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First published online 26 November 2008
doi: 10.1242/dev.029587
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1 Department of Veterinary Anatomy, The University of Tokyo, Yayoi 1-1-1,
Bunkyoku, Tokyo 113-8657, Japan.
2 Department of Anatomy, Kyorin University School of Medicine, Shinkawa 6-20-2,
Mitaka, Tokyo 181-8611, Japan.
3 Department of Aging Intervention, National Institute for Longevity Sciences,
National Center for Geriatrics and Gerontology, Gengo 36-3, Morioka-cho, Obu,
Aichi 474-8511, Japan.
4 Department of Molecular Biology, Graduate School of Medicine, Kyushu
University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan.
5 Institute for Molecular Bioscience, The University of Queensland, St Lucia,
Brisbane, QLD 4072, Australia.
* Author for correspondence (e-mail: aykanai{at}mail.ecc.u-tokyo.ac.jp)
Accepted 23 October 2008
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Sry, Sox9, Fgf9, Wnt4, Sertoli cells, Sex differentiation, Mouse
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Gubbay et al.,
1990; Koopman et al.,
1991). Without Sry action the supporting cells
differentiate into granulosa cells
(Albrecht and Eicher, 2001),
resulting in ovarian development. During mouse embryogenesis, Sry is
transiently expressed from 12 to 24 tail somites (ts) - around 11.0-12.0 days
post-coitum (dpc) - in the supporting cell lineage. This expression occurs in
a center-to-pole wave pattern along the anteroposterior (AP) axis of the
indifferent XY gonads (Bullejos and
Koopman, 2001; Albrecht and
Eicher, 2001). An autosomal gene, Sox9, also required for
testis determination (Bishop et al.,
2000; Vidal et al.,
2001; Chaboissier et al.,
2004; Barrionuevo et al.,
2006), is upregulated in the supporting cells in the same
center-to-pole pattern shortly after the onset of Sry expression,
apparently in response to Sry
(Sekido et al., 2004;
Kidokoro et al., 2005;
Wilhelm et al., 2005;
Sekido and Lovell-Badge,
2008). However, unlike Sry, Sox9 continues to be
expressed in Sertoli cells throughout testis development
(Morais da Silva et al., 1996;
Kent et al., 1996). It is
believed that continuous Sox9 expression is involved in directing
subsequent testis differentiation and development (for reviews, see
Brennan and Capel, 2004;
Kanai et al., 2005;
Polanco and Koopman,
2007).
Recently, we demonstrated that induced, ubiquitous expression of an
Sry transgene in the entire gonadal area earlier than normal
Sry expression does not result in any advance in timing or ectopic
activation of Sox9 expression
(Kidokoro et al., 2005). This
finding indicates that the testis-initiation program immediately downstream of
Sry action is tightly regulated and that supporting cells need to
achieve a competent state to respond to Sry. However, the molecular
mechanism and the time window within which SRY must act to induce a switch
from the female to the male pathway in gonadal supporting cells are
unknown.
In order to resolve these questions, we established a heat shock-inducible Sry transgenic (Tg) mouse system that allows the induction of testis development in cultured XX genital ridges at various time points during development. By using this Sry-inducible system, we demonstrate for the first time that the ability of Sry to determine the testis fate is limited to approximately 12 to 15 ts (11.0-11.25 dpc), a time window of only 6 hours. We found that this time window is delimited by the competing actions of FGF9 and WNT4 signaling. Our findings indicate an unexpectedly narrow time window during which Sry must act to initiate and maintain Sox9 expression in developing XY gonads to induce testis formation.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Although most XX transgenic (Tg) mice with the HSP-Sry transgene
showed an XX-male sex reversal in normal breeding (e.g. #40 and #46), we
successfully established one HSP-Sry transgenic line (#44; ICR
background) in which all XX Tg mice develop into normal fertile females (all
Tg mice were viable and fertile with no obvious abnormalities). We refer to
#44 line of HSP-Sry as `Tg' in this paper. In this line, the
transgene was transmitted at an expected Mendelian ratio in both matings
between the Tg males and wild-type females (HSP-Sry versus wild type:
219 versus 245 males in XY, 252 versus 229 females in XX, total 945) and
matings between wild-type males and Tg females (HSP-Sry versus wild
type: 14 versus 13 males in XY, 11 versus 14 females in XX, total 52). We also
obtained Wnt4+/- mice from the Jackson Laboratory (Bar
Harbor, ME). They were crossed with this HSP-Sry Tg line, and then
maintained on a mixed 129/ICR genetic background. The sex and genotype of each
animal were determined by PCR as described previously
(Kidokoro et al., 2005;
Mizusaki et al., 2003). Animal
experiments were conducted in accordance with the Guidelines for Animal Use
and Experimentation set out by the University of Tokyo.
Heat-shock (HS) treatment and organ culture
Embryos at 9.5-16.5 dpc were collected from pregnant female mice
pre-treated with aspirin (Fawcett et al.,
1997). From 10.5 to 12.5 dpc, the tail somites of each embryo were
counted for accurate staging. Using tail somite stages, 10.5 dpc corresponds
to approximately 8 ts, 11.5 dpc to 18 ts, and 12.5 dpc to 30 ts
(Hacker et al., 1995). Genital
ridges were isolated in cold Dulbecco's Modified Eagle's Medium (DMEM; Sigma).
In all samples, one genital ridge (left) of each pair was treated with heat
shock (HS; 43°C for 10 minutes), while the other genital ridge (right) was
used as a control. HS treatment did not exert any appreciable negative effects
on testis formation in the wild-type XY genital ridges in vitro [number of the
abnormal testis explants (e.g. atrophy and defective cord formation) per total
HS-treated XY explants: 4/37 explants at 12-14 ts and 0/40 explants at 15-18
ts]. In some experiments, we HS-treated whole embryos and other various organs
(43°C for 10-15 minutes in thin-wall PCR tubes). All samples except for
whole embryos were cultured on ISOPORE membrane filters (Millipore) in DMEM
containing 10% horse serum or fetal calf serum at 37°C for appropriate
periods (2 hours to 4 days), as described previously
(Hiramatsu et al., 2003). Some
genital ridges were cultured in 10% horse serum-DMEM supplemented with FGF9
(Recombinant human fibroblast growth factor 9, Sigma; 100 ng/ml) and/or sFRP2
(Recombinant mouse secreted frizzled-related protein 2, R&D Systems; 1.5
µg/ml; medium change every 24 hours). Whole embryos were cultured using a
rotating-bottle system in DMEM containing 50% rat serum at 37°C. All
explants were subjected to histological, immunohistochemical and RT-PCR
analyses as described below.
Histology and immunohistochemistry
Cultured explants were fixed in Bouin's solution, 4% paraformaldehyde (PFA)
or 10% formaldehyde containing 2% Ca(CH3COO)2, and
routinely embedded in paraffin. De-paraffinized sections (4 µm in
thickness) were subjected to conventional histological (Hematoxylin-Eosin,
Periodic Acid Schiff) and immunohistochemical staining. Testis cord formation
in the XX Tg explants was histologically evaluated at each tail somite stage
(10-24 ts).
For immunohistochemical staining, sections were incubated with anti-SRY
(1:50 dilution) (Wilhelm et al.,
2005), anti-SOX9 (1:250 dilution)
(Kent et al., 1996;
Kidokoro et al., 2005),
anti-SCP3 (1:250 dilution) (Chuma and
Nakatsuji, 2001) (kindly provided by Drs Shinichiro Chuma and
Norio Nakatsuji, Kyoto University, Japan), anti-SF1/Ad4Bp, anti-3βHSD
(1:1000 and 1:2000 dilution, respectively)
(Ikeda et al., 2001),
anti-laminin (1:400 dilution; ICN Pharmaceuticals) or anti-MIS (1:100
dilution; sc-6886; Santa Cruz Biotechnology, CA) antibody at 4°C for 12
hours. The reaction was visualized with biotin-conjugated secondary antibody
in combination with the Elite ABC Kit (Vector Laboratories, CA).
For quantitative analysis of the number of SOX9-positive cells, the cell
number per area (cell number per mm2) was calculated in three
longitudinal sagittal sections per explant, as described previously
(Kidokoro et al., 2005).
Proliferation assay and immunofluorescence
The genital ridges were isolated at 11.0 dpc (12-13 ts) and 11.5 dpc (18-19
ts), and then treated with or without HS. To detect proliferating cells, all
explants were cultured in the presence of BrdU (bromodeoxyuridine, 10 µM;
Sigma) for 3 hours following the pre-culture for 9 hours as described above
(total 12-hour culture). The PFA-fixed explants were then used for double
staining of BrdU and SF1/Ad4Bp. BrdU labeling was first visualized by using
mouse anti-BrdU antibody (Dako Cytomation) in combination with anti-mouse
IgG-AF488 (Molecular Probes). Explants were then stained with anti-SF1/Ad4Bp
antibody in combination with anti-rabbit IgG-AF594 (Molecular Probes). After
being counterstained with DAPI, the numbers of BrdU-positive cells in the
coelomic epithelium were calculated in three longitudinal sagittal sections
per explant. Finally, the proliferation index (%) of coelomic epithelial cells
was estimated in the explants (n=5).
Mesonephric cell migration assay
Gonads were separated from the XX Tg or XY/XX wild-type genital ridges
treated with HS at 11.5 dpc (18-19 ts). GFP-positive mesonephroi were
dissected from the XY embryos at 11.5 dpc (Green mice; SLC, Japan). HS-treated
gonads were assembled with GFP-positive mesonephros and cultured on 1.5% agar
blocks in 10% horse serum-DMEM for 48 hours
(Martineau et al., 1997).
Whole-mount in situ hybridization
Whole-mount in situ hybridization was performed on 4% PFA-fixed cultured
explants at 70°C for 16 hours as described previously
(Hiramatsu et al., 2003). RNA
probes for Sry (Bullejos and
Koopman, 2001), Fgf9
(Colvin et al., 2001) and
Wnt4 (Mizusaki et al.,
2003) were used in this study.
Quantitative RT-PCR analyses
Total RNA was extracted from the genital ridges using Trizol reagent
(Invitrogen Life Technologies, CA). After treatment with DNase I for 30
minutes, RNA was reverse transcribed using random primers with a Superscript
III cDNA Synthesis Kit (Invitrogen Life Technologies, CA), following the
manufacturer's instructions. A reverse-transcriptase-free reaction was
performed as control.
In RT-PCR for Sry expression, Sry and Gapdh
(control) were amplified as described previously
(Kidokoro et al., 2005). For
Sox9, Fgf9 and Wnt4 expression, specific primers and
fluorogenic probes were purchased from Applied Biosystems (Assays-on-Demand,
Applied Biosystems, CA). Amplification of the Gapdh gene was used to
standardize the amount of RNA in each reaction mixture (Taqman control
reagents). PCR was performed using an ABI Prism 7900HT sequence detector as
described previously (Kidokoro et al.,
2005). The expression levels represented the relative expression
levels of each marker gene per Gapdh amplicon ratio
(mean±s.e.m.).
Statistical analysis
Quantitative data of both gene expression and cell number were analyzed by
Student's t-test. For the stage-dependent differences in frequency of
XX/testis formation, statistical analysis was based on the Fisher exact test
(two tailed) using the StarView statistical program.
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RESULTS |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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). In situ hybridization analysis of XX Tg embryos showed no
detectable expression of the Sry transgene in various tissues,
including the genital ridges (left, Fig.
1A). HS treatment of whole embryo and organ cultures promoted
strong Sry expression within several hours in whole embryos/various
organs of this line (right, Fig.
1A). Anti-SRY immunohistochemical analyses confirmed that the
transgene-derived SRY protein was ubiquitously expressed in the somatic cells
of various HS-treated tissues (Fig.
1B), whereas only weak signals were detectable in germ cells
(arrowheads; Ov and Ts, Fig.
1B).
Next, we examined in detail the consequences of inducing Sry at
different time points in ex vivo gonad cultures. In wild-type genital ridges
explanted at 12 ts (approximately 11.0 dpc), endogenous Sry
expression started in the center of the gonad, expanded throughout the whole
gonadal area after 12 hours (12 h), and disappeared by 24 hours (24 h) in
culture (Fig. 1C), which is
consistent with the center-to-pole wave pattern of endogenous Sry
expression in vivo (Bullejos and Koopman,
2001). Similarly, genital ridges of XX Tg embryos were explanted
at 12 ts and either left untreated or exposed to HS treatment. Control
cultures without HS treatment showed no Sry expression at any time
point investigated. By contrast, HS treatment induced strong Sry
expression within 3 hours (3h; Fig.
1C, left panel). This expression was rapidly reduced to barely
detectable levels by 6 hours after HS treatment (6h;
Fig. 1C, middle panel).
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;
Kidokoro et al.,
2005). At this stage, about half of the explants
displayed ovarian development, while the remainder developed into typical
ovotestes with testicular tissue, shown by SOX9-positive Sertoli cells, in the
central region and ovarian tissue, with SCP3-positive oocytes, at the poles
(Fig. 2A, middle panel),
similar to that seen in B6-YDOM sex reversal models
(Eicher et al., 1982).
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Sox9 expression is induced but not maintained by ectopic Sry beyond the critical time window
In order to gain further insight into the cause of this narrow window of
SRY action required to determine the testis fate, the time course of SOX9
expression was examined in SRY-induced XX Tg gonads within and beyond this
critical time point (15 ts; Fig.
3). In the SRY-induced gonads at 12 ts (on time), SOX9-positive
cells were first detected after 6 hours in culture, had rapidly increased in
number by 9 hours, and were maintained at similar numbers 12 and 24 hours
after Sry induction (Fig.
3A, left panels). In explants at 24 ts (
24 hours after
endogenous Sry induction), no appreciable SOX9 expression was
detected throughout the culture period
(Fig. 3A, right panels). These
results support the above data showing testis formation in explants induced
for Sry expression at 12 ts, but ovarian development in explants
initiated after the critical period (see
Table 1).
Surprisingly, in genital ridges explanted at 18 to 21 ts, a stage at which
all explants display ovarian development at later stages, SOX9-positive cells
were detectable after 6 hours in culture and had increased in number by 9
hours (Fig. 3A, middle panels).
However, SOX9 expression levels were rapidly reduced to an undetectable level,
resulting in no SOX9-positive cells after 24 hours. Quantitative real-time
RT-PCR analysis confirmed transient Sox9 upregulation 6 hours after
Sry induction (P<0.01, Student's t-test;
Fig. 3B). This 6-hour interval
between HS-dependent Sry induction and Sox9 upregulation
reflects the in vivo expression patterns of these genes (approximately a
4-hour-time lag) (Sekido et al.,
2004; Kidokoro et al.,
2005; Wilhelm et al.,
2005), taking into account a time lag for the recovery from HS
stress. Immunohistochemistry demonstrated that the transient SOX9 expression
occurred in the presumptive supporting cells, as shown by SF1/Ad4Bp (NR5A1)
staining (Fig. 3C), even though
transgenic Sry is expressed ubiquitously. These data demonstrate that
Sry expression in itself does not necessarily result in the
upregulation of Sox9, but that the cellular environment plays an
important role. Sox9 is induced only in the supporting cells of the
XX Tg gonads at 12 to 21 ts by ectopic Sry expression, which
coincides roughly with the end of Sry expression in developing XY
gonads. Sry expression that has been artificially delayed by more
than 6 hours is not capable of maintaining sufficiently high levels of
Sox9 expression, which consequently results in ovarian
development.
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14 ts)
(Matoba et al., 2005),
mesonephric cell migration (15 ts)
(Tilmann and Capel, 1999) and
the proliferation of coelomic epithelial cells (14-15 ts)
(Schmahl et al., 2000;
Schmahl et al., 2004), were
shown to occur just at or immediately after this period and to play crucial
roles in the subsequent establishment of SOX9 expression in pre-Sertoli
cells.
First, the testis-specific glycogenesis of pre-Sertoli cells, which is
likely to occur cell-autonomously immediately downstream of SRY action
(Matoba et al., 2005), was
examined in SRY-induced and non-induced XX Tg gonads at 18 ts (11.5 dpc;
12-hour delay). A comparative analysis of PAS (Periodic Acid Schiff) reaction
and anti-SOX9 staining revealed that glycogen accumulation is properly induced
in the SOX9-positive supporting cells in SRY-induced explants
(Fig. 4A).
Next, we performed a mesonephric cell migration assay
(Martineau et al., 1997) by
combining HS-treated XX Tg gonad and GFP-positive XY mesonephros at 18 ts. No
explants of non-induced, as well as SRY-induced, XX Tg gonads showed an
appreciable contribution of GFP-positive mesonephric cells to the gonadal area
after 2 days in culture (n=4; Fig.
4B, upper panels). This is clearly in contrast to the high
contribution of GFP-positive mesonephric cells in XY wild-type gonads at 18 ts
and in SRY-induced XX Tg gonads explanted at 13 ts
(Fig. 4B, bottom panels).
Finally, anti-BrdU immunohistochemistry demonstrated proliferating somatic cells at and near the coelomic epithelium of both SRY-induced and non-induced XX Tg explants at 18 ts (Fig. 4C, upper panels). Quantification of this staining revealed no significant difference between these two treatments (Fig. 4D, third and fourth bars). Moreover, the numbers of proliferating cells were similar to those seen in the non-induced XX Tg gonads at 12-13 ts (Fig. 4C, bottom left panel; Fig. 4D, first bar). By contrast, the XX Tg gonads that were isolated and HS-treated at 12-13 ts (Fig. 4C, bottom right panel; Fig. 4D, second bar) showed a significantly higher number of proliferating cells at the coelomic epithelium than did those of all other treatments. These findings indicate that delayed Sry induction is not capable of inducing the early testis-specific mesonephric cell migration and increased proliferation within the coelomic epithelium.
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;
Schmahl et al., 2004).
Moreover, recent genetic analysis has indicated that the balance between two
opposing pathways involving Fgf9 and Wnt4 could affect the
establishment of SOX9 expression in supporting cells at later stages
(Kim et al., 2006). Although
both Fgf9 and Wnt4 are expressed in similar patterns in XX
and XY gonads at early phases of the sex-determining period, their
sex-dimorphic expression (i.e. high Fgf9/low Wnt4 expression
in males, but low Fgf9/high Wnt4 in females) becomes evident
around at 18 ts (Mizusaki et al.,
2003; Schmahl et al.,
2004). In XX Tg gonads HS treated at 12-13 ts (on time),
Sry induction resulted in the male-specific pattern of Fgf9
and Wnt4 expression (Fig.
5A, left panel). By contrast, in Sry-induced XX Tg gonads
dissected at 18 ts (12-hour delay), we could not find any changes in either
Fgf9 or Wnt4 expression when compared with expression in the
non-induced control explants (Fig.
5A, right panels). No significant changes between SRY-induced and
non-induced gonads at 18 ts were confirmed by quantitative real-time RT-PCR
(Fig. 5B). Our data indicate
that delayed Sry induction is not capable of switching the XX gonad
from the female- to the male-specific expression patterns of Fgf9 and
Wnt4.
A forced reversal from the female- to the male-specific patterns of FGF9/WNT4 signaling states can rescue the defective maintenance of SOX9 expression caused by delayed SRY induction
In order to clarify the possible contribution of female-type
Fgf9/Wnt4 expression in defining this critical time window
of SRY action, we next examined the effects of the exogenous addition of FGF9
and/or a WNT4 antagonist, the secreted frizzled-related protein 2 (sFRP2)
(Lee et al., 2000), on the
maintenance of SOX9 expression in XX Tg gonads Sry induced at 18 ts
(Fig. 5C,D). The addition of
FGF9 and sFRP2 did not cause any appreciable changes in the SOX9 expression
pattern in control XY and XX wild-type gonads at 18 ts (see Fig. S3 in the
supplementary material), or in the XX Tg gonads explanted at 18 ts and
incubated for 9 hours after Sry induction (9h;
Fig. 5C,D). After 24 hours, the
WNT4 inhibitor sFRP2 alone could not restore the defective maintenance of SOX9
expression in XX Tg gonads, although the addition of FGF9 alone resulted in a
few SOX9-positive cells (see Fig. S4 in the supplementary material). By
contrast, the addition of FGF9 and sFRP2 together restored the maintenance of
SOX9 expression for up to 72 hours in culture
(Fig. 5C,D), although the
number of SOX9-positive cells was approximately one-third of those in
SRY-induced XX Tg gonads at 12-13 ts (Fig.
5D; see also Fig. S3B in the supplementary material). Moreover,
prolonged SOX9 expression led to subsequent Leydig cell differentiation in all
of the XX Tg explants Sry induced at 18 ts, as judged by 3βHSD
expression (Fig. 5E). These
data indicate that a forced male-specific pattern of the imbalance between
FGF9 and WNT4 signals can counteract the defective maintenance of SOX9
expression caused by delayed Sry activation, leading to the
establishment of Sertoli and Leydig cells at later stages.
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In non-treated (HS-) Wnt4+/- explants dissected at 18-19 ts, neither SOX9 expression nor cord formation was detected after 72 hours in culture (Fig. 6A,B; right lower panels), which was similar to what was observed in non-treated and Sry-induced XX Tg explants of the Wnt4+/+ wild-type littermates (Fig. 6; left panels). Interestingly, in all XX Wnt4+/- explants Sry-induced at 18-19 ts (12-hour delay), SOX9 expression was maintained at 72 hours in culture (n=3; Fig. 6A; right upper panel). In these Wnt4+/- explants, well-defined testis cords were also induced in their gonadal region (Fig. 6B; right upper panel). These data indicate that the loss of one allele of the Wnt4 gene can rescue the failure of the Sertoli cell establishment and testis cord formation caused by delayed Sry induction. This further implies that the reduced Wnt4 activity prolongs the critical time window of SRY action that is required to determine the testis fate in developing XX gonads.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Schmahl et al., 2004;
Kim et al., 2006). These
findings, therefore, indicate that SRY action during the initial 6-hour phase
is crucial to switch from the female- to the male-specific patterns of
FGF9/WNT4 signaling in developing gonads. The overarching importance of SRY
action during this initial phase (12 to 15 ts) is corroborated by the finding
that a delay in Sry expression during this critical period in
B6-XYPOS mice results in XY sex reversal
(Bullejos and Koopman,
2005).
In this study, we demonstrated that this narrow critical time window
required to determine testis fate is non-cell-autonomously defined by the
ability to engage the FGF9 signaling state required for Sertoli cell
establishment (Colvin et al.,
2001; Schmahl et al.,
2004) and by the competing action of WNT4 signaling that promotes
the female pathway (Vainio et al.,
1999; Kim et al.,
2006; Ottolenghi et al.,
2007). We showed that delayed SRY induction is not capable of
switching the gonad from the female- to the male-specific expression patterns
of Fgf9 and Wnt4. A forced male-specific pattern of the
imbalance between these two signals can counteract the defective maintenance
of SOX9 expression, leading to the establishment of Sertoli cells at later
stages. Moreover, we genetically demonstrated that reduced Wnt4
activity can rescue the failure of the Sertoli cell establishment and testis
cord formation caused by delayed Sry induction. Because the
sex-dimorphic expression of Fgf9 and Wnt4 becomes evident in
the gonadal area by 11.5 dpc (Mizusaki et
al., 2003; Schmahl et al.,
2004), the FGF9/WNT4 signal state that progresses the female
pathway is likely to define the end of the time window of SRY action required
to determine testis fate in developing XX gonads. These data also provide
clear evidence to support the hypothesis by Kim et al. that sex determination
is controlled by mutually antagonistic signals between FGF9 and WNT4 in the
gonadal field of mouse embryos (Kim et
al., 2006).
In contrast to the narrow time window of Sry required to ensure
testis development, the present data showed the wider time window of
cell-autonomous SRY action that is required to initiate pre-Sertoli cell
differentiation in developing XX gonads
(Fig. 6, purple arrow). In
Sry-induced XX gonads, SOX9 was shown to be, although transiently,
activated in the presumptive supporting cells during 12 to 21 ts (11.0-11.75
dpc), which coincides roughly with the period of endogenous Sry
expression in developing XY gonads
(Bullejos and Koopman, 2001).
These findings indicate that XX gonads, as well as XY gonads, maintain the
ability to initiate Sox9 activation upon Sry expression
during the sex differentiation period. Although recent microarray data has
revealed the initiation of a robust female-specific genetic program, including
high Wnt4 expression, as early as 11.5 dpc (18 ts)
(Nef et al., 2005;
Beverdam and Koopman, 2006), it
was shown that Foxl2, a granulosa cell marker gene implicated in
ovarian determination (Crisponi et al.,
2001; Schmidt et al.,
2004; Uda et al.,
2004; Ottolenghi et al.,
2005; Ottolenghi et al.,
2007), starts to be upregulated in a female-specific manner at
around 12.5 dpc (30 ts) (Loffler et
al., 2003; Schmidt et al.,
2004). Taken together, the present results suggest that the major
population of XX supporting cells maintains the sexually undifferentiated and
bipotential states by 21 ts. The rapid loss of the potency to initiate
SRY-dependent Sox9 activation at around 23-24 ts is one of the
earliest cellular events of pre-granulosa cell differentiation in developing
XX gonads (Fig. 6, red
arrow).
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Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/1/129/DC1
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Chaboissier, M. C., Kobayashi, A., Vidal, V. I., Lutzkendorf,
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