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First published online 8 April 2004
doi: 10.1242/dev.01090
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1 Brookdale Department Molecular, Cell and Developmental Biology, Mount Sinai
Medical School, 1 G Levy Place, New York, NY 10029, USA
2 Department of Pathology and OB/GYN, Columbia University, 630 West 168th
Street, New York, NY 10032, USA
Author for correspondence (e-mail:
david.sassoon{at}mssm.edu)
Accepted 22 January 2004
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: Wnt, Uterus, Mouse, DES, Glands
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). The murine FRT is immature at birth and consists of two
tubes of simple columnar epithelia surrounded by a mesenchymal sheath fused at
the level of the cervix. Distinct cytodifferentiation occurs during postnatal
development and differentiation is complete 2 weeks after birth. Uterine horns
develop postnatally to form an external myometrium surrounding the mesenchymal
(stromal) compartment which contains glands. By contrast, the vagina and
cervix do not develop glands and the luminal epithelium undergoes a transition
from simple columnar to squamous (stratified) morphology. Experiments in which
neonatal epithelium from any part of the FRT is recombined with presumptive
uterine or vaginal mesenchyme reveals that the epithelium is developmentally
plastic and adopts either a uterine (simple columnar) or vaginal
(squamous/stratified) epithelial fate dependent upon the origin of the
mesenchyme (Cunha, 1976;
Kurita et al., 2001). Recent
results using grafts between estrogen receptor 
(Esr1) mutant
and wild-type FRTs demonstrates that Esr1 function is required in the
mesenchyme but not in the epithelium to mediate estrogen-mediated responses in
the epithelium (Cooke et al.,
1997; Kurita et al.,
2000). Taken together, these studies demonstrate that FRT
mesenchyme delivers developmental and estrogenic signals to the
epithelium.
Wnt genes encode secreted glycoproteins that regulate cell and tissue
growth and differentiation (Polakis,
2000) and activate multiple signaling pathways through the
frizzled receptors and the cytoplasmic signaling protein, dishevelled
(Pandur et al., 2002). We
identified three members of the Wnt gene family (Wnt4, Wnt5a, Wnt7a)
that are expressed at high levels in the adult FRT throughout development
(Miller et al., 1998b). At
birth, Wnt4 expression is restricted to the uterine mesenchyme. By
contrast, Wnt5a is distributed throughout the mesenchyme of the
uterus, cervix and vagina, whereas Wnt7a is in the epithelium. During
postnatal differentiation, Wnt5a and Wnt7a become restricted
to the uterine horns, whereas Wnt4 expression is activated in the
stratified epithelium of the cervix and the vagina
(Miller et al., 1998b). We
noted that the levels and the sites of Wnt expression fluctuate
during estrous, suggesting a continued role in the adult
(Miller et al., 1998b).
Mice corresponding to all three Wnt genes expressed in the FRT have been
generated. Wnt4 mutants fail to form Müllerian ducts and die at
birth due to numerous defects, thus an analysis of how Wnt4 contributes to
later FRT development is unknown (Vainio
et al., 1999). Wnt7a mutants are viable and exhibit
malformations in the FRT including shortened and uncoiled oviducts,
hypoplastic uterine horns and a vaginal septum
(Miller and Sassoon, 1998;
Parr and McMahon, 1998). The
uterus is most affected with a marked reduction in the stromal compartment,
accompanied by a lack of uterine glands and a disorganized myometrium. In
addition, Wnt7a mutant uterine epithelium fails to maintain a normal
columnar phenotype and becomes stratified upon puberty
(Miller and Sassoon, 1998). It
was subsequently demonstrated that fetal diethylstilbestrol (DES) exposure
transiently represses Wnt7a expression in the Müllerian ducts
and is sufficient to recapitulate the Wnt7a mutant FRT phenotypes
providing a molecular basis for environmental endocrine disruption
(Miller et al., 1998a).
In this study, we examined the role of Wnt5a, which is expressed
in the FRT mesenchyme, and thus is a good candidate as a potential mediator of
mesenchymal-epithelial interactions
(Miller et al., 1998b;
Pavlova et al., 1994).
Wnt5a mutants die at birth due to a failure to complete
anteroposterior body axis development
(Yamaguchi et al., 1999). In
order to circumvent the neonatal lethality, we grafted neonatal mutant FRT
tissue into adult hosts to assess postnatal potential and phenotypes. We find
that although the oviduct, uterine and cervical compartments of the FRT
develop in the absence of Wnt5a, the mutant uterus fails to form
glands that are essential for adult function. In addition, we demonstrate that
Wnt5a is required for the complete repertoire of estrogen-mediated
cellular and molecular responses. Furthermore, Wnt5a participates in
a regulatory loop with Wnt7a and is required for correct regulation
of Hoxa10 and Hoxa11, which control anteroposterior
patterning of the FRT (Benson et al.,
1996; Hsieh-Li et al.,
1995). These data shed light upon the mechanism by which uterine
stroma mediates both developmental and estrogen-mediated changes in uterine
epithelium and reveal that Wnt5a is required in these processes.
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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) and
maintained in a C57BL6/SV129 mixed background. Neonates were obtained after
delivery or C-section on day 19 of gestation. Wnt7a mutant mice were
obtained from B. Parr and A. McMahon (Parr
and McMahon, 1998) and maintained in a SV129 background.
Lef1 mutant mice were kindly obtained from R. Grosschedl
(van Genderen et al., 1994)
and were maintained in a C57BL6/SV129 mixed background. Nude mice in a C57BL6
background were purchased intact or ovariectomized from Taconic, Germantown,
NY. All procedures for handling of mice, housing and maintenance were
performed according to approved institutional guidelines. All surgical
procedures were prior approved by the institution according to NIH
guidelines.
Tissue recombination and renal capsule grafting
Wild-type and mutant neonate FRT fragments were grafted under the renal
capsule of each kidney of the same adult female nude host to ensure identical
hormonal conditions. Grafting procedures were performed as previously
described (Cunha, 1976). Adult
female hosts were ovariectomized 3-4 weeks prior to grafting where indicated.
Separation of the neonatal epithelium from the mesenchyme for recombination
between wild-type and Wnt5a mutant tissues was performed as
previously described (Bigsby et al.,
1986). Diethylstilbestrol (DES) administration was delivered i.p.
suspended in saline between day 18 to 20 post graft implantation following a
previously described protocol (Miller et
al., 1998a). Host FRT and neonate grafts were harvested 24 hours
after the last injection on day 21, i.e. a developmental stage equivalent to
3-week-old FRT, and were fixed o/n in 4% PAF 4°C and processed for
paraffin histology.
Retroviral expression vectors
Wnt cDNAs encoding HA tagged Wnt4 and Wnt5a were inserted
in QCX backbone vectors derived from MLV retrovirus and produced as previously
described (Julius et al.,
2000; Shimizu et al.,
1997). Retroviral supernatants were concentrated by
ultracentrifugation, 2 hours at 100 g in a Beckman SW28 rotor.
Titer was estimated to 1x106 infection unit by lacZ
staining of NIH3T3 cells infected with a parallel QC-lacZ prep. Western blot
and in situ immunofluorescence using anti-HAtag high affinity, rat monoclonal
antibody (3F10) from Roche Diagnostics (Mannheim) were performed on infected
NIH3T3 to confirm the synthesis of the Wnt factors by the retrovirus. Neonate
uterine fragments were infected overnight in retroviral supernatant
resuspended in DMEM/20% FCS/4 µg/ml hexadimethrine bromide (H9268,
Sigma).
In situ hybridization
In situ hybridizations for the Wnt, Hoxa and Msx1 were
performed as previously described (Miller
and Sassoon, 1998). Esr1 and Pgr probes were
kindly provided by G. Cunha. RNA probes were labeled with 35S-UTP.
Black and white dark field images were converted to using Adobe Photoshop to
allow superimposing upon phase contrast images.
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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60 pups or 16.53% versus
25%), indicating loss of mutant fetuses in utero. Of 40 mutant pups, 17 were
males and 19 females revealing no gender bias in survival during gestation. We
could not determine the sex of 4 pups because of extreme reduction in
posterior development. The anterior Müllerian-derived structures
(oviducts and uterine horns) could easily be identified, whereas posterior
derived structures (cervix and vagina) were absent
(Fig. 1A,B). The uterine horns
are either fused at the midline (Fig.
2) or terminate as a blind pouch
(Fig. 1B). The Wnt5a
mutant uterine horns have an undulated lumen and show a 60 to 90% reduction in
length when compared with wild types (see also left panels in
Fig. 2). The oviducts are less
affected and we observe correct narrowing of the anterior uterine horn at the
level of the uterotubal junction with the oviduct. The anterior border of
expression for Hoxa10 defines the site of the uterotubal junction
(Benson et al., 1996). Using
whole-mount in situ hybridization, we confirmed the position of the uterotubal
junction in the Wnt5a mutant and in the wild type
(Fig. 1). Expression of
Wnt7a and Msx1 is detected in the epithelium of the Wnt5a
mutant FRT (Fig. 1E-H) whereas
the expression of Wnt4 is only slightly reduced when compared with wild-type
uterus and is not affected in the ureters
(Fig. 1I,J). The expression of
the Wnt5a mutant transcript is not affected in the absence of
Wnt5a in the FRT (Fig.
1K,L) as previously seen for the Wnt5a mutant limb bud
(Yamaguchi et al., 1999). We
conclude that loss of Wnt5a affects posterior growth of the
Müllerian duct.
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).
Smooth muscle myosin heavy chain in situ hybridization revealed correct
differentiation of smooth muscle layers in the Wnt5a mutant (data not
shown). We observed grafts that developed stratified epithelium in two out of
five independent Wnt5a mutant grafts which were derived from the most
posterior portion of the neonate Wnt5a mutant Müllerian ducts
(Fig. 3A-C). The morphological
columnar-to-squamous junction of the epithelium was accompanied at the
molecular level by the correct boundary of Msx1 and Wnt7a
expression and activation of Wnt4 in the stratified epithelium
(Fig. 3D-F; data not shown).
This result reveals the potential to form a cervix in the Wnt5a
mutant despite the lack of a morphologically defined cervix at birth.
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; Branham et al.,
1985b; Gray et al.,
2001). As normal (postnatal) glandulargenesis occurs in the
immature uterus in the absence of high levels of circulating steroid hormones,
we placed grafts into female hosts that were ovariectomized 2 weeks prior to
the grafting procedures. Under these conditions, numerous and normal appearing
glands developed in all the grafts (n=18;
Fig. 4A). In addition, the
tissue organization of the grafts and the luminal folds were indistinguishable
from uteri in situ at an equivalent stage of post-natal development (3 weeks).
Thus, this procedure for obtaining normal morphogenesis and maturation of
wild-type grafted uterine tissues allows for assessment of the outcome of
mutant FRT development, particularly in the case of perinatal lethality. Using
ovariectomized hosts, 10 out of 12 grafts derived from eight independent
Wnt5a mutant mice did not develop glands whereas the remaining two
grafts developed very few glands (Fig.
4B; data not shown). The overall morphology of the Wnt5a
mutant grafts is otherwise normal, although the smooth muscle layers are
moderately thinner compared with the wild-type grafts.
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), suggesting a potential genetic interaction between
Wnt7a and Wnt5a. However, we had analyzed the Wnt7a
uterine phenotype from samples obtained directly from postnatal mutant
females. To compare directly our results under the same experimental
conditions, we tested Wnt7a mutant uterine grafts in ovariectomized
hosts. The Wnt7a mutant uterine grafts (n=3) fail to develop
glands in ovariectomized hosts and recapitulate the myometrial and epithelial
phenotypes that have been reported in our previous studies
(Miller and Sassoon, 1998)
(Fig. 4C).
Lef1, a mediator of canonical Wnt signaling and gland formation, is not required for uterine glandulargenesis
Lef1 is a transcription factor that interacts with ß-catenin
and mediates the canonical Wnt pathway
(McKendry et al., 1997).
Lef1 is expressed in the Müllerian duct mesenchyme
(Oosterwegel et al., 1993);
however, Lef1 mutant mice die several days after birth prior to
cytodifferentiation of the FRT and uterine gland formation. As Lef1
is required for glandulargenesis in the mammary gland and mediates numerous
epithelial-mesenchymal interactions during development
(van Genderen et al., 1994),
we evaluated the potential role of Lef1 in uterine postnatal
development using grafting procedures. We find that Lef1 is not
required for uterine development. Moreover, glandulargenesis proceeds normally
in Lef1 mutant grafts (n=2). In addition we generated grafts
(n=2) from double Wnt7a/Lef1 mutants that are
indistinguishable from Wnt7a mutant grafts
(Fig. 4D,E). Taken together,
these results show that Wnt5a and Wnt7a are required for
gland formation in the uterus and participate in a signaling pathway that does
not require Lef1.
Wnt5a is required in the stroma to induce gland formation
Uterine gland formation initiates on postnatal days 7-9 (P7-9). and they
continue to grow and increase in number until puberty. By P15, Wnt7a
is expressed exclusively in luminal epithelium but not in glandular epithelium
(Fig. 4G). Wnt7a is
also expressed in the deep folds of the luminal epithelium that start to form
between P5 and P7 (Brody and Cunha,
1989). In wild-type grafts, we observe a sharp boundary of
Wnt7a expression at the transition between luminal and glandular
epithelium (Fig. 4J).
Wnt4 expression is restricted to the stroma between the luminal
epithelium and adjacent uterine glands
(Fig. 4I). Wnt5a
expression is abundant throughout the stroma that extends from the
subepithelial region up to the inner smooth muscle layer. This domain of
expression includes stroma surrounding the folding luminal epithelium and more
distal glands (Fig. 4H). In
addition, low but detectable levels of Wnt5a expression are observed
in luminal and glandular epithelium. These patterns of expression suggest that
Wnt7a and Wnt5a act in juxtaposed compartments to control
gland formation. The sharp boundary of Wnt7a expression at the site
of glandular invagination suggests a mechanism whereby Wnt7a is
repressed locally to allow luminal epithelium to participate in gland
formation. If this model is correct, then Wnt5a may provide a
permissive environment for proper regulation of Wnt7a. To determine
if Wnt5a expression is required in the stroma to mediate gland
formation, we performed recombinant graft experiments using wild-type and
Wnt5a mutant (–/–) uterine fragments
(Fig. 5A). When wild-type
stroma is recombined with wild-type epithelium or Wnt5a mutant
epithelium, recombinant grafts form glands
(Fig. 5B,D). When
Wnt5a mutant stroma is recombined with Wnt5a mutant or
wild-type epithelium, no glands developed, except for one mutant
mesenchyme/wild-type epithelium graft that developed a single gland
(Fig. 5C,E; data not shown).
These results demonstrate that Wnt5a is required in the stroma for
glandulargenesis.
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Wnt5a is required for Wnt7a and Hoxa repression by DES
We evaluated the morphological and cellular responses of the Wnt5a
mutant uterine graft to the potent estrogen, DES (diethylstilbestrol), which
normally elicits pronounced cellular and molecular changes in uterine tissue.
Grafts were allowed to grow in ovariectomized hosts for 18-20 days followed by
three daily injections of DES or saline followed 24 hours by harvesting the
grafts. Wild-type (n=3) and Wnt5a+/– grafts
(n=6) responded to DES exposure with the typical changes associated
with the estrogenic response, i.e. hypertrophy and hyperplasia of the luminal
and glandular epithelial cells and the distension of the stromal compartment
which is associated with the changes in vascular permeability that occur upon
estrogenic compounds exposure (Korach and
McLachlan, 1995) (Fig. 6,
compare B with A). All Wnt5a mutant grafts (n=4)
exposed to DES responded by a normal increase in cellularity and thickness of
the epithelial compartment (although no glandular response is measured)
(Fig. 6D,H). The average
luminal epithelium height was assayed in three independent grafts of each
genotype. The epithelium height changed from 19.4±3.8 µm in the
absence of DES to 38.4±0.2 µm after DES exposure in the
Wnt5a mutant graft (compare Fig.
6G,H), showing no significant difference with the wild-type
grafts, 20.1±1.5 µm in saline conditions to 38.3±5.1 µm
after DES exposure (Fig. 6E,F). By contrast, the global response of the Wnt5a mutant grafts was
abnormal in appearance. The uterine walls of the mutant grafts did not enlarge
in response to DES, but instead underwent an unusual dilation. In addition, we
note that two out of the four Wnt5a mutant DES exposed grafts did not
display stromal edema (Fig. 6D;
data not shown).
|
;
Miller et al., 1998a) are not
repressed by DES exposure in the Wnt5a mutant grafts
(Fig. 6M-T, compare P with N and T with
R). To determine if prolonged exposure to high levels of a synthetic estrogen adequately reproduces the endogenous regulation of Wnt7a and the Hoxa genes by Wnt5a, we analyzed uterine grafts grown in intact cycling hosts that were sacrificed at different period of the estrous cycle. Expression patterns of Wnt7a, Hoxa10 and Hoxa11 are normal in Wnt5 mutant grafts harvested at diestrus from the host, when levels of estrogen are low and levels of progesterone are high (Fig. 7A). During proestrus, when the levels of estrogen are high and the levels of progesterone are low, we find that Wnt7a levels remain high in Wnt5a mutants (Fig. 7B). Similarly, Hoxa10 and Hoxa11 remain high during proestrus in the Wnt5a mutant grafts. These results show that estrogen mediated repression of Wnt7a in the epithelium and repression of Hoxa10 and Hoxa11 in the stroma is dependent upon Wnt5a expression.
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|
). We found that Pgr is repressed by DES in the
ovariectomized adult host consistent with previously reported observations
(Fig. 8K,L). In contrast to
adult host uteri, Pgr is not repressed by DES in the epithelium of
control grafts, rather it is repressed in the stroma and activated in the
epithelium and smooth muscle layers (Fig.
8N). The differences in regulation of Pgr between the
3-week-old wild-type grafts and the adult host may reflect the previously
noted differences in the estrogenic response between prepubertal females and
sexually mature females (Korach and
McLachlan, 1995); however, we note that Pgr gene
expression and regulation in the Wnt5a mutant grafts is identical to
control grafts (Fig. 8O,P).
These results show that key aspects of the genomic response to estrogenic
signals is preserved in the Wnt5a mutant. We then analyzed
Wnt5a expression following DES exposure. Wnt5a transcripts
are restricted primarily to the stroma in control grafts grown in
mock-injected ovariectomized hosts (Fig.
8G), whereas Wnt5a mutant transcripts are present in both
the stroma and epithelium in mutant grafts
(Fig. 8I). DES exposure
increases Wnt5a levels in the epithelium and the smooth muscle in
both control and Wnt5a mutant grafts
(Fig. 8H,J) revealing that
Wnt5a signaling is not required for the regulation of its own gene
product by estrogen. Therefore, in contrast to Wnt7a, which is
primarily repressed by DES exposure, Wnt5a undergoes a spatial change
in expression similar to the situation observed with Esr1. This
result and the fact that Esr1 itself is correctly regulated in the
Wnt5a mutant (Fig.
8A-F) suggest that Wnt5a regulation by estrogenic stimuli
is genetically downstream of Esr1.
To determine if genes expressed in the epithelium other than Wnt7a
are misregulated in the Wnt5a mutant, we analyzed the regulation of
Msx1, a homeobox gene whose expression is maintained specifically in
the luminal and glandular uterine epithelium of the adult
(Pavlova et al., 1994). We
found that DES represses Msx1 in the Wnt5a mutant grafts as
in control grafts (Fig. 8Q-T)
indicating that Msx1 regulation by estrogen is Wnt5a
independent in marked contrast to what is seen with other patterning genes
examined in this study. Thus, Msx1 represents a potential
developmental and hormone-sensitive pathway that is not subject to control by
Wnt genes.
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Discussion |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Miller et al., 1998b;
Pavlova et al., 1994;
Taylor et al., 1997;
Ying and Zhao, 2000). Mouse
mutants generated to the Hoxa10, Hoxa11 and Wnt7a
genes reveal a requirement for these genes during postnatal development
(Benson et al., 1996;
Hsieh-Li et al., 1995;
Miller and Sassoon, 1998;
Parr and McMahon, 1998). In
cases where gene mutation leads to perinatal lethality, postnatal FRT
development has not been examined. Using grafting techniques as a means to
circumvent the neonatal lethality of the Wnt5a mutant, we find that
Wnt5a is required to appropriately establish the development of the
posterior region of the FRT. Wnt5a mutant FRTs have short and coiled
uterine horns of normal diameter and lack defined cervical/vaginal structures.
These findings are in contrast to our previous observations for the
Wnt7a mutant FRT, which shows complete posterior development whereas
the uterine horns are atrophic (Miller and
Sassoon, 1998) (this study). Although the Wnt5a and
Wnt7a phenotypes differ, they share specific characteristics
described for the FRT of different Hox gene mutants
(Fig. 9A). In the
Hoxa13 mutant, the caudal region of the Müllerian ducts does not
develop (Warot et al., 1997).
In fact other aspects of the phenotypes of posterior Hoxa gene
mutants and Wnt5a mutant are similar as the growth of the genital
tubercle and the limb buds is also severely affected in the double
Hoxa13/d13 mutant as in the Wnt5a mutant
(Warot et al., 1997). Taken
together, the phenotypic similarities of Wnt5a and
Hoxa13/d13 mutant FRTs suggest that they may act in a common pathway
during development to regulate posterior growth of the Müllerian ducts
(Fig. 9A). Whereas a phenotypic
similarity between Wnt5a and posterior Hoxa mutant FRTs is
found, we note that the atrophic uterine horns of the Wnt7a mutants
and the reduction of the glandular and stromal compartments in the adult
resemble the phenotypes described for the Hoxa11 and
Hoxa10/Hoxa11 transheterozygotes mutant FRTs
(Branford et al., 2000)
(Fig. 9A). We reported
previously that Hoxa10 and Hoxa11 expression is normal at
birth in the Wnt7a mutant FRT; however, Hoxa10/11 expression
is not maintained in the mature FRT
(Miller and Sassoon, 1998).
Similarly, the expression of Wnt7a is normal in the Hoxa11
mutant neonates and subsequently declines (data not shown). Therefore
Wnt7a and Hoxa11 are independently activated but then
maintain expression of each other. These genetic analyses suggest that in
addition to their homeotic functions along the anteroposterior axis
(Benson et al., 1996;
Branford et al., 2000;
Hsieh-Li et al., 1995),
Hoxa10 and Hoxa11 participate in a common morphogenetic
pathway with Wnt7a that directs growth along the radial axis of
uterine horn and subsequent stromal/epithelial differentiations required to
generate the glandular compartment (Fig.
9A).
|
). Analyses of the Wnt7a and Wnt5a mutants
demonstrate the requirement of both genes in glandulargenesis of the uterus
(Miller and Sassoon, 1998)
(this study). The fact that Wnt7a is expressed in uterine epithelium
and that Wnt5a is expressed in uterine stroma is consistent with
longstanding observations that cytodifferentiation of the uterus requires
epithelial-mesenchymal paracrine interactions. Although Wnt5a is
expressed throughout the uterine mesenchyme, we observed that Wnt7a
is downregulated specifically in the invaginating epithelium that gives rise
to the glands during postnatal development. Based upon these observations, we
propose that highly regionalized repression of Wnt7a is required to
allow luminal epithelium to change fate, invaginate and form glands and that
Wnt5a is required for this downregulation (see model in
Fig. 9B). Although global
hormonal repression of Wnt7a and local repression of Wnt7a
during pre-pubertal development may well reflect completely different
pathways, we find that Wnt5a is required for downregulation of
Wnt7a in response to DES.
The complete loss or transient repression of Wnt7a expression
during perinatal FRT development leads to global disorganization of the
uterine epithelium and a disruption of gland formation later in adult life
(Miller et al., 1998a;
Miller and Sassoon, 1998).
This is in contrast to Wnt5a mutant FRT, which maintains a normal
columnar epithelial phenotype but still fails to form glands. These
observations suggest that Wnt7a is required to maintain a columnar
epithelial phenotype and if downregulation of Wnt7a is blocked, gland
formation will not occur as seen in the Wnt5a mutant. Alternatively,
if Wnt7a expression is disrupted, epithelial cells may attempt to
participate in gland formation giving rise to an abnormal multilayered
epithelium that is not permissive for gland formation. Chimeric analyses in
mice has shown that uterine glands are monoclonal in origin
(Lipschutz et al., 1999),
raising the intriguing possibility that the repression of Wnt7a may
occur in a single cell that then gives rise to a gland (see model in
Fig. 9B). We note that
Wnt5a is expressed throughout the mesenchyme, suggesting that an
additional factor may cooperate with Wnt5a to restrict
glandulargenesis at specific sites of the luminal epithelium. Alternatively,
it is possible that Wnt7a repression is a stochastic event that
occurs in a unique cell and that Wnt5a is simply required for
subsequent growth. Experiments to address these models are in progress.
Neither Wnt7a nor Wnt5a has been clearly linked to the
canonical Wnt signaling pathway that requires members of the
lef1/tcf1 family. In the chick limb bud, ß-catenin and
Lef1 retroviral infections induce morphogenetic outcomes similar to
Wnt3a infection and distinct from Wnt7a overexpression
(Kengaku et al., 1998).
Wnt5a has been implicated in Ca2+ signaling and has been
demonstrated to antagonize canonical Wnt signaling
(Miller et al., 1999;
Topol et al., 2003). Our
results demonstrate that Lef1 is dispensable for uterine
morphogenesis and gland formation, suggesting that canonical Wnt signaling is
not required for Wnt signaling in the uterus. We note that Tcf1 is
also expressed in the uterus and may rescue the lack of Lef1 in the
uterus, although it does not do so in many other structures dependent upon
epithelial-mesenchymal interactions previously examined. The early embryonic
lethality of the double Tcf1/Lef1 mutants precludes analyses of FRT
development in the double mutant (Galceran
et al., 1999).
Wnt5a is required for downregulation of Wnt7a and Hoxa genes by estrogenic stimuli
It has been demonstrated that estrogen induction of uterine epithelial
proliferation is dependent upon Esr1 expression in the stroma which
then signals via an unknown ligand to the epithelium
(Cooke et al., 1997;
Kurita et al., 2000). We have
demonstrated previously that DES represses Wnt7a in the neonate FRT
(Miller et al., 1998a) and it
was later demonstrated that Wnt7a repression requires the expression
of Esr1 in the FRT (Couse et al.,
2001). We observe here that downregulation of Wnt7a and
Hoxa10 and Hoxa11 genes by estrogens is abolished in absence
of Wnt5a (Figs 6,
7). However, Wnt5a
mutant uterine grafts undergo an abnormal dilation and show an increase in
epithelial thickness following DES exposure. Based on these results, we
propose that there are Wnt5a dependant and independent responses to
estrogenic stimulation (Fig.
9C). Factors such as Msx1 may be part of a
Wnt-independent regulatory response to estrogen as shown in this
study. Candidate factors that link Wnt5a to estrogenic signaling may
include Wnt7a and Hoxa genes that are misregulated in the
Wnt5a mutant FRT. Wnt7a and Hoxa genes are developmental
factors required for normal morphogenesis of the FRT
(Branford et al., 2000;
Gendron et al., 1997;
Hsieh-Li et al., 1995;
Miller and Sassoon, 1998;
Parr and McMahon, 1998;
Warot et al., 1997), and are
expressed throughout adult life (Benson et
al., 1996; Lim et al.,
1999; Ma et al.,
1998; Miller et al.,
1998b; Pavlova et al.,
1994). Expression of Hoxa10 in the uterus is required for
successful embryo implantation through the regulation of PGE2 receptors
subtypes EP3 and EP4 (Benson et al.,
1996; Lim et al.,
1999). The expression of genes involved in Wnt signaling is
modified during the implantation period
(Kao et al., 2002;
Paria et al., 2001;
Pavlova et al., 1994). We note
that Wnt7a mutant females are sterile although their ovaries are
functional following transplantation into wild-type recipients
(Parr and McMahon, 1998).
Taken together, these data implicate uterine Wnt gene expression as crucial
regulators of uterine adult function.
A system for the analysis of lethal mutant FRTs
We found that wild-type neonate uterine grafts grown in cycling hosts show
highly impaired and delayed gland formation. By contrast, neonate uterine
fragments grown in ovariectomized hosts develop normally and form uterine
glands. We conclude that precocious exposure to endogenous adult levels of
ovarian hormones is sufficient to disrupt crucial perinatal patterning events
in the FRT. Indeed, precocious exposure to DES, 17 ß-estradiol, progestin
or tamoxifen alter FRT morphogenesis and glandulargenesis
(Branham et al., 1985a;
Branham et al., 1985b;
Cunha et al., 1991;
Gray et al., 2001). The
mechanisms underlying how hormonal teratogens permanently alter FRT
development have not been completely elucidated; however, these studies
support a model whereby precocious exposure to estrogens exerts a teratogenic
effect upon the FRT through a perturbation of patterning gene expression in
the FRT and a permanent change in gene regulation in response to hormone
challenge.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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