|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 24 October 2007
doi: 10.1242/dev.010249
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Division of Stem Cell Biology and Developmental Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
* Author for correspondence (e-mail: sguioli{at}nimr.mrc.ac.uk)
Accepted 11 September 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Sex determination, Pitx2, Left-right asymmetry, Chick embryo
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Raya and Belmonte, 2006;
Shiratori and Hamada, 2006).
However, early cues established from the midline that lead to stable
differences in gene expression - for example, between left and right lateral
plate mesoderm - can either be followed, leading to appropriate L-R
patterning, or they can be overridden to permit symmetrical development. For
example, in the absence of a protective signal provided by retinoic acid, the
usually regular formation of somites occurs asymmetrically
(Morales et al., 2007;
Vermot and Pourquie, 2005). If
both somitic mesoderm and lateral plate mesoderm (LPM) are subject to
mechanisms promoting asymmetry, then this will also be true of intermediate
mesoderm. Most intermediate mesoderm-derived structures usually develop with a
considerable degree of symmetry. This is true of the urogenital system, which
develops bilaterally. However, in all birds, with the exception of a few
predatory species (Kinsky,
1971), female gonadal development is unusual in that only one
functional ovary persists. This is normally on the left, while on the right
side the ovary fails to differentiate and undergoes a degenerative fate along
with its associated reproductive tract. Adult male birds have bilateral
testes. There must, therefore, be a mechanism in females that actively
promotes or permits L-R differences to occur, in contrast to one in males that
overrides or ignores any underlying L-R asymmetry, if indeed the latter is
present.
The indifferent genital ridges arise on the ventromedial surface of the
mesonephroi at around 3 days of embryonic development (E3) [equivalent to
Hamburger and Hamilton (HH) stage 20
(Hamburger and Hamilton,
1992)], whereas sex-specific differentiation of the gonads becomes
apparent from E6.5 (HH29-30). In ZZ embryos, both testes differentiate into
functional organs with the inner medulla organised into seminiferous cords
containing germ cells. In ZW embryos, an asymmetry between left and right
gonads becomes very evident after E8 (HH34). While the left gonad develops a
thick cortical layer with cords budding towards the medulla and enclosing most
germ cells, the right fails to differentiate its epithelial layer into a
cortex and all the germ cells remain in the medulla that, as in the left
ovary, is made of degenerated cords. No further development occurs in the
right gonad, which eventually loses germ cells and becomes vestigial
(Carlon and Stahl, 1985).
Apart from the obvious functional asymmetry associated with ovarian
development, one morphological difference between left and right gonads during
embryonic development has been described: the left germinal epithelium is
thicker than that on the right. This L-R difference is evident in both ZZ and
ZW embryos within the indifferent genital ridges at HH24-25 and is maintained
in both sexes through the initial steps of sex determination (HH28-30)
(Carlon and Stahl, 1985). A few
genes were also found to have L-R asymmetric expression patterns during
ovarian development (Andrews et al.,
1997; Gonzalez-Moran,
2005; Hoshino et al.,
2005; Reed and Sinclair,
2002; Yoshioka et al.,
2005), and one, Bmp7, has been shown to express
asymmetrically in the indifferent ridges of both sexes
(Hoshino et al., 2005).
However, there have been no data linking any asymmetrically expressed factor
to gonadal phenotype and to subsequent ovarian fate. Importantly, relatively
little attention has been paid to the observation that asymmetries occur in
both sexes from the indifferent stages of genital ridge development. These
might indicate a convergence of the chick sex determination/differentiation
pathway with the lateralisation signals that initiate L-R asymmetry within the
early embryo.
In the last few years several studies have explored how these complex
signals are interpreted at the level of single organs. One of the key genes of
the L-R pathway found at the interphase between specification of the L-R
biasing signal and its translation into asymmetric organ morphogenesis is
Pitx2. This is expressed in the left LPM of mouse, chick,
Xenopus and zebrafish, but its expression is also found and persists
in organs undergoing asymmetric morphogenesis
(Dagle et al., 2003;
Gormley and Nascone-Yoder,
2003; Piedra et al.,
1998; Schweickert et al.,
2000; St Amand et al.,
1998; Yoshioka et al.,
1998). Misexpression of Pitx2 in the right LPM is
sufficient to alter the situs of heart, gut and embryonic rotation
(Campione et al., 1999;
Essner et al., 2000;
Logan et al., 1998;
Ryan et al., 1998).
Loss-of-function experiments also support the idea that Pitx2 plays
important roles in the local generation of asymmetry within multiple organs
(Ai et al., 2006;
Dagle et al., 2003;
Gormley and Nascone-Yoder,
2003; Kitamura et al.,
1999; Liu et al.,
2002; Shiratori et al.,
2006). Moreover, the analysis of Pitx2 conditional and
hypomorphic mutations in mouse has shown that proper asymmetric morphogenesis
of heart, lung and duodenum requires organ-specific thresholds of PITX2
activity, indicating the existence of organ-intrinsic mechanisms regulating
asymmetric morphogenesis dependent upon dosage of PITX2
(Gage et al., 1999;
Liu et al., 2001). Despite the
enormous progress made towards the identification of the cascade of genetic
interactions controlling the establishment and the propagation of L-R
asymmetry signals in various vertebrates, there are no data addressing the
importance of the lateralisation signal in chick gonad development.
We show here that perturbations of L-R signals also result in changes in the L-R asymmetry of the chick gonads. Furthermore, Pitx2 is expressed in the left gonad and misexpression of Pitx2 to the right is sufficient to induce symmetric development of the gonads as left isomers in both ZZ and ZW embryos. This transformation is sufficient to rescue the degenerative fate of the right ovary.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
In-ovo drug treatment
At 0 hours of incubation, 5 ml albumen were removed from each egg and 2 ml
were mixed with 400 µl Hank's Buffered Salt Solution (HBSS) with or without
-lindane (Sigma) (40 µl from a 40 mg/ml stock in DMSO), and
re-injected into the egg. Eggs were left to develop at 37.5°C until E7-8.5
(HH31-35).
RCAS(A) virus in-ovo infection
RCAS(A)Pitx2a virus (gift of Malcolm Logan, NIMR, London, UK) and control
RCAS(A) virus expressing alkaline phosphatase (AP) were prepared by infection
of the DF1 chick cell line as described
(Logan et al., 1998). Eggs
were infected at HH8-10 by injecting virus on the right side posterior to the
last somite with a glass capillary needle and an Inject+matic pico-pump.
Infected eggs were screened at E7 (HH31) and E12.5 (HH38-39). E7 (HH31) gonads
were processed for Pitx2 whole-mount in situ hybridisation. With some
gonads, only the posterior part was analysed for Pitx2 expression by
whole-mount, and the anterior part was sectioned and analysed for other
markers. Both WEF and HS eggs were used; HS eggs showed infection efficiencies
that were consistently 3-4 times higher than those of WEF eggs.
Generation of RCAS(A)-Pitx2c construct and in-ovo electroporation
A chick Pitx2c-specific fragment was generated by PCR (Pfu Turbo,
Stratagene) using the primers 5'-CCCAAGCTTGCGCTCCTTCTCCCGTCAGCC-3'
and 5'-CTGGAGCTCCTGCGGCCTCGGGGCTGGAG-3' on cDNA generated from
E6.5 (HH29-30) gonadal RNA. This was cloned into pBluescript SKII-
together with the 3' common part of Pitx2 fused to a triple
HA-tag to obtain cPitx2c-HA. The full-length cDNA was cloned into the RCAS(A)
retroviral vector. RCAS(A)Pitx2c DNA (1 µg/µl) was injected together
with a plasmid ubiquitously expressing GFP into the right coelomic cavity of
HH15-17 embryos (HS eggs), using a glass capillary needle and an Inject+matic
pico-pump and then electroporated to the dorsal coelomic epithelium as
described (Guioli et al.,
2007).
Antibodies, immunohistochemistry and in situ hybridisation
The following antibodies were used: mouse monoclonals N-cadherin (1:200;
Zymed) and fibronectin [1:1000; Developmental Studies Hybridoma Bank (DSHB)],
rat monoclonal ER
(1:200) (Greene
et al., 1984) and rabbit polyclonals LHX9
(Liem et al., 1997) and DMRT1
(1:1500). DMRT1 antibody was raised against the peptide PSIPSRGHLESTSDL from
the chick protein and its specificity checked by comparing the protein
expression pattern against the RNA expression pattern. Whole-mount and section
in situ hybridisation protocols were as described
(De Grandi et al., 2000;
Dunwoodie et al., 1997).
Probes specific for Pitx2a and c were generated by PCR of
the specific N-terminal-encoding portion; the Pitx2 generic probe
includes sequences common to all isoforms. Urogenital ridges for
histochemistry were fixed in 4% paraformaldehyde (PFA) at 4°C for 2 hours,
rinsed in PBS at room temperature (RT), transferred to 30% sucrose/PBS
overnight at 4°C, then embedded in OCT and stored at -80oC.
Cryosections were rinsed 3x 5 minutes in PBS, blocked in PBS/0.1% Tween
20 (PBST), 0.5% BSA, 2% sheep serum for 2 hours at RT. Hybridisation with
primary antibodies was at 4°C in blocking solution overnight, with the
exception of ER (overnight at 37°C). After 3x 10-minute
washes in PBST at RT, sections were incubated with secondary antibodies
(1:400) in PBST for 2 hours at RT, washed (3x 10 minutes in PBST) and
mounted in Vectashield (Vector) containing DAPI.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) and
asymmetric gene expression (Hoshino et
al., 2005; Reed and Sinclair,
2002), all concern the epithelium. We therefore investigated this
further, by analysing typical epithelial markers, and by revisiting the
expression profile of gonad-specific markers, paying particular attention to
their epithelial pattern. Fibronectin, one of the main components of basement
membranes (Larsen et al.,
2006), displayed a striking asymmetry. At E5 (HH26-27), the
staining was similar within the two gonads, where it marked the basal laminae
of the primitive medullary cords and germinal epithelium. By E6 (HH28), around
the time of sex determination, fibronectin was still around the medullary
cords on both sides, but a thick, although discontinuous, deposit was now
present at the interface between epithelium and medulla only on the left. This
difference was conspicuous in both differentiating testes and ovaries at E7
(HH31) and until E9 (HH35). By E12 (HH38), only the ovary maintained high
levels of fibronectin within the expanding left cortical layer and at the
interface with the medulla, whereas in the ZZ embryo the left-sided expression
was downregulated to levels similar to those of the right
(Fig. 1). These data reveal
that the left and right epithelia have distinct properties with respect to
laying down their basal lamina.
Next we found that the adhesion molecule N-cadherin (also known as cadherin
2) (Gumbiner, 2005) was highly
expressed in the epithelial cells along the genital ridge and adjacent dorsal
mesentery, but not along the mesonephros. As the protein localised to the cell
membrane, it clearly marked the polarity of the cells and the difference in
thickness between left and right. By E7 (HH31), in both sexes the right
epithelium had changed polarity, from columnar to flattened, while the left
maintained its columnar shape, although having thickened to become stratified
in the female. By E12 (HH38) in the male, the left epithelium was mostly
flattened (Fig. 1).
|
). Its expression is very specific, being restricted to the
gonads and, in chick, the Mullerian ducts
(De Grandi et al., 2000;
Raymond et al., 2000;
Smith et al., 1999). We
noticed that in both sexes DMRT1 was within the medulla of both gonads, and in
the epithelial cells of the left (Fig.
1). This asymmetry was already evident at E5 (HH26-27) and
maintained until E7 (HH31). After E7.5 (HH32), in the female the expression
within the left epithelial cells was downregulated (the positive cells at E12
in Fig. 1 are germ cells),
whereas in the male, epithelial cells were still weakly positive at E10 (HH36)
and a few were detected even at E12 (HH38) at the edges of the gonad
epithelium.
Several reports have described an asymmetric pattern of expression of
estrogen receptor alpha (ER; also known as ESR1), but with slightly
different results (Andrews et al.,
1997; Gonzalez-Moran,
2005; Nakabayashi et al.,
1998). ER is a nuclear receptor that controls gene
transcription in response to estrogens. Its function in chick ovarian
development has not yet been clarified, but much evidence suggests that it is
essential for normal ovarian differentiation, as this is very much dependent
on estrogens (Elbrecht and Smith,
1992; Kagami and Tomita,
1990). We found that ER RNA is expressed from E6
(HH29) in both sexes, symmetrically within left and right medulla and
asymmetrically in the epithelium, being only on the left (data not shown). The
protein had a distribution similar to the RNA, but in the male it was very
faint and mainly cytoplasmic. By contrast, in the female, ER was
nuclear and abundantly expressed by E7 (HH31)
(Fig. 1). The L-R asymmetry in
the female was even more evident by double staining with an antibody against
LHX9, another transcription factor essential for gonadal development, at least
in the mouse (Birk et al.,
2000; Mazaud et al.,
2002). In the chick, this marks the epithelium and its derivative
cortical layer as well as a few medullary cells. We found that ER and
LHX9 co-localise only in cortical cells of the left gonad. After E7.5 (HH32),
the female cortical expression of ER increased relative to that in the
medulla and, at E12 (HH38), although still seen in the epithelium and cortex,
only very faint expression of ER was detected in both the right and
left medulla (Fig. 1).
These data provide clear evidence that the left and right gonads are asymmetric in both sexes from the indifferent stage. Furthermore, both sexes maintain gonadal asymmetric features well beyond the start of sex determination. It is therefore reasonable to propose that both sex determination pathways might be influenced by lateralisation signals. Our panel of markers provide readout of left and right identity; we have therefore used it as a tool to assess the `situs' of the developing gonads in a series of functional experiments aimed to test the influence of the L-R pathway on gonadal development in ZZ and ZW embryos.
The L-R pathway controlling visceral organ situs interferes with the pathway regulating gonadal morphogenesis
Gap junction communication (GJC) in the chick blastoderm is required to
establish early differences between left and right sides. Consequently,
exposure to pharmacological compounds interfering with the junctions can cause
heterotaxia or even complete situs inversus
(Levin and Mercola, 1999).
Lindane is a proven inhibitor of GJC and relatively well tolerated by chick
embryos in culture. We therefore added lindane in ovo prior to incubation and
examined the embryos at E7-8 (HH31-35) for defects in the asymmetric
development of the gut, as an indication of L-R pathway disturbances. Out of
76 embryos, six displayed clear inversion or malformation of gut looping. Five
of these were analysed further for gonadal phenotype by assessing the
expression profile of our panel of asymmetry markers. One embryo with complete
situs inversus displayed gonads with complete L-R reversion of the cortical
features (Fig. 2A). Of the four
embryos with gut heterotaxia, one developed gonads with the usual L-R
differences (data not shown), whereas the other three had symmetrical features
as if they were left isomers (Fig.
2B,C). These data suggest that the signals controlling visceral
organ asymmetries also control the `situs' of the gonads.
|
Several Pitx2 isoforms have been isolated that differ in the
region 5' to the DNA-binding domain
(Cox et al., 2002;
Essner et al., 2000;
Schweickert et al., 2000;
Yu et al., 2001). Two main
isoforms, Pitx2a and Pitx2c, have been described in the
chick (Yu et al., 2001);
therefore, we also used in situ probes specific for the `a' and `c' regions.
Only Pitx2c was detected in the developing left gonad
(Fig. 3B). RT-PCR for
Pitx2a and Pitx2c using RNA from E6-6.5 (HH29) left gonads
confirmed the expression of Pitx2c, but also revealed a very faint
band for Pitx2a (data not shown). These results suggest that the main
isoform expressed within the developing gonad is Pitx2c, the isoform
usually found correlated with L-R asymmetry
(Schweickert et al., 2000;
Yu et al., 2001).
We next examined Pitx2 expression in the embryos treated with lindane. Pitx2 was expressed in the right gonad of the embryo with complete situs inversus, but in both gonads of the three embryos with gut heterotaxia and gonadal left isomerism (Fig. 3C). So, in all samples the expression of Pitx2 was found on the side(s) with `left' characteristics, indicating that Pitx2 might have a direct, instructive role in the situs-specific morphogenesis of the gonads.
Misexpression of Pitx2 to the right side at stage HH10 induces gonadal left isomerism
In order to misexpress Pitx2 in the right gonad from as early as
the gonad forms (HH20), we infected the right side of embryos at stage HH8-10
using RCAS virus expressing Pitx2a (RCAS-Pitx2a). Pilot experiments
with a control virus expressing alkaline phosphatase (RCAS-AP) indeed showed
that injection at HH8-10 posterior to the last somite results in the variable
infection of tissues located between forelimb and hindlimb. These include body
wall, mesonephros, gonad, dorsal mesentery and hindgut (data not shown).
Embryos from three independent experiments were examined at E7-7.5 (HH30-32). No embryo was identified showing any gross defect of heart or gut looping. This was expected, as no AP staining was observed within heart, foregut and midgut from the pilot or parallel control infections with RCAS-AP. In a proportion of RCAS-Pitx2a-infected embryos (16/98 using WEF eggs and 10/17 using HS eggs) the gonads showed bilateral expression of Pitx2 (by in situ hybridisation), although the relative intensity varied from being much weaker to even slightly more intense on the right compared with the left side. In some instances, the expression pattern on the right was discontinuous along the anterior-posterior (A-P) axis (data not shown). A small number of control embryos (3/87 WEF eggs and 1/8 HS eggs) expressed bilateral Pitx2.
The anterior portion of twelve pairs of gonads infected with RCAS-Pitx2 and
expressing bilateral Pitx2 within the posterior half, were analysed
with the battery of asymmetry markers. In eight samples, the right epithelium
had acquired features of a left epithelium with fibronectin accumulation
between epithelium and medulla and epithelial cells remaining columnar and
DMRT1-positive. In the ZW samples, the right cortical layer expressed
ER in addition to that on the left
(Fig. 4A,B). Indeed, it was no
longer possible to distinguish the right from the left side, indicating that
the gonads were undergoing symmetric development. In the other four samples -
three males and one female - the right gonad showed an ambiguous phenotype
with mixed features of right and left epithelium in terms of cell shape, DMRT1
and fibronectin expression (Fig.
4C); in the female, we did not detect ER by immunostaining.
In order to investigate this variability we reanalysed Pitx2
expression on sections from the anterior part of all twelve pairs of gonads.
The eight left isomers displayed clear bilateral Pitx2 staining,
whereas the four right gonads with an ambiguous phenotype displayed very
patchy staining, mostly at the edge with the mesentery
(Fig. 4A-C). These data show
that the gonad situs is not randomised following the induction of
Pitx2 bilateral expression, but there is instead a strong correlation
between the level of expression of Pitx2 within the epithelium and
leftness, indicating that PITX2 directs the morphogenesis of the
gonad towards the left differentiation pathway. Distinct left characteristics
might also respond with different sensitivities to PITX2.
|
In order to ascertain whether the left isomerism visualised at E7 (HH30-32)
was translating into functional left isomerism, we screened some infected
embryos at E12-13 (HH38-39). At these stages in ZW embryos, the degeneration
of the right gonad is well underway and the left gonad has developed cortical
cords embedding most germ cells. Out of 11 ZW embryos screened, six displayed
a right ovary generally shorter than the left along the A-P axis, but
resembling the left in the other two dimensions. These pairs were analysed for
the expression of ER to assess the status of the cortical somatic
cells. In two cases, both left and right gonads showed similar expression of
ER and LHX9 within the epithelium at the cortical surface, and a weaker
staining in some somatic cells of the cortical cords
(Fig. 5). The expression of the
meiotic marker H2AX [phosphorylated histone H2AX (also known as H2AFX)]
was also investigated to assess both the localisation of germ cells and their
stage of maturation. Several studies of mouse meiosis have established that
H2AX marks condensing chromosomes in leptotene/zygotene stages of
prophase (Mahadevaiah et al.,
2001). We found that in chick, H2AX is normally expressed
in germ cells of the ovary at E12-13 (HH38-39)
(Fig. 5A). Most of the positive
cells were in the cortical cords of the left ovary, with a few additional
cells scattered in the medulla on both sides. In our experiments, the two
gonad pairs with symmetric expression of ER also displayed a
H2AX pattern that was identical on left and right. This demonstrates
the ability of the right gonad to form a niche for the germ cells that is
equivalent to the one provided by the left
(Fig. 5B). Three out of the
four remaining pairs had an ambiguous phenotype. Meiotic germ cells were
clustered in one or a few discrete cortical sites, sometimes extruding over
the rest of the ovarian surface (Fig.
5C,D). These discrete areas were also double positive for
ER and LHX9 within the epithelium, as if they were pieces of left
cortex. This indicates that specific cortical sites within these right gonads
were undergoing differentiation (presumably responding to estrogens) and
attempting to provide a niche for meiotic germ cell (i.e. oocyte) development.
All six samples were tested for Pitx2 expression on sections adjacent
to those analysed for the cortical markers
(Fig. 5). Pitx2 was
found along the right epithelium of the two left isomers. In the ambiguous
samples containing discrete cortical clusters of germ cells, Pitx2
was found in the portion of epithelium overlaying the sites of cortical
differentiation, again strongly indicating that Pitx2 interacts with
the ovarian pathway to direct the differentiation of the ovarian cortex.
Pitx2 misexpression in the right genital ridge is sufficient to induce cortical differentiation
Next we asked whether it was possible to revert the identity of the right
gonad after this had already been set. We infected the right dorsal coelomic
epithelium at HH15-17 by in-ovo electroporation of RCAS-Pitx2 DNA, mostly
using the Pitx2c isoform. This procedure allows a few tissues to be
targeted, including gonads, dorsal mesentery, mesonephros and Mullerian ducts
(Guioli et al., 2007). In
order to visualise the transfected area, the DNA was co-injected with a
plasmid ubiquitously expressing GFP. The embryos were allowed to develop until
E7-8 (HH31-34) and those with high levels of GFP expression within the gonads
were analysed with the battery of asymmetry markers. In some gonads,
epithelial expression of DMRT1 and deposits of fibronectin at the interface
with the medulla were found on the right side, widespread along the A-P axis
(Fig. 6A-C); in others, they
were restricted to portions of the gonad in accordance with the distribution
of GFP expression (data not shown). Epithelial expression of ER was
also found as discrete patches of cells
(Fig. 6A,B) or more widespread
(Fig. 6C) within the surface
epithelium. In some infected gonads, the epithelium appeared rough, with
`humps' extruding in the coelomic cavity and/or lack of a proper border with
the dorsal mesentery as if the gonad were fused to it. However, epithelial
features of left identity were also present in these samples
(Fig. 6D). We took advantage of
the HA-tag fused to Pitx2 in the construct to analyse the actual
extent of infection in different samples. Ectopic Pitx2 was found, at
variable levels, in those tissues surrounded by dorsal coelomic epithelium,
including gonads, mesonephros, dorsal mesentery and Mullerian duct. Areas
along the A-P axis that were negative for HA within the gonad and mesentery
had, as expected, right identity (data not shown). HA-positive samples or
portions of it were associated with some degree of right-to-left
transformation, as described above. These gonads were mosaic for HA-positive
and HA-negative cells within the epithelium and medulla
(Fig. 6B,D). Groups of adjacent
epithelial cells expressing high levels of ectopic Pitx2 were often
found in the `humps' (Fig. 6D).
Similar results were obtained by misexpressing the Pitx2a isoform
(data not shown). These data suggest that PITX2 expression in the right gonad
after the start of gonadogenesis is sufficient to induce right-to-left changes
within the gonad, but also that the location and levels of PITX2 within the
gonadal cells might be important for proper differentiation. The process of
extrusion could indeed result in portions of differentiating epithelium being
lost. Moreover, those samples lacking a proper border between gonad and
mesentery displayed high levels of ectopic PITX2 within the mesentery and the
medial part of the gonad, suggesting that the correct dosage of PITX2 is
required within both tissues for appropriate gonadal development according to
the left pathway.
|
|
and LHX9. Expression analysis for the HA-tag
revealed ectopic PITX2 widespread within both epithelium and medulla. The
surface of these right gonads appeared rough, similar to the E7-8
electroporated samples. These data indicate that sex differentiation is
sensitive to the dosage of PITX2 and that its expression is sufficient to both
direct and redirect the morphogenesis of the gonad according to the left sex
differentiation pathway and to produce a cortical layer containing meiotic
germ cells. |
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), three different endpoints of L-R patterning are possible:
(1) directional looping of midline structures, notably the heart and gut,
which generates an asymmetry within the organ as well as in its placement; (2)
unilateral regression and/or persistence of structures that begin bilaterally,
such as the spleen and the sixth pair of aortic arch arteries that persist
only on the left; and (3) lateralisation of structures that begin
symmetrically, such as the liver and lungs. Gonadal development in the chick
would appear to fall into the second category, with the important difference
that the endpoint is sex-specific.
Our data provide clear evidence that left and right gonads have distinct
morphological and molecular characteristics in both sexes from the indifferent
stage. Furthermore, these asymmetric features are maintained in both sexes
well beyond the start of sex determination, making unlikely a model whereby
the asymmetric fate of left and right ovaries is dependent on an asymmetry
established within the ovarian pathway itself. Both sex differentiation
pathways have to deal with the initial L-R differences and whereas the male
pathway can overcome them, the female pathway cannot. We found no evidence for
L-R differences within the medulla; instead, the epithelia display
qualitatively distinct properties. Because testis cords differentiate within
the medulla, and this is central to male development, it is possible that L-R
differences within the epithelia have little impact on this process. Indeed,
whereas in the mouse the gonad coelomic epithelial cells are the source of
Sertoli cells and therefore play an essential role in cord formation, this is
not the case in chick, where the cords appear to form from the reorganisation
of the primitive sex cords with no contribution from the coelomic epithelial
cells (Carlon and Stahl, 1985;
Sekido and Lovell-Badge,
2007). Moreover, at around E12 (HH38), testis expression of
Pitx2 is almost completely downregulated and the left epithelium has
mostly flattened similar to the right side, suggesting that the male pathway
has lost its epithelial L-R asymmetry in favour of right isomerism.
|
|
;
Kagami and Tomita, 1990). This
is closely followed by the thickening of the left germinal epithelium, which
later elaborates the cortex (Carlon and
Stahl, 1985). The right epithelium, presumably owing to the
absence of ER, is unlikely to respond to estrogens. In fact, this is
the only part of the gonads of either sex shown to lack target cells for
estrogens (Gasc, 1980),
providing a simple explanation for its inability to differentiate further.
We show that the gonad situs is dependent upon the signalling pathway
controlling the establishment of the L-R body axis and, moreover, that
Pitx2 has a role in the lateralisation of the gonad and its
morphogenesis. The gonads, at least in chick, add to the list of bilateral
organs, such as the lungs, whose morphogenesis is directly linked to the
lateralisation pathway (Shiratori and
Hamada, 2006). In mammals, normal gonadal development leads to a
pair of functional testes or ovaries. However, asymmetry is evident in
disorders of sex differentiation leading to hermaphroditism, with ovaries
being more common on the left side and testes and ovotestes on the right in
humans, and, with opposite tendency, in mice
(Mittwoch, 2001).
Pitx2 is expressed in mouse gonads, but this appears to be both
male-specific and symmetrical (Coveney et
al., 2007). If the lateralisation signals exert any influence,
this effect is evidently buffered in normal development by the sex
determination/differentiation pathways. In chick, however, the L-R pathway via
Pitx2 has a direct, instructive role at organ level. Pitx2
instructs the epithelium to differentiate according to a `left' sex
differentiation pathway. We show not only that there is always a correlation
between the expression of Pitx2 within the epithelium and a display
of left identity, but also that the misexpression of Pitx2 to the
right side changes the fate of the gonad. First, Pitx2 misexpression
to the right at stage HH10 is sufficient to transform the right gonad into the
mirror image of the left gonad. In males, this means the formation of testes
with polarised left and right germinal epithelium and deposits of fibronectin
well beyond the start of sex differentiation. In females, this allows the
formation of a pair of ovaries with a stratified cortex containing most germ
cells. As the RCAS virus infection leads to expression in the mesonephros,
gonad and mesentery, we expected changes in gonadal fate to be the direct
result of ectopic PITX2a in the gonad from the start of its formation.
However, the finding that endogenous Pitx2c is expressed in the
transformed gonads and adjacent mesentery in a precise pattern similar to that
of the left side, suggests a more complex picture, whereby ectopic PITX2a
induces PITX2c and then either the latter or both together induce a
right-to-left transformation. A similar result was obtained in
Xenopus, where ectopic expression of Pitx2b in the early
whole embryo or animal cap explant induced Pitx2c expression in the
right side of the heart (Schweickert et
al., 2000). We also show that misexpression of Pitx2
directed to the coelomic epithelium after the start of gonad development
induces a right-to-left change. This means that Pitx2 is sufficient
to direct and redirect gonadal development towards a `left' sex
determination/differentiation pathway. PITX2 activity leads to the expression
of all the genes in our asymmetry marker panel, including those likely to have
a role in sex differentiation, notably ER. PITX2 also regulates
Bmp7, which is active in the left gonad from its formation
(Hoshino et al., 2005), as
expression of the gene is induced in the right gonad of embryos infected with
the Pitx2-expressing virus either at HH8-10 or HH15 (see Fig. S1 in
the supplementary material).
It has been reported that correct levels of PITX2 are essential for normal
morphogenesis of organs such as heart, lung and duodenum, and that each organ
has a different dosage requirement (Liu et
al., 2001). This appears to be true also for chick gonads, as L-R
epithelial asymmetry markers may well have different threshold sensitivities
to PITX2 activity. For example, in some E7 infected gonads, all of the
epithelium expresses DMRT1, whereas only patches of cells are
ER-positive. Moreover, it is evident that too much PITX2 is
deleterious. In the infected gonads, the positive cells, both within medulla
and cortical regions, express ectopic PITX2 at different levels. We observed
that groups of highly expressing epithelial cells tend to forms humps
extruding into the coelomic cavity where they may subsequently be lost. This
phenotype is visible at E7, but also at E12, and might explain why we observe
a discontinuous cortex in the E12 electroporated samples.
In conclusion, our results suggest a model whereby, in response to the signals that initiate L-R axis development, asymmetric expression of Pitx2 in the gonadal coelomic epithelium, which is derived from LPM, confers `leftness' to the resulting gonad, which also includes intermediate mesoderm. This permits development to continue towards an ovary in a female or a testis in a male. By contrast, `rightness' only allows testis development to continue. Furthermore, it seems likely that the ability of the left epithelium to respond to estrogens, made as a consequence of gene activity in the ovarian-determining pathway, is involved in promoting ovary development. It now seems clear that the two processes of sex determination and L-R asymmetry interact, rather than one being dependent on the other. This needs to be borne in mind when testing candidates for sex-determining genes in birds, and when exploring evolutionary relationships between species showing asymmetry in gonadal development and those that do not.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/23/4199/DC1
|
ACKNOWLEDGMENTS |
|---|
antibody;
DSHB for the anti-fibronectin antibody developed by D. M. Fambrough; and J. M.
Turner and C. E. Scott for critical reading of the manuscript. The work was
funded by the UK Medical Research Council. |
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Ai, D., Liu, W., Ma, L., Dong, F., Lu, M. F., Wang, D., Verzi, M. P., Cai, C., Gage, P. J., Evans, S. et al. (2006). Pitx2 regulates cardiac left-right asymmetry by patterning second cardiac lineage-derived myocardium. Dev. Biol. 296,437 -449.[CrossRef][Medline]
Andrews, J. E., Smith, C. A. and Sinclair, A. H. (1997). Sites of estrogen receptor and aromatase expression in the chicken embryo. Gen. Comp. Endocrinol. 108,182 -190.[CrossRef][Medline]
Birk, O. S., Casiano, D. E., Wassif, C. A., Cogliati, T., Zhao, L., Zhao, Y., Grinberg, A., Huang, S., Kreidberg, J. A., Parker, K. L. et al. (2000). The LIM homeobox gene Lhx9 is essential for mouse gonad formation. Nature 403,909 -913.[CrossRef][Medline]
Campione, M., Steinbeisser, H., Schweickert, A., Deissler, K., van Bebber, F., Lowe, L. A., Nowotschin, S., Viebahn, C., Haffter, P., Kuehn, M. R. et al. (1999). The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping. Development 126,1225 -1234.[Abstract]
Carlon, N. and Stahl, A. (1985). Origin of the somatic components in chick embryonic gonads. Arch. Anat. Microsc. Morphol. Exp. 74,52 -59.[Medline]
Coveney, D., Ross, A. J., Slone, J. D. and Capel, B. (2007). A microarray analysis of the XX Wnt4 mutant gonad targeted at the identification of genes involved in testis vascular differentiation. Gene Expr. Patterns 7, 82-92.[CrossRef][Medline]
Cox, C. J., Espinoza, H. M., McWilliams, B., Chappell, K.,
Morton, L., Hjalt, T. A., Semina, E. V. and Amendt, B. A.
(2002). Differential regulation of gene expression by PITX2
isoforms. J. Biol. Chem.
277,25001
-25010.
Dagle, J. M., Sabel, J. L., Littig, J. L., Sutherland, L. B., Kolker, S. J. and Weeks, D. L. (2003). Pitx2c attenuation results in cardiac defects and abnormalities of intestinal orientation in developing Xenopus laevis. Dev. Biol. 262,268 -281.[CrossRef][Medline]
De Grandi, A., Calvari, V., Bertini, V., Bulfone, A., Peverali, G., Camerino, G., Borsani, G. and Guioli, S. (2000). The expression pattern of a mouse doublesex-related gene is consistent with a role in gonadal differentiation. Mech. Dev. 90,323 -326.[CrossRef][Medline]
Dunwoodie, S. L., Henrique, D., Harrison, S. M. and Beddington, R. S. (1997). Mouse Dll3: a novel divergent Delta gene which may complement the function of other Delta homologues during early pattern formation in the mouse embryo. Development 124,3065 -3076.[Abstract]
Elbrecht, A. and Smith, R. G. (1992). Aromatase
enzyme activity and sex determination in chickens.
Science 255,467
-470.
Essner, J. J., Branford, W. W., Zhang, J. and Yost, H. J. (2000). Mesendoderm and left-right brain, heart and gut development are differentially regulated by pitx2 isoforms. Development 127,1081 -1093.[Abstract]
Gage, P. J., Suh, H. and Camper, S. A. (1999). Dosage requirement of Pitx2 for development of multiple organs. Development 126,4643 -4651.[Abstract]
Gasc, J. M. (1980). Estrogen target cells in gonads of the chicken embryo during sexual differentiation. J. Embryol. Exp. Morphol. 55,331 -342.[Medline]
Gonzalez-Moran, M. G. (2005). Immunohistochemical detection of estrogen receptor alpha in the growing and regressing ovaries of newly hatched chicks. J. Mol. Hist. 36,147 -155.[CrossRef]
Gormley, J. P. and Nascone-Yoder, N. M. (2003). Left and right contributions to the Xenopus heart: implications for asymmetric morphogenesis. Dev. Genes Evol. 213,390 -398.[CrossRef][Medline]
Greene, G. L., Sobel, N. B., King, W. J. and Jensen, E. V. (1984). Immunochemical studies of estrogen receptors. J. Steroid Biochem. 20,51 -56.[CrossRef][Medline]
Guioli, S., Sekido, R. and Lovell-Badge, R. (2007). The origin of the Mullerian duct in chick and mouse. Dev. Biol. 302,389 -398.[CrossRef][Medline]
Gumbiner, B. M. (2005). Regulation of cadherin-mediated adhesion in morphogenesis. Nat. Rev. Mol. Cell Biol. 6,622 -634.[Medline]
Hamburger, V. and Hamilton, H. L. (1992). A series of normal stages in the development of the chick embryo. 1951. Dev. Dyn. 195,231 -272.[Medline]
Hoshino, A., Koide, M., Ono, T. and Yasugi, S. (2005). Sex-specific and left-right asymmetric expression pattern of Bmp7 in the gonad of normal and sex-reversed chicken embryos. Dev. Growth Differ. 47,65 -74.[CrossRef][Medline]
Kagami, H. and Tomita, T. (1990). Genetic and morphological studies on the right gonad of ovariectomized chickens. Jpn. Poult. Sci. 27,111 -121.
Kinsky, F. C. (1971). The consistent presence of paired ovaries in the Kiwi (Apteryx) with some discussion of this condition in other birds. J. Ornithol. 112,334 -357.[CrossRef]
Kitamura, K., Miura, H., Miyagawa-Tomita, S., Yanazawa, M., Katoh-Fukui, Y., Suzuki, R., Ohuchi, H., Suehiro, A., Motegi, Y., Nakahara, Y. et al. (1999). Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra- and periocular mesoderm and right pulmonary isomerism. Development 126,5749 -5758.[Abstract]
Koopman, P. and Loffler, K. A. (2003). Sex determination: the fishy tale of Dmrt1. Curr. Biol. 13,R177 -R179.[CrossRef][Medline]
Larsen, M., Artym, V. V., Green, J. A. and Yamada, K. M. (2006). The matrix reorganized: extracellular matrix remodeling and integrin signaling. Curr. Opin. Cell Biol. 18,463 -471.[CrossRef][Medline]
Levin, M. (2005). Left-right asymmetry in embryonic development: a comprehensive review. Mech. Dev. 122,3 -25.[CrossRef][Medline]
Levin, M. and Mercola, M. (1999). Gap junction-mediated transfer of left-right patterning signals in the early chick blastoderm is upstream of Shh asymmetry in the node. Development 126,4703 -4714.[Abstract]
Liem, K. F., Jr, Tremml, G. and Jessell, T. M. (1997). A role for the roof plate and its resident TGFbeta-related proteins in neuronal patterning in the dorsal spinal cord. Cell 91,127 -138.[CrossRef][Medline]
Liu, C., Liu, W., Lu, M. F., Brown, N. A. and Martin, J. F.
(2001). Regulation of left-right asymmetry by thresholds of
Pitx2c activity. Development
128,2039
-2048.
Liu, C., Liu, W., Palie, J., Lu, M. F., Brown, N. A. and Martin,
J. F. (2002). Pitx2c patterns anterior myocardium and aortic
arch vessels and is required for local cell movement into atrioventricular
cushions. Development
129,5081
-5091.
Logan, M., Pagan-Westphal, S. M., Smith, D. M., Paganessi, L. and Tabin, C. J. (1998). The transcription factor Pitx2 mediates situs-specific morphogenesis in response to left-right asymmetric signals. Cell 94,307 -317.[CrossRef][Medline]
Mahadevaiah, S. K., Turner, J. M., Baudat, F., Rogakou, E. P., de Boer, P., Blanco-Rodriguez, J., Jasin, M., Keeney, S., Bonner, W. M. and Burgoyne, P. S. (2001). Recombinational DNA double-strand breaks in mice precede synapsis. Nat. Genet. 27,271 -276.[CrossRef][Medline]
Mazaud, S., Oreal, E., Guigon, C. J., Carre-Eusebe, D. and Magre, S. (2002). Lhx9 expression during gonadal morphogenesis as related to the state of cell differentiation. Gene Expr. Patterns 2,373 -377.[CrossRef][Medline]
Mittwoch, U. (2001). Genetics of mammalian sex determination: some unloved exceptions. J. Exp. Zool. 290,484 -489.[CrossRef][Medline]
Morales, A. V., Acloque, H., Ocana, O. H., de Frutos, C. A., Gold, V. and Nieto, M. A. (2007). Snail genes at the crossroads of symmetric and asymmetric processes in the developing mesoderm. EMBO Rep. 8,104 -109.[CrossRef][Medline]
Nakabayashi, O., Kikuchi, H., Kikuchi, T. and Mizuno, S. (1998). Differential expression of genes for aromatase and estrogen receptor during the gonadal development in chicken embryos. J. Mol. Endocrinol. 20,193 -202.[Abstract]
Piedra, M. E., Icardo, J. M., Albajar, M., Rodriguez-Rey, J. C. and Ros, M. A. (1998). Pitx2 participates in the late phase of the pathway controlling left-right asymmetry. Cell 94,319 -324.[CrossRef][Medline]
Ramsdell, A. F. (2005). Left-right asymmetry and congenital cardiac defects: getting to the heart of the matter in vertebrate left-right axis determination. Dev. Biol. 288, 1-20.[CrossRef][Medline]
Raya, A. and Belmonte, J. C. (2006). Left-right asymmetry in the vertebrate embryo: from early information to higher-level integration. Nat. Rev. Genet. 7, 283-293.[CrossRef][Medline]
Raymond, C. S., Murphy, M. W., O'Sullivan, M. G., Bardwell, V.
J. and Zarkower, D. (2000). Dmrt1, a gene related to worm and
fly sexual regulators, is required for mammalian testis differentiation.
Genes Dev. 14,2587
-2595.
Reed, K. J. and Sinclair, A. H. (2002). FET-1: a novel W-linked, female specific gene up-regulated in the embryonic chicken ovary. Mech. Dev. 119,S87 -S90.[CrossRef][Medline]
Ryan, A. K., Blumberg, B., Rodriguez-Esteban, C., Yonei-Tamura, S., Tamura, K., Tsukui, T., de la Pena, J., Sabbagh, W., Greenwald, J., Choe, S. et al. (1998). Pitx2 determines left-right asymmetry of internal organs in vertebrates. Nature 394,545 -551.[CrossRef][Medline]
Schweickert, A., Campione, M., Steinbeisser, H. and Blum, M. (2000). Pitx2 isoforms: involvement of Pitx2c but not Pitx2a or Pitx2b in vertebrate left-right asymmetry. Mech. Dev. 90, 41-51.[CrossRef][Medline]
Sekido, R. and Lovell-Badge, R. (2007). Mechanisms of gonadal morphogenesis are not conserved between chick and mouse. Dev. Biol. 302,132 -142.[CrossRef][Medline]
Shiratori, H. and Hamada, H. (2006). The
left-right axis in the mouse: from origin to morphology.
Development 133,2095
-2104.
Shiratori, H., Yashiro, K., Shen, M. M. and Hamada, H.
(2006). Conserved regulation and role of Pitx2 in situs-specific
morphogenesis of visceral organs. Development
133,3015
-3025.
Smith, C. A., McClive, P. J., Western, P. S., Reed, K. J. and Sinclair, A. H. (1999). Conservation of a sex-determining gene. Nature 402,601 -602.[Medline]
St Amand, T. R., Ra, J., Zhang, Y., Hu, Y., Baber, S. I., Qiu, M. and Chen, Y. (1998). Cloning and expression pattern of chicken Pitx2: a new component in the SHH signaling pathway controlling embryonic heart looping. Biochem. Biophys. Res. Commun. 247,100 -105.[CrossRef][Medline]
Vermot, J. and Pourquie, O. (2005). Retinoic acid coordinates somitogenesis and left-right patterning in vertebrate embryos. Nature 435,215 -220.[CrossRef][Medline]
Yoshioka, H., Meno, C., Koshiba, K., Sugihara, M., Itoh, H., Ishimaru, Y., Inoue, T., Ohuchi, H., Semina, E. V., Murray, J. C. et al. (1998). Pitx2, a bicoid-type homeobox gene, is involved in a lefty-signaling pathway in determination of left-right asymmetry. Cell 94,299 -305.[CrossRef][Medline]
Yoshioka, H., Ishimaru, Y., Sugiyama, N., Tsunekawa, N., Noce, T., Kasahara, M. and Morohashi, K. (2005). Mesonephric FGF signaling is associated with the development of sexually indifferent gonadal primordium in chick embryos. Dev. Biol. 280,150 -161.[CrossRef][Medline]
Yu, X., St Amand, T. R., Wang, S., Li, G., Zhang, Y., Hu, Y. P., Nguyen, L., Qiu, M. S. and Chen, Y. P. (2001). Differential expression and functional analysis of Pitx2 isoforms in regulation of heart looping in the chick. Development 128,1005 -1013.[Abstract]
| ||||||||||||||||||||||||||||||||||||
