1 Department of Anatomy, University of California, San Francisco, CA 94143-0452,
USA
2 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
* Author for correspondence (e-mail: kurita{at}itsa.ucsf.edu)
Accepted 15 December 2003
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SUMMARY |
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Key words: Columnar-squamous transformation, Müllerian duct, Endocrine disruptor, Uterus, Estrogen receptor
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Introduction |
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Columnar and squamous epithelia are dramatically different. The major
functions of columnar epithelium are absorption and secretion, while
stratified squamous epithelia form barriers. In addition, cytoskeletal and
cell-adhesion molecules are different in columnar versus squamous epithelia.
For example, cytokeratins 5 and 14 are expressed in squamous epithelial cells,
and are essential to maintain the integrity of squamous epithelium
(Chan et al., 1994;
Ehrlich et al., 1995
;
Hutton et al., 1998
;
Rugg et al., 1994
). It is not
understood how squamous and columnar epithelia differentiate from their
embryonic precursors. The female reproductive tract is an excellent model with
which to study the program of epithelial differentiation because it is lined
with two distinct types of epithelia that differentiate from a common
precursor. The Müllerian vagina, cervix, uterus and oviduct develop from
the embryonic Müllerian duct, which is composed of a uniform layer of
pseudo-stratified columnar epithelial cells. In the mouse, the Müllerian
duct epithelium undergoes organ-specific morphogenetic changes during
postnatal development induced by uterine and vaginal mesenchyme
(Cunha, 1976
;
Kurita et al., 2001a
). In the
uterus, the epithelium gives rise to columnar luminal and glandular epithelia.
In the Müllerian vagina and cervix, the columnar epithelium transforms
into a stratified squamous epithelium. In adulthood, columnar uterine and
squamous cervicovaginal epithelia meet at the squamocolumnar junction (SCJ) in
the cervix. In the mouse, epithelial cells of the Müllerian duct are
fully capable of being induced by heterotypic mesenchyme to undergo uterine or
vaginal differentiation prior to 7 days postnatal, after which this
developmental plasticity is gradually lost. By adulthood, most uterine and
cervicovaginal epithelial cells do not change their phenotype in response to
induction by heterotypic mesenchyme (Cunha,
1976
; Kurita et al.,
2001a
).
Historically, uterine and vaginal epithelial phenotypes have been judged by
histology. However, 17ß-estradiol (E2) and progesterone
(P4) modify epithelial morphology in the uterus and vagina, and
thus the effects of ovarian steroids must be always considered. For example,
uterine epithelium can stratify as a result of hyper-proliferation in response
to E2. In this case, the stratification is reversible, and does not
involve expression of squamous-epithelial markers
(Kurita et al., 2001a). The
uterus of progesterone receptor (PR) knockout mice shows a stratified
epithelial phenotype due to hyperplasia caused by unopposed estrogen action
(Lydon et al., 1995
), which is
due to loss of PR in the stromal cells
(Kurita et al., 1998
). Thus,
epithelial stratification (histology) per se is not the most reliable marker
distinguishing uterine versus cervicovaginal epithelia. Unequivocal
identification of cervicovaginal epithelial differentiation can be achieved by
examination of squamous markers such as K14, which are not modified by steroid
hormones (Kurita et al.,
2001a
). Likewise, uterine epithelial differentiation is best
assessed by estrogen-independent expression of PR, which is a unique feature
of rodent uterine epithelium (Kurita et
al., 2000
). In this study, we used multiple markers to assess
differentiation of uterine and cervicovaginal epithelia.
We report the crucial role of p63 as an identity switch in differentiation
of Müllerian duct epithelium. P63 (KET, p51A, p51B, p40 or p73L) is a
homologue of the p53 tumor suppressor gene
(Yang et al., 1998).
p63/ mice have skin defects and lack organs
arising from epidermis such as mammary and salivary glands
(Mills et al., 1999
;
Yang et al., 1999
). As
development of uterus, cervix and vagina occurs mostly during postnatal
stages, the phenotype of p63/ mice in the
mature female reproductive tract is unknown because of newborn lethality.
Through rescue of p63/ cervicovaginal
rudiments by grafting, we have shown that cervicovaginal epithelium of
p63/ mice expresses the full spectrum of
uterine epithelial markers. In this study, we describe the ontogeny of p63 in
the mouse female reproductive tract and demonstrate a key role for p63 in
DES-induced cervicovaginal adenosis
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Materials and methods |
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ER/ mice on a C57BL6J/129Svj mixed
genetic background were produced and genotyped as described previously
(Lubahn et al., 1993
).
For the UtE/VgM recombination experiment with
ER/ mice, hosts were ovariectomized
at the time of grafting and 25 mg DES Silastic-capsules were implanted into
half of the hosts. The capsules were removed 2 weeks after the grafting, and
tissues were harvested 2 weeks after removal of the DES-capsule.
The detailed protocol for tissue recombination has been described
previously (Kurita et al.,
2001a). Results were based upon analysis of 8-11 tissue
recombinants/group from at least three independent experiments.
Primary culture of uterine epithelial cells
The methods for epithelial separation and primary culture have been
described previously (Kurita et al.,
2000). Briefly, epithelial cells were embedded in a 1:1 mixture of
growth factor reduced Matrigel (BD Bioscience, Franklin Lakes, NJ) and rat
tail collagen, and cultured in a 1:1 mixture of DME and Ham's F-12 media
(Gibco, Gaithersburg, NY) with transferrin (5 µg/ml) (Sigma) and insulin
(10 µg/ml) (Gibco).
Immunohistochemistry (IHC)
Methods for IHC have been described
(Kurita et al., 1998). Mouse
monoclonal antibodies were used at the following concentrations:
anti-ER
1D5 (1:50), anti-cytokeratin 10 (1:25, DAKO, Carpenteria, CA),
anti-p63 4A4 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA), anti-p63 Ab2
(1:100, Lab Vision, Fremont, CA), anti-Ki67 (1:100, Novacastra Laboratories,
Burlingame, CA), anti-K8 LE41 (1:5) and anti-K14 LE001 (1:5, gift from Dr E.
B. Lane, University of Dundee, Dundee, UK). Rabbit and goat polyclonal
antibodies were used at the following concentrations: anti-P-cadherin goat
(1:100) and anti-p130 rabbit (1:200) (Santa Cruz Biotechnology), anti-PR
rabbit (1:100, DAKO) and anti-involucrin rabbit (1:2000, Covance, Princeton,
NJ). Anti-K19 rabbit monoclonal antibody (1:1) was obtained from Dr Robert
Pytela, UCSF, San Francisco, CA. Positive signals were visualized as brown
precipitates utilizing 3,3'-diaminobenzidine tetra-hydrochloride
(Sigma).
P63-positive epithelial index
To determine the p63-positive epithelial index, images of p63-IHC were
captured with a DC330 camera (Dage-MTI, Michigan City, IN), interfaced with a
computer. Lengths of basement membrane were measured on the images with Scion
Image 1.62a (Scion, Frederick, MD), and the length of basement membrane
associating with p63-positive cells against the length of total basement
membrane was calculated. Approximately 2000-14,000 µm of basement membrane
was analyzed for each tissue recombinant.
Neonatal DES-treatment
Neonatal CD-1, BALB/c and C57B6 mice and neonatal mice from
ER+/ breeding cages were treated with DES by
the following protocol previously described
(McLachlan et al., 1980
).
Briefly, newborn mice were injected subcutaneously with 2 µg DES in 20
µl corn oil or 20 µl corn oil from day 1. The neonatal mice received DES
or oil every 24 hours for 5 or 7 days. mice were sacrificed at P1, P2, P3, P4,
P5, P7, P10, P14, P21, P35 and P60. mice to be harvested at P60 were
ovariectomized at P35 and treated with 125 ng E2 in 100 µl corn
oil or 100 µl corn oil alone everyday for 3 days before termination.
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Results |
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Although E2 and/or P4 regulate histodifferentiation and functional cytodifferentiation of UtE and CVE in adulthood, E2 and/or P4 did not modify the adult expression pattern of p63 in the female reproductive tract. Therefore, p63 was always expressed in CVE but not in UtE (Fig. 1L-N). E2-treatment stimulated proliferation and caused stratification of UtE (Fig. 1N, blue arrows), but the uterine epithelial cells remained negative for p63 and K14 (not shown), which were always expressed in adult CVE.
Expression of p63 in the Müllerian duct epithelium is induced by vaginal mesenchyme
Tissue recombinants were constructed with newborn uterine (UtM) or
Müllerian-vaginal mesenchyme (VgM) plus UtE or Müllerian-vaginal
epithelium (VgE) derived from either P1 neonatal or adult BALB/c mice. When
VgE from neonatal mice was recombined with UtM (VgE+UtM), the epithelial
tissue developed a columnar uterine phenotype as previously reported
(Cunha, 1976;
Kurita et al., 2001a
)
(Fig. 2A). Even though a
substantial portion of Müllerian-vaginal epithelial cells expressed p63
at the time of tissue recombination (Fig.
1E), the entire epithelium became negative for p63 in VgE+UtM
tissue recombinants after 1 month of growth
(Fig. 2A). Thus, UtM turned-off
p63 expression in VgE. When neonatal-UtE was recombined with VgM (UtE+VgM), a
squamous vaginal epithelium developed that was positive for p63
(Fig. 2B). Homotypic
recombinants (UtE+UtM and VgE+VgM) behaved as expected
(Fig. 2C,D). Thus, based upon
p63 ontogeny and tissue recombination studies, p63 expression is induced in
Müllerian duct epithelial cells by VgM during embryonic-neonatal
development.
|
Phenotype of p63/ mice
The rudiments of uterus, cervix and Müllerian vagina were rescued from
p63/, wild-type (p63+/+)
and heterozygous (p63+/) embryos (E16-18) by
grafting. Uterine and cervicovaginal phenotypes of p63+/+
and p63+/ mice were indistinguishable by all
criteria examined. Thus, both p63+/+ and
p63+/ progeny were referred as
p63+. In uterine grafts of
p63/ mice, the epithelium appeared
completely normal in morphology and expression of differentiation markers
(Table 1). In cervicovaginal
and Müllerian-vaginal grafts from p63/
mice, the epithelium failed to differentiate into squamous and instead formed
a uterine-like columnar epithelium (Fig.
3). Moreover, in the
p63/cervicovaginal grafts deep epithelial
invaginations or glands were always observed
(Fig. 3A,B, black arrows). The
glandular epithelium in the
p63/cervicovaginal grafts became
hyperplastic and multilayered in response to E2-treatment
(Fig. 3B). However, markers for
squamous differentiation (Fig.
3C-F) or keratinization (Fig.
3G-J) were never detected in
p63/ CVE
(Table I). Although negative
for cervicovaginal differentiation markers,
p63/ CVE expressed markers common to both
uterine and cervicovaginal epithelia such as ER (Esr1 Mouse
Genome Informatics) and p130 (Fig.
3K-N, Table 1).
Most importantly, p63/ CVE expressed a high level of
PR in the absence of E2 [Fig.
3, compare p63+ (O) and
p63/ (P)], which is a feature unique to
rodent UtE. Unlike normal UtE, which downregulates PR in response to
E2, the epithelium of p63/
cervicovaginal grafts remained strongly positive for PR when treated with
E2 (Fig. 3R).
However, this pattern of PR expression is actually identical to that of
adult-UtE + VgM tissue recombinants (Table
1). It is known that PR in UtE is downregulated by an action of
uterine stroma induced by E2
(Kurita et al., 2000
), and the
stromal activity to downregulate PR in UtE is specific for uterine stroma.
Therefore, when adult-UtE is recombined with VgM, the adult-UtE retained the
original uterine phenotype and expressed high levels of PR in the absence of
E2, but the UtE also strongly expressed PR in the presence of
E2 (Kurita et al.,
2001a
). In conclusion, the profile of differentiation markers for
p63/ CVE was identical to that of normal UtE
for the all criteria examined (Table
1). Hence, in the absence of p63, Müllerian CVE
differentiated into UtE.
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|
Uterine mesenchyme is required to induce uterine epithelial differentiation
As CVE differentiated into UtE in the absence of p63, it appeared that the
uterine phenotype might be the default differentiation endpoint for embryonic
Müllerian duct epithelium, suggesting that induction by UtM may not be
required for differentiation of UtE. Whether embryonic UtE requires
instructive induction from UtM to establish the proper uterine marker
expression pattern was tested in vitro and in vivo.
At E18, UtE did not express PR (Fig.
4A) or ER (Fig.
4B). When undifferentiated UtE from E18 embryos (E18-UtE) was
cultured in an extracellular matrix for 7 days, only a small subset of the
cultured epithelial cells expressed PR
(Fig. 4E), which was expressed
in the entire UtE in situ by P5 (Fig.
4C). In addition, whereas ER
was undetectable in UtE in
situ at P5 (Fig. 4D), ER
was expressed in a substantial proportion of cultured E18-UtE cultured to the
age equivalent of P5 (Fig. 4F).
The comparison of PR and ER
expression in vivo versus in vitro
demonstrates that the gene expression pattern of developing UtE is strongly
affected by presence or absence of UtM (compare
Fig. 4C,D with E,F). By
contrast, when adult (2-month-old) UtE were cultured under the same conditions
as the E18-UtE, the adult-UtE retained its original expression pattern of PR
and ER
(Fig. 5G,H).
Thus, the absence of PR expression in the cultured E18-UtE was not due to the
culture conditions.
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P63 expression in DES-induced epithelial lesions in uterus, cervix and vagina
Cervicovaginal adenosis consists of focal areas of columnar surface
epithelium or simple columnar submucosal glands in the cervix or vagina, which
are normally lined with squamous epithelium
(Robboy et al., 1986). In the
cervix and Müllerian vagina of neonatally DES-treated mice cervicovaginal
adenosis appeared as deep-inclusions or glands with openings into the lumen of
cervicovaginal canal (Fig.
5A-D), or simple columnar surface epithelium within the vaginal or
cervical canal (Fig. 5E-G). In
DES-induced cervicovaginal adenosis, squamous differentiation markers were not
expressed (Table 1). Instead,
the columnar adenosis lesions expressed high levels of PR in the absence of
E2 (Fig. 5C,G) and
other markers indicative of UtE. Indeed, cervicovaginal adenosis expressed the
full spectrum of uterine markers (Table
I).
In DES-induced uterine SQM of the mouse, the metaplastic foci
(Fig. 5H-J, red arrows)
expressed p63, K14 (Fig. 5H,I),
K19, P-cadherin and involucrin (Table
1). In the metaplastic squamous epithelium, PR was expressed in
the presence of E2 (Table
1), but was undetectable in the absence of E2
(Fig. 4J, red arrows). Taken
together, all of these markers of uterine SQM are identical to that of CVE. In
both cervicovaginal adenosis and uterine squamous lesions, the epithelium
expressed both ER (Fig.
5D) and p130 (Table
1). Thus, based upon the morphology and the expression of all
markers tested (Table 1),
cervicovaginal adenosis is not persistent undifferentiated Müllerian duct
epithelium, but the development of UtE in the cervix and vagina. Likewise,
uterine SQM appears to be the development of CVE in the uterus. Although
uterine SQM was always associated with expression of p63, whether p63 was
essential for the development of uterine SQM was not clear. To answer the
question, uterus from E17.5 p63/ and p63+
embryos were grafted into untreated or DES-treated nude mice. Expression of
squamous differentiation markers was examined 2 weeks after the grafting. In
the absence of DES, no epithelial cells expressed squamous markers in the
p63/ and p63+ uterine
grafts. With DES-treatment, p63- (Fig.
5K) and K14- (Fig.
5L)-positive basal epithelial cells were detected in four out of
12 p63+ uterine grafts, but these markers were not detected in 16
p63/ uterine grafts
(Fig. 5M). In the presence of
DES, p63/ uterine grafts showed
stratification of epithelium (Fig.
5M, black arrow), but the stratified epithelial cells were
negative for K14 and involucrin (not shown). Therefore, p63 is not just a
marker but is essential for development of uterine SQM.
P63 expression in the development of cervicovaginal adenosis
The ontogeny of p63 expression was examined during treatment of neonatal
mice with DES. In 5-day-old oil-treated mice (control), a continuous layer of
p63-positive basal epithelial cells extended from the Müllerian vagina
into the cervical canal (Fig.
6A). However, when neonatal wild-type mice were treated with DES
from P1 through P5, induction of p63 was greatly inhibited in the upper part
of Müllerian vagina and the cervix. At P5, this was manifest as large
gaps in the p63-positive basal epithelial layer in the fornix and common
cervical canal (Fig. 6B, green
arrows), which are the primary site for development of cervicovaginal adenosis
(Forsberg and Kalland, 1981).
Two days after the last DES-injection (P7)
(Fig. 6D), most of the gaps in
the p63-positive layer in the CVE were filled-in with p63-positive cells.
However, small patches of p63-negative epithelial cells in the fornix and
common cervical canal (Fig.
6D,F, green arrows) persisted into adulthood as adenosis
(Fig. 6G).
|
DES disrupts p63 expression in CVE via ER in the epithelial cells
ER+/+, ER
+/
and ER
/ mice were treated with DES
from P1 to P5. Confirming the results described above, DES disrupted p63
expression in CVE in ER
+/+ and
ER
+/ mice. By contrast, the p63-positive
basal layer of cervicovaginal epithelial cells developed normally in
DES-treated ER
/ mice
(Fig. 6C), which was identical
to that of the oil-treated ER
+/+ control
(Fig. 6A). Therefore, DES
action on p63 in developing CVE requires signaling through ER
.
In the cervix and Müllerian vagina, ER is highly expressed in
both epithelial and mesenchymal cells by P3
(Kurita et al., 2001a
).
Therefore, DES action may be mediated by ER
in either epithelial and/or
mesenchymal cells. To test whether DES disrupts induction of p63 via ER
in epithelial or mesenchymal cells, the four types of tissue recombinants were
constructed with UtE and VgM from ER
+/+ and
ER
/ mice
(ER
+/+
UtE+ER
+/+ VgM,
ER
+/+
UtE+ER
/ VgM,
ER
/
UtE+ER
+/+ VgM and
ER
/
UtE+ER
/ VgM) and grafted into
ovariectomized female nude mice. In untreated hosts, a p63-positive squamous
basal epithelial layer formed in all four types of UtE+VgM tissue recombinants
(Fig. 7A, parts a and b, Fig.
7B) indicative of normal cervicovaginal epithelial
differentiation. The basal cells were also strongly positive for K14 (not
shown). When hosts were treated with DES, squamous basal cells were not
detected in the UtE+VgM tissue recombinants prepared with
ER
+/+ UtE (ER
+/+
UtE+ER
+/+ VgM and ER
+/+
UtE+ER
/ VgM) as assessed by histology
and K14 expression (not shown), indicating that DES had inhibited
cervicovaginal differentiation (Fig. 7A,
parts c and e). By contrast, when
ER
/ UtE were used to construct the
tissue recombinants (ER
/
UtE+ER
+/+ VgM and
ER
/
UtE+ER
/ VgM tissue recombinants), a
p63-positive squamous basal epithelial layer was induced even when the hosts
were treated with DES (Fig. 7A, parts d and
f, red arrows). These results clearly demonstrate that DES acts
via ER
in the epithelial cells to inhibit induction of p63 in CVE. DES
action via mesenchymal ER
does not inhibit p63 expression and squamous
differentiation of CVE.
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Discussion |
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This process seems to be a general principle among many organs. Besides female reproductive tract, we have studied the ontogeny of p63 in other developing mouse organs. For all organs examined, expression of p63 precedes the expression of squamous markers and morphological changes (90° nuclear polarity change) by several days. Squamous differentiation appears to consist of at least three steps: induction of p63, stabilization of p63 and subsequent squamous differentiation. As proposed above, expression of p63 itself does not transform columnar epithelium into squamous immediately, but p63 expression appears to be an essential step for squamous differentiation in general, because epithelial cells in the forestomach and seminal vesicle of p63/ mice also failed to undergo squamous differentiation (T.K., unpublished).
Role of p63and epithelial-mesenchymal tissue interaction in normal and abnormal Müllerian duct development
Normal development (Fig. 8B, part a)
In embryo, the Müllerian duct is composed of uniform, undifferentiated
columnar epithelial cells (i.e. stage 1 in
Fig. 8A). UtM and CVM already
have acquired their organ-specific inductive identities. Therefore, squamous
epithelial differentiation inducing signals (red arrows) are expressed only in
the CVM, whereas common uterine epithelial differentiation inducing signals
(blue arrows) are expressed throughout the cervicovaginal and uterine regions
of the Müllerian duct. In response to induction by CVM (red arrows),
epithelial cells in the cervicovaginal area express p63 (P1-5). Subsequently,
the p63-positive epithelial cells differentiate into squamous CVE as
instructed by CVM. Because the signals inducing squamous differentiation are
expressed only in CVM, epithelium in the uterus never expresses p63 and
differentiates into columnar UtE, which requires mesenchymal inductive
activity (blue arrows). By P5, the adult expression pattern of p63 is
established in CVE and UtE, but the status of p63 and thus the developmental
fate of the epithelial cells can still be altered by heterotypic mesenchyme
(stage 2 in Fig. 8A). By P7, a
substantial percentage of uterine and vaginal epithelial cells are insensitive
to heterotypic uterine or cervicovaginal mesenchymal induction
(Cunha, 1976), and the uterine
and cervicovaginal epithelial phenotypes become irreversibly determined in the
first few weeks of postnatal development. Accordingly, in adulthood almost all
uterine and cervicovaginal epithelial cells cannot be re-programmed by uterine
or vaginal mesenchyme to express an alternative epithelial phenotype (stage 3
in Fig. 8A).
P63/ mice (Fig. 8B, part b)
In the p63/ mice, mesenchymal activity to
induce uterine (blue arrows) and cervicovaginal (red) epithelial
differentiation is normal. However, as the Müllerian duct epithelial
cells cannot express p63, the entire Müllerian duct epithelium
differentiates into UtE both in the uterine and cervicovaginal anlagen.
Neonatal DES treatment (Fig. 8B, part c)
DES binds to ER in the Müllerian duct epithelial cells and
blocks expression of p63, which is a prerequisite for subsequent squamous
differentiation. As a result, p63 is induced in only a subpopulation of
cervicovaginal epithelial cells, even though the mesenchymal signals to induce
squamous epithelial differentiation (red arrow) are not affected by DES. Like
most developmental processes, the timing of mesenchymal specification of
uterine and cervicovaginal epithelial differentiation is tightly regulated.
When DES treatment is stopped at P5, most epithelial cells in cervix and
vagina respond to the mesenchymal signals (red arrows) and express p63 by P7.
However, at least some Müllerian duct epithelial cells have lost
sensitivity to the mesenchymal signals, as epithelial cell fate is already
irreversibly fixed in some epithelial cells by this stage. These p63-negative
epithelial cells in cervix and vagina have missed the time window to express
p63, and thus, remain simple columnar and differentiate into UtE, which
persists into adulthood as cervicovaginal adenosis. The tissue recombinant
studies using ER
+/+ and
ER
/ UtE and VgM demonstrate that DES
inhibits expression of p63 in Müllerian duct epithelial cells through
epithelial ER
. Because DES elicits abnormal epithelial differentiation
via epithelial ER
, epithelial (and not mesenchymal/stromal) regulatory
molecules are clearly the immediate targets of DES action in inducing
adenosis. However, our model does not exclude a subordinate role of
mesenchymal genes such as homeobox genes in the development of adenosis.
The correct and full expression of uterine epithelial markers in the
p63/ CVE implies the expression of uterine
inductivity in the CVM as well as in the UtM. However, the molecular signals
comprising uterine inductive activity may not be identical in CVM versus UtM.
Many developmental regulatory genes have been detected only in the uterine
regions of Müllerian duct, e.g. Wnt and Hox genes
(Ma et al., 1998;
Miller et al., 1998b
;
Pavlova et al., 1994
). Our
data do not exclude involvement of these molecules in normal uterine
epithelial development. However, it is unlikely that a single molecule
regulates all aspects of uterine epithelial differentiation, and instead a
balance of several genes may be important for induction of normal UtE. CVM
appears to express a reasonably complete combination of signals for inducing
uterine epithelial differentiation based upon analysis of
p63/ cervix and Müllerian vagina. The
squamous differentiation inducing activity in CVM is also likely to be a
combination of multiple factors.
Wnt7a has been suggested as an immediate target of DES in development of
vaginal adenosis because Wnt7a was downregulated by DES in the Müllerian
duct, and the Wnt7a/ mouse developed
`vaginal adenosis' (Miller et al.,
1998a). However, in the paper by Miller et al., the term
`adenosis' was used to describe `epithelial inclusions'. The glands in the
Wnt7a/ mouse appeared to be lined by
keratinized squamous epithelium, which is not strictly adenosis. Such glands
seem to be absent in younger Wnt7a/ mice
(more than 4 months old), which is also not in agreement with the phenotype of
neonatally DES-treated mice. Therefore, it is not clear if downregulation of
Wnt7a plays a role in development of columnar epithelium in cervix/vagina.
Wnt7a may be upstream of p63, or the glands observed in
Wnt7a/ mice may have possibly developed via
a different mechanism from DES-induced adenosis.
In DES-daughters, cervical/vaginal clear cell adenocarcinoma is rare, even
though cervical/vaginal adenosis is commonly found. Although adenosis is
thought to be the substrate from which clear cell adenocarcinoma develops, the
mechanism by which adenosis develops into adenocarcinoma is unclear. We have
shown that DES actions on CVM do not play a role in formation of adenosis.
However, growth, cell death and differentiation of epithelial tissue in the
female reproductive tract are regulated by stromal cells during embryogenesis
as well as adult period (Buchanan et al.,
1998; Cooke et al.,
1997
; Kurita et al.,
2001b
; Kurita et al.,
1998
). Thus, it is likely that the DES-caused changes in the
stromal cells also play important roles in the subsequent development of
cervicovaginal adenocarcinoma.
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ACKNOWLEDGMENTS |
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