302 (2007) 132–142 www.elsevier.com/locate/ydbio

Mechanisms of gonadal morphogenesis are not conserved between chick and mouse ⁎ Ryohei Sekido , Robin Lovell-Badge

Division of Developmental Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK Received for publication 3 June 2006; revised 16 August 2006; accepted 5 September 2006 Available online 9 September 2006

Abstract

To understand mechanisms of sex determination, it is important to know the lineage relationships of cells comprising the gonads. For example, in mice, the Y-linked gene Sry triggers differentiation of Sertoli cells from a cell population originating in the coelomic epithelium overlying the nascent gonad that also gives rise to uncharacterised interstitial cells. In contrast, little is known about origins of somatic cell types in the chick testis, where there is no Sry gene and sex determination depends on a ZZ male/ZW mechanism. To investigate this, we performed fate mapping experiments in ovo, labelling at indifferent stages the coelomic epithelium by electroporation with a lacZ reporter gene and the underlying nephrogenous (or mesonephric) with chemical dyes. After sex differentiation, LacZ-positive cells were exclusively outside testis cords and were 3βHSD-negative, indicating that the coelomic epithelium contributes only to non-steroidogenic interstitial cells. However, we detected dye-labelled cells both inside and outside the cords. The former were AMH-positive while some of the latter were 3βHSD- positive, showing that nephrogenous mesenchyme contributes to both Sertoli cells and steroidogenic cells. This is the first demonstration via lineage analysis that steroidogenic cells originate from nephrogenous mesenchyme, but the revelation that Sertoli cells have different origins between chick and mouse suggests that, during evolution, mechanisms of gonad morphogenesis may diverge alongside those of sex determination. © 2006 Elsevier Inc. All rights reserved.

Keywords: Cell migration; Testis; Lineage labelling; Sertoli cells; Leydig cells; Sex determination

Introduction sex in reptiles such as the turtle and alligator (Bull and Vogt, 1979; Ferguson and Joanen, 1982), while in fish, many different Awide variety of environmental and genetic sex-determining mechanisms have been described, ranging from strict ESD to (ESD and GSD) mechanisms are known to exist amongst GSD, with the Y-linked testis determining gene DMY in medaka vertebrate species. In most mammals, where sex is strictly being an example of the latter (Matsuda et al., 2002). determined by the genotype of the gametes to give XY males Despite the fact that different determinants trigger testis and XX , the Y-linked gene Sry is a dominant male formation amongst vertebrates, the adult testis is very similar in determinant acting within somatic cells of the indifferent gonad morphology, cellular organisation and physiology. The semi- (or genital ridge) to initiate testis development. In birds, sex is niferous tubules comprise germ cells and their supporting cells, also dependent on the gametes, but with a ZZ male/ZW female the Sertoli cells, surrounded by a layer of peritubular myoid system. This may rely on a dominant female determinant on the cells. These are a direct elaboration from the testis cords found in W, a Z-linked gene dosage mechanism or a combination of both, the embryonic testis. Leydig cells are the steroidogenic cells but despite a number of candidates, a critical sex-determining present in the interstitium between the cords along with blood gene has yet to be identified. In many lower vertebrates, either vessels and other miscellaneous connective tissue cells. Several males and females are genetically identical or chromosome cell migration events are known to be required to establish testis constitution is not always a strict determinant. For example, the morphology in the mouse. Somatic cells from the underlying temperature during egg incubation influences the phenotypic mesonephros may also contribute to the early gonad, but the supporting cell lineage was shown by DiI labelling of single ⁎ Corresponding author. Fax: +44 20 8816 2009. cells to be derived from the coelomic epithelium that covers the E-mail address: [email protected] (R. Sekido). genital ridge. Immunohistochemistry revealed that laminin-1 is

0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.09.007 R. Sekido, R. Lovell-Badge / Developmental Biology 302 (2007) 132–142 133 discontinuously expressed in a layer underneath the coelomic through the hindgut, dorsal mesentery and the mesonephros, epithelium at 11.5 days post coitum (dpc), and this may allow while in birds much of their passage to the genital ridge is via the cells to migrate into the gonad from the coelomic epithelium blood stream (Ginsburg and Eya-Giladi, 1987; Nieuwkoop and (Karl and Capel, 1998). This migration begins in a sex- Satasurya, 1979). Therefore, the question remains as to how independent manner, although it becomes more pronounced in much testis development is conserved amongst vertebrates. male gonads coincident with SRY activity. Some labelled cells In the chick, the genital system begins to form with the contribute to Sertoli cells, and some stay outside the cords, either thickening of the intermediate around the nephric because they are prevented from doing so by basement region, termed the nephrogenous mesenchyme. Based on fate membrane laid down between peritubular myoid and Sertoli mapping experiments using quail–chick chimaeras, the cells or because they are a distinct lineage fated to remain in the between somite 18 and 21 of stage interstitium. The latter is more consistent with data obtained 14 (day 2) embryos is the main source of gonadal cells (Rodemer using expression from an Sry reporter transgene as a short-term et al., 1986). The enlarged nephrogenous mesenchyme hangs lineage label (Sekido et al., 2004). Subsequently, at 12.5 dpc down into the peritoneal cavity or the coelom to form the when Sry expression has declined to almost undetectable levels, nephrogenic ridge and contributes to the mesonephros (after continuous expression of laminin-1 under the coelomic which it can be referred to as the mesonephric mesenchyme) and epithelium appears to physically block further cell migration metanephros after stage 18 (day 3). Primordial germ cells (Karl and Capel, 1998). Moreover, it has been reported, using a migrating via the vascular system from the germinal crescent similar DiI labelling and technique, that Sertoli cells originate colonise the ridge by day 3.5. The gonads develop “primitive sex from the coelomic epithelium in the turtle (Yao et al., 2004). cords” in the ridge toward the end of day 5, but there is no visual From these data, it seemed likely that there would be a conserved difference between male and female gonads until around day 7. cellular origin for Sertoli cells during vertebrate evolution. After the indifferent stages, the differentiate into the With respect to other gonadal cell types in the mouse, co- seminiferous tubules of the testis and the medullar cords of the culture of an 11.5 dpc genital ridge with a mesonephros in which ovary. Both the left and right gonads continue to develop in the a reporter gene is expressed revealed that mesonephric cells also male, while in the female, the left gonad develops into the ovary migrate into the gonad and contribute to connective tissues and the right regresses (Bellairs and Osmond, 1998). including peritubular myoid cells, vascular endothelial cells and As in the mouse, it has been proposed that gonadal somatic interstitial cells. This process is male-specific and occurs after cells in the chick arise from either the mesonephros or the SRY action (Buehr et al., 1993; Martineau et al., 1997). Similar coelomic epithelium. Early histological studies showed that co-culture experiments revealed that cells derived from the mobilised mesonephric cells or mesonephric blastema became mesonephros can also contribute to Leydig cells (Merchant- incorporated into the gonadal primitive medulla, leading to the Larios and Moreno-Mendoza, 1998; Nishino et al., 2001). suggestion that the somatic cells of the gonad originate from the However, some of these may normally be adrenal progenitors mesonephros (Carlon et al., 1983). In another experiment, in (Jeays-Ward et al., 2003) and Leydig cells still differentiate in which the elongation of the nephric duct was blocked by gonads cultured without the mesonephros, suggesting that most inserting a barrier into the intermediate mesoderm at day 2, the steroidogenic precursors are already present within the genital formation of the mesonephros was not induced beyond the ridge by 11.5 dpc (Merchant-Larios et al., 1993). It is possible barrier but the gonad still developed, although it was small. This that they enter at an earlier stage from the mesonephros, from a suggested that mesonephric cells are not required for the pool of cells that also give rise to steroidogenic cells of the formation of the gonad and implied that the gonadal somatic adrenal. Male-specific cell migration from the mesonephros has cells may originate from the coelomic epithelium (Bishop- also recently demonstrated to occur when chicken gonads are Calame, 1966; Merchant-Larios et al., 1984). These contradic- co-cultured with quail mesonephros at stage 29 (as described by tory data had not been resolved, mainly because cell-type- Hamburger and Hamilton, 1951), corresponding to just after specific lineage analysis was missing. 6 days of incubation (day 6). In this experiment, the quail In this report, we have investigated the cellular origin of mesonephric cells contribute to interstitial cells in the male Sertoli cells and steroidogenic cells in the chick using lineage chicken gonads although the identity of these cells remains analysis combined with cell-type-specific markers. These unknown (Smith et al., 2005). The origin of Leydig cells has experiments reveal that both Sertoli cells and steroidogenic therefore remained uncertain in both mouse or chick. cells are derived from the nephrogenous mesenchyme but not Although adult testis morphology is similar amongst verte- the coelomic epithelium. brate species, it may be unwise to extrapolate from what is known about gonad morphogenesis in the mouse, in which many Results of these studies have been conducted, to other species. In the mouse, cords are never apparent in the XX gonad, while in some Primitive sex cords are formed early in both male and female other mammalian species and in other vertebrates, early or gonads primitive cord formation has been described to occur in the gonads of both sexes, although these then degenerate in the To visualise the formation of the sex cords, we performed female. Moreover, it is already known in mammals that germ laminin-1 immunostaining in developing chick gonads. Lami- cells enter the gonads via migration from the base of the allantois, nin-1 was found to be continuously expressed directly 134 R. Sekido, R. Lovell-Badge / Developmental Biology 302 (2007) 132–142 underneath the coelomic epithelium at stage 17 (day 3) (Fig. 1A). observed between sexes by this stage (female data not shown). The coelomic epithelium thickens to form the nephrogenic ridge By day 5, the nephrogenic ridge protrudes into the coelomic at day 4 and laminin-1 expression starts to become discontinuous cavity and laminin-1 expression under the coelomic epithelium under the coelomic epithelium (Fig. 1B). No difference was was almost undetectable in the ridge domain. This presumably

Fig. 1. Sex cord formation during gonadal development. Right (R) and left (L) gonads of both sexes were immunostained for laminin-1 (green) with DAPI (blue) from day 2.5 to day 13. (A) Laminin-1 is continuously expressed underneath the coelomic epithelium in the male embryo at stage 17 (day 2.5). (B) The nephrogenic ridge is formed at day 4, and laminin-1 expression starts to be discontinuous. (C, D) At day 5, the ridge becomes thicker to form indifferent gonads and laminin-1 expression is lost under the coelomic epithelium specifically in the region of the genital ridges in both females (C) and males (D) (arrows). (E, F) At day 6, laminin-1 expression remains absent from the coelomic epithelium surrounding the gonads, but is still seen at their edges (arrows). Within the body of the gonads of both sexes, the primitive sex cords start to become obvious with the appearance of some laminin-1 staining in this region. By this stage, no obvious differences are seen between female and male (E, F). (G, H) At day 7, laminin-1 is expressed again in medullary cords and on the border between medulla and cortical region of female (G) and male (H) gonads. The right gonad of the female starts to regress. (I, J) At day 8, the medulla and cortical region are completely discernible by laminin-1 expression and medullary cords are well organised in both the female (I) and male (J). (K) At day 13, double-immunostaining for laminin-1 and AMH (red) reveals that peritubular myoid cells are not observed in the testis cords at day 13. (L, M) Electron microscopic analysis shows no obvious peritubular myoid cells in embryonic male gonads. The basal lamina is already recognised at the outline of the testis cords at day 7. Elongated interstitial mesenchymal cells are seen at the boundary between testis cords, but they are not characteristic of peritubular myoid cells (L). The testis cords are well organised at day 14, and the basal lamina surrounding the cords has become very evident. Germ cells, with large round nuclei, are observed adjacent to Sertoli cells. Although there are many cells outside the testis cords, none shows the close association typical of peritubular myoid cells (M). Coelomic epithelium (ce), cortical region (cr), medulla (md), Sertoli cells (S), germ cells (G), interstitial cells (I). Basal lamina (black arrows). Scale bars in A–K, 50 μm and in L and M, 2 μm. R. Sekido, R. Lovell-Badge / Developmental Biology 302 (2007) 132–142 135 allows cells to migrate from the epithelium into the gonads. No epithelium. For fate mapping, a lacZ reporter plasmid carrying obvious laminin-1-positive primitive sex cords were seen within a nonspecific promoter/enhancer was injected into the right the gonads at this stage (Figs. 1C, D). At day 6, laminin-1 coelomic cavity at stages 17–18 (day 3) together with a small expression remains discontinuous. The primitive sex cords amount of an EGFP expression plasmid. The DNA was become laminin-1-positive in both sexes, although they are subsequently electroporated from the ventral to dorsal side in disorganised (Figs. 1E, F). At day 7, the primitive sex cords start order to incorporate it into the surface layer of the coelomic to differentiate into medullary or testis (seminiferous) cords in the epithelium covering the intermediate mesoderm of the gonadal male, although the right gonad starts to regress in the female. region as well as the (Fig. 2A). The Laminin-1 expression is then found on the edge of medullary inclusion of the EGFP plasmid allowed selection of those cords and also between the medulla and cortical region, except embryos in which the electroporation had been successful for for the right gonad of the female (Figs. 1G, H). At day 8, the subsequent analysis. medulla and cortical region were completely separated by We electroporated 96 embryos in total and dissected them laminin-1 expression and medullary cords were fully developed at various stages. 30–40% of embryos died within 24 h after (Figs. 1I, J). The peritubular myoid cells, which are thin crescent- electroporation and were discarded. At day 4, X-gal staining shaped cells surrounding the testis cords in adult testis, were not was observed throughout the entire coelomic epithelium and observed at day 7 (Fig. 1L) or even at days 13–14 (Figs. 1K, M). lateral plate in both male (n=3) and female (n=3) embryos This is in contrast to mouse embryonic gonads, where they are (Figs. 3A, B and data not shown). At day 6, when the apparent as soon as the testis cords form at 12.5 dpc. indifferent gonad was clearly recognisable, X-gal staining was evident within both female (n=4) (Fig. 3C) and male (n=4) The coelomic epithelial cells migrate into the gonads gonads (Fig. 3D). A significant difference between mouse and chick gonadogenesis is the extent of asymmetric gonadal While it has been reported that coelomic epithelial cells development. The left and the right gonads show similar migrate into the developing gonads in the mouse (Karl and development in the mouse, whereas in the female chick, the Capel, 1998), the fate of the coelomic epithelial cells during right gonad is developmentally retarded when compared to the chick gonadogenesis has not been addressed. This is partly left. Therefore, we also carried out several experiments because of technical difficulties in labelling the coelomic electroporating the left coelomic epithelium (35 embryos), epithelium in ovo. To overcome these, we applied in ovo but no difference was observed in cell migration events. This electroporation to embryos before gonadal development had result implies that the asymmetry is not a result of differences begun, in a way that specifically labels the coelomic in migration.

Fig. 2. Strategies for labelling the coelomic epithelium and the nephrogenous mesenchyme in ovo. Various stages of chicken embryos corresponding to approximately day 2–3 were used for the labelling experiments. (A) For labelling the coelomic epithelium, lacZ/EGFP reporter plasmids are injected into the coelome of stage 14–18 embryos and electroporated from the ventral to the dorsal side. (B) For labelling the nephrogenous mesenchyme, a single somite between 18 and 21 of stage 15–16 embryos is microsurgically opened to expose the nephrogenous mesenchyme and DiI or MitoTracker solution is injected into the incision. CE, coelomic epithelium; EN, ; ND, nephric duct; NM, nephrogenous mesenchyme. Details of the numbers of experiments and embryos are shown in Supplementary Table 1. 136 R. Sekido, R. Lovell-Badge / Developmental Biology 302 (2007) 132–142

electroporation from stage 17–18 to stage 14–15 (41 embryos), but again, no LacZ-positive cells were found inside the testis cords (n=5) (data not shown). These results reveal that Sertoli cells are not derived from the coelomic epithelium, implying a different origin of Sertoli cells between chick and mouse. The interstitial cells are composed of steroidogenic cells and non-steroidogenic cells. Many different enzymes involved in steroid synthesis have been identified. For example, 3β- hydroxysteroid dehydrogenase (3βHSD) is expressed in both sexes, while P450 aromatase (AROM), which converts testosterone to estradiol, is specific to the female gonad (Nakabayashi et al., 1998). To determine whether the LacZ- labelled coelomic epithelial cells contribute to steroidogenic cells, we compared LacZ expression with 3βHSD expression in the male and with AROM in the female at day 8. We chose AROM as a female steroidogenic cell marker instead of 3βHSD because the antibody for AROM gives stronger signals than that for 3βHSD. We found that LacZ does not co-localise with either 3βHSD (n=5) (Fig. 4C) in the male or AROM in the female gonad (n=4) (Fig. 4D), indicating that the coelomic epithelial cells contribute only to non-steroidogenic interstitial cells. These LacZ-positive cells are present in the cortical region, but their fate is unknown.

Nephrogenous mesenchymal cells migrate into the gonads and contribute to Sertoli cells and steroidogenic cells

Since the gonad is formed by expanding the nephrogenic ridge into the coelomic cavity, the nephrogenous mesenchyme is thought to be another source of gonadal somatic cells. To Fig. 3. Cell migration of the coelomic epithelial cells. (A, B) At day 4, 24 h after investigate this, we labelled the nephrogenous mesenchyme electroporation, embryos were harvested and stained for lacZ activity (A) and with a lipophilic molecule, DiI, which is incorporated into sectioned (B). X-gal staining was detected in the coelomic epithelium (ce) and plasma membrane. A single somite between 18 and 21 at stages the lateral plate mesoderm (lp) on the right side of the embryo (R) but not on 15–16 was microsurgically incised to expose the nephrogenous the control side (L). mn, mesonephros. Embryos sometimes show X-gal mesenchyme and DiI was injected into the incision (86 embryos staining in the gut, when the injection was deep enough to reach the endoderm (arrow). (C, D) Most of X-gal staining accumulated in the gonads (g) of both on the right and 32 on the left) (Fig. 2B). To make sure that the female (C) and male (D) embryos. Section of the gonad (inset in D) indicates labelling was in the correct position, some embryos were that the staining is not only on the surface but also inside the gonad. harvested 2 h after Dil labelling (n=4) (Fig. 5A). After sectioning, DiI signal was detected in the nephric tissue The coelomic epithelial cells contribute to non-steroidogenic including the nephrogenous mesenchyme (Fig. 5B). At day 8, interstitial cells but not to Sertoli cells the signal was observed at the anterior tip of the gonad as well as the adrenal gland in the male (n=4) (Fig. 5C) and the female In the mouse, cells migrating from the coelomic epithelium (n=5) (Fig. 5D). These results appear to show that cell into the gonad can give rise to Sertoli cells. To clarify whether migration from the mesonephros is not a male-specific event in this is also the case in the chick, we immunostained the chick, unlike in the mouse and in contradiction to the data electroporated gonads with an antibody to the shown by Smith et al. (2005) for the chick. However, we are marker SOX9 and AMH, together with an antibody to LacZ in labelling cells at a much earlier stage, which implies that there order to identify migrating cells. These Sertoli cell markers and may be two phases of cell migration, an early sex-independent LacZ were already mutually exclusive at day 8 (n =5) phase followed after a pause by one that is male-specific. (Supplementary Figs. 1A, B). At day 13, LacZ-positive cells Towards identifying the types of cell that were DiI-positive inside the gonad were located within the interstitial area, but not in the testis, we performed immunostaining for AMH at day 8. at all in the testis cords (n=4)(Fig. 4A). With regard to timing, Although amounts of DiI were reduced as a result of cell there could be a critical point for cell migration beyond which division occurring over the 5 days since the injection, it was Sertoli cell progenitors might not be labelled. Indeed in the nevertheless observed on the surface of AMH-positive cells, mouse, cells within the coelomic epithelium only contribute to showing that Sertoli cells originate from the nephrogenous Sertoli cells when they have been labelled prior to 11.5 dpc mesenchyme in the chick (n=4)(Fig. 5E). DiI signal was also (Karl and Capel, 1998). Therefore, we shifted the timing of the detected on interstitial cells (Fig. 5F), but we could not R. Sekido, R. Lovell-Badge / Developmental Biology 302 (2007) 132–142 137

Fig. 4. The coelomic epithelial cells contribute to non-steroidogenic interstitial cells. Labelled coelomic epithelial cells were detected by an antibody against LacZ (green), and nuclei were stained with DAPI (blue). (A, B) Co-immunostaining with SOX9 (red) (A) and AMH (red) (B), as Sertoli cell markers, shows that LacZ- positive cells are exclusively outside the testis cords (dotted lines) at day 13. (B, C) LacZ-positive cells do not co-localise with either 3βHSD (red) in the male (B) or AROM (red) in the female (C) gonads at day 8. Broken lines indicate the border between medulla (md) and cortical region (cr). LacZ-positive cells are also seen in cortical region, but their cell identity is unknown. determine whether they are steroidogenic because DiI was been shown to originate from the coelomic epithelium in the removed by treatment with the organic solvent required for mouse. This is surprising, particularly given the central 3βHSD immunostaining (see Materials and methods). To importance of Sertoli cells to testis function in all vertebrates. circumvent this problem, we used a fixable chemical dye, Both Sertoli and steroidogenic cells also appear to differentiate MitoTracker, which passively diffuses across the plasma prior to the migration and/or differentiation of peritubular membrane and accumulates in mitochondria (36 on each myoid cells in the chick, whereas the latter are thought side). It gave speckled signals in the cytoplasm of the important for cord formation and for maintenance of testicular mesonephric mesenchyme at day 5 (Supplementary Fig. 2A). cell types in the mouse. We also find that steroidogenic cells in At day 8, although the number of MitoTracker-positive both the chick ovary and testis have their origins in the mitochondria also decreased during cell division, as with the underlying nephrogenous mesenchyme. Although suspected, DiI labelling experiment, we were able to detect co-localisation this had not been demonstrated formally for either the mouse or of MitoTracker with 3βHSD in the male (n=6)(Figs. 6A, B, C) chick, and it may be a site of origin that is conserved amongst and with AROM in the female gonad (n=5) (Figs. 6D, E, F). other vertebrates. However, the steroidogenic cells differentiate These data reveal that Sertoli and steroidogenic cells both much earlier in the chick ovary than is apparent in the mouse. originate from the nephrogenous mesenchyme in the chick. These differences in cell migration and relative timing of differentiation may parallel the evolution of sex-determining Discussion mechanisms and are likely to be informative with respect to defining the underlying molecular processes. In this study, we provide evidence that development of the testis in the chick proceeds in a manner distinct from that in the Origin of supporting and steroidogenic cells mouse (Fig. 7). Cell migration, which contributes various cell types to the body of the gonad, occurs from the coelomic In mammals, sex determination is tightly linked with Sertoli epithelium and the mesonephros in both species. However, we cell differentiation because SRY acts in Sertoli cell precursors. find that Sertoli cells are derived from the nephrogenous Sox9 is expressed at a low level in both sexes at early stages (mesonephric) mesenchyme in the chick, whereas they have and is up-regulated in the male, where it becomes a Sertoli cell 138 R. Sekido, R. Lovell-Badge / Developmental Biology 302 (2007) 132–142

Fig. 5. The nephrogenous mesenchymal cells contribute to Sertoli cells. (A) Whole-mount view of the embryo dissected 2 h after DiI injection shows a single somite was labelled (arrow). (B) Section of the embryo shown in A confirms that DiI was injected into the intermediate mesoderm including the nephrogenous mesenchyme. Nuclei were stained with DAPI (blue). e, endoderm; nd, nephric duct; nt, neural tube; s, somite. (C, D) At day 8, DiI-positive nephrogenous mesenchymal cells migrated to the anterior part of the gonad (g) and the adrenal gland (a) in both ZW (C) and ZZ (D) embryos. (E, F) Two examples of immunostaining for AMH (green) at day 8 reveal DiI (arrowheads) localised on the plasma membrane of AMH-positive cells (Ea, b). DiI was also seen in AMH-negative cells outside the testis cords (F). Nuclei were stained with DAPI (blue). Dotted lines indicate the outline of the testis cords. marker, probably as a result of SRY expression, and down- level), before going off in the female, and this must be regulated in the female (Morais da Silva et al., 1996). In independent of the expression of Sox9 (Oreal et al., 1998). contrast, Sox9 is expressed exclusively in the male gonad in the Therefore, the strict importance of Sertoli cell differentiation for chick (Oreal et al., 1998; Smith et al., 1999c). Furthermore, in sex determination in the chick is unclear. On the other hand, the mouse, SOX9 activates Amh expression, which is while sex steroid hormones are important for sexual differen- completely male-specific in the embryo, while in the chick, tiation in both birds and mammals, they also appear to act Amh is expressed in both sexes early (although at a low within, or very close to, the process of sex determination in R. Sekido, R. Lovell-Badge / Developmental Biology 302 (2007) 132–142 139

Fig. 6. The nephrogenous mesenchymal cells also contribute to steroidogenic cells. MitoTracker was injected into the nephrogenous mesenchyme of ZW and ZZ embryos at stage 15–16, with the same method as shown for DiI in Fig. 5. At day 8, the resulting testes or ovaries were sectioned and examined for MitoTracker signals. These were observed as scattered dots in the male (A) and female (D) gonads (red, arrowheads). The sections were then immunostained with 3βHSD (green) (B) or AROM (green) (E), together with DAPI (blue). Merged images show that some of MitoTracker signals co-localised with 3βHSD in the male (C) and AROM in the female (F) gonads. Higher magnifications of each co-localisation are shown in Supplementary Figs. 2B, C. birds. Thus, female to male sex reversal is observed when the of the cells (Yoshioka et al., 2005). Our data support this activity of P450 aromatase is inhibited by a drug at indifferent idea and, for the first time, identify a source of embryonic embryonic stages (Elbrecht and Smith, 1992). However, no sex steroidogenic cells in a vertebrate. reversal was observed in XX mice homozygous for a targeted Expression studies during mouse testis formation revealed mutation in P450 aromatase gene (cyp19)(Fisher et al., 1998). that coelomic epithelial cells express SF-1 (Schmahl et al., This suggests that differentiation of the steroidogenic cell 2000), some of which become SRY-positive shortly after they lineage may be more important than the supporting cell lineage migrate into the gonads. SRY-positive cells exclusively for sex determination in the chick. differentiate into Sertoli cells later, although SRY expression The molecular mechanisms underlying initiation of both is transient and no longer detected when Sertoli cells are Sertoli and Leydig cell differentiation are poorly understood, in morphologically recognisable (Albrecht and Eicher, 2001; part because of uncertainties about the origins of these cell Bullejos and Koopman, 2001; Sekido et al., 2004). Combining types. The orphan nuclear receptor SF-1 (Nr5a1) is thought to these data suggests that SRY is only expressed after SF-1- be important for the differentiation of both steroidogenic and positive cells have left the coelomic epithelium and made the supporting cells in mammals. Homozygous null mutations in decision to be Sertoli cell precursors rather than this interstitial Sf-1 impair development of both the gonad and the adrenal cell type. As mentioned above, SF-1 is expressed in the gland (Luo et al., 1994). It was originally identified as a nephrogenous mesenchyme, but not in the coelomic epithelium transcription factor regulating steroidogenic P450 genes and is in the chick (Oréal et al., 2002; Yoshioka et al., 2005). Assuming expressed in tissues producing steroid hormones such as the that the molecular players are conserved, this is consistent with pituitary, hypothalamus, gonad and adrenal cortex (Lala et al., our observation that Sertoli cells are of mesenchymal origin in 1992; Morohashi et al., 1992). SF-1 expression studies in the rat the chick. suggested that both the gonadal and adrenal steroidogenic cells The mesonephric kidney is formed around day 3 and originate from the same SF-1-positive cell population in the functions up to day 15 in chick embryos while if it functions at early urogenital ridge (Hatano et al., 1996). In the chick, SF-1 is all in the mouse it would be for a very brief period only as the also expressed by steroidogenic cells of the gonad and adrenal mesonephric tubules degenerate very soon after their formation. gland (Smith et al., 1999b), raising the possibility that a similar This might raise the possibility that functionality is somehow SF-1-positive cell population could be the origin of both. connected to the different origin of Sertoli cells. On the other Yoshioka et al. have recently shown that SF-1 expression starts hand, the mesonephros functions as an embryonic kidney in at stage 19 in a specific domain of the nephrogenous some mammals such as the sheep and cat (Davies and Davies, mesenchyme, and by stage 21, the expression has split into 1950; Tiedeman, 1976) and indeed the elephant is reported to dorsal and ventral domains. They assumed that the former have nephrostomes in the mesonephric kidney similar to those domain corresponds to the adrenal primordia while the latter present in birds and reptiles (Gaeth et al., 1999). Perhaps, it is gives cells that colonise the gonad, but they did not trace the fate worth exploring the origins of Sertoli cells in these other 140 R. Sekido, R. Lovell-Badge / Developmental Biology 302 (2007) 132–142

Fig. 7. Comparison of testis formation between the mouse and the chick. (A) In the mouse, cells from both the coelomic epithelium (blue) and the mesonephric mesenchyme (red) migrate into the gonads after 10.5 dpc. At 12.5 dpc, when the testis cords are formed, the coelomic epithelium gives rise to Sertoli cells and unknown interstitial cells as described by Karl and Capel (1998), while the mesonephric mesenchyme contributes to peritubular myoid cells, vascular endothelial cells and also unknown interstitial cells as previously described (Buehr et al., 1993; Martineau et al., 1997). (B) In the chick, cells from both the coelomic epithelium and the nephrogenous mesenchyme, from which the mesonephric mesenchyme arises, also migrate into the gonads, but Sertoli cells have different origin. Thus, the nephrogenous mesenchyme gives rise to Sertoli cells, steroidogenic cells and unidentified interstitial cells, while the coelomic epithelium apparently contributes to any non-steroidogenic interstitial cells. mammals. However, Sertoli cells were found to be derived from the interstitial cells in the male gonads. In their experiment, the coelomic epithelium in the turtle (Yao et al., 2004). quail mesonephroi were co-cultured with stage 29 chicken Consequently, there is no obvious correlation between the gonads, where steroidogenic cells are already present at least in functional development of the mesonephros and Sertoli cell the female, suggesting that steroidogenic cells enter the gonad origins amongst species. It seems more likely that the earlier than this stage (Smith et al., 2005). Alternatively, we may mechanisms of gonad morphogenesis diverge in parallel with have been labelling cells too early in our experiments since those of sex determination. mouse mesonephric cells only migrate into the genital ridge after Sertoli cells begin to differentiate. Different lineage tracing Other gonadal cell types methods are required to address this.

Mesonephric cells contribute to peritubular myoid and A new assay for candidate sex-determining genes in the vascular endothelial cells in the mouse, while in the chick chick these cells are not evident in embryonic testis at least by days 13–14. At this time, the mesonephros and gonad are well One of the reasons that sex determination is less understood developed, with other cell types expressing functional markers, in birds than in mammals is that a crucial sex-determining gene such as AMH and 3βHSD. Peritubular myoid and vascular analogous to mammalian Sry/SRY has not been identified either endothelial cells are both evident in the mature testis in the on the Z or W chromosome. Several genes have been identified chick, but our data do not reveal their source. We found as candidates for the female-determining gene on the Z unidentified non-steroidogenic interstitial cells derived from chromosome, such as ASW/Wpkci (W chromosome-linked both the coelomic epithelium and nephrogenous mesenchyme, PKC inhibitor/interacting protein) (Hori et al., 2000; O'Neill which could contribute to the connective tissues at later stages. et al., 2000) and FET-1 (Female expressed transcript 1) (Reed Smith et al. also reported that mesonephric cells contribute to and Sinclair, 2003). Another possible mechanism is dosage of R. Sekido, R. Lovell-Badge / Developmental Biology 302 (2007) 132–142 141 the Z chromosome. Dmrt1, a member of the DM domain Acknowledgments genes, is thought to be a candidate gene on the Z chromosome and is expressed higher in male gonads than in female We sincerely thank Nobue Itasaski for teaching us to culture (Raymond et al., 1999; Smith et al., 1999a). However, there and manipulate chick embryos and also for critical comments has so far been no functional test carried out for these candidate and insightful ideas throughout the experiments. We are also sex-determining genes. This is partly because of the lack of in indebted to Liz Hirst for generating and helping to interpret the vivo or in ovo assay system during chick gonadal develop- electron microscopy data. We are grateful to Bill Buaas, Silvana ment. Gene manipulations such as transgenesis or gene Guioli and James Turner for careful reading of the manuscript. targeting are still difficult techniques in the chick. Transient For providing us materials, we thank Harold Erickson (laminin- gene expression in ovo such as by electroporation or virus 1 antibody), Nobuhiro Harada (AROM antibody), Ian Mason infection is also not so easy for the gonad once it has formed (3β-HSD antibody) and Danièle Carré-Eusèbe (AMH anti- because it develops too deep inside the embryo for efficient body). This work was supported by the MRC and Louis Jeantet gene delivery. Even if it was possible to access to the gonad Foundation. R.S. was a recipient of EMBO and HFSP long term from outside the embryo, it may be necessary to target a fellowships. specific cell type. Here, we have not only developed methods that may help to introduce genes, but, critically, we have also Appendix A. Supplementary data revealed the origins of gonadal somatic cells prior to sex determination. This should facilitate future studies on the role Supplementary data associated with this article can be found, of specific genes in sex determination. in the online version, at doi:10.1016/j.ydbio.2006.09.007.

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