Wnt4 overexpression disrupts normal testicular
vasculature and inhibits testosterone synthesis by
repressing steroidogenic factor 1??-catenin synergy
Brian K. Jordan*†, Jennifer H.-C. Shen†‡, Robert Olaso†‡§, Holly A. Ingraham‡¶, and Eric Vilain*
*Departments of Human Genetics, Pediatrics, and Urology, University of California School of Medicine, Los Angeles, CA 90095-7088; and‡Department of
Physiology, Graduate Program in Biomedical Sciences, University of California, San Francisco, CA 94143-0444
Communicated by Melvin M. Grumbach, University of California, San Francisco, CA, July 17, 2003 (received for review March 6, 2003)
Genetic studies in mice suggest that Wnt4 signaling antagonizes
expression of male hormones and effectively blocks male devel-
opment in the female embryo. We recently identified an XY
intersex patient carrying a chromosomal duplication of the WNT4
locus and proposed that this patient’s feminization arises from an
increased dosage of WNT4. To test this hypothesis, a transgenic
mouse was generated with a large genomic P1 containing the
human WNT4. Although a complete male to female intersex
phenotype was not observed in WNT4 transgenic male mice, a
dramatic reduction in steroidogenic acute regulatory protein
was detected consistent with the marked reduction in serum and
testicular androgen levels. Furthermore, a mild reduction of germ
cells and a disorganized vascular system were observed in testes
of WNT4 transgenic males. Consistent with these in vivo data,
Wnt4 repressed steroidogenesis in adrenocortical and Leydig cell
lines, as evidenced by reduced progesterone secretion and 3?-
hydroxysteroid dehydrogenase activity. In vitro studies showed
that Wnt4 antagonizes the functional synergy observed between
the major effector of the Wnt signaling pathway, ?-catenin
and steroidogenic factor 1, and chromatin immunoprecipitation
showed that Wnt4 attenuates recruitment of ?-catenin to the
steroidogenic acute regulatory protein promoter. Our findings
suggest a model in which Wnt4 acts as an anti-male factor by
disrupting recruitment of ?-catenin at or near steroidogenic factor
1 binding sites present in multiple steroidogenic genes.
genes direct testicular or ovarian development. Although the
precise genetic interactions responsible for testicular differen-
tiation remain unclear, both human and mouse mutants have
shown that several genes, including Sry, Sox9, SF-1, Dax1, and
Wnt4, positively or negatively regulate male sexual development.
In male mice, expression of Sry at embryonic day 10.5 triggers
several distinct morphological events, including differentiation
of Sertoli cells, migration of mesonephric cells into the gonad,
and reorganization of the gonadal vasculature (1). After expres-
sion of Sry, Sox9 is up-regulated in male mouse gonads at
embryonic day 11.5. In turn, Sox9 controls expression of target
genes, including SF-1 (2), an orphan nuclear hormone receptor
that plays a crucial role in regulating expression of three
male-specific hormones: testosterone, Mu ¨llerian inhibiting sub-
stance, and Insl-3. Coordinated action of these three hormones
is required for the normal male gonadal and reproductive tract
development (3). The overlapping and sexually dimorphic ex-
pression patterns of Sry, Sox9, and steroidogenic factor 1 (SF-1)
in the developing testis suggest that these genes are needed to
direct male sexual differentiation. Moreover, loss-of-function
mutations in each of these genes result in a male to female
human intersex phenotype.
Conversely, two genes, Dax1 and Wnt4, are thought to antag-
onize male development in a dosage-dependent manner. Dax1,
an orphan nuclear receptor, antagonizes an attenuated Sry allele
when overexpressed (4, 5). Furthermore, several in vitro studies
n the mammalian embryo, male and female urogenital ridges
are morphologically indistinct until sex-linked and autosomal
have shown that Dax1 represses SF-1-mediated gene activation
(6, 7). However, loss-of-function Dax1 mutants exhibit relatively
normal sexual development in both male and female mice (8). In
contrast, whereas both male and female Wnt4-knockout mice
exhibit similar defects in kidney development and in adrenal
function (9, 10), gonadal development and steroidogenic func-
tion are affected exclusively in the Wnt4?/?females and not in
Wnt4?/?males (11). Wnt4-null females are masculinized as
demonstrated by the absence of Mu ¨llerian ducts (presumptive
female reproductive tract) and the presence of Wolffian ducts
(presumptive male reproductive tract). In addition, mutant
Wnt4?/?females express steroidogenic enzymes required for
production of testosterone, which are normally repressed in the
female ovary; these include 3?-hydroxysteroid dehydrogenase
(3?-HSD) and 17?-hydroxylase (Cyp17) (11). Collectively, these
data suggest that Wnt4 normally functions to repress gonadal
androgen biosynthesis in females.
were originally identified as mammalian homologues of the
Drosophila wingless gene. This family of signaling factors func-
tions in a paracrine manner to effect a number of developmental
changes, including kidney development and angiogenesis (9,
12–14). Wnts bind to members of the Frizzled family of cell-
surface receptors and are known to use at least three separate
signaling pathways to effect gene expression (15). It is generally
presumed that the molecular details elucidated for Wnt1 sig-
naling will apply directly to other Wnts, including Wnt4. How-
ever, the specifics of Wnt4 signaling in gonadal or kidney
development remain unclear.
Recently, we identified an XY female with ambiguous geni-
talia carrying a large duplication of chromosome 1p35 including
phenotype results from a gain of WNT4 function. However,
because other duplicated genes are present within this chromo-
somal region, their contributions could not be ruled out. To
investigate the hypothesis that Wnt4 normally represses andro-
gen synthesis in females, we generated a gain-of-function mouse
model by using a P1 clone carrying the human WNT4 gene
controlled by its endogenous promoter. On the basis of our in
vivo and in vitro data, we propose that the high levels of Wnt4
expression directly antagonize the normal functions of SF-1 in
the female embryo.
Materials and Methods
Generation of WNT4 Transgenics. A clone (P145) containing the
Abbreviations: SF-1, steroidogenic factor 1; 3?-HSD, 3?-hydroxysteroid dehydrogenase;
immunoprecipitation; SRB1, scavenger receptor class B type 1.
†B.K.J., J.H.-C.S., and R.O. contributed equally to this work.
§Present address: Laboratoire de Neuroge ´ne ´tique Mole ´culaire, 91057 Evry Cedex, France.
¶To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2003 by The National Academy of Sciences of the USA
September 16, 2003 ?
vol. 100 ?
was isolated from a P1 library (Incyte Genomics, Palo Alto, CA).
Transgenic founders were generated in CB6F1 and C57BL?6
backgrounds (University of California Transgenic Facility,
Irvine). Genotyping for sex and transgene was performed
by PCR and Southern blotting as described (16, 17). A 1.6-kb
genomic fragment specific for WNT4 exons 3–5 was used as
probe. RNA extracted from adult mice gonads and kidneys was
analyzed qualitatively by RT-PCR for transgene expression as
Cell Culture and Transfections. The wild-type and hemagglutinin
epitope (HA)-tagged SF-1 plasmids were generated as described
(18). The mouse ?-catenin expression vector was provided by
R. Grosschedl (19), and the mouse axin plasmid was provided
by F. Costantini (Columbia University, New York). The mouse
Wnt4HA, human ?-CATENIN-S37A-HA expression vectors,
and RatB1a cells were kindly provided by M. Julius (20). The
StAR-LUC was provided by D. M. Stocco (21); the human
3?-HSD LUC was provided by S. Mellon (University of Cali-
fornia, San Francisco).
Human embryonic kidney cells (HEK293S), mouse adreno-
cortical cells (Y1), RatB1a fibroblasts (RB1), and human colo-
rectal adenocarcinoma cells (SW480) were maintained in
DMEM supplemented with antibiotics and serum (HEK293S,
5% FBS?5% calf serum; Y1, 2.5% FBS?15% horse serum; RB1,
2.5% FBS?7.5% calf serum; SW480, 10% FBS). The mouse
Leydig tumor cells (MA10) were cultured in Waymouth medium
supplemented with 15% horse serum, 20 mM Hepes, and 50
mg?ml gentamycin. Transient transfections were performed
with calcium phosphate (Specialty Media, Lavellette, NJ) for
HEK293S cells or with FuGENE 6 (Roche Molecular Biochemi-
cals) for Y1 cells. For coculture experiments, RB1 cells
were treated with or without 1 mM sodium butyrate for 24 h
before culturing with MA10 cells. Luciferase activity was deter-
mined as described (2). All transfections were performed in
triplicate and normalized with ?-galactosidase activity (carried
by pCMV-?Gal, 0.1 ?g) for transfection efficiency. TRIzol
reagent (GIBCO?BRL) was used to extract total RNA from
coculture cells; primers and RT-PCR conditions were as de-
scribed (2, 22, 23).
Western Blotting Analyses and Immunohistochemistry. Total cell
lysate (10–40 ?g) was separated by electrophoresis on SDS?
polyacrylamide gels, transferred to a nitrocellulose membrane,
blocked in 3% milk, blotted overnight at 4°C with primary
antibodies, washed, and incubated with either sheep anti-rabbit
IgG coupled to horseradish peroxidase (1:10,000) or goat anti-
mouse IgG coupled to horseradish peroxidase (1:10,000) for 1–2
h at room temperature. Rabbit anti-SRB1 (1:20,000, Novus
Biologicals, Littleton, CO), monoclonal antibodies against
?-catenin (1:10,000, BD Biosciences) or against the HA tag on
Wnt4HA (1:2,000, Covance) were used. Detection of SF-1 and
steroidogenic acute regulatory protein (StAR) proteins was as
described (24). Blots were developed by using a chemilumines-
cence kit (ECL, Amersham Biosciences). Sections of paraffin-
embedded testes were probed with c-kit antibody (1:2,000, Santa
Cruz Biotechnology) to mark germ and Leydig cells.
Steroid Measurements. Progesterone concentrations in culture
medium were determined by using an125I RIA kit (ICN). All
samples were run in duplicate. The cytochemical assay for
3?-HSD activity in MA10 cells was previously described (25).
The blue enzymatic 3?-HSD signal in each transfected well was
analyzed with the NIH IMAGE program. Serum testosterone
concentrations were analyzed by Esoterix Endocrinology (Cala-
basas Hills, CA). Mice were killed by cervical dislocation and
immediately dissected to access the heart. After an incision was
made in the right atrium, blood was collected as it pooled in the
thoracic cavity. For testicular testosterone measurement, testes
were homogenized in PBS and extracted with acetone (1:10
ratio). Excess acetone was allowed to evaporate in 37°C water
bath for 5–8 h; the remaining supernatant was subjected to
testosterone measurement with an125I RIA kit (ICN). The
Student’s t test was used for all statistical analyses in this study.
Chromatin Immunoprecipitation (ChIP) Assay. HEK293S cells were
cotransfected with SF-1 (2 ?g), ?-CATENIN S37A (2 ?g), and
the StAR promoter–reporter plasmid (4 ?g) with or without
Wnt4 (2 ?g) in 150-mm dishes. ChIP assays were carried out as
and resuspended in buffer. The resulting lysate was sonicated for
3 min on ice and cleared by centrifugation. Samples were
immunoprecipitated with the anti-?-catenin antibody (BD Bio-
sciences) and washed. Cross-linking was reversed on the bound
immunocomplex, and the reaction was subjected to 30 cycles of
PCR at 57°C for the StAR promoter and at 55°C for the control
HSP70 promoter gene expression. The following primers were
used: StAR-FOR, 5?-GCGAGGACAGGGCTTGAAGT-3?;
StAR-REV, 5?-CGCAGATCAAGTGCGCTGCC-3?; HSP70-
FOR, 5?-GGATCCAGTGTTCCGTTTCC-3?; and HSP70-
Lectin Perfusion and Confocal Microscopy. Adult male mice were
anesthetized with i.p. injections of 350 ?l of 2.5% avertin
solution, followed by injection of 125 ?l of FITC-labeled lectin
(from Lycopersicon esculentum, Vector Laboratories) into the
femoral vein. Mice were perfused with 1% paraformaldehyde,
and testes were embedded in 7% SeaPlaque agarose (Biowhit-
taker). Sections (100 ?m) were mounted on slides and counter-
stained to label cell nuclei with ToPro3 in VectaShield mounting
solution (Vector Laboratories). CONFOCAL ASSISTANT was used
to reconstruct the confocal images.
Overexpression of WNT4 in Mice Disrupts Testicular Vasculature and
Testosterone Synthesis. To explore the effect of gain of WNT4
function on gonadal development and function, we generated
transgenic mice carrying the human WNT4 gene with its endog-
enous regulatory sequences (Fig. 1A). The protein encoded by
human WNT4 shares 99% identity with mouse Wnt4. Among six
identified transgenic founders, only one line, 90-2, expressed the
transgene appropriately in the kidney and gonad (data not
shown). Southern blot hybridization with a transgene-specific
probe indicated that line 90-2 carries two copies of the WNT4
transgene (Fig. 1B). Although analysis of the human WNT4
protein expression is not possible because of the lack of a reliable
anti-Wnt4 antibody, semiquantitative RT-PCR analyses showed
similar gonadal expression of both the human WNT4 and
endogenous mouse Wnt4 transcripts (Fig. 1C).
We examined the testicular vasculature, given that this is a
major characteristic feature of the testis, and found the vascu-
lature to be disrupted in WNT4 transgenic males. While the
testicular artery was present on the surface of both transgenic
and wild-type testes, the number of collateral vessels branching
off this artery was markedly reduced in transgenic animals (Fig.
1 D and E). To examine the internal vasculature, we perfused
adult males with an FITC-labeled lectin that binds specifically to
endothelial cells of blood vessels. In contrast to the highly
organized vasculature observed in wild-type testes, blood vessels
in the transgenic testes seemed disorganized and failed to
encircle the seminiferous tubules (Fig. 1 F–I). In addition, the
internal vasculature of transgenic testes showed an increase in
vascular branching with thread-like projections. This defect was
restricted to testes; all other vascular development appeared
normal (data not shown).
Previous studies suggested that loss of Wnt4 function in mice
Jordan et al.
September 16, 2003 ?
vol. 100 ?
no. 19 ?
leads to abnormal testosterone synthesis in embryonic ovaries
(11). Here, in WNT4 transgenic male mice, total serum and
testicular testosterone concentrations were significantly lower
than in wild-type littermates (Fig. 2 A and C). Lowered testicular
androgen levels suggest that a reduction in serum testosterone is
not due to the abnormal testicular vasculature, but instead
represents a primary defect in androgen synthesis in WNT4
transgenic testes. The physiological effects of low testosterone
were evident by both a weight reduction (Fig. 2B) and altered
morphology of the androgen-sensitive organ, seminal vesicles.
Seminal vesicles in WNT4 transgenic males appeared underde-
veloped, lacking the deep invaginations characteristic of the
wild-type organ (Fig. 2 D and E). Testicular histology in the
transgenic animals also revealed elongation of seminiferous
tubules in all planes of sections, and thinning of the epithelium
with a moderate reduction in round spermatids; these features
are consistent with low testosterone (Fig. 2 F and G). WNT4
transgenic males are fertile despite these low levels of testoster-
one. We also noted that the fertility and ovarian vasculature are
normal in WNT4 transgenic females (data not shown). This
as an anti-male factor and that overexpression of Wnt4 in the
female gonad does not interfere with ovarian function.
The inhibitory effects of Wnt4 on testosterone synthesis were
tested in steroidogenic Leydig MA10 cells by coculturing with an
inducible Wnt4-expressing fibroblast RatB1a (RB1) cell line. In
these cocultures, 3?-HSD was measured by a cytochemical
acid (25). We noted a significant reduction in 3?-HSD activity
(40%, Fig. 3A) and transcripts (Fig. 3B) in MA10 cells after
Wnt4 induction compared with control cocultures (without
butyric acid, Fig. 3A). Moreover, coculturing MA10 cells with
increasing ratios of Wnt4-expressing RB1 cells resulted in a
dose-dependent inhibition of 3?-HSD (Fig. 3C). Collectively,
these cellular studies support our in vivo findings that an
increased dosage of Wnt4 lowers testosterone in male mice.
?-Catenin Enhances SF-1-Mediated Transcription. Given that Wnt4
repressed steroidogenesis in MA10 cells and in transgenic mice,
we investigated how ?-catenin, a downstream effector of Wnt
signaling, might affect SF-1 action. We tested the activities of
SF-1 and ?-catenin on a reporter driven by five tandem SF-1
response elements, 5x-SF-1RE LUC (27). Cotransfection of
SF-1 and the constitutively active ?-catenin S37A dramatically
increased SF-1 reporter construct activity 10-fold over the
activity induced by SF-1 alone (Fig. 4A). Enhancement of SF-1
activity by ?-catenin increased in a dose-dependent manner and
was observed also with wild-type ?-catenin, although increased
concentrations of wild-type ?-catenin plasmid were needed to
achieve the same effect. A fully intact SF-1 was required for
enhancement by ?-catenin; SF-1 mutants in the DNA-binding
domain (Fig. 4B) or in the activation function 2 domain failed to
show this effect (data not shown). Furthermore, overexpression
of axin, a direct inhibitor of ?-catenin, attenuated the functional
synergism between SF-1 and ?-catenin (Fig. 4B).
This functional synergism between SF-1 and ?-catenin was
the genomes of transgenic mice as shown by Southern blotting. Lane 1 shows
the WNT4 hybridization signal of a transgenic mouse. Lanes 2 and 3 are
negative controls containing DNA from nontransgenic littermates. Lanes 4
and 5 are positive controls containing the same amount of human genomic
control (upper bands) and WNT4 transgene (lane 1, lower band) and the
endogenous mouse Wnt4 (lane 2, lower bands) from transgenic (Tg, Upper)
and wild-type (Wt, Lower) testes. Lanes 3 and 4 are minus RT controls corre-
sponding to lanes 1 and 2, respectively. (D and E) On the surface testicular
vasculature of transgenic males (E), a marked reduction in the number of
blood vessels and in the degree of vessel branching was observed compared
with the testes of wild-type littermates (D). (F–I) Confocal images of the
internal testicular vasculature (labeled with lectin, a green fluorescent endo-
thelial cell marker) showing organized vasculature in a wild-type testis (F and
H) and disorganized vascular branching in a WNT4 transgenic testis (G and I).
(F and G, ?40; H and I, ?100.)
Human WNT4 construct in transgenic mice. (A) Schematic represen-
adult males compared with age-matched wild-type (Wt) controls (A;**, P ? 0.01). (B) Seminal vesicles weigh significantly less in WNT4 transgenic males than
in the wild type (**, P ? 0.01). (C) Testicular testosterone was analyzed from three different pairs of age-matched (12 or 18 months) wild-type and transgenic
testes. Wild-type testosterone level (ranges from 12 to 40 ng per testis) is taken as 100% for each pair. (D and E). The deep invaginations (white arrows)
characteristic of wild-type seminal vesicles (D) are absent from the seminal vesicles of transgenic males (E). (F and G) Immunohistostaining of c-kit, a germ and
Leydig cell marker, on equivalent testes sections (identical orientation) revealed the abnormal elongation and thinning of the seminiferous tubules epithelium
in transgenic animals (G) compared with the wild type (F). (D and E, ?2; F and G, ?100.)
www.pnas.org?cgi?doi?10.1073?pnas.1834480100Jordan et al.
further tested in mouse adrenocortical Y1 cells by measuring the
basal levels of progesterone secretion. By contrast to HEK293S
cells, which are negative for both SF-1 and ?-catenin, Y1 cells
express modest amounts of ?-catenin and high levels of SF-1
(data not shown and ref. 2). Transfection of the ?-catenin S37A
mutant alone resulted in a slight increase in progesterone
secretion. Overexpression of both SF-1 and ?-catenin resulted in
a significant increase in hormone secretion, whereas cotransfec-
tion of a Wnt4 expression vector inhibited progesterone secre-
tion below basal levels (Fig. 4C). Thus, these results in Y1 cells
are similar to those obtained in MA10 cells, showing that
steroidogenesis is enhanced by SF-1 and ?-catenin and repressed
Wnt4 Represses ?-Catenin Enhancement of SF-1-Mediated Transcrip-
tion. Repression of the SF-1??-catenin functional interaction by
Wnt4 was tested further on three different SF-1-responsive
reporter constructs. These included the tandem SF-1-responsive
elements (5x-SF-1RE LUC) used above, the proximal promoter
of 3?-HSD (3?-HSD LUC), and the proximal promoter of StAR
(StAR-LUC). StAR shuttles cholesterol into the mitochondria
and is therefore a rate-limiting step in steroidogenesis. Exoge-
nous SF-1, Wnt4, and ?-catenin were transfected in HEK293S
cells. For all three reporters, we observed increased activity with
cotransfection of both SF-1 and ?-catenin (Fig. 5), although this
increase was much less robust with the 3?-HSD LUC reporter.
By contrast, Wnt4 attenuated the SF-1??-catenin activity, but
not SF-1 activity alone.
To further investigate Wnt4-induced repression in vivo, we
analyzed the expression of SF-1, ?-catenin, StAR, and scavenger
receptor class B type 1 (SRB1) in the WNT4 transgenic testes.
Both StAR and SRB1 are transcriptionally regulated by SF-1,
and SRB1 is the predominant cell-surface receptor that supplies
serum cholesterol into cytoplasm of steroidogenic tissues (28). A
significant decrease in StAR protein expression was observed in
3?-HSD expression was determined in MA10 cells cocultured with either RB1
cells (?RB1) or RB1 cells secreting Wnt4 (?RB1?Wnt4). Representative plates
of blue reflects 3?-HSD activity and is reduced after coculture of MA10 cells
with RB1 cells expressing Wnt4 (Left). The signal intensity for 3?-HSD was
quantifiedby NIH IMAGE(Right,bargraph;**,P?0.01).(Inset)HA-taggedWnt4
acid, which induces Wnt4 protein expression. (B) RT-PCR analysis of Wnt4 and
3?-HSD mRNA expression in MA10-RB1 coculture. ?-Actin was used as a
control to indicate the relative RNA input. (C) Dose-dependent reduction of
3?-HSD expression in MA10-RB1 coculture assay. For every point showing the
ratio of RB1 to MA10 cells (x axis), 3?-HSD was measured in MA10 cells
cocultured with either RB1 cells (?RB1) or RB1 cells expressing Wnt4 (?RB1?
Wnt4). Values obtained for MA10 cells cocultured with control RB1 cells are
taken as 100%.
promoter, and this activation is repressed by the Wnt4 signaling pathway. (A)
The effects of ?-catenin on SF-1 transcriptional activity are shown on a
reporter containing five copies of SF-1 response element (5x-SF1-RE LUC, 0.2
?g). SF-1 expression plasmid (0.2 ?g) was cotransfected with increasing
amounts of either ?-catenin S37A (10–200 ng, gray bars) or wild-type ?-cate-
nin (0.2–2 ?g, black bars) in HEK293S cells. Plus (?) and minus (?) indicate the
presence or absence of transfected plasmids. Luciferase activity is shown as
fold activation, where transfection of reporter alone (5x-SF1-RE LUC) is taken
as 1-fold. (Inset) Protein expression of ?-catenin, detected by using an anti-
?-catenin antibody, is shown when empty vector (?) or HA-tagged ?-catenin
S37A expression plasmid (?) was transfected into HEK293S cells. Endogenous
?-catenin from SW480 cell lysate was used as positive control (SW, far left
lane). (B) ?-Catenin enhancement of SF-1 activity was not observed with an
SF-1 mutant deleted of DNA-binding domain (SF-1 ?DBD) or after cotransfec-
tion of axin (0.2 ?g) in HEK293S cells. (C) (Left) Progesterone levels in cultured
medium were measured after Y1 cells were transfected with different com-
binations of SF-1, ?-catenin S37A, or Wnt4 expression plasmids (0.2 ?g each;
*, P ? 0.05). (Right) Protein expression of transfected ?-catenin S37A (?,
Upper), Wnt4 (?, Lower), and empty expression vector (?) is shown for Y1
?-Catenin enhances SF-1-mediated activation of SF-1-responsive
promoters. Luciferase activities of three different SF-1-responsive promoters
(A, 5x-SF1-RE LUC; B, 3?-HSD LUC; C, StAR LUC, 0.2 ?g each) were determined
in HEK293S cells transfected with different combinations of SF-1, ?-catenin
S37A, and Wnt4 expression plasmids (all plasmids 0.2 ?g, except 0.4 ?g of
Wnt4 in C, far right bar). Plus (?) and minus (?) indicate the presence or
absence of transfected plasmids. (Inset) Protein expression of Wnt4 is shown
when empty vector (?) or HA-tagged Wnt4 expression plasmid (?) was
transfected in HEK293S cells.
Wnt4 inhibits the SF-1 and ?-catenin synergism on SF-1 target gene
Jordan et al.
September 16, 2003 ?
vol. 100 ?
no. 19 ?
WNT4 transgenic adult testes compared with wild-type litter-
mates, whereas no obvious differences were noted for SF-1,
?-catenin, and SRB1 (Fig. 6A). The lowered StAR levels are
consistent with reduced testosterone observed in the WNT4
transgenic mice. To further elucidate the repression by Wnt4
signaling on StAR expression, ChIP assays were carried out in
HEK293S cells transfected with SF-1??-catenin and the StAR
sites in the proximal StAR promoter, we found that recruitment
of ?-catenin to the StAR promoter reporter is attenuated
markedly after activation of Wnt4 signaling (Fig. 6B).
In the last decade, new insights into mammalian sexual differ-
entiation have suggested that the morphological fate of the
indifferent gonad depends not on a single testis-determining
gene but on a delicate balance between genes that promote or
inhibit testis development (29). On the basis of human and
mouse genetic studies, Dax1 and Wnt4 are both proposed to
counteract male development. Our recent identification of an
XY female carrying a duplication including the WNT4 locus (16)
strengthens the hypothesis that WNT4 functions as an anti-male
factor and is consistent with the masculinized phenotype of
female embryos observed in Wnt4-null mice. In the present
study, our transgenic mouse model showed that WNT4 overex-
pression interferes with the normal development of male go-
nadal vasculature and with testosterone biosynthesis. In vitro
studies strongly suggest that Wnt4 suppresses steroid biosynthe-
sis by antagonizing SF-1-mediated gene transcription and dis-
rupting the functional synergism between SF-1 and ?-catenin.
Our findings lead to the proposal that Wnt4 signaling disrupts
recruitment of ?-catenin at or near SF-1 binding sites within
multiple steroidogenic promoters.
Our results demonstrate that overexpression of WNT4 in male
mice disrupts normal testicular vasculature and function, but
does not lead to an XY sex-reversed phenotype observed in a
human patient carrying a duplication of the WNT4 locus (16).
However, it should be noted that among the four known XY
human patients with duplications of chromosome 1p, and who
presumably overexpress WNT4, symptoms range from isolated
cryptorchidism to severe genital ambiguity. Thus, the phenotype
in our one WNT4 transgenic mouse line may simply recapitulate
the milder symptoms exhibited by human patients, or may reflect
the fact that other duplicated genes present in this region are
required to observe a fully XY sex-reversed phenotype. Dis-
crepant phenotypes between engineered mouse mutants and
human patients have been observed for other genes involved in
sex determination. In an analogous situation, duplication of the
DSS region containing DAX1 leads to XY female sex reversal in
humans, whereas Dax1 transgenic mice exhibit only a minor
delay in testicular development (4). Moreover, the XY intersex
phenotype observed in heterozygous SOX9 and SF-1 human
patients is not exhibited by either SF-1 or Sox9 heterozygous
targeted mice (24, 30). These results could imply that humans
and mice differ in their mechanisms controlling sexual develop-
ment or that they may differ in their dosage sensitivity to gene
products because of genetic backgrounds. Indeed, in Dax1
transgenic mice, an overt intersex phenotype was observed only
in a weakened Sry allelic background (4). At present, it is still
unclear whether altering the genetic background would exacer-
bate the gonadal phenotype observed in WNT4 transgenic male
mice. However, it is of interest to note that all other WNT4
transgenic founders generated on a pure C57BL?6 background
were infertile (data not shown); whether these infertile WNT4
transgenic founders mimic the more severe phenotypes found in
some WNT4-duplication XY individuals remains unknown.
In the developing testis, the formation of the coelomic vessel
and the concurrent reorganization of the existing gonadal vas-
culature mark one of the earliest sex-specific morphological
events (1). The finding that WNT4 transgenic mice exhibit a
disruption in the testicular vasculature suggests that WNT4
signaling influences this male-specific process during early go-
nadogenesis. Our findings are reminiscent of other studies
linking Wnt signaling and vascular development. For example,
Wnt2 has been shown to play a role in the vascularization of the
placenta (12), and members of the Frizzled receptors for Wnts,
FZD4 and Fzd5, have been implicated in normal angiogenesis of
the retina and in the vascularization of the yolk sac, respectively
(13, 14). In WNT4 transgenic testes, the increased internal
vasculature contrasts the decreased branching of the testicular
artery and might suggest that WNT4 primarily influences
branching of small vessels. Consistent with this notion, Wnt4
signaling has been shown to stimulate the side branching of
terminal ducts, but not major ducts, in mammary glands (31).
The in vivo and in vitro disruption of steroidogenesis by
overexpressing Wnt4 implies that this signaling factor dampens
expression of some proteins, such as StAR, which are required
for steroid biosynthesis. Here, we focused on the possibility that
Wnt4 signaling might repress the activity of SF-1, as this orphan
nuclear hormone receptor is known to regulate many steroido-
genic enzymes in conjunction with other regulatory factors. The
canonical Wnt signaling pathway is known to affect the nuclear
association of ?-catenin and members of the TCF?LEF family to
effect gene expression (15). Our analyses of the functional
SF-1 is tethered to DNA, ?-catenin is recruited. Using a
mammalian two-hybrid Lex-A fusion system, we found that the
hinge and N-terminal portion of the ligand-binding domain of
SF-1 are required for this functional interaction (data not
shown). Previous studies have shown a ligand-dependent func-
tional and physical association between the androgen receptor
and ?-catenin (32–34). However, a robust interaction between
SF-1 and ?-catenin was not observed in our hands, despite
repeated attempts to show direct interaction. Instead, we found
SRB1 (82 kDa), StAR (30 and 37 kDa), and actin (48 kDa) was analyzed from
controls. Decreased expression of both the functional (37-kDa) and the
cleaved (30-kDa) StAR proteins was observed in WNT4 transgenic mice. Pro-
tein molecular weight markers are as indicated on the left. (B) A schematic
diagram of the StAR proximal promoter is shown with the two SF-1 response
tional initiation site (?1) (21). Arrows indicate the primers used for PCR after
ChIP. Two independent ChIP experiments are shown (I and II) in HEK293S cells
after cotransfection of StAR LUC, SF-1, and ?-catenin (?SF1??CAT) with
(?Wnt4) or without Wnt4 (?Wnt4). Cells transfected with StAR LUC reporter
and empty expression plasmids were used as controls (C, left lanes). Purified
HEK293S DNA was immunoprecipitated without (input, Top) or with anti-?-
catenin antibodies (Middle). HSP70 primers were used as controls to show
nonspecific DNA binding to the anti-?-catenin antibody (HSP70, Bottom).
Wnt4 reduces ?-catenin recruitment to the proximal StAR promoter.
www.pnas.org?cgi?doi?10.1073?pnas.1834480100Jordan et al.
a weak, but reproducible, interaction between SF-1 and ?-cate-
nin (data not shown), comparable to the modest interaction
reported by Mizusaki et al. (35). The weak association between
SF-1 and ?-catenin may reflect the lack of an SF-1 ligand or,
alternatively, may suggest that additional bridging factors are
needed for the integrity of the SF-1??-catenin complex.
Although our data show that Wnt4 attenuates ?-catenin
recruitment to an SF-1 target promoter, the precise molecular
nature for this inhibitory effect remains unclear. Clearly, Wnt4
could activate repressors of SF-1, such as Dax1, as recently
proposed (35). However, our studies support a Wnt4 repression
that is independent of Dax1 because neither Y1 nor HEK293S
cells express endogenous Dax1 (data not shown). Other poten-
tial targets affected by Wnt4 signaling might include Sox pro-
teins; we found that Sox3, Sox8, and Sox9 markedly inhibit the
SF-1??-catenin functional synergy (data not shown). These data
are consistent with the known interference of the canonical
Wnt??-catenin pathway by Sox proteins via direct interaction
with ?-catenin (36, 37). The use of a noncanonical signaling
pathway by Wnt4, as proposed in the original classification of
Wnts (38), suggests that Wnt4 may antagonize the canonical
Wnt??-catenin signaling pathway as shown in other systems (39).
Activation of these noncanonical pathways could disrupt the
SF-1??-catenin functional interaction either by destabilizing the
?-catenin protein or by disrupting SF-1 DNA binding. However,
endogenous ?-catenin was not decreased after Wnt4 overex-
pression (data not shown), and there is little evidence that
?-catenin competes directly for SF-1 DNA binding. Clearly,
further experiments are needed to delineate the precise molec-
ular mechanisms that account for Wnt4 repression of SF-1-
We thank Drs. Rudi Grosschedl and Martin Julius for plasmids, Drs. W.
Miller, S. Mellon, S. Akana, M. Desclozeaux, B. Cheyette, N. Yehya, and
L. Iruela-Arispe for useful discussion, and Dr. E. Delot for insightful
comments on the manuscript. This work was supported by a French
Association pour la Recherche sur le Cancer Fellowship (to R.O.), a
University of California, San Francisco, Graduate Opportunity fellow-
ship (to J.H.S.), an American Heart Association grant, a National
Institute of Child Health and Human Development RO1 Grant, and a
National Institute of Child Health and Human Development Research
Career Development Award (to H.A.I.), a Genomic Analysis and
Interpretation National Human Genome Research Institute Training
Grant (to B.K.J.), National Institute of Child Health and Human
Development RO1 Grant HD044513, and March of Dimes Grant
5-FY01-441 (to E.V.).
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Jordan et al.
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vol. 100 ?
no. 19 ?