BIOLOGY OF REPRODUCTION 74, 195–201 (2006)
Published online before print 5 October 2005.
Homozygous Inactivation of Sox9 Causes Complete XY Sex Reversal in Mice1
Francisco Barrionuevo,5Stefan Bagheri-Fam,3,5Ju ¨rgen Klattig,4,6Ralf Kist,5,7Makoto M. Taketo,8
Christoph Englert,4,6and Gerd Scherer2,5
Institute of Human Genetics and Anthropology,5University of Freiburg, D-79106 Freiburg, Germany
Institute of Physiological Chemistry I,6Biocenter, University of Wu ¨rzburg, Am Hubland, D-97074 Wu ¨rzburg, Germany
Institute of Human Genetics,7International Centre for Life, University of Newcastle upon Tyne, Newcastle upon Tyne
NE1 3BZ, United Kingdom
Department of Pharmacology,8Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
In the presence of the Y-chromosomal gene Sry, the
bipotential mouse gonads develop as testes rather than as
ovaries. The autosomal gene Sox9, a likely and possibly direct
Sry target, can induce testis development in the absence of Sry.
Sox9 is thus sufficient but not necessarily essential for testis
induction. Mutational inactivation of one allele of SOX9/Sox9
causes sex reversal in humans but not in mice. Because Sox9–/–
embryos die around Embryonic Day 11.5 (E11.5) at the onset of
testicular morphogenesis, differentiation of the mutant XY gonad
can be analyzed only ex vivo in organ culture. We have
therefore conditionally inactivated both Sox9 alleles in the
gonadal anlagen using the CRE/loxP recombination system,
whereby CRE recombinase is under control of the cytokeratin 19
promoter. Analysis of resulting Sox9–/–XY gonads up to E15.5
reveals immediate, complete sex reversal, as shown by
expression of the early ovary-specific markers Wnt4 and Foxl2
and by lack of testis cord and Leydig cell formation. Sry
expression in mutant XY gonads indicates that downregulation
of Wnt4 and Foxl2 is dependent on Sox9 rather than on Sry. Our
results provide in vivo proof that, in contrast to the situation in
humans, complete XY sex reversal in mice requires inactivation
of both Sox9 alleles and that Sox9 is essential for testogenesis in
developmental biology, gene regulation, ovary, Sertoli cells, testis
Mammalian testis determination is triggered by the Y-
chromosomal testis-determining factor SRY. In its presence,
the bipotential gonadal anlagen differentiate into testes, while
in its absence, ovaries develop. Whereas the testis-inducing
function of SRY has been unambiguously demonstrated ,
its direct target gene or genes still await identification. As one
likely SRY target, the Sry-related gene Sox9 has emerged. In
humans, heterozygous SOX9 mutations cause partial or
complete XY sex reversal in the context of the skeletal
malformation syndrome campomelic dysplasia [2, 3], and
duplication of the chromosomal region 17q23.1-q24.3
encompassing the SOX9 locus was present in an XX
individual with female-to-male sex reversal . During
gonadogenesis of the mouse, Sox9 is initially expressed in
both sexes, with expression decreasing in the developing
ovary and strongly increasing in the developing testis,
concomitant with the peak of Sry expression at Embryonic
Day 11.5 (E11.5) [5, 6]. Furthermore, SOX9 has been shown
to colocalize with SRY in the nucleus of Sertoli cell
precursors as early as E11.5 , consistent with the
hypothesis that Sox9 is a direct, and possibly the only, target
of SRY . Sox9 can also substitute for Sry as a testis-
determining factor, as ectopic expression of a Sox9 transgene
 and mutational upregulation of Sox9 expression  cause
testis development in XX mice. These latter data show that
Sox9 is sufficient for testis induction but do not prove that it
is essential for this process.
In contrast to the situation in humans, heterozygous Sox9
mutations do not cause XY sex reversal in mice . As
mouse embryos homozygously mutant for Sox9 die at E11.5 at
the onset of testicular morphogenesis, the fate of the mutant
XY gonad could be studied only ex vivo in organ culture,
revealing no signs of testis cord formation after 3 days in
culture . To follow the development of Sox9–/–XY gonads
in vivo and during the entire phase of gonadogenesis,
a conditional, gonad-specific knockout of Sox9 is needed. A
Cre transgene under control of an Sf1 (steroidogenic factor 1,
also known as Nr5a1) regulatory element has been used for
this purpose, but because of inefficient and/or late Cre-
mediated deletion of the Sox9floxallele, mutant gonads always
showed some sex cord formation . By using the Ck19:Cre
mouse line, where the CRE recombinase is under control of
the cytokeratin 19 promoter , we have achieved complete,
homozygous inactivation of Sox9 at the initial stage of XY
gonadal development, providing in vivo evidence for an
essential role of Sox9 in testogenesis.
MATERIALS AND METHODS
Sox9flox/þmice, originally on a 129P2/OlaHsd 3 C57BL/6 mixed genetic
background , have been backcrossed to C57BL/6 for three generations.
These N3 mice were made Sox9flox/floxby sister-brother matings and crossed
with the Ck19:Cre transgenic mouse line . Resultant Cre/þ;Sox9flox/þ
offspring were backcrossed to Sox9flox/floxmice to obtain Cre/þ;Sox9flox/flox
mice. Embryos at E11.5 were staged by counting the number of tail somites
posterior to the hind-limb bud. Older embryos were staged according to the day
of plug formation. PCR was used on tail tip or yolk sac DNA for genotyping.
1Supported by the Plan de Perfeccionamiento de Doctores de la Junta
de Andalucı ´a to F.B. and by grants from the Deutsche Forschungsge-
meinschaft to C.E. (En 280/6–1) and G.S. (Sche 194/15–1þ2).
2Correspondence: Gerd Scherer, Institute of Human Genetics and
Anthropology, Breisacherstr. 33, D-79106 Freiburg, Germany.
FAX: 49 761 270 7041; e-mail: email@example.com
3Current address: Prince Henry’s Institute of Medical Research, Monash
Medical Centre, 246 Clayton Rd., Melbourne, Victoria 3168, Australia.
4Current address: Leibniz Institute for Age Research-Fritz Lipmann
Institute e.V. (FLI), Beutenbergstr. 11, D-07745 Jena, Germany.
Received: 22 July 2005.
First decision: 24 August 2005.
Accepted: 26 September 2005.
? 2006 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
Primers and PCR conditions for the Ck19:Cre allele , for the Sox9 and
Sox9floxalleles , and for Sry  were used as previously described.
The experiments with animals were performed at the Animal Facility of the
Institute of Human Genetics and Anthropology, Freiburg, Germany. The
animals were housed under a 12L:12D cycle with free access to standard mouse
chow and tap water. All experimental procedures complied with the rules of the
German Animal Welfare Law and were licensed by the local authorities. This is
in accordance with the International Guiding Principles for Biomedical
Research Involving Animals.
Histology, Immunohistochemistry, and In
For histology, embryos from E11.5 to E15.5 were collected in PBS, fixed in
Serra (ethanol: 37% formaldehyde:acetic acid, 6:3:1), embedded in paraffin,
and sectioned at 7 lm. Staining of sections with hematoxylin and eosin
followed standard techniques.
For immunohistochemistry, sections were deparaffinized in xylene and
hydrated through descending ethanols, and antigens were retrieved in sodium
citrate buffer. Sections were blocked in 5% serum from the species where the
secondary antibody was raised for 2 h and incubated with primary antibody at
48C overnight. Rabbit anti-SOX9 (1:200; kind gift of M. Wegner), goat anti-
DMC1 (Santa-Cruz; 1:200), and rabbit anti-SF1 (1:1000; kind gift of K.
Morohashi) were used as primary antibodies. After washing with PBS þ 0.1%
Tween20, sections were incubated in secondary biotinylated antibodies (Vector
Laboratories) for 1 h at room temperature. After washing, sections were
incubated with avidin-fluorescein (Vector Laboratories) for 30 min at room
temperature, counterstained with DAPI, and mounted with Vectashield
mounting medium (Vector Laboratories). For double immunostaining,
following SOX9 immunohistochemistry, sections were treated with an
Avidin/Biotin Blocking Kit (Vector Laboratories), and immunostained using
goat anti-AMH antibody (1:200; Santa Cruz) and Avidin-Texas Red (Vector
Laboratories). Section in situ hybridization with a probe for Wnt4  was
performed as described , with some modifications.
For histology and immunohistochemistry, a minimum of four and up to 10
mutant gonads were analyzed for each time point and marker; for in situ
hybridization, four mutant gonads were analyzed for each time point.
Quantitative RT-PCR Analysis
For real-time RT-PCR analysis, total RNA from single pairs of urogenital
ridges (mesonephros þ gonad) or from individual urogenital ridges was
isolated using the Absolutely RNA Microprep Kit (Stratagene), including
a DNase I treatment, and eluted in 30 ll. Subsequently, an aliquot of 10 ll
RNA was reverse transcribed with SuperScript II RNase H–Reverse
Transcriptase (Invitrogen) and oligo-dT primers in a total volume of 20 ll.
To verify the absence of genomic DNA contamination, an aliquot of each RNA
sample was used for PCR without reverse transcription, as Sry and Foxl2 are
intronless genes. Primer pairs for all other genes covered at least one intron.
One microliter of cDNA was used for PCR analysis employing the QuantiTect
SYBR green real-time PCR kit (Qiagen) on a Biorad iCycler in a 96-well
format. Because of the limited amount of material and to ensure that each
sample could be subjected to at least two independent experiments, all samples
were measured as duplicates. A standard curve was generated for each gene
using serial dilutions of a cDNA pool from several male embryos with 13–23
tail somites. Expression levels were determined in one plate for all samples
simultaneously and normalized to the corresponding amounts of Tbp (TATA
box binding protein) cDNA. Control experiments using additional housekeep-
ing genes (beta-actin, glyceraldehyde-3-phosphate dehydrogenase, and hypo-
xanthine-guanine-phosphoribosyl transferase) had shown that Tbp expression
was not affected by Sox9 inactivation. Analysis was done on a total of five
mutant samples of E11.5 together with six male and four female controls. For
stage E12.5, two independent experiments using two (experiment 1) and four
(experiment 2) samples of each genotype (mutant and control males and
females) were performed. Since the two experiments were done at different
times, the results could not be pooled.
The following primers were used: for Tbp, GGC CTC TCA GAA GCA
TCA CTA and GCC AAG CCC TGA GCA TAA (see http://medgen31.ru-
g.ac.be/primerdatabase/); for Sox9, CGG AGG AAG TCG GTG AAG A and
GTC GGT TTT GGG AGT GGT G; for Sry, GCA AAC AGC TTT GTG GTC
AA and GGA AAA GGG GAT GAA ATG GT; for Amh, ACC CTT CAA
CCA AGC AGA GA and CCT CAG GCT CCA GGG ACA; and for Foxl2,
GCA GAA GCC CCC GTA CTC and ATG CTA TTC TGC CAG CCC TTC.
All primers (except for Tbp) were designed using the Primer3 program (http://
Ck19:Cre;Sox9flox/floxXY Embryos Show Absence of Testis
Differentiation and of Mu ¨llerian Duct Regression
We used the CRE/loxP system to generate a homozygous
knockout of Sox9 during early gonadal development. For this,
we crossed our Sox9floxmouse line, where exons 2 and 3 of
Sox9 are flanked by loxP sites , with the Ck19:Cre mouse
line . This line expresses the CRE recombinase throughout
the early postimplantation mouse embryo, including the
embryonic ectoderm, mesoderm, and definitive endoderm
. Relevant to the present work, intense lacZ staining of
E10.5 and of E11.5 urogenital ridges in the CRE reporter line
R26R  reveals that the Ck19:Cre transgene must be
efficiently expressed in this tissue already before the
upregulation of Sox9 expression in XY embryos (see
Supplemental Fig. 1 [available online at http://www.
biolreprod.org]). Cre/þ;Sox9flox/þanimals survive, are fertile,
and can be backcrossed to Sox9flox/floxmice to obtain Cre/
þ;Sox9flox/floxoffspring. As revealed by SOX9 immunohisto-
chemistry at E8.5 to E11.5, embryos with the latter genotype
show moderate to severe reduction of SOX9-positive cells in
the otic placode/vesicle, premigratory and early migratory
cranial neural crest cells (CNCCs), migrating sklerotomal cells,
and heart endocardial cushions and thus display reduction in
CNCC-derived tissue and in all cartilage anlagen and heart
hypoplasia (F. Barrionuevo, unpublished). As a consequence of
these various defects, Cre/þ;Sox9flox/floxembryos show
significant early embryonic lethality, with only about one
eighth of the expected number of embryos recovered after
E11.5. Whereas Cre/þ;Sox9flox/floxXX and Cre/þ;Sox9flox/þ
XY embryos show normal ovary and testis development,
respectively (not shown), Cre/þ;Sox9flox/floxXY embryos
(hereafter termed mutants) show complete sex reversal. The
failure of testis differentiation is already apparent at E12.5
when testis cords first become visible in the control male gonad
(Fig. 1A), while no signs of testis differentiation are observed
in the mutant gonad, which has the morphology of a control
female gonad (Fig. 1, B and C). This difference is more
pronounced at later stages when control male gonads show
distinct testis cords, while mutant gonads remain in the
differentiation stage typical of female gonads up to E15.5,
the latest stage analyzed (Fig. 1, D–I).
The absence of testicular function in the mutant gonad is
also apparent from the differentiation status of the sexual ducts.
At E15.5, in the control male, secretion of AMH (anti-
Mu ¨llerian hormone) from testicular Sertoli cells has initiated
the degeneration of the Mu ¨llerian duct, while testosterone
produced from testicular Leydig cells has stimulated differen-
tiation of the Wolffian duct, which shows a well-developed
epithelium (Fig. 1J). In the control female as well as in the
mutant, the Mu ¨llerian duct displays no signs of degeneration
and presents with an open lumen, while the Wolffian duct has
started to degenerate (Fig. 1, K and L).
Immunohistochemistry Reveals Presence of Female-
Specific Markers in XY Mutant Gonads
To characterize the mutant gonads further, immunohisto-
chemistry was performed. Double staining for SOX9 and
AMH, an early Sertoli cell marker regulated by SOX9 [21, 22],
gives signals for both markers within the testicular cords in
E13.5 control male gonads (Fig. 2A) but not in control female
or mutant gonads (Fig. 2, B and C). Staining with an antibody
against SF1, a key regulator of steroidogenesis that is
BARRIONUEVO ET AL.
expressed in the gonads of both sexes from E9.5 on, gradually
increasing its expression until E14.5 and subsequently down-
regulated in ovaries but not in testes , reveals strong
expression in interstitial Leydig cells surrounding the testis
cords and weak expression in Sertoli cells in E13.5 control
male gonads (Fig. 2D). The different SF1 expression pattern
characteristic of E13.5 control female gonads  (Fig. 2E) is
also shown by the mutant gonad (Fig. 2F). If E15.5 gonadal
sections are stained with an antibody against DMC1, a marker
for female germ cells at the stage of meiotic arrest , the
control male gonad gives no signal (Fig. 2G), while the mutant
gonad gives a strong signal similar to that seen in the control
female gonad (Fig. 2, H and I).
One E13.5 and one E15.5 mutant embryo, out of 11
mutant embryos analyzed histologically, had a completely
sex-reversed gonad on one side and an ovotestis on the
contralateral side, revealing areas with and areas without testis
cords (Fig. 3, A and D). These ovotestes most likely resulted
from mosaic expression of the Ck19:Cre transgene, a phe-
nomenon observed previously [13, 19, 26]. Immunohisto-
chemical analysis shows that the Sertoli cell markers SOX9
and AMH are expressed only in regions of the ovotestes that
contain regularly formed testis cords and in nearby clusters of
cells that may constitute incompletely formed testis cords
(Fig. 3, B and E). Likewise, the Leydig cell marker SF1 is
expressed only in these regions (Fig. 3, C and F), while the
region devoid of testis cords in the E15.5 ovotestis stains
positive for the oocyte marker DMC1 (Fig. 3G). This
confirms that Sox9 is needed for Sertoli cell formation 
and that Leydig cells can differentiate only in the neighbor-
hood of testis cords.
Gene Expression Analyses Corroborate Complete Sex
Reversal of Ck19:Cre;Sox9flox/floxXY Gonads
To quantify the expression levels of selected sex-de-
termination or -differentiation genes, we performed real-time
quantitative RT-PCR using RNA isolated from mutant and
from control gonads. At around E11.5, mutant gonads show
low Sox9 expression levels in the range of XX control gonads
that is drastically below the level seen in XY control gonads
(Fig. 4A, left). Also, mutant gonads display no measurable
Amh expression at stages when control male gonads begin to
express Amh (Fig. 4A, right). Figure 4A also indicates that
a threshold of Sox9 has to be reached to initiate Amh
expression, in accordance with the known role of Sox9 in this
process [21, 22]. At E12.5, mutant embryos display even
lower Sox9 expression levels when compared to female
controls (Fig. 4B, left). The ovary-specific marker Foxl2 that
is required for granulosa cell differentiation starts to be
expressed in female gonads around E12.5 . Accordingly,
we detect significant E12.5 Foxl2 expression in the mutant and
control female but only very weak expression in the control
male gonads (Fig. 4B, middle), whereas the mirror-image
expression pattern is seen for Amh in the same gonads (Fig.
4B, right). Wild-type Sry expression starts at E10.5, peaks at
E11.5, and disappears around E12.5 . In our analysis,
control and mutant XY gonads show similar Sry levels at
E11.5 (Fig. 4C, left). Thus, Sry expression is independent of
Sox9 dosage, illustrating that Sry acts upstream of Sox9. At
E12.5, Sry is downregulated in control male gonads but
remains at least 5-fold higher in the mutant XY gonads (Fig.
4C, right). Similarly, at E13.5, 4.5-fold higher Sry levels were
gonads and sexual ducts. Starting at E12.5,
testis cords (arrows) are visible in the
control male gonad (A); no signs of differ-
entiation can be observed in the control
female (B) and mutant gonad (C). At E13.5,
control male gonads present with clearly
visible testis cords (D), whereas control
female (E) and mutant (F) gonads show no
sign of differentiation. This can also be
observed at E15.5 (G–I). At E15.5, in the
control male, the Mu ¨llerian duct (md) has
started to degenerate (J), while the epithe-
lium of the Wolffian duct (wd) is well
developed. In the control female (K), the
Mu ¨llerian duct presents no signs of de-
generation, whereas the Wolffian is re-
gressing. Mutant ducts develop like the
control female ducts (L). Transverse sec-
tions; bar ¼ 80 lm for A–C and 50 lm for
Histology of control and mutant
COMPLETE XY SEX REVERSAL IN SOX9–/–MICE
detected in Sox9 mutant embryos compared to male controls
(data not shown).
Wnt4 Is Expressed in XY Mutant Gonads
Wnt4 is an early ovary-specific marker that is expressed at
E11.0 in the genital ridges of both sexes, becoming down-
regulated in the male and maintained in the female gonad ,
where it inhibits endothelial and steroidogenic cell migration
into the developing ovary . Because Wnt4 is also expressed
in the mesonephros that was not removed from the urogenital
ridges used for real-time PCR, Wnt4 expression was analyzed
by RNA in situ hybridization. At E11.5, the Wnt4 signal is
observed only in the mesonephros of the male urogenital ridge
(Fig. 5A), while in control female and mutant gonads, a clear
signal is also visible in the gonadal compartment of the
urogenital ridge (Fig. 5, B and C). At later stages, the control
male gonad shows only weak background signals for Wnt4,
while control female and mutant gonads continue to show
significant Wnt4 expression (Fig. 5, D–I). At these later stages,
continuous Wnt4 expression in the mesonephroi and expression
in the developing kidney is observed in both sexes, as
We show here that by use of the Ck19:Cre line, complete,
homozygous inactivation of a conditional Sox9 allele could be
achieved at the earliest stages of gonadal development. In
contrast to constitutively inactivated Sox9–/–embryos, which
die at E11.5 at the onset of testicular morphogenesis, some of
the Ck19:Cre;Sox9flox/floxembryos survive up to at least E15.5.
This allowed us to follow the fate of Sox9–/–XY gonads in vivo
throughout this entire phase of testicular development and
circumvented the necessity for organ culture as in the case of
constitutively inactivated Sox9–/–XY gonads . Both our in
vivo study and the ex vivo organ culture study by Chaboissier
et al.  show that, in contrast to the situation in humans,
complete XY sex reversal in mice requires inactivation of both
Sox9 alleles and that Sox9 is essential for testis induction to
A detailed study of the spatial and temporal activity of the
CRE recombinase expressed from the Ck19 locus revealed that
and mutant gonads. A–C) Double immu-
nohistochemistry with antibodies against
SOX9 (green-yellow signal, nuclear) and
AMH (red signal, cytoplasmatic) at E13.5. In
the control male (A), fluorescent signals are
observed for both antibodies within the
testis cords but no signals in the control
female (B) and the mutant gonad (C). D–F)
Immunohistochemistry with antibody
against SF1 at E13.5. SF1 is strongly ex-
pressed in Leydig cells surrounding testis
cords in the control male gonad (D),
displaying a distinctly different staining
pattern in the control female and mutant
gonads (E and F, respectively). G–I) Immu-
nohistochemistry with antibody against
DMC1, a marker for meiotic oocytes, at
E15.5. Control male gonad shows no signal
(G), whereas both control female (H) and
mutant gonads (I) show clear signals.
Transverse sections; bar ¼ 100 lm for A–I.
Immunohistochemistry of control
mice. A–C) E13.5 ovotestis. Hematoxylin-
eosin(HE)-stained section shows a small
area with testis cords and a larger area
devoid of testis cords (A). Immunohisto-
chemistry shows that testis cords express
male markers SOX9 and AMH (B) and that
surrounding regions express SF1 in Leydig
cells (C). D–G) E15.5 ovotestis. HE staining
shows regions with and without testis cords
(D). E–G) Regions corresponding to the area
boxed in D from parallel sections. Testicular
portion stains positive for SOX9 þ AMH (E)
and SF1 (F), ovarian portion stains positive
for DMC1 (G). Arrowheads point to testis
cords. Transverse sections; bar in C ¼ 100
lm for A–C; in D, 50 lm; bar in G ¼ 100
lm for E–G.
Ovotestes in XY Cre/þ;Sox9flox/flox
BARRIONUEVO ET AL.
CRE reaches functional levels already between E4.5 and E5.5
throughout the epiblast and that, by E7.5, b-galactosidase-
positive cells of the R26R reporter line used were distributed
throughout the entire embryo proper, including the embryonic
ectoderm, mesoderm, and definitive endoderm . Corre-
spondingly, we observed Ck19:Cre-mediated inactivation of
Sox9 not only in gonads but also in many other tissues, causing
significant early embryonic lethality, as mentioned in Results.
It may be argued that Sox9 inactivation in these additional
tissues would have an indirect effect on gonad development.
However, the fact that Sry expression in the mutant gonads
initiates normally as shown by quantitative RT-PCR strongly
suggests that the gonadal anlagen form properly and that
development along the ovarian pathway is based solely on
Sox9 inactivation in the gonad itself.
Another aspect of the Ck19:Cre allele is that it can cause
recombination of a loxP-flanked allele in a mosaic pattern [13,
19, 26]. In the detailed study by Means et al. , the extent of
CRE-mediated recombination of the R26R lacZ reporter allele
was shown to vary from embryo to embryo, with about two
thirds of the embryos exhibiting b-galactosidase activity in
75%–100% of cells and about one third of embryos in 25%–
75% of cells; however, the tissue distribution was consistent.
We observed the formation of a unilateral ovotestis in only two
out of 11 mutant embryos (22 gonads) analyzed histologically,
indicating that the degree of gonadal mosaicism in our cross
was rather low.
Efficient CRE-mediated excision of the Sox9 alleles in
mutant embryos is also demonstrated by our real time RT-PCR
analysis. At E11.5, Sox9 expression in the mutant gonads was
not completely extinguished and was similar to the low levels
detected in female controls, whereas at E12.5, Sox9 expression
in the mutants was even lower than in female embryos [5, 6].
This indicates that CRE-mediated inactivation of Sox9 was not
mutant gonads at E11.5. B) Analysis of Sox9 (left), the female-specific gene Foxl2 (middle), and the male-specific gene Amh (right) in control and mutant
gonads at E12.5. C) Determination of Sry expression levels at E11.5 and E12.5. In case of E12.5 (in B and C), the left bars correspond to experiment 1, the
right bars to experiment 2, performed with two and four samples of each genotype, respectively. All experiments were performed at least twice, yielding
virtually identical results. In each case, the result of one measurement is shown. Numbers on the y-axis refer to relative expression levels. Note that to
define a point of reference, the mean values for male controls are set as 1. Error bars denote standard deviation.
Gene expression analysis of control and mutant gonads by real-time PCR. A) Analysis of Sox9 (left) and Amh (right) expression in control and
COMPLETE XY SEX REVERSAL IN SOX9–/–MICE
complete but was efficient enough to reduce Sox9 expression to
a level that causes complete, immediate XY sex reversal.
Expression analyses by real time RT-PCR have been
performed using five mutant samples of E11.5 and two and
four mutant samples of E12.5, respectively. As exemplified by
Sry, gene expression during gonad development and differen-
tiation is a very dynamic and stage-dependent process. In our
analysis this can be observed in case of the Sox9 and Amh
expression in male E11.5 controls, which shows a linear
correlation with developmental stage, indicated by the number
of tail somites. This dynamic gene expression might also be an
explanation for the variability between the two experiments for
E12.5, in which case the embryos were staged according to plug
formation. Lower Sox9 and Foxl2 and higher Sry levels in the
mutant gonads of the second experiment might be due to
a slightly earlier developmental stage of the respective embryos.
We found that, in contrast to wild-type male gonads, Sry
expression is not turned off at E12.5 (and E13.5) but remains
high in mutant XY gonads. These findings are in line with the
observation by Chaboissier and coworkers  of persistent
Sry expression in E13.5 XY gonads of Sf1:Cre;Sox9flox/flox
mice with low levels of Sox9. It appears that one function of
SOX9 is to downregulate Sry expression, directly or indirectly,
after it has itself been upregulated by SRY.
The fact that the mutant XY gonads analyzed by us express
Wnt4 and Foxl2 in a female-specific manner even though Sry
expression persists beyond E11.5 furthermore indicates that
downregulation of these early ovary-specific markers in wild-
type XY gonads is dependent not on SRY but rather on SOX9
or on a downstream target of SOX9, or on SOX9 and on
a downstream target of SRY with which SOX9 may interact in
this process. Our results also indicate that Sox9 may be
involved in the rapid downregulation, between E11.0 and
E11.5, of Wnt4, the earliest known female-specific gonadal
marker. A recent report by Qin and Bishop  arrives at the
same conclusion. In humans, an additional copy of WNT4 has
been implicated in dosage-dependent XY sex reversal .
Based on our data, one could speculate that this sex reversal is
caused by the extra dosage of WNT4 overriding the repressive
action of SOX9, which normally downregulates WNT4 below
a certain threshold level to allow testis development to proceed.
Under this hypothesis, the XY sex reversal resulting from
SOX9 haploinsufficiency in humans may, at least in part, be
caused by the inability of the reduced amount of SOX9 protein
to downregulate WNT4 below the critical threshold level. On
the other hand, it has been reported that Sertoli cell
differentiation is compromised in Wnt4 mutant testes and that
Wnt4 acts downstream of Sry and upstream of Sox9 .
Together with the data presented here, it seems possible that
both Sry and Wnt4 are required for Sox9 upregulation and that
for normal testis development to proceed, Wnt4 subsequently
has to be downregulated. This effect appears to be mediated by
SOX9, either directly or indirectly.
In conclusion, by crossing our mouse line carrying
a conditional Sox9 allele with the Ck19:Cre line, complete,
homozygous inactivation of Sox9 was achieved at the earliest
stages of gonadal development, resulting in XY sex reversal.
Thus, even though the Ck19:Cre line expresses the CRE
recombinase in many embryonic tissues in addition to the
gonadal anlagen, this line may nevertheless prove to be
a valuable tool for the effective, early conditional inactivation
of other genes in the sex determination pathway.
We thank Kenichiro Morohashi and Michael Wegner for generous gifts
of antibodies, Katrin Wieland and Ju ¨rgen Zimmer for dedicated technical
assistance, and Ulrike Dohrmann for breeding Sox9floxmice to homo-
zygozity and for comments on the manuscript.
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COMPLETE XY SEX REVERSAL IN SOX9–/–MICE