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
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ABSTRACT
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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.
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INTRODUCTION
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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.
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RESULTS
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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. 1A
) 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. 1B
, 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.
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To identify the cis-element(s), several overlapping
oligonucleotides ranging from -117 to -40 bp were synthesized (Fig. 2A
and Table 1
) and used for electrophoretic mobility
shift assays (EMSAs) using a nuclear extract prepared from Y-1 cells.
As shown in Fig. 2B
, 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. 2C
), 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. 3A
). Three oligonucleotides, DaxEY-91Md,
Me, and Mf, did not compete out the complex formation (Fig. 3B
),
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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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 ( Ad4BP/SF-1) was mixed with nuclear extracts
before the addition of the probe. Arrowhead indicates a
DNA-protein complex.
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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 DaxAd42 (Fig. 4A
). 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 (DaxAd41) (Fig. 4A
). Although the
reported sequence of the human gene did not cover the region containing
DaxAd41, comparison of the nucleotide sequences between the two
animals showed that nucleotide sequences corresponding to DaxAd42
were identical. In addition, the newly identified DaxAd43 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. 4B
, 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. DaxAd41, DaxAd42, and
DaxAd43 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. 1A ) 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.
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To determine which Ad4/SF-1 sites are responsible for
Ad4BP/SF-1-mediated transcription, we transfected the deletion
constructs indicated in Fig. 1A
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 DaxAd41
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 DaxAd41 and DaxAd42 sequences. Finally, DaxCAT40
lacking all Ad4/SF-1 sites, DaxAd41, DaxAd42, and DaxAd43,
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. 5A
. The impaired
abilities as the binding sequence for Ad4BP/SF-1 were confirmed by
EMSAs. As shown in Fig. 5B
, the binding activities of the mutated
oligonucleotides, DaxAd41M and DaxAd43M, were significantly
decreased when they were used as probes. A significant decrease was
also observed with DaxAd42M, 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 DaxAd41, -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 1 ) containing intact (DaxAd41, -2,
and -3) and mutated Ad4/SF-1 sites (DaxAd41M, -2M, and -3M) were
performed. The nucleotide sequence of DaxAd43 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
( 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.
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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. 5C
, the CAT activity of
DaxCAT1M, which contained a disrupted DaxAd41, was not diminished
compared with that of the intact construct, DaxCAT540. In contrast, the
CAT activity of DaxCAT2M, which contained a disrupted DaxAd42,
decreased to 48% relative to the intact construct. Similarly,
disruption of DaxAd43 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
3853%. In addition, further reduction of CAT activity was noted by
disruption of all binding sites (DaxAd41, DaxAd42, and
DaxAd43).
As pointed out above, mutation at DaxAd42 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 DaxAd42. As indicated in Fig. 6A
, we tentatively designate the Ad4/SF-1
sites located at 5'- and 3'-sides DaxAd42a and DaxAd42b,
respectively. To determine the functional contribution of each
Ad4/SF-1 site, we prepared an additional CAT reporter construct,
DaxCAT2bM, which contained disrupted DaxAd42b. The transcriptional
activity of DaxCAT2bM driven by Ad4BP/SF-1 was compared with that of
DaxCAT2aM, which was identical to DaxCAT2M in Fig. 5
. As indicated in
Fig. 6B
, 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 DaxAd42bM, it was retained as confirmed by EMSA,
probably due to the intact DaxAd42a. A complete disappearance of the
binding activity was achieved by disruption of both DaxAd42a and
DaxAd42b (data not shown). Taken together, these results clearly
indicate that at least three Ad4/SF-1 sites, DaxAd42a, DaxAd42b,
and DaxAd43, 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 DaxAd42
A, DaxAd42a (shaded) is identical to DaxAd42, while
DaxAd42b (boxed) is another Ad4/SF-1 site pointed out
by Yu et al. (47 ). DaxAd42aM and DaxAd42bM
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. 6A 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.
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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. 7A
, 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. 7B
).

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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. CH,
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.
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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. 7C
). DAX-1 immunoreactive cells were mainly localized
in the zona glomerulosa (g in Fig. 7D
), while a few cells were stained
weakly in the zonae fasciculata and reticularis (f and r in Fig. 7D
).
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. 7E
), while the same cell types were stained with the
antibody to DAX-1 (Fig. 7F
). However, the intensity of staining was
stronger in Sertoli cells in the seminiferous tubule labeled as s1 than
in seminiferous tubule s2 in Fig. 7F
. 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 IVII.
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. 7
, 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. 8B
) compared with the wild
type (Fig. 8A
). When probed with Dax-1, a weak but clear signal was
detected in the urogenital ridge of the wild type (Fig. 8C
). In
contrast, any signal higher than the background level was not observed
in KO fetuses (Fig. 8D
). 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. 9
, 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. 9C
) but was undetectable in KO fetuses
(Fig. 9D
).

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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.
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Subsequently, brains from newborn mice and fetuses were subjected to
the following immunohistochemical analyses. As shown in Fig. 10A
, 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. 10B
). When the same region was stained with Dax-1
antibody, some but not all VMH cells were immunoreactive in the wild
type (Fig. 10C
). In addition, Dax-1 immunoreactive cells were detected
in the arcuate nuclei (arrows in Fig. 10C
). However, no
staining was observed in the VMH and arcuate nuclei of KO mice (Fig. 10D
). 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. 10
, E and G). However, such immunoreactive
cells were not present in the corresponding areas of KO mice (Fig. 10
, 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. 11A
) and E14.5
fetuses (Fig. 11E
) were immunoreactive for Ad4BP/SF-1. As expected, no
signal was detected in the pituitary of KO mice (Fig. 11B
) and fetuses
(Fig. 11F
). With regard to the expression of Dax-1, it was detected in
the pituitary of wild-type newborn mice (Fig. 11C
) and fetus (Fig. 11G
). However, Dax-1 expression was decreased significantly in KO
pituitary of newborn and fetuses (Fig. 11
, 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. 11
, 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 (AD) and sagittal sections of the
E14.5 fetal (EH) 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 AD, 50 µm in panels EH.
|
|

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Figure 11. Immunohistochemical Detection of Dax-1 in the
Pituitary
Sections of the pituitary glands from newborn (AD), E14.5 fetuses
(EH), 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 AH; 20
µm in I and J.
|
|
 |
DISCUSSION
|
---|
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 DaxAd42a and DaxAd42b 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 DaxAd43. Functional analyses of
various Ad4/SF-1 sites, DaxAd41, DaxAd42a, DaxAd42b, and
DaxAd43, demonstrated that mainly three, DaxAd42a, DaxAd42b, and
DaxAd43, 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, DaxAd41
contributed to a lower extent to the transcription. However, it seems
that the use of cultured cells hindered the intrinsic activity of
DaxAd41.
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, DaxAd43 overlapped with the
region. Therefore, assuming that Ad4BP/SF-1 does not recognize
DaxAd43 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
|
---|
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. 5A
and 6A
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 Hams 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. 3A
and 6A
and Table 1
. Double-stranded oligonucleotides containing
5'-protruding ends were labeled by [
-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 2030 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.
 |
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