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First published online May 30, 2007
doi: 10.1242/10.1242/dev.004390
1 Section of Gene Function and Regulation, Institute of Cancer Research, 237
Fulham Road, London SW3 6JB, UK.
2 Neural Development Unit, Institute of Child Health, University College London,
London, UK.
* Author for correspondence (e-mail: amanda.swain{at}icr.ac.uk)
Accepted 4 April 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Adrenal development, Gene dosage, Organogenesis, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Val et al., 2003), suggests
that these two tissues originate from a so-called adrenogonadal primordium
(AGP) present at embryonic day 9 (E9.0) in mouse
(Hatano et al., 1996;
Ikeda et al., 1994). This is
consistent with the absence of both adrenal glands and gonads in mice mutant
for the transcription factor Wt1, although Wt1 was not found in the
adrenal glands but was expressed in the coelomic epithelium of the urogenital
region at E9.0 (Moore et al.,
1999; Moore et al.,
1998; Vidal and Schedl,
2000). As development proceeds, the adrenal cortical primordium
separates from the gonadal primordium in the rostral region of the AGP and
undergoes an adrenal cortical-specific differentiation programme. This is
characterised by expression of adrenal steroidogenic enzymes, zonation of
their expression and establishment of hormonal sensitivity
(Morohashi, 1997). The
innermost region of the adrenal gland, the medulla, is composed of chromaffin
cells that derive from neural crest cells that invade the adrenal cortical
primordium during development.
Most mutant mouse models in which a defect in adrenal development is
observed also show impaired gonad development
(Else and Hammer, 2005). This
is the case for genetic ablation of factors such as Sf-1
(Luo et al., 1994), Pbx1
(Schnabel et al., 2003), M33
(Cbx2 - Mouse Genome Informatics)
(Katoh-Fukui et al., 2005) and
Odd1 (Osr1 - Mouse Genome Informatics)
(Wang et al., 2005). Although
these observations conform to the idea of a common developmental origin of
these two tissues, these models do not provide insight into the developmental
and molecular mechanisms that underlie the specification of one or other
primordium from the AGP.
Interestingly, mice mutant for the transcription co-factor Cited2 were
reported to have no adrenal gland but apparently normal gonads at E17.5
(Bamforth et al., 2001),
suggesting that Cited2 might participate in adrenal development. Studies have
shown that Cited2 is also involved in the development of the heart
and neural tube and is implicated in left-right patterning through interaction
with the transcription factor Tfap2 (Tcfap2a - Mouse Genome Informatics) on
the Pitx2 promoter (Bamforth et
al., 2001; Bamforth et al.,
2004; Bragança et al.,
2003; Barbera et al.,
2002; Weninger et al.,
2005; Yin et al.,
2002). The Cited2 protein lacks DNA-binding properties and has
been shown to function as a transcription coactivator for the nuclear hormone
receptors Ppar
and Ppar
in vitro, although the physiological
significance of such an interaction is still unclear
(Tien et al., 2004). Cited2
has also been proposed to repress Hif1 transcription activity by
competing for the CH1-binding domain on CBP/p300 (Crebbp - Mouse Genome
Informatics) (Bhattacharya et al.,
1999; Freedman et al.,
2003).
Here, we have investigated the role of Cited2 in adrenal development. We provide molecular and genetic evidence that Cited2 acts in the AGP, interacting with the transcription factor Wt1 to promote adrenal cortical primordium development from the AGP. Analysis of Sf-1 expression in Cited2-/- and Cited+/- Wt1+/- embryos by quantitative real-time PCR indicates a model in which the interaction between Cited2 and Wt1 results in an increase in the expression of Sf-1 above a threshold required for adrenal cortical development.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Probes for
Cyp11a1 (P450scc), Sf-1, Sox9, Wnt4 and
Dax1 have been described previously
(Ikeda et al., 1996;
Kent et al., 1996;
Swain et al., 1996;
Vainio et al., 1999). The
Cited2 probe was synthesized from a fragment of Cited2 cDNA
(positions 4 to 561) subcloned from AA023157 (Image clone 456049) into
pT7T3-PacI. Probes for Lim1, Wt1, Hoxb9 and tyrosine hydroxylase
(Th) were synthesized directly from PCR templates, which included the
sequences encoding the T7 RNA polymerase promoter. PCR fragments were made
from MF1 mouse genomic DNA (Lim1, Wt1, Hoxb9) or E18.5 brain cDNAs
(Th) using the following primers (F, forward; R, reverse):
Lim1, 5'-CACTGCAAGAAACCGGCAATG-3' (F) and
5'-GTAATACGACTCACTATAGGGTTGCAGCCCGCACAGTGC-3' (R); Wt1,
5'-TTTGAGGGGTCCTGACACGG-3' (F) and
5'-GTAATACGACTCACTATAGGGCTGGCCTTCCAGGTTTCCAG-3' (R);
Hoxb9, 5'-CCACTCTCCTGGTGTTAAGA-3' (F) and
5'-GTAATACGACTCACTATAGGGAGACTTGTCTTGCTGGTG-3' (R); and for
Th, 5'-CCGTGCAGCCCTACCAAG-3' (F) and
5'-GTAATACGACTCACTATAGGGGTAGAATACAGCATGAAGGGC-3' (R). These PCR
fragments were isolated and used as templates in a DIG labelling reaction
using T7 RNA polymerase (Val et al.,
2006).
Mice
Cited2 mutant mice have been described previously and were bred on
a C57/Bl6 strain background (Barbera et
al., 2002). Unless otherwise stated, the phenotype of
Cited2+/- mice was identical to
Cited2+/+ mice and therefore these mice were sometimes
used as wild-type control samples. Wt1 mutant mice were kindly
provided by Dr Andreas Schedl (INSERM U470, Nice, France) and were initially
created by Kreidberg et al. (Kreidberg et
al., 1993). Sf-1 mutant mice were kindly provided by Dr
Lovell-Badge (NIMR, London, UK) and were initially created by Luo et al.
(Luo et al., 1994). Both lines
were bred on a mixed strain background. The expression studies were performed
on embryonic tissue from MF1 mice. For early development, staging of the
embryos was determined by counting the number of tail somites (ts). E10,
E10.5, E11, E11.5 and E12 were considered equivalent to approximately 5ts,
10ts, 14ts, 18ts and 21ts, respectively. Older embryos were staged by limb
morphology.
Immunohistochemistry
Laminin was detected on cryosections (10 µm) or on whole-mount
urogenital regions using anti-laminin antibody (1/400, L9393, Sigma).
Secondary fluorescent antibodies were obtained from Molecular Probes and used
at 1/1000 dilution.
Apoptosis and proliferation
Apoptosis was detected by incubating dissected embryos in the presence of
Lysotracker Red (Molecular Probes) (Zucker
et al., 1999) according to the protocol of Schmahl and Capel
(Schmahl and Capel, 2003). For
proliferation analysis, pregnant mice were injected with Bromodeoxyuridine
(BrdU) (50 mg/kg) 2 hours before culling. Incorporated BrdU was detected on
cryosections after acid treatment using a 1/50 dilution of a monoclonal
anti-BrdU antibody (clone BMC 9318, Roche). For double immunohistochemistry
with Sf-1 and BrdU antibodies, sections were incubated with Sf-1 antibody
(rabbit anti-Sf-1, 1/4000, a kind gift from K. Morohashi, NIBB, Okasaki,
Japan) prior to acid treatment. After washes, the primary antibody was
detected with the Vectastain ABC Amplification Kit (Vector Labs) according to
the manufacturer's instructions. Peroxidase activity was detected with the
fluorescent substrate TSA (Pierce) and sections were then processed through
acid treatment and BrdU detection.
Immunoprecipitation
Total proteins (500 µg) from transfected 293A cells or nuclear proteins
from M15 cells were immunoprecipitated overnight with anti-Wt1 antibody (1.6
µg, Wt1 C-19, SC-192, Santa Cruz), anti-ß-galactosidase antibody (1.6
µg, AB1211, Chemicon), non-immune rabbit IgG (1.6 µg, SC-2027, Santa
Cruz), anti-Sf-1 antibody (1.6 µg, 07-618, Upstate/Millipore) or
anti-Cited2 antibody (10 µg, clone JA22, Abcam). Immunoprecipitates were
collected on protein A- or protein G-sepharose. After four washes in PBS in
the presence of 0.1% Triton X-100, protease inhibitors (Complete Mix, Roche)
and 150 mM NaCl, immunocomplexes were eluted in Laemmli buffer and analysed by
western blotting.
GST pull-down
GST fusions of the mouse Wt1(-/-) protein were provided by Dr Jonathan
Licht and are described in Johnstone et al.
(Johnstone et al., 1996).
Recombinant proteins were prepared and attached to glutathione-sepharose 4B
beads (GE Healthcare) according to a standard protocol. Mouse Cited2 protein
(AA023157) was translated in vitro in the presence of
35S-methionine using the TNT T7/T3-Coupled Reticulocyte Lysate Kit
(Promega). 35S-labelled Cited2 protein was incubated with the beads
in binding buffer (20 mM Hepes pH 7.4, 100 mM NaCl, 0.1% Triton X-100, 0.2
mg/ml BSA, 2 mM dithiothreitol, 10 µM ZnCl2, 2 mM EDTA, 1 mM
PMSF, 1xcomplete protease inhibitor mix) for 2 hours at 4°C.
Glutathione-sepharose beads were then washed six times in PBS in the presence
of 0.2% Triton X-100 and 150 mM NaCl, and resuspended in Laemmli buffer. Bound
proteins were recovered by boiling for 5 minutes and subjected to SDS-PAGE
analysis. After drying the gel, proteins were visualised by Coomassie Blue
staining and Cited2 was detected by autoradiography.
Adrenal surface measurement
After WISH for Sf-1 (Fig.
5) or Hoxb9 (Fig.
7), images of all the samples were taken at the same
magnification, processed into ImageJ software and the surface of the adrenal
gland was measured using the `measure' tool. Experiments were performed on at
least three independent samples and results are expressed as a percentage of
the average surface of the wild-type adrenal±s.d.
Real-time PCR
For real-time RT-PCR, E10.5 urogenital regions (8-11ts) or E11.5 gonads
were microdissected, based on the in situ expression pattern of the genes
analysed: at E10.5, the urogenital region was dissected away from the rest of
the embryo and at E11.5 the gonad and mesonephroi were dissected away from the
dorsal aorta and adrenal region. Total mRNAs were extracted using the
RNAqueous-Micro Kit (Ambion). cDNA synthesis was primed with oligo(dT) and
performed with Superscript II reverse transcriptase (Invitrogen). Sf-1,
Wt1, Sox9, Cyp11a1 and Hprt1 mRNA accumulation was quantified by
real-time PCR using the Taqman system (Applied Biosystems). The premade and
pretested primer/probe sets that were used were: Sf-1, Mm00446826_m1;
Wt1, Mm00460570_m1; Sox9, Mm00448840_m1; Cyp11a1,
Mm00490735_m1; Hprt1, Mm00446968_m1. Relative mRNA accumulation was
determined by the 
Ct method in which Hprt1 was used as
the normaliser and one of the wild-type samples was used as the baseline
value. Results are expressed as a percentage of the mRNA accumulation in
wild-type embryos and represent the mean values obtained with at least four
urogenital regions or four gonads for each genotype±s.d.
Cell transfections
C2C12 cells were maintained in DMEM with 10% foetal calf serum and passaged
at 50-60% confluence to prevent differentiation. Cells were transfected with
JetPEI (Autogen Bioclear) 20 hours after seeding in six-well plates (Falcon)
at a density of 2x105 cells per well. Transfections were
performed with 1 µg of pGL3-Sf1P, or the Wt1-responsive element mutant
pGL3-Sf1Pm (Wilhelm and Englert,
2002), or pGL3-amphiregulin
(Lee et al., 1999) reporter
genes and 50 ng of RcCMV-Wt1(-/-) (Kim et
al., 1999) alone or in combination with 50 ng of pCMV5-Cited2
(Cited2 sequence derived from IMAGE clone AA023157 and cloned into the
EcoRI/HindIII sites of pCMV5). Amounts of DNA and
constitutive promoter were kept constant by addition of pCMV5 empty vector.
Luciferase activity was assayed 24 hours after transfection using Genofax A
reagent (Yelen, France) and was normalised to the activity of 2 ng of
co-transfected pRLSV40 vector coding for renilla. Renilla activity was assayed
with Genofax C reagent (Yelen, France). Each experiment was performed in
triplicate and repeated at least three times. All data are expressed as
mean±s.d. Western blotting confirmed that Wt1 expression levels were
not affected by Cited2 co-transfection (data not shown).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). To
study the early stages of adrenal development in these mice, we analysed the
expression of Sf-1, which marks adrenal cortical cells at all stages
of development, by whole-mount in situ hybridisation (WISH) followed by
sections of the urogenital region (Fig.
1A). At E10.5, when the adrenal primordium is first apparent in
wild-type embryos, we observed a lack of Sf-1 expression in this
region. This lack of expression was also seen at later stages and for other
adrenal cortical markers including Cyp11a1, Dax1 (Nr0b1 -
Mouse Genome Informatics) and Wnt4
(Fig. 2 and data not shown). We
also investigated adrenal development by WISH for Hoxb9, a recently
identified marker of adrenal steroidogenic cells that is not known to be
regulated by Sf-1 (Zubair et al.,
2006). At E12, the adrenal primordium appeared as a distinct and
organised Hoxb9 expression-positive structure in the wild-type
embryo. By contrast, such organisation was not apparent in the
Cited2-/- mutant embryo, although a few Hoxb9
expression-positive cells were found in the presumptive adrenal area. The
developing gonad of the mutant embryo showed Sf-1 expression,
although the levels appeared to be decreased at E10-11
(Fig. 1A). These studies show
that development of the adrenal cortical primordium is markedly impaired in
Cited2-/- embryos.
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), they hypothesised that the neural crest phenotype could
account for impaired adrenal cortical development
(Bamforth et al., 2001).
However, previous reports (Bland et al.,
2004; Gut et al.,
2005) and our observations showed that although neural crest cells
were found in the urogenital region at E11.5 and E12.5, it was not until E13
that these cells were found near the presumptive adrenal area
(Fig. 1B). This late stage of
neural crest cell migration indicated that the adrenal cortical phenotype in
the mutant mice was unlikely to result from a neural crest developmental
defect. Analysis of expression of the neural crest marker Mash1
(Ascl1 - Mouse Genome Informatics) in the E13 urogenital region of
Cited2-/- embryos showed that neural crest cells were
still present in the urogenital region, although the numbers were reduced.
This observation was confirmed by WISH for the chromaffin cell marker tyrosine
hydroxylase (Th) at E13.5 (Fig.
1C). Altogether, this indicated that the adrenal cortical
development failure was independent of the neural crest phenotype. Consistent
with this, Erbb3-/- mice, which are completely devoid of
neural crest cells in the urogenital region, show development of an adrenal
cortex, although it becomes disorganised at later stages
(Britsch et al., 1998).
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Gonad phenotype in Cited2-/- embryos
Because of the early adrenal phenotype in Cited2-/-
embryos and the common developmental origin of the gonad and adrenal cortex,
we analysed the differentiation of the gonad in these mutant embryos by WISH
for expression of gonadal markers (Fig.
2A). Expression levels of Sox9, a Sertoli cell marker and
of Cyp11a1, a steroidogenic cell marker, were decreased in
Cited2-/- XY gonads at E11.5, but their expression levels
were normal at E13.5. Sf-1 expression levels were mildly decreased in
Cited2-/- gonads at E11.5 and appeared normal at E13.5.
These in situ observations were confirmed by real-time RT-PCR on E11.5 gonads
(Fig. 2B). Expression of
Sox9 and Cyp11a1 was decreased in the mutant to 37% and 54%
of wild-type levels, respectively. Sf-1 expression was also decreased
in the mutant, albeit to a lesser extent (64% of wild-type levels). A similar
decrease in Cyp11a1 and Sf-1 was observed in the XX mutant
E11.5 gonad (data not shown). These data show that early differentiation of
the gonads in Cited2-/- embryos is impaired, but that at
later stages the gonads recover to develop normally. This is consistent with
the higher levels of Cited2 expression observed at the early stages
of AGP development, which decreased as gonad development occurred
(Fig. 1D).
Proliferation and apoptosis are not altered at the early stages of AGP development in Cited2-/- embryos
Previous studies had implicated Cited2 in the control of apoptosis and cell
proliferation during embryonic development
(Kranc et al., 2003;
Barbera et al., 2002;
Yin et al., 2002). Therefore,
we investigated whether an increase in apoptosis or a decrease in cell
proliferation could account for impaired adrenal cortex development in
Cited2-/- embryos. Apoptosis was analysed by Lysotracker
Red incorporation (Fig. 3A) or
TUNEL assays (data not shown). There was no difference in apoptosis rates at
E10.5 between Cited2-/- and the wild type. However,
apoptotic cells were detected in the presumptive adrenal area of
Cited2-/- embryos at E12.5
(Fig. 3A). Analysis of cell
proliferation by in vivo BrdU incorporation and double immunohistochemistry
with BrdU and anti-laminin or anti-Sf-1 antibodies in urogenital regions at
E10.5 (Fig. 3B) and E11.5 (data
not shown) showed no difference between wild-type and mutant embryos at these
stages.
Genetic interaction between Cited2 and Wt1 in the urogenital region
Detailed analysis of the urogenital region in Cited2-/-
embryos uncovered an additional phenotype characterised by abnormal
development of the mesonephric tubules. Analysis of Lim1
(Lhx1 - Mouse Genome Informatics) and laminin expression, to
highlight the tubules within the mesonephros, showed that the caudal tubules,
which are not connected to the Wolffian duct, failed to develop in the mutant
embryos (Fig. 4A).
Interestingly, this phenotype was also observed in Wt1 mutant
embryos, which also lack adrenal glands and gonads
(Sainio et al., 1997). This
similarity in phenotypes within the adrenal and mesonephric tubules in
Cited2 and Wt1 mutant embryos suggested that these factors
are part of a common molecular pathway within the AGP. WISH analysis of
Cited2 and Wt1 expression showed that these genes are
coexpressed in the coelomic epithelium of E10 embryos and within some
mesonephric tubules, although their general expression patterns in the
urogenital region were not identical. Once the adrenal primordium had formed,
however, their expression patterns differed in that Cited2, but not
Wt1, was expressed in adrenal cortical cells
(Fig. 4B).
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Physical interaction between Cited2 and Wt1
Our data demonstrated a genetic interaction between Cited2 and Wt1. To
establish whether there was a physical interaction between these two proteins
we performed immunoprecipitation and GST pull-down experiments
(Fig. 6). Endogenous Wt1
protein from M15 cells, which are derived from mouse embryonic mesonephros,
was immunoprecipitated with an anti-Wt1 antibody raised against the C-terminus
of the protein. Western blot analysis showed that endogenous Cited2 protein
was present in the immunoprecipitate isolated with Wt1 antibody, but not in an
immunoprecipitate isolated with a control anti-ß-galactosidase antibody
(Fig. 6A). This indicated that
endogenous Wt1 and Cited2 proteins physically interact in M15 cells. The
transcripts of Wt1 undergo two main alternative splicing events. The first
introduces an extra exon 5 encoding 17 amino acids that can alter co-factor
recruitment. The second introduces a KTS motif between the third and fourth
zinc-fingers of the DNA-binding domain; this is thought to prevent
transcriptional activity and to promote interaction with the RNA-splicing
machinery (Hastie, 2001). To
determine whether Cited2 interacted with these Wt1 isoforms, we performed
immunoprecipitation assays on human embryonic kidney 293A cell lines,
transfected with expression plasmids for Cited2 and either the Wt1(-/-)
(lacking the 17 aa and KTS motifs) or Wt1(+/+) (harbouring both the 17 aa and
KTS motifs) isoforms. In these experiments, Cited2 was shown to co-precipitate
with the two isoforms of Wt1 (Fig.
6B, top left panel). Specificity of the interaction was confirmed
with non-immune isotype-matched rabbit IgG and Sf-1 antibody
(Fig. 6B, bottom left panel).
In reciprocal experiments, both Wt1 isoforms were found in the
immunoprecipitate formed with a monoclonal antibody raised against Cited2
(Fig. 6B, right panel). This
showed that Cited2 interacted with Wt1(-/-) and Wt1(+/+) proteins, although
the level of interaction with the non-DNA-binding Wt1(+/+) isoform was
consistently lower than that with the DNA-binding Wt1(-/-) isoform
(Fig. 6B). These observations
were extended by GST pull-down experiments in which GST fusions of the
full-length Wt1(-/-) protein, its N-terminal transactivation domain or its
zinc-finger DNA-binding domain were incubated with in vitro translated,
radioactively labelled Cited2 protein. In these experiments, Cited2 interacted
with full-length protein or the DNA-binding domain but not with the N-terminal
domain of Wt1 (Fig. 6C).
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|
).
As Sf-1 is required for the development of adrenals and gonads
(Luo et al., 1994;
Val et al., 2003), we analysed
Sf-1 expression levels in the AGP of Cited2-/-
embryos. Careful examination of WISH experiments suggested that Sf-1
expression was markedly reduced in the urogenital ridge of E10.5 mutant
embryos (Fig. 7A and
Fig. 1A). To confirm these
observations, urogenital regions from E10.5 wild-type and mutant embryos were
microdissected, mRNAs were extracted and Sf-1 expression levels were
determined by quantitative real-time RT-PCR
(Fig. 7B). Strikingly,
Sf-1 mRNA accumulation in Cited2-/- embryos was
decreased to 36% (±6%) of wild-type levels. Wt1 mRNA levels
were not affected in Cited2-/- mutant embryos, confirming
that there was no general defect in urogenital development. To confirm that
the levels of Sf-1 expression in the urogenital region in early
embryos were coordinately controlled by Wt1 and Cited2, we measured
Sf-1 mRNA accumulation in E10.5 Cited2+/-
Wt1+/- embryos (Fig.
7C). Sf-1 levels were higher than in
Cited2-/- embryos, but were decreased to 45% of wild-type
levels. Heterozygosity for either Cited2 or Wt1 led to a
mild decrease in Sf-1 mRNA accumulation, which was only statistically
significant in Wt1+/- embryos. We thus concluded that
Sf-1 levels were correlated to the severity of the adrenal phenotype.
This strongly suggested that the marked decrease in Sf-1 expression
in Cited2-/- embryos could account for the adrenal
phenotype.
|
; Bland et al.,
2000). Strikingly, Cited2+/-
Sf-1+/- embryos displayed drastically reduced adrenal
development at this stage. These results show that these two factors act in
the same pathway.
Cited2 stimulates Wt1 transcriptional activity at the Sf-1 promoter
To establish a functional interaction between Cited2 and Wt1 at Sf-1
regulatory regions, we performed co-transfection experiments in C2C12 cells
that express low endogenous levels of Wt1, Cited2 and Sf-1 (data not shown).
We elected to use the 674 bp Sf-1 promoter fragment previously shown
to be active in the urogenital ridge of transgenic mice
(Wilhelm and Englert, 2002).
As expected, Wt1 transfection caused a mild increase in Sf-1 promoter
activity (1.7-fold), consistent with previously published observations
(Fig. 7E). Cited2 alone was
also able to induce Sf-1 promoter activity, presumably through
endogenous Wt1 protein in C2C12 cells. Consistent with our in vivo
observations, co-transfection of Cited2 and Wt1 led to a further increase in
Sf-1 promoter activity (3.0-fold), with Cited2 causing a 1.7-fold
increase in Wt1 transcriptional activity. This effect of Cited2, although
relatively mild, is consistent with previous reports on its activity on
Tfap2-mediated transcription and with the effect of Cited2 on Sf-1
levels in vivo (Bamforth et al.,
2001; Bamforth et al.,
2004). Mutation of the four Wt1-responsive elements within the
Sf-1 promoter induced a drastic decrease in its activity, in line
with endogenous Wt1 expression in C2C12 cells. Importantly, this also
abrogated the effect of Cited2 on Wt1-mediated transcription, indicating that
Cited2 function at the Sf-1 promoter is dependent on Wt1.
We then evaluated the outcome of the functional interaction between Wt1 and
Cited2 at the amphiregulin promoter, a known Wt1 target gene
(Lee et al., 1999). Activity
of the amphiregulin promoter was markedly induced by Wt1 transfection in C2C12
cells (38 fold). In contrast to the Sf-1 promoter, co-transfection of
Cited2 induced a decrease in Wt1-mediated amphiregulin promoter activity
(Fig. 7E). This indicated that
the effect of Cited2 on Wt1 activity was dependent on the promoter
context.
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DISCUSSION |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Barbera et al.,
2002; Yin et al.,
2002). Although a previous report hypothesised that the absence of
the adrenal gland in Cited2-/- embryos was dependent on
impaired neural crest development (Bamforth
et al., 2001), we provide compelling evidence for a new
physiological role for Cited2 in the development of the adrenal cortical
anlagen from the AGP that is independent of the neural crest phenotype. The
following aspects of our data strongly suggest that Cited2 plays a direct role
in the AGP to promote adrenal cortical development by upregulating Sf-1
expression. (1) Cited2 and Sf-1 were colocalised in the AGP
prior to adrenal development. (2) As observed in Sf-1-/-
mice, there was no overt adrenal differentiation in
Cited2-/- embryos, but apoptotic cells were found in the
presumptive adrenal area at E12.5 (Bland et
al., 2004; Luo et al.,
1994). (3) Sf-1 expression was downregulated to 36% of
wild-type levels in the Cited2 mutant AGP at E10.5, prior to adrenal
differentiation. (4) Cited2+/- Sf-1+/- embryos
displayed reduced adrenal development as compared with either
Sf-1+/- or Cited2+/- embryos. (5) We
showed a genetic, physical and functional interaction between Cited2 and Wt1
that resulted in a direct stimulation of Sf-1 promoter activity and
an increase in Sf-1 dosage in the AGP.
Sf-1 dosage has been associated with different adrenal and gonadal
phenotypes in mice and humans. Sf-1 mutant mice show no gonad or
adrenal development, whereas Sf-1+/- mice show a marked
impairment in early adrenal development, which gives rise to a smaller adrenal
gland at early stages of development (Bland
et al., 2004; Bland et al.,
2000). Sf-1 haploinsufficiency also affects early mouse
gonadal differentiation, but sex determination occurs normally and both male
and female mice are fertile (Park et al.,
2005). In this study, we show a direct correlation between levels
of Sf-1 expression in the AGP and induction of adrenal and gonad
development. With doses of Sf-1 expression in the AGP at 36% of
wild-type levels, as seen in the Cited2-/- embryos,
adrenal development does not occur but gonad development is initiated,
although differentiation is impaired. Increasing the dose of Sf-1
expression, as seen in Cited2+/-
Wt1+/- mice, is enough to ensure adrenal specification,
although the organ is smaller. This indicates that adrenal development is more
sensitive to Sf-1 dosage than gonadal development in mouse. Consistent with
this, transgenic overexpression of Sf-1 rescued the gonad but not the adrenal
defect in Sf-1-/- mice, although the transgenic constructs
were expressed in both tissues in wild-type animals
(Fatchiyah et al., 2006).
Therefore, our data strongly suggest that Sf-1 dosage has to reach a critical
threshold in the AGP to trigger adrenal development and that Cited2 is
required to raise Sf-1 expression above this threshold. Strain background has
been shown to affect gene levels in the gonad during sex determination
(Albrecht et al., 2003).
Consistent with this, in our real-time RT-PCR experiments we observed a higher
level (144±15%) of Sf-1 expression in the AGP in wild-type embryos on
the mixed background derived from our breeding of Wt1 and
Cited2 heterozygous mice than on the C57/Bl6 background. Although
this will affect our relative values of Sf-1 expression in mutant versus
wild-type alleles (Sf-1 levels in Cited2+/-
Wt1+/- embryos were 65%, instead of 45%, of C57/Bl6
wild-type levels), the reduction observed in Sf-1 levels always correlated
with the Cited2 and Wt1 mutant alleles. Altogether, these
studies highlight the importance of gene dosage and the factors that modulate
gene dosage (co-factors and/or strain background) in specific tissues and at
critical times in embryonic development, in order to ensure induction of organ
formation.
Consistent with the phenotype of Sf-1-/- embryos
(Bland et al., 2004;
Luo et al., 1994), our data
show the late appearance of apoptotic cells in the presumptive adrenal area.
Although genetic ablation of Cited2 has been shown to induce apoptosis in the
neuroectoderm (Bamforth et al.,
2001; Martinez Barbera et al., 2002), it is unclear whether
apoptosis in the presumptive adrenal region is a primary effect of the lack of
Cited2, or whether cells in this area require adrenal cortical cells
for their survival.
Wt1 is a transcription regulator that can either activate or repress
transcription depending on the cellular context
(Roberts, 2005). Wt1 was
previously shown to positively regulate Sf-1 expression in vitro and
in the AGP of transgenic mice (Wilhelm and
Englert, 2002). In this paper we provide genetic and molecular
evidence that Cited2 interacts with Wt1 in order to stimulate Sf-1
gene transcription through its 5' regulatory sequences. This indicates
that Cited2 can be considered a bona fide transcriptional co-factor for Wt1.
Although Cited2 exerted a positive effect on Wt1-mediated transcription from
the Sf-1 promoter in C2C12 cells, it had no effect in other cell
types (data not shown). Furthermore, we show that Cited2 repressed Wt1
activation of the amphiregulin promoter, another Wt1 target gene
(Lee et al., 1999). This shows
that Cited2 can function either as a coactivator or a co-repressor of Wt1
transcriptional activity, depending on the promoter and cellular context. A
similar effect has been observed for the co-factor Par-4 (Pawr - Mouse Genome
Informatics), in that it acts as a coactivator of the Wt1 isoforms containing
the alternatively spliced 17 amino acid motif, and as a co-repressor of the
isoforms lacking this motif (Johnstone et
al., 1996; Richard et al.,
2001). However, in our studies, we did not observe any difference
in the interaction of Cited2 and the different isoforms of Wt1 that contained
or lacked the 17 amino acid motif (data not shown). Future work will focus on
identifying the factors that cooperate with Cited2 and Wt1 to achieve correct
spatiotemporal expression of Sf-1 in the AGP.
The hypothesis that the adrenal cortex and gonads share a common precursor
cell population initially stemmed from the analysis of Sf-1 expression at
early stages of urogenital development, and was further reinforced by the
presence of both adrenal and gonadal developmental defects in a number of
mouse mutant models or human pathologies
(Else and Hammer, 2005).
Although Wt1 is never found in the overtly differentiated adrenal cortex, we
demonstrate that Cited2 and Wt1 genetically interact to promote adrenal
development. This indicates that the interaction between Cited2 and Wt1 has to
occur at a stage prior to overt adrenal differentiation, when the two factors
are coexpressed in the AGP. Consistent with this idea, we also show an early
defect in gonad development. Therefore, our data further substantiate the idea
that adrenal cortex and gonads originate from the AGP and show that Cited2 and
Wt1 act at the earliest stages of adrenal differentiation, inside the AGP.
Whereas the promoter fragment used in this study was shown to drive Sf-1
expression in the AGP (Wilhelm and
Englert, 2002), Zubair et al. recently identified a fetal
adrenal-specific enhancer (FAdE) in the fourth intron of Sf-1. This
enhancer was inactive in the adrenogonadal primordium, but its activity became
evident in the adrenal anlagen as soon as it separated from the AGP
(Zubair et al., 2006).
Consistent with the absence of Wt1 expression in the overtly differentiated
adrenal anlagen, our computer analysis of the FAdE sequence using the
Genomatix suite
(www.genomatix.de)
did not show any Wt1-responsive elements. Based on our data, we suggest that
expression of Sf-1 in the precursors of the adrenal cortex is first
induced in the AGP at the level of its 5' regulatory sequences through
the interaction between Cited2 and Wt1, and that the FAdE enhancer element
then takes over after overt adrenal cortex differentiation. Whether Cited2
also cooperates with transcription factors bound to the FAdE element remains
open to investigation.
Our studies demonstrate the essential role of Cited2 and Wt1 in modulating Sf-1 expression in the AGP and promoting adrenocortical development in mouse. It would therefore be interesting to determine if mutations in CITED2 can be linked to adrenal and/or gonadal deficiencies in humans.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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