Dax-1 as One of the Target Genes of Ad4BP/SF-1

Ken Kawabe, Tatsuji Shikayama, Hisae Tsuboi, Sanae Oka, Koichi Oba, Toshihiko Yanase, Hajime Nawata and Ken-ichirou Morohashi

Department of Developmental Biology (K.K., T.S., H.T., S.O., K-i.M.) National Institute for Basic Biology Department of Molecular Biomechanics (K-i.M.) School of Life Science The Graduate University for Advanced Studies Core Research for Evolutional Science and Technology Japan Science and Technology (K-i.M.) Myodaiji-cho, Okazaki 444-8585, Japan
Third Department of Internal Medicine (K.K., K.O., T.Y., H.N.) Faculty of Medicine and Department of Molecular Biology (T.S.) Graduate School of Medical Science Kyushu University Higashi-ku, Fukuoka 812-8582


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The DAX-1 (also known as AHC) gene encodes an unusual member of the nuclear hormone receptor superfamily. DAX-1 plays a critical role during gonadal and adrenal differentiation since mutations of the human DAX-1 gene cause X-linked adrenal hypoplasia congenita associated with hypogonadotropic hypogonadism. In recent studies, DAX-1 was reported to function as a transcriptional suppressor of Ad4BP/SF-1, a critical transcription factor in gonadal and adrenal differentiation. With respect to implication of Ad4BP/SF-1 in the transcriptional regulation of the DAX-1 gene, inconsistent findings have been previously reported. We investigated the upstream region of the mouse Dax-1 (also known as Ahch) gene and identified a novel Ad4/SF-1 site by transient transfection and electrophoretic mobility shift assays. In addition, immunohistochemical analyses with a specific antibody to Dax-1 indicated the presence of immunoreactive cells in steroidogenic tissues, pituitary gland, and hypothalamus. Although the distributions of Dax-1 and Ad4BP/SF-1 were very similar, they were not completely identical. The expression of Dax-1 was significantly impaired in knock-out mice of the Ftz-f1 gene, which encodes Ad4BP/SF-1. Taken together, our findings indicate that Ad4BP/SF-1 controls the transcription of the Dax-1 gene.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Several studies have indicated the possible involvement of genes encoding DNA-binding proteins, such as WT-1 (1, 2, 3), SRY (4, 5), Ad4BP/SF-1 (6, 7, 8), SOX9 (9, 10, 11), and DAX-1 (also known as AHC) (12, 13), in the differentiation of steroidogenic tissues. Among these genes, WT-1, SRY, SOX9, and DAX-1 have been identified as responsible genes for various human diseases characterized by impaired differentiation of steroidogenic tissues, gonads, and/or adrenal glands (14, 15, 16). These findings indicate that these genes play a crucial role in the differentiation of steroidogenic tissues, although the functional role(s) of gene products remains to be elucidated. In addition to the above factors, Ad4BP/SF-1 was originally identified as the steroidogenic tissue-specific transcription factor that regulates the steroidogenic cytochrome P450 (CYP) genes (17, 18, 19, 20). Subsequent gene disruption studies indicated the important role of Ad4BP/SF-1 in the differentiation of steroidogenic tissues by demonstrating developmental deficits of such tissues in affected animals (21, 22, 23). However, the exact role(s) of these factors in the differentiation process of steroidogenic tissues remains to be identified.

Several research groups have focused on this topic and valuable observations have accumulated so far. With regard to the target genes of Ad4BP/SF-1, genes functionally related to steroidogenesis such as the steroidogenic CYP (17, 18, 19, 24, 25, 26, 27, 28, 29, 30), 3ß-hydroxysteroid dehydrogenase (31), and steroidogenic acute regulatory protein (StAR) (32, 33) genes have been identified. Müllerian inhibitory substance (MIS) gene, a key factor for the development of male reproductive tissues, is regulated by Ad4BP/SF-1 (34). Interestingly, the transcription of these genes is regulated cooperatively by the transcription factors described above. DAX-1, for instance, is likely to act as a suppressor of Ad4BP/SF-1-mediated transcription by a direct interaction with Ad4BP/SF-1 (35, 36) or by binding to the stem-loop structure of DNA formed in the promoter region (37). In addition, recent studies have shown that WT-1 (38), GATA-4 (39), and Sox9 (40) activate MIS gene transcription by showing synergistic effects on Ad4BP/SF-1-mediated transcription.

The transcriptional regulation of these transcription factor genes has also been investigated. A number of these studies have shown that the Ftz-f1 gene coding for Ad4BP/SF-1 is regulated by an E box (41) and a binding factor, USF (upstream stimulatory factor) (42, 43). Investigations of the DAX-1 gene transcriptional regulation have also been reported, although conflicting results have been reported regarding regulation by Ad4BP/SF-1 (44, 45, 46, 47). In the present study, we investigated the upstream region of the mouse Dax-1 (also known as Ahch) gene and identified a novel Ad4/SF-1 site, which is recognized by Ad4BP/SF-1. The activity of the newly identified Ad4/SF-1 site was confirmed by transient transfection assays using cultured cell lines. Furthermore, using immunohistochemical analyses with antibodies specific for Dax-1 and Ad4BP/SF-1, we demonstrated that the tissue distribution of Dax-1 is highly similar to that of Ad4BP/SF-1 but not completely overlapped. Finally, Dax-1 expression was investigated using tissues of Ftz-f1 gene disrupted mice, which demonstrated that the Dax-1 gene is under the control of Ad4BP/SF-1 in vivo.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of a Novel Ad4/SF-1 Site
Several studies have investigated the transcriptional regulation of the Dax-1 gene. However, results of these studies showed inconsistent conclusions with respect to the participation of Ad4BP/SF-1 in this process (44, 45, 46, 47). To examine the involvement of Ad4BP/SF-1 in Dax-1 gene regulation, we investigated the 2.5-kb upstream region of mouse Dax-1 gene. A series of deletion mutants of the upstream region were constructed with a chloramphenicol acetyltransferase (CAT) reporter gene (Fig. 1AGo) and used for transfection into two lines of steroidogenic cells, Y-1 mouse adrenocortical and R2C rat Leydig tumor cells, both of which express Ad4BP/SF-1 as confirmed by immunoblotting (data not shown). As shown in Fig. 1BGo, CAT activities in these cells did not decrease by truncation of the upstream down to -117 bp. In contrast, the activity decreased significantly by further truncation to -40 bp, strongly suggesting the presence of cis-element(s) between -117 and -40 bp.



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Figure 1. Transcriptional Activity of the Mouse Dax-1 Gene Promoter

A, CAT reporter gene constructs carrying various lengths of the upstream region of Dax-1 gene were prepared as described in Materials and Methods. Hd, HindIII; EV, EcoRV; Nh, NheI; Ms, MscI; Fs, FspI. B, CAT reporter gene plasmids (2 µg) were transfected into Y-1 and R2C cells, and the cells were subjected to CAT assays. CAT activity was expressed relative to that of DaxCAT540 and shown as mean ± SD values of triplicate transfections.

 
To identify the cis-element(s), several overlapping oligonucleotides ranging from -117 to -40 bp were synthesized (Fig. 2AGo and Table 1Go) and used for electrophoretic mobility shift assays (EMSAs) using a nuclear extract prepared from Y-1 cells. As shown in Fig. 2BGo, a DNA-protein complex was detected only when DaxEY-91 was used as the probe. As a control, we used an oligonucleotide, HSC-a, which contains a representative Ad4/SF-1 site lying in the human CYP11A (P450SCC) gene promoter (24). Interestingly, the location of the signal with DaxEX-91 was almost identical to that of the signal with HSC-a. The complex formed with DaxEY-91 was inhibited by the addition of excess amounts of unlabeled HSC-a as well as DaxEY-91 (Fig. 2CGo), suggesting the presence of an Ad4/SF-1 site. To identify the exact nucleotides between -91 and -67 bp that are responsible for the binding, competition assays were conducted using a series of oligonucleotides carrying a three-base substitution (DaxEY-91 Ma to Mf listed in Fig. 3AGo). Three oligonucleotides, DaxEY-91Md, Me, and Mf, did not compete out the complex formation (Fig. 3BGo), indicating that the sequence, 5'-CGCCCTTGT-3', is involved in the binding. Finally, this complex disappeared in the presence of an antiserum to Ad4BP/SF-1. As we described previously (24), Ad4BP/SF-1 was able to recognize PuPuAGGTCA and PyCAAGGPyPyPu. The newly identified sequence (5'-ACAAGGGCG-3' in the antisense strand) was quite similar to the latter consensus sequence. Considered together, it was concluded that a novel Ad4/SF-1 site is present in the region.



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Figure 2. Identification of cis-Element(s) between -117 and -40 bp

A, Seven oligonucleotides were synthesized to cover a region from -117 to -40 bp and used as probes for EMSAs. B and C, EMSAs were performed with 32P-labeled oligonucleotides indicated in panel A. The probes (P.) were incubated with 5 µg nuclear extract prepared from Y-1 cells, and the mixtures were subjected to electrophoresis. In competition experiments, 50-fold molar excess amounts of unlabeled oligonucleotides (C.) were used. Arrowheads indicate DNA-protein complexes. HSC-a is an oligonucleotide containing a representative Ad4/SF-1 site lying in the promoter region of the human CYP11A gene.

 

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Table 1. Oligonucleotides Used for EMSAs

 


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Figure 3. Identification of Ad4/SF-1 Site

A, Nucleotide sequences of oligonucleotides containing nucleotide substitutions. Each oligonucleotide from DaxEY-91Ma to DaxEY-91Mf contains a three-nucleotide substitution indicated by underlined letters. B, Competition of DNA-protein complex formation by the mutated oligonucleotides listed in panel A. 32P-Labeled DaxEY-91 and 50-fold molar excess amounts of unlabeled oligonucleotides were used as the probe (P.) and competitors (C.), respectively. The antiserum to Ad4BP/SF-1 ({alpha}Ad4BP/SF-1) was mixed with nuclear extracts before the addition of the probe. Arrowhead indicates a DNA-protein complex.

 
Activation of Dax-1 Gene Transcription by Ad4BP/SF-1
In previous studies (44, 45, 46), a single Ad4/SF-1 site was confirmed to be located upstream of the mouse and human DAX-1 genes. The Ad4/SF-1 site of the mouse gene located at -128 to -121 bp was tentatively designated DaxAd4–2 (Fig. 4AGo). Yu et al. (47) recently reported another Ad4/SF-1 site present at -122 to -115 (47). In addition, based on the nucleotide sequence, we detected a possible Ad4/SF-1 site at -330 to -326 bp (DaxAd4–1) (Fig. 4AGo). Although the reported sequence of the human gene did not cover the region containing DaxAd4–1, comparison of the nucleotide sequences between the two animals showed that nucleotide sequences corresponding to DaxAd4–2 were identical. In addition, the newly identified DaxAd4–3 at -80 to -72 bp was highly conserved. To determine the function of these Ad4/SF-1 sites, we investigated the effect of Ad4BP/SF-1 on the transcription of DaxCAT2.5K. DaxCAT2.5K was transfected with various amounts of RSV/Ad4BP into CV-1 cells in which no endogenous Ad4BP/SF-1 was detected (data not shown). As shown in Fig. 4BGo, the transcriptional activity was elevated dose dependently by the addition of the expression vector.



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Figure 4. Location of Ad4/SF-1 Sites in the Mouse Dax-1 Gene Promoter and Their Functions

A, Nucleotide sequences of mouse (upper) and human (lower) DAX-1 genes are aligned. Identical nucleotides between the two genes are shown by asterisks. A part of nucleotide sequences of the first exon and deduced amino acid sequences are shown in bold letters. The transcription initiation site of the mouse gene is indicated by a large asterisk. DaxAd4–1, DaxAd4–2, and DaxAd4–3 are indicated. Arrows in DaxCAT540, DaxCAT265, DaxCAT117, and DaxCAT40 indicate their truncated positions. Numbers on the right are relative to the transcription initiation site. B, Transcriptional activation of Dax-1 gene by Ad4BP/SF-1. Increasing amounts (0, 0.5, 1.0, and 2.5 µg) of the expression vector for Ad4BP/SF-1 (RSV/Ad4BP) were cotransfected with DaxCAT2.5K (0.5 µg) into CV-1 cells. C, Effect of RSV/Ad4BP on the deletion series of DaxCAT plasmids. The deletion plasmid carrying various lengths of the Dax-1 gene promoter (Fig. 1AGo) and pSV00CAT (control plasmid for CAT constructs) were transfected into CV-1 cells in the presence or absence of RSV/Ad4BP. CAT activities compared with those of DaxCAT2.5K in the absence of RSV/Ad4BP are shown as mean ± SD of triplicate transfections.

 
To determine which Ad4/SF-1 sites are responsible for Ad4BP/SF-1-mediated transcription, we transfected the deletion constructs indicated in Fig. 1AGo with the expression vector. CAT activities elevated by Ad4BP/SF-1 were increased gradually until the promoter was truncated down to -540 bp (DaxCAT540), which preserved the three Ad4/SF-1 sites. When DaxCAT265 lacking DaxAd4–1 was transfected, the CAT activity decreased to 80% of DaxCAT540. Further truncation to -117 bp reduced the activity to 56% of DaxCAT540. These changes in CAT activities were likely to be caused by deletion of DaxAd4–1 and DaxAd4–2 sequences. Finally, DaxCAT40 lacking all Ad4/SF-1 sites, DaxAd4–1, DaxAd4–2, and DaxAd4–3, abolished the remaining CAT activity.

Dax-1 Gene Transcription Controlled by Multiple Ad4/SF-1 Sites
The contribution of multiple Ad4/SF-1 sites was investigated by using CAT reporter constructs carrying nucleotide substitution as illustrated in Fig. 5AGo. The impaired abilities as the binding sequence for Ad4BP/SF-1 were confirmed by EMSAs. As shown in Fig. 5BGo, the binding activities of the mutated oligonucleotides, DaxAd4–1M and DaxAd4–3M, were significantly decreased when they were used as probes. A significant decrease was also observed with DaxAd4–2M, although a weak but clear signal remained to be detectable. Similarly, they failed to function as efficient competitors. Thus, the intrinsic activities of Ad4/SF-1 sites were impaired by these mutations.



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Figure 5. Functional Analyses of Multiple Ad4/SF-1 Sites in the Dax-1 Gene Promoter

A, Schematic presentation of CAT plasmids carrying mutated Ad4/SF-1 sites. To disrupt Ad4/SF-1 sites in DaxCAT540, the boxed nucleotides in DaxAd4–1, -2, and -3 were substituted as indicated by underlines. The CAT plasmids, DaxCAT1M to DaxCAT123M, have disrupted Ad4/SF-1 site(s) indicated by crosses. B, EMSAs with oligonucleotides (Table 1Go) containing intact (DaxAd4–1, -2, and -3) and mutated Ad4/SF-1 sites (DaxAd4–1M, -2M, and -3M) were performed. The nucleotide sequence of DaxAd4–3 is identical to that of DaxEY-91. 32P-labeled oligonucleotides (P.) and 25-fold molar excess amounts of unlabeled oligonucleotides (C.) were used as probes and competitors, respectively. Antiserum to Ad4BP/SF-1 ({alpha}Ad4BP/SF-1) was mixed with Y-1 cell nuclear extracts before the addition of the probe. Arrowhead indicates a DNA-protein complex. C, Effects of disruption of Ad4/SF-1 sites. CAT plasmids indicated in A (0.5 µg) were transfected in the absence or presence of RSV/Ad4BP (2.5 µg) into CV-1 cells. CAT activities relative to the intact plasmid (DaxCAT540) are shown as the mean ± SD of triplicate transfections.

 
CAT constructs carrying these mutations were cotransfected with RSV/Ad4BP into CV-1 cells, and the effect of the nucleotide substitution was investigated. As shown in Fig. 5CGo, the CAT activity of DaxCAT1M, which contained a disrupted DaxAd4–1, was not diminished compared with that of the intact construct, DaxCAT540. In contrast, the CAT activity of DaxCAT2M, which contained a disrupted DaxAd4–2, decreased to 48% relative to the intact construct. Similarly, disruption of DaxAd4–3 to make DaxCAT3M reduced CAT activity to 39%. Constructs carrying two disrupted Ad4/SF-1 sites of all combinations, DaxCAT12M, DaxCAT13M, and DaxCAT23M, showed a diminished activity of 38–53%. In addition, further reduction of CAT activity was noted by disruption of all binding sites (DaxAd4–1, DaxAd4–2, and DaxAd4–3).

As pointed out above, mutation at DaxAd4–2 did not result in the complete disappearance of its intrinsic binding activity to Ad4BP/SF-1. Recently, Yu et al. (47) showed the presence of another Ad4/SF-1 site that overlapped with DaxAd4–2. As indicated in Fig. 6AGo, we tentatively designate the Ad4/SF-1 sites located at 5'- and 3'-sides DaxAd4–2a and DaxAd4–2b, respectively. To determine the functional contribution of each Ad4/SF-1 site, we prepared an additional CAT reporter construct, DaxCAT2bM, which contained disrupted DaxAd4–2b. The transcriptional activity of DaxCAT2bM driven by Ad4BP/SF-1 was compared with that of DaxCAT2aM, which was identical to DaxCAT2M in Fig. 5Go. As indicated in Fig. 6BGo, DaxCAT2bM showed reduced activity (to 45%) compared with that of DaxCAT540. The reduction rate was slightly stronger than that with DaxCAT2aM. As a control, DaxCAT12ab3M carrying all mutated Ad4/SF-1 sites exhibited a reduction in activity to 40%. With respect to the binding activity of DaxAd4–2bM, it was retained as confirmed by EMSA, probably due to the intact DaxAd4–2a. A complete disappearance of the binding activity was achieved by disruption of both DaxAd4–2a and DaxAd4–2b (data not shown). Taken together, these results clearly indicate that at least three Ad4/SF-1 sites, DaxAd4–2a, DaxAd4–2b, and DaxAd4–3, are involved in Ad4BP/SF-1-mediated transcription of the mouse Dax-1 gene and that they contribute almost equally to the transcriptional activity.



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Figure 6. Functional Analyses of an Ad4/SF-1 Site Overlapped with DaxAd4–2

A, DaxAd4–2a (shaded) is identical to DaxAd4–2, while DaxAd4–2b (boxed) is another Ad4/SF-1 site pointed out by Yu et al. (47 ). DaxAd4–2aM and DaxAd4–2bM contain two- and three-nucleotide substitutions indicated by underline, respectively. B, DaxCAT2aM, DaxCAT2bM, and DaxCAT12ab3M (0.5 µg) containing the nucleotide substitutions indicated in Fig. 6AGo were transfected in the absence or presence of RSV/Ad4BP (2.5 µg) into CV-1 cells. CAT activities relative to the intact plasmid (DaxCAT540) are shown as the mean ± SD of triplicate transfections.

 
Expression of DAX-1 in Steroidogenic Tissues
The results described above indicated that the Dax-1 gene is one of the target genes of Ad4BP/SF-1. If such regulation is functional in vivo, an overlapped expression between the two factors should be observed. Therefore, we compared in the next step the distribution of DAX-1 with that of Ad4BP/SF-1 in adult rat tissues using specific antibodies. Since it has already been shown that Ad4BP/SF-1 is expressed in steroidogenic tissues such as the gonads and adrenal glands (48, 49), the expression of DAX-1 was examined in these tissues. Before immunohistochemical studies, the specificity of the antibody was investigated by immunoblot analysis. As shown in Fig. 7AGo, a single band was detected at the expected molecular weight in CV-1 cells to which an expression vector for Dax-1 was transfected. In contrast, untransfected CV-1 cells gave no signal. Immunoblot analysis was further performed using several mouse tissues, indicating that a specific signal was detected in the adrenal glands and testes but not in the nonsteroidogenic liver, kidney, lung, and spleen. Likewise, a specific signal was also observed when the steroidogenic tissues (adrenal glands, testes, and ovaries) of rats were subjected to analysis (Fig. 7BGo).



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Figure 7. Expression and Localization of DAX-1 and Ad4BP/SF-1 in Steroidogenic Tissues

A and B, Immunoblot analyses were performed with an antibody to Dax-1. Cell and tissue lysates were prepared from CV-1 cells transfected (Dax-1) or untransfected (-) with RSV/mDax-1 (A), from the adrenal gland (Ad), liver (Liv), kidney (Kd), lung (Lun), spleen (Sp), and testis (Tes) of adult mice (A), and from the adrenal gland (Ad), testis (Tes), and ovary (Ov) of adult rats (B). Lysates of CV-1 cells (10 µg), mouse tissues (50 µg), and rat tissues (100 µg) were used for immunoblot analyses with anti-Dax-1 antibody. The locations of molecular weight markers (Bio-Rad Laboratories, Inc. Hercules, CA) are indicated on the left of each panel. Arrows indicate the signals for Dax-1. C–H, Representative stainings of adult rat adrenal glands (C and D), testes (E and F), and ovaries (G and H) with anti-Ad4BP/SF-1 (C, E, and G) and anti-DAX-1 antibodies (D, F, and H). Immunohistochemical staining was performed as described in Materials and Methods. g, Zona glomerulosa; f, zona fasciculata; r, zona reticularis; s1 and s2, seminiferous tubules; f1, f2, and f3, ovarian follicles; cl, corpus luteum. Bars, 100 µm in C, D, G, and H; 50 µm in E and F.

 
Using antibodies to DAX-1 and Ad4BP/SF-1, we examined and compared immunohistochemically the expressions of the two binding proteins in steroidogenic tissues. As described previously (48, 49), all adrenocortical cells in the adrenal glands were immunoreactive for Ad4BP/SF-1 (Fig. 7CGo). DAX-1 immunoreactive cells were mainly localized in the zona glomerulosa (g in Fig. 7DGo), while a few cells were stained weakly in the zonae fasciculata and reticularis (f and r in Fig. 7DGo). The adrenal medulla was negative for DAX-1 (data not shown). In the testis, the expression of Ad4BP/SF-1 was observed in Sertoli and Leydig cells (Fig. 7EGo), while the same cell types were stained with the antibody to DAX-1 (Fig. 7FGo). However, the intensity of staining was stronger in Sertoli cells in the seminiferous tubule labeled as s1 than in seminiferous tubule s2 in Fig. 7FGo. Staining of Sertoli cells at a level similar to the background was also observed in other tubules (data not shown). It is well known that different stages of germ cell differentiation proceed simultaneously at different locations in seminiferous tubules. Based on the morphological study, less expression in Sertoli cells was observed in seminiferous tubules at stages I–VII. Interestingly, the expression increased rapidly to the highest level at stage VIII and decreased gradually thereafter until stage XIV (data not shown). The expression profile clarified by the present morphological study was fairly consistent with that obtained by Western blot analysis (50). In the ovary, Ad4BP/SF-1 was expressed in the granulosa, theca, and corpus luteum cells whereas DAX-1 was expressed in the same cell types with the exception of corpus luteum (Fig. 7Go, G and H). DAX-1 staining profile of the granulosa and theca cells was quite different from that of Ad4BP/SF-1. Previous studies showed that all granulosa cells in healthy follicles, but not in atretic follicles, expressed Ad4BP/SF-1 (51). In this study, all granulosa cells of follicles labeled f1, f2, and f3 were uniformly stained by the antibody to Ad4BP/SF-1. Interestingly, however, DAX-1 was never detected in the follicle f2. In addition, the expression of DAX-1 was significantly distinct in granulosa cells in follicle f1. Such inconsistent expression of the two factors was also observed in theca cells. Although all theca cells were immunoreactive for Ad4BP/SF-1, a certain proportion of these cells were immunoreactive for DAX-1. Immunohistochemical analysis with mouse tissues revealed that the distribution of Dax-1 in the testis and ovary was quite similar to that in the corresponding rat tissues. In the case of the adrenal cortex, however, it could not be concluded definitely if the staining profile of the rat adrenal gland was identical to that of the mouse tissue because of a high background covering the entire adrenal cortex (data not shown).

Expression of Dax-1 in Ftz-f1 Gene-Disrupted Mice
As described above, DAX-1 was expressed in certain cell populations in which Ad4BP/SF-1 was expressed. The overlapping distribution of these two factors supports the conclusion drawn from the promoter analysis that the Dax-1 gene is controlled by Ad4BP/SF-1. Further evidence was obtained with the following analysis using Ftz-f1 gene-disrupted (KO) mice. Whole-mount in situ hybridization and immunohistochemical analysis were carried out with tissues from the wild type and Ftz-f1 KO mice. As demonstrated in previous studies (21, 22, 23), the gonads failed to develop in Ftz-f1 KO mice at the early stages of ontogeny. In fact, at 12.5 gestational days (E12.5), degeneration of the whole part of the genital ridge was clearly observed. In contrast, such deficit was not observed in E10.5 fetuses. Therefore, whole-mount in situ hybridization probed by Ad4BP/SF-1 and Dax-1 was performed with the developing urogenital ridges of E10.5 fetuses. A significantly reduced amount of Ad4BP/SF-1 mRNA was detected in the urogenital ridge of KO fetuses (Fig. 8BGo) compared with the wild type (Fig. 8AGo). When probed with Dax-1, a weak but clear signal was detected in the urogenital ridge of the wild type (Fig. 8CGo). In contrast, any signal higher than the background level was not observed in KO fetuses (Fig. 8DGo). Previous studies showed that the mRNA and protein products of the Ftz-f1 gene were detected in the ventromedial hypothalamic nucleus (VMH) and pituitary gland (22, 52, 53). In the next step, we examined the expression of Dax-1 in these tissues of E14.5 KO fetuses. As shown in Fig. 9Go, A and B, mRNA coding for Ad4BP/SF-1 was clearly detected in the developing VMH of the wild-type and KO fetuses of E14.5, although the amount was significantly higher in the wild type. Similar to the gonads, the expression of Dax-1 was detected in wild-type (Fig. 9CGo) but was undetectable in KO fetuses (Fig. 9DGo).



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Figure 8. Expression of Dax-1 mRNA in the Developing Gonads

Whole-mount in situ hybridization was carried out with the developing gonads at E10.5. Genital ridges were prepared from wild-type (A and C) and Ftz-f1 gene-disrupted (B and D) fetuses and subjected to whole-mount in situ hybridization probed with Ad4BP/SF-1 (A and B) or Dax-1 (C and D). Arrows indicate genital ridges.

 


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Figure 9. Expression of Dax-1 mRNA in Developing VMH

Whole-mount in situ hybridization was carried out using the developing VMH at E14.5. Brains were prepared from wild-type (A and C) and Ftz-f1 gene-disrupted (B and D) fetuses and subjected to whole-mount in situ hybridization probed with Ad4BP/SF-1 (A and B) and Dax-1 (C and D). Arrows indicate the developing VMH.

 
Subsequently, brains from newborn mice and fetuses were subjected to the following immunohistochemical analyses. As shown in Fig. 10AGo, Ad4BP/SF-1 was immunohistochemically detected in the VMH of the wild-type newborn mice, whereas the signal was undetectable in the corresponding region of KO mice (Fig. 10BGo). When the same region was stained with Dax-1 antibody, some but not all VMH cells were immunoreactive in the wild type (Fig. 10CGo). In addition, Dax-1 immunoreactive cells were detected in the arcuate nuclei (arrows in Fig. 10CGo). However, no staining was observed in the VMH and arcuate nuclei of KO mice (Fig. 10DGo). Similar immunohistochemical analyses were performed using the developing brain of E14.5 fetuses. Certain areas of the developing diencephalon of the wild type contained immunoreactive cells for Ad4BP/SF-1 and Dax-1 (Fig. 10Go, E and G). However, such immunoreactive cells were not present in the corresponding areas of KO mice (Fig. 10Go, F and H). Finally, the expressions of the two factors were examined in the pituitary glands. Certain cells in the pituitary glands of the newborn wild-type (Fig. 11AGo) and E14.5 fetuses (Fig. 11EGo) were immunoreactive for Ad4BP/SF-1. As expected, no signal was detected in the pituitary of KO mice (Fig. 11BGo) and fetuses (Fig. 11FGo). With regard to the expression of Dax-1, it was detected in the pituitary of wild-type newborn mice (Fig. 11CGo) and fetus (Fig. 11GGo). However, Dax-1 expression was decreased significantly in KO pituitary of newborn and fetuses (Fig. 11Go, D and H). Since Ad4BP/SF-1 was reported to be expressed specifically in gonadotrophs of the pituitary (22, 52), we also investigated whether Dax-1 was expressed in the same trophs by double immunostaining with an antibody to FSHß. As shown in Fig. 11Go, I and J, certain population of pituitary trophs were immunoreactive for Dax-1, and almost all of them seemed to have FSHß-immunoreactive cytoplasms.



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Figure 10. Immunohistochemical Detection of Dax-1 in the Hypothalamic Nucleus

Coronal sections of the newborn (A–D) and sagittal sections of the E14.5 fetal (E–H) brains were prepared from wild-type (A, C, E, and G) and Ftz-f1 gene-disrupted (B, D, F, and H) mice. They were stained with anti-Ad4BP/SF-1 antiserum (A, B, E, and F) or anti-Dax-1 antibody (C, D, G, and H) as described in Materials and Methods. Regions encircled by arrowheads (A and C) indicate VMH. Inset in panel C is enlargement of Dax-1 immunoreactive cells in VMH. Arrows in panel C indicate Dax-1-immunoreactive cells in the arcuate nucleus. p, Pituitary. Bars; 100 µm in panels A–D, 50 µm in panels E–H.

 


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Figure 11. Immunohistochemical Detection of Dax-1 in the Pituitary

Sections of the pituitary glands from newborn (A–D), E14.5 fetuses (E–H), and adult mice (I and J) were prepared from wild-type (A, C, E, G, I, and J) and Ftz-f1 gene-disrupted (B, D, F, and H) mice. They were stained with anti-Ad4BP/SF-1 antiserum (A, B, E, and F) or anti-Dax-1 antibody (C, D, G, and H). The adult tissue was used for staining with the anti-Dax-1 antibody (I) and for double-label staining with the anti-Dax-1 and anti-FSHß antibodies (J) as described in Materials and Methods. Bars, 50 µm in panels A–H; 20 µm in I and J.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transcriptional Regulation of Dax-1 Gene
The results reported by Ikeda et al. (45) and Yu et al. (47) using the murine Dax-1 gene promoter suggested the presence of an unknown cis-element in addition to Ad4/SF-1 sites corresponding to DaxAd4–2a and DaxAd4–2b within 100 bp upstream of the transcription initiation site. However, the exact sequence responsible for the transcriptional activity was not determined in the above studies. In the present study, we confirmed that deletion of the same region resulted in a large decrease of transcriptional activity and subsequently confirmed the presence of a novel Ad4/SF-1 site corresponding to DaxAd4–3. Functional analyses of various Ad4/SF-1 sites, DaxAd4–1, DaxAd4–2a, DaxAd4–2b, and DaxAd4–3, demonstrated that mainly three, DaxAd4–2a, DaxAd4–2b, and DaxAd4–3, mediated Ad4BP/SF-1 dependent transcription of the gene. The transcriptional activity of the gene was largely attributed to the function of these Ad4/SF-1 sites, indicating that Ad4BP/SF-1 acts as a crucial transcription factor for the regulation of Dax-1 gene. In spite of the ability to bind to Ad4BP/SF-1, DaxAd4–1 contributed to a lower extent to the transcription. However, it seems that the use of cultured cells hindered the intrinsic activity of DaxAd4–1.

It was reported that the transcription of Dax-1 gene was inhibited by its own product through binding to a hairpin structure (37). In fact, Dax-1 and StAR genes have potential site(s) to form the hairpin structure in their promoter regions and thereby the gene transcription was suppressed in a Dax-1-dependent fashion. Interestingly, DaxAd4–3 overlapped with the region. Therefore, assuming that Ad4BP/SF-1 does not recognize DaxAd4–3 when the region forms a hairpin structure, it is reasonable to suggest that conformational transition between a double helix and a hairpin structure is critical for the transcriptional control of Dax-1 gene. With respect to the transcriptional suppression by Dax-1, another mechanism has been proposed (35, 36) in which a direct interaction between Ad4BP/SF-1 and DAX-1 is necessary. Interestingly, such interaction leads to recruitment of the nuclear receptor corepressor (N-CoR) and results in a marked decrease of transcriptional activation by Ad4BP/SF-1 (36). Whatever the mechanism, the transcription of Dax-1 gene is likely to be under the control of coordinated functions of Ad4BP/SF-1 and Dax-1.

Unique Expression Profile of DAX-1
In the present study, we compared the distribution of immunoreactive cells for Ad4BP/SF-1 and Dax-1 in steroidogenic tissues. In all these tissues, there was a significant overlap in the distribution of the two factors, which is consistent with the findings of in situ hybridization (45). However, Dax-1 expression was not observed in all cells immunoreactive for Ad4BP/SF-1. Such inconsistent expression between the two factors has already been reported in the testis (50). Namely, the amount of Dax-1 expressed in Sertoli cells varied in a fashion synchronized with spermatogenesis in spite of the constant expression of Ad4BP/SF-1. In the present study, the presence of a particular cell type immunoreactive for Ad4BP/SF-1, but not for Dax-1, was identified in other steroidogenic tissues. In the adrenal cortex, the majority of cells constituting the zonae fasciculata and reticularis were negative for Dax-1. It is well known that each of the three zones of the adrenal cortex is comprised of functionally and morphologically distinct cell types. In fact, among six forms of cytochrome P450s implicated in steroid hormone synthesis, a unique set of the P450s is expressed in each zone, and thereby distinct types of steroid hormones are synthesized and secreted from each zone. Considering the function of Dax-1 as a suppressor of Ad4BP/SF-1-mediated transcription, the unique distribution of Dax-1 might be essential for the distinct physiological functions of the three zones. In support of this conclusion, the expression of P450SCC catalyzing the side chain cleavage of cholesterol is stronger in the zonae fasciculata and reticularis than in zona glomerulosa (54).

In the ovary, the expression of Dax-1 in the granulosa cells varied among follicles. One follicle contained granulosa cells that were all negative for Dax-1, while another follicle contained granulosa cells with variable staining intensity. It was also shown that a particular type of granulosa (cumulus) cells surrounding an oocyte in an antral follicle was less stained than granulosa cells located near the basement membrane. Interestingly, the expression profile of the MIS gene reported thus far is roughly complementary to that of Dax-1. Namely, stronger signals were observed in the cumulus cells than peripheral granulosa cells of the antral follicle (55, 56). Since MIS gene transcription is activated by Ad4BP/SF-1 and suppressed by Dax-1 (34, 38), it is possible that the heterogeneity of the MIS gene expression in the granulosa cells is caused by the unique distribution of Dax-1. Recently, Swain et al. (57, 58) indicated that the amount of Dax-1 in sexually differentiating gonads is crucial for a proper sex differentiation. Considering these findings, it is likely that both the expression level and distribution of Dax-1 are crucial for gonadal function and sex differentiation.

All our immunohistochemical studies showed that Dax-1 is localized to nuclei in adult tissues. In contrast, a previous report by Majdic and Saunders (59) showed cytoplasmic localization of DAX-1 in interstitial cells of human and rat fetal testes. In sharp contrast, our studies in fetal steroidogenic tissues showed nuclear localization similar to adult tissues (our unpublished data). This discrepancy is likely to be due to specificity of the antibodies used in the two studies. In fact, our antibody recognized Sertoli as well as Leydig cells in the fetal testis, whereas the antibody used by Majdic and Saunders (59) gave no signal in the former cells. Alternatively, subcellular localization of Dax-1 might vary according to physiological conditions.

Impaired Expression of Dax-1 in Ftz-f1 Gene KO Mice
In the present study, we initially demonstrated the involvement of Ad4BP/SF-1 in the transcriptional regulation of Dax-1 gene based on reporter gene assays in cultured cells. Therefore, it seemed to be important to confirm that such regulation is functional in vivo. In these studies, we postulated that the expression of Dax-1 should be significantly impaired in the Ftz-f1 gene-disrupted mouse because of the absence of the upstream regulator, Ad4BP/SF-1. In this regard, it was critical to demonstrate whether the cells in which Ad4BP/SF-1 is expressed in the wild type survive in the KO mouse (21, 22, 23). In fact, it was clearly shown that the absence of Ad4BP/SF-1 in KO mice resulted in total agenesis of steroidogenic tissues from the early stages of ontogeny. Therefore, the urogenital region of fetuses was investigated at E10.5, since degeneration was not detected morphologically. The mRNA transcript from the disrupted Ftz-f1 gene was detected in KO fetuses although at a small amount compared with the wild type, indicating that the cells comprising the urogenital ridge were present at the gestational day. With respect to VMH, cells expressing the mRNA were also detected in E14.5 fetuses although, as in the case of the gonad, the signal for the mRNA in KO fetuses was very weak compared with the wild type. Likewise, Ikeda et al. (45, 53) reported the persistent presence of the developing genital ridge and VMH in KO mouse fetuses. The presence of gonadotrophs in the pituitary of the KO mouse was also confirmed by Ikeda et al. (53) and Shinoda et al. (22), although the ability of these trophs to produce gonadotropin was markedly reduced. Considered together, we concluded that the gonad and VMH were present in KO fetuses until 10.5 and 14.5 gestational days, respectively, while the gonadotroph was present until birth. By using these tissues, examination of Dax-1 expression at mRNA and/or protein levels showed that the expression was clearly reduced in comparison with the corresponding tissues of the wild type. Thus, the results of our in vivo studies confirmed the findings established in in vitro studies using cultured cells.

In contrast to the present findings, Ikeda et al. (45) showed that Dax-1 expression was maintained in the gonads and VMH of E11.5 KO and E18.5 KO fetuses, respectively, which was detected by in situ hybridization analysis using 35S-labeled probes. In the present study, we used a digoxygenin-labeled probe for whole-mount in situ analysis. A comparison of the two probes indicates that they are not substantially different from each other except for the labeling materials. In general, 35S-labeled probes are more efficient than digoxygenin-labeled probes with respect to sensitivity, thus explaining the detection of a small amount of Dax-1 mRNA with 35S-labeled probe. In fact, a careful examination of their in situ results shows that the amount of Dax-1 mRNA was significantly low in KO fetuses. Therefore, their results seem to agree with our conclusion that Ad4BP/SF-1 acts as a critical regulator of the Dax-1 gene.

The detection of a novel Ad4/SF-1 site in the Dax-1 gene promoter in the present study indicates that the gene is one of the target genes of Ad4BP/SF-1. In fact, the distribution of the two factors closely overlapped in steroidogenic tissues, pituitary, and VMH. Nevertheless, the expression profile of Dax-1 was not completely identical to that of Ad4BP/SF-1. In addition to the results described in the present study, sexually dimorphic expressions of these factors in fetal gonads were also noticed to be nonidentical (7, 57). These distinct profiles of expression do not seem to be incompatible with our conclusion that the Dax-1 gene is under the control of Ad4BP/SF-1. As an adequate explanation, it is possible that Ad4BP/SF-1-mediated transcription of the Dax-1 gene is modulated by other transcription factors. In fact, such factors are incorporated in the MIS (38, 39, 40), steroidogenic CYP (60, 61, 62, 63), and LHß (64) transcription in which Ad4BP/SF-1 is absolutely needed. Alternatively, it is also possible that the transcriptional activity of Ad4BP/SF-1 may be regulated by protein modification (65) and ligand binding (66, 67). Further studies are necessary to understand their biological functions as transcription factors and the regulatory mechanisms of the Ftz-f1 and Dax-1 gene expression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cloning of Mouse Dax-1 Gene and cDNA
By screening of a mouse 129J genomic library (Stratagene, La Jolla, CA) with a 32P-labeled human DAX-1 gene fragment (68), three positive clones were obtained. The regions containing 5'-upstream and two exons were mapped and sequenced. Since Dax-1 gene was divided into two exons by an intron, the full-length cDNA was obtained by deleting the intron as follows. Two oligonucleotide primers from the 340th to the 346th amino acid in the first exon (5'-GCCACAGAGCAGCCACAGATG-3') and 3'-untranslated region in the second exon (5'-TGCACTTGAGTGACATATGCC-3') were used for RT-PCR to synthesize a coding fragment from the 340th amino acid to the termination codon. The PCR product was digested with SacII, and the resulting fragment containing 3'-downstream from the SacII site was ligated with a SacII-digested fragment containing the amino-terminal coding region.

Construction of Plasmids for Promoter Analyses
A 2.5-kb long fragment containing the 5'-upstream region from the initiation methionine of Dax-1 gene was subcloned into pSV00CAT (69) to produce DaxCAT2.5K. The resultant CAT constructs were used subsequently to generate a series of deletion constructs by digestion or partial digestion at restriction enzyme sites. For the construction of recombinant plasmids (DaxCAT1.6K, DaxCAT 540, DaxCAT265, DaxCAT117, and DaxCAT40), the EcoRV site at 1.6 kb, NheI site at 540 bp, NheI site at 265 bp, MscI site at 117 bp, and FspI site at 40 bp upstream from the transcriptional initiation site were used. PCR-based site-directed mutagenesis was performed to construct plasmids bearing mutated Ad4/SF-1 sites. PCR was performed with several mutated primers listed in Figs. 5AGo and 6AGo using DaxCAT540 as the template. An expression vector for Dax-1 (RSV/mDax-1) was constructed by insertion of the full-length Dax-1 cDNA into the blunt-ended SpeI site of pRc/RSV (Invitrogen, San Diego, CA). The expression vector for Ad4BP/SF-1 (RSV/Ad4BP) was described previously (25).

Cell Culture and Transient Transfection
CV-1 monkey kidney fibroblast and Y-1 mouse adrenocortical tumor cells were maintained as described previously (70, 71). R2C rat Leydig tumor cells were maintained in Ham’s F-10 medium supplemented with 2.5% FBS (Whittaker, Walkersville, MD) and 15% horse serum (Hazleton Laboratories, Lenexa, KS) at 37 C in a humidified atmosphere containing 5% CO2 and 95% air. One day before transfection, CV-1, Y-1, and R2C cells were plated at densities of 7 x 105, 5 x 105, and 1 x 106 cells per 60-mm dish, respectively. The reporter plasmids were transfected into the cultured cells with LipofectAMINE Reagent according to the protocol provided by the manufacturer (Life Technologies, Inc., Rockville, MD). To monitor the efficiency of transfection, 0.3 µg of an expression vector for luciferase (RSV/luc) was used as an internal control. Cells were harvested 48 h after lipofection and subjected to CAT assays using 1-deoxy-[dichloroacetyl-1-14C] chloramphenicol (54 mCi/mmol) (Amersham International, Amersham, UK). Luciferase assays were performed (25) using a luminometer, Microlumat LB96P (EG&G Berthold, Bad Wildbad, Germany). All transfection experiments were performed at least three times.

Electrophoretic Mobility Shift Assays
Oligonucleotides used in this study are listed in Figs. 3AGo and 6AGo and Table 1Go. Double-stranded oligonucleotides containing 5'-protruding ends were labeled by [{alpha}-32P]dCTP (110 TBq/mmol) (NEN Life Science Products, Boston, MA) with Klenow fragment (Toyobo Co., Tokyo, Japan) and used for gel mobility shift assays as probes. Nuclear extracts were prepared from Y-1 cells as described (24). In competition experiments, 25- to 50-fold excess amounts of unlabeled oligonucleotides were mixed with the labeled probes and then added to the nuclear extracts. An antiserum to Ad4BP/SF-1 was mixed with the nuclear extracts before the addition of the labeled probes. The gel mobility shift assay was performed using the method described previously by Morohashi et al. (24).

Antibody Preparation and Immunoblot Analysis
A prokaryotic expression vector for Dax-1 (pET-11b/mDax-1) was constructed by insertion of the full-length Dax-1 cDNA into the blunt-ended BamHI site of pET-11b (Stratagene, La Jolla, CA). Recombinant mouse Dax-1 protein was prepared by the procedure described previously (25). Guinea pigs were immunized with the purified Dax-1 in Ribi adjuvant according to the protocol provided by the manufacturer (Ribi ImmunoChem Research Inc., Hamilton, MT). The specific antibody to Dax-1 was purified using Formyl-cellulofine (Chisso Co., Tokyo, Japan) conjugated with the purified Dax-1.

For SDS-PAGE, tissues and cultured cells were homogenized in 50 mM Tris-HCl (pH 7.5) containing 2% SDS, and cellular DNAs were subsequently disrupted by sonication. Proteins in the homogenates were separated by SDS-PAGE and blotted onto a nitrocellulose filter as described previously (25). For immunodetection of Dax-1, the nitrocellulose filter was incubated with the primary antibodies and then peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech, Arlington Heights, IL) as described (25). ECL Western blot reagents (Amersham Pharmacia Biotech) were used for detection.

Immunohistochemical Analyses
For immunohistochemical analysis, the adrenal glands, testes, and ovaries were prepared from adult rats. The whole brains and pituitary glands were prepared from wild-type and Ftz-f1 gene-disrupted fetuses (E14.5), newborn, and adult mice. They were fixed with 4% paraformaldehyde (PFA) in PBS at 4 C, dehydrated, and embedded in paraffin wax. Sections (7 µm thick) were mounted on slides and used for immunostaining as described previously (54). The guinea pig antibody to mouse Dax-1, rabbit antiserum to bovine Ad4BP/SF-1, and rabbit antiserum to mouse FSHß (Biogenesis, Poole, UK) were used as the primary antibodies. Biotinylated antiguinea pig IgG (Vector Laboratories, Inc., Burlingame, CA) and antirabbit IgG (Nichirei Co., Tokyo, Japan) were used as the secondary antibodies. Antibody-antigen complexes were detected by the streptavidin-biotin-peroxidase or alkaline phosphatase method using Histofine kit (Nichirei Co.). When double-label immunohistochemistry was performed with the pituitary section, the first immunoreaction with anti-Dax-1 antibody was colored by peroxidase with 3, 3'-diaminobenzidine tetrahydrochloride. After the color reaction stopped, the second immunoreaction with anti-FSHß antibody was colored by alkaline phosphatase with 4-nitro-blue-tetrazolium-chloride and 5-bromo-4-chloro-3-indolyl-phosphate.

Whole Mount in Situ Hybridization
SacI/SacI and MluI/BamHI fragments were separately cloned into pBluescript (Stratagene) as probes for Ad4BP/SF-1 and Dax-1, respectively. Digoxigenin-labeled antisense and sense probes were prepared according to the instructions provided by the manufacturer (Roche Molecular Biochemicals, Mannheim, Germany). Whole-mount in situ hybridization was performed as described previously (72) with minor modifications. Wild-type and Ftz-f1 gene-disrupted mouse fetuses (E10.5 and E14.5) were fixed with 4% PFA in PBS at 4 C, dehydrated, and stored at 4 C until use. The developing gonads and brains were dissected, followed by rehydration and washing twice with PBS containing 0.1% Triton X-100 (PBST). After treatment with 10 µg/ml proteinase K for 20–30 min at room temperature, the samples were refixed with 0.2% glutaraldehyde-4% PFA for 20 min followed by washing twice with PBST. The samples were incubated for more than 2 h at 65 C with prehybridization solution containing 50% formaldehyde, 5 x SSC, 2% blocking powder (Roche Molecular Biochemicals), 0.1% Triton X-100, 0.5% 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPS), 1 mg/ml yeast RNA, 5 mM EDTA, and 50 µg/ml heparin. They were then used for hybridization at 65 C overnight in a prehybridization solution with digoxigenin-labeled probes (< 1 µg/ml). Washing was performed at 65 C in solution 1 (50% formamide, 5 x SSC, 0.1% Triton X-100, and 0.5% CHAPS), a 3:1 mixture of solution 1 and 2x SSC, a 1:1 mixture of solution 1 and 2x SSC, and a 1:3 mixture of solution 1 and 2x SSC successively. This was followed by further washing with 2x SSC containing 0.1% CHAPS and 0.2x SSC containing 0.1% CHAPS at 65 C. After changing the solution to TBTX (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Triton X-100), samples were blocked with 10% sheep serum and 2% BSA in TBTX for a few hours to prevent nonspecific binding of antidigoxigenin antibody. Samples were then incubated overnight at 4 C with an alkaline phosphatase-conjugated antidigoxigenin antibody preabsorbed with embryo powder. Before the color reaction, samples were washed with TBTX for several hours at room temperature. The samples were incubated with 4-nitro-blue-tetrazolium-chloride and 5-bromo-4-chloro-3-indolyl-phosphate in NTMT (100 mM NaCl, 100 mM Tris-HCl, pH 9.5, 50 mM MgCl2, and 0.1% Tween-20). After completion of the color reaction, the samples were washed several times with PBS containing 1% Triton X-100 and fixed with 4% PFA in PBXT.


    ACKNOWLEDGMENTS
 
We thank Dr. En Li (Harvard Medical School, Boston, MA) for kindly supplying the Ftz-f1 gene-disrupted mice and Prof. Yoshitake Nishimune (Osaka University, Suita, Japan) for his help in determining the stage of seminiferous tubules.


    FOOTNOTES
 
Address requests for reprints to: Ken-ichirou Morohashi, Ph.D., Department of Developmental Biology, National Institute for Basic Biology, Myodaiji-cho, Okazaki 444-8585, Japan.

This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and grants from Sumitomo Foundation, Suzuken Memorial Foundation, Uehara Memorial Foundation, and Asahi Glass Foundation.

Received for publication November 30, 1998. Revision received April 21, 1999. Accepted for publication April 29, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Call KM, Glaser T, Ito CY, Buckler JA, Pelletier J, Haber DA, Rose EA, Kral A, Yeger H, Lewis WH, Jones C, Housman DE 1990 Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms’ tumor locus. Cell 60:509–520[Medline]
  2. Gessler M, Poustka A, Cavenee W, Neve RL, Orkin SH, Bruns GAP 1990 Homozygous deletion in Wilms’ tumors of a zinc-finger gene identified by chromosome jumping. Nature 343:774–778[CrossRef][Medline]
  3. Pelletier J, Bruening W, Kashtan CE, Mauer SM, Manivel JC, Striegel JE, Houghton DC, Junien C, Habib R, Fouser L, Fine RN, Silverman BL, Haber, DH, Housman D 1991 Germline mutations in the Wilms’ tumor suppressor gene are associated with abnormal urogenital development in Denys-Drash syndrome. Cell 67:437–447[Medline]
  4. Gubbay J, Collingnon J, Koopman P, Capel B, Economou A, Münsterberg A, Vivian N, Goodfellow P, Lovell-Badge R 1990 A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature 346:245–250[CrossRef][Medline]
  5. Koopman P, Gubbay J, Vivian N, Goodfellow P, Lovell-Badge R 1991 Male development of chromosomally female mice transgenic for Sry. Nature 351:117–121[CrossRef][Medline]
  6. Ikeda Y, Shen W, Ingraham HA, Parker KL 1994 Developmental expression of mouse steroidogenic factor-1, an essential regulator of the steroid hydroxylases. Mol Endocrinol 8:654–662[Abstract]
  7. Hatano O, Takayama K, Imai T, Waterman MR, Takakusu A, Omura T, Morohashi K 1994 Sex-dependent expression of a transcription factor, Ad4BP, regulating steroidogenic P-450 genes in the gonads during prenatal and postnatal rat development. Development 120:2787–2797[Abstract/Free Full Text]
  8. Hatano O, Takakusu A, Nomura M, Morohashi K 1996 Identical origin of adrenal cortex and gonad revealed by expression profiles of Ad4BP/SF-1. Genes Cells 1:663–671[Abstract/Free Full Text]
  9. Wanger T, Wirth J, Meyer J, Zabel B, Held M, Zimmer J, PasantesJ, Bricarelli FD, Keutel J, Hustert E, Wolf U, Tommerup N, Schempp W, Scherer G 1994 Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 79:1111–1120[Medline]
  10. Kent J, Wheatley SC, Andrews JE, Sinclair AH, Koopman P 1996 A male-specific role for SOX9 in vertebrate sex determination. Development 122:2813–2822[Abstract/Free Full Text]
  11. Silva SMD, Hacker A, Harley V, Goodfellow P, Swain A, Lovell-Badge R 1996 Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds. Nat Genet 14:62–67[Medline]
  12. Zanaria E, Muscatelli F, Bardoni B, Strom TM, Guioli S, Guo W, Lalli E, Moser C, Walker AP, McCabe ERB, Meitinger T, Monaco AP, Sassone-Corsi P, Camerino G 1994 An unusual member of the nuclear hormone receptor superfamily responsible for X-linked adrenal hypoplasia congenita. Nature 372:635–641[CrossRef][Medline]
  13. Muscatelli F, Strom TM, Walker AP, Zanaria E, Recan D, Meindl A, Bardoni B, Guioli S, Zehetner G, Rabl W, Schwarz HP, Kaplan J-C, Camerino G, Meitinger T, Monaco AP 1994 Mutations in the DAX-1 gene give rise to both X-linked adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Nature 372:672–676[CrossRef][Medline]
  14. Morohashi K 1997 The ontogeny of the steroidogenic tissues. Genes Cells 2:95–106[Abstract/Free Full Text]
  15. Parker KL, Scimmer BP 1997 Steroidogenic factor 1: A key determinant of endocrine development and function. Endocr Rev 18:361–377[Abstract/Free Full Text]
  16. Yu RN, Acherman JC, Ito M, Jameson JL 1998 The role of DAX-1 in reproduction. Trends Endocrinol Metab 9:169–1175[CrossRef]
  17. Honda S, Morohashi K, Omura T 1990 Novel cAMP regulatory elements in the promoter region of bovine P-450(11ß) gene. J Biochem 108:1042–1049[Abstract]
  18. Rice DA, Kirkman MS, Aitken LD, Mouw AR, Schimmer BP, Parker KL 1990 Analysis of the promoter region of the gene encoding mouse cholesterol side-chain cleavage enzyme. J Biol Chem 265:11713–11720[Abstract/Free Full Text]
  19. Parker KL, Schimmer BP 1993 Transcriptional regulation of the adrenal steroidogenic enzymes. Trends Endocrinol Metab 4:46–50
  20. Morohashi K, Omura T 1996 Ad4BP/SF-1, a transcription factor essential for the transcription of steroidogenic cytochrome P450 genes and for the establishment of the reproductive function. FASEB J 10:1569–1577[Abstract/Free Full Text]
  21. Luo X, Ikeda Y, Parker KL 1994 A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77:481–490[Medline]
  22. Shinoda K, Lei H, Yoshii H, Nomura M, Nagano M, Shiba H, Sasaki H, Osawa Y, Ninomiya Y, Niwa O, Morohashi K, Li E 1995 Developmental defects of the ventromedial hypothalamic nucleus and pituitary gonadotroph in the Ftz-F1 disrupted mice. Dev Dynam 204:22–29[Medline]
  23. Sadovsky Y, Crawford PA, Woodson KG, Polish JA, Clements MA, Tourtellotte LA, Simburger K, Milbrandt J 1995 Mice deficient in the orphan receptor steroidogenic factor 1 lack adrenal glands and gonads but express P450 side-chain-cleavage enzyme in the placenta and have normal embryonic serum levels of corticosteroids. Proc Natl Acad Sci USA 92:10939–10943[Abstract]
  24. Morohashi K, Honda H, Inamata Y, Handa H, Omura T 1992 A common trans-acting factor, Ad4-binding protein, to the promoters of steroidogenic P-450s. J Biol Chem 267:17913–17919[Abstract/Free Full Text]
  25. Morohashi K, Zanger UM, Honda S, Hara M, Waterman MR, Omura T 1993 Activation of CYP11A and CYP11B gene promoters by the steroidogenic cell-specific transcription factor, Ad4BP. Mol Endocrinol 7:1196–1204[Abstract]
  26. Fitzpatrick SL, Richards JS 1993 Cis-acting elements of the rat aromatase promoter required for cyclic adenosine 3',5'-monophosphate induction in ovarian granulosa cells and constitutive expression in R2C Leydig cells. Mol Endocrinol 7:341–354[Abstract]
  27. Watanabe N, Kitazume M, Fujisawa J, Yoshida M, Fujii-Kuriyama Y 1993 A novel cAMP-dependent regulatory region including a sequence like the cAMP-responsive element, far upstream of the human CYP21A2 gene. Eur J Biochem 214:521–531[Abstract]
  28. Takayama K, Morohashi K, Honda S, Hara N, Omura T 1994 Contribution of Ad4BP, a steroidogenic cell-specific transcription factor, to regulation of the human CYP11A and bovine CYP11B genes through their distal promoters. J Biochem 116:193–203[Abstract]
  29. Michael MD, Kilgore M, Morohashi K, Simpson MR 1995 Ad4BP/SF-1 regulates cyclic AMP-induced transcription from the proximal promoter (PII) of the human aromatase P450(CYP19) gene in the ovary. J Biol Chem 270:13561–13566[Abstract/Free Full Text]
  30. Bakke M, Lund J 1995 Mutually exclusive interactions of two nuclear orphan receptors determine activity of a cyclic adenosine 3',5'-monophosphate-responsive sequence in the bovine CYP17 gene. Mol Endocrinol 9:327–339[Abstract]
  31. Leers-Sucheta S, Morohashi K, Mason JI, Melner MH 1997 Synergistic activation of the human type II 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4 isomerase promoter by the transcription factor steroidogenic factor 1/Adrenal 4-binding protein and phorbol ester. J Biol Chem 272:7960–7967[Abstract/Free Full Text]
  32. Caron KM, Ikeda Y, Soo S-C, Stocco DM, Parker KL, Clark BJ 1997 Characterization of promoter region of the mouse gene encoding the steroidogenic acute regulatory protein. Mol Endocrinol 11:138–147[Abstract/Free Full Text]
  33. Sugawara T, Kiriakidou M, McAllister JM, Kallen CB, Straus III JF 1997 Multiple steroidogenic factor 1 binding elements in the human steroidogenic acute regulatory protein gene 5'-franking region are required for maximal promoter activity and cyclic AMP responsiveness. Biochemistry 36:7249–7255[CrossRef][Medline]
  34. Giuili G, Shen W-H, Ingraham H 1997 The nuclear receptor SF-1 mediates sexually dimorphic expression of Mullerian inhibiting substance, in vivo. Development 124:1799–1806[Abstract/Free Full Text]
  35. Ito M, Yu R, Jameson JL 1997 DAX-1 inhibits SF-1-mediated transactivation via a carboxy-terminal domain that is deleted in adrenal hypoplasia congenita. Mol Cell Biol 17:1476–1483[Abstract]
  36. Crawford PA, Dorn C, Sadovsky Y, Milbrandt J 1998 Nuclear receptor DAX-1 recruits nuclear receptor corepressor N-CoR to steroidogenic factor 1. Mol Cell Biol 18:2949–2956[Abstract/Free Full Text]
  37. Zazopoulos E, Lalli E, Stocco DM, Sassone-Corsi P 1997 DNA binding and transcriptional repression by DAX-1 blocks steroidogenesis. Nature 390:311–315[CrossRef][Medline]
  38. Nachtigal MW, Hirokawa Y, Enyeart-VanHouten DL, Flanagan JN, Hammer GD, Ingraham HA 1998 Wilms’ tumor 1 and Dax-1 modulate the orphan nuclear receptor SF-1 in sex-specific gene expression. Cell 93:445–454[Medline]
  39. Viger RS, Mertineit C, Trasler JM, Nemer M 1998 Transcription factor GATA-4 is expressed in a sexually dimorphic pattern during mouse gonadal development and is a potent activator of the Müllerian inhibiting substance promoter. Development 125:2665–2675[Abstract/Free Full Text]
  40. Barbara PDS, Bonneaud N, Boizet B, Desclozeaux M, Moniot B, Sudbeck P, Scherer G, Poulat F, Berta P 1998 Direct interaction of SRY-related protein SOX9 and steroidogenic factor 1 regulates transcription of the human anti-Müllerian hormone gene. Mol Cell Biol 18:6653–6665[Abstract/Free Full Text]
  41. Nomura M, Bartsch S, Nawata H, Omura T, Morohashi K 1995 An E box element is required for the expression of the ad4bp gene, a mammalian homologue of ftz-f1 gene, which is essential for adrenal and gonadal development. J Biol Chem 270:7453–7461[Abstract/Free Full Text]
  42. Woodson KG, Crawford PA, Sadovsky Y, Milbrandt J 1997 Characterization of the promoter of SF-1, an orphan nuclear receptor required for adrenal and gonadal development. Mol Endocrinol 11:117–126[Abstract/Free Full Text]
  43. Harris AN, Mellon PL 1998 The basic helix-loop-helix, lucine zipper transcription factor, USF (upstream stimulatory factor), is a key regulator of SF-1 (steroidogenic factor-1) gene expression in pituitary gonadotrope and steroidogenic cells. Mol Endocrinol 12:714–726[Abstract/Free Full Text]
  44. Burris TP, Guo W, Le T, McCabe RB 1995 Identification of a putative steroidogenic factor-1 response element in the DAX-1 promoter. Biochem Biophys Res Commun 214:576–581[CrossRef][Medline]
  45. Ikeda Y, Swain A, Weber TJ, Hentges KE, Zanaria E, Lalli E, Tamai KT, Sassone-Corsi P, Lovell-Badge R, Camerino G, Parker KL 1996 Steroidogenic factor-1 and Dax-1 colocalize in multiple cell lineages: potential links in endocrine development. Mol Endocrinol 10:1261–1272[Abstract]
  46. Vilain E, Guo W, Zhang Y-H, McCabe ERB 1997 DAX1 gene expression upregulated by steroidogenic factor 1 in an adrenocortical carcinoma cell line. Biochem Mol Med 61:1–8[CrossRef][Medline]
  47. Yu RN, Ito M, Jameson JL 1998 The murine Dax-1 promoter is stimulated by SF-1 (steroidogenic factor-1) and inhibited by COUP-TF (chicken ovalbumin upstream promoter-transcription factor) via a composite nuclear receptor-regulatory element. Mol Endocrinol 12:1010–1022[Abstract/Free Full Text]
  48. Ikeda Y, Lala DS, Luo X, Kim E, Moisan M-P, Parker KL 1993 Characterization of the mouse FTZ-F1 gene, which encodes a key regulator of steroid hydroxylase gene expression. Mol Endocrinol 7:852–860[Abstract]
  49. Morohashi K, Iida H, Nomura M, Hatano O, Honda S, Tsukiyama M, Niwa O, Hara T, Takakusu A, Shibata Y, Omura T 1994 Functional difference between Ad4BP and ELP, and their distributions in steroidogenic tissues. Mol Endocrinol 8:643–653[Abstract]
  50. Tamai KT, MonacoL, Alastalo T-P, Lalli E, Parvinen M, Sassone-Corsi P 1996 Hormonal and developmental regulation of DAX-1 expression in Sertoli cells. Mol Endocrinol 10:1561–1569[Abstract]
  51. Takayama K, Sasano H, Fukaya T, Morohashi K, Suzuki T, Tamura M, Costa MJ, Yajima A 1995 Immunohistochemical localization of Ad4-binding protein with correlation to steroidogenic enzyme expression in cycling human ovaries and sex cord stromal tumors. J Clin Endocrinol Metab 80:2815–2821[Abstract]
  52. Ingraham HA, Lala DS, Ikeda Y, Luo X, Shen W-H, Nachtingal MW, Abbud R, Nilson JH, Parker KL 1994 The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Gene Dev 8:2302–2312[Abstract]
  53. Ikeda Y, Luo X, Abbud R, Nilson JH, Parker KL 1995 The nuclear receptor steroidogenic factor 1 is essential for the formation of the ventromedial hypothalamic nucleus. Mol Endocrinol 9:478–486[Abstract]
  54. Nomura M, Kawabe K, Matsushita S, Oka S, Hatano O, Harada N, Nawata H, Morohashi K 1998 Adrenocortical and gonadal expression of the mammalian Ftz-F1 gene encoding Ad4BP/SF-1 is independent of pituitary control. J Biochem 124:217–224[Abstract]
  55. Ueno S, Takahashi M, Manganaro TF, Ragin RC, Donahoe PK 1989 Cellular localization of Müllerian inhibiting substance in the developing rat ovary. Endocrinology 124:1000–1006[Abstract]
  56. Münsterberg A, Lovell-Badge R 1991 Expression of the mouse anti-Müllerian hormone gene suggests a role in both male and female sexual differentiation. Development 113:613–624[Abstract]
  57. Swain A, Zanaria E, Hacker A, Lovell-Badge R, Camerino G 1996 Mouse Dax-1 expression is consistent with a role in sex determination as well as in adrenal and hypothalamus function. Nat Genet 12:404–409[Medline]
  58. Swain A, Narvaez V, Burgoyne P, Camerino G, Lovell-Badge R 1998 Dax-1 antagonizes Sry action in mammalian sex determination. Nature 391:761–767[CrossRef][Medline]
  59. Majdic G, Saunders PTK 1996 Differential patterns of expression of DAX-1 and steroidogenic factor-1 (SF-1) in the fetal rat testis. Endocrinology 137:3586–3589[Abstract]
  60. Liu Z, Simpson ER 1997 Steroidogenic factor 1 (SF-1) and SP1 are required for regulation of bovine CYP11A gene expression in bovine luteal cells and adrenal Y1 cells. Mol Endocrinol 11:127–137[Abstract/Free Full Text]
  61. Carlone D, Richards JS 1997 Functional interaction, phosphorylation, and levels of 3',5'-cyclic adenosine monophosphate-regulatory element binding protein and steroidogenic factor-1 mediate hormone-regulated and constitutive expression of aromatase in granulosa cells. Mol Endocrinol 11:292–304[Abstract/Free Full Text]
  62. Zhang P, Mellon SH 1997 Multiple orphan nuclear receptors converge to regulate rat P450c17 gene transcription: novel mechanisms for orphan nuclear receptor action. Mol Endocrinol 11:891–904[Abstract/Free Full Text]
  63. Bischof LJ, Kagawa N, Moskow JJ, Takahashi Y, Iwamatsu A, Buchberg AM, Waterman MR 1998 Members of the Meis1 and Pbx homeodomain protein families cooperatively bind a cAMP-responsive sequence (CRS1) from bovine CYP17. J Biol Chem 273:7941–7948[Abstract/Free Full Text]
  64. Lee LL, Sadovsky Y, Swirnoff AH, Polish JA, Goda P, Gavrilian G, Milbrant J 1996 Luteinizing hormone deficiency and female infertility in mice lacking the transcription factor NGFI-A (Egr-1). Science 273:1219–1221[Abstract]
  65. Zhang P, Mellon SH 1996 The orphan nuclear receptor steroidogenic factor-1 regulates the cyclic adenosine 3',5'-monophosphate-mediated transcriptional activation of rat cytochrome P450c17 (17{alpha}-hydroxylase/c17–20 lyase). Mol Endocrinol 10:147–158[Abstract]
  66. Lala DS, Syka PM, Lazarchik SB, Mangelsdorf DJ, Parker KL, Heyman RA 1997 Activation of the orphan nuclear receptor steroidogenic factor 1 by oxysterol. Proc Natl Acad Sci USA 94:4895–4900[Abstract/Free Full Text]
  67. Mellon S, Bair SR 1998 25-Hydroxycholesterol is not a ligand for the orphan nuclear receptor steroidogenic factor-1 (SF-1). Endocrinology 139:3026–3029[Abstract/Free Full Text]
  68. Yanase T, Takayanagi R, Oba K, Nishi Y, Ohe K, Nawata H 1996 New mutation of DAX-1 genes in two Japanese patients with X-linked congenital adrenal hypoplasia and hypogonadotropic hypogonadism. J Clin Endocrinol Metab 81:530–535[Abstract]
  69. Araki E, Shimada F, Shiciri M, Mori M, Ebina Y 1988 pSV00CAT: low background CAT plasmid. Nucleic Acids Res 16:1627[Medline]
  70. Morohashi K, Nonaka Y, Kirita S, Hatano O, Takakusu A, Okamoto M, Omura T 1990 Enzymatic activities of P-450(11ß)s expressed by two cDNAs in COS-7 cells. J Biochem 107:635–640[Abstract]
  71. Kirita S, Hashimoto T, Kitajima M, Honda S, Morohashi K, Omura T 1990 Structural analysis of multiple bovine P-450(11ß) genes and their promoter activities. J Biochem 108:1030–1041[Abstract]
  72. Hogan BLM, Beddington RSP, Costantini F, Lacy E 1994 Manipulating the Mouse Embryo, ed. 3. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p 325