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First published online 16 October 2008
doi: 10.1242/dev.024653
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Department of Genetics, Dartmouth Medical School, Hanover, NH 03755, USA.
Author for correspondence
(sergei.g.tevosian{at}dartmouth.edu)
Accepted 15 September 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Fog2 (Zfpm2), Gata4, Dkk1, Ovary, β-catenin, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Swain and Lovell-Badge, 1999;
Wilhelm et al., 2007). By
contrast, genetic mechanisms of development remain enigmatic for mammalian
females. The idea that ovarian differentiation requires its own set of genes
and occurs shortly after (Eicher and
Washburn, 1986), or even precedes
(McElreavey et al., 1993),
that of the testes was put forward a number of years ago. However, sexually
dimorphic expression in the developing ovary was not identified until fairly
recently. This new evidence clearly demonstrates that the female pathway of
development engages a number of dedicated genes. The two alternative sex fates
are thought to emerge through the antagonistic activities of sex-specific
transcription factors in a restricted number of gonadal cells. This initial
cell-fate decision is further expanded by extracellular non-cell-autonomous
signals that promote one developmental program, while at the same time
suppressing the other (Brennan and Capel,
2004; Capel, 2006;
Kim and Capel, 2006;
Kim et al., 2006).
We have previously demonstrated an in vivo requirement for the
transcription factor GATA4 and its co-factor FOG2 (ZFPM2 - Mouse Genome
Informatics) in testis differentiation
(Tevosian et al., 2002).
Gata4ki/ki mutants [ki is a V217G mutation in GATA4 that
specifically cripples the interaction between GATA4 and FOG proteins
(Crispino et al., 2001)], as
well as Fog2-null embryos
(Tevosian et al., 2000),
exhibit a profound early block in testis differentiation. Here, we demonstrate
that a deficiency in GATA4-FOG2 interaction leads to a block in ovarian
development coincidental with a drastic alteration in the female gene
expression program.
To avoid referring each time to both FOG2 null and GATA4ki mutants we will sometimes refer to them collectively as `GATA4/FOG2' mutants (and to the phenotype as the `GATA4-FOG2 interaction/complex loss'). This is justified, as the abrogation of GATA4-FOG2 interaction by a Gata4ki mutation (Gata4ki/ki) or Fog2 loss (Fog2-/-) results in equivalent defects in mouse gonadal differentiation in every experiment we have performed so far. It is formally possible, however, that these mutations have non-overlapping roles in gonadogenesis (for example, FOG2 could have a GATA4-independent function; and the `ki' mutation in GATA4 also renders it incapable of interacting with FOG1). Hence, we performed experiments with both mutants to eliminate this possibility. Importantly, the Gata4ki mutation is not a Gata4 loss of function; deletion of Gata4 gene in gonads may have a different outcome that does not necessarily phenocopy Fog2 gene loss or the Gata4ki/ki phenotype.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Tevosian et al., 2000). These
strains, as well as Wnt4+/- mice
(Vainio et al., 1999), were
maintained on the C57BL/6 background. β-catenin (Ctnnb1) mutant
mice were obtained from the Jackson Laboratory. The Flk-1-lacZ strain
has been genotyped, as previously described
(Shalaby et al., 1997). The
Dkk1+/- genotype was determined using primers dkk1
(5'-CTTCCGACACACAAACACTCCC-3') and dkk1rev
(5'-GTAAACCAAACTCTCGTTCAGC-3'). Sf1-Cre mice were
genotyped with Cre-specific primers as previously described
(Bingham et al., 2006).
Axin2lacZ mice (Yu et
al., 2005) were obtained from the EMMA repository.
Affymetrix microarray analysis of gene expression
Gonad-mesonephros complexes were dissected from E12.5 XX wild-type and
Gata4ki/ki mutant embryos and Affymetrix oligonucleotide
arrays were used for RNA expression analysis
(Chee et al., 1996;
Lipshutz et al., 1999). The
array experiment was performed by Dartmouth Genomic and Microarray Laboratory
according to a standard protocol. The microarray data have been deposited at
the GEO database (GSE11314) and were analyzed using Gene Traffic (Iobion
Informatics).
In situ hybridization
In situ hybridization (ISH) analysis was carried out essentially as
previously described (Manuylov et al.,
2007a). Sox9, Mis (Amh), Wnt4, Cyp17a1,
Hsd3b1 and Cyp11a1 RNA probes have been described
(Tevosian et al., 2002); the
Irx3-fragment-containing vector was a gift of Dr Nef
(Nef et al., 2005) and the
Gng13-containing vector was a gift of Dr Arango
(Fujino et al., 2007). Other
probes were generated with cDNA obtained from the embryonic total or gonadal
RNA by RT-PCR (Table 1).
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; Smagulova et al.,
2008). The relative expression level for each sample was
determined in the same run and was expressed as the ratio of the quantity of
RNA of interest to that of a control RNA (Gapdh). Gene-specific
primers and probes were designed using Primer Express (Perkin Elmer); primer
sequences are shown in Table 2
and are also available upon request.
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H2AX (Upstate;
1:300); rabbit anti-β-catenin (Sigma; 1:300); rabbit anti-SYN1 (a gift of
Drs Moens and Spyropoulos, York University, Toronto, Canada; 1:300) and rabbit
anti-Cre (a gift of Dr Ernst, Dartmouth, Hanover, USA; 1:1000). Secondary
antibodies (Invitrogen) were used at 1:500. All antibodies were diluted in
Antibody Diluent Solution (Dako). The slides were mounted in Vectashield with
DAPI (4',6-diamidino-2-phenylindole, Vector Labs) and photographed. The
confocal analysis of anti-PECAM1-stained gonads was performed as described
(Manuylov et al., 2007a).
Germ cell depletion
Germ cell depletion was performed as described
(Yao et al., 2003). The
depletion was confirmed by Oct4 ISH and alkaline phosphatase
staining. To generate W/Wv embryos depleted of germ cells,
male mice carrying the c-Kit mutation, dominant white spotting
(W), were mated to females carrying viable dominant spotting
(Wv) (Mintz and
Russell, 1957). Double heterozygotes depleted of germ cells were
identified by immunostaining one gonad from each pair with the germ cell
marker PECAM1.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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12-fold). The Fst
gene encodes a secreted protein that blocks the function of multiple members
of the TGFβ superfamily (Patel,
1998). Although Fst expression in embryonic gonads had
been reported a number of years ago
(Feijen et al., 1994), its
sexually dimorphic pattern was not appreciated until recently
(Menke and Page, 2002). Both
in situ hybridization (ISH) with an antisense Fst probe
(Fig. 1A,B) and real-time
RT-PCR (Fig. 1L) confirmed the
loss of Fst expression in Gata4/Fog2 mutants.
Fst is a downstream component of Wnt4 signaling
(Yao et al., 2004).
Wnt4 controls Fst and Bmp2 (bone morphogenetic
protein 2) expression in embryonic ovaries; in Wnt4-null XX E12.5
gonads, expression of both Fst and Bmp2 is lost. The
Wnt4-Fst pathway opposes the formation of the male-specific
vasculature in the female and ensures the survival of meiotic germ cells; this
constitutes the first established signaling pathway important for early
development of the mammalian ovary (Yao et
al., 2004). Wnt4 expression was lost in the gonad of
E12.5 XX Fog2-null mutants (Fig.
1D). Importantly, although E12.5 Gata4/Fog2
mutant XX gonads did not express Wnt4, weak but detectable
Wnt4 expression reappeared in the E13.5 mutant gonads
(Fig. 1F). Wnt4
expression is also observed in XY E13.5-14.5 Fog2 mutant gonads
(Tevosian et al., 2002).
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). In
summary, loss of GATA4-FOG2 interaction in the XX gonad results in the loss of
Fst expression and a failure to activate Wnt4 in the gonad
at E12.5; however, residual Wnt4 expression appears to be sufficient
for maintaining the normal level of Bmp2.
GATA4/FOG2 loss affects multiple aspects of early ovarian differentiation
In addition to affecting Wnt4 and Fst expression, as
described above, loss of the GATA4-FOG2 complex disrupts the expression of
numerous other genes that have been implicated in ovarian development.
Expression of the dimorphically expressed genes, Sprr2d (small
proline-rich 2d) (Beverdam and Koopman,
2006) and Foxl2 (forkhead box L2), was lost in
Gata4/Fog2 mutants (Fig.
2A-I) and expression of Gng13 (guanine nucleotide binding
protein, gamma 13) (Beverdam and Koopman,
2006; Fujino et al.,
2007) was strongly downregulated
(Fig. 2J,K). By contrast,
Sf1 (Nr5a1 - Mouse Genome Informatics) expression does not
require the GATA4-FOG2 complex (Fig.
2L,M) (see Tevosian et al.,
2002).
The current view of mammalian sex determination emphasizes the notion that
the two alternative fates, female and male, arise as closely intertwined
parities that are determined by antagonistic activities
(Kim and Capel, 2006); hence,
suppression of one developmental program could result in the emergence of the
other. Examination of XX gonads with GATA4/FOG2 loss showed no signs of testis
cord formation (see Fig. S1 in the supplementary material) and markers of
Sertoli cell differentiation (Sox9, Mis and Dhh) were absent
(Fig. 3A,B). The GATA4-FOG2
transcription complex is required for Sox9 activation and testis
differentiation (Manuylov et al.,
2007a; Tevosian et al.,
2002), so it is not surprising that the loss of GATA4-FOG2
interaction does not result in the activation of Sertoli cell differentiation
in the XX mutant gonad. Despite the absence of Sertoli cell differentiation,
Gata4/Fog2 mutants selectively expressed some steroidogenic
genes commonly associated with the embryonic testis
(Fig. 3A,B and see Fig. S2 in
the supplementary material). Similarly, expression of inhibin alpha
(Inha) has been associated with embryonic testes rather than ovarian
development in several vertebrate species (e.g.
Majdic et al., 1997;
Safi et al., 2001); the
regulation of Inha expression by GATA factors has been documented in
cultured cells (Robert et al.,
2006). The GATA4-FOG2 complex functions to repress Inha
expression in the developing ovary (Fig.
3C). In the control E12.5 gonads, Inha expression was
much stronger in the male sample, whereas both XX and XY
Fog2-/- gonads robustly expressed Inha. In
summary, similar to the recently reported Rspo1-/-
mutation (Chassot et al.,
2008; Tomizuka et al.,
2008), analysis of the Gata4/Fog2 mutants
revealed a complex blend (rather than exclusive dominance) of female- and
male-specific expression in the XX gonads with a compromised female
program.
|
;
Brennan et al., 2003;
Chassot et al., 2008;
Jeays-Ward et al., 2003;
Yao et al., 2004). To examine
the vascular development in Gata4/Fog2 XX mutants, we
introduced the Flk1-lacZ allele (Flk is also known as
Kdr - Mouse Genome Informatics)
(Shalaby et al., 1997) into
the crosses and used an X-Gal staining assay to `develop' the vasculature as
previously described (Tevosian et al.,
2000) (Fig. 3D). We
noted that the vascular system in both E12.5 XY and XX mutants developed
similarly to the control XX gonad (ovary) and lacked the coelomic
male-specific vessel that is clearly observed in the normal testis
(Fig. 3D, arrows).
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Wnt pathway genes in the Gata4/Fog2 mutants
At present, a preponderance of data defines DKK1 function mainly within the
context of the antagonism of canonical Wnt/β-catenin signaling
(Mukhopadhyay et al., 2001)
(reviewed by Kikuchi et al.,
2007; Niehrs,
2006). The Axin2lacZ reporter has proven to be
effective in monitoring the activity of the β-catenin signaling pathway
in the ovary (Chassot et al.,
2008); Axin2lacZ animals carry the
β-galactosidase (lacZ) gene in (knocked-in) the Axin2
locus (Yu et al., 2005).
Axin2 is considered to be one of the two (the other being
Sp5, see below) candidates for a `universal' Wnt target gene
(Clevers, 2006). Although the
Axin2 expression level in embryonic gonads is low (data not shown), a
sensitive X-Gal assay in the E13.5 gonads of Axin2lacZ
embryos clearly shows activation of this gene in ovaries but not testes
(Fig. 5A,B), as reported
previously (Chassot et al.,
2008). To examine the Axin2lacZ expression
upon Fog2 loss, we generated XX Fog2-null embryos with an
Axin2lacZ reporter. In the XX Fog2-null gonads,
Axin2lacZ expression was lost
(Fig. 5A-D).
A list of Wnt/β-catenin target genes is available from the Wnt
homepage
(http://www.stanford.edu/~rnusse/wntwindow.html).
We compared this list to the list of genes differentially expressed in
Gata4ki/ki mutants. Although transcriptional outputs of
the Wnt pathway are thought to be cell-specific
(Clevers, 2006), one of the
best `universal' Wnt/β-catenin target genes is considered to be
Sp5 (Clevers, 2006;
Weidinger et al., 2005).
Microarray analysis detected the downregulation of Sp5 expression in
the Gata4ki/ki mutant (3-fold). Likewise, expression
of the other Wnt target gene that encodes a transcription factor,
Irx3 (Braun et al.,
2003), is reduced. ISH confirmed that Sp5 and
Irx3 are downregulated in XX E13.5 mutants with GATA4-FOG2 complex
loss (Fig. 5E-H). This decrease
in the Sp5 and Irx3 levels in the mutants was confirmed by
qRT-PCR (Fig. 5M). Both
Sp5 and Irx3 are expressed in an XX-enriched, sexually
dimorphic manner (Bouma et al.,
2007a; Jorgensen and Gao,
2005; Nef et al.,
2005).
Recent work has established that Rspo genes, another gene family that
activates the β-catenin pathway, also play a role in female sexual
development (Chassot et al.,
2008; Parma et al.,
2006; Tomizuka et al.,
2008). Rspo1 expression is normal in
Gata4ki/ki and Fog2 mutants
(Fig. 5I-M).
GATA4-FOG2 and WNT4 regulate a partially overlapping set of genes
Our data suggest that in Gata4/Fog2 mutants, gonadal
Wnt4 expression is strongly downregulated during the critical time
(E12.5) for ovarian development, as is the WNT4 target gene Fst. At
the same time, the expression of another WNT4 gonadal target, Bmp2,
remains unchanged. To examine whether the GATA4-FOG2 and WNT4 pathways overlap
with respect to any other gene targets, we performed qRT-PCR in the
Wnt4 XX mutants (Fig.
5N). Both Fst and Bmp2 were severely
downregulated in the absence of Wnt4, as previously reported
(Yao et al., 2004). The
targets of canonical β-catenin signaling, Sp5 and Irx3,
were also downregulated in the Wnt4 mutants. However, Irx3
expression was reduced to a greater extent in the Wnt4-null than in
the Gata4/Fog2 XX mutant gonads
(Fig. 5, compare Irx3
in M and N). By contrast, Dkk1 was not upregulated upon Wnt4
loss. Similarly, another target of GATA4-FOG2 regulation, Foxl2, was
expressed normally in the Wnt4-null gonads
(Fig. 5N).
|
). To
investigate the localization of DKK1 protein in the developing gonads, we
performed immunofluorescence analysis. In accordance with the RNA expression
data (Fig. 4), DKK1 staining in
the E12.5 gonads was faint and appeared marginally enhanced in the testes
(Fig. 6A, arrowheads) as
compared with the ovaries (Fig.
6B). By contrast, we observed intense DKK1 staining in both XY and
XX mutant gonads, as predicted from our ISH experiments
(Fig. 6C,D). No staining was
observed in the gonads of XY Dkk1-/- embryos
(Fig. 6E).
DKK1 acts cell-autonomously in the somatic cells of the developing ovary
Immunofluorescence analysis demonstrated that DKK1 accumulates in the
vicinity of germ cells (Fig.
6C,D); the localization of DKK1 in other settings, at or near
plasma membranes, has been reported previously
(Caneparo et al., 2007;
Maekawa et al., 2005;
Mao et al., 2002). This
finding was unexpected, as GATA4 and FOG2 are not expressed in germ cells
during embryogenesis. Although this expression pattern could be indicative of
germ cell-derived DKK1, DKK1 is a secreted protein and so its accumulation
pattern does not necessarily reflect its expression origin. To investigate the
origin of gonadal Dkk1 expression we used busulfan, an alkylating
agent that can be used in rodents to deplete embryonic gonads of germ cells
(Menke and Page, 2002;
Merchant, 1975). Staining for
expression of the germ cell-specific POU transcription factor Oct4
(Pou5f1 - Mouse Genome Informatics) confirmed that most germ cells
were eliminated in the busulfan-treated E12.5 XX gonads
(Fig. 6F,G). By contrast, the
expression of Dkk1 was not affected by busulfan in either the control
XY sample (Fig. 6H,I) or in XX
Gata4/Fog2 mutants (Fig.
6J-M). Therefore, Dkk1 expression is not dependent on the
presence of germ cells.
DKK1 acts through binding to the LRP receptors (LRP5 or LRP6) with high
affinity (Bafico et al., 2001;
Mao et al., 2001;
Semenov et al., 2001).
Lrp6 (but not Lrp5, data not shown) is expressed in the
developing gonad at E13.5 (Fig.
6N) and its expression was unaffected in E13.5 XX
W/Wv (Kit-mutant) germ cell-deficient gonads
(Fig. 6O). In summary, these
data suggest that somatic cells in the ovaries are the primary source of
Dkk1 expression as well as being the recipients of canonical
β-catenin signaling.
|
|
H2AX (Fig. 7B-D).
Similarly, qRT-PCR analysis of Stra8
(Baltus et al., 2006) and
Scp1 (Sycp1 - Mouse Genome Informatics)
(Dobson et al., 1994)
expression detected no difference between the control and XX
Gata4/Fog2 mutant samples
(Fig. 7E). Not surprisingly, XY
germ cells in the Gata4/Fog2 mutants upregulated
Stra8 and Scp1 expression similarly to their XX
counterparts, as male differentiation is blocked in these embryos
(Tevosian et al., 2002)
(Fig. 7E). As
Gata4/Fog2 mutants are embryonic lethal at E13.5-14.5,
further development of the XX germ cells was not analyzed.
Analysis of the sexual differentiation phenotype in doubly homozygous mutant mice
We reasoned that if an abnormally high level of DKK1 in
Gata4/Fog2 mutants results in the downregulation of
canonical β-catenin pathway targets
(Fig. 5), then these same genes
could be activated in XX Dkk1-/- embryonic gonads. Indeed,
we observed that several canonical β-catenin pathway targets (Irx3,
Sp5 and Wnt9a) are upregulated in E12.5
Dkk1-/- ovaries (Fig.
8A,C). Interestingly, in addition to a `β-catenin set', the
expression of Foxl2 was also increased; this gene has not previously
been described as a target for canonical β-catenin signaling
(Fig. 8A,C). These data support
the previous assertion, by us and others, that the canonical β-catenin
pathway functions during ovarian development and, additionally, identify the
Foxl2 gene as a novel target of the canonical β-catenin pathway
in the ovary.
|
; Maatouk et al.,
2008; Tomizuka et al.,
2008), a profound block in female development observed upon
GATA4/FOG2 loss could be explained solely by a dramatic increase in its
secreted inhibitor, DKK1, in Gata4/Fog2 mutants.
Alternatively, an intact GATA4-FOG2 complex could be independently (i.e.
regardless of its role in Dkk1 repression) required for ovarian
differentiation and development. To examine XX gonads that are incapable of
activating Dkk1 expression in response to the ablation of the
GATA4-FOG2 complex, doubly homozygous Dkk1-/-;
Fog2-/- and Dkk1-/-; Gata4ki/ki
embryos were generated. We isolated RNA/cDNA from the E12.5 XX control and
mutant gonad samples and examined the expression of genes that we have
previously confirmed as targets of GATA4-FOG2 regulation in the ovary. As
double mutants are no longer capable of upregulating Dkk1, we
predicted that the expression of a subset of GATA4-FOG2-dependent genes,
previously inhibited through DKK1, would be (at least partially) restored. The
expression of Sp5, Irx3 and Wnt9a increased in the double
mutants as compared with Fog2 mutants, whereas Inha
expression decreased (Fig. 8C).
We conclude that the GATA4-FOG2 complex is required to establish the requisite
normal level of canonical β-catenin signaling in the ovary by repressing
Dkk1. By contrast, Fst and Foxl2 expression in the
XX Gata4/Fog2 mutants was not restored by deleting
Dkk1, and neither was Wnt4 expression at E12.5
(Fig. 8B,C). These data argue
that the GATA4-FOG2 complex regulates these genes independently of its role in
maintaining the normal level of canonical β-catenin signaling through
repressing Dkk1.
Analysis of the sexual differentiation phenotype in mutants with somatic cell loss of β-catenin
The GATA4-FOG2 transcription complex is required for maintaining a normal
level of β-catenin signaling; however, it is also essential for
maintaining ovary-specific Wnt4 expression. Either of these
GATA4-FOG2-dependent genes (β-catenin or Wnt4) could potentially
regulate Fst transcription. Fst levels do not increase in
Dkk1-/- XX gonads and Fst expression was not
restored in the Dkk1-/-; Fog2-/- double mutants
(Fig. 8C). To independently
assess the contribution of canonical β-catenin signaling to ovarian
Fst expression, we performed a conditional excision of the
β-catenin gene in the ovary. A Cre line of mice based on the BAC
harboring the Sf1 locus has recently been described
(Bingham et al., 2006). This
Sf1-Cre is robustly expressed during early gonadogenesis (see Fig. S3
in the supplementary material) and hence is ideal to excise β-catenin.
Sf1-Cre excision led to the loss of somatic β-catenin expression
(see Fig. S4 in the supplementary material) and to a 7-fold reduction in
the Fst expression level in the XX gonads, whereas Gata4,
Fog2 and Dkk1 were not affected
(Fig. 9A,C). This experiment
demonstrates that β-catenin regulates Fst transcription without
affecting the GATA4-FOG2 level. In addition to Fst, loss of gonadal
β-catenin affected the expression of many other genes. The canonical
β-catenin target, Irx3, and the essential regulators of ovarian
development, Foxl2 and Wnt4, were severely downregulated
(Fig. 9A and see Fig. S5 in the
supplementary material). Loss of Wnt4 and Fst expression was
likely to be responsible for a dramatic reduction in the survival of female
germ cells in the XX E18.5 β-catenin mutant gonads
(Fig. 9D,E) (see
Vainio et al., 1999;
Yao et al., 2004).
|
;
Gonzalez-Sancho et al., 2005;
Niida et al., 2004),
Dkk1 expression was not affected
(Fig. 9C). β-catenin
deletion in the ovary did not result in the activation of the alternative male
pathway (sex reversal), as shown by the lack of expression of the Sertoli
cell-specific genes Sox9, Mis and Dhh at either E13.5 or
E18.5 (Fig. 9B; see Fig. S6 in
the supplementary material; data not shown). Similar to the
Gata4/Fog2 mutants, the expression of some male-specific
genes was derepressed in β-catenin mutants (e.g. Cyp11a1 and
Inha, Fig. 9B,C).
However, in contrast to the Gata4/Fog2 mutants (but similar
to the XX Rspo1-null mutation), XX gonads with β-catenin loss
developed a coelomic vessel (Fig.
10H,K). In summary, β-catenin is required for normal ovarian
development, whereas testis development in the absence of β-catenin
proceeds apparently as normal (Fig.
10G and see Fig. S6 in the supplementary material) (see
Chang et al., 2008). |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). In
the interim period, work by many laboratories has uncovered a number of
sexually dimorphic genes in the developing mouse ovary and a functional
relationship between them is beginning to emerge (e.g.
Bouma et al., 2007a;
Chassot et al., 2008;
Jorgensen and Gao, 2005;
Menke et al., 2003;
Menke and Page, 2002;
Nef et al., 2005;
Yao et al., 2004). This new
body of data has provided a sufficient foundation to integrate the GATA4-FOG2
complex into the transcriptional cascade orchestrating ovarian development in
mammals.
|
), it
is primarily Wnt4 that has been associated with sexual development in
mammals. Wnt4-null females are masculinized, as demonstrated by the
absence of Müllerian ducts and the retention of Wolffian ducts
(Vainio et al., 1999).
Wnt4 is also required to repress steroidogenic and vascular
endothelial cell migration into the developing XX gonad; absence of
Wnt4 leads to both ectopic steroid (e.g. testosterone) production and
formation of a male-specific coelomic blood vessel
(Heikkila et al., 2005;
Jeays-Ward et al., 2003). Four
known XY human subjects with duplications of the chromosome 1p35 that includes
the WNT4 locus exhibit symptoms that range from isolated
cryptorchidism to severe genital ambiguity
(Jordan et al., 2001;
Jordan et al., 2003). A recent
report demonstrates that in the ovary, Wnt4 partially acts through
canonical β-catenin signaling
(Chassot et al., 2008).
Our analysis of Gata4/Fog2 mutant ovaries revealed a
dramatic activation of Dkk1 expression in the absence of GATA4-FOG2
interaction, concomitant with the downregulation of genes linked to canonical
Wnt/β-catenin signaling. DKK1, the founding member of the DKK family
(Krupnik et al., 1999;
Monaghan et al., 1999), is a
secreted protein and a potent Wnt signaling inhibitor
(Glinka et al., 1998). It
binds to the LRP receptors (LRP5 or LRP6) and prevents interaction between the
Wnt ligand and the Fz-LRP receptor complex
(Bafico et al., 2001;
Mao et al., 2001;
Semenov et al., 2001). The
mechanism of Dkk1 activation as a result of GATA4/FOG2 loss is not
clear. The c-JUN transcription factor and JNK signaling have been reported to
positively regulate Dkk1 transcription
(Colla et al., 2007;
Grotewold and Ruther, 2002);
however, this is an unlikely explanation because the c-Jun RNA level
is unaffected in Gata4/Fog2 XX mutant gonads, and an active
form of JNK is undetectable (data not shown).
GATA4-FOG2 complex loss affects multiple aspects of ovarian development
One of the genes requiring the GATA4-FOG2 complex is Foxl2. FOXL2,
a forkhead transcription factor, is essential for reproductive development in
females (reviewed by Uhlenhaut and Treier,
2006). In mice [as in humans
(Cocquet et al., 2002)]
Foxl2 is one of the earliest genes expressed in a female-specific
fashion (Loffler et al., 2003)
and Foxl2 homozygous mutants recapitulate female infertility in
humans (Schmidt et al., 2004).
Although Foxl2 is required in granulosa cell function in postnatal
ovaries, embryonic ovarian development initiates and proceeds apparently
normally in its absence (Ottolenghi et
al., 2005; Schmidt et al.,
2004). Importantly, in contrast to the loss of Foxl2
expression in Gata4/Fog2 XX mutants
(Fig. 2) and in β-catenin
deficiency (Fig. 9 and see Fig.
S5 in the supplementary material), Foxl2 expression is normal in the
Wnt4-null (Fig. 5N)
and Rspo1-null (Chassot et al.,
2008) mutants, underscoring the specific requirement for the
GATA4-FOG2 complex and β-catenin protein in the control of Foxl2
transcription.
Although GATA4-FOG2 complex loss affects several key elements of the
ovarian gene expression program, some dimorphically expressed ovarian genes,
such as Bmp2, retain their wild-type levels. Unexpectedly,
Bmp2 levels remain unchanged in the XX E12.5
Gata4/Fog2 mutants despite the loss of gonadal Wnt4
expression; Wnt4 is epistatic to Bmp2 and in the
Wnt4-null XX gonads Bmp2 expression is dramatically reduced
(Fig. 5N)
(Yao et al., 2004). The
mesonephric expression of Wnt4 that persists in
Gata4/Fog2 mutants (e.g.
Fig. 1C-F) could be responsible
for maintaining the wild-type level of gonadal Bmp2. Alternatively,
it is possible that the early (E11.5) expression of Wnt4 that is
independent of GATA4-FOG2 regulation is sufficient to trigger the activation
of Bmp2 transcription.
Expression of Rspo1 is also normal in Gata4/Fog2
mutants (Fig. 5). It was
proposed that Rspo1 functions to relieve the DKK1-imposed inhibition
of the β-catenin pathway by antagonizing DKK1-dependent LRP6 receptor
internalization (Binnerts et al.,
2007). Our data suggest that in the XX gonads of the
Gata4/Fog2 mutants, excessive DKK1 is no longer adequately
antagonized by normal concentrations of the RSPO1 protein. This results in an
outcome similar to that of Rspo1 deficiency in that a downregulation
of female-specific Wnt4 expression is observed, whereas Bmp2
is unaffected (Fig. 1)
(Chassot et al., 2008).
Fst expression requires multiple regulatory inputs
Gata4/Fog2, Wnt4 and Rspo1 mutations all
converge on the β-catenin signaling pathway and Fst expression
is severely reduced or lost in these mutants. Hence, it was tempting to
speculate that Fst expression critically depends on the nuclear
β-catenin pathway in the ovary. The regulation of an Fst
promoter by canonical β-catenin signaling has been reported in cell
culture (Miyanaga and Shimasaki,
1993). Mutation of the putative TCF binding site (CTTTGAT) at -223
to -217 relative to the start of Fst transcription led to the
abrogation of the WNT3A response (Willert
et al., 2002).
A conditional knockout of the β-catenin gene in the gonad results in a
drastic reduction in Fst expression, validating the essential
requirement for canonical β-catenin signaling in Fst regulation
in vivo. A recent report on the Rspo1 knockout
(Chassot et al., 2008) also
suggests that RSPO1 regulates Fst expression through β-catenin;
constitutively active β-catenin is sufficient to rescue the ovarian
development of the Rspo1-null mice, although Fst expression
was not directly examined in the rescued ovaries. By contrast, Fst
expression is not upregulated in Dkk1-/- gonads and is not
restored in the Fog2 mutant by Dkk1 ablation. This
demonstrates that, in addition to its reliance on intact β-catenin
signaling, Fst also requires a functional GATA4-FOG2 complex for its
expression. Whether WNT4 regulates Fst expression via (or
independently of) β-catenin is currently unclear and will require
restoring (or ectopically stabilizing) β-catenin signaling in the
Wnt4-null gonad.
A pivotal role for the GATA4-FOG2 complex in sexual differentiation
Previous work demonstrated the importance of the GATA4 and FOG2 proteins in
testis development. Loss of GATA4-FOG2 interaction leads to a block in the
male pathway because the upregulation of Sox9 gene expression, which
is necessary for testicular development, does not occur in mutant XY gonads
(Tevosian et al., 2002).
Moreover, a threshold concentration of the functional GATA4-FOG2 complex is
required to mount an adequate Sox9 expression level that will tip the
scale towards testis differentiation (Bouma
et al., 2007b; Manuylov et
al., 2007a). We have now shown that interaction between these
protein partners is also required for the ovarian pathway.
As Gata4 and Fog2 are expressed in gonads of both sexes,
their involvement in both testicular and ovarian development is not entirely
surprising. The most parsimonious explanation is that GATA4 and FOG2 control
the developmental program or programs common to both fates. The block in the
proliferation or survival of the pre-Sertoli/pre-granulosa cells could, in
principle, account for the observed loss of gene expression. FOG2-GATA4
function is required to maintain sufficient numbers of SOX9-positive cells in
the developing testis (Bouma et al.,
2007b; Manuylov et al.,
2007a). However, a reduction in cell number is unlikely to play a
major role in the ovarian pathway block; in this respect, the dramatic
increase in coelomic epithelial proliferation in XY E11.5 gonads is not
detected in XX gonads (Schmahl et al.,
2000). Correspondingly, we observed no significant reduction in
proliferation, as assessed by staining for phosphorylated histone H3 and the
proliferation-associated protein Ki67 (MKI67), in the E11.5-12.5 XX gonads of
the Gata4/Fog2 mutants; the TUNEL assay did not register an
increase in apoptosis either. Moreover, whereas Fog2
haploinsufficiency leads to a measurable decrease in the number of
SOX9-positive cells in the testis
(Manuylov et al., 2007a), we
observe no decrease in the number of FOXL2-positive cells in
Fog2+/- ovaries (data not shown). Finally, the normal
expression of the early ovarian markers Bmp2
(Fig. 1) and Rspo1
(Fig. 5), which are expressed
by the somatic support cells (Bouma et al.,
2007a; Chassot et al.,
2008), argues strongly against a generalized block in the
proliferation or survival of a pre-granulosa cell population.
Loss-of-function mutations in male-specific genes such as Sox9
(Chaboissier et al., 2004) or
Fgf9 (Colvin et al.,
2001; Kim et al.,
2006) result in activation of the ovarian-specific expression
pattern. Similarly, loss-of-function mutations in ovarian-specific genes
(Wnt4, Fst, Rspo1 and now β-catenin; Figs
9 and
10) launch the expression of
the testis-specific program in the XX gonad (for example, ectopic formation of
a male-specific coelomic blood vessel is observed in these knockouts). By
contrast, a `battle of the sexes' that lacks its GATA4 and FOG2 pieces results
in an earlier tie, as neither side can win. Loss of
Gata4/Fog2 does not, however, preclude the initiation of
female-specific development of germ cells: in Gata4/Fog2
mutants (XX or XY), germ cells enter meiotic prophase normally, beginning at
around E13.5 (Fig. 7).
Many of the crucial events in gonadal (especially ovarian) development can only be realized postnatally, and conditional deletion of Gata4 and Fog2 will be required to analyze the mutants after the time of birth. This will be informative for gaining further insight into the function of GATA4-FOG2 in ovarian development, as only once the mutant XX cells have had to march through the major competence test of folliculogenesis can the function of the early ovarian genes be truly exposed.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/22/3731/DC1
|
ACKNOWLEDGMENTS |
|---|
) prior to
publication. This work was supported by a grant to S.G.T. from the National
Institutes of Health (R01HD42751 NICHD). |
Footnotes |
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|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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