|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 30 April 2008
doi: 10.1242/dev.020099
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Comparative Biology and Experimental Medicine, Faculty of
Veterinary Medicine, The University of Calgary, 3330 Hospital Drive NW,
Calgary, AB, T2N 4N1, Canada.
2 Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Str. 10, 13092
Berlin, Germany.
Author for correspondence (e-mail:
jcross{at}ucalgary.ca)
Accepted 31 March 2008
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Chorion, Gcm1, Labyrinth, Placenta, Syncytin, Syncytiotrophoblast, Trophoblast, Mouse
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
), a flat plate of trophoblast cells that otherwise contains
multipotent trophoblast stem (TS) cells
(Uy et al., 2002). The
occlusion of the ectoplacental cavity around E8.0 then brings cells from the
bottom of the ectoplacental cone in contact with the distal side of the
chorion, which results in the downregulation of the TS cell markers Cdx2,
Eomes and Esrrb within the chorionic trophoblast
(Beck et al., 1995;
Hancock et al., 1999;
Pettersson et al., 1996) and a
loss of TS cell potential from chorionic ectoderm
(Uy et al., 2002). Shortly
after ectoplacental cavity occlusion, the allantois, which grows out from the
posterior end of the embryo, makes contact with and attaches to the basal side
of the chorion (reviewed by Hemberger and
Cross, 2001; Rossant and
Cross, 2001). Thereafter, folding of the chorion into simple
branches initiates at sites of Gcm1 expression
(Anson-Cartwright et al.,
2000). Allantoic mesoderm with its associated blood vessels fills
into the epithelial branches (Cross,
2000). Gcm1 expression is subsequently confined to the
tips of elongating branches (Cross et al.,
2006), the site where syncytiotrophoblast differentiation is
thought to begin (Hernandez-Verdun,
1974). Continued branching morphogenesis ultimately produces a
complex villous structure providing the large surface area that is required
for nutrient exchange to support fetal growth past E10.5. The loss of
Gcm1 results in failure to initiate branching morphogenesis following
chorioallantoic attachment
(Anson-Cartwright et al., 2000;
Schreiber et al., 2000).
The maternal and fetal blood spaces within the labyrinth are separated by
three layers of trophoblast cells (tri-chorial) and by a layer of fetal
endothelial cells (Enders,
1965; Hernandez-Verdun,
1974). The trilaminar trophoblast includes a single layer of
mononuclear sinusoidal trophoblast giant cells (S-TGCs) that line the maternal
blood sinusoids (Coan et al.,
2005; Simmons and Cross,
2005; Simmons et al.,
2007), and two layers of syncytiotrophoblast, SynT-I and -II, the
latter of which is in contact with fetal endothelial cells
(Simmons and Cross, 2005;
Watson and Cross, 2005). The
S-TGCs are secretory in nature, expressing hormones such as placental lactogen
II (Campbell et al., 1989;
Dai et al., 2000;
Deb et al., 1991;
Ishida et al., 2004;
Lee et al., 2003;
Sahgal et al., 2000;
Simmons and Cross, 2005;
Simmons et al., 2007), and are
therefore likely to have a primary endocrine function. S-TGCs are loosely
attached to the underlying syncytial layers via desmosomal adhesions and
contain fenestrations to allow the SynT-I cells direct access to maternal
blood (Coan et al., 2005;
Davies and Glasser, 1968;
Hernandez-Verdun, 1974).
cathepsin Q (Ctsq) has recently been shown to be an exclusive marker
of these cells in the mature labyrinth
(Simmons et al., 2007). The
syncytiotrophoblast cell layers are multinucleated, forming as a result of
trophoblast cell-cell fusion, are very thin and function in nutrient transport
(Enders, 1965;
Hernandez-Verdun, 1974;
Jollie, 1964;
Snell and Stevens, 1966). The
two syncytiotrophoblast layers are tightly adhered to one another through
tight junctions, are clearly different in their cellular composition as
observed by electron microscopy, and are situated on basement membranes
overlying the fetal capillary endothelium
(Coan et al., 2005;
Davies and Glasser, 1968;
Enders, 1965;
Hernandez-Verdun, 1974;
Jollie, 1964). In addition to
the trophoblast cells separating the maternal and fetal blood spaces,
histological examination of the mid-gestation labyrinth also reveals abundant
`pillars' of spongiotrophoblast that extend inwards, as well as clusters of
tightly packed cuboidal cells that resemble the morphology of chorion
trophoblast cells at earlier stages. These latter cells are hypothesized to be
labyrinth progenitors, but there is little data to directly support this
idea.
Mouse mutants have provided significant insights into the details of
placental formation, especially into the mechanisms of chorioallantoic
attachment (reviewed by Watson and Cross,
2005) and initiation of branching morphogenesis and
syncytiotrophoblast differentiation [Gcm1 mutants
(Anson-Cartwright et al., 2000;
Schreiber et al., 2000)].
However, little molecular detail is known about how the trilaminar trophoblast
structure is formed, despite a large number of mutants that manifest labyrinth
phenotypes. A major problem to date has been the difficulty in discerning the
three trophoblast cell layers at the light microscopy level and the lack of
markers that distinguish them. We have previously observed that Gcm1
is not uniformly expressed in all syncytiotrophoblast cells at E14.5, and
expression appears to be closer to fetal blood spaces than to maternal blood
spaces, suggesting localization to the SynT-II cells
(Cross et al., 2006). In this
study, we expanded the search for layer-specific markers and used them as
tools to define the developmental origins of the three differentiated
trophoblast cell layers in the mature labyrinth. Based on our findings, we
propose a model for how early patterning and cell specification within the
flat chorion lay down the progenitors for the trilaminar trophoblast structure
of the labyrinth.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
)
on a CD1 background were crossed to obtain mutant Gcm1-/-
conceptuses. Cebpa+/-;Cebpb+/- double
heterozygotes (Begay et al.,
2004) were crossed to obtain Cebpa-/-,
Cebpb-/- and
Cebpa-/-;Cebpb-/- double-knockout
embryos. Implantation sites were dissected at E6.5 through E14.5, with noon of
the day of the vaginal plug defined as E0.5. Genotyping was performed as
previously described. Animals were housed under normal light conditions (12
hours light/12 hours dark) with free access to food and water. All animal
procedures were carried out in accordance with the University of Calgary
Animal Care Committee. For Cebpa;Cebpb mutant mice, all
procedures were carried out in accordance with Max Delbrueck Center for
Molecular Medicine animal facility standards.
Histology
For histology, whole uteri (E6.5-7.5), isolated implantation sites
(E8.5-10.5), or whole dissected placentas (E14.5) were fixed overnight at
4°C in 4% paraformaldehyde (PFA), processed through a sucrose gradient and
embedded in OCT compound (Sakura Finetek, Torrence, CA) for preparation of
frozen sections. For ultrathin resin histology, implantation sites were fixed
overnight in 4% PFA/0.2% glutaraldehyde and embedded in JB-4 epoxy resin
according to the manufacturer's instructions (Electron Microscopy Sciences,
Hatfield, PA). Sections (2 µm) were then cut using glass knives on a Leica
RM2265 microtome and stained with Tissue Epoxy Stain (Electron Microscopy
Sciences) according to the manufacturer's instructions.
Probes and plasmids
The cDNA probe for Gcm1 has been described
(Basyuk et al., 1999). The
following cDNAs were generously provided: Esx1 (Dr Richard Behringer,
University of Texas, M. D. Anderson Cancer Center, Houston, TX), Dlx3
(Dr Kathleen Mahon, Baylor College of Medicine, Houston, TX) and
Nr6a1 (Dr Austin Cooney, Baylor College of Medicine, Houston, TX).
cDNA probes for Cebpa, Syna and Synb were generated by
RT-PCR using the following primers: Cebpa forward,
5'-CGCTGGTGATCAAACAAGAG-3' and reverse,
5'-GTCACTGGTCAACTCCAGCA-3'; Syna forward,
5'-TTGCAATCACACCTTTCAGC-3' and reverse,
5'-TGGTGTCCACAGACAGGGTA-3'; Synb forward,
5'-CTTTCCACCACCCATACGTT-3' and reverse,
5'-TGACCTTGAAGTGGGTAGGG-3'. Amplicons were cloned into pGEM-T easy
(Promega, Madison, WI) and verified by sequencing.
In situ hybridization
Frozen sections (8-10 µm) were adhered to Super Frost Plus (VWR
International, West Chester, PA) slides and stored at -80°C until used. In
situ hybridization was carried out as described
(Simmons et al., 2007) with
some modifications. Briefly, digoxigenin (DIG) and fluorescein cRNA probes
were generated from plasmids according to the manufacturer's instructions
(Roche, Laval, Quebec, Canada). Sections were rehydrated in PBS, post-fixed in
4% PFA, treated with proteinase K (15 µg/ml for 5 minutes at room
temperature), acetylated for 10 minutes (acetic anhydride, 0.25%; Sigma
Aldrich, Oakville, Ontario, Canada) and hybridized with DIG-labeled probes
overnight at 65°C (for double ISH, fluorescein-labeled probes were also
added at this stage). Hybridization buffer contained 1x salts (200 mM
NaCl, 13 mM Tris, 5 mM sodium phosphate monobasic, 5 mM sodium phosphate
dibasic, 5 mM EDTA), 50% formamide, 10% (w/v) dextran sulfate, 1 mg/ml yeast
tRNA (Sigma Aldrich), 1x Denhardt's [1% (w/v) bovine serum albumin, 1%
(w/v) Ficoll, 1% (w/v) polyvinylpyrrolidone], and cRNA probe (final dilution
of 1:2000 from reaction with 1 µg template DNA). Post-hybridization washes
were followed by an RNase treatment [400 mM NaCl, 10 mM Tris (pH 7.5), 5 mM
EDTA, 20 µg/ml RNase A]. After blocking, sections were incubated overnight
in blocking solution containing anti-DIG antibody (Sigma Aldrich) at 1:2500
dilution. Color was developed using NBT/BCIP according to the manufacturer's
instructions (Promega). For double in situ hybridizations, the anti-DIG
antibody conjugated to alkaline phosphatase was inactivated at 65°C in
maleic acid buffer for 30 minutes, followed by 30 minutes in 0.1 M glycine (pH
2.2). Sections were blocked again for 1 hour and incubated overnight with
anti-fluorescein antibody (1:2500; Roche) at 4°C. After washing, color was
developed using INT/BCIP (Roche) until a brown precipitate was visible. In
some cases, slides were counterstained with Nuclear Fast Red. For single
NBT/BCIP in situ hybridization, slides were dehydrated and cleared in xylene
and mounted in Cytoseal Mounting Medium (VWR International, West Chester, PA).
For in situ hybridizations containing brown INT/BCIP precipitate (xylene and
alcohol soluble), sections were mounted first under Crystal Mount Aqueous
Mounting Medium (Sigma Aldrich) to form a barrier, then coverslipped using the
xylene-based Cytoseal Mounting Medium. Photographs were taken promptly before
fading of the INT/BCIP precipitate occurred.
Trophoblast stem cell cultures
Wild-type trophoblast stem (TS) cells (Rs26 line) were provided by Dr J.
Rossant (Tanaka et al., 1998).
Gcm1-/- and wild-type TS cell lines were derived from
blastocysts isolated from Gcm1+/- intercrosses as
previously described (Tanaka et al.,
1998). TS cells were cultured and processed as described
(Simmons et al., 2007).
Northern blot analysis
Total RNA from TS cell cultures was isolated using QIAshredder and RNeasy
columns (Qiagen, Mississauga, Ontario, Canada) following the manufacturer's
instructions. Ten µg of total RNA was separated on a 1.1% formaldehyde
agarose gel, blotted onto GeneScreen nylon membrane (Perkin Elmer, Shelton,
CT) and UV cross-linked. Random-primed DNA labeling of cDNA probes was carried
out with 25 µCi [32P]dCTP and probes were isolated on Sephadex
G-50 columns (Amersham Biosciences, Baie d'Urfe, Quebec, Canada).
Hybridizations were performed at 60°C overnight in hybridization buffer as
described (Church and Gilbert,
1984). Following post-hybridization washes, signals were detected
by exposure to BioMax MR film (Kodak, New Haven, CT) at -80°C.
|
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). In order
to identify other markers that might distinguish the three differentiated
trophoblast cell types in the labyrinth, we investigated the expression of
genes encoding Dlx3 (Dlx3), Esx1 (Esx1), Gcnf
(Nr6a1), C/EBP
(Cebpa) and the murine endogenous
retrovirus envelope proteins syncytin A (Syna) and syncytin B
(Synb), all of which have all been used as markers for labyrinth
trophoblast (Fig. 1).
Expression of both Dlx3 and Nr6a1 was predominantly detected
within the ectoplacental cone at E7.5, broadly in the chorion at E8.5,
although signal was somewhat stronger in the upper chorion trophoblast cells,
and thereafter was broadly in the labyrinth without indication of
layer-specificity (Fig. 1).
Esx1 was detectable within the chorion at E7.5 and the basal chorion
at E8.5, but then more broadly (Fig.
1). Cebpb was detectable in both ectoplacental cone and
chorion cells as well as within the decidua at E7.5, throughout the chorion at
E8.5 and more broadly throughout trophoblast cell subtypes, such as
spongiotrophoblast and labyrinth trophoblast layers, later in gestation (E12.5
and thereafter). Within the developing labyrinth, Cebpb expression
was similar to that of Dlx3, Nr6a1 and Esx1
(Fig. 1). In contrast to these
genes, expression of Syna, Synb and Cebpa was not detectable
at E7.5. However, their expression appeared starting by 
E8.5. We detected
both Cebpa and Synb expression in a pattern similar to
Gcm1, confined to clusters of trophoblast cells at the
chorioallantoic interface and in some cases in cells at the initial branch
points, suggesting they are expressed in the same cells as Gcm1
(Fig. 1). By E9.0 and 14.5,
Gcm1, Cebpa and Synb were detected in elongated trophoblast
cells adjacent to the fetal endothelial cells, consistent with SynT-II
expression. By contrast, Syna was detectable at E8.5 in cells at the
apical side of the chorion, closer to the maternal blood sinusoids and by
E14.5 in cells closer to the maternal blood sinusoids and yet not in S-TGCs
(Fig. 1).
|
), to ascertain whether unstained areas indicative of SynT-I
cells could be detected between the positive signals for these two genes.
Indeed, a clear unstained cell layer was observed between
Ctsq+ S-TGCs and Synb+ SynT-II cells
(Fig. 2C, black arrow), in the
position of cells that express Syna
(Fig. 2B, brown arrow).
The E8.5 chorion shows patterns of Syna-, Gcm1/Synb/Cebpa- and Hand1-expressing cells
In addition to the exclusive patterns at E14.5, Syna and
Gcm1/Synb/Cebpa expression patterns were also
non-overlapping at earlier stages of development
(Fig. 2D,E and data not shown).
Gcm1, Synb and Cebpa expression was co-localized in clusters
of cells at the basal side of the chorion
(Fig. 3A) and in SynT-II cells
of the labyrinth (Fig. 1,
Fig. 2B-E and data not shown).
By contrast, Syna expression was detected initially in cells at the
top of the chorion overlying
Gcm1/Cebpa/Synb+ cells and was later
restricted to SynT-I cells (Fig.
1, Fig. 2B-E).
Cells lining the maternal sinusoids, which appear directly above the chorion
at E8.5, are lined by cells that presumably become the S-TGC layer. Although
S-TGCs express Ctsq (Simmons et
al., 2007), this marker is restricted to S-TGCs of the mature
labyrinth (after E12.5). S-TGCs in the mature labyrinth express Hand1
(Simmons et al., 2007) and we
found that trophoblast cells facing the maternal sinusoids at E8.5 also
express Hand1 (Fig.
3A). Hand1 is expressed in several different trophoblast
cell subtypes including all TGC subtypes, ectoplacental cone and both apical
and basal cells in the chorion. The apical chorion cells were Pl1
(Prl3d1)-negative, indicating that they are not simply parietal TGCs,
but are more likely to be early S-TGCs. Importantly, Syna+
cells were almost always separated from maternal sinusoids by at least one
layer of cells, predominately Hand1+
(Fig. 3B), although some
Syna+ cells without an overlying
Hand1+ cell layer were rarely observed. The orientation of
Hand1+/Pl1- cells lining the maternal
sinusoids, of Syna+ cells along the apical side of the
chorion and of Gcm1/Cebpa/Synb+ cells at
the leading edge of the E8.5 chorion coincides with the subsequent
organization of the trilaminar trophoblast layer in the mature placenta.
Electron microscopy (Hernandez-Verdun,
1974) or even histology on ultrathin plastic resin sections
(Fig. 3C) also reveals unique
populations of chorionic trophoblast (with different morphology) that
correspond to the three future layers of the trilaminar structure.
Gcm1 regulates Synb and Cebpa but not Syna
Because expression of Gcm1, Cebpa and Synb were
co-localized in both the chorion and subsequently in SynT-II cells, we
examined the expression of these genes in Gcm1 mutant placentas.
Neither Cebpa nor Synb expression was detected at E8.5 (data
not shown) or E9.5 (Fig. 4) in
Gcm1 mutants, indicating that both genes are downstream of
Gcm1. Some limited Cebpa expression was seen in a few
Gcm1 mutant placentas by E9.5, although expression was both much
reduced and atypically localized (data not shown). The induction of
Syna expression was unaffected in Gcm1 mutants, consistent
with expression in a different chorionic trophoblast population and no obvious
interdependence of these cell layers. However, the morphology and/or
organization of Syna+ cells in Gcm1 mutants
resembled that of wild-type chorions at earlier developmental stages,
consistent with the block to branching morphogenesis known to occur in
Gcm1 mutants (Anson-Cartwright et
al., 2000; Schreiber et al.,
2000). Cebpa;Cebpb compound mutants manifest a
similar phenotype to Gcm1 mutants, although some initial branching
morphogenesis can occasionally be seen
(Begay et al., 2004). Neither
Synb nor Gcm1 expression was altered in Cebpa
mutants, Cebpb mutants or Cebpa;Cebpb compound
mutants (Fig. 4). Therefore,
Gcm1 is an upstream regulator of both Cebpa and Synb, but
Cebpa (or Cebpb) is not an upstream regulator of Synb.
|
E7.5, expression of
Synb and Cebpa was only evident closer to E8.5 (data not
shown), consistent with their being downstream of Gcm1. Syna
expression, by contrast, was not always detectable in our E8.5 samples, which
was likely to result from variation in when mating occurred versus the
consistency in sampling times (data not shown). This indicates that
Syna expression begins at E8.5 or slightly after, clearly after
Gcm1, Cebpa and Synb. This raises the possibility that
specification of SynT-I and S-TGC precursors within the chorion might require
interaction with, or signals from, SynT-II precursors
(Gcm1/Cebpa/Synb+ cells). Earlier
studies of labyrinth morphogenesis by electron microscopy indicated that
SynT-I formation, the fusion of trophoblast cells into the first syncytial
layer, was dependent upon interaction with SynT-II cells, now known to be
Gcm1/Cebpa/Synb+
(Hernandez-Verdun, 1974). In
addition, there is no evidence of trophoblast cell-cell fusion in
Gcm1 mutants (Anson-Cartwright et
al., 2000). However, gene expression studies in Gcm1
mutants clearly show Hand1+ S-TGC precursors and
Syna+ SynT-I precursors in the absence of
Gcm1+ cells or branching morphogenesis
(Fig. 5). In addition,
differentiating Gcm1-/- TS cell cultures also demonstrate
the ability to form Syna+ and Ctsq+
cells. Contrary to what we observed in situ, some Cebpa and
Synb expression could be detected in cultured
Gcm1-/- TS cells, although the expression patterns were
significantly altered over the course of differentiation as compared with
wild-type TS cells, suggesting that Gcm1 does have a role in regulating
Synb and Cepba in vitro, albeit not as the sole regulator
(Fig. 5) |
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Enders,
1965; Hernandez-Verdun,
1974; Jollie,
1964). However, very little progress has been made in
understanding the developmental origins of the three cell layers. There were
several key unanswered questions. (1) How early in development do the three
cell layers become distinguishable from the multipotent TS cells of the
chorion/extraembryonic ectoderm? (2) Do the three cell types have a common
progenitor and, if so, is it simply TS cells of the chorion/extraembryonic
ectoderm that are present up to at least the early post-implantation stages
(Tanaka et al., 1998;
Uy et al., 2002)? (3) How do
the two layers of syncytiotrophoblast form so that they remain separate from
each other during cell-cell fusion within their respective layers? This is of
interest because other hemochorial placental animals, including humans and
guinea pigs, contain only one layer of syncytiotrophoblast. (4) What is the
sequence of differentiation events and do the cell layers develop
autonomously? Our studies here have identified several genes that are
differentially expressed in the three trophoblast cell layers and have helped
to lead us to answers to these questions.
|
;
Basyuk et al., 1999),
Dlx3 (Berghorn et al.,
2005; Morasso et al.,
1999), Nr6a1 (Morasso
et al., 1999; Schreiber et
al., 2000; Susens et al.,
1997), Tead3
(Anson-Cartwright et al., 2000;
Jacquemin et al., 1998),
Tcfeb (Steingrimsson et al.,
1998; Tompers et al.,
2005; Voss et al.,
2000), Esx1 (Li et
al., 1997; Morasso et al.,
1999; Voss et al.,
2000), Hand1 (Scott
et al., 2000; Simmons et al.,
2007) and Syna and Synb
(Dupressoir et al., 2005). Of
these genes, some show layer-specific expression that has allowed insight into
the early developmental origins of the three cell types (see summary in
Fig. 6). Specifically,
Hand1 is expressed in S-TGCs
(Simmons et al., 2007),
Syna is restricted to SynT-I cells, and Gcm1, Cebpa and
Synb are all expressed in SynT-II cells in the mature labyrinth.
These genes begin their expression in the chorion at E8.5 and, even from
the earliest detection, their patterns are non-overlapping. Specifically, at
E8.5, Hand1 is expressed in cells at the apical side of the chorion
facing the maternal sinuses; Syna is expressed in cells just below
the Hand1-positive cells; and
Gcm1/Cebpa/Synb are expressed in clusters of cells
on the basal side of the chorion at the interface with the allantois. These
molecular data are supported by electron microscopy
(Hernandez-Verdun, 1974) and
ultrathin epoxy resin histology (this study) that reveal distinct cellular
morphologies of the different layers of the chorion at E8.5, well before
morphogenesis and syncytial fusion begin. The presence of these chorion cell
populations suggests that the chorion is already patterned into distinct cell
types by E8.5, establishing the precursors of the S-TGC, SynT-I and SynT-II
cell layers in the mature labyrinth.
The patterns for the layer-specific genes imply that the three
differentiated cell types have distinct precursors. Gcm1 is the only
one of the marker genes that is expressed prior to E8.5, but it is unlikely to
be a marker of a common progenitor because Gcm1-expressing cells are
post-mitotic in the chorion at E8.5 (Cross
et al., 2006) and ectopic Gcm1 expression in TS cells promotes
cell cycle exit (Hughes et al.,
2004). It has been hypothesized that the basal-most layer of the
chorion at E8.5 is derived from the extraembryonic ectoderm at E7.5, whereas
the upper layers of the chorion originate from the basal ectoplacental cone
based on similarity of cell ultrastructure and reaction to various fixatives
apparent by electron microscopy
(Hernandez-Verdun, 1974).
These layers come together after the collapse of the ectoplacental cavity
around E8.0-8.5. The expression patterns of Dlx3, Nr6a1 and
Esx1 also attest to the divergent developmental origins of cells at
the apical and basal sides of the E8.5 chorion. At E7.5, Dlx3 and
Nr6a1 are expressed in ectoplacental cone cells, whereas
Esx1 is expressed in the chorion, and by E8.5 they have distinct
apical (Dlx3 and Nr6a1) and basal (Esx1) bias.
Consistent with the lack of a common precursor cell type in the chorion,
the ability to derive TS cell lines from the chorion declines after E8.5
(Uy et al., 2002). However, it
is clear that there must be significant ongoing cell contribution to labyrinth
growth because the number of trophoblast cells in the mature labyrinth is
likely to exceed the number of cells in the chorion at E8.5. In addition, cell
proliferation does occur in the chorion
(Cross et al., 2006). The
Syna- and Gcm1/Cebpa/Synb-expressing cell
layers at E8.5 are separated by several layers of cells that do not express
any of the marker genes. These cells could represent a population of
fusion-competent trophoblast cells, perhaps differentially expressing the
receptors for the syncytins, and/or be a reserve of still-proliferating
progenitors (either multipotent or layer-restricted). Proliferative cells are
known to be located closer to the chorioallantoic interface, adjacent to
Gcm1-positive cells (Cross et
al., 2006). A possible marker of these cells is Rhox4b
(Ehox), a gene expressed in extraembryonic ectoderm early in
development and later in clusters of proliferating trophoblast preferentially
located near the chorionic plate (A. Davies, D.R.C.N., D.G.S., E.
Mariusdottir, J. G. Matyas and J.C.C., unpublished). The
Syna-negative Gcm1/Cebpa/Synb-negative
cells in the E8.5 chorion are small, tightly packed and cuboidal. Cells with
this same morphology do persist into mid-gestation in clusters throughout the
labyrinth. Interestingly, even as early as E9, they are no longer present
between the Syna-positive and the
Gcm1/Cebpa/Synb-positive cell layers. This might
simply be the result of the invaginating cell shape change and movements of
the Gcm1-positive cells (Cross et
al., 2006) that push up towards the apical side of the chorion
(Hernandez-Verdun, 1974).
|
|
).
These data indicate that they utilize different receptors and/or cell
machinery to promote cell-cell fusion. Differential use of the fusogenic
proteins within each syncytial layer is a possible way to ensure the formation
of two independent syncytial layers. For this model to work, the different
Syncytin receptors may or may not have unique localization patterns.
Our data now outline a fairly comprehensive view of the sequence of events
leading to differentiation of the three trophoblast cell layers in the
labyrinth (Fig. 6). Events
start with the onset of expression of Gcm1 in clusters of cells in
the extraembryonic ectoderm at E7.5 that persists in the basal chorion at
E8.5, when Cebpa and Synb expression appears in the same
cells. The temporal pattern as well as the analysis of Gcm1 and
Cebpa;Cebpb compound-mutant placentas indicate that
Gcm1 induces Cebpa and Synb. Although C/EBP
is a transcription factor, it is not required for Synb expression and
therefore its target genes are unclear. It is still unclear whether Gcm1
regulates the Cebpa and Synb genes directly or indirectly.
However, Gcm1 is a transcriptional activator
(Akiyama et al., 1996;
Chang et al., 2005;
Schreiber et al., 1997;
Schubert et al., 2004) and
both Cebpa and Synb contain Gcm1 binding sites within their
regulatory regions. Within 10 kb upstream of the Cebpa
transcriptional start site, there are seven predicted Gcm1 binding sites at
-9256, -7558, -6335, -5842, -5275, -5231 and -1861, whereas within the same
region upstream of Synb, there are eight at -6496, -3178, -2656,
-2634, -1864, -1767, -866 and -769. It is important to note that although
expression of Cebpa and Synb was undetectable in
Gcm1 mutants at E8.5, there was a small number of
Cebpa-positive cells by E9.5 and the genes were also detectable in
Gcm1 mutant TS cells, albeit in abnormal patterns. This suggests that
Gcm1 is not essential for Cebpa and Synb transcription per
se, but clearly is for their full expression and in the correct pattern.
Only after the establishment of the Gcm1/Cebpa/Synb pattern in the
basal chorion, does Syna expression become detectable in the apical
chorion, immediately below the Hand1-expressing cells that line the
maternal blood spaces (Fig. 6).
Neither the induction nor the maintenance of Syna or Hand1
requires the presence of Gcm1/Cebpa/Synb-expressing
cells, as both the Syna and Hand1 patterns are properly
specified in Gcm1 and Cebpa;Cebpb mutant placentas.
However, fusion of the Syna-positive cells to form SynT-I cells does
appear to require some sort of interaction with
Gcm1/Cepba/Synb-positive cells as suggested by two
lines of evidence. First, syncytiotrophoblast formation is not present in
Gcm1 mutant placentas
(Anson-Cartwright et al.,
2000). Second, fusion of trophoblast cells into the SynT-I
syncytium occurs only after the SynT-II cells make contact
(Hernandez-Verdun, 1974).
Gcm1 mutants do not survive past E10.5 and, therefore, it is
unclear whether the Hand1-positive cells in them would be able to
differentiate into S-TGCs. Interestingly, Cyr61 mutant placentas,
which contain few fetal vessels within the labyrinth, have a single layer of
syncytiotrophoblast in areas containing only maternal sinuses
(Mo et al., 2002). This
implies that single layers of SynT can form. However, the cell lineage origin
of the single SynT layer is unknown. Cyr61 placentas do contain areas
where both maternal and fetal vessels are separated by a normal trilaminar
trophoblast barrier. It is unclear whether the single layer of SynT observed
in Cyr61 mutants is continuous with, and potentially originates from,
SynT located in areas where there is a normal trilaminar barrier between fetal
and maternal blood compartments. As there is some normal branching
morphogenesis in these mice, albeit much reduced, it could provide the
necessary signals to induce the lateral fusion of SynT-I cells. What is clear
from the Cyr61 mutants is that in the absence of any branching
morphogenesis, as is the case with Gcm1 mutants, cell-cell fusion in
either layer is blocked.
To date over 125 mouse mutants have been generated that manifest defects in
placental development and function, and the majority of placental phenotypes
involve very poorly characterized defects in the labyrinth (reviewed by
Watson and Cross, 2005).
Markers for S-TGC (Hand1, Ctsq), SynT-I (Syna) and SynT-II
(Gcm1, Cebpa, Synb) should allow for greater insights into which cell
types are affected, increasing our understanding of labyrinth morphogenesis.
However, it is worth pointing out that there are some limitations to the
markers that we have to date. First, Ctsq expression is not evident
throughout the S-TGC population until after E12.5
(Simmons et al., 2007).
Hand1 is expressed in the presumptive S-TGC precursors as early as
E8.5, in the apical chorion and in S-TGCs in the mature labyrinth, but
Hand1 is not a specific marker of this lineage because it is
expressed in all TGC subtypes as well as the upper ectoplacental cone and some
cells within the chorion itself. Second, expression of the SynT layer-specific
genes Gcm1, Cebpa, Synb and Syna, is not uniformly detected
in each of their respective populations by mid-gestation and is ultimately
downregulated between E14.5 and E16.5. This makes these markers of limited use
in studying late-stage placentas. Because these genes are thought not to be
just markers, but also functional for syncytiotrophoblast differentiation,
their downregulation is consistent with the slowed expansion of the labyrinth
layer towards the end of gestation.
In summary, the labyrinth is the most complex compartment of the rodent placenta in terms of its functions, the number of mouse mutants with defects in it, and the number of different cell types and cellular interactions that are present. The current studies will significantly advance our understanding of the labyrinth through provision of new markers and methods for assessing its structure and through insights into how the trilaminar trophoblast structure that forms the maternal-fetal interface forms.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Akiyama, Y., Hosoya, T., Poole, A. M. and Hotta, Y.
(1996). The gcm-motif: a novel DNA-binding motif conserved in
Drosophila and mammals. Proc. Natl. Acad. Sci. USA
93,14912
-14916.
Anson-Cartwright, L., Dawson, K., Holmyard, D., Fisher, S. J.,
Lazzarini, R. A. and Cross, J. C. (2000). The glial cells
missing-1 protein is essential for branching morphogenesis in the
chorioallantoic placenta. Nat. Genet.
25,311
-314.[CrossRef][Medline]
Basyuk, E., Cross, J. C., Corbin, J., Nakayama, H., Hunter, P.,
Nait-Oumesmar, B. and Lazzarini, R. A. (1999). Murine Gcm1
gene is expressed in a subset of placental trophoblast cells. Dev.
Dyn. 214,303
-311.[CrossRef][Medline]
Beck, F., Erler, T., Russell, A. and James, R.
(1995). Expression of Cdx-2 in the mouse embryo and placenta:
possible role in patterning of the extra-embryonic membranes. Dev.
Dyn. 204,219
-227.[Medline]
Begay, V., Smink, J. and Leutz, A. (2004).
Essential requirement of CCAAT/enhancer binding proteins in embryogenesis.
Mol. Cell. Biol. 24,9744
-9751.
Berghorn, K. A., Clark, P. A., Encarnacion, B., Deregis, C. J.,
Folger, J. K., Morasso, M. I., Soares, M. J., Wolfe, M. W. and Roberson, M.
S. (2005). Developmental expression of the homeobox protein
Distal-less 3 and its relationship to progesterone production in mouse
placenta. J. Endocrinol.
186,315
-323.
Campbell, W. J., Deb, S., Kwok, S. C., Joslin, J. A. and Soares,
M. J. (1989). Differential expression of placental
lactogen-II and prolactin-like protein-A in the rat chorioallantoic placenta.
Endocrinology 125,1565
-1574.
Chang, C. W., Chuang, H. C., Yu, C., Yao, T. P. and Chen, H.
(2005). Stimulation of GCMa transcriptional activity by cyclic
AMP/protein kinase A signaling is attributed to CBP-mediated acetylation of
GCMa. Mol. Cell. Biol.
25,8401
-8414.
Church, G. M. and Gilbert, W. (1984). Genomic
sequencing. Proc. Natl. Acad. Sci. USA
81,1991
-1995.
Coan, P. M., Ferguson-Smith, A. C. and Burton, G. J.
(2005). Ultrastructural changes in the interhaemal membrane and
junctional zone of the murine chorioallantoic placenta across gestation.
J. Anat. 207,783
-796.[CrossRef][Medline]
Cross, J. C. (2000). Genetic insights into
trophoblast differentiation and placental morphogenesis. Semin.
Cell Dev. Biol. 11,105
-113.[CrossRef][Medline]
Cross, J. C., Nakano, H., Natale, D. R., Simmons, D. G. and
Watson, E. D. (2006). Branching morphogenesis during
development of placental villi. Differentiation
74,393
-401.[CrossRef][Medline]
Dai, G., Wang, D., Liu, B., Kasik, J. W., Muller, H., White, R.
A., Hummel, G. S. and Soares, M. J. (2000). Three novel
paralogs of the rodent prolactin gene family. J.
Endocrinol. 166,63
-75.[Abstract]
Davies, J. and Glasser, S. R. (1968).
Histological and fine structural observations on the placenta of the rat.
Acta Anat. Basel 69,542
-608.[Medline]
Deb, S., Faria, T. N., Roby, K. F., Larsen, D., Kwok, S. C.,
Talamantes, F. and Soares, M. J. (1991). Identification and
characterization of a new member of the prolactin family, placental lactogen-I
variant. J. Biol. Chem.
266,1605
-1610.
Dupressoir, A., Marceau, G., Vernochet, C., Benit, L.,
Kanellopoulos, C., Sapin, V. and Heidmann, T. (2005).
Syncytin-A and syncytin-B, two fusogenic placenta-specific murine envelope
genes of retroviral origin conserved in Muridae. Proc. Natl. Acad.
Sci. USA 102,725
-730.
Enders, A. C. (1965). A comparative study of
the fine structure of the trophoblast in several hemochorial placentas.
Am. J. Anat. 116,29
-67.[CrossRef][Medline]
Hancock, S. N., Agulnik, S. I., Silver, L. M. and Papaioannou,
V. E. (1999). Mapping and expression analysis of the mouse
ortholog of Xenopus Eomesodermin. Mech. Dev.
81,205
-208.[CrossRef][Medline]
Hemberger, M. and Cross, J. C. (2001). Genes
governing placental development. Trends Endocrinol.
Metab. 12,162
-168.[CrossRef][Medline]
Hernandez-Verdun, D. (1974). Morphogenesis of
the syncytium in the mouse placenta. Ultrastructural study. Cell
Tissue Res. 148,381
-396.[Medline]
Hughes, M., Dobric, N., Scott, I. C., Su, L., Starovic, M.,
St-Pierre, B., Egan, S. E., Kingdom, J. C. and Cross, J. C.
(2004). The Hand1, Stra13 and Gcm1 transcription factors override
FGF signaling to promote terminal differentiation of trophoblast stem cells.
Dev. Biol. 271,362
-371.[CrossRef][Medline]
Ishida, M., Ono, K., Taguchi, S., Ohashi, S., Naito, J.,
Horiguchi, K. and Harigaya, T. (2004). Cathepsin gene
expression in mouse placenta during the latter half of pregnancy.
J. Reprod. Dev. 50,515
-523.[CrossRef][Medline]
Jacquemin, P., Sapin, V., Alsat, E., Evain-Brion, D., Dolle, P.
and Davidson, I. (1998). Differential expression of the TEF
family of transcription factors in the murine placenta and during
differentiation of primary human trophoblasts in vitro. Dev.
Dyn. 212,423
-436.[CrossRef][Medline]
Jollie, W. P. (1964). Fine structural changes
in placental labyrinth of the rat with increasing gestational age.
J. Ultrastruct. Res. 10,27
-47.[CrossRef][Medline]
Lee, C. K., Moon, D. H., Shin, C. S., Kim, H., Yoon, Y. D.,
Kang, H. S., Lee, B. J. and Kang, S. G. (2003). Circadian
expression of Mel1a and PL-II genes in placenta: effects of melatonin on the
PL-II gene expression in the rat placenta. Mol. Cell.
Endocrinol. 200,57
-66.[CrossRef][Medline]
Li, Y., Lemaire, P. and Behringer, R. R.
(1997). Esx1, a novel X chromosome-linked homeobox gene expressed
in mouse extraembryonic tissues and male germ cells. Dev.
Biol. 188,85
-95.[CrossRef][Medline]
Mo, F., Muntean, A. G., Chen, C., Stolz, D. B., Watkins, S. C.
and Lau, L. F. (2002). CYR61 (CCN1) is essential for
placental development and vascular integrity. Mol. Cell.
Biol. 22,8709
-8720.
Morasso, M. I., Grinberg, A., Robinson, G., Sargent, T. D. and
Mahon, K. A. (1999). Placental failure in mice lacking the
homeobox gene Dlx3. Proc. Natl. Acad. Sci. USA
96,162
-167.
Pettersson, K., Svensson, K., Mattsson, R., Carlsson, B.,
Ohlsson, R. and Berkenstam, A. (1996). Expression of a novel
member of estrogen response element-binding nuclear receptors is restricted to
the early stages of chorion formation during mouse embryogenesis.
Mech. Dev. 54,211
-223.[CrossRef][Medline]
Rossant, J. and Cross, J. C. (2001). Placental
development: lessons from mouse mutants. Nat. Rev.
Genet. 2,538
-548.[Medline]
Sahgal, N., Knipp, G. T., Liu, B., Chapman, B. M., Dai, G. and
Soares, M. J. (2000). Identification of two new nonclassical
members of the rat prolactin family. J. Mol.
Endocrinol. 24,95
-108.[Abstract]
Schreiber, J., Sock, E. and Wegner, M. (1997).
The regulator of early gliogenesis glial cells missing is a transcription
factor with a novel type of DNA-binding domain. Proc. Natl. Acad.
Sci. USA 94,4739
-4744.
Schreiber, J., Riethmacher-Sonnenberg, E., Riethmacher, D.,
Tuerk, E. E., Enderich, J., Bosl, M. R. and Wegner, M.
(2000). Placental failure in mice lacking the mammalian homolog
of glial cells missing, GCMa. Mol. Cell. Biol.
20,2466
-2474.
Schubert, S. W., Kardash, E., Khan, M. A., Cheusova, T., Kilian,
K., Wegner, M. and Hashemolhosseini, S. (2004). Interaction,
cooperative promoter modulation, and renal colocalization of GCMa and Pitx2.
J. Biol. Chem. 279,50358
-50365.
Scott, I. C., Anson-Cartwright, L., Riley, P., Reda, D. and
Cross, J. C. (2000). The HAND1 basic helix-loop-helix
transcription factor regulates trophoblast differentiation via multiple
mechanisms. Mol. Cell. Biol.
20,530
-541.
Simmons, D. G. and Cross, J. C. (2005).
Determinants of trophoblast lineage and cell subtype specification in the
mouse placenta. Dev. Biol.
284, 12-24.[CrossRef][Medline]
Simmons, D. G., Fortier, A. L. and Cross, J. C.
(2007). Diverse subtypes and developmental origins of trophoblast
giant cells in the mouse placenta. Dev. Biol.
304,567
-578.[CrossRef][Medline]
Snell, G. D. and Stevens, L. C. (1966). Early
embryology. In Biology of the Laboratory Mouse (ed. E.
L. Green), pp. 205-245. New York:
McGraw-Hill.
Steingrimsson, E., Tessarollo, L., Reid, S. W., Jenkins, N. A.
and Copeland, N. G. (1998). The bHLH-Zip transcription factor
Tfeb is essential for placental vascularization.
Development 125,4607
-4616.[Abstract]
Susens, U., Aguiluz, J. B., Evans, R. M. and Borgmeyer, U.
(1997). The germ cell nuclear factor mGCNF is expressed in the
developing nervous system. Dev. Neurosci.
19,410
-420.[Medline]
Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A. and
Rossant, J. (1998). Promotion of trophoblast stem cell
proliferation by FGF4. Science
282,2072
-2075.
Tompers, D. M., Foreman, R. K., Wang, Q., Kumanova, M. and
Labosky, P. A. (2005). Foxd3 is required in the trophoblast
progenitor cell lineage of the mouse embryo. Dev.
Biol. 285,126
-137.[CrossRef][Medline]
Uy, G. D., Downs, K. M. and Gardner, R. L.
(2002). Inhibition of trophoblast stem cell potential in
chorionic ectoderm coincides with occlusion of the ectoplacental cavity in the
mouse. Development 129,3913
-3924.[Medline]
Voss, A. K., Thomas, T. and Gruss, P. (2000).
Mice lacking HSP90beta fail to develop a placental labyrinth.
Development 127,1
-11.[Abstract]
Watson, E. D. and Cross, J. C. (2005).
Development of structures and transport functions in the mouse placenta.
Physiology Bethesda 20,180
-193.[CrossRef][Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  K. Forbes, G. West, R. Garside, J. D. Aplin, and M. Westwood The Protein-Tyrosine Phosphatase, Src Homology-2 Domain Containing Protein Tyrosine Phosphatase-2, Is a Crucial Mediator of Exogenous Insulin-Like Growth Factor Signaling to Human Trophoblast Endocrinology, October 1, 2009; 150(10): 4744 - 4754. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Kawamura, N. Kawamura, W. Sato, J. Fukuda, J. Kumagai, and T. Tanaka Brain-Derived Neurotrophic Factor Promotes Implantation and Subsequent Placental Development by Stimulating Trophoblast Cell Growth and Survival Endocrinology, August 1, 2009; 150(8): 3774 - 3782. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Dupressoir, C. Vernochet, O. Bawa, F. Harper, G. Pierron, P. Opolon, and T. Heidmann From the Cover: Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene PNAS, July 21, 2009; 106(29): 12127 - 12132. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. N. Sferruzzi-Perri, A. M. Macpherson, C. T. Roberts, and S. A. Robertson Csf2 Null Mutation Alters Placental Gene Expression and Trophoblast Glycogen Cell and Giant Cell Abundance in Mice Biol Reprod, July 1, 2009; 81(1): 207 - 221. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Nadeau, S. Guillemette, L.-F. Belanger, O. Jacob, S. Roy, and J. Charron Map2k1 and Map2k2 genes contribute to the normal development of syncytiotrophoblasts during placentation Development, April 15, 2009; 136(8): 1363 - 1374. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
