DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE 3383
Development 137, 3383-3391 (2010) doi:10.1242/dev.050195
© 2010. Published by The Company of Biologists Ltd
Disorganized epithelial polarity and excess trophectoderm
cell fate in preimplantation embryos lacking E-cadherin
Robert Odell Stephenson1,2, Yojiro Yamanaka3 and Janet Rossant1,2,*
SUMMARY
The first two cell lineages in the mouse, the surface trophectoderm (TE) and inner cell mass (ICM), are morphologically
distinguishable by E3.5, with the outer TE forming a polarized epithelial layer enclosing the apolar ICM. We show here that in
mouse embryos completely lacking both maternal and zygotic E-cadherin (cadherin 1), the normal epithelial morphology of
outside cells is disrupted, but individual cells still initiate TE- and ICM-like fates. A larger proportion of cells than normal showed
expression of TE markers such as Cdx2, suggesting that formation of an organized epithelium is not necessary for TE-specific gene
expression. Individual cells in such embryos still generated an apical domain that correlated with elevated Cdx2 expression. We
also show that repolarization can occur in isolated early ICMs from both wild-type and Cdx2 mutant embryos, indicating that
Cdx2 is not required for initiating polarity. The results demonstrate that epithelial integrity mediated by E-cadherin is not
required for Cdx2 expression, but is essential for the normal allocation of TE and ICM cells. They also show that Cdx2 expression is
strongly linked to apical membrane polarization.
INTRODUCTION
At E3.5, the mouse embryo consists of two cell types, the outside
trophectoderm (TE), which generates the embryonic portion of the
placenta, and the inner cell mass (ICM), which later forms the
embryo itself along with other extraembryonic structures, including
the yolk sac endoderm (Yamanaka et al., 2006). The mechanisms
by which these two cell lineages emanate from what is likely to be
a uniform and totipotent population of cells at the 8-cell stage is a
continuous focus of research. In this paper, we focus upon the roles
of cell adhesion and apical-basal polarity in the divergence of these
two cell populations.
At the 8-cell stage a number of morphological changes occur
that are believed to be important for the divergence of the first two
cell lineages. The first of these is compaction, which involves a
smoothening of the surface of the embryo, associated with an
increase in intercellular adhesion mediated by cadherin 1 (Cdh1;
hereafter referred to as E-cadherin) (Shirayoshi et al., 1983).
Concomitant with compaction, individual blastomeres undergo
apical-basal polarization (Ducibella et al., 1977; Johnson and Maro,
1986). This involves the formation of an apical microvillusenriched polar region on the outer surface of the embryo associated
with proteins including the atypical protein kinase (aPKC) isoforms
PKC and PKC/ (Prkcz and Prkci), Par6 and ezrin (Ducibella et
al., 1977; Louvet et al., 1996; Pauken and Capco, 2000; Reeve and
Ziomek, 1981; Vinot et al., 2005). E-cadherin is involved in the
formation of adherens junctions at points of cell-cell contact, which
1
Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8,
Canada. 2Program in Developmental and Stem Cell Biology, The Hospital for Sick
Children Research Institute, Toronto, ON M5G 1X8, Canada. 3Rosalind and Morris
Goodman Cancer Research Centre, Department of Human Genetics, Faculty of
Medicine, McGill University, Montreal, QC H3A 1A3, Canada.
*Author for correspondence ([email protected])
Accepted 4 August 2010
are regions enriched in proteins such as Lgl, Jam1 (F11r – Mouse
Genome Informatics) and Par1 (Mark2 – Mouse Genome
Informatics) (Thomas et al., 2004; Vinot et al., 2005; Yamanaka et
al., 2003; Yamanaka et al., 2006).
Establishment of apical-basal polarity is thought to permit
polarized divisions of blastomeres, whereby daughter cells
inheriting the apical pole remain at the embryo surface and apolar
progeny become enclosed within the embryo (Johnson and
Ziomek, 1983; Johnson and Ziomek, 1981). By the 32-cell stage
(E3.0), the outside polar cells strengthen their intercellular adhesion
by formation of tight junctions so as to generate an epithelial
monolayer, which will form the TE (Fleming et al., 2000). This
epithelium ensures a seal that allows the formation of the blastocoel
cavity and the generation of a blastocyst. The inside apolar cells,
now the ICM, are compressed to one side of the cavity.
Several lineage-specific transcription factors have been
identified that play important roles in TE and ICM fate. Expression
of the transcription factor Oct4, although ubiquitous early on,
becomes restricted to the ICM after E3.5 (Dietrich and Hiiragi,
2007; Palmieri et al., 1994). Embryos lacking Pou5f1, which
encodes Oct4, generate an ICM that expresses TE markers (Nichols
et al., 1998; Ralston et al., 2010). Cdx2 expression, by contrast,
becomes restricted exclusively to outside TE progenitors prior to
Oct4 restriction at ~E3.5 (Dietrich and Hiiragi, 2007; Niwa et al.,
2005; Ralston and Rossant, 2008). Loss of Cdx2 leads to the
ectopic expression of ICM markers in the TE and an inability to
sustain TE development (Strumpf et al., 2005). The early restriction
of Cdx2 expression, along with its role in inhibiting the expression
of ICM-specific transcription factors in the TE, indicate that Cdx2
is an essential factor for the divergence of TE and ICM lineages.
Cdx2 expression itself is regulated by the transcription factor Tead4
and its co-activator partner Yap (Yap1 – Mouse Genome
Informatics) (Nishioka et al., 2009; Nishioka et al., 2008; Yagi et
al., 2007). Although Tead4 is expressed in all cells of the
preimplantation embryo, nuclear Yap is restricted to the developing
TE, thus restricting Cdx2 expression to these outside cells. Yap
DEVELOPMENT
KEY WORDS: Trophectoderm, Inner cell mass, Blastocyst, Polarity, Polarization, Apical, Basolateral, E-cadherin, Cdh1, Cdx2, Yap, PKCzeta,
Jam1, Lgl, Par6, P-cadherin, Mouse
3384 RESEARCH ARTICLE
MATERIALS AND METHODS
Mouse lines and genotyping
Cdh1tm2Kem mice (with a floxed allele of Cdh1) (Boussadia et al., 2002)
were backcrossed to ICR mice for four generations and intercrossed to
generate mice homozygous for the floxed allele. Mice lacking zygotic,
maternal or both maternal and zygotic E-cadherin were generated as
shown in Fig. S1 in the supplementary material. Additional mice used
in this study include wild-type ICR, Tg(Zp3-cre)3Mrt mice carrying the
Zp3-cre transgene (Lewandoski et al., 1997) and Cdx2tm1Fbe mice, which
carry a null allele of Cdx2 (Chawengsaksophak et al., 1997). Genotyping
of mice was performed on DNA extracted from ear punches or
blastocysts using the RedExtract-N-Amp Kit (Sigma). DNA preparation
for ears was performed as per manufacturer’s specifications. Blastocysts
were lysed in 4.4 l Extraction and 1.1 l of Tissue Preparation
solutions from the RedExtract-N-Amp Kit for 30 minutes at 25°C then
30 minutes at 56°C; then, 4.4 l Neutralization Buffer was added after
incubation at 95°C. To determine the presence of the Zp3-cre transgene,
a primer pair specific for cre was used: Zp3-creF 5⬘-TGATGAGGTTCGCAAGAACC-3⬘ and Zp3-creR 5⬘-CCATGAGTGAACGAACCTGG-3⬘. PCRs for the presence of the Cdh1tm2Kem and Cdx2tm1Fbe
alleles were performed as described previously (Boussadia et al., 2002;
Chawengsaksophak et al., 1997).
Embryo collection and culture
Embryos were collected from timed natural matings. Preimplantation
embryos were collected by flushing dissected oviducts or uteri with M2
medium (Specialty Media, Chemicon). Embryos and ICMs were cultured
in 15 l drops of KSOM under mineral oil at 37°C in a CO2 incubator for
specified times, if not examined immediately (Nagy et al., 2003).
ICM isolation
Zona pellucidae were removed with acid-Tyrode’s solution. Embryos were
allowed to recover for 2 hours and were then treated with non-adsorbed
rabbit anti-mouse lymphocyte serum (Cedarlane) diluted 1/8 in KSOM (50
l) for 20 minutes in a CO2 incubator at 37°C. Embryos were washed six
times with KSOM and then added to Alexa 488 anti-rabbit antibody for 10
minutes in a CO2 incubator at 37°C. Embryos were washed six times with
KSOM and then added to standard guinea pig complement (Cedarlane)
diluted 1/4 in KSOM (70 l) for 8 minutes in a CO2 incubator at 37°C.
Finally, embryos were washed six times in M2 medium and dead cells
were removed from ICMs by pipetting through a fine pulled glass needle.
Efficiently isolated ICMs, i.e. those lacking fluorescence upon examination
under a microscope, were then cultured in KSOM to the time points
indicated.
Whole-mount immunofluorescence
Embryos were fixed in 4% formaldehyde at room temperature for 30
minutes, washed three times in PBS containing 0.1% Triton X-100,
permeabilized for 30 minutes in 0.25% Triton X-100 and then blocked
in PBS with 10% foetal calf serum overnight at 4°C. For claudin 6 and
Na/K ATPase b1 immunohistochemistry, embryos were fixed for 15
minutes in 10% trichloroacetic acid at 4°C. Primary antibodies were
diluted in blocking solution and embryos were incubated in this solution
overnight at 4°C (19-20 hours). Embryos were then washed three times
in PBS and incubated in secondary antibodies diluted 1/400. Primary
antibodies were: mouse anti-Cdx2 (1/400, CDX-88, Biogenex), rabbit
anti-Cdx2 (1/400) (Chawengsaksophak et al., 1997), rabbit anti-Nanog
(1/200, Cosmobio), mouse anti-Oct4 (1/100, Santa Cruz), goat antihuman GATA6 (1/200, R&D Systems), goat anti-claudin 6 (1/200, Santa
Cruz), rat anti-Jam1 (1/400, HyCult), rabbit anti-Lgl1 (Plant et al.,
2003), mouse anti-Na/K ATPase b1 (1/1000, Upstate), goat antiP-cadherin (1/200, R&D Systems), rabbit anti-PKC (1/400, Santa
Cruz), rat anti-Uvomorulin (E-cadherin) (1/100, Sigma); rabbit antiCdx2 was only used for experiments involving co-staining with mouse
anti-Oct4, otherwise mouse anti-Cdx2 was used. Secondary antibodies
included: Alexa 488 goat anti-mouse and anti-rabbit, Alexa 488 donkey
anti-goat, Alexa 546 goat anti-mouse and anti-rabbit, and Alexa 647
donkey anti-mouse (all Molecular Probes). Dylight 549 donkey antirabbit (Jackson ImmunoResearch) was only used for reactions when
embryos were co-stained for Gata6 and Nanog. Nuclei were labeled
using either Hoechst 33342 (2.5 g/ml, Molecular Probes) or Draq5
(1/400, Alexis Biochemicals).
DEVELOPMENT
nuclear localization has been shown to be regulated by
phosphorylation and cytoplasmic sequestration mediated by the
Hippo pathway member Lats2 (Nishioka et al., 2009).
The close association between restriction of Cdx2 expression and
formation of the outer polarized epithelium raises the question of
whether there is a causal link between cell polarity, epithelium
formation and TE cell fate. Previous work has demonstrated that Ecadherin is the primary mediator of cell adhesion in the
preimplantation embryo and that embryos lacking zygotic E-cadherin
are preimplantation lethal (De Vries et al., 2004; Larue et al., 1994;
Ohsugi et al., 1997) with failure to form an organized blastocyst.
However, some cells in mutants lacking zygotic E-cadherin still
express Krt8, which is usually a marker of TE fate (Ohsugi et al.,
1997). Other research has demonstrated a link between apical-basal
polarization and cell allocation. Notably, disruption of the activity of
Par3 and PKCproteins localized to the apical pole can direct the
progeny of early blastomeres preferentially to the inside of the
embryo (Plusa et al., 2005). Although fate markers were not
examined in these embryos, these observations suggest that
polarization is likely to be upstream of lineage-specific transcription
factor expression. In Cdx2 mutants, initial polarization occurs but the
outer polarized epithelium fails to be maintained, suggesting that
Cdx2 is not required to initiate polarity (Strumpf et al., 2005).
Although this study found no evidence for a maternal contribution
of Cdx2, a recent study suggests that maternal Cdx2 is present in the
early embryo. Furthermore, knockdown of maternal Cdx2 suggested
that it regulates the expression level of polarity proteins in early
blastomeres (Jedrusik et al., 2010). Also, when isolated inside apolar
cells repolarize (Eckert et al., 2004; Johnson and Ziomek, 1983) they
re-express Cdx2 (Suwinska et al., 2008), again supporting a close
correlation between polarity and lineage specification. A recent study
suggested that increased expression of Cdx2 could polarize cells and
bias them towards TE fate, whereas reduced expression of Cdx2 led
to blastomeres being preferentially allocated to the ICM (Jedrusik et
al., 2008; Ralston and Rossant, 2008; Strumpf et al., 2005).
However, no similar bias was observed when Cdx2 mutant
blastomeres were mixed with wild-type cells in chimaeric aggregates
(Ralston and Rossant, 2008). It is therefore still unclear whether
polarization drives the TE differentiation program or Cdx2
expression drives morphogenetic changes associated with TE fate.
The focus of the current study was to further clarify the
relationship between formation of an organized polar epithelium,
apical-basal polarity and lineage segregation. We generated mouse
embryos completely lacking both maternal and zygotic E-cadherin
and showed that they fail to form an organized outer polar
epithelium and, subsequently, an organized blastocyst. However,
the majority of cells showed expression of TE markers such as
Cdx2, suggesting that formation of an organized epithelium is not
necessary for TE-specific gene expression. Individual cells in such
embryos still generated an apical membrane domain that correlated
with elevated Cdx2 expression. We also showed that repolarization
can occur in isolated early ICMs from both wild-type and Cdx2
mutant embryos, indicating that Cdx2 is not required for initiating
polarity. These results demonstrate that Cdx2 expression does not
require the formation of an organized epithelium, but is strongly
linked to intracellular polarization.
Development 137 (20)
Confocal microscopy and image analysis
Embryos were mounted in PBS containing 0.1% Triton X-100 in wells of
Secure Seal spacers (Molecular Probes) and placed between two cover
glasses for imaging. Images were captured using a Zeiss Axiovert 200
inverted microscope equipped with either a Hamamatsu Orca AG CCD
camera and Quorum spinning disk confocal scan head or a Zeiss LSM510
META emission scan head. The spinning disk confocal was driven by
Volocity Acquisition software (Perkin Elmer), and the laser-scanning
microscope was driven with the LSM510 software package (Zeiss).
Images were taken using a 25⫻ or 30⫻ water-immersion objective.
Settings were unchanged within a given session to permit direct
comparison of expression levels among embryos stained in the same
batch. Image series were taken with 0.5 m between z-sections.
Expression levels were estimated from Cdx2, Oct4 and Yap
immunohistochemistry by measuring average pixel intensity and dividing
by corresponding values for Hoechst using ImageJ software. Cells were
binned as expressing strong, weak or no Cdx2 by eye for half of control
embryos (wild type or unmanipulated) and expression levels were
recorded for each cell to determine bin ranges. Cells in remaining controls
were binned based exclusively on expression levels and bins were
validated by visual inspection. Scoring of PKC and Par6 localization was
carried out blind from Cdx2 expression data.
Time-lapse imaging was performed on a Zeiss Axiovert inverted
microscope equipped with an environment controller as previously
described (Yamanaka et al., 2010). Embryos were placed in a glass-bottom
dish (MatTek) in KSOM covered with mineral oil. A 20⫻ dry (NA0.75)
objective lens and Axiocam MRm (1388⫻1044 pixels) camera were used.
Images were taken every 15 minutes for 48 hours.
RESULTS
E-cadherin is not required for TE differentiation
To investigate the roles of compaction and cell adhesion in the
generation of the ICM and TE, we used mouse embryos totally
deficient in E-cadherin. Crossing a floxed allele of Cdh1 created in
the Kemler laboratory (Boussadia et al., 2002) with mice expressing
oocyte-specific Zp3-cre (Lewandoski et al., 1997) we generated
mutants lacking maternal E-cadherin, zygotic E-cadherin or both
maternal and zygotic E-cadherin (see Fig. S1 in the supplementary
material for crosses and images of embryos). As previously reported,
embryos lacking maternal E-cadherin compacted late, but otherwise
formed normal blastocysts (De Vries et al., 2004). Embryos lacking
zygotic E-cadherin compacted normally, but consisted of loosely
associated cells by E3.5, as shown previously (Larue et al., 1994). In
the absence of both maternal and zygotic E-cadherin (mz mutants),
embryos never compacted but developed as a loose aggregate of
cells (see below). Preliminary E-cadherin mutant crosses
demonstrated that although most embryos divided normally to E3.5
(12/14 wild-type embryos, 30/37 heterozygous embryos, 18/23
maternal mutants, 20/28 zygotic E-cadherin mutants and 21/32 mz
E-cadherin mutants showed little cell death and normal cell
numbers), some never developed past the 8- to 16-cell stage, before
the decision to form TE or ICM (see Table S1 in the supplementary
material). Similarly, a delay of cell division is observed in embryos
cultured in the presence of E-cadherin-blocking antibody and in
calcium-free medium (Dietrich and Hiiragi, 2007; Ducibella et al.,
1975). This indicates that E-cadherin might have a role in early cell
division. However, mutant embryos that survived past this stage
showed little effect with regard to total cell number (Fig. 1G). Since
the focus of our current study is on the role of E-cadherin in cell fate,
we excluded developmentally compromised embryos from the
experiments described below.
When the zona pellucida was removed from mz mutants, there
was still some residual cell adhesion (not shown) even though the
absence of E-cadherin was confirmed by immunohistochemistry in
RESEARCH ARTICLE 3385
confocal sections (see Fig. S2 in the supplementary material).
Indeed, we found that cadherin 3 (also known as P-cadherin),
which is normally present at cell-cell junctions in wild-type
embryos at E3.5, was also present at residual cell-cell junctions in
E-cadherin mutants (see Fig. S3A,B,C in the supplementary
material). We further found claudin 6, a component of tight
junctions, at a few of these residual cell-cell junctions in mz
mutants, suggesting that residual cell adhesion in E-cadherin
mutants could permit the formation of tight junctions (see Fig.
S3D-F in the supplementary material).
To determine the effects of loss of E-cadherin on cell fate, we
examined the expression of the TE- and ICM-specific transcription
factors Cdx2 and Oct4 in E-cadherin mutant embryos. As
expression of Oct4 is not fully restricted to the ICM in wild-type
embryos by E3.5, we flushed embryos from the uterus at E3.5 and
cultured them for one additional day before fixation (Dietrich and
Hiiragi, 2007; Ralston and Rossant, 2008; Strumpf et al., 2005).
Expression of Oct4 and Cdx2 in heterozygous and maternal mutant
embryos was virtually indistinguishable from that in wild-type
embryos. There was a clear restriction of expression of Oct4 and
Cdx2 to the ICM and TE, respectively (Fig. 1A-C). Examination
of zygotic and mz mutant embryos revealed that Cdx2 and Oct4
expression was mutually exclusive in most cases, although cells
expressing these markers were not necessarily arranged in the usual
spatial manner (Fig. 1D,E, arrowheads and arrows, respectively;
see also Fig. S4 in the supplementary material). This indicates that
cells in E-cadherin mutants can adopt a TE-like fate despite the fact
that these embryos formed neither a blastocoel cavity nor organized
TE epithelia. Some Cdx2 and Oct4 co-expressing cells persisted in
zygotic and mz mutants (Fig. 1D,E, asterisks; see Fig. S4 in the
supplementary material). These could represent cells that were
developmentally delayed and had not yet resolved expression of
these fate markers. When we counted the number of Cdx2-positive
cells in embryos of all genotypes, we found a significant increase
in the proportion of Cdx2-positive cells in both zygotic and mz
mutant embryos in comparison to wild type and heterozygotes (Fig.
1F). In the most extreme cases, almost all of the cells in these
embryos were Cdx2 positive (4 of 8 zygotic mutant embryos had
greater than 95% Cdx2-positive cells and 5 of 7 mz mutant
embryos had over 95% Cdx2-positive cells). Importantly, there was
no difference in total cell numbers between embryos of all
genotypes (among embryos that developed past the 16-cell stage),
indicating that there was unlikely to be a difference in cell
proliferation or death (Fig. 1G). Categorization of Cdx2-expressing
cells as strongly or weakly positive by quantitation of fluorescence
levels revealed that the increased proportion of Cdx2-expressing
cells in zygotic and mz mutants was primarily due to an increase
in the number of weakly positive cells (Fig. 1F). This suggested
that the absence of cell adhesion prevented the downregulation of
the early, widespread, low-level expression of Cdx2 observed in
normal development (Dietrich and Hiiragi, 2007; Ralston and
Rossant, 2008).
To confirm that Cdx2-positive cells in these embryos constituted
cells initiating a TE fate, we examined the expression of the TEspecific intermediate filament protein Krt8 with respect to Cdx2
(Fig. 1H-J). We scored cells in mutant and wild-type embryos as
Krt8- or Cdx2-positive and then overlaid the datasets, counting the
number of co-expressing cells. This analysis revealed that
expression of Krt8 was tightly linked to Cdx2 in both wild-type and
mutant embryos. Only infrequently was Krt8 expressed on its own
(Fig. 1K). Notably, the proportion of Krt8 and Cdx2 doublepositive cells was significantly greater in mz and zygotic mutant
DEVELOPMENT
Morphology and cell fate in blastocyst
3386 RESEARCH ARTICLE
Development 137 (20)
Fig. 1. E-cadherin is not required for initiation of TE fate. (A-E)Mouse embryos flushed from uteri at E3.5 and cultured for 24 hours. (A)Wild
type (WT), (B) E-cadherin heterozygous, (C) maternal E-cadherin mutant, (D) zygotic E-cadherin mutant, (E) maternal and zygotic (mz) E-cadherin
mutant. Cdx2 in red, Oct4 in green. Arrows indicate Oct4-expressing cells, arrowheads indicate Cdx2-expressing cells and asterisks indicate Oct4
and Cdx2 co-expressing cells. Scale bars: 20m. (F)Average percentage Cdx2-positive cells per embryo for each genotype. Dark red represents
strongly expressing cells and pink, weakly expressing cells. To ensure clearer resolution of Cdx2 levels between trophectoderm (TE) and inner cell
mass (ICM), embryos were flushed at E3.5 and cultured for 6 hours before fixation. Standard deviation is expressed as deviation for all Cdx2positive cells, weak and strong. *P<0.01 by Student’s t-test assuming equal variances with Bonferroni correction for multiple comparisons.
(G)Average number of cells per embryo for each genotype in F. Note that only a random subset of heterozygous embryos were used for this data,
to balance sample sizes. (H-J)Wild-type, zygotic mutant and mz mutant embryos flushed from uteri at E3.5 and cultured for 6 hours. Draq5 nuclear
stain is in blue, Krt8 in green, Cdx2 in red. (K)Cdx2 and Krt8 co-expression in embryos. n128, 234 and 196 total cells for wild type, zygotic
mutants and mz mutants, respectively (from 2, 4 and 3 embryos). There was no significant difference between wild-type and mutant embryos in
the proportion of cells expressing exclusively Cdx2 or Krt8 (2 comparing cells expressing either Cdx2 or Krt8 alone versus all other cells, P>0.5). The
proportion of Krt8 and Cdx2 double-positive cells was significantly greater in mz and zygotic mutant embryos than in wild-type embryos (as
determined by 2, P<0.01 for wild type versus zygotic mutants and wild type versus mz mutants).
their correct positioning in the embryo are dependent upon Ecadherin-mediated cell adhesion and the development of a normal
outer epithelium.
Polarization is altered in E-cadherin mutants at E3.5
Earlier work demonstrated that when E-cadherin-mediated cell
adhesion is blocked by antibody treatment, cells in preimplantation
embryos can still polarize as judged by concanavalin-A binding to
the apical pole (Shirayoshi et al., 1983). However, this analysis did
not assess whether basolateral polarization was affected, nor whether
E-cadherin function was completely lost. We therefore examined cell
polarity in E-cadherin mutant embryos using current molecular
markers of apical and basolateral polarity. Specifically, we examined
the distribution of the apical protein PKC and the basolateral
proteins Na/K ATPase b1, Jam1 and Lgl1 in E3.5 (blastocyst stage)
embryos. In wild-type and heterozygous embryos, PKC was
strongly enriched in the apical membrane domain of TE cells,
whereas ICM cells showed a cytoplasmic distribution with weak
DEVELOPMENT
embryos than in wild-type embryos (Fig. 1K). This suggests that
more cells in E-cadherin mutants were initiating TE fate than in
wild-type embryos. We also confirmed that when Cdx2-negative
cells were present, they were Nanog- or Gata6-positive (see Fig.
S5 in the supplementary material, arrows and arrowheads,
respectively), confirming that Cdx2-negative cells in mz and
zygotic mutants were adopting ICM fates.
Interestingly, many Cdx2-positive cells were found in the
interior of mutant embryos. Whereas only 4% of wild-type inner
cells were strongly Cdx2 positive, 51% and 44% of zygotic and mz
mutant inside cells were strongly Cdx2 positive, respectively.
Conversely, whereas Cdx2-negative cells never appeared on the
surface of wild-type and heterozygous embryos, they were found
in zygotic mutant embryos (6/29 Cdx2-negative cells were outside
in 8 embryos) and in mz mutant embryos (3/9 Cdx2-negative cells
were outside in 7 embryos). Thus, although compaction and
epithelial organization are not absolutely required for the adoption
of TE or ICM cell fate, the correct ratio of the two cell types and
Morphology and cell fate in blastocyst
RESEARCH ARTICLE 3387
membrane enrichment (Fig. 2A⬘), similar to previous reports (Eckert
et al., 2004; Plusa et al., 2005; Ralston and Rossant, 2008). The Na/K
ATPase b1 subunit and Jam1 were localized to cell-cell contacts of
the ICM and were absent from the apical surface of TE (Fig. 2A⬙; see
Fig. S6A in the supplementary material), as previously reported
(Madan et al., 2007; Thomas et al., 2004). Similarly, we found that
Lgl1, another protein localized to the basolateral membrane in other
epithelia, was associated specifically with the basolateral domain in
the blastocyst TE (see Fig. S6B in the supplementary material)
(Hutterer et al., 2004; Yamanaka et al., 2003; Yamanaka et al., 2006).
In both zygotic and mz E-cadherin mutants, PKC was strongly
enriched in the membrane and lost from the cytoplasm of the
majority of cells, similar to what is observed in wild-type TE cells
(Fig. 2B⬘,C⬘). In contrast to wild-type TE cells, however, PKC was
enriched throughout the majority of the outer cell membrane in
zygotic and mz mutant cells, rather than in a specific apical domain.
The Na/K ATPase b1 subunit, Jam1 and Lgl1 were still found at the
surface in mutant cells; however, their localization overlapped with
membrane-enriched PKC (Fig. 2B⬘,B⬙,C⬘,C⬙). Strikingly, these
basolateral markers also accumulated in either small puncta or large
vacuole-like structures within the cell (Fig. 2B⬙,C⬙; see Fig.
S6A⬘,A⬙,B⬘,B⬙ in the supplementary material). Thus, in the absence
of E-cadherin-mediated adhesion, proteins that normally form the
basolateral domain tend to be retained in vacuoles within the cell
(Fig. 2D). When these markers were trafficked to the cell surface they
tended to colocalize with PKC, suggesting that sorting of apical and
basolateral proteins could be disrupted in E-cadherin mutants.
We found a significant increase in the proportion of cells with
membrane enrichment of the apical protein PKC and a paucity of
PKC in the cytoplasm in E-cadherin mutants. In wild-type embryos,
64% of cells had membrane-enriched PKC, whereas 87% of
zygotic mutant cells and 88% of mz mutant cells had membraneenriched PKC (Student’s t-test, P<0.01; n7, 8 and 7 embryos,
respectively; see Fig. S7A in the supplementary material). We then
scored cells as Cdx2 positive or negative and overlaid these data with
those for PKC expression. Cells with membrane-enriched PKC of
wild-type, zygotic mutant and mz mutant embryos were almost all
Cdx2 positive (99%, 98% and 99%, respectively). Some cells
DEVELOPMENT
Fig. 2. Localization of polarity proteins in E-cadherin mutants. (A-C⬙) Immunohistochemistry for PKC in red (A⬘,B⬘,C⬘) and Na/K ATPase b1 in
green (A⬙,B⬙,C⬙) in (A) wild-type, (B) zygotic E-cadherin mutant and (C) mz E-cadherin mutant mouse embryos. The boxed regions are shown at
higher magnification beneath. Arrows indicate membrane-enrichment of PKC. Arrowheads show enrichment of Na/K ATPase b1. All nuclei are
marked with Hoechst nuclear stain (blue). Scale bars: 20m. (D)Model for polarization in E-cadherin mutant cells. The apical domain appears to
form; however, proper specification of the basolateral domain is disrupted. Apical and basolateral proteins tend to overlap in mutant cells and
basolateral proteins also accumulate in intracellular puncta and vacuole-like structures. (E)Cdx2 expression levels (arbitrary units) in cells with
membrane-enriched PKC (red) and lacking this enrichment (gray). Cdx2 expression levels were significantly higher in cells with membrane-enriched
PKC than in cells without membrane-enriched PKC in wild-type, zygotic mutant and mz mutant embryos (Student’s t-test, *P<0.01 for all
genotypes). For wild type: n5 embryos, 190 cells with membrane-enriched PKC, 95 without. For zygotic mutants: n8 embryos, 432 cells with
membrane-enriched PKC, 50 without. For mz mutants: n4 embryos, 147 cells with membrane-enriched PKC, 33 without.
3388 RESEARCH ARTICLE
Development 137 (20)
lacking PKC enrichment also expressed Cdx2 (16% of wild-type,
67% of zygotic mutant and 89% of mz mutant apolar cells).
However, examination of the intensity of Cdx2 expression
demonstrated that Cdx2 expression levels were significantly higher
in cells with membrane-enriched PKC in comparison to those
without in wild-type, zygotic mutant and mz mutant embryos (Fig.
2E). Given that Cdx2-expressing cells are still observed in Ecadherin mutants, the formation of a restricted apical domain per se
is not necessary for Cdx2 expression. However, apical membrane
specification itself could still be important for TE specification, as
expression of apically localized markers and Cdx2 expression remain
tightly linked in E-cadherin mutants.
Examination of Yap localization in cells with and without
membrane-enriched PKC in E-cadherin mutants revealed that Yap
was localized to the nuclei of cells with membrane-enriched PKC
and to the cytoplasm of cells without membrane-enriched PKC,
independent of their spatial distribution in the embryo (Fig.
3B⬘,C⬘,D,E). Strikingly, Yap was enriched in the cytoplasm of cells
lacking membrane-enriched PKC even when they were found on
the surface of E-cadherin mutant embryos (Fig. 3B⬘,C⬘, arrows).
This indicates that localization of apical markers, and not just cell
position, is important for Yap nuclear localization. There was also
a greater number of cells with nuclear localized Yap in E-cadherin
mutants (62% of cells in wild-type embryos had nuclear localized
Yap, in comparison to 87% and 82% for zygotic and mz mutants,
respectively; see Fig. S7B in the supplementary material). Thus,
increased Yap activity could account for the increased proportion
of Cdx2-positive cells in these embryos.
Polarization anticipates Cdx2 expression and
upregulation in isolated ICMs
Clearly, intracellular polarization can occur in the absence of Ecadherin, but the normal segregation of the apical and basolateral
domains is disrupted. Apical membrane marker localization is
associated with strong Cdx2 expression, suggesting that apical
polarity might drive upregulation of Cdx2. To test this further we
examined whether polarization precedes Cdx2 upregulation in other
contexts. Recent work from our laboratory has demonstrated that
polarization of early blastomeres precedes upregulation of Cdx2
expression in undisturbed embryos (Ralston and Rossant, 2008). We
tested whether the same was true in a situation in which de novo
regeneration of TE occurs. When ICMs are immunosurgically
isolated from early blastocysts, they normally regenerate a TE layer
on their surface after culture (Hogan and Tilly, 1978; Spindle, 1978).
It was also recently demonstrated that cells on the surface of
aggregates formed from inner cells isolated from 16- and 32-cell
embryos can initiate Cdx2 expression and generate an apparently
morphologically normal TE (Suwinska et al., 2008).
To examine the relationship between polarization and Cdx2
expression in this context, we isolated ICMs at the ~32-cell stage,
when embryos had either no blastocoel or the cavity constituted less
than half the total embryo volume (late morula stage or early
blastocyst stage). Isolation and screening for efficiently isolated ICMs
took ~1 hour. ICMs were fixed immediately after isolation and
screening or after culture for 6, 12 or 30 hours. For ICMs cultured for
0, 6 and 12 hours, polarization was scored by enrichment of Par6 in
the cell surface membrane, as this is a clearer marker of polarization
DEVELOPMENT
Fig. 3. Membrane enrichment of PKC is associated with nuclear Yap in E-cadherin mutant cells. (A-C⬙) Yap (A⬘-C⬘) localization in wild-type
(A-A⬙), zygotic E-cadherin mutant (B-B⬙) and mz E-cadherin mutant (C-C⬙) mouse embryos stained for PKC(A-C) or nuclei (A⬙-C⬙). Arrowheads
indicate cells with membrane-enriched PKC and nuclear Yap. Arrows indicate cells lacking membrane-enriched PKC that have cytoplasmic Yap.
(D)Yap localization score (arbitrary units) in cells with membrane-enriched PKC (red) and those lacking enrichment (gray) of wild-type and zygotic
mutant embryos imaged in the same session. For wild type: n2 embryos, 64 cells with membrane-enriched PKC, 61 without. For zygotic mutants:
n6 embryos, 311 cells with membrane-enriched PKC, 36 without. (E)Yap nuclear enrichment score in wild-type and mz mutant embryos imaged
in the same session. For wild type: n3 embryos, 128 cells with membrane-enriched PKC, 67 without. For mz mutants: n3 embryos, 132 cells
with membrane-enriched PKC, 23 without. Similar to in wild-type embryos, in zygotic and mz mutants there was a significant difference in Yap
nuclear enrichment between cells with membrane-enriched PKC and those without (Student’s t-test, *P<0.01 for all genotypes). Yap localization
score was calculated by subtracting the intensity of cytoplasmic Yap expression from nuclear expression as determined by immunohistochemistry.
Scores above zero indicate enrichment of Yap in the nucleus, whereas scores below zero indicate enrichment of Yap in the cytoplasm.
Morphology and cell fate in blastocyst
RESEARCH ARTICLE 3389
Fig. 4. Cdx2 expression is linked to cell polarization in isolated
ICMs. (A-E⬙) ICMs immediately after isolation (A,B) and after 6 (C), 12
(D) and 30 (E) hours of culture. After 30 hours of culture only one ICM
did not express Cdx2 in all of its outside cells. These cells were polar but
morphologically resembled primitive endoderm, suggesting few cells on
the surface of ICMs had failed to regenerate TE (these are Cdx2negative polar cells in F). Immunohistochemistry for Cdx2 in green,
Par6 and PKC in red and Hoechst nuclear stain in blue. (A⬘-E⬘) Signal
for Cdx2 only. (A⬙-E⬙) Magnified views of boxed regions in A-E.
(B⬙-D⬙) Polar cells (as determined by Par6 enrichment in the plasma
membrane) are indicated by arrows. (E⬙)Arrow indicates polar cell with
membrane-enriched PKC. Scale bars: 20m. (F,G)Cdx2 expression in
(F) polar cells and (G) apolar cells of isolated ICMs after 0, 6, 12 and
30 hours of culture. Strong Cdx2-positive cells are represented in dark
green, weak Cdx2-positive cells in light green and Cdx2-negative cells
in black. For 0, 6, 12 and 30 hours, n7, 6, 4 and 6 ICMs, respectively.
than PKC at earlier time points (Fig. 4A-D). For embryos cultured
for 30 hours, polarization was determined by strong enrichment of
PKC in the membrane and paucity of PKC in the cytoplasm (Fig.
4E). Cells were blindly scored for Cdx2 expression and the two
datasets were overlaid. Surprisingly, we found that already
immediately after isolation, 32% of cells had begun to repolarize. This
proportion increased to 70% after 30 hours of culture. As expected,
the proportion of polar cells expressing Cdx2 also increased over time
(Fig. 4F). Immediately after isolation, half the polar cells (n18 polar
cells) were still Cdx2 negative, but by 30 hours almost all polar cells
(86%, n45 cells) showed strong Cdx2 expression (Fig. 4F). These
results clearly demonstrate that polarization can anticipate Cdx2
expression. Thus, as in E-cadherin mutants, there is a tight relationship
between apical domain formation and strong Cdx2 expression.
Cdx2 expression is not required for repolarization
in isolated ICMs
To test whether Cdx2 expression is required for repolarization of
ICM cells and regeneration of TE, we isolated and cultured the
early ICMs of Cdx2 mutant embryos as described above. After 30
DISCUSSION
We have investigated in detail the role that E-cadherin-mediated
cell adhesion plays in the restriction of cell fate in preimplantation
mouse embryos. We demonstrate that loss of both maternal and
zygotic E-cadherin leads to both the absence of normal segregation
of apical and basolateral domains and a failure to generate a
normal, organized epithelium at the blastocyst stage. However,
cells in mutant embryos can still initiate TE and ICM fate, although
the normal ratio of these cell types is disturbed. In E-cadherin
mutants more cells express Cdx2 than in wild-type embryos,
suggesting that E-cadherin-mediated cell adhesion is important for
the restriction of Cdx2 expression and TE fate. Moreover, the
normal spatial distribution of TE- and ICM-like cells to the outside
and the inside of the blastocyst, respectively, did not occur. This
result extends an earlier study demonstrating that delaying
blastocyst formation by temporarily blocking E-cadherin activity
with antibodies biases cells in blastocysts towards TE fate
(Shirayoshi et al., 1983).
Lats kinase is required to repress the expression of Cdx2 via
phosphorylation and nuclear exclusion of Yap (Nishioka et al.,
2009) in the preimplantation embryo. Lats kinase is activated by
upstream Hippo signaling in mammalian cells and Hippo signaling
itself is dependent upon cell-cell contact (Zhao et al., 2007). Thus,
lack of E-cadherin-mediated cell contact could lead to reduced
Hippo signaling and account for the increased proportion of Cdx2positive cells compared with wild-type embryos. Consistent with
this, we observed nuclear localization of Yap in Cdx2-positive cells
throughout E-cadherin mutant embryos, suggestive of reduced
Hippo signaling. However, in the few remaining apolar cells
observed, Yap was enriched in the cytoplasm, even when these
cells were found on the surface of the embryo. This indicates that
apical membrane polarization, and not just reduced cell contact, is
important for repression of Hippo signaling.
The polarization hypothesis states that outer cells that polarize
de novo or inherit an apical domain through cell division will
become TE, whereas inside apolar cells occupy the ICM (Johnson
and McConnell, 2004; Johnson and Ziomek, 1981; Yamanaka et
al., 2006). Previous work has shown that early blastomeres can
polarize in the absence of E-cadherin-mediated cell adhesion
(Shirayoshi et al., 1983; Ziomek and Johnson, 1980). We
confirmed in this study that, even in the complete absence of Ecadherin, cells still express apical markers on their surface,
although they were more broadly distributed across the cell surface
than in wild-type TE. We also found that although basolateral
markers could accumulate on the cell surface, their distribution
partially overlapped with that of apical markers. This suggests a
defect in the basolateral sorting machinery. Indeed, interference
with the transport of basolateral proteins by disrupting clathrindependent traffic can lead to an accumulation of basolateral
DEVELOPMENT
hours in culture, we found that mutant ICMs lacking Cdx2
expression regenerated a cell layer on their surface that resembled
TE (77% of all mutants compared with 79% of wild-type ICMs
and 93% of heterozygous ICMs; n13, 14 and 14 ICMs for each
genotype, respectively). This cell layer was polarized as judged by
PKC localization and was negative for the primitive endoderm
marker Gata6 (Fig. 5). In some cases, embryos even generated
blastocoels (54% of all mutants formed at least a small cavity,
compared with 57% and 79% of all wild-type and heterozygous
ICMs, respectively; Fig. 5). This result demonstrates that Cdx2
expression is not required for polarization of ICM cells, nor for
regeneration of a morphologically normal TE-like epithelial layer.
3390 RESEARCH ARTICLE
Development 137 (20)
Fig. 5. Cells in Cdx2-null ICMs can polarize. (A)Intact
Cdx2-null mouse embryo. (B)Cdx2 heterozygous mutant
isolated ICM. (C)Cdx2-null isolated ICM. Cdx2 in dark
blue, Gata6 in green, PKC in red and Hoechst stain in
blue. ICMs were isolated at E3.25 in order to reduce
mortality due to immaturity of the epithelium. Scale
bars: 20m.
maternally loaded transcript in this study implicated maternally
derived Cdx2 protein in cell survival, polarization and even TE
lineage specification (Jedrusik et al., 2010). Based on these results
it would be informative to examine maternal Cdx2-null embryos
for similar defects. Interestingly, TE regeneration is only found in
ICMs isolated from early blastocysts, a time at which Cdx2
expression is still apparent at low levels within the ICM (Dietrich
and Hiiragi, 2007; Ralston and Rossant, 2008); if ICMs are isolated
from later blastocysts, a primitive endoderm layer is generated on
their surface, rather than a TE layer (Handyside, 1978; Hogan and
Tilly, 1978; Spindle, 1978). Since most Cdx2-null ICMs still
regenerate a TE-like epithelium, we conclude that although Cdx2
expression is required to maintain TE, it is not the sole factor
determining whether ICM cells retain the ability to regenerate TE.
The results of this study demonstrate that cells of the
preimplantation embryo can initiate TE fate in the absence of
epithelium formation and E-cadherin-mediated cell adhesion. They
also demonstrate that these processes are important for the restriction
of apical membrane formation and for the correct segregation of TE
and ICM cell fates. Finally, these results provide support for a role
for apical membrane polarization in the upregulation and/or
maintenance of Cdx2 expression. Although Cdx2 expression can
initiate prior to polarization (Dietrich and Hiiragi, 2007; Ralston and
Rossant, 2008), maintenance and upregulation of expression appear
to occur only in polarized cells. Future work will need to address
how apical membrane polarization acts to facilitate Cdx2 expression;
for example, whether it facilitates/represses signaling involved in
Cdx2 expression, or whether it is involved in the segregation of a
determinant required for expression.
Acknowledgements
We thank members of the J.R. laboratory, especially Jorge Cabezas for animal
care and Andres Nieto and Valerie Prideaux for technical support; Amy Ralston
for suggestions and Brian Cox and Fredrick Lanner for critical reading of this
manuscript; Rolf Kemler for E-cadherin floxed mice, Gail Martin for Zp3-cre
mice and Tony Pawson for antibodies to Lgl1. This work was supported by the
Canadian Institutes of Health Research.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.050195/-/DC1
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DEVELOPMENT
Morphology and cell fate in blastocyst