1 INSERM U470, Centre de Biochimie, Parc Valrose, 06108 Nice Cedex 2,
France
2 Program in Developmental Biology, Baylor College of Medicine, Houston, TX
77030, USA
3 Department of Molecular Genetics, University of Texas, MD Anderson Cancer
Center, Houston, TX 77030, USA
4 Departments of Endocrinology and of Cell Biology, Utrecht University, 3584-CH
Utrecht, The Netherlands
5 Institute for Biochemistry, University of Erlangen, D-91054 Erlangen,
Germany
* Author for correspondence (e-mail: schedl{at}unice.fr)
Accepted 7 January 2004
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SUMMARY |
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Key words: Sex determination, Gonads, Conditional gene targeting, Mouse
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Introduction |
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Campomelic dysplasia is caused by heterozygous loss-of-function mutations
typically affecting the DNA-binding or transcriptional-activation domain (HMG
box) of SOX9 (Foster et al.,
1994; Wagner et al.,
1994
). In addition, translocations or inversions with breakpoints
several hundred kilobases (kb) upstream of SOX9 have been identified
(Wagner et al., 1994
;
Wirth et al., 1996
), and
transgenic analysis suggested that upstream sequences contain important
regulatory elements for the SOX9 gene
(Bishop et al., 2000
;
Wunderle et al., 1998
).
Studies in vitro and in vivo have demonstrated that SOX9 can bind and
bend DNA, and can act as a transcriptional activator on target promoters
(Bell et al., 1997
;
Lefebvre et al., 1997
).
Consistent with these findings, mutations in the transactivation domain at the
C terminus have been found in individuals with campomelic dysplasia
(Sudbeck et al., 1996
).
Moreover, recent in vitro data suggest that this gene may have an additional
function during pre-mRNA splicing (Ohe et
al., 2002
).
Sex determination in the mouse occurs at E11.5, when an initially
bipotential gonad becomes determined to develop either along the male or
female axis depending on the presence or absence of the Y chromosome-encoded
gene Sry. The expression of Sry is highly dynamic: very low
levels of Sry transcripts can be detected as early as E10.5, which
then sharply increase to a peak at E11.5, only to become repressed again by
E12 (Hacker et al., 1995;
Jeske et al., 1995
). The
regulation of Sry is largely unknown, but it has been shown that the
Wilms' tumour suppressor Wt1 is required for the presence of high
levels of Sry RNA (Hammes et al.,
2001
; Hossain and Saunders,
2001
). Moreover, steroidogenic factor 1 (Sf1; Nr5a1 Mouse
Genome Informatics) seems to be able to activate the Sry promoter at
least in pig (Pilon et al.,
2003
).
One of the first genes activated in male gonads after the expression of
Sry is Sox9. Although small amounts of cytoplasmic Sox9 can
be detected in both male and female gonads at E10.5, the production of high
levels of nuclear Sox9 protein is triggered through the expression of Sry in
XY gonads (de Santa Barbara et al.,
2000; Morais da Silva et al.,
1996
). Based on this early appearance of Sox9 after the
onset of Sry, its association with sex reversal in man, and as it can
functionally substitute for Sry in transgenic mice
(Vidal et al., 2001
), it has
been proposed that Sry directly regulates Sox9
(Canning and Lovell-Badge,
2002
).
Sox9 can function as a transcriptional activator and there is strong
evidence that it regulates the Mullerian-inhibiting substance Mis
(Amh Mouse Genome Informatics). First, cotransfection
experiments show that Sox9 can bind and transactivate the Mis
promoter, at least in vitro (De Santa
Barbara et al., 1998). Second, mutations in the potential
Sox9-binding site upstream of the Mis coding sequence result in a
lack of Mis expression in vivo
(Arango et al., 1999
).
Interestingly, a recent in vitro study by Schepers et al. indicated that Sox8,
a close homologue of Sox9, can also activate the Mis promoter, albeit
to a somewhat lower extent (Schepers et
al., 2003
). Sox8 also shows a Sertoli cell-specific
expression pattern in the developing testis, with an onset of expression at
E12, just after the activation of Sox9. However, homozygous
Sox8 knock-out mice did not show a gonadal phenotype, suggesting
either functional redundancy with another gene or that Sox8 does not play a
role in the development of this organ
(Sock et al., 2001
).
Heterozygous mutations in the human SOX9 gene are sufficient to
induce sex reversal. By contrast, sex determination in heterozygous
Sox9 knock-out mice occurs normally, suggesting that this pathway is
less sensitive to gene dosage in mice than in humans
(Bi et al., 2001). We were
interested in investigating the role of Sox8 and Sox9 during
sex determination and testis differentiation in more detail. The perinatal
lethal phenotype of heterozygous Sox9 knock-out mice precludes the
generation of homozygous null animals using standard genetic approaches. To
overcome this problem we have generated a conditional knock-out allele of
Sox9 (Sox9flox)
(Akiyama et al., 2002
). Using
transgenic lines expressing the Cre recombinase either in the germline or
within the developing gonad, we show that Sox9 is essential for
Sertoli cell differentiation and seminiferous tubule formation. The Sertoli
cell-specific markers Mis and Sox8 directly depend on
Sox9 levels, whereas high Sry expression persists in
Sox9 knock-out mice, indicating that Sox9 activation leads
to the downregulation of this gene. Finally, experiments with
Sox8/Sox9 double knock-out mice suggest that Sox8 reinforces
Sox9 function in testis formation.
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Materials and methods |
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Mouse strains and genetic background
Sox8 and Sox9 knock-out mice were kept on a mixed
129/C57Bl6 background. The Sf1:Cre transgene was injected into
fertilized oocytes derived from C57Bl6/CBA females mated with C57Bl6/CBA
males, or with males homozygous for the Sox9flox allele
(mixed 129/C57Bl6 background). Prm1:Cre and Zp3:Cre mice were maintained on a
129/C57Bl6 mixed background and C57Bl6 inbred background, respectively. Mice
homozygous for the Sox8 mutation and heterozygous for the
Sox9 deletion (Fig. 9)
were generated using either the Prm1:Cre or the deleter:Cre strain
(Schwenk et al., 1995). Both
crosses gave similar results.
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Histological analysis
Visceral organs were dissected free of the urogenital system that remained
associated with the carcass, fixed overnight at 4°C in Bouin's solution
and embedded in paraffin wax. Sections were cut at 5-6 µm thickness and
stained with haematoxylin and eosin.
Whole mount in situ hybridization
Embryos were fixed with 4% paraformaldehyde (PFA) in 1xPBS at 4°C
overnight. Further processing of embryos and in situ hybridization were
carried out essentially as described
(Wilkinson, 1992).
Sox9 riboprobes were synthesized according to Morais da Silva et al.
(Morais da Silva et al.,
1996
).
Quantitative RT-PCR analysis
Individual urogenital ridges (mesonephros+gonad) were dissected in PBS from
E13.5 embryos and immediately frozen at -70°C. RNA was prepared using the
Absolutely RNA RT-PCR kit (Stratagene). Each sample was divided into two
aliquots, one of which was reverse transcribed using the MMLV (Gibco),
following the manufacturers instructions. The second aliquot was used as a
control (RT-PCR without reverse transcription) to identify samples with DNA
contamination that could not be included in the analysis of the expression
from the intron-less Sry gene. Primers and probes for all other genes
were designed to cover introns. All real time PCR assays were carried out
using the LC-Faststart DNA Master kit (Roche). A standard curve for each gene
was generated using serial dilutions of cDNA. Relative expression levels of
each sample were determined in the same run and normalized by measuring the
amount of Gapdh (Gapd Mouse Genome Informatics)
cDNA.
Primers for real-time PCR analysis were:
Gapdh cDNA, 5'-ATTCAACGGCACAGTCAAGG-3' and 5'-TGGATGCAGGGATGATGTTC-3', hybridization probes 5'-CCAGAAGACTGTGGATGGCCCCT-X and LC-Red640-TGGAAAGCTGTGGCGTGATGGC-p;
Sox9, 5'-GACAAGCGGAGGCCGAA-3' and 5'-CCAGCTTGCACGTCGGTT-3', hybridization probes 5'-CTTGCAGCGCCTTGAAGATAGCATTAG-X and LC-Red640-GAGATGTGAGTCTGTTCCGTGGCCTC-p; and
Mis, 5'-GTCCTACATCTGGCTGAAGTGAT-3' and 5'-CCGAGTAGGGCAGAGGTTCT-3' (overlapped the junction between exon 1 and 2, and exon 2 and 3, respectively), hybridization probes 5'-GGCCCTGTTAGTGCTATACTCTGGACC-X and LC-Red640-GCCCCCAGGTCACAGTCACAGG-p.
We also used the following primers:
Sf1, 5'-TTCTGAGAGCCCGCTAGCC-3' and 5'-CCTCGTCGTACGAGTAGTCCATG-3', hybridization probes 5'-CCTGGTGTCCAGTGTCCACCCT-X and LC-Red640-TCCGGCTGAGAATTCTCCTTCCG-p;
Sox8, 5'-CAGAGCTCAGCAAGACCCTA-3' and 5'-GGGTGGTGGCCCAGTT-3', hybridization probes 5'-TTACAAATACCAGCCAAGGCGAAG-X and LC-Red640-AAGAGTGTGAAGACTGGCCGGAGC-p; and
Sry, 5'-AGCCTATGTGTAGTTCCTTGGTC-3' and 5'-TGCATAAGGAGTCACATTTTGCT-3', hybridization probes 5'-CAATCTGGCAGTTGAGTTAATGTGCAGAT-X and LC-Red640-CCATTCATTCATCCCACATATACTTGCCC-p.
Detection of the linear Sry transcript was performed as described
(Toyooka et al., 1998). Each
sample of RNA was checked for DNA contamination using the same amount of
starting material without reverse transcription.
Immunohistochemistry
Tissues were isolated at E15.5, fixed for 2 hours in 4% PFA, washed twice
for 5 minutes in 1xPBS and, after equilibration in 30% sucrose, frozen
in OCT on dry ice. Sectioning and staining were carried out as described by
Hammes et al. (Hammes et al.,
2001). The following dilutions of primary antibodies were used:
Sox9 (provided by Michael Wegner), 1:500; Mis (C-20, cat# sc-6886, Santa Cruz
Biotechnology), 1:100; Wt1 (C-19, cat# sc-192 Santa Cruz Biotechnology),
1:100; and laminin (L-9393 Sigma), 1:50.
For laminin staining of cultured gonads, tissues were fixed with 4% PFA
overnight and paraffin embedded. Sections were generated at 7 µm. After
re-hydration, slides were placed in boiling 10 mM sodium citrate (pH 6.0) in a
microwave oven for 20 minutes for antigen retrieval, and soaked in 3% hydrogen
peroxide in methanol for 10 minutes at room temperature to block endogenous
peroxidases. Immunodetection was performed by Vectastain Elite ABC Kit
(PK-6100, Vector Laboratories) and substrate development by AEC Substrate
System (K3464, DakoCytomation). Laminin -1 antibody (M-20, cat# sc-6017
Santa Cruz Biotechnology) was used as the primary antibody at a 1:350
dilution.
In vitro organ cultures
The culture medium was Dulbecco's modified Eagle's medium (DMEM) with 10%
fetal calf serum, 0.1 mM 2-mercaptoethanol, 2 mM glutamine, 0.5 mM pyruvate,
100 units/ml penicillin and 0.05 mg/ml streptomycin
(McLaren and Southee, 1997).
The urogenital organs from Sox9flox/
and
Sox9
/
embryos were dissected in the culture
media containing 20 mM HEPES. The isolated organs were cultured on
polycarbonate membranes (Transwell #3403, Coster, NY) coated with 1% agarose
in phosphate-buffered saline (PBS), with 500 µl of culture media per well
at 37°C with 5% CO2 in air for 2 or 3 days. After culture the
organs were fixed in 4% PFA at 4°C overnight. For histological analysis,
the fixed tissues were paraffin waxembedded and sectioned at 7 µm.
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Results |
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Sox9 ablation in developing gonads interferes with Sertoli cell differentiation
To address Sox9 function in gonad development in vivo we deleted
the Sox9flox allele in a tissue specific manner. To direct
Cre expression to the developing gonad, we chose regulatory regions of the
Sf1 gene, which in vivo is expressed in the gonadal primordium from
E9.5 onwards. Using a 674 bp promoter fragment that has previously been shown
to direct gonad-specific expression in transgenic mice
(Wilhelm and Englert, 2002),
17 Sf1:Cre transgenic animals were generated. Expression in a number
of these transgenic lines was early/ectopic, which resulted in complete
deletion of the loxP-flanked Sox9 gene and concomitant
perinatal lethality in the heterozygous state (Sf1:Cre;
Sox9flox/+). However, bigenic mice from several lines survived
and line Sf1:Cre (5) was selected for all following analysis based on
its efficient ablation of Sox9 expression
(Fig. 1).
|
At E13.5, male testis development can be easily recognized by the formation of sex cords (Fig. 2B). In XY Sf1:Cre; Sox9flox/Sox9flox gonads we always detected some sex cord formation, although they appeared highly abnormal in a proportion of animals (Fig. 2C). Older embryos (E18.5) showed testicular descent, indicating the production of sufficient male hormones in these mice (data not shown). To analyze the effect of Sox9 ablation on testicular differentiation we performed histological analysis at day E13.5, E15.5 and E18.5. All XY gonads analyzed showed the development of male sex cords, and the general organization of germ cells within the seminiferous tubules seemed to be normal. However, gonads appeared to have a reduced number of seminiferous cords and the outline of these cords was irregular (Fig. 3G,H).
|
|
Molecular analysis of Sox9 knock-out gonads
To investigate the effect of Sox9 ablation on the expression of
genes involved in the sex determination process, we performed real-time PCR
analyses with RNA isolated from individual E13.5 urogenital ridges. First we
analyzed the expression of the Mullerian-inhibiting substance
(Mis/Amh), which has been suggested to be a direct target of
Sox9 (Arango et al.,
1999; De Santa Barbara et al.,
1998
). Indeed, Mis expression in our conditional
knock-out mice showed a linear relationship with Sox9 expression
(Fig. 4A; r=0.9905;
P<0.0001), supporting the theory that Mis represents a
direct target of this transcription factor. Similarly, Sox8, which
belongs to the same subgroup of Sox genes as Sox9, and which also
shows Sertoli cell-specific expression from E12.5 days onwards
(Schepers et al., 2003
), was
directly dependent on the level of Sox9
(Fig. 4B; r=0.8444;
P<0.0001). Remarkably, even in cases where Sox9
expression was as low as that of wild-type female gonads, both the expression
of Mis and Sox8 in XY knock-out gonads was still higher than
in XX tissues. By contrast, expression of Sf1, which in vitro can be
activated by the Sox9 protein (Shen and
Ingraham, 2002
), did not show a clear dependence on Sox9
expression levels (Fig. 4C;
P=0.27), which may partly be due to the expression of Sf1 in
other cell types, including progenitor cells and Leydig cells.
|
Homozygous deletion of Sox9 interferes with sex cord formation and the expression of male specific markers
Although our real-time PCR analysis at E13.5 suggested that, at least in
some gonads, Sox9 expression was very low and comparable to the level
of expression in female gonads, we could not exclude the possibility that the
lack of sex reversal in our tissue-specific knock-out animals was due to
incomplete deletion of Sox9 at the time of sex determination (E11.5).
To address this possibility we generated homozygous knock-out animals, making
use of germ-line specific Cre transgenic mouse lines. Males carrying the
spermatogenesis-specific Prm1:Cre transgene
(O'Gorman et al., 1997) on a
floxed Sox9 background (Prm1:Cre;
Sox9flox/Sox9flox) were crossed with female
Sox9flox/Sox9flox mice transgenic for the
oocyte-specific ZP3:Cre transgene (de
Vries et al., 2000
) (Fig.
5). Unfortunately, homozygous Sox9 knock-out animals die
at E11.5 as a result of cardiac failure
(Fig. 6A-D) (H. Akiyama et al.,
unpublished), which prohibited the in vivo analysis of the fate of XY gonads
in these embryos at later stages. Hence, to investigate the function of
Sox9 during sex determination, gonads were isolated at E11.5, placed
into organ culture and analyzed after two to three days in culture using in
situ hybridization or immunohistochemistry, respectively.
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Discussion |
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The fact that we did not observe a complete sex reversal in our
tissue-specific knock-out mice using the Sf1:Cre transgene was
probably due to incomplete inactivation of Sox9 at the time of sex
determination, suggesting that the remaining small amounts of this protein may
have been sufficient to activate male-specific genes. However, these
variations in Sox9 levels allowed us to draw a number of important
conclusions. First, using real-time PCR analysis, we could relate the
expression of varying amounts of Sox9 with the expression of several
genes known to be involved in the process of sexual differentiation. This
analysis demonstrated a clear dependence of Mis expression on
Sox9 levels, thus supporting in vivo the findings of earlier studies
that described Mis as a direct transcriptional target of Sox9
(Arango et al., 1999;
De Santa Barbara et al., 1998
).
Similarly, the transcription factor Sox8 directly depends on
expression levels of Sox9. At present we cannot clearly distinguish
whether this dependence is due to a direct regulation of Sox8 by
Sox9, or whether it simply reflects a reduction of the number of Sertoli cells
in knock-out animals.
In contrast to the direct dependence of Mis and Sox8 on
Sox9 expression levels, Sry expression was higher in gonads
with low levels of Sox9. At present there is no evidence for a
repressive function of the Sox9 protein, and the downregulation of
Sry may occur through the interaction of Sox9 with other factors, or
through the activation of a transcriptional repressor. The fact that
Sry levels were only increased in cases where Sox9
expression dropped below a certain threshold may suggest that persistent
expression is an indirect event, possibly because of a continuous presence of
Sry-positive Sertoli precursor cells originating from the coelomic
epithelium (Karl and Capel,
1998; Schmahl et al.,
2000
) that fail to differentiate.
The incomplete inactivation of Sox9 in our tissue-specific analysis also allowed us to look at the differentiation of testes with reduced expression levels of Sox9 at later stages of development. In our histological analysis of E18.5 XY gonads, we observed meiotic gonocytes, a hallmark of female differentiation. This may suggest that Sox9 is important to maintain a signal that suppresses meiosis in XY gonocytes. Further analysis may allow us to identify this signal.
Maybe the most surprising finding in our studies was the absence of sex
reversal in tissue-specific knock-out animals (Sf1:Cre;
Sox9flox/Sox9flox), despite the significant
reduction of Sox9 expression levels. This suggests that Sox9
expression levels are much less critical in mice than in humans. By contrast,
the additional deletion of at least one knock-out allele Sox8 can
lead to XY male to female sex reversal, suggesting that important functions of
Sox9 can be taken over by its close homologue Sox8. While
this study was in progress, a paper by Schepers et al. was published
describing Sox8 as a potential activator of the Mis promoter
(Schepers et al., 2003). The
authors demonstrated that although Sox8 can activate the Mis promoter
region, this activation was much weaker than that seen following Sox9
induction. Based on their studies, Schepers et al.
(Schepers et al., 2003
)
speculated that Sox8 expression in the gonad might only represent an
evolutionary remnant of a duplicated gene, which is in the process of adopting
new functions. However, another possible explanation would be that
upregulation of Sox8 by Sox9 (directly or indirectly) soon after the
commencement of sex determination is used to reinforce and imprint male sex
determination and testis differentiation on the developing gonad. This
hypothesis is further strengthened by the fact that Sox8 expression
seems to depend on Sox9 levels rather than the expression of Sry, which may
suggest that the male-specific regulatory elements of Sox8 were
acquired independently of that of Sox9.
When interpreting results in a tissue-specific knock-out it is important to
consider the inactivation of Sox9 on a cellular level. In any given cell, Sox9
is either wild-type (100%), heterozygous (50%) or homozygous (0%) for the
mutation. Cells that are heterozygous for the Sox9 knock-out allele
seem to differentiate normally into Sertoli cells (at least in mice), as
heterozygous mutants do not show a gonadal phenotype
(Bi et al., 2001). However, in
the case of an additional heterozygous or homozygous deletion of
Sox8, the overall Sox8/Sox9 gene dosage in a given cell would be
further reduced, which may interfere with the activation of their
transcriptional targets and, as a consequence, block the differentiation of
this cell into a Sertoli cell.
Taken together, our data suggest the following model. Sox9 is activated through the expression of Sry, and is both essential and sufficient to induce testis formation. Sox9, either directly or indirectly, represses expression of Sry and activates Sox8. A certain threshold of Sox8 and Sox9, or Sox9 on its own, is then required for gonadal precursor cells to differentiate into Sertoli cells.
Our finding that Sox8 may reinforce Sox9 function during gonad formation may also have an impact on human genetics. Only 75% of XY individuals with campomelic dysplasia show sex reversal. The reason for this variable penetrance of the phenotype is presently unknown. Our data suggest that the gonadal phenotype in individuals with campomelic dysplasia may be influenced by the expression of SOX8, which could be variable because of either mutations at the SOX8 locus or polymorphism of SOX8, which influence its expression level or function. Moreover, given its ability to at least partially take over SOX9 function, it is conceivable that ectopic activation of SOX8 in XX gonads may cause sex reversal in human patients similar to findings for the SOX9 gene.
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ACKNOWLEDGMENTS |
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