Spatially dynamic expression of Sry in mouse genital ridges

Authors

  • Monica Bullejos,

    1. Centre for Molecular and Cellular Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia
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  • Peter Koopman

    Corresponding author
    1. Centre for Molecular and Cellular Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia
    • Centre for Molecular and Cellular Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia
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Abstract

We have studied the spatial dynamics of Sry transcription in the genital ridges of mouse embryos. We find that Sry is expressed in a dynamic wave that emanates from the central and/or anterior regions, extends subsequently to both poles, and ends in the caudal pole. This dynamism may explain the relative positioning of ovarian and testicular tissue seen in ovotestes in mice. Since direct regulatory targets of SRY ought to be expressed in a corresponding or complimentary wave, our observations pave the way for identification of target genes. Sry is expressed in internal cells but not in coelomic surface epithelial cells, indicating that its effect on proliferation of surface cells is achieved non-cell-autonomously. The cellular dynamism of Sry expression revealed in this study thus provides important insights into both the cellular and molecular mode of action of SRY, and how perturbations in Sry expression can lead to anomalies of sexual development. © 2001 Wiley-Liss, Inc.

INTRODUCTION

Sry is the only Y chromosomal gene necessary for male sex determination in eutherian mammals (Koopman et al., 1991). Expression of Sry initiates a network of gene activity that transforms the undifferentiated gonad, or genital ridge, into a testis. Once the testis differentiates, anti-Müllerian hormone (AMH) and testosterone secreted by Sertoli and Leydig cells, respectively, are responsible for directing the development of male secondary sex characteristics. In the absence of Sry, or when Sry is defective, the gonad differentiates as an ovary, and the sexual phenotype is female.

Since the discovery of Sry as the testis-determining factor (Sinclair et al., 1990; Gubbay et al., 1990), much has been learned about this gene and corresponding protein (reviewed by Koopman, 1999). Sry encodes a transcription factor that binds to, and bends, specific DNA sequences (Harley et al., 1992; Giese et al., 1994; Pontiggia et al., 1995). Some evidence has emerged that SRY protein may directly activate (Dubin and Ostrer, 1994) or repress (McElreavey et al., 1993) transcription of its target genes, perhaps in concert with other proteins (Poulat et al., 1997; Zhang et al., 1999; Bowles et al., 1999). Sry expression appears to have several cellular consequences in the genital ridges, including induction of cell proliferation (Schmahl et al., 2000), stimulation of ingression of mesonephric cells, and subsequent cord formation (Buehr et al., 1993; Merchant-Larios et al., 1993; Merchant-Larios and Moreno-Mendoza, 1998; Martineau et al., 1997; Capel et al., 1999; Tilmann and Capel, 1999), and induction of Sertoli cell differentiation (Burgoyne et al., 1988; Palmer and Burgoyne, 1991; Patek et al., 1991). This last role is known to involve a cell-autonomous mechanism in keeping with the proposed role of SRY as a transcription factor (Burgoyne et al., 1988). In addition, the observation that Sertoli cells are not exclusively XY in XX↔XY chimeras suggests an additional, non-cell-autonomous role in recruiting cells to the Sertoli cell fate (Palmer and Burgoyne, 1991).

Despite these insights, major questions relating to the molecular mode of action of SRY, and the identity of its targets, remain unanswered. In order to illuminate these issues, it is important to fully characterize Sry expression. In mice, Sry expression is limited to a window of approximately two days prior to overt differentiation of the testis. Due to low levels of expression and the small size of the genital ridges, information about Sry expression during mouse sex determination has come mainly from sensitive RNA detection methods such as RT-PCR and RNase protection (Koopman et al., 1990; Hacker et al., 1995; Jeske et al., 1995; Jeske et al., 1996). Using RNase protection, Hacker et al. (1995) delimited the window of Sry expression from the 11/12 tail somite (ts) stage (about 10.5 days post coitum, dpc) to just after 27 ts (about 12.5 dpc), peaking at 18 ts (about 11.5 dpc). These data are consistent with the RT-PCR data of Koopman et al. (1990) and Jeske et al. (1996, 1995), although the high sensitivity of RT-PCR extended this window to 10.25–13.25 dpc (Jeske et al., 1996). While these techniques have proven sufficiently powerful to elucidate the timing and overall level of Sry expression, they are not suitable for resolving the spatial dynamics of Sry expression within the genital ridge.

In a study of Dax1 expression during mouse sexual development, Swain et al. (1998) provided evidence from whole mount in situ hybridization that Sry is expressed most strongly at the posterior poles of the genital ridges at 11.5dpc, and suggested that an anteroposterior wave of Sry expression might occur. We have now used whole mount in situ hybridization to examine in detail the spatial distribution of Sry transcripts at a range of developmental timepoints. The results obtained reveal a number of important and surprising aspects of Sry expression in the genital ridges.

RESULTS AND DISCUSSION

We observed that Sry is not expressed uniformly along the length of the genital ridges (Fig. 1). Typically, no expression was detected before the 14 ts stage (Fig. 1A), when expression was found to initiate in the central and anterior portions of the genital ridges (Fig. 1B). By 15 ts, expression was seen in the anterior 2/3 of the urogenital ridge, and in some samples was stronger in the centre than anteriorly (Figs. 1C, 2A). Between 16 and 18 ts, the domain of Sry expression had expanded to include both poles of the genital ridges (Fig. 1D). Transcript levels increased to reach a maximum around 18 ts (Fig. 1E). Sry expression levels subsequently declined, first in the central region (19 ts) and shortly after also at the anterior pole (20 ts), while remaining high at the posterior pole (Fig. 1F, G). The central portion of the gonad was negative for Sry expression by 22 ts (Fig. 1H). This extinction subsequently encompassed the anterior poles of the genital ridges, leaving expression only in the most posterior tip of the male gonad by 24 ts (Fig. 1I). Expression ceased entirely by 26 ts (not shown).

Figure 1.

Spatially dynamic expression pattern of Sry in mouse genital ridges. Wholemount in situ hybridization of male urogenital ridges at 12 (A), 14 (B), 15 (C), 16 (D), 18 (E), 20 (F), 21 (G), 22 (H), and 24 (I) tail somites (ts). The anterior poles of the genital ridges are shown at the top of each panel.

Figure 2.

Cryosections of the wholemount in situ hybridizations shown in Figure 1. A: 15 ts (Fig. 1C); B: 20 ts (Fig. 1F); C: 21 ts (Fig. 1G), showing that Sry expression is confined to inner cells of the genital ridge at all stages. Anterior poles are shown left, coelomic surface upward. Scale bar = 50 μm

No signal was detected on XX gonads at any stage (data not shown), nor on early (Fig. 1A) or later-stage (not shown) XY gonads. These observations indicate that the probe is specific for Sry and does not detect transcripts of related genes such as Sox9 expressed in the genital ridges, and that no signal was generated through endogenous enzymatic activity in the somatic or germ cell components of the genital ridges.

While the dynamic pattern of expression was reproducible, the exact timing of events was found to vary between embryos and between experiments. The profile of Sry expression appeared to vary in timing by ±2 ts within any experiment relative to the profile described above, apparently due to differences between embryos. In addition, experimental variables between experiments, such as probe labeling efficiency and incubation conditions, affected absolute signal levels and apparent duration of expression. For example, although Figure 1 shows a window of Sry expression from 14 to 24 ts, in other experiments we were able to observe expression from 11 to 27 ts (similar to the window found by RNase protection and RT-PCR) by extending the colour development incubation step in the in situ hybridization (data not shown). However, the combination of stronger specific signal and higher background under these extended incubation conditions obscured the dynamic spatial, temporal, and quantitative expression pattern of Sry.

In cryosections of male urogenital ridges after wholemount in situ hybridization, Sry expression was observed in the internal cell population, with no evidence of expression in coelomic surface epithelial cells at any stage studied (from 15 to 21 ts; Fig. 2). It is known that at least some Sertoli cells develop from a population of cells that ingress from the coelomic epithelium. This coelomic cell migration is not Sry-dependent, as it occurs in both XX and XY genital ridges. In XY gonads, individual cells from the coelomic epithelium can give rise to multiple lineages, including Sertoli and interstitial cells, indicating that cell fate restriction occurs in the migrating cell population (Karl and Capel, 1998). Up-regulation of Sry expression may, therefore, coincide with the restriction of cell fate in these cells. Alternatively, Sry could fulfill its cell-autonomous function in a subpopulation of internal mesenchymal cells, which then recruit some of the migrating coelomic epithelial cells to differentiate as Sertoli cells by a non cell-autonomous mechanism. Indeed, both scenarios are possible, and further definition of the cell population (migrating coelomic epithelial cells or “gonadal” cells) in which Sry expression is initiated requires more detailed studies using cell-specific markers. The internal location of Sry-expressing cells within the genital ridges suggests that the Sry-dependent proliferation of coelomic epithelium cells (Schmahl et al., 2000) also occurs via a non-cell-autonomous mechanism.

We observed no substantial or consistent difference in timing or levels of expression between the left and right genital ridges. This is significant in view of asymmetry in the distribution of testicular tissue in mouse hermaphrodites (Eicher and Washburn 1983; Washburn and Eicher, 1983), and documented cases of asymmetry between the left and right gonads in humans and chickens (Mittwoch, 1976, 1998, 2000; Mittwoch and Mahadevaiah, 1980; van Niekerk and Retief, 1981; Johnson et al., 1984; Krob et al., 1994). We conclude that differences in expression between the left and right gonads may be too subtle to be observed by this method, or do not occur in mice, or involve testis-determining genes other than Sry.

Our data indicate that Sry is expressed in a dynamic wave during the development of the genital ridges in mice. In contrast to the suggestion of Swain and colleagues (1998), our data suggest that this wave emanates either from the centre of the genital ridges, or simultaneously from the central and anterior regions. Clearly, not all parts of the genital ridge are exposed to Sry transcripts at the same time, nor for the same length of time. Further, the period for which any individual cell is exposed to significant levels of Sry transcripts, and hence SRY protein, is likely to be shorter than previously believed. These features of Sry expression should be taken into account in any model that tries to explain the cellular and molecular mechanism of action of Sry.

We propose that the dynamic expression pattern of Sry combines with a dynamic window of competence among cell populations in the developing gonad to respond to SRY signaling. Support for this model can be found in the position of testicular and ovarian tissue seen in ovotestes in mice. Ovotestes can occur when certain Mus domesticus-derived Y chromosomes (YDom) are transferred onto the C57BL/6J (B6) inbred mouse strain background (Eicher et al., 1982, 1996; Washburn and Eicher, 1989), due to reduction in the expression levels of SryDom and inability to initiate normal cell migration when present on the B6 background (Nagamine et al., 1999; Albrecht et al., 2000). In these cases the ovotestes commonly comprise testicular material in the central region flanked by ovarian tissue at the poles (Eicher et al., 1980, 1995). A mismatch between the dynamics of SryDom expression and dynamics of B6 cellular competence may result in the threshold of SRY activity being reached only in the central region of the gonad during the window of competence.

In some previous studies of gene expression in mouse genital ridges, changes in total expression levels of expression of Sry and other genes have been measured and graphed as a function of time, and this information used to propose regulatory relationships between different genes (e.g., Hacker et al., 1995; Viger et al., 1998). Such changes could be caused by fluctuations in the number of cells that express a given gene, or by changes in the level of expression per cell. Our data indicate that both parameters change with time, and an awareness of these dynamics may assist in identifying possible regulatory targets of SRY. Genes responding directly to SRY might be expected to show a corresponding, or indeed complimentary, wave of expression in the genital ridges.

In conclusion, the cellular dynamism of Sry expression revealed in this study provides insights into both the cellular and molecular mode of action of Sry, and how perturbations in Sry expression may lead to anomalies of sexual development.

EXPERIMENTAL PROCEDURES

Staging, Dissection, and Genotyping of Fetal Mouse Gonads

Timed matings were produced by housing outbred Swiss female mice with males carrying an X-linked GFP marker (Hadjantonakis et al., 1998). In these matings, the fetuses can be sexed under a fluorescent lamp (Univers-al MAA-02, BSL, Budapest). Female fetuses express the GFP marker while males do not.

Fetuses were dissected between 10.5 and 12.5 days post coitum (dpc), where 0.5 dpc is defined as noon on the day of plug. For more accurate staging, the tail somite (ts) stage of the embryo was determined by counting the number of somites posterior to the hind limb (Hacker et al., 1995). Using this method, 10.5 dpc corresponds to approximately 8 ts, 11.5 dpc to 18 ts, and 12.5 dpc to 30 ts.

Wholemount In Situ Hybridization

Wholemount in situ hybridization was carried out as described (Hargrave and Koopman, 2000), using the Sry fragment p422.04 as a probe (Gubbay et al., 1990). Stained wholemounts were cryosectioned as described (Xu and Wilkinson, 1998).

Acknowledgements

We thank Blanche Capel, Jo Bowles, Kelly Loffler, and Andrew Sinclair for critical comments on the manuscript. We gratefully acknowledge the assistance of Jo Bowles and Paul Addison for GFP mouse breeding and histology. M.B. is supported by the Secretaría de Estado de Universidades, Investigación y Desarrollo, Spain. P.K. is an Australian Research Council Senior Research Fellow.