreement with this finding, a recent tissue culture study of mouse fetal gonad revealed that human recombinant FGF9 failed to activate the expression of Sertoli marker genes (Sox9, Mis, and Dhh) (Schmahl et al., 2004). Taken together, the results indicated that FGF9 is essential but not capable of activation of Sertoli cell differentiation. Another yet unknown male-specific factor seems to cooperate with FGF9 to ensure the process of testicular differentiation. Consistent with this assumption, Schmahl et al. (2004) recently described that the sexually dimorphic property of extracellular matrix is crucial for testicular differentiation. Indeed, they reported that the extracellular matrix exhibits a high affinity to FGF9 and is distributed abundantly in the male gonad more than the female. Considering that the male-specific presence of the extracellular matrix contributes to testicular formation, it is conceivable that the misexpressed FGF9 failed to reverse the gonadal sex from a female to male. In the present study, we found and characterized a novel expression domain for FGF9 in the developing mesonephros. In addition to its function related to testicular differentiation, FGF9 expressed in this domain is involved in the

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