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First published online 16 January 2008
doi: 10.1242/dev.012856
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,
1 Department of Natural Sciences, Hyogo University of Teacher Education, 942-1,
Shimokume, Kato, Hyogo 673-1494, Japan.
2 Division for Sex Differentiation, National Institute for Basic Biology,
National Institutes of Natural Sciences, Myodaijicho, Okazaki, Aichi 444-8787,
Japan.
3 Department of Anatomy and Developmental Biology, Graduate School of Medicine,
Chiba University, Chiba 260-8670, Japan.
4 Retinoid Research, Departments of Chemistry and Biology, Allergan, Irvine, CA
92623, USA.
Author for correspondence (e-mail:
moro{at}nibb.ac.jp)
Accepted 3 December 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in the right
ovarian primordium. Ad4BP/SF-1 expressed in the left ovarian primordium
asymmetrically upregulates cyclin D1 to stimulate cell proliferation. These
data suggest that early asymmetric expression of PITX2 leads to
asymmetric ovarian development through up- or downregulation of RALDH2,
Ad4BP/SF-1, estrogen receptor and cyclin D1.
Key words: Pitx2, Asymmetry, Chick, Estrogen, Ovary
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Raya and Belmonte,
2006). PITX2, a member of the conserved bicoid-type
homeobox gene family (Gage et al.,
1999), has been implicated in the establishment of L-R asymmetry
through its expression in the left lateral plate mesoderm. Indeed,
Pitx2-knockout mice exhibit abnormal visceral organ asymmetry
(Lin et al., 1999;
Lu et al., 1999).
In mammals, the gonads develop bilaterally through the orchestrated action
of a number of genes (Ross and Capel,
2005; Swain and Lovell-Badge,
1999). Many of these genes have been identified through the study
of knockout mice and patients suffering from gonadal developmental
abnormalities. However, no gonad defects identified so far have exhibited
clear laterality, and no genes involved in gonad development are expressed
with L-R asymmetry. Unlike mammals, most female birds develop ovaries only on
the left side, while males develop bilateral testes. During early
developmental stages before sexual differentiation, chick embryonic gonads
show no obvious morphological L-R asymmetry and consist of two components, the
cortex and medulla (Smith and Sinclair,
2004). After sexual differentiation, testicular development occurs
bilaterally in the male (genetically ZZ). The testicular cords appear in the
medulla where Sertoli and Leydig cells differentiate, while the cortex
regresses and eventually disappears. In female birds (genetically ZW), the
left cortex proliferates and develops into the ovary, whereas the right cortex
disappears (Smith and Sinclair,
2004). Studies of chick gonad development implicate estrogen in
asymmetric ovarian development. Indeed, estrogen receptor
(ER) is expressed in the left but not the right cortex
(Nakabayashi et al., 1998) of
both sexes (Andrews et al.,
1997). However, it has not been known how asymmetric
ER expression is induced and whether or not asymmetric
estrogen signaling is related to asymmetric ovarian development.
Retinoic acid (RA) plays a variety of roles throughout development
(Niederreither et al., 1999;
Sakai et al., 2001) and
functions by binding to the nuclear receptors RAR and RXR to regulate gene
expression. The synthesis and distribution of RA is controlled by the
coordinated expression of genes encoding RA-synthesizing retinaldehyde
dehydrogenases (RALDH1, RALDH2 and RALDH3)
(Zhao et al., 1996;
Niederreither et al., 1997;
Swindell et al., 1999;
Mic et al., 2000;
Niederreither et al., 2002)
and RA-metabolizing cytochrome P450 proteins (CYP26A1, CYP26B1 and
CYP26C1) (Fujii et al.,
1997; Swindell et al.,
1999; MacLean et al.,
2001; Tahayato et al.,
2003; Reijntjes et al.,
2004). Because RA is involved in cell growth and differentiation,
cell-cycle accelerator and suppressor genes have been studied as potential RA
targets (Chen and Ross, 2004;
Dragnev et al., 2004;
Guidoboni et al., 2005).
Here, we investigate the roles of RALDH2, PITX2, ER and
Ad4BP/SF-1 (Ad4-binding protein/steroidogenic factor 1) in L-R
asymmetric ovarian development in chicks. We demonstrate that a genetic
cascade including these factors differentially regulates cyclin D1 gene
expression in the left and right cortices and eventually causes asymmetric
cell proliferation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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);
RALDH2 and CYP26A1 from Dr Eichele
(Swindell et al., 1999);
RALDH1 and RALDH3 from Dr Noda
(Suzuki et al., 2000);
RARβ from Dr Nohno (Nohno et
al., 1991); CYP26B1 (clone ID: ChEST239e5),
RXR (clone ID: ChEST142D11), RXRβ (clone ID:
ChEST102B17), RXR
(clone ID: ChEST248B14), LHX9
(clone ID: ChEST664O12) and cyclin D1 (clone ID: ChEST223D23) from the Medical
Research Centre Geneservice (Cambridge, UK); and RAR (clone
ID: pgp1n.pk009.m12) from the Delaware Biotechnology Institute, University of
Delaware (Newark, DE). cDNA encoding mouse Pitx2c was supplied by Dr Semina
(Semina et al., 1996). Chick
cDNA for PITX2a was amplified by 5'-RACE (Invitrogen, Carlsbad,
CA) with the primer shown in Table
1. Chick cDNA for PITX2c, RAR and
ER was amplified with primers listed in
Table 1. As the probe for
PITX2a contains a region common to PITX2b, the probe detects
both PITX2a and PITX2b. These cDNAs were cloned into pCR
II-TOPO vector (Invitrogen) and digoxigenin-labeled probes were prepared
according to the manufacturer's instructions (Roche Diagnostics, Penzberg,
Germany). Whole-mount in situ hybridization was performed as described
(Yoshioka et al., 1998). After
staining, the embryos were embedded in 2% gellan gum followed by sectioning
with a microslicer (Dosaka EM, Kyoto, Japan).
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). Three embryos at stage 27 [embryonic day 5-5.5 (E5-5.5)]
and 29 (E6-6.5) were fixed with 4% paraformaldehyde (PFA)/PBS and
cross-sectioned. Five sections containing the gonad were randomly selected for
each embryo and were labeled with 4',6-diamidino-2-phenylindole (DAPI)
and mouse anti-cytokeratin monoclonal antibody (1:100; Lab Vision Corp., part
of Thermo Fisher Scientific, Fremont, CA) for 1 hour at 37°C to visualize
the cortical region (Oreal et al.,
1998). To detect cytokeratin, Alexa-Fluor-555-labeled goat
anti-mouse antiserum (1:200; Molecular Probes, Willow Creek Road, Eugene, OR)
was used for secondary labeling. DAPI-positive nuclei in the cortical
(cytokeratin-positive) and medullary (cytokeratin-negative) regions were
counted and summed across all five sections for each embryo. The mean and
standard deviation were determined using data from all three embryos. The
relative cell numbers are presented as described in the figure legends.
Bromodeoxyuridine (BrdU)-labeling of chick embryos and detection of the
BrdU-labeled cells were basically performed as described elsewhere
(Yoshioka et al., 2005). In
brief, 1 µl BrdU solution (10 mg/ml in H2O) was injected into
untreated or manipulated (as described below) embryos through the vitelline
vein at stage 27 or 29. After 1 hour incubation, the embryos were fixed with
4% PFA/PBS and cross-sectioned. Three embryos were used for each experiment.
Five sections containing the gonad were randomly selected for each embryo and
were subjected to immunofluorescence. The sections were incubated with sheep
anti-BrdU antiserum (1:300; Exalpha Biological, Watertown, MA) and mouse
anti-cytokeratin monoclonal antibody for 1 hour at 37°C.
Alexa-Fluor-488-labeled donkey anti-sheep antiserum (1:200; Molecular Probes)
and Alexa-Fluor-555-labeled goat anti-mouse antiserum were used for secondary
detection. The number of BrdU-positive and total DAPI-stained cells in the
cortical and medullary regions was determined. Statistical analysis and
calculation of relative cell numbers are described above.
Manipulation of chick embryos
Manipulation of chick embryos of both sexes was performed as described
previously (Yoshioka et al.,
2005). To construct Pitx2-en, the repressor domain of
Drosophila engrailed (amino acids 1-296)
(Jaynes and O'Farrell, 1991)
was fused to the carboxy-terminus of Pitx2c. cDNAs for Pitx2,
Pitx2-en, Ad4BP/SF-1 and alkaline phosphatase (AP) were cloned
into the avian viral vector RCASBP (A) and transfected into chick embryonic
fibroblasts to generate virus-producing cells. Pitx2, Ad4BP/SF-1 and
AP-expressing cells were implanted into the right lateral plate
mesoderm of stage 11-12 embryos at the level of the presumptive gonad, while
the Pitx2-en and AP-expressing cells were implanted into the
left presumptive gonad (see Fig. S1A in the supplementary material). After
being incubated for 72 (stage 27) or 96 hours (stage 29), the embryos were
fixed with 4% PFA-PBS and subjected to whole-mount in situ hybridization or
the cell proliferation assay described above. AG1-X2 ion-exchange resin beads
(150-200 µm diameter; BioRad Laboratories, Hercules, CA) were soaked in 10
µM all-trans-RA (Wako Pure Chemical Industries, Osaka, Japan) or
12.7 mM retinoid antagonist AGN 193109 (Allergan, Irvine, CA)
(Mercader et al., 2000)
dissolved in dimethylsulphoxide (DMSO). The beads were washed with PBS, and
implanted into the coelomic epithelium of the left or right presumptive gonad
region at stage 18-19 (see Fig. S1B in the supplementary material). After 48
(stage 27) or 72 hours'(stage 29) incubation, the embryos were subjected to
whole-mount in situ hybridization or the cell proliferation assay.
Sexing of tissue samples
The sex of chick embryos was determined by the presence or absence of the W
chromosome. To detect the W chromosome, the W-specific XhoI repeat
was amplified by PCR with genomic DNA as a template. β-actin was
amplified as a control (Kent et al.,
1996).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As differences in cell number can arise from differential cell
proliferation and/or cell death, we examined cell proliferation by BrdU
incorporation in embryos at stage 27 (Fig.
1C) and 29 (Fig.
1D). BrdU-positive proliferating cells were visualized by
immunofluorescence (Fig. 1C,D,
green) and the cortical region of the developing gonad was visualized by
anti-cytokeratin staining (Fig.
1C,D, red) (Oreal et al.,
1998). The number of BrdU-positive cells in the left cortices of
ZW and ZZ embryos were 1.8-fold and 1.6-fold higher, respectively, than in the
right cortices at stage 27 (Fig.
1E), and this bias persisted through stage 29. By contrast, there
was no significant asymmetry observed in the BrdU incorporation in the
medullae during the same time period (Fig.
1F). Next, we examined whether apoptotic cell death occurs
asymmetrically in the developing gonad. Only a few TUNEL-positive cells were
detected on both sides of the cortex and medulla, and their numbers did not
increase between stages 27 and 29 in either sex (see Fig. S2G,H in the
supplementary material). Taken together, these data indicate that increased
proliferation in the left cortex, rather than increased apoptosis in the right
cortex, is primarily responsible for the observed asymmetric cortical
development.
Although the gonad is still structurally symmetric at stage 27, we
hypothesized that some genes are expressed asymmetrically at this stage. As RA
signaling has been implicated in cell proliferation, we examined the
expression of genes involved in RA signaling (RALDH1, RALDH2, RALDH3,
CYP26A1, CYP26B1, RAR, RARβ, RAR,
RXR, RXRβ and RXR). In addition,
we examined the expression of genes implicated in establishing the L-R
asymmetric body plan (PITX2a, PITX2b and PITX2c).
Interestingly, RALDH2 and CYP26A1 were expressed
asymmetrically in a mutually exclusive fashion at stage 27. RALDH2
was expressed in the right cortex (Fig.
1G), whereas CYP26A1 was expressed in the left cortex and
in both sides of the medullae (Fig.
1H). RAR (Fig.
1I) and RXR (data not shown) expression was
stronger in the right cortex than in the left, and was not detected in the
medullae, suggesting that the right cortex is activated by RA signaling.
PITX2c was expressed only in the left cortex, showing a good
correlation with a previous report (Fig.
1J) (Logan et al.,
1998). As there is genetic evidence that Ad4BP/SF-1 and
LHX9 are essential for gonad development
(Birk et al., 2000;
Luo et al., 1994), we also
examined their expression. The pattern of Ad4BP/SF-1 expression was
similar to that of CYP26A1 (Fig.
1K), whereas LHX9 was expressed symmetrically
(Fig. 1L). Interestingly,
asymmetric gene expression occurred only in the cortex in both sexes. The
other genes examined were not expressed in the gonad at stage 27.
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As the L-R cortical asymmetry seen at stages 27-29 is caused by increased cell proliferation rather than cell death, we examined whether RA signaling affected cortical cell proliferation. After BrdU was injected into bead-implanted embryos, we counted the number of BrdU-positive cells at stage 27. Interestingly, the number of BrdU-positive cells decreased in the left cortex implanted with RA beads compared with DMSO-treated embryos (Fig. 2G,H,O), but there was no corresponding effect in the medulla (Fig. 2P). Conversely, the number of BrdU-positive cells increased in the right cortex implanted with the RAA beads (Fig. 2I,J,Q), with no effect on the medullary cell number (Fig. 2R). We next examined the morphologic effects of bead implantation. By stage 29, the thickness of the left cortex, as visualized by cytokeratin immunostaining, was reduced in the RA-bead-implanted embryos (Fig. 2K,L), while the right cortex thickened following RAA bead implantation (Fig. 2M,N).
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We implanted cells producing either Pitx2- or Pitx2-en-encoding viruses at stage 11-12 into the presumptive right or left gonad, respectively. Because the viruses were replication competent, transgene expression expanded throughout the gonad region (Fig. 3A,D,G). Operated chick embryos were then incubated to stage 27, and the expression of RALDH2 and Ad4BP/SF-1 was examined. Exogenous expression of Pitx2 led to reduced expression of RALDH2 in the right cortex (Fig. 3B, arrowhead; male=11/14, female=12/16), and increased expression of Ad4BP/SF-1 (Fig. 3C, arrowhead; male=10/14, female=12/15). By contrast, expression of Pitx2-en increased expression of RALDH2 in the left cortex (Fig. 3E, arrowhead; male=8/11, female=10/13), and reduced expression of Ad4BP/SF-1 (Fig. 3F, arrowhead; male=8/10, female=12/15). A control virus encoding AP did not affect expression of either RALDH2 (Fig. 3H; male=0/13, female=0/15) or Ad4BP/SF-1 (Fig. 3I; male=0/14, female=0/15).
Pitx2 is required for cell-type-specific proliferation during
cardiac outflow tract (Kioussi et al.,
2002) and pituitary gland
(Kioussi et al., 2002;
Zhu and Rosenfeld, 2004)
development. Thus, we examined the effect of Pitx2 and
Pitx2-en on BrdU incorporation in the developing gonad. When cells
producing Pitx2-encoding viruses were implanted into the right
presumptive gonad region, the number of BrdU-positive cells increased by
2.0-fold in the right cortex at stage 27
(Fig. 3J,R), but proliferation
in the medulla was not affected (Fig.
3S). By contrast, the number of BrdU-positive cells decreased in
left cortices implanted with Pitx2-en virus-producing cells
(Fig. 3L,T), whereas the
medulla was not affected (Fig.
3U). Exogenous AP expression did not affect cell
proliferation (Fig. 3K,M,R-U).
Structurally, right cortices implanted with Pitx2 virus-producing
cells thickened by stage 29 (Fig.
3N,O), whereas Pitx2-en-implanted left cortices were
thinner (Fig. 3P,Q).
Likewise, we examined the effect of exogenous Ad4BP/SF-1 expression on the right gonad cortex (Fig. 4A). Expression of PITX2c nor RALDH2 was unaffected following implantation of Ad4BP/SF-1 virus-producing cells (data not shown). However, the number of BrdU-positive cells in the right cortex increased by 1.8-fold (Fig. 4B,C,F) and the cortical layer thickened (Fig. 4D,E). Proliferation of the right medulla was unaffected (Fig. 4G).
Regulation of cyclin D1 gene expression
Our data indicate that PITX2, RA and Ad4BP/SF-1 are
involved in asymmetric L-R cortical cell proliferation. In order to determine
the mechanism by which these factors act, we examined their effect on the
expression of the cell cycle regulators cyclin D1 and D2. At stage 27, cyclin
D1 was expressed asymmetrically in the left cortex and bilaterally in the
medulla (Fig. 5A), whereas
cyclin D2 was transiently expressed on both sides of the cortex and
disappeared by stage 29 (data not shown). As the expression of cyclin D1
overlapped with that of CYP26A1 and Ad4BP/SF-1, we examined
whether production of RA, PITX2 and Ad4BP/SF-1 leads to asymmetric cyclin D1
expression. RA and RAA beads were implanted as described. At stage 27, the
expression of cyclin D1 was significantly downregulated in the
RA-bead-implanted left cortices (Fig.
5B, arrowhead; male=5/8, female=6/10). Expression in the medulla
was unaffected. By contrast, implantation of RAA beads in the right cortex
stimulated expression of cyclin D1 (Fig.
5C, arrowhead; male=5/7, female=7/10). Embryos implanted with DMSO
beads (Fig. 5D; male=0/9,
female=0/9) resembled untreated embryos. When Pitx2 virus-producing
cells were implanted, cyclin D1 was upregulated in the right cortex
(Fig. 5E, arrowhead; male=5/8,
female=6/8), but forced expression of Pitx2-en reduced the expression
of cyclin D1 in the left cortex (Fig.
5F, arrowhead; male=5/9, female=5/10). When cells producing
Ad4BP/SF-1-encoding virus were implanted, the expression of cyclin D1
was upregulated in the right cortex (Fig.
5G, arrowhead; male=6/8, female=7/9). Finally, implantation of
cells producing a control AP-virus did not affect the expression of
cyclin D1 (Fig. 5H; male=0/7,
female=0/9). These results indicate that cyclin D1 is negatively regulated by
RA and positively regulated by PITX2 and Ad4BP/SF-1 in the developing gonad,
although it remains to clarify whether the regulations are direct or
indirect.
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and CYP26A1; Romanoff,
1960), we examined the expression of ER at stage
27. As shown in Fig. 6A,
ER was expressed asymmetrically in the left but not right
gonadal cortex, and symmetrically in both sides of the medulla
(Andrews et al., 1997;
Smith et al., 1997). This
expression was identical in males and females (data not shown). When RA beads
were implanted in the left developing gonad, the expression of
ER was significantly reduced in the left cortex
(Fig. 6B, arrowhead; male=5/6,
female=8/9). Conversely, RAA-bead-implantation in the right developing gonad
increased the expression of ER in the right cortex
(Fig. 6C, arrowhead; male=5/7,
female=7/9). Implantation of DMSO beads had no effect on the expression of
ER (Fig. 6D;
male=0/6, female=0/7). Subsequently, we examined the effect of Ad4BP/SF-1 on
ER expression by implanting cells with
Ad4BP/SF-1-encoding viruses in the right cortex. There was no change
in cortical ER expression in animals implanted with
Ad4BP/SF-1-expressing cells (Fig.
6E; male=0/6, female=0/7) or control AP-expressing cells
(Fig. 6F; male=0/5,
female=0/5). These data suggest that asymmetric RA signaling in the developing
chick gonad regulates asymmetric ER expression, whereas
Ad4BP/SF-1 is not involved in ER expression. We further
investigated whether or not estrogen regulates expression of RALDH2,
PITX2 and Ad4BP/SF-1 by implanting estrogen-coated beads, but
found no effect of estrogen on the expression of these genes (data not
shown).
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RA signaling has been shown to activate CYP26 gene expression
during the development of several tissues
(Swindell et al., 1999;
Moreno and Kintner, 2004;
Yashiro et al., 2004). Here,
we show that CYP26A1 expression is induced by RA signaling in the
developing gonad. Interestingly, however, CYP26A1 is not expressed in
the right cortex. As the right cortex produces RA via RALDH2 and expresses
RAR (Fig. 1I)
and RXR (data not shown), RA signaling is likely to be active
there. Therefore, suppression of CYP26A1 expression in the right
cortex must be mediated by an unknown mechanism independent of RA
signaling.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Just before
sex differentiation, the gonad cortex begins to develop morphological
asymmetry, with the right cortex becoming thinner and the left cortex
thickening. Given that the cortex contributes significantly to ovarian
development, it seemed likely that the right gonad loses the ability to
develop into an ovary at this stage. Thus, we focused on this step and
investigated the molecular mechanism by which L-R asymmetry is induced in the
gonadal cortices.
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show
asymmetric expression at the left cortex. Interestingly, the asymmetric
expression of these genes occurs only in the cortex, suggesting that the
initial step in L-R asymmetric ovarian development is dominated by
differential expression of genes in the cortex. To investigate the functional
relationship between these L-R asymmetric factors, we performed various
experiments to test how forced gene expression or chemical treatment affects
laterality of the developing gonads. Our data indicate that in the developing
left cortex, PITX2 suppresses RALDH2 expression, leading to reduced
RA signaling and increased Ad4BP/SF-1 and ER
expression. Subsequently, Ad4BP/SF-1 upregulates cyclin D1 expression and
promotes cell proliferation. In the right cortex, lack of PITX2 results in
upregulation of RALDH2, which in turn activates RA synthesis. RA then
suppresses cell proliferation through downregulation of Ad4BP/SF-1
and cyclin D1 (Fig. 7). Our
current studies successfully demonstrate a functional relationship between the
above components, although it still remains to be determined whether the
regulation is direct or indirect. It would be interesting to examine the later development of gonads implanted with Pitx2, Pitx2-en and Ad4BP/SF-1-expressing cells and RA and RAA beads. However, the operated embryos die at around stage 30, when L-R asymmetric ovarian development and gonadal sex differentiation begin, so we were not able to examine gonadal development in these animals. As operated embryos die even when control AP-expressing cells and DMSO beads are implanted, it appears that the embryos die from physical damage resulting from the operation itself. Novel techniques that cause less damage to the embryo are therefore necessary to address this question.
In the present study, we show that the genes involved in the initial L-R
asymmetric development of the gonads are expressed similarly in both sexes,
and initial morphogenetic events occur independent of sex
(Fig. 7). Indeed, the medulla
develops bilaterally, whereas the cortex develops only on the left in both
sexes. This left-sided cortical development is thought to be important for
left-sided ovarian development. Thereafter, the gonads display sexually
dimorphic development, with bilateral testicular development in the male and
left-sided ovarian development in the female. As estrogen activates
development of the gonadal cortex into the ovary
(Scheib, 1983), and estrogen
is synthesized only in the female, left-sided ovarian development is achieved
in the female but not the male. By contrast, as it is the medulla that
contributes most to development of the testis, the testis develops bilaterally
in males.
Mechanisms for L-R asymmetric cell proliferation
During the establishment of L-R asymmetry in ovarian development, cell
proliferation appears to be accelerated in the left cortex and decelerated in
the right cortex. Our data demonstrate that Ad4BP/SF-1 activates cyclin D1
transcription, which probably accelerates cell proliferation. Interestingly,
LRH1 (NR5A2), which is highly homologous to Ad4BP/SF-1, activates cyclin D1
gene transcription through its direct interaction with β-catenin
(Botrugno et al., 2004). Like
LRH1, Ad4BP/SF-1 can interact directly with β-catenin to synergistically
activate transcription (Mizusaki et al.,
2003). Therefore, it is possible that Ad4BP/SF-1 functions in a
similar manner to activate cyclin D1 gene transcription. Moreover, LRH1
activates cyclin E1 gene transcription by binding directly to its promoter. As
the nucleotide sequence recognized by LRH1 is almost identical to that bound
by Ad4BP/SF-1, Ad4BP/SF-1 may also regulate cyclin E1 gene transcription. In
addition to the possible activation of cyclin D1 by Ad4BP/SF-1, Pitx2 also
directly upregulates mouse cyclin D1 transcription
(Kioussi et al., 2002;
Zhu and Rosenfeld, 2004). The
chick cyclin D1 promoter contains a putative PITX2-binding site, suggesting
that PITX2 may also directly activate chick cyclin D1 gene expression.
Moreover, the synergistic activation of LHβ transcription by Pitx2 and
Ad4BP/SF-1 (Tremblay et al.,
2000) suggests that cyclin D1 gene transcription might be
synergistically regulated by these two factors. However, more work is
necessary to determine the precise mechanism by which these factors
interact.
The deceleration of cell proliferation in the right cortex appears to be
mediated by inhibition of cyclin D1 expression by RA. Consistent with our
present observations, other labs have shown that RA inhibits cyclin D1, cyclin
E1 and cyclin-dependent kinase 6 in cultured cell lines
(Balasubramanian et al., 2004;
Chen and Ross, 2004;
Sah et al., 2002). In addition
to downregulating these cell cycle accelerators, RA stimulates the expression
of several cell cycle inhibitors, including cyclin-dependent kinase inhibitor,
p27Kip1 and p21Cip1
(Balasubramanian et al., 2004;
Buzzard et al., 2003;
Chen and Ross, 2004;
Guidoboni et al., 2005). This
is further supported by studies of Raldh2 (also known as
Aldh1a2 - Mouse Genome Informatics) knockout mice
(Lai et al., 2003), which
exhibit reduced expression of p27Kip1 (Cdkn1b) and
p21Cip1 (Cdkn1a), and increased proliferation of endothelial
cells. These findings are consistent with our current observations in the
developing gonad.
Interestingly, a novel role for RA was recently observed in the mouse
embryonic gonad. Typically, germ cells enter meiosis in the embryonic ovary,
whereas meiosis is retarded in the embryonic testis. Increased
Cyp26b1 expression in the male gonad leads to decreased RA relative
to the female gonad, and the different levels of RA appear to regulate the
sex-specific timing of meiotic initiation in germ cells
(Bowles et al., 2006;
Koubova et al., 2006). We did
not observe sex-specific expression of RALDH2 or CYP26 in
the chick gonad, but the meiotic regulation of these genes by RA could occur
at a later stage, when sexual differentiation has already occurred.
Estrogen signaling and L-R asymmetric ovarian development
Previous work has shown that in ovo inhibition of estrogen synthesis by
aromatase inhibitor induces bilateral female-to-male sex reversal
(Elbrecht and Smith, 1992;
Hudson et al., 2005;
Smith et al., 2003;
Vaillant et al., 2001a;
Vaillant et al., 2001b). By
contrast, although in ovo estrogen administration induces partial
male-to-female sex reversal (ovotestis), it occurs only in the left gonad
(Nakabayashi et al., 1998;
Romanoff, 1960). This
differential effect of estrogen has been thought to be due to the preformed
differential potential of the left and right gonadal cortices
(Mittwoch, 1998). Based on
these observations, it was proposed that the differential potential of the
cortices is established independently of estrogen signaling
(Nakabayashi et al., 1998).
Our present study demonstrates that RA signaling triggers two events in the
right cortex: loss of responsiveness to estrogen through suppression of
ER expression, and inhibition of cell proliferation through
suppression of Ad4BP/SF-1 and thus cyclin D1 expression. As the left
cortex retains both responsiveness to estrogen and the ability to undergo cell
proliferation, it is not surprising that exogenous estrogen induces ovotestis
only on the left side.
L-R asymmetric ovarian development is interesting from both an evolutionary and developmental standpoint. During L-R axis formation, L-R asymmetry at the node is transmitted to the lateral plate mesoderm by upregulation of PITX2 expression on the left side, which is thought to result in asymmetric visceral organ development. Although the correlation between the L-R asymmetry of the body plan and ovarian development remains to be investigated, we have shown that PITX2, RA signaling, Ad4BP/SF-1, estrogen signaling and cyclin D1 are involved in the process of asymmetric ovarian development in the chick.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/4/677/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Department of Molecular Biology, Graduate School of
Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka
812-8582, Japan
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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