Dax1 Expression Is Dependent on Steroidogenic Factor 1 in the Developing Gonad
Christine Hoyle1,
Veronica Narvaez1,
Graham Alldus,
Robin Lovell-Badge and
Amanda Swain
Section of Gene Function and Regulation (C.H., G.A., A.S.), Chester Beatty Laboratories, Institute of Cancer Research, London SW3 6JB; and Division of Developmental Genetics (V.N., R.L.-B.), Medical Research Council National Institute for Medical Research, London NW7 1AA, United Kingdom; and Facultad de Ciencias (V.N.), Universidad Autonoma del Estado de Morelos, Cuernavaca, Morelos 62210, Mexico
Address all correspondence and requests for reprints to: Dr. Amanda Swain, Section of Gene Function and Regulation, Institute of Cancer Research, 237 Fulham Road, London, United Kingdom SW3 6JB. E-mail: aswain{at}icr.ac.uk.
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ABSTRACT
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The nuclear hormone receptor DAX1 has been implicated in mammalian gonad development and sex determination. The expression of the gene in the gonad follows a dynamic pattern in time and place in the embryo and the adult. We have undertaken the first in vivo study of the regulation of Dax1 expression. Using a transgenic mouse approach we have identified a novel 500-bp region 4 kb upstream of the mouse Dax1 start codon that is essential for LacZ reporter gene expression in the embryonic gonad. Within this region, a highly conserved steroidogenic factor 1 (SF1) consensus-binding site is necessary to direct LacZ expression to the embryonic gonad implicating SF1 in the regulation of Dax1 in the developing gonad. Consistent with this, Dax1 is expressed at much reduced levels in gonads of embryos that are deficient in SF1. In addition, our results show that SF1 consensus-binding sites close to the start of Dax1 transcription are important in regulating levels of expression in the developing gonad. These studies have identified the critical in vivo regulatory region for expression of Dax1 in the early gonad and provide novel information on how a specific enhancer element acts in different cell types at different stages of development.
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INTRODUCTION
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THE ORPHAN NUCLEAR hormone receptors steroidogenic factor 1 (SF1) and DAX1 have been shown to be important in the development of the reproductive axis in mouse and man. A heterozygous mutation in SF1 in humans causes adrenal failure and XY sex reversal (1). Mice lacking a functional SF1 gene show complete failure of adrenal and gonad development and abnormal pituitary and hypothalamus development (2, 3, 4). Loss of function mutations in DAX1, which is X-linked, cause hypogonadotropic hypogonadism and adrenal hypoplasia congenita characterized by adrenal failure in XY individuals (5, 6). An inactivating mutation in Dax1 in mice causes abnormal adrenal development in both sexes and a spermatogenic defect in the male (7). DAX1 has also been implicated in mammalian sex determination, in which it acts antagonistically to SRY, the testis-determining gene. Humans with a duplication of the region of the X chromosome that contains DAX1 show XY sex reversal, and XY transgenic mice that overexpress Dax1 in the developing gonad show a delay in testis development or sex reversal with certain genetic backgrounds (8, 9). The related phenotypes associated with DAX1 and SF1 have led to the proposal that these factors might act in the same molecular pathway to determine the proper development of reproductive organs.
Consistent with their shared function, SF1 and DAX1 have similar expression patterns during embryonic and adult life, although these patterns are not identical. In the mouse embryo, SF1 is expressed in the gonads and adrenals as soon as these organs begin to develop, and it precedes Dax1 expression (10, 11). Dax1 and SF1 expression in the adrenal are then associated with the cortical cells, both during embryogenesis and in the adult (11, 12, 13, 14, 15). In the developing gonad, SF1 and Dax1 are coexpressed at high levels in both sexes at the same time that SRY acts in the male to trigger testis development (11, 15). However, as gonad development proceeds, SF1 and Dax1 expression patterns diverge. SF1 expression is higher in the testis than in the ovary, whereas Dax1 expression is down-regulated in the male and stays on in the ovary (9, 11, 14, 15, 16). In the adult, both genes have been reported to be expressed in the Leydig and Sertoli cells of the testis and in the theca and follicle cells of the ovary (11, 12, 13, 14, 15, 15, 17, 18).
DAX1 is a novel member of the nuclear hormone receptor superfamily in that it does not possess a DNA-binding domain containing zinc finger motifs found in classic family members such as SF1. Instead, DAX1 contains a novel domain in the N-terminal region made up of three and one half repeats of a motif of unknown function. DAX1 has been proposed to act as an inhibitor of transcription (for review see Ref. 19). In vitro studies have shown that DAX1 can silence transcription directly or inhibit activation of transcription by SF1 (20, 21). The inhibitory domain of DAX1 has been mapped to the carboxy-terminal region, which is deleted or mutated in patients with adrenal hypoplasia congenital. This suggests that one of the roles of DAX1 during adrenal and gonad development is to modulate the activity of SF1.
To date, all studies on the regulation of Dax1 expression have been done in vitro. In these studies, SF1 and WT1 have been implicated in the regulation of Dax1 transcription. WT1 has been found to stimulate expression of reporter constructs containing up to 200 bp of Dax1 DNA in transient expression assays, and WT1 consensus binding sites were identified close to the start of transcription (22). SF1 was also found to stimulate reporter constructs containing up to 540 bp of Dax1 DNA in transient expression assays in cells lacking endogenous SF1 (18, 23). Within this region of Dax1 an SF1 consensus binding site was identified around 120 bp upstream of the start of transcription, but mutation of this site did not abolish SF1 stimulation of expression of these constructs (15, 18, 23, 24). Three other SF1 consensus binding sites were identified in this region, and it was shown that three of the four sites contributed in equal measure to the stimulation by SF1 of these reporter constructs (18, 23). Consistent with the role of SF1 in the regulation of Dax1 expression, the levels of Dax1 in the gonads and ventral medial hypothalamus of SF1-deficient mice were found to be reduced when compared with wild-type levels (15, 18).
Although the in vitro promoter studies identified factors important in Dax1 transcription, they may not reflect the in vivo situation and cannot address the regulation of Dax1 during embryogenesis. In this study we identified the in vivo promoter element for Dax1 expression in the developing gonad using a mouse transgenic approach. This element is located 4 kb upstream of the start codon of Dax1 and contains an SF1 consensus-binding site, which is important for the expression in the developing, but not the adult, gonad.
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RESULTS
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Definition of a Gonad Promoter Element in Dax1 Regulatory Sequences
Previously we reported that transgenic mice containing a construct with 11 kb of DNA upstream of the Dax1 start codon with the LacZ gene introduced in frame into the Dax1 open reading frame (called Dax11kbLacZ) showed consistent ß-galactosidase activity in the developing and adult gonads (9). Around 70% (12 of 17) of transgenic embryos showed LacZ expression in the gonad in a pattern that was consistent with the endogenous Dax1 gene expression. During embryogenesis, ß-galactosidase activity first appeared around 11 d of development in both male and female genital ridges. At later stages of development, LacZ expression was absent from the testis, except in a region abutting the mesonephros that is thought to give rise to the rete testis, but it persisted within the ovary. There was no LacZ expression elsewhere within the embryo except for a domain within the caudal neural tube, which expressed low levels in many, but not all, transgenic embryos with gonadal expression. This domain was found not to be a site of Dax1 expression as determined by ribonuclease (RNase) protection (data not shown). No consistent LacZ expression was found in any other known sites of Dax1 expression, such as the developing adrenals, hypothalamus, and pituitary, when this construct was used.
To further characterize this gonad promoter element, a series of constructs were made that carried deletions within the 11-kb Dax1 fragment (Fig. 1
). Transgenic embryos carrying these deleted constructs were assayed at 11.512.5 d of embryonic development for ß-galactosidase activity. Constructs with up to 5.4 kb of upstream Dax1 DNA showed LacZ expression in the developing gonad of transgenic embryos, but those with 4 kb and 3 kb of Dax1 DNA did not. These studies showed that a 1.4-kb region located between 5.4 kb and 4 kb away from the Dax1 start codon was essential for embryonic gonadal expression of the LacZ gene (Fig. 1
). In general, the levels of LacZ expression in these transgenics were similar to that of the Dax11kbLacZ construct. However, in the case of the construct with 5.7 kb of Dax1 DNA, the levels of LacZ expression in the embryonic gonad were consistently lower than those obtained with the other constructs. This could be due to the uncovering of repressive elements in the DNA that are usually inactivated by neighboring DNA sequences, which are missing in this construct.

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Figure 1. Mapping of the Regulatory Elements of Dax1 Expression in Transgenic Mice
The structure of the mouse Dax1 locus is shown at the top of the figure. The structures of the constructs used to create transgenic mice are shown below, and the amount of Dax1 genomic DNA upstream of the start of translation is indicated. The column of numbers shows the number of transgenic embryos derived from different integration events that showed ß-galactosidase activity compared with the total number of transgenic embryos that were analyzed. The right-hand column indicates the relative levels of ß-galactosidase activity seen in the embryos that were positive. *, The LacZ activity in this embryo was not specific to the gonad.
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To further delineate the gonad-specific element and to determine whether this region was sufficient to direct expression to the developing gonad, a 500-bp fragment derived from the 1.4-kb region was introduced upstream of the LacZ gene driven by a minimal promoter [Fig. 1
(this 500-bp fragment will be referred to as region A)]. This promoter, which is derived from the mouse ß-globin gene, lacks tissue-specific elements and has been used to define enhancer regions in other genes that are expressed during embryogenesis (for example see Ref. 25). Four of nine transgenic embryos carrying this construct (called 1XminLacZ) showed ß-galactosidase activity in the developing gonad. However, the level of LacZ expression and the number of expressing cells in the gonad of these transgenic embryos were consistently low (Fig. 2A
). In contrast, when six copies of region A were placed upstream of the minimal promoter (in a construct called 6XminLacZ), much higher levels of expression were found in the developing gonads of four of seven transgenic embryos (Fig. 2B
). Unlike the results obtained with the Dax11kbLacZ construct, consistent sites of ectopic LacZ expression, including the mesonephric ducts and kidney, were found in transgenic embryos carrying either the 1XminLacZ or 6XminLacZ constructs (Fig. 2B
). This type of ectopic expression cannot be due to transgene integration sites, but is probably due either to the loss of repressor elements or to the accidental combination of enhancer and promoter sequences present in hybrid constructs of this sort.

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Figure 2. The 500-bp Regulatory Element Is Sufficient to Drive ß-Galactosidase Activity to the Embryonic Gonad
A, Dissected gonad and mesonephros from a transgenic animal with the 1XminLacZ construct stained for ß-galactosidase activity. The arrows indicate the cells of the gonad that are positive for ß-galactosidase activity. B, Gonad, mesonephros, and kidney from a 12.5-dpc transgenic male with the 6XminLacZ construct (a and b are derived from the same embryo) stained for ß-galactosidase activity. C, Dissected gonad and mesonephros from 14.5-dpc male embryos (a and b) and female embryos (c, d, and e) with the 6XminLacZ (a, c, and d) and the Dax11kbLacZ (b and e) constructs. All embryos in C are derived from independent integration events. Different tissues are denoted by letters in each figure: g, gonad; t, testis; o, ovary; m, mesonephros; k, kidney.
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ß-Galactosidase activity was also analyzed at later stages of gonad development in embryos with the 6XminLacZ construct. At 14.5 d of development, the levels of LacZ expression were reduced in both the ovary and testis (nine embryos were analyzed: six males and three females). In the ovary, in contrast to the expression seen with the Dax11kbLacZ construct (Fig. 2C
, panel e), only weak ß-galactosidase activity was found, with the highest levels present in the region abutting the mesonephros (Fig. 2C
, panels c and d). In the testis, four of six embryos carrying the 6XminLacZ construct showed a few faint LacZ-positive cells within the gonad (Fig. 2C
, panel a). These were not seen with the Dax11kbLacZ construct (Fig. 2C
, panel b). In addition, the expression in the region between the testis and mesonephros was missing or very low in the 6XminLacZ embryos (Fig. 2C
, panel a). This suggests that region A is necessary and sufficient for the initiation of Dax1 expression in the embryonic gonad but not for the maintenance at later stages of development.
Regulation of Dax1 Expression in the Developing Gonad by SF1
The sequence of region A was determined and compared with that of the human DAX1 gene. A 200-bp region of high homology was identified (86% identical) (Fig. 3
). This region was located around 4 kb upstream of the DAX1 start codon in the human gene. Analysis of the sequence of the 200-bp region of homology revealed a perfect SF1 consensus-binding site that was identical in both species [Fig. 3
(this site will be referred to as SF14kb)]. To determine whether this site was necessary for LacZ expression in the developing gonad, a 10-bp deletion that included this site was engineered into the Dax11kbLacZ construct (called Dax11kbdelLacZ) (Fig. 4
). Nine of 12 transgenic embryos with this construct showed no LacZ expression in the developing gonad, and in the remaining three cases the level of expression was weaker than that of the Dax11kbLacZ construct. In some cases, the ectopic expression in the caudal neural tube characteristic of the Dax11kbLacZ construct was observed but without the gonadal expression (Fig. 4
, panels a and b).

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Figure 3. Alignment of the Human and Mouse Sequences from the Delineated Regulatory Region in DAX1 DNA
The perfect SF1 consensus-binding site, which is deleted in the Dax11kbdelLacZ construct and mutated in the Dax11kbmutLacZ construct, is highlighted in bold and boxed. Putative GATA binding site is indicated by underlines. The nucleotide numbering is relative to the translation initiation start for both human and mouse.
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Figure 4. A Deletion and Mutation of a Conserved SF1 Consensus-Binding Site in the Dax1 Regulatory Element Abolishes ß-Galactosidase Activity in the Developing Gonad
The structures and ß-galactosidase activity in the transgenic 11- to 12-dpc embryos and gonads of the Dax11kbdelLacZ (a and b) and Dax11kbmutLacZ (c and d) constructs are shown.
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To directly implicate SF1 in the regulation of Dax1, we engineered a 2-bp change known to abolish SF1 binding, into the SF14kb consensus-binding site, within the Dax11kbLacZ construct (to give Dax11kbmutLacZ). These same changes, two Gs were replaced by two Ts, have been used to implicate SF1 in the regulation of the Amh gene in the developing testis (26, 27). No LacZ expression was observed in the embryonic gonad of the four transgenic animals that were analyzed, although two of the embryos showed the characteristic ectopic expression in the spinal cord (Fig. 4
, c and d). These data suggest that binding of SF1 to the defined consensus site is necessary for embryonic gonadal expression of Dax1.
SF1 consensus binding sites located close to the start of transcription have been shown to be important for Dax1 expression in vitro (15, 18, 23, 24). The results presented above show that these sites are not sufficient to direct LacZ expression to the developing gonad in transgenic embryos. However, they might contribute to the regulation of Dax1 expression by region A. To investigate whether these proximal SF1 binding sites have any role in the regulation of expression of Dax1 in the developing gonad, we engineered mutations in all four of these sites within the Dax11kbLacZ construct (18). This construct, Dax11kb12ab3MlacZ, was used to create transgenic animals, and embryos were analyzed at 11.512.5 d post coitus [dpc (six females and three males)] and 14.5 dpc (six females and two males). In all cases, LacZ expression was identical with that of the Dax11kbLacZ construct (two females and two males at 11.512.5 dpc and three females and two males at 14.5 dpc showed LacZ expression; data not shown). However, at 11.512.5 dpc, but not at later stages, the level of expression and number of expressing cells was found to be consistently lower and comparable to that of the 1XminLacZ construct (Fig. 2A
). These results show that the proximal SF1 binding sites act in the gonad as quantitative regulators in the initiation of Dax1 expression, but have no role in its maintenance.
To further implicate SF1 in the regulation of the Dax1 gene, we measured levels of Dax1 RNA in the gonads of SF1 homozygous and heterozygous mutant embryos (2). We used two techniques, RNase protection assays, which are quantitative but lack cellular resolution, and whole-mount in situ hybridization, which is semiquantitative but allows a signal to be seen in individual cells. Although genital ridges begin to develop in the absence of SF1, they degenerate after 11.5 dpc. We therefore looked at or before 11.5 dpc. Using both techniques we see very low levels of Dax1 in the homozygous mutant embryonic gonads (Fig. 5
). Similar results have been found by others, but as the gonads are degenerating at this stage it is hard to establish whether the reduction in transcript levels is due to absence of tissue or a genuine reduction in Dax1 expression (15, 18). Interestingly, the levels of Dax1 in the gonads of embryos heterozygous for the SF1 mutation were reduced by a third when compared with wild-type gonads (Fig. 5
). The whole-mount in situ hybridization showed that the reduction in Dax1 levels in the heterozygous gonads was due to a decrease in both the number of Dax1-expressing cells and in the level of Dax1 expression per cell (Fig. 5
). Whole-mount in situ hybridization with another gonadal marker, Amh, showed no similar decrease in expression in heterozygous embryos when compared with wild-type embryos (data not shown). This suggests that the difference in expression seen for Dax1 was gene specific and not due to a general effect of the lack of SF1 on the development of the gonad.

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Figure 5. Dax1 Expression Is Down-Regulated in the Developing Gonad of Embryos That Are Homozygous and Heterozygous for a Mutation in the SF1 Gene
A, RNase protection analysis on RNA from one pair of gonads and mesonephros from 11.5-dpc embryos that were wild type (lane 4), heterozygous (lane 5), or homozygous (lane 6) for a mutation in SF1 and from Escherichia coli tRNA (lane 3). RNA samples were hybridized to Dax1 (lane 2) and Sap62 (lane 1) antisense radioactively labeled probes. B, Whole-mount in situ hybridization using a Dax1-specific antisense probe on dissected gonad and mesonephros from embryos that were homozygous (a), heterozygous (b), and wild type (c) for a mutation in SF1.
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Regulation of Dax1 Expression in Adult Gonads
Transgenic mice containing the Dax11kbLacZ construct showed ß-galactosidase activity in the adult gonad. In the testis, LacZ expression was seen in some Sertoli cells and in the ovary in stromal, theca, and some follicle cells (Fig. 6
). A high level of ß-galactosidase activity was also apparent in Leydig cells within the adult testis. However, this level of activity did not correlate with the presence of the transgene and therefore reflects some endogenous enzyme activity detected under the experimental conditions of our assay. When the same promoter element was used to drive a different gene, transcripts were detected at high levels in some Sertoli cells but very low levels were found in Leydig cells (data not shown). To compare the promoter elements needed for embryonic and adult gonadal Dax1 expression, transgenic lines were made with either the 6XminLacZ construct or the Dax11kbdelLacZ construct. Two transgenic lines containing the 6XminlacZ construct were analyzed. In adult testes, both lines showed ß-galactosidase activity within testicular cords, but in a more punctate pattern than that seen with the original Dax11kbLacZ construct, whereas the pattern in the adult ovary was similar to that obtained with Dax11kbLacZ. However, the level of activity in the two 6XminlacZ lines was significantly lower in both the developing and adult gonad (data not shown). Nevertheless, these data show that region A is sufficient to drive LacZ expression to the adult as well as the embryonic gonad. Interestingly, four transgenic lines with the Dax11kbdelLacZ construct showed ß-galactosidase activity in the ovary in a pattern similar to that of the Dax11kbLacZ construct, and three of these lines showed a punctate staining in some Sertoli cells of the adult testis (Fig. 6
). Of these four lines, three completely lacked ß-galactosidase activity in the developing gonad, whereas the remainder showed very weak staining. These data shows that the SF14kb consensus site in region A is necessary for the expression of LacZ in the developing, but not in the adult, gonad.

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Figure 6. LacZ Expression in the Adult Gonad of Transgenic Embryos
ß-Galactosidase activity seen in the ovary (a and c) and testis (b and d) of the Dax11kbLacZ (a and b) and Dax11kbdelLacZ (c and d).
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DISCUSSION
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Using a transgenic mouse approach we have identified the promoter element responsible for tissue-specific expression of Dax1 in the developing gonad. A 500-bp element located 4 kb upstream of the Dax1 start codon was necessary to direct LacZ expression to the embryonic gonad. In addition, when this 500-bp element was linked to a heterologous minimal promoter, LacZ reporter gene activity was detected in the developing gonads of transgenic animals showing that this element was sufficient for the initiation of Dax1 expression at this site. These results extend those obtained by others using in vitro assays, which indicated the presence of important regulatory elements close to the Dax1 promoter (15, 18, 22, 23, 24). However, our data show that these proximal elements are not sufficient to drive expression in vivo, either in the gonad or in any other known site of Dax1 expression during embryonic development, such as the adrenals, pituitary, and hypothalamus. This suggests that there are different regulatory elements for each site of Dax1 expression during embryogenesis. This modular type of regulation has been seen in many genes that are expressed in various different tissues of the embryo (for example, see Ref. 28).
Our results implicate SF1 in the regulation of Dax1 expression because SF1-deficient mice show reduced levels of Dax1 expression. Our studies show that one SF1 consensus-binding site (SF14kb) found in region A is required to initiate expression in the developing gonad. However, our results also show that to achieve high levels of Dax1 expression at this stage, multiple SF1 consensus binding sites found proximal to the start of transcription are needed. These results are consistent with studies on the regulation of the Amh gene where SF1 has been shown to be involved and might act through several binding sites (27, 29). It is interesting to note that the levels of expression of the 1XminLacZ was increased dramatically when region A was multimerized to give 6XminLacZ. This suggests that quantitative regulation of expression is achieved through SF1 binding to multiple consensus sites.
Although the 6XminLacZ construct showed consistent levels of LacZ expression in the developing gonad, these levels were dramatically reduced in both the ovary and testis at later stages of development. This suggests that region A is needed for initiation of Dax1 expression, but maintenance of expression requires additional promoter elements found outside this region. The proximal SF1 consensus binding sites cannot be involved in this maintenance promoter element, as mutation of these sites does not affect levels of LacZ expression at later stages of gonad development. Therefore, our results are consistent with the proposal that SF1 is required for the initiation of Dax1 expression in early gonad development, where both genes are highly expressed, but not in the maintenance of expression at later stages of development where their expression patterns differ. This proposal explains the paradox of why Dax1 expression is maintained in the fetal ovary, which expresses only low levels of SF1, whereas it is turned off in the testis, which expresses high levels. This conclusion also provides an answer to a second paradox: how could SF1 regulate the expression of its own antagonist? The requirement of SF1 for the initiation of Dax1 expression, but not for its maintenance, provides a mechanism to ensure the presence of a modulator the continuing expression of which is then dependent on other factors.
The requirements for Dax1 expression were found to be different among the various cell types of the embryonic and adult gonad. Although region A was sufficient to direct LacZ expression to both stages, the developing gonad required the SF14kb consensus-binding site for expression, whereas the adult gonad did not. SF1 and Dax1 expression overlap in the adult testis and ovary but their patterns are not identical (18). These results suggest that if SF1 activates Dax1 expression in the adult gonad, it must form part of a different transcription complex to that within the embryonic gonad. Other conserved putative binding sites for SF1 are present, both within region A (see Fig. 3
) and close to the Dax1 transcriptional start site (15, 18, 23, 24). These would all be present in the construct with the 10-bp deletion. Like most transcription factors, SF1 acts within the context of other factors, binding to DNA as a complex, to regulate target gene expression. The combinations of factors required in different cell types at different stages need not be the same. Therefore, the relative importance of any specific enhancer element will also vary.
The structure of the ligand-binding domain of SF1 suggests that it might require a ligand for activation of transcription. However, no clear in vivo ligand has been identified. In vitro studies have suggested that interaction of SF1 with other transcription factors might alleviate the need for a ligand (30). The transcription factors that cooperate with SF1 in the regulation of Dax1 in the embryonic gonad are unknown. SOX9 has been shown to cooperate with SF1 to regulate the Amh promoter, but this factor is unlikely to activate the Dax1 promoter. High levels of Dax1 precede high levels of SOX9 in the testis, whereas in the ovary SOX9 is absent after 11 d of development (27, 31, 32, 33). WT1 has been shown to activate Dax1 expression in vitro and is present in the developing urogenital ridge in both sexes (22). No obvious conserved WT1 binding sites can be observed in the defined Dax1 regulatory element. However, Nachtigal et al. (34) have shown that WT1 can act as a cofactor by synergizing SF1 transcriptional activation in vitro without binding DNA. GATA-4 is another possible candidate involved in Dax1 regulation as it is expressed in the developing gonad of both sexes. Moreover, a conserved GATA consensus-binding site is found in region A (see Fig. 3
). Further studies are needed to determine which factors are involved in the regulation of Dax1 in the developing gonad. The present study will allow the testing of candidates that will be relevant to the situation in vivo.
The studies presented in this paper show the value of studying the regulation of gene expression in transgenic mice, as we have defined in vivo regulatory elements that were additional to those defined by in vitro studies. Our results suggest that in the developing gonad, two promoter elements act in concert to achieve proper expression of Dax1: an upstream element that provides tissue specificity and a downstream element that provides the required levels of expression. Our results also suggest that SF1 has a role in both these promoter elements. Other transcription factors are also involved in this process, and these studies can now serve as a template for future characterization of these factors, which will provide further information on in vivo transcription factor complexes important in gonad development and sex determination.
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MATERIALS AND METHODS
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Constructs for Transgenics
All sequence numbers are distances to the start of translation.
Dax11kbLacZ.
Details of this construct were described by Swain et al. (9).
Dax11kbdelLacZ.
The 10-bp deletion in the Dax11kbLacZ construct was engineered by digestion with restriction enzymes BstEII (-4,275) and MscI (-4,268). The digested DNA was treated with mung bean nuclease and the blunt ends were ligated.
Dax11kbmutLacZ.
A 10-bp linker containing the changes in the SF1 consensus binding site was ligated to the Dax11klacZ construct digested with MscI (-4,268) and BstEII (-4,275) and treated with Klenow.
Dax11kb12ab3LacZ.
The Nhe (-566) to NcoI (-2) fragment from Dax11kbLacZ was replaced by the identical fragment from DaxCAT12ab3M, kindly given to us by Ken Morohashi. Therefore, this construct contains identical mutations in all four SF1 binding sites (DaxAd41, 42a, 42b, and 43) to those described by Kawabe et al. (18).
1XminLacZ and 6XminLacZ.
An RsaI (-4,676) to PstI (-4,163) 513-bp fragment was introduced into the BGZ40 plasmid obtained from the laboratory of R. Krumlauf (25) upstream of the human minimal ß-globin promoter, which is linked to the LacZ gene.
Transgenic Mice
Vector sequences were removed from the DNA that was used for injection by enzyme digestion and separation by gel electrophoresis. The fragments for pronuclear injections were purified either by the Wizard PCR prep kit from Promega Corp. (Madison, WI) or by treatment with AgarACE from Promega Corp. Pronuclear injections were performed on fertilized mouse eggs from an intercross of F1 hybrids (C57BL6xCBA). Transgenic embryos were determined by PCR using primers that were specific to Dax1 and LacZ sequences (see Ref. 9). Transgenic males were mated to F1 females to determine ß-galactosidase activity in the embryos of these lines.
LacZ Expression
Staining for ß-galactosidase activity was performed as described previously (9). Briefly, postimplantation embryos were fixed in 2% paraformaldehyde/0.1% glutaraldehyde for 30 min and then washed in PBS and incubated for different times at 37 C in staining solution. Fixed adult testis and ovaries were left overnight in 30% sucrose, embedded in optimum cutting temperature compound, and frozen on dry ice. Cryostat sections were incubated overnight at 37 C in staining solution, washed in PBS, and mounted.
Whole-Mount in Situ Hybridization and RNase Protection Assay
These were performed using the same Dax1-specific probes as described previously (9).
Human DAX1 Gene Sequence
The genomic sequence was obtained from the National Center for Biotechnology Information Web site (accession no. AC005926).
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ACKNOWLEDGMENTS
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We thank members of the National Institute for Medical Research and Institute of Cancer Research Biological Services Units for invaluable help with mouse breeding, and other members of our laboratories for helpful discussions. We thank Ken Morohashi for the DaxCAT12ab3M plasmid.
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FOOTNOTES
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This work was supported by the Medical Research Council and Louis Jeantet Foundation, and by the Mexican Government and British Council (to V.N.).
1 These authors contributed equally to this work. 
Abbreviations: dpc, Days post coitus; RNase, ribonuclease; SF1, steroidogenic factor 1.
Received for publication March 20, 2001.
Accepted for publication December 21, 2001.
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