Human Molecular Genetics, 2008, Vol. 17, No. 19
doi:10.1093/hmg/ddn193
Advance Access published on July 9, 2008
2949 – 2955
Stabilization of b-catenin in XY gonads
causes male-to-female sex-reversal
Danielle M. Maatouk1, Leo DiNapoli1,{, Ashley Alvers1, Keith L. Parker2, Makoto M. Taketo3
and Blanche Capel1,
1
Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA, 2Department of Internal
Medicine and Pharmacology, University of Texas Southwestern, Dallas, TX 75390, USA and 3Department of
Pharmacology, Graduate School of Medicine, Kyoto University, Yoshida-Konoé-cho, Sakyo, Kyoto 606-8501, Japan
Received February 27, 2008; Revised and Accepted July 3, 2008
During mammalian sex determination, expression of the Y-linked gene Sry shifts the bipotential gonad
toward a testicular fate by upregulating a feed-forward loop between FGF9 and SOX9 to establish
SOX9 expression in somatic cells. We previously proposed that these signals are mutually antagonistic
with counteracting signals in XX gonads and that a shift in the balance of these factors leads to either
male or female development. Evidence in mice and humans suggests that the male pathway is opposed by
the expression of two signals, WNT4 and R-SPONDIN-1 (RSPO1), that promote the ovarian fate and block
testis development. Both of these ligands can activate the canonical Wnt signaling pathway. Duplication
of the distal portion of chromosome 1p, which includes both WNT4 and RSPO1, overrides the male program
and causes male-to-female sex reversal in XY patients. To determine whether activation of b-catenin is sufficient to block the testis pathway, we have ectopically expressed a stabilized form of b-catenin in the somatic
cells of XY gonads. Our results show that activation of b-catenin in otherwise normal XY mice effectively
disrupts the male program and results in male-to-female sex-reversal. The identification of b-catenin as
a key pro-ovarian and anti-testis signaling molecule will further our understanding of the mechanisms
controlling sex determination and the molecular mechanisms that lead to sex-reversal.
INTRODUCTION
The murine gonad forms at 10.5 days post-coitum (dpc) and
is initially indistinguishable between XY and XX embryos.
At this stage, the cells of the gonad are bipotential and can
follow either the male or female pathway. Many lines of
evidence suggest that sex determination in mammals centers
on a cell fate decision in the supporting cells of the gonad.
These cells can either become Sertoli cells in the testis, or
their ovarian counterpart, follicle cells. Sex determination
occurs in XY embryos when Sry is expressed in supporting
cell precursors, which leads to the rapid upregulation of
SOX9. Establishment of SOX9 expression in this precursor
population triggers a fate decision in these cells, which leads
to Sertoli cell differentiation (reviewed in 1).
SOX9 performs all known downstream functions of SRY,
and its expression is sufficient to cause complete femaleto-male sex reversal of XX embryos (2,3). In XY gonads,
expression of SRY and SOX9 leads to increased
proliferation of the coelomic epithelium, reorganization of
somatic cells into testis cord structures and migration of endothelial cells into the gonad from the mesonephros, forming a
male-specific blood vessel along the surface of the gonad
(reviewed in 4).
At a time when male embryos undergo rapid differentiation,
reorganization of the female gonad is subtle, with few obvious
changes. To date, a female sex-determining gene has not been
identified. Several years ago mice lacking Wnt4 were generated; these mice exhibited a partial female-to-male sexreversal, but lacked embryonic testis cords (5). Consistent
with this result, overexpression of Wnt4 alone in XY gonads
does not result in sex-reversal (6,7), suggesting that Wnt4 is
not the sole female sex-determining gene and that additional
factors are required to effectively disrupt the male pathway
and promote female development.
†
To whom correspondence should be addressed. Tel: þ1 9196846390; Fax: þ1 9196683467; Email: [email protected]
Present address: Cato Research, Ltd, Durham, NC 27713, USA.
# The Author 2008. Published by Oxford University Press. All rights reserved.
For Permissions, please email: [email protected]
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Human Molecular Genetics, 2008, Vol. 17, No. 19
Recent findings implicated an additional gene in female
sex-determination. Human patients carrying a mutation in
the R-SPONDIN1 (RSPO1) gene exhibited male-to-female
sex reversal (8). Similar to WNT ligands, RSPO proteins
also can affect the canonical WNT signaling pathway. Mice
lacking Rspo1 exhibit partial female-to-male sex-reversal,
similar to mice lacking Wnt4, suggesting that these genes act
within the same pathway to activate b-catenin (9,10). The phenotype of Rspo1 mutant ovaries could be rescued by stabilized
b-catenin expression in ovarian somatic cells, suggesting that
b-catenin acts downstream of Rspo1 to block testis development in XX gonads (9). Interestingly, an XY human patient
with a duplication of chromosome 1p, which includes
both the WNT4 and RSPO1 loci, showed male-to-female
sex-reversal (11), suggesting that the elevation of signals
downstream of WNT4 and RSPO1 may be sufficient to override the testis pathway.
To test the hypothesis that b-catenin signaling antagonizes
the establishment of the testis pathway, we ectopically
expressed a dominant-stable allele of b-catenin in XX and
XY gonads. Here, we show that stabilization of b-catenin in
XY gonads is sufficient to disrupt the male pathway and
promote ovarian development. These results support a role
for b-catenin in sex determination and provide a molecular
mechanism to account for XY sex-reversal cases that result
from gain-of-function mutations.
RESULTS
Stabilization of b-catenin causes male-to-female
sex reversal
To determine whether b-catenin can disrupt male development
and drive female development in XY gonads, we have
taken advantage of two previously generated mouse lines.
The Sf1-cre transgenic mice express Cre recombinase in the
gonadal somatic cells of both sexes 11.5 dpc (12). A cross
between this transgenic and Catnblox(ex3) (b-cat fl.ex3)
mice (13) results in double heterozygotes (b-cat fl.ex3/þ;
Sf1-cre), in which b-catenin is stabilized in gonadal somatic
cells. Litters from this cross yielded approximately equal
numbers of XX and XY mice (52 and 48%, respectively;
n ¼ 6 litters). However, on the basis of the phenotype of the
external genitalia, 70% of the progeny appeared female and
only 30% appeared male. We found that 100% of XY
b-cat fl.ex3/þ; Sf1-cre mice had completely feminized external
genitalia and were indistinguishable from XX wild type and
XX b-cat fl.ex3/þ; Sf1-cre littermates (Fig. 1, row 1). Internal
reproductive tracts of XY b-cat fl.ex3/þ; Sf1-cre mice lacked
male organs and instead resembled those of females (Fig. 1,
row 2). The sex-reversed gonads were located near the
kidneys similar to wild-type ovaries, and reproductive tracts
were found to have a uterus and vagina at P0 and 3 weeks
after birth.
Histological analysis at P0, prior to the formation of ovarian
follicles, showed that both wild-type and XX b-cat fl.ex3/þ;
Sf1-cre ovaries contained many meiotic germ cells (Supplementary Material, Fig. S1). Wild-type male testes had
numerous cords containing germ cells. In contrast, XY
b-cat fl.ex3/þ; Sf1-cre gonads morphologically resembled
Figure 1. Stabilization of b-catenin in XY gonads leads to sex-reversal. Litters
from crosses of b-catfl.ex3/þ and Sf1-cre mice were inspected for sex-reversal.
Genotypes are indicated above each column. Row 1 shows external genitalia
of 3-week-old mice. Reproductive tracts were dissected from newborn (P0)
pups (row 2). o, ovary; ov, oviduct; u, uterus; t, testis; epi, epididymis; sr, sexreversed gonad. H&E staining was performed on gonads from 3-week-old
mice and is shown at 10 (row 3).
ovaries and completely lacked testis cords. No germ cells
were detected at this stage. At 3 weeks of age, histological sections of wild-type female ovaries showed numerous ovarian
follicles (Fig. 1, row 3). XX b-cat fl.ex3/þ; Sf1-cre mice also
contained ovarian follicles; however, large hemorrhagic
areas were present, resembling the previously described
phenotype of b-cat fl.ex3/þ; Amhr2-Cre mice (14). Although
wild-type male testes had cords containing many germ cells,
XY b-cat fl.ex3/þ; Sf1-cre gonads structurally resembled
ovaries at P1, but lacked follicles, likely due to the absence
of germ cells.
Stabilization of b-catenin in XY gonads disrupts
formation of testis cords
We next examined the effect of stabilized b-catenin
expression on the initiation of the testis pathway during the
fetal stages when sex determination occurs. Despite the lack
of testis cords, a coelomic vessel is formed normally along
the surface of the XY b-cat fl.ex3/þ; Sf1-cre gonad (Fig. 2,
white arrow). Additionally, the size of the gonad resembled
a wild-type XY gonad, suggesting that the early proliferation
of the coelomic epithelium, which occurs specifically in XY
gonads, occurs in XY b-cat fl.ex3/þ; Sf1-cre gonads. Both of
these aspects of the testis pathway normally occur downstream
of SOX9.
Stabilization of b-catenin had a dramatic effect on SOX9
expression (Fig. 2A). At 11.5 dpc, minor differences in
SOX9 expression were detectable between XY control and
b-cat fl.ex3/þ; Sf1-cre gonads. However, SOX9 expression
rapidly declined and was limited to a small number of cells
between 12.5 and 13.5 dpc. At 12.5 and 13.5 dpc, testis
cords had organized and were clearly apparent in the XY wildtype control gonads; however, XY b-cat fl.ex3/þ; Sf1-cre
gonads lacked testis cord structure. (We previously showed
that expression of the Sf1-cre transgene at 11.5 dpc is
restricted to a small number of cells (15)). The delayed
Human Molecular Genetics, 2008, Vol. 17, No. 19
Figure 2. Male development is disrupted by the stabilization of b-catenin. (A)
XY control (row 1) and XY b-cat fl.ex3/þ; Sf1-cre gonads (row 2) at 11.5, 12.5
and 13.5 dpc were immunostained for the Sertoli-cell marker, SOX9 (green)
and PECAM1 (red), which marks germ and endothelial cells. Confocal
microscopy images were taken at 40 for 11.5 and 12.5 dpc samples and
20 for 13.5 dpc. A white arrow marks the coelomic vessel in the XY
b-cat fl.ex3/þ; Sf1-cre sample. (B) XY control and XY b-cat fl.ex3/þ; Sf1-cre
gonads were collected at 12.5 dpc and immunostained for AMH (blue) and
b-catenin (red). White arrowheads mark background staining of blood cells.
Images were taken at 40. In images where the mesonephros is present, a
white dotted line marks the gonad-mesonephric border, and all images are
oriented with the coelomic surface of the gonad up. The scale bar represents
100 mm.
stabilization of b-catenin could account for the transient
accumulation of SOX9 at 11.5 dpc. Chaboissier et al. (16)
have shown that delayed deletion of Sox9 with an independent
Sf1-cre transgene sometimes allows for the formation of the
coelomic vessel, suggesting that transient expression of
SOX9 is sufficient to initiate the signals that drive vessel
formation.
To confirm the ectopic stabilization of b-catenin in XY
b-cat fl.ex3/þ; Sf1-cre gonads, we examined b-catenin
expression at 12.5 dpc. In control XY gonads, b-catenin
was expressed on germ cell membranes and some Sertoli
cell membranes. In the XY b-cat fl.ex3/þ; Sf1-cre gonads,
b-catenin was extensively expressed throughout the gonad in
somatic cell cytoplasm and nuclei (Fig. 2B). Additionally,
immunostaining for the Sertoli cell marker anti-Mullerian
hormone (AMH) showed a rapid loss of AMH expression in
the mutant gonads. Therefore, stabilization of b-catenin in
XY b-cat fl.ex3/þ; Sf1-cre gonads disrupted the establishment
of the Sertoli cell lineage, marked by the loss of both SOX9
and AMH.
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Figure 3. Activation of b-catenin by in vitro culture with LiCl. (A) 11.5 dpc
XY gonads carrying an Sox9-ECFP transgene were cultured in control media,
or media containing LiCl for 24 h. Gonads were immunostained for PECAM1
to mark germ cells (red), b-catenin (blue) and Sox9-ECFP expression is shown
in green (marking Sertoli cells). Confocal images were taken at 40. A white
dotted line marks the gonad/mesonephros border. (B) XY gonads were cultured in 50 mM LiCl and then analyzed for sex-reversal based on the presence
of a coelomic vasculature, SOX9 expression and size. Samples were judged as
either male (blue), partial male (gray) or female (pink). Examples of the range
of cultured samples are pictured in (C). Embryos were staged by counting tail
somites (27). The number of samples analyzed at each stage is printed within
each column. The scale bar represents 50 mm.
In order to investigate the effect of earlier stabilization of
b-catenin in all cells of the gonad and mesonephros, we
performed in vitro culture of whole gonads in the presence
of a b-catenin activator. Lithium chloride (LiCl), an inhibitor
of GSK3b, activates b-catenin by blocking the pathway which
leads to its phosphorylation and subsequent degradation.
When XY gonads carrying an ECFP transgene under the
control of the Sox9 promoter (Sox9-ECFP) were cultured in
control media, a male-specific blood vessel was formed normally as well as testis cords consisting of Sox9-ECFP positive
cells surrounding germ cells. b-catenin was expressed in germ
cell membranes (Fig. 3A). When treated with 50 mM LiCl, XY
gonads expressed b-catenin throughout the gonad, and the
Sox9-ECFP reporter was rapidly downregulated. This downregulation of Sox9 is consistent with the rapid downregulation
of SOX9 in XY b-cat fl.ex3/þ; Sf1-cre gonads.
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Human Molecular Genetics, 2008, Vol. 17, No. 19
Figure 4. Ovarian development is initiated in XY gonads with stabilized
b-catenin. Litters from crosses of b-cat fl.ex3/þ and Sf1-cre mice were analyzed for FOXL2 expression, a marker of pregranulosa cells. Gonads from
12.5 and 13.5 dpc embryos were immunostained for FOXL2 (green) and
PECAM1 (red), which marks germ and endothelial cells. At 12.5 dpc, few
FOXL2 positive cells were observed in XY b-cat fl.ex3/þ; Sf1-cre gonads
(white arrows). However, by 13.5 dpc, the number of FOXL2 positive
cells was similar to wild-type XX gonads. Confocal microscopy images
are shown at 40 for 12.5 dpc and 20 at 13.5 dpc with a 40 inset. The
white dotted line marks the gonad-mesonephric border when present. The
scale bar represents 50 mm.
Sex-reversal due to exposure to LiCl in culture was variable
in our samples; therefore, in vitro culture of gonads with or
without LiCl was initiated between 16 and 20 tail somites
(11.5 dpc), to examine the effects of LiCl at different
stages. XX and XY gonads were cultured in the presence of
50 mM LiCl or in control medium for 24 h and then assayed
for three sexually dimorphic characteristics: the presence of
a coelomic vessel, expression of SOX9 and gonad size. LiCltreated XX gonads appeared normal or similar to XX control
gonads (data not shown). XY gonads cultured in LiCl lacked
an organized coelomic vessel, had decreased numbers of
SOX9 expressing cells and resembled female gonads in size;
these effects were more severe with cultures of the earliest
staged gonads (Fig. 3B and C). Although we cannot rule out
the possibility that the effects of inhibiting GSK3b, independent of b-catenin activation, cause some of the observed
results, the similarities with the in vivo results suggest that
stabilization of b-catenin is sufficient to disrupt male sex
determination. Additionally, this effect is more severe when
b-catenin activation occurs early in all cells of the XY
gonad and is not limited to the cells that express the Sf1-cre
transgene.
Sex-reversed XY gonads upregulate markers
of ovarian development
To determine whether b-catenin activation is sufficient to
induce an ovarian cell fate in XY cells, we examined the
expression of ovarian markers. FOXL2 is expressed specifically in XX somatic cells starting at 12.5 dpc and is a
marker of pre-granulosa cells (17). Examination of FOXL2
in 12.5 and 13.5 dpc XX and XX b-cat fl.ex3/þ; Sf1-cre
gonads showed a wild-type expression pattern, with FOXL2
expressed in the nuclei of somatic cells (Fig. 4). Interestingly,
FOXL2 appeared to be expressed in more cells of XX
b-cat fl.ex3/þ; Sf1-cre gonads, compared with XX controls.
Figure 5. Ovarian markers are upregulated in XY gonads expressing stabilized
b-catenin. (A) In situ hybridization for Bmp2 (row 1) at 13.5 dpc shows
expression only in XX and XY b-cat fl.ex3; Sf1-cre gonads. Similarly,
meiotic germ cells, identified by gH2AX (green) and PECAM1 (red) immunostaining (row 2), are found only in XX and XY b-cat fl.ex3; Sf1-cre
gonads. Immunostained samples were imaged using confocal microscopy
and are shown at 20. The inset shows an enlarged view of germ cells.
Scale bar represents 20 mm. (B) qPCR analysis of Rspo1, Wnt4 and Fst in
13.5 XY b-cat fl.ex3/þ; Sf1-cre versus XY controls. All results were normalized to b-actin. qPCR was performed in triplicate, and each bar represents
the average of three biological replicates.
Additionally, mutant female gonads were larger than XX controls. Although no staining was detected in XY gonads, XY
b-cat fl.ex3/þ; Sf1-cre gonads showed a small number of
cells with nuclear FOXL2 staining at 12.5 dpc and large
numbers of FOXL2-expressing cells by 13.5 dpc.
We analyzed two additional aspects of ovarian development
in the XY b-cat fl.ex3/þ; Sf1-cre gonads: expression of XX
somatic cell markers and germ cell entry into meiosis. By in
situ hybridization, Bmp2 (18) was detected in the coelomic
domain of wild-type and XY b-cat fl.ex3/þ; Sf1-cre gonads
at 14.5 dpc (Fig. 5A). The expression of Rspo1, Wnt4 and
Follistatin (Fst) (18) was examined by comparing mRNA
expression levels between 13.5 dpc control and XY
b-cat fl.ex3/þ; Sf1-cre gonads (Fig. 5B). By quantitative PCR
(qPCR), Rspo1 expression was found to be lower in mutant
gonads, compared with controls, whereas Wnt4 and Fst
expression was upregulated. We next examined germ cell
entry into meiosis. At 15.5 dpc, we detected nuclear accumulation of gH2AX (a characteristic of meiotic germ cells)
in XY b-cat fl.ex3/þ; Sf1-cre gonads, similar to XX controls
Human Molecular Genetics, 2008, Vol. 17, No. 19
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(Fig. 5A). As germ cell differentiation depends on the somatic
environment and is independent of the germ cell’s own sex
chromosomes (19 – 21), these results suggest that stabilization
of b-catenin in somatic cells of XY gonads results in the
activation of the ovarian pathway.
Ectopic stabilization of b-catenin rescues
the Wnt4 mutant phenotype
Wnt4 is necessary for ovarian development and for antagonizing aspects of male development. In XX Wnt4 null
gonads, a coelomic vessel forms and steroidogenic cells
are present, both normal characteristics of male, and not
female gonads (6). To determine whether WNT4 signals
through the canonical WNT signaling pathway, we attempted
to rescue the partial sex-reversal of XX Wnt4 null
gonads by stabilization of b-catenin. XX control and Wnt4
null gonads were explanted at 11.5 dpc and cultured in
the presence of LiCl-containing medium, or in control
medium (Fig. 6). After 24 h of culture, male-specific
vasculature was apparent in XX gonads lacking Wnt4.
However, XX Wnt4 null gonads treated with LiCl lacked a
coelomic vessel. These results suggest that stabilization of
b-catenin at 11.5 dpc throughout the Wnt4-/- gonad and
mesonephros can directly or indirectly rescue the Wnt4
mutant phenotype and block the formation of the male
coelomic vessel.
Figure 6. Stabilization of b-catenin rescues the Wnt4 mutant phenotype
observed in XX gonads. Litters from crosses of Wnt4þ/- mice were dissected,
and gonads placed in droplet cultures with or without 50 mM LiCl. After 24 h
of culture, gonads were immunostained for PECAM1 (red) to mark germ
and endothelial cells. Samples were incubated in DAPI to mark nuclei
(blue). Confocal microscopy images are shown at 40. The white dotted
line marks the gonad-mesonephric border. The scale bar represents 50 mm.
DISCUSSION
In the XX gonad, two secreted ligands, WNT4 and RSPO1, are
capable of activating the b-catenin canonical signaling
pathway, and loss of either Wnt4 or Rspo1 in mice results
in a partial sex-reversal (5,9,10). The existence of human
sex-reversed XY males carrying chromosomal duplications
suggests that overexpression of female sex-determining
genes could lead to sex-reversal. Attempts to sex reverse
XY mice by overexpression of Wnt4 have been unsuccessful,
suggesting that Wnt4 alone is insufficient to override the male
pathway (6,7). However, an XY patient carrying a duplication
that includes WNT4 and RSPO1 exhibited sex-reversal (11),
leading to the hypothesis that upregulation of both of these
ligands may be required to antagonize male development.
Here, we show that stabilization of b-catenin, the downstream
effector of WNT4 and RSPO1 signaling, was sufficient to
disrupt the male pathway in XY gonads. This led to a loss
of SOX9 and AMH expression, a failure of testis cord
formation and the increased expression of several ovarian
somatic cell markers including FOXL2, Bmp2, Wnt4 and
Fst, suggesting that the somatic lineages have switched from
a male to female fate.
Although we observed male-to-female sex-reversal of XY
b-cat fl.ex3/þ; Sf1-cre gonads, a male-specific vasculature was
formed. However, when b-catenin was stabilized in XY
gonads cultured in the presence of LiCl, formation of the
coelomic vasculature was disrupted. This discrepancy is likely
related to the timing of b-catenin stabilization relative to the
time of establishment of the male program. SOX9 expression
is sufficient to initiate all subsequent aspects of testis differen-
tiation, including the migration of mesonephric endothelial
cells (2,3). Transient SOX9 expression in XY b-cat fl.ex3/þ;
Sf1-cre gonads may initiate the male vascular program, in
contrast to the case in early LiCl-treated XY samples, where
SOX9 expression is absent. However, we cannot rule out the
possibility that b-catenin signaling has a more direct role in
vascular development. Wnt4 has been shown to block the
migration of endothelial cells into XX gonads (6). Thus, the
early stabilization of b-catenin in LiCl-treated gonads may
have a more direct effect on blocking endothelial migration.
XX gonads lacking Wnt4 exhibit a partial sex-reversal and lose
expression of ovarian somatic markers including Fst and Bmp2
(5,18). As these genes are thought to be downstream of Wnt4
signaling, we hypothesized that they would be upregulated by
b-catenin signaling. Additionally, gonads lacking Rspo1 show
decreased Wnt4 expression, suggesting that b-catenin signaling
might also positively regulate Wnt4 (9,10). We examined
expression of these genes in XY b-cat fl.ex3/þ; Sf1-cre gonads
and found that increased b-catenin signaling leads to increased
expression of Wnt4, Fst and Bmp2. However, Rspo1 was downregulated, suggesting that its expression is negatively correlated
with high levels of b-catenin signaling. Additionally, we
observed that both XX and XY b-cat fl.ex3/þ; Sf1-cre gonads
were larger than controls. This might suggest that b-catenin
promotes the survival or proliferation of an ovarian somatic
cell population. Consistent with this, we observed a larger
number of FOXL2-expressing cells in XX b-cat fl.ex3/þ;
Sf1-cre gonads when compared with XX controls (Fig. 4). In
wild-type gonads, a negative-feedback loop may regulate
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Human Molecular Genetics, 2008, Vol. 17, No. 19
levels of b-catenin to maintain the size of the ovary; this
regulation would be lost when b-catenin is artificially stabilized,
leading to the increased size. However, more work is required to
test the role of b-catenin on patterning of ovarian somatic cells.
Previously, we proposed a model in which an antagonistic
relationship between SOX9 and WNT4 exists within the bipotential gonad (22). This relationship is likely to be indirect
as WNT4 is an extracellular ligand and SOX9 is a nuclear
transcription factor. Our data suggest that b-catenin mediates
this antagonistic relationship. Similarly, Chang et al. (23)
recently performed a similar experiment in which b-catenin
was stabilized specifically in Sertoli cells. Although
b-catenin stabilization occurred later than in our mouse
model (13.5 dpc), they also observed a decrease in the
SOX9 expression by E14.5. Stabilized b-catenin could
antagonize SOX9 in several ways. As b-catenin from the
recombined b-cat fl.ex3 allele cannot be degraded, b-catenin
can translocate to the nucleus and could interact directly with
SOX9, compete for a binding partner or target site or bind to
the Sox9 promoter to negatively regulate its expression. A negative interaction between SOX9 and b-catenin has previously
been documented during chondrocyte differentiation, in which
heterodimerization of SOX9 and b-catenin leads to the
mutual degradation of both proteins (24).
Our work strongly suggests that antagonism between SOX9
and b-catenin is the molecular mechanism through which the
fate of the supporting cell lineage in the gonad is established
to drive male or female sex determination. This competition
is pushed toward the ovarian pathway by RSPO1 and WNT4,
which act to establish b-catenin as the dominant signal in XX
gonads. This provides a potential mechanistic explanation for
how male-to-female sex-reversal can occur in XY patients
who lack mutations in genes required for the activation of the
male program; stabilization of b-catenin counteracts the
effects of SRY by destabilizing its only known downstream
target, SOX9.
MATERIALS AND METHODS
Mice and matings
The Sf1-cre transgenic (12), the Wnt4 deletion strain (5) and
the Catnblox(ex3) (b-cat fl.ex3) (13) mouse lines were maintained
on the C57BL/6 background. To generate mice expressing the
stabilized form of b-catenin specifically in the gonad, Sf1-cre
males were mated to b-cat fl.ex3 females. Mice carrying a
single copy of the Sf1-cre transgene, or a single copy of the
b-cat fl.ex3 allele, were indistinguishable from wild-types
and used interchangeably as controls. Mutant mice were
designated as b-cat fl.ex3/þ; Sf1-cre.
Immunohistochemistry, histology
and in situ hybridization
Following timed matings, gonads were dissected from embryos
and fixed overnight at 48C in 4% paraformaldehyde. Samples
were immunostained as whole mounts using the following antibodies: SOX9 (1:200; a gift of F. Poulat, Institut deGenetique
Humaine, Montpelier, France), FOXL2 (1:250; a gift of
Reiner Veitia, Paris, France), PECAM1 (1:250; BD BioScience,
San Jose, CA, USA), gH2AX (1:100; Calbiochem, San Diego,
CA, USA), b-catenin (1:2000; Sigma, St Louis, MO, USA) and
AMH (1:500; Santa Cruz, Santa Cruz, CA, USA). Double
immunohistochemistry was detected by Cy3- and Cy5-conjugated secondary antibodies (1:500; Jackson ImmunoResearch
Laboratories, West Grove, PA, USA) and imaged using confocal scanning microscopy. For gH2AX, b-catenin and AMH
staining, samples were embedded in OCT and sectioned at
12–14 mm before staining.
For histology, samples were fixed in Bouin’s fixative
overnight at 48C, dehydrated through an ethanol series
and embedded in paraffin. Samples were sectioned at
5– 7 mm and then stained with hematoxylin and eosin (H&E).
For in situ hybridization, samples were fixed overnight
at 48C in 4% paraformaldehyde and processed as described
(25). A digoxigenin-labeled RNA probe was detected by
using an alkaline phosphatase-conjugated anti-digoxigenin
antibody (1:1000; Roche, Indianapolis, IL, USA).
Quantitative RT-PCR
Total RNA was extracted from E13.0–13.5 dpc gonads (separated from mesonephroi) using Trizol (Invitrogen, Carlsbad,
CA, USA). Gonads of the same genotype were pooled for
each RNA preparation (between 4 and 10). The following was
performed on three independent pools of RNA (biological replicates): RNA was DNase-treated (Sigma) and converted to cDNA
using an iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA,
USA), according to the manufacturer’s protocol. Quantitative
PCR was performed in triplicate using SensiMix Plus SYBRþ
Fluorescein (Quantace, Norwood, MA, USA) and run on the
iCyclerTM Thermal Cycler (Bio-Rad). PCR conditions were as
follows: 958C for 3 min and 30 s (one cycle); 958C for 30 s,
558C for 45 s, 728C for 45 s (40 cycles) and 728C for 5 min
(one cycle). Primer sequences are listed 50 –30 : Fst F-AA
ACCTACCGCAACGAATGTG, R-GGTCACACAGTAGGCA
TTATTGGTC; b-actin F-GGCTGTATTCCCCTCCATCG,
R-CCAGTTGGTAACAATGCCATGT; Wnt4 F-AGCCGGG
CACTCATGAATCT, R-GCACGCCAGCACGTCTTTAC and
Rspo1 F-GTCTATCTTGGGGGTGGTTC, R-AGGGGTGGTC
TCTTGCTAA.
Organ culture using the droplet method
Organ cultures were performed using a modified version of
a previously described protocol (26). Briefly, gonad/
mesonephros complexes were cultured at 378C with 5%
CO2/95% air in a 30 ml droplet of Dulbecco’s modified
Eagle’s medium (Gibco), supplemented with 10% fetal calf
serum (Cambrex), and 50 mg/ml ampicillin with or without
50 mM LiCl. Gonads were collected from embryos at stages
between 16 and 23 tail somites (27) and cultured for 24 h.
Contralateral gonads were used as controls, and genetic sex
was determined by PCR genotyping of embryonic tail
samples (28). Two gonad/mesonephros complexes were dissected and immediately placed in round droplets of media
on opposite sides of an inverted lid of a 5 cm Petri dish.
Using a pipette tip, the droplets were spread out until the
gonad/mesonephros complex lay on its side and the surface
tension of the medium just held the gonads in place against
the dish. The droplet was approximately the size of a dime
Human Molecular Genetics, 2008, Vol. 17, No. 19
once spread out. This dish was then floated on water in a
larger, covered 10 cm Petri dish.
Treated gonads were scored relative to untreated XX and
XY controls based on the presence or absence of the coelomic
vessel (based on PECAM staining), levels of SOX9 protein
(using an antibody to detect SOX9) and size. When a disrupted
vasculature or intermediate levels of SOX9 postive cells were
observed, those characteristics were considered ‘partial male’.
Although size was a continuous variable, the LiCl treated
gonads were considered a ‘male’ size when the width of the
gonad (from the mesonephros to the coelomic surface)
closely resembled control XY gonads; gonads were considered
‘female’ when they were observed to be smaller and closer in
size to control XX gonads (see Fig. 3A for comparison).
SUPPLEMENTARY MATERIAL
Supplementary Material is available at HMG Online.
ACKNOWLEDGEMENTS
We would like to acknowledge Yuna Kim for her helpful suggestions and Reiner Veitia and Francis Poulat for contributing
valuable antibody reagents. The project described was
supported by grant number F32HD055791 (to D.M.M.) from
the National Institute of Child Health and Human Development. The content is solely the responsibility of the authors
and does not necessarily represent the official views of the
National Institute of Child Health and Human Development
or the National Institutes of Health.
Conflict of Interest statement. None declared.
FUNDING
Funding was provided by the National Institutes of
Health (HL63054 and HD39963 to B.C. and DK54480 and
HD046743 to K.L.P.).
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