1 The Jackson Laboratory, Bar Harbor, ME 04609, USA
2 Department of Medicine Genetics Program, and Department of Genetics and
Genomics, Boston University School of Medicine, Boston, MA 02118, USA
* Authors for correspondence (e-mail: jbouma{at}jax.org and eme{at}jax.org)
Accepted 5 May 2005
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SUMMARY |
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Key words: Testis development, Nr0b1, Sry, Fetal gonad, Autosomal modifier, Mouse
![]() |
Introduction |
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It was originally thought that DAX1 was an ovarian determining
gene, based on sex reversal data in humans
(Bardoni et al., 1994;
Swain et al., 1996
). More
recent studies, however, have challenged this view and suggested that
Dax1 is involved in testicular development. Three studies have dealt
with loss of Dax1 function. The first report investigated gonad
development in 129Sv/J mice containing a Dax1 exon 2 (ligand-binding
domain) deletion (hereafter designated Dax1-). Dax1-/Y adult
males had smaller than normal sized testes with impaired testicular germinal
epithelial development and eventual germ cell loss
(Yu et al., 1998
). By
contrast, homozygous Dax1- mice were fertile females and appeared to
have no reproductive problems. A second study examined testicular development
in fetal 129Sv/J Dax1-/Y mice
(Meeks et al., 2003a
). Testis
development appeared to progress normally until E13.5, when some testis cords
appeared disorganized and incomplete. The third study examined the effects of
the Dax1- allele in mice containing a mixed genetic background and a
Mus domesticus poschiavinus Y chromosome (YPOS)
(Meeks et al., 2003b
).
Combining a `weaker' Sry (sex-determining region of chromosome Y)
allele on YPOS (Eicher et al.,
1995
) and Dax1- caused sex reversal in
Dax1-/YPOS mice.
Here, we report that Dax1 has an early, essential role in fetal
testis development. Experiments involving mice from the two inbred strains
C57BL/6JEi (B6) and DBA/2J (D2) were used. The B6 strain was chosen because B6
XY mice are exceptionally sensitive to disturbances in the early events of
testicular development and thus provide a sensitized genetic test system for
identifying genes that are important for primary (gonadal) sex determination
(Eicher and Washburn, 1986;
Eicher et al., 1996
;
Eicher et al., 1982
). The D2
strain was chosen because D2 XY mice develop normal testes under genetic
circumstances in which B6 XY mice develop ovaries or ovotestes
(Eicher et al., 1996
). We
found that ovaries developed in B6 Dax1-/Y mice (i.e. these mice are
completely sex reversed), whereas testes developed in D2 and F1
Dax1-/Y mice. Multi-gene expression analysis indicated that
Sox9 expression was not upregulated in B6 Dax1-/Y fetal
gonads even though Sry was expressed at the correct time and at
appropriate levels. Ovarian development in B6 Dax1-/Y mice was
prevented if Sry expression was increased by addition of a multicopy
Sry transgene. Finally, experiments to map B6-derived genes involved
in B6-DAX1 sex reversal identified a modifier gene located on distal
chromosome (Chr) 4. We conclude that Dax1 functions upstream of
Sox9 in the testis development pathway and hypothesize that DAX1, SRY
and TDA1, a protein encoded by the Chr 4 gene (testis-determining autosomal 1,
symbolized Tda1), are required for the upregulation of Sox9
expression in the somatic supporting cell lineage precursors, a molecular
event required for these cells to initiate differentiation as Sertoli
cells.
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Materials and methods |
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A D2-YPOS consomic strain was constructed by mating a B6 XYPOS hermaphrodite to a D2 female. Successive backcrosses involving XYPOS males mated to D2 females resulted in the D2-YPOS consomic strain. A D2-Dax1- congenic strain was produced by transferring Dax1- onto the D2 strain background using successive backcrossing.
A coisogenic B6 strain that is homozygous for the white-bellied agouti (AW) allele at the agouti gene and carries the tabby-6J (EdaTa-6J) mutation at the X-linked ectodysplasin-A (Eda) gene was used to efficiently identify Dax1-/Y backcross females at weaning in linkage analyses crosses. (Hereafter, EdaTa-6J is designated Ta6J.) Ta6J/+, Aw/Aw (or Aw/a) mice have a striped coat, whereas +/+ and +/Y, Aw/Aw (or Aw/a) mice have a non-striped coat. Thus, in the cross involving F1 Dax1-/+ females mated to Ta6J/Y, Aw/Aw males, XY (i.e. Dax1-/Y) females can be distinguished from XX (i.e. Ta6J/+) females because they lack a striped coat. (Males of the C57BL/6J-AW EdaTa-6J strain were provided by the Mouse Mutant Resource program of The Jackson Laboratory.)
Sry transgene rescue
To determine if increased Sry expression rescued gonadal sex
reversal in B6 Dax1-/Y mice, two approaches were used. The first used
B6 XYAKR,Sxr mice (Albrecht et
al., 2003). The YAKR,Sxr chromosome contains two copies
of Sry, an endogenous (AKR/J strain-derived) copy located on the
short arm and a second (RIII strain-derived) copy (i.e. Sxr) located in a
duplicated segment of the short arm that is transposed distal to the
pseudoautosomal region of the long arm of the Y chromosome. The second
approach used B6 XYAKR mice carrying a multicopy transgene,
designated TgN(Sry-129)2Ei (Tg2) (Washburn
et al., 2001
), containing the 129-derived Sry gene
(Koopman, 2001
). Our
unpublished data indicate that
84 copies of Sry are present in
Tg2 carriers and that the relative expression level of Sry in the
testes of XYAKR Tg2 fetuses is
16-fold higher than in the
testes of E12 XYAKR fetuses. Of the possible genotypes obtained in
these crosses, three were of interest: Dax1-/YAKR,
Dax1-/YAKR,Sxr and Dax1-/YAKR Tg2.
Genotyping
Genotyping was accomplished using lysate obtained by incubation of a small
piece of tissue overnight in lysis buffer [0.05 M KCl, 0.05 M Tris at (pH
8.3), 0.1 mg/ml gelatin, 0.45% Nonident P-40, 0.45% Tween and 60 µg/ml
Proteinase K] at 55°C. Primer sequences, PCR amplification cycles and
generated amplicon sizes are available (see Table S1 in the supplementary
material).
Staging of fetal gonads
Timed matings were performed to provide a rough estimate of fetal age.
Because gonad development progresses rapidly in mice and the developmental
stage of individual fetuses within a litter may differ, a more accurate
assessment of fetal age was employed. Fetuses younger than embryonic day (E)
13 were staged by counting tail somites (ts) distal to the hindlimbs (e.g.
28 ts corresponds to E12.5) (Hacker
et al., 1995
). Fetuses E13 and older were staged according to
fore- and hindlimb morphology (Theiler,
1989
).
Whole-mount immunohistochemistry
Whole-mount immunohistochemical analysis was performed as described
previously (Albrecht and Eicher,
2001). Briefly, E12.5 and E13.5 gonad-mesonephros complexes were
fixed overnight at 4°C in 4% paraformaldehyde, followed by a 24 hour
incubation in blocking buffer (1% BSA, 0.1% saponin, 0.02% sodium azide in
PBS) at 4°C. Samples were incubated with appropriate primary and secondary
antibodies diluted in blocking buffer for 24 hours for each antibody. Primary
antibodies included GATA4 (C-20, goat polyclonal, Santa Cruz Biotechnology;
1:500), PECAM1 (rat monoclonal, BD PharMingen; 1:100), WT1 (mouse monoclonal,
DakoCytomation; 1:300), AMH (rabbit polyclonal, gift from Dr Natalie Josso
(Rey et al., 1996
); 1:250),
SF1 (rabbit polyclonal, gift from Dr Ken-ichirou Morohashi
(Morohashi et al., 1993
);
1:1000) and SOX9 [rabbit polyclonal, gift from Dr Francis Poulat
(Gasca et al., 2002
); 1:1000].
Cy3-, Cy5-conjugated (Jackson ImmunoResearch; 1:500) or Alexa
Fluor-488-conjugated (Molecular Probes; 1:750) secondary antibodies were used
for visualization.
Fluorescently labeled samples were mounted in Slowfade-Light Antifade (Molecular Probes). Images were obtained using a Leica TCS-NT laser-scanning confocal microscope, and assembled using ImageJ software version 1.32v (http://rsb.nih.gov/ij/) and Adobe Photoshop v7.
Real-time RT-PCR
Gonads from fetuses ranging in age from E10 to E14 were used for real time
RT-PCR analysis. At E10 and E10.5, tissue containing urogenital ridges was
used. At E11.5 and E12, isolated gonad-mesonephros complexes were used. At
E12.5, E13 and E14, gonads were carefully dissected free of the mesonephros
and used. To prevent RNA degradation, tissues were collected using dissection
instruments cleaned with RnaseZap wipes (Ambion, Austin, TX). Tissues were
homogenized in lysis buffer containing ß-mercaptoethanol (Qiagen RNAeasy
kit, Qiagen, Valencia, CA) and stored at -80°C until further use.
|
Changes in relative gene expression between cDNA samples were determined
using version 2 of the statistical algorithm `Global Pattern Recognition' (GPR
v2.0) (Akilesh et al., 2003).
GPR v2.0 assigns a GPR score to each gene, indicating the fraction of
normalizer genes to which the gene is found to be significantly different
(P<0.05). Genes with a GPR score of at least 0.4 were considered
expressed at significantly different levels between cDNA samples. In addition,
GPR v2.0 uses a modified version of the recently published geometric averaging
algorithm (geNorm) to calculate fold changes in relative gene expression
(Vandesompele et al., 2002
).
Fold changes were determined based on the geometric mean of the 10 best
normalizers (most stable) (Vandesompele et
al., 2002
), instead of calculating fold changes based on a single
normalizer.
Modifier gene mapping
To map the modifier genes involved in B6-DAX1 XY sex reversal, B6
Dax1-/+ females were mated to D2 males and the resulting
(B6xD2)F1 Dax1-/+ female offspring were backcrossed to B6
Dax1+/Y males. Fetal gonads were analyzed between E14.5 and E16
(Theiler, 1989), a
developmental time period when ovarian and testicular tissue is easily
distinguishable within single gonads (see
Eicher et al., 1980
;
Eicher et al., 1996
). Gross
morphological inspection was accomplished by viewing and photographing each
gonad using an inverted light microscope. Each gonad was classified as an
ovary, a testis or an ovotestis (a gonad that contains both ovarian and
testicular tissue). In addition, a small sample of somatic tissue was used to
determine the Dax1 genotype and presence of the Y chromosome. The
remainder of each fetus was frozen for later DNA isolation. A total of 123
backcross fetuses were genotyped as Dax1-/Y and constitute the mice
used for linkage analysis.
A genome scan was performed using a set of single nucleotide polymorphic
(SNP) markers that differ between B6 and D2 and are located at 10-20 Mb
intervals along the length of each autosome
(Petkov et al., 2004
). SNP
typing was performed by Kbiosciences (Hertfordshire, UK). (The list of SNPs
used is available in Table S2 in the supplementary material.) Linkage analyses
were carried out using the pseudomarker software package version 1.06
(Sen and Churchill, 2001
)
(http://www.jax.org/staff/churchill/labsite/software).
Two versions of the data were analyzed. The first consisted of the 123
Dax1-/Y fetuses backcross mice, scored as 0, 0.5 and 1 for male,
hermaphrodite and female, respectively. The classification of hermaphrodite
included mice containing an ovary accompanied by an ovotestis, a testis
accompanied by an ovotestis, or a pair of ovotestes. The second linkage
analysis involved only the 19 male and 17 female fetuses, scored as a binary
trait.
A three-stage analysis strategy
(Sugiyama et al., 2001) was
used to identify the genetic loci underlying sex reversal in B6
Dax1-/Y mice. We first employed interval mapping
(Lander and Botstein, 1989
) to
identify QTLs with main effects and then carried out an exhaustive pair-wise
search (Sen and Churchill,
2001
) to identify epistatic interactions among loci. Permutation
analysis (Churchill and Doerge,
1994
) was used to establish significance thresholds and loci that
exceed the 95% genome-wide adjusted level are reported. The third stage of
analysis constructed a multi-locus regression model that includes simultaneous
effects of all significant loci. We initialized the model construction by
selecting all significant (P<0.05) and suggestive
(P<0.63) QTLs and interactions, and then removed terms using a
backward elimination strategy until each model term was significant at the
P<0.001 level. The multi-locus regression model is available in
Table S3 in the supplementary material.
Further confirmation for involvement of a distal Chr 4 locus in B6-DAX1 sex reversal was obtained by analyzing 32 additional Dax1-/Y females, produced by mating (B6xD2)F1 Dax1-/+ females to B6 or B6 Ta6J/Y, AW/AW males. All offspring obtained from B6 males were analyzed for the Dax1 alleles and the Y chromosome. In the case of offspring obtained from Ta6J/Y males, only females lacking Ta-striping (i.e. that did not inherit the X chromosome from their father) were typed for Dax1 and the Y chromosome. For both crosses, a Dax1-/Y mouse was classified as a female if both gonads had the shape and position (just below the kidneys) of normal ovaries, and the internal and external genitalia were consistent with normal female anatomy. In the cross involving normal B6 males, 10.5% of the offspring were Dax1-/Y females. In the cross involving B6 Ta6J/Y, AW/AW males, 10.6% of the Dax1-/Y offspring were females. These results are similar to the 13.8% Dax1-/Y fetal females obtained in the initial mapping cross.
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Results |
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Meeks and co-workers had reported that the combined presence of a
Dax1- allele and a `weaker' Sry allele causes sex reversal
in XYPOS mice (Meeks et al.,
2003b). Because genetic background influences whether sex reversal
occurs in XYPOS mice (Eicher et
al., 1995
; Eicher et al.,
1996
), we tested if presence of D2-derived genes would influence
testis development in Dax1-/YPOS mice. We mated B6
Dax1-/+ females to D2 XYPOS males and analyzed the
offspring at weaning: the 26 Dax1-/YPOS offspring
presented as females, whereas the 23 Dax1+/YPOS offspring
presented as males. We also backcrossed F1 Dax1-/+ females to D2
XYPOS males and analyzed the offspring at weaning: the 17
Dax1-/YPOS offspring presented as females whereas the 15
Dax1+/YPOS offspring presented as males. We conclude that
the combined presence of the SryPOS allele and the
Dax1- allele cause sex reversal in XY mice.
|
Multi-gene expression profiling
Real-time RT-PCR analysis was performed to examine the gene expression
profile of B6 Dax1-/Y fetal gonads compared with normal B6 XX and XY
gonads between E10 and E14 (see Materials and methods). With the exception of
Sry, gene expression in gonads of fetal B6 Dax1-/Y mice
followed the ovarian developmental pattern
(Table 1). For example, five
genes (Adamts19, Bmp2, Emx2, Fgfr2 and Fst), normally
expressed at higher levels in B6 XX gonads compared to XY gonads
(Bouma et al., 2004;
Menke and Page, 2002
), were
expressed in B6 Dax1-/Y gonads at levels comparable with those in B6
XX gonads. Alternatively, 12 genes (Aard, Amh, Cbln1, Cbln4, Cst9,
Cyp11a1, Cyp17a1, Cyp26b1, Dhh, Hhip, Sox9 and Tdl), normally
expressed at higher levels in B6 XY compared with XX gonads
(Bouma et al., 2004
;
Menke and Page, 2002
), were
expressed at levels comparable with those in B6 XX gonads. Sry
expression levels were similar in B6 Dax1+/Y and Dax1-/Y
gonads until E12.5, after which Sry expression was downregulated in
Dax1+/Y testes but not in Dax1-/Y ovaries
(Fig. 3). Whereas Sry
was below the detection limit of our assay in E14 Dax1+/Y testes,
Sry expression in E14 Dax1-/Y ovaries remained unchanged
relative to the expression level measured in E12.5 and E13 Dax1-/Y
ovaries. Sry transcripts were not detected in the ovaries of B6
Dax1-/Y newborn mice (data not shown), indicating that Sry
expression was extinguished between E14 and birth. We conclude that B6 mice
require Dax1 for Sry to initiate testicular development.
|
The second approach used an Sry transgene (Tg2)
(Washburn et al., 2001)
derived from the 129 Sry gene
(Koopman et al., 1991
). Tg2
contains all the necessary cis-acting sequences required for testicular tissue
development because all XX Tg2 mice develop exclusively testicular tissue. Tg2
contains 84 copies of Sry and results in a 16-fold increase in
Sry expression in the fetal gonads of E12 B6 XYAKR Tg2
mice (data not shown). We mated B6 Dax1-/+ females to B6
XYAKR Tg2 males and analyzed the offspring at weaning. The 12
Dax1-/YAKR offspring lacking Tg2 developed as females,
which agreed with the previous finding that the AKR Y chromosome does not
correct B6-DAX1 sex reversal. By contrast, the 20 B6
Dax1-/YAKR Tg2 offspring presented as males, and the
subset of males tested were fertile. Real-time RT-PCR analysis indicated that
Sox9 is upregulated in E12 B6 Dax1-/YAKR Tg2 and
Dax1+/+ Tg2 fetal gonads, and the relative expression levels in both
are similar to expression levels found in B6 Dax1+/Y fetal gonads
(data not shown).
We also examined GATA4 and PECAM1 expression in gonads of B6 Dax1-/YAKR Tg2 mice at E13.5 using whole-mount immunohistochemistry and confocal microscopy (Fig. 4). Gonad morphology in B6 Dax1-/YAKR Tg2 testes was indistinguishable from control B6 Dax1+/YAKR testes. We conclude that testis development in B6 Dax1-/YAKR mice is rescued if multiple copies of Sry are present.
Identification of modifier loci involved in B6-DAX1 XY sex reversal
To map the chromosomal location of B6-derived genes involved in B6-DAX1 XY
sex reversal, (B6xD2)F1 Dax1-/+ females were mated to B6 males,
and the gonadal phenotype of 123 Dax1-/Y fetuses was assessed at
E14.5 to E16: 19 (13.8%) contained exclusively testicular tissue, 17 (15.5%)
contained exclusively ovarian tissue and 87 (70.7%) contained both ovarian and
testicular tissue (ovotestis), with 22 containing an ovary and an ovotestis,
52 containing two ovotestes and 13 containing a testis and ovotestis. A genome
scan involving 160 SNP (single nucleotide polymorphic) loci detected two
chromosomes with peak LOD scores that approached the 0.05 genome-wide
threshold (threshold LOD=3.15) (Fig.
5A; see Table S2 in the supplementary material). Chromosome (Chr)
4 contained a single peak (LOD=3.06) distal to marker rs3718220. The Chr 1 LOD
profile was bimodal with highest peak (LOD=2.61) proximal to maker rs3697376
and a second peak (LOD=2.19) distal to marker rs3664528. Suggestive peaks also
were noted on Chrs 5, 6, 16, and 17.
|
To confirm the Chr 4 location of a major B6-derived modifier, we obtained 32 additional Dax1-/Y backcross adult females (see Materials and methods). A genome scan again identified a Chr 4 locus located between markers rs4224709 and rs371820. A summary of data for these 32 females and the original 17 Dax1-/Y fetuses is presented in Table 2.
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Discussion |
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Dax1 is required for fetal testis development in B6 mice
Data presented here indicate that a mutant Dax1 gene causes
ovarian development in B6 XY mice. By contrast, presence of the same mutant
Dax1 gene causes no major impact on testicular development in
(B6xD2)F1 XY mice, given that these males are fertile. Together, these
findings demonstrate that Dax1 is crucial for B6 fetal testis
differentiation and suggest that B6-derived autosomal genes play a role in
B6-DAX1 sex reversal.
Previously, Meeks and collaborators reported that the mutant Dax1-
allele in combination with the `weaker' SryPOS allele
causes XY sex reversal (Meeks et al.,
2003b). In their study, however, it was unclear if the observed
sex reversal in Dax1-/YPOS mice was influenced by the
genetic background of the mice used or the combined presence of Dax1-
and SryPOS. To test if the genetic background was
important, both Dax1- and SryPOS were tested in
B6 (B6xD2)F1 and backcross Dax1-/YPOS mice (produced
by mating Dax1-/+ F1 females to D2-YPOS males). In all
three cases, the Dax1-/YPOS mice were female. We conclude
that SryPOS is unable to initiate testicular development
in Dax1-/YPOS mice even when D2 autosomal genes are
present.
Identification of an important modifier locus
Genetic mapping revealed that a locus on distal Chr 4 and two loci on Chr 1
are involved in B6-DAX1 sex reversal. In addition, an interactive effect was
observed between Chr 1 and Chr 8. The finding of a QTL (quantitative trait
locus) on distal Chr 4 is significant. We previously mapped an autosomal
testis-determining locus (Tda1) to distal Chr 4 in studies involving
B6-YPOS sex reversal (Eicher et
al., 1996) and the results involving B6-DAX1 sex reversal suggest
that both inherited sex reversals involve the same Chr 4 locus. Clearly, one
or more genes located within this Chr 4 QTL play a major role in sex
determination and differentiation.
The nature of the interaction between Chr 1 and Chr 8 on the sex reversal phenotype is a classical `masking' epistasis in which the effect of Chr 1 is only observed when the Chr 8 locus carries a D2 allele. This observation may help in the further dissection of this complex trait by narrowing the field of candidate genes to those with products that can manifest such interactions.
Interestingly, the Wnt4 gene resides in a region of Chr4 that
overlaps with Tda1, thus Tda1 maybe Wnt4. In
addition, recent experiments show that Wnt4, which initially was
demonstrated to play an important role in ovary development, is also needed
for testis development (Vainio et al.,
1999; Jeays-Ward et al.,
2003
; Jeays-Ward et al.,
2004
; Yao et al.,
2004
). Moreover a number of studies have implicated Wnt4,
acting through ß-catenin and SF1, in the regulation of Dax1
expression (Jordan et al.,
2001
; Jordan et al.,
2003
; Mizusaki et al.,
2003
). The phenotypic differences observed between
Wnt4-/- XY and B6 Dax1-/Y mice (abnormal testes
and ovaries, respectively), however, suggest that factors, such as genetic
background (Wnt4-/- mice contained a mixed 129Sv, CBA, B6
genetic background) or primary cell type affected [Wnt4 is involved
in steroidogenic and endothelial cell migration from the mesonephros
(Jeays-Ward et al., 2003
)],
are responsible for these phenotypic differences. Future experiments using
B6-Wnt4- mice will address these issues.
B6-DAX1 sex reversal is due to lack of Sox9 up-regulation
Whole-mount immunohistochemical and real time RT-PCR analyses revealed that
B6 Dax1-/Y fetal gonads initiate development as ovaries rather than
testes. Moreover, these data suggest that the testis determining pathway is
interrupted at a very early stage because expression of testis-specific genes,
other than Sry, was not observed. Significantly, Sry
expression was initiated at the correct time and at normal levels in
Dax1-/Y fetal gonads. Expression of Sox9 is necessary for
Sertoli cell differentiation and accumulating evidence suggests that
Sox9 is the immediate downstream target of SRY (reviewed by
Canning and Lovell-Badge,
2002; Lovell-Badge et al.,
2002
). For example, expression of Sox9 driven by
Wt1 regulatory sequences initiates testicular differentiation in XX
mice (Vidal et al., 2001
), and
SOX9 and Sry are co-expressed in pre-Sertoli cells with SOX9
expression initiating slightly after Sry expression
(Sekido et al., 2004
). Taken
together, these data suggest that Dax1 participates either in
parallel with, or immediately downstream of, Sry to activate
Sox9 expression and the male sex-determination genetic cascade.
Absence of Sox9 upregulation in B6 Dax1-/Y gonads allows the
supporting cell precursors to differentiate as granulosa cells.
Normally, Sry expression is downregulated after E12.5 in XY gonads
(Hacker et al., 1995;
Jeske et al., 1995
), including
B6 XY gonads (Bouma et al.,
2004
). However, Sry transcript levels were not
downregulated in E13 and E14 B6 Dax1-/Y gonads. Recent results
suggest that the upregulation of Sox9 in pre-Sertoli cells is
responsible for downregulation and eventual silencing of Sry
(Chaboissier et al., 2004
;
Morais da Silva et al., 1996
).
Because Sox9 expression is not upregulated in Dax1-/Y
gonads, the finding that Sry is not immediately downregulated in
these gonads is consistent with this idea. It is not clear how Sry
expression is eventually extinguished in Dax1-/Y gonads. Perhaps in
the absence of Sox9, Sry expression is downregulated by itself, given
that Sox genes appear to recognize similar DNA-binding sites
(Bergstrom et al., 2000
;
Bowles et al., 2000
).
|
Model for upregulation of Sox9 in pre-Sertoli cells
We propose that the upregulation of Sox9 in XY mice depends on the
correct dose (i.e. expression level) of Sry, Dax1 and one or more
autosomal genes, including Tda1 located on Chr 4
(Fig. 6). We suggest that
testicular development in B6 mice is sensitive to perturbations in the
expression levels of these genes, and that alterations in the expression of
these genes interfere with the upregulation of Sox9, leading to
failure of testicular development. The findings that complete gonadal sex
reversal occurs in B6 Dax1-/Y (this study),
Dax1-/YPOS (Meeks et
al., 2003b) (this study) and B6 XYPOS
(Eicher et al., 1996
) mice are
compatible with this hypothesis, given that in each case two of the three
genes (i.e. Sry, Dax1 and Tda1) either malfunction and/or
are expressed at suboptimal levels. For example, if the protein encoded by the
B6-derived Tda1 gene is less efficient at facilitating the
upregulation of Sox9, even when expression levels of Sry are
normal, homozygosity for the B6 Tda1 allele together with the absence
of DAX1 will fail to increase Sox9 expression to the levels needed
for Sertoli cell differentiation and the supporting cell precursors will
initiate development as granulosa rather than Sertoli cells. Similarly,
absence of DAX1 and presence of a `weak' SryPOS allele
also will prevent the upregulation of Sox9 in
Dax1-/YPOS mice, leading to granulosa rather than Sertoli
cell differentiation. The importance of correct dose is further apparent from
an investigation using transgenic mice carrying extra copies of Dax1:
depending on the Sry allele present, increasing Dax1
expression in XY mice caused delayed testicular cord development or partial
development of ovarian tissue by antagonizing Sry action
(Swain et al., 1998
).
In conclusion, our findings provide evidence that Dax1 is essential for normal B6 XY fetal testis development. Furthermore, the importance of B6 mice as a genetic model for identifying novel gonadal sex-determining genes is demonstrated by the identification of an important locus on distal Chr 4 (Tda1) involved in at least two mouse XY sex reversal models, B6 Dax1-/Y and B6 XYPOS. Finally, a model is presented in which correct doses of Dax1, Sry and Tda1 are required for upregulation of Sox9 in precursor somatic support cells, an event essential for fetal testis development and differentiation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/13/3045/DC1
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ACKNOWLEDGMENTS |
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