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First published online 23 March 2005
doi: 10.1242/dev.01801
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1 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University
Avenue, Toronto M5G 1X5, Ontario, Canada
2 Department of Biochemistry, University of Leicester, Leicester LE1 7RH,
UK
3 Department of Molecular and Medical Genetics, University of Toronto, Toronto
M5S 1A8, Ontario, Canada
Author for correspondence (e-mail:
rossant{at}mshri.on.ca)
Accepted 22 February 2005
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SUMMARY |
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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
REFERENCES |
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Key words: Trophoblast, Stem cells, Pou5f1, Oct4, Eomesodermin, Nanog, Mouse
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Introduction |
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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
REFERENCES |
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;
Pratt et al., 1982;
Reeve and Ziomek, 1981). In
subsequent cell divisions, outer cells remain polarized and generate the TE
epithelium. During blastocyst formation, the TE cells form a transporting
epithelium, with functional adherens and tight junctions
(Fleming et al., 2001). The
activity of sodium pumps (Na+, K+-ATPases) leads to
establishment of ionic gradients across the TE and accumulation of fluid in
the forming blastocoel cavity (Watson,
1992). In addition, aquaporins contribute to water movements
across the TE, leading to blastocoel expansion
(Barcroft et al., 2003). After
hatching from the zona pellucida, a subpopulation of TE cells – the
mural TE cells – exhibit increased protrusive activity and undergo
changes in cell polarity through relocalization of proteins such as integrin
5ß1 and integrin 7ß1 from basal to apical domains
(reviewed by Sutherland et al., 2003). At this stage, cells in the ICM have
segregated into the epiblast, or embryonic lineage, and the primitive endoderm
(PE), or extra-embryonic endoderm lineage.
Following implantation, cells overlying the ICM – the polar TE
– continue to proliferate and form the extra-embryonic ectoderm (ExE)
that contains trophoblast stem (TS) cells
(Tanaka et al., 1998) and the
diploid ectoplacental cone (EPC), while the mural cells cease division and
form trophoblast giant cells. Further differentiation of the trophoblast
lineage generates the labyrinth, spongiotrophoblast and glycogen cells of the
mature chorioallantoic placenta (Rossant
and Cross, 2001).
Identification of the molecular components required for the initial
segregation of the TE and ICM lineages has been elusive, as few mutations have
been found to cause early lineage-specific defects. Oct4 (Pou5f1), a POU
domain transcription factor (TF), is required for maintenance of ICM fate and
pluripotency of ES cells (Nichols et al.,
1998; Niwa et al.,
2000). Oct4 is expressed in all blastomeres of the cleavage stage
embryo, but becomes restricted to the ICM after initiation of blastocyst
formation (Palmieri et al.,
1994). Homozygous mutant Oct4 embryos develop to the
blastocyst stage, but their isolated ICM cells express trophectoderm markers
when outgrown in vitro (Nichols et al.,
1998). Furthermore, conditional repression of Oct4 in ES
cells leads to differentiation into trophoblast morphology and an increase in
expression of trophoblast-specific markers
(Niwa et al., 2000;
Hay et al., 2004). Culturing
these cells under conditions that promote trophoblast proliferation generated
cells apparently equivalent to TS cells
(Niwa et al., 2000).
Nanog, a homeobox gene, is also expressed in the ICM at 3.5 days
post-coitum (dpc) and becomes epiblast-specific in the implanting blastocyst.
Nanog maintains ES cell pluripotency independent of LIF signalling, and in the
absence of Nanog, ES cells and ICMs both differentiate into extra-embryonic
endoderm (Chambers et al.,
2003; Mitsui et al.,
2003). Thus, Nanog has been implicated in repressing the
extra-embryonic endoderm or PE fate, while Oct4 may function as a repressor of
the trophoblast cell fate.
Only a few TFs show TE-specific expression at the blastocyst stage
(Beck et al., 1995;
Hancock et al., 1999;
Luo et al., 1997;
Rossant et al., 1998;
Russ et al., 2000), and none
so far has shown absence of TE formation when mutated
(Guillemot et al., 1994;
Luo et al., 1997;
Russ et al., 2000). It has
been proposed that the TE develops by default in the absence of Oct4
(Pesce and Scholer, 2001).
However, TE differentiation begins prior to downregulation of Oct4 in the
outer cells of the nascent blastocyst, suggesting that there should also be
positive acting factors promoting TE fate. Cdx2, a caudal-type homeodomain TF,
has been reported to be specifically expressed in TE at blastocyst stage, and
expression is maintained within the proliferating ExE
(Beck et al., 1995).
Heterozygous Cdx2 mutants show homeotic transformation defects and
homozygous mutants die at the peri-implantation stage
(Chawengsaksophak et al., 1997;
Tamai et al., 1999).
Eomesodermin (Eomes), a T-box TF, is also expressed specifically in the TE at
the blastocyst stage, and, like Cdx2, is expressed at later stages in the ExE
(Ciruna and Rossant, 1999;
Hancock et al., 1999;
Russ et al., 2000).
Eomes mutants have also been reported to show early defects in
trophoblast proliferation (Russ et al.,
2000).
In this paper, we compare the Cdx2-/- and Eomes-/- mutant phenotypes in more detail, and show that Cdx2 mutant blastocysts fail to maintain trophoblast differentiation and fail to implant. Interestingly, loss of Cdx2 is associated with failure to downregulate Oct4 and Nanog in outer cells of the blastocyst and results in subsequent death of outer cells. By contrast, Eomes mutants form blastocysts, display ICM-restricted Oct4 expression and TE-specific Cdx2 expression, but trophoblast does not differentiate further. Thus, Cdx2 is the earliest TF identified so far to be involved in specification of TE fate, and Cdx2 is required for repression of Oct4/Nanog and normal blastocyst development.
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Materials and methods |
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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
REFERENCES |
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)
were maintained by crossing to outbred ICR strain mice. Embryos were collected
from intercrossed Cdx2 or Eomes heterozygotes. Genotyping
was performed on individually isolated embryos directly or after observation
in culture or following in situ hybridization or antibody staining. All
genotyping was performed blind by PCR with primers that produced both mutant
and wild-type bands, thus ensuring unequivocal identification of genotypes.
The conditions of PCR genotyping of mice and embryos for Cdx2
targeted mutation Cdx2tm1Fbe were as previously described
(Chawengsaksophak et al.,
1997). Eomes targeted mutants were genotyped using the
following primers (see also Fig.
8A): primer A, (forward primer)
5'-GAAAGCGCCTGTCTCCAGCACCC-3'; primer B, (reverse primer)
5'-AACACTCCTGCGTCCTCCAGTCAC-3'; primer C, (Neor forward
primer) 5'-CAGCGCATCGCCTCTATCCGCC-3'. A 440 bp amplification
product is generated from the mutant allele between primer C and B; a 350 bp
product is amplified from the wild-type allele between primers A and B.
RNA preparation and RT-PCR
RNA was isolated from single embryos as described
(Chomczynski and Sacchi, 1987).
cDNA was reverse transcribed using Superscript II reverse transcriptase
according to manufacturer (Invitrogen). cDNA was diluted 1/10 for
semi-quantitative PCR. Minimum number of cycles required for amplification by
a given primer set was determined on cDNA from single wild-type whole-embryo
RNA preparation cultured under the experimental conditions tested.
Primers used were as follows: ß-actin, (forward) 5'-ggcccagagcaagagaggtatcc-3' and (reverse) 5'-acgcacgatttccctctcagc-3' (30 cycles; product size 460 bp); Cdx2, (forward) 5'-gcagtccctaggaagccaagtga-3' and (reverse) 5'-ctctcggagagcccaagtgtg-3' (35 cycles; product size 162 bp); Fgfr2, (forward) 5'-gacaagcccaccaactgcacc-3' and (reverse) 5'-cgtcccctgaagaacaagagc-3' (40 cycles; product size 217 bp); Hand1, (forward) 5'-atgaacctcgtgggcaggta-3' and (reverse) 5'-tcactggtttagctccagcg-3' (40 cycles; product size 550 bp); Eomes, (forward) 5'-gtgacagagacggtgtggagg-3' and (reverse) 5'-agaggaggccgttggtctgtgg-3' (35 cycles; products sizes 350 bp and 304 bp); Pl1 (Csh1 – Mouse Genome Informatics) (forward) 5'-atcttctcagaaatgcagctg-3' and (reverse) 5'-gatcattgctttcagaaggtc-3' (40 cycles; product size 336 bp).
In situ hybridization
Fluorescent in situ hybridization using digoxigenin- or FITC-labelled RNA
probes was performed according to protocol available on the Rossant laboratory
website:
http://www.mshri.on.ca/rossant/protocols/doubleFluor.html.
RNA antisense probes were in vitro transcribed from the following mouse cDNA
templates: Cdx2 (939 bp; Dr Peter Traber, PA); Oct4 (1336
bp; Dr Hitoshi Niwa, Japan); Nanog (981 bp; Dr Austin Smith, UK).
Immunohistochemistry
Immunohistochemistry protocols can be found on the Rossant laboratory
website
(http://www.mshri.on.ca/rossant/protocols/immunoStain.html).
The following antibodies were used at the following dilutions:
affinity-purified polyclonal rabbit anti-Cdx2 C-term and CNL (gift of Dr
Edmond Rings) (Rings et al.,
2001) 1:500-1:1000 of 0.6 mg immunoglobulin/ml [specificity of
anti-Cdx2 CNL was confirmed in blocking experiments as described by Silberg et
al. (Silberg et al., 2000)];
monoclonal anti-Cdx2 (CDX2-88, BioGenex, CA, USA) 1:200; monoclonal mouse
anti-Oct4 (C10; Santa Cruz Biotechnology) 1:100; rabbit anti-Nanog
(Mitsui et al., 2003; Dr
Yamanaka, NAIST, Japan) 1:400; monoclonal rat anti-integrin 7
(undiluted CA5; gift of Dr Ann Sutherland, University of Virginia)
(Klaffky et al., 2001); rabbit
anti-mouse ZO-1+ 1:250; rabbit anti-ZO-1- at 1:250 (both gifts
of Dr Bhavwanti Sheth, UK); and rat anti-E-cadherin (Sigma) 1:500. Secondary
antibodies (Cy3 or Cy5-donkey anti-mouse; biotin or Cy3-donkey anti-rabbit;
Cy5-donkey anti-rat (Jackson ImmunoResearch Laboratories) were used at
1:300-1:400, and Cy5-streptavidin at 1:1000. To visualize nuclei, embryos were
incubated in YOYO-1 or YOYO-3 (Molecular Probes) at 1:400-1:1000 dilution with
10-20 µg/ml RNAse A, for 15-30 minutes at room temperature.
Embryo culture
To follow development in vitro, eight-cell stage embryos were flushed from
oviducts in M2, treated with acidic Tyrode's to remove zonae pellucidae
(Nagy et al., 2003). The
embryos were then cultured in microdrops of KSOM-AA under mineral oil for 48
hours at 37°C, 5% CO2 and transferred into microdrops of RPMI
1640 containing 0.1% BSA and 100 µM non-essential amino acids for an
additional 24 hours. For trophoblast outgrowth formation, 3.5 dpc blastocysts
from heterozygous Cdx2 or Eomes intercrosses were
individually cultured in KSOM-AA overnight and then transferred into RPMI 1640
containing 20% FCS in four-well tissue culture dishes (Nunc, Denmark)
untreated or pre-coated with 0.1% gelatin (Sigma), fibronectin (from bovine
plasma, Sigma; 20 µg/ml in PBS) or laminin (Sigma; 25 µg/ml in PBS).
Outgrowth formation was monitored over 72-120 hours. For digital time-lapse
microscopy, embryos were collected and cultured in KSOM-AA in glass-bottom
dishes (MatTek, USA) overlaid with light mineral oil and imaged using the
Zeiss Axiovert 200 inverted microscope with Incubator XL, Heating Insert P and
CO2 controller. Temperature and CO2 were set to
37.5°C and 5.5%, respectively. DIC images were recorded, with halogen lamp
voltage (<2.5 V), every 30 minutes using AxioCam MRm with Axiovision 3.1
software.
TS cell culture and derivation
Derivation of trophoblast stem (TS) cell lines from blastocysts from
heterozygous Cdx2 or Eomes intercrosses was performed as
previously described (Tanaka et al.,
1998) and as detailed at
http://www.mshri.on.ca/rossant/protocols/TScells.html.
Genotyping of cell lines was performed by PCR (for Cdx2) or confirmed
by Southern analysis (for Eomes).
TUNEL staining
Eight-cell embryos were cultured in KSOM-AA for 48 hours, then fixed in 4%
paraformaldehyde in PBS for 1 hour at room temperature, and washed in PBS+0.1%
Tween 20, incubated in TUNEL reaction mixture (Roche) for 60 minutes at
37°C and washed as above. The total number of FITC-labelled/TUNEL-positive
nuclei in each embryo was scored as well as their distribution in the ICM or
TE by counting multiple stacked optical sections.
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Results |
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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
REFERENCES |
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). To gain
better insight into the role of Cdx2 in early development of the
trophoblast lineage, we followed the spatial and temporal dynamics of Cdx2
expression from the early morula stage to 5.5 dpc
(Fig. 1). By RT-PCR analysis,
Cdx2 mRNA was first detected at the eight-cell stage (not shown), and
Cdx2 protein was first clearly detected in the nucleus and cytoplasm of the
outer cells of early morula (
16 cells, n=4;
Fig. 1A,B) and late morula
stages (25 cells, n=4; Fig.
1C,D). Some low level of expression was seen cytoplasmically
throughout the rest of the embryo. Interestingly, at these stages Oct4 protein
is present equally in all cells of the embryo, and thus is co-expressed with
Cdx2 in the outer cells (Fig.
1D). At the early blastocyst stage, Cdx2 expression was more
restricted to the outer cell layer (3.25 dpc n=2;
Fig. 1E). By the expanded
blastocyst stage, Cdx2 protein was exclusively localized to the TE nuclei (3.5
dpc, n=4; Fig. 1F). At
4.5 dpc, by the onset of implantation, Cdx2 protein levels appeared to be
reduced in the mural TE, but maintained in the polar TE cells directly
overlying the ICM (n=3; Fig.
1G). At later stages, Cdx2 expression became confined to the polar
TE (nascent ExE) cells directly overlaying the epiblast (4.75 dpc,
n=2; Fig. 1H). By 5.5
dpc both epiblast and ExE have expanded, and intense Cdx2 expression spanned
the first three or four cell rows in the ExE adjacent to the epiblast
(n=2; Fig. 1I). Cdx2
expression is thus associated with the initiation of TE fate, and later
specifically delineates the cells of the trophoblast lineage that give rise to
TS cells in the peri-implantation embryo
(Corson et al., 2003;
Tanaka et al., 1998). A
similar expression profile was found for Cdx2 mRNA (see Fig. S1 in
the supplementary material).
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Cdx2-/- blastocysts fail to maintain blastocoel
Previous analysis revealed that Cdx2 homozygous mutant embryos die
around the time of implantation. Dissection and genotyping of
post-implantation stages revealed neither evidence of
Cdx2-/- mutants nor any empty deciduae, which is
indicative of death prior to implantation
(Chawengsaksophak et al.,
1997). We examined the morphology of embryos from
Cdx2+/– intercrosses by dissection from the uterus
at 4.5 dpc. Cdx2+/+ or Cdx2+/–
blastocysts were recovered and were fully expanded and hatched from their
zonae pellucida by this stage (n=10). By contrast,
Cdx2-/- embryos recovered were still enclosed in their
zonae, and had little or no blastocoelic cavity (n=4). Blastocyst
formation was monitored by culturing zona-free eight-cell stage embryos from
Cdx2+/– intercrosses over a time course of 72 hours
(Fig. 2). Between 24 and 48
hours, all embryos had a blastocoel. However, by 72 hours all Cdx2
mutants (n=4) showed no blastocoelic cavity and surface morphology
was rough. Live imaging of litters from Cdx2 +/–
intercrosses revealed that the blastocoel of Cdx2 mutants initially
expanded, but began to collapse around the time that non-mutant littermates
hatch from the zona (three mutants, 18 non-mutants; see Movie 1 in the
supplementary material). This collapse was accompanied by a morphological
change in the TE, as cells acquired a rounded, non-epithelial appearance.
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– and ZO-1+ (encoded by Tjp1) incorporate
into tight junctions early and late during TE formation, respectively
(Sheth et al., 1997).
E-cadherin localization to the basolateral adherens junctions precedes tight
junction formation (reviewed by Fleming et
al., 2000; Fleming et al.,
2001). Both tight and adherens junctions appeared grossly normal
in early blastocysts of both mutant and non-mutant genotype, as assessed by
expression of ZO-1+ (four mutants, 15 non-mutants;
Fig. 3A,B), ZO-1- (six
mutants, 21 non-mutants; not shown), and E-cadherin (10 mutants, 46
non-mutants; Fig. 3C,D). By
contrast, both tight and adherens junctions appeared abnormal in most mutants
by the late blastocyst stage. In particular, ZO-1+ appeared patchy or
diffuse compared with littermates (7/8 mutants; 12 non-mutants;
Fig. 3E,F), while ZO-1-
was diffuse (3/3 mutants, five non-mutants; not shown). E-cadherin expression
was still observed in Cdx2-/- blastocysts but appeared to
be mislocalized basally in some cells of the TE (6/6 mutants, 13 non-mutants;
Fig. 3G,H). These observations
suggest that the polarity and integrity of the TE epithelium, while initially
normal, is not maintained in the absence of zygotic Cdx2.
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7 (Itga7) (Klaffky et al.,
2001) was undetectable in abnormal, collapsed Cdx2 mutant
at 4.5 dpc (Fig. 4E).
Interestingly, Oct4 appeared to be expressed in all cells of
Cdx2-/- embryos at this stage
(Fig. 4E; see below).
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; Russ et al.,
2000), although the expression levels of Eomes were
markedly lower in Cdx2 presumptive null embryos than in littermates.
The expression of Hand1 and Pl1 (Csh – Mouse
Genome Informatics) (Cross et al.,
1995; Faria et al.,
1991), markers of differentiated trophoblast giant cells, was
undetectable in Cdx2-/- embryos, although these markers
were detected in Cdx2+/+ and
Cdx2+/– embryos
(Fig. 4J). Altogether,
TE-specific marker analysis indicated that, although blastocyst formation
begins in Cdx2-/- embryos, trophoblast differentiation is
compromised beyond the expanded blastocyst stage.
Cdx2 homozygous mutants fail to form trophoblast giant cells or TS cell lines in vitro
To address whether, under in vitro culture conditions, the TE of
Cdx2-/- embryos could be promoted to differentiate,
zona-free 3.5 dpc blastocysts from Cdx2+/–
intercrosses were cultured in serum-containing medium or specific
extracellular matrix substrates (Fig.
4G-I). Although Cdx2+/– or
Cdx2+/+ blastocysts attached and initiated TE outgrowth
within 24-36 hours after plating in serum-containing medium,
Cdx2-/- blastocysts failed to attach regardless of the
extracellular matrix substrate used (fibronectin, gelatin, or laminin; see
Table 1). By 72 hours of
culture, Cdx2+/– or Cdx2+/+
blastocysts formed both ICM and trophoblast outgrowths, the latter containing
trophoblast giant cells with typical large nuclei and cytoplasm
(Fig. 4G). By contrast,
Cdx2-/- embryos showed no attachment and giant cell
outgrowth, with only occasional parietal endoderm-like cells attached to the
substrate. Embryos survived and grew into structures resembling embryoid
bodies (Fig. 4H,I). Indeed, ES
cells can be derived from Cdx2-/- blastocysts
(Chawengsaksophak et al.,
2004), indicating that ICM development is not affected in these
embryos. When we attempted to derive TS cell lines
(Tanaka et al., 1998) from
blastocysts obtained from Cdx2+/– intercrosses, 22
TS cell lines were obtained from a total of 36 blastocysts initially cultured.
However, none were Cdx2-/- by genotype. Thus,
Cdx2 is required for all aspects of early TE development, both
diploid proliferation and polyploid giant cell development.
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; Mitsui et al.,
2003). If true, this would suggest that loss of Cdx2
leads to ectopic activation of Nanog in outside cells. Using antibody
to detect Nanog protein, we found detectable levels of nuclear Nanog in all
cells of 16-cell (n=13) and 32-cell (n=8)
Cdx2+/+ and Cdx2+/– embryos
(Fig. 6A; not shown). Nanog was
restricted to the ICM of Cdx2+/+ and
Cdx2+/– embryos by the early blastocyst stage
(n=10; Fig. 6C). At
this stage, Nanog was detected at equivalent levels in TE and ICM cells in
Cdx2-/- embryos (n=5),
(Fig. 6C,D). However, no
difference in Nanog expression was observed between non-mutant and
Cdx2-/- embryos (n=3) at earlier stages
(Fig. 6A,B and not shown).
These results indicate that Cdx2 is also required to ensure downregulation of
Nanog, as well as Oct4, in outer cells beginning around the early blastocyst
stage.
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|
; Russ et al.,
2000), including the TE of the blastocyst stage
(Hancock et al., 1999).
Previous analysis of Eomes knockout embryos has shown that
Eomes-/- embryos survive to 6.5-7.5 dpc in utero, but
display a blastocyst-like morphology and fail to form trophoblast outgrowths
in vitro (Russ et al., 2000).
However, the exact stage of trophoblast development affected by loss of
Eomes was not clear. We generated a targeted allele of Eomes
(Fig. 8A) and confirmed that
blastocyst-like embryos could be identified in a quarter of the deciduae from
Eomes+/– intercrosses
(Fig. 8D,H). Thus,
Eomes mutants, unlike Cdx2 mutants, can induce a decidual
response. To evaluate the degree of TE differentiation in
Eomes-/- embryos we examined the expression of TE-specific
markers (Fig. 8). Embryos from
Eomes+/– intercrosses were dissected at 4.5-4.75 dpc
and Oct4 and Cdx2 expression analyzed. TE-specific
expression of Cdx2 and ICM-restricted expression of Oct4
were observed in Eomes-/- embryos (n=2),
comparable with the expression pattern in Eomes+/+ and
Eomes+/– littermates (n=3;
Fig. 8B,F). However, despite
the relatively normal appearance of the Eomes mutant blastocysts, TE
differentiation at the time of implantation was compromised, as measured by
the expression of integrin 7, which was undetectable in the
Eomes-/- blastocysts (two mutants, three non-mutants;
Fig. 8C,G).
To determine whether the TE of Eomes-/- embryos can
differentiate in vitro, blastocysts from Eomes+/–
intercrosses were assayed for trophoblast outgrowth formation
(Fig. 8E,I). As previously
reported (Russ et al., 2000),
Eomes-/- blastocysts failed to attach and form a TE
outgrowth (Fig. 8I).
Morphologically we observed that the Eomes-/- blastocysts
remained as fully expanded blastocysts after 96 hours of culture, unlike
Cdx2-/- embryos, which eventually collapse. We examined
the expression of additional TE markers by a semi-quantitative RT-PCR analysis
from individual in vitro cultured blastocysts from
Eomes+/– intercrosses, as described above for
Cdx2 analysis. Analysis was performed on embryos at 24 hours, 96
hours and 120 hours of culture (Fig.
8J). The analysis indicated that in the absence of Eomes,
Cdx2 and Fgfr2 are still expressed
(Beck et al., 1995;
Haffner-Krausz et al., 1999).
However, the expression of Hand1 and Pl1
(Cross et al., 1995;
Faria et al., 1991), markers
of differentiated trophoblast giant cells, was undetectable in Eomes
presumptive null embryos (Fig.
8J). We also attempted to derive TS cell lines from blastocysts
obtained from Eomes+/– intercrosses
(Tanaka et al., 1998).
Twenty-one TS cell lines were derived from a total of 36 blastocysts initially
cultured. However, none was Eomes-/- by genotype.
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Discussion |
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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
REFERENCES |
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Examination of markers of TE differentiation in non-attached mutant embryos
revealed no expression of Hand1 and Pl1, markers of
trophoblast giant cells. The trophectoderm differentiation marker Pl1
can be detected in cultured wild-type blastocysts, even if they do not form
outgrowths (Nieder and Nagy,
1991). Thus, the failure of cultured Cdx2-/-
blastocysts to express Pl1 is not secondary to outgrowth failure but
represents a block in trophectoderm differentiation. Expression of the
trophoblast stem cell marker, Eomes, was also strongly reduced and TS
cell lines could not be derived from Cdx2-/- embryos,
suggesting a block in TS cell formation or self-renewal. As both stem cell and
giant cell fate are blocked by absence of Cdx2, this TF must play a
key role in early maintenance of the integrity and function of the blastocyst
TE.
Cdx2 is required for lineage-restricted expression of Oct4 and Nanog
In Cdx2-/- embryos, Oct4 and Nanog
are not downregulated in outer cells, and persist in these cells even by 4.5
dpc. Thus, it appears that Cdx2 plays a primary role in specifying
the fate of the trophectoderm cells by restricting the expression of
Oct4 and Nanog to the ICM. One explanation for the failure
of TE development in Cdx2-/- mutants could then be that
the outside cells initiate blastocyst epithelium formation but do not properly
specify TE fate. Retention of pluripotency-associated markers in the outer
cells might indicate transformation of outer cells to a more ICM-like
phenotype. However, it is not clear whether, in the absence of Cdx2,
outside cells are actually converted to ICM cells. Rather, increased levels of
TUNEL staining in the outer cells of Cdx2 mutants suggest that the
misexpression of Oct4 and Nanog is incompatible with maintenance of the TE
phenotype, leading to subsequent cell death. The remaining ICM cells in 4.5
dpc Cdx2-/- blastocysts can continue to develop into
embryoid body type structures with distinct epiblast and primitive endoderm
layers. Moreover, it is possible to generate Cdx2-/- ES
cells and to derive early somite embryos from them
(Chawengsaksophak et al.,
2004).
Cdx2 expression marks TE precursors prior to blastocyst formation
Cdx2 expression is restricted to prospective TE cells prior to
restriction of Oct4/Nanog to the ICM, consistent with a role in
downregulation of Oct4/Nanog in the TE. The timing and expression
domains of Cdx2 and Oct4/Nanog in normal embryos, and the
upregulation of Oct4/Nanog in Cdx2 mutants might suggest
that Cdx2 restriction to outer cells is the primary driver of the
divergence of ICM and TE lineages. Consistent with this, Niwa et al. show that
overexpression of Cdx2 is sufficient to drive differentiation of ES
cells into trophoblast cells, but Cdx2 is not necessary for
trophoblast differentiation if Oct4 is directly downregulated (H.
Niwa, unpublished).
Early segregation of Cdx2 to the outer cells of the morula and
early blastocyst may thus be key for initiating TE/ICM specification. Whether
Cdx2 can directly regulate Oct4/Nanog at the transcriptional level or
acts post-transcriptionally is currently unknown. What drives the segregation
of Cdx2 to the outside cells also remains to be determined, although
the similar restricted expression pattern of Fgfr2 in the outside
cells (Haffner-Krausz et al.,
1999) and the known role for this signalling pathway in promoting
TE development (Chai et al.,
1998) suggests that FGF signalling may be important upstream of
Cdx2.
Why does blastocoel initiate in the absence of Cdx2?
Although Cdx2 has a pivotal role in the development of a fully
functional TE and downregulation of the pluripotency-associated genes,
blastocoel initiation does not require zygotic Cdx2. Nor does it seem
that TE initiation can be explained by persistent maternal Cdx2 protein, as
little or no Cdx2 protein was observed in Cdx2-/- morulae
and early blastocysts.
Other factors could support the initial development of the TE epithelium in
the absence of Cdx2. Two additional members of the Caudal-type TF
family, Cdx1 and Cdx4, are found in the mouse. Previous analyses indicated
that the three Cdx family members share partially overlapping expression
patterns and functions along the embryonic axis, but only Cdx2 is
expressed in the postimplantation trophoblast, making redundancy with other
Cdx genes unlikely (Beck et al.,
1995; Gamer and Wright,
1993; Meyer and Gruss,
1993; van den Akker et al.,
2002).
Eomes is expressed in the TE of the blastocyst, and persists in
the proximal region of the ExE in early post-implantation stages. Similarity
of expression with Cdx2 might suggest overlapping functions. However,
we show that both phenotypic and expression analysis place Cdx2
upstream of zygotic Eomes in TE development and make it unlikely that
the two genes are acting redundantly in the initial specification of the TE
epithelium. Unlike Cdx2 mutant embryos, Eomes-/-
blastocysts cause a decidual reaction and show full expansion and maintenance
of the blastocoel in vitro, indicative of a functional TE
(Russ et al., 2000) (this
study). Cdx2 is expressed normally and Oct4 expression is
segregated to the ICM, indicating that Eomes is not required for
initial ICM/TE separation. However, neither trophoblast giant cells nor TS
cells can develop from Eomes mutants and markers of TE
differentiation are lost. This does not exclude overlapping functions for the
two genes later in TS cell development, as may be indicated by the failure to
obtain TS cells in both cases. Elucidation of their later roles will require
timed conditional inactivation studies.
In conclusion, our studies have uncovered that Cdx2 and Eomes are key TFs required at distinct stages during early TE lineage development. Eomes is required for TE differentiation and proliferation beyond the expanded blastocyst stage, while Cdx2 is the earliest TE-specific TF essential for TE function and establishment of the trophoblast lineage, as well as for the lineage restricted expression of the pluripotency markers, Oct4 and Nanog. Therefore, cell fate specification in the preimplantation embryo relies on positive acting TFs in both the ICM and TE lineages. Elucidation of the regulatory mechanisms that underlie the restricted expression and feedback loops between these TFs during morula stages should shed light on how cell fate specification is initiated in the preimplantation embryo.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/9/2093/DC1
* Present address: Department of Biochemistry and Molecular Biology,
University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA 
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REFERENCES |
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TOP
SUMMARY
Introduction
Materials and methods
Results
Discussion
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