|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 3 October 2007
doi: 10.1242/dev.010223
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Laboratory of Molecular Growth Regulation, National Institute of Child Health
and Human Development, National Institutes of Health, 6 Center Drive,
Bethesda, MD 20892-2753, USA.
2 Section on Molecular Neurobiology, National Institute of Child Health and
Human Development, National Institutes of Health, 35 Lincoln Drive, Bethesda,
MD 20892-3714, USA.
Author for correspondence (e-mail:
buonanno{at}mail.nih.gov)
Accepted 8 August 2007
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Cdx2, Oct4, Eomes, Embryonic stem cell, Trophoblast stem cell, Morula, Blastocyst
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Kunath et al.,
2004). In vitro, it is the trophectoderm that gives rise to
trophoblast stem (TS) cells, and the ICM that gives rise to embryonic stem
(ES) cells.
Presumably, selective expression of one or more specific genes in the
blastomeres of preimplantation embryos triggers their differentiation into
either trophectoderm or ICM. For example, embryos deficient in the POU-domain
transcription factor OCT4 survive through the morula stage, but cannot form an
ICM and fail to give rise to embryonic stem cells in vitro
(Nichols et al., 1998). OCT4
prevents trophectoderm and perhaps somatic-cell differentiation of the ICM in
addition to being crucial for maintaining the pluripotent state during
embryonic development. Moreover, in mouse embryonic stem cells, the relative
amount of OCT4 ultimately determines cell fate
(Boiani and Scholer, 2005). The
counterpart to OCT4 is the caudal-type homeodomain transcription factor CDX2,
a protein that is required for specification and differentiation of the
trophectoderm (Strumpf et al.,
2005). CDX2-deficient embryos form blastocysts but fail to
implant, and genes such as Oct4 (also known as Pou5f1 -
Mouse Genome Informatics) and Nanog, that are normally expressed only
in the ICM, are ectopically expressed in the outer cells of the blastocyst,
resulting in eventual death of the embryo. The cellular concentration of OCT4
relative to CDX2 appears to determine which of the totipotent blastomeres will
become trophectoderm and which will become ICM
(Niwa et al., 2005). This
implies that OCT4 and CDX2 constitute the `prime movers' in establishing the
first cell-type-specific lineages during mammalian development, although how
the fate of each blastomere is determined remains speculative. Hence, other
genes involved in specifying trophectoderm and ICM may act upstream of either
Oct4/Pou5f1 or Cdx2.
Zygotic gene expression in the mouse begins at the 2-cell stage in
development (Nothias et al.,
1995; Schultz,
2002). Microinjection of DNA into mouse preimplantation embryos
revealed that their ability to utilize enhancers to activate transcription
also appeared with formation of a 2-cell embryo, and that the most effective
enhancers contained one or more DNA binding sites for a TEAD transcription
factor (Kaneko and DePamphilis,
1998). In placental mammals, there are four TEAD family members
characterized by a highly conserved, virtually identical 72 amino acid TEA DNA
binding domain. Furthermore, the amino acid sequences in the C-terminal halves
of these proteins share 80% to 87% similarity. This conservation of structure
reflects the ability of mammalian TEAD proteins to bind the same
transcriptional co-activator proteins
(Mahoney et al., 2005;
Vassilev et al., 2001), and to
substitute for TEA DNA binding domain proteins in other organisms
(Deshpande et al., 1997).
Most, if not all, embryonic and extraembryonic tissues express at least one
of the TEAD genes during mammalian development
(Kaneko and DePamphilis,
1998). However, Tead2 (also known as Tef4 and
ETF) (Jacquemin et al.,
1996; Kaneko et al.,
1997; Yasunami et al.,
1995) and Tead4 (also known as Tef3, Tefr and
Etfr2) (Jacquemin et al.,
1996; Yasunami et al.,
1996; Yockey et al.,
1996) are the only two Tead genes expressed at significant levels
in preimplantation mouse embryos (Kaneko
et al., 1997) (this report). Thus, TEAD2 and TEAD4, like OCT4 and
CDX2, are among the earliest transcription factors expressed during mammalian
development, suggesting that they too play critical roles in the early stages
of development. Therefore, to investigate this further the Tead2
(Kaneko et al., 2007) and
Tead4 (this work) genes were each inactivated by site-specific
recombination, with the expectation that they would also be required for
preimplantation development. Surprisingly, inactivation of the Tead2
gene significantly increased the risk of exencephaly (a defect in neural tube
closure), but did not otherwise prevent development of viable adults. By
contrast, inactivation of Tead4 resulted in a preimplantation lethal
phenotype.
|
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
). Mice
were screened by PCR to verify germline transmission, using primers TEAD4.20,
TEAD4.27, and TEAD4.28 (Fig.
1E) or P1, P2 and P4 (Fig.
1D). Size of PCR products were as follows: primers TEAD4.20,
TEAD4.28 and TEAD4.27, 294 bp for the wild-type and 480 bp for KO allele;
P1/P2, 550 bp for the wild-type allele; P2/P4, 650 bp for the KO allele;
P1/P3, 400 bp for the conditional allele. PCR genotyping of the Meox2-Cre
transgene was performed as described previously
(Tallquist and Soriano,
2000). Adult animals were genotyped from tail clip DNA. Cell lines were genotyped after multiple passages in the absence of primary MEF feeder cells. Blastocysts were genotyped after isolation, outgrowth assays, or immunostaining using DNA lysis buffer containing 50 mM Tris-HCl pH 8.0, 0.5% Triton X-100, 200 µg/ml proteinase K. The embryos were digested at 55°C for 2 hours and the proteinase K was inactivated at 95°C for 5 minutes.
In situ hybridization
Uteri from wild-type embryonic day 6.5 (E6.5) pregnant mice were collected,
separated into individual placentas and fixed for 24 hours in freshly prepared
4% paraformaldehyde in PBS (pH 7.4). Tissues were then dehydrated, embedded in
paraffin, and 10 µm thick sections cut. Consecutive sections were
hybridized to an in vitro-transcribed 33P-labeled cRNA probe for
TEAD4, as described previously (Wilkinson
and Nieto, 1993), but with greater stringency by hybridizing
overnight at 54°C. Probe templates were made by RT-PCR from E15 mouse hind
limb total RNA and cloned into pGEM-T (Promega). Primers used for
amplification were TEAD4.1 and TEAD4.2
(Table 1).
|
). For
blastocyst outgrowth experiments, E3.5 embryos from Tead4
heterozygous matings were cultured on 0.1% gelatin-treated plates in RPMI1640
containing 20% ES-qualified FBS (Chemicon) and standard supplements for up to
5 days.
RNA preparation, real-time PCR and RT-PCR
Total RNA was isolated from preimplantation embryos using RNeasy micro
columns (Qiagen). For real-time PCR, RNA was reverse-transcribed using TaqMan
reagents (Applied Biosystems). Tead4 and Gapdh probes were
obtained from Applied Biosystems. Quantification of gene expression was
performed using an ABI Prism 7000 sequence detection system. For RT-PCR, RNA
was collected from individual embryos and reverse transcribed using the
SuperScript first-strand synthesis system (Invitrogen). Primers are given in
Table 1.
Immunofluorescent staining
Immunofluorescent staining was performed as described previously
(Strumpf et al., 2005) using
primary antibodies against CDX2 (Biogenex), OCT4 (Santa Cruz Biotechnologies)
and CDH1 (Sigma), and visualized with Alexa Fluor 488 goat anti-mouse
secondary antibody (Invitrogen) or FITC goat anti-rat secondary antibody
(Zymed). Embryos were then examined using a Zeiss LSM510 confocal
microscope.
Cell lines and embryoid bodies
TS cell lines and trophoblast giant (TG) cells were generated from E3.5
embryos of Tead4 heterozygous matings as described previously
(Tanaka et al., 1998). After
several passages, the TS cells were removed from MEFs and genotyped for
Tead4. TS cells lines were similarly generated from E2.5 embryos,
except that the embryos were treated with acidic Tyrode's solution to remove
their zonae pellucidae, placed in wells containing a small droplet of calcium-
and magnesium-free PBS, allowed to adhere to the surface of the dish, and then
gently seeded into the wells in culture medium. ES cells lines were generated
from E2.5 morulae as described previously
(Tesar, 2005). Embryoid bodies
were produced as described Kaneko et al.
(Kaneko et al., 2004).
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
) and
Tead4 (this report) by targeting their TEA DNA-binding domains. The murine Tead4 gene encompasses 42.7 kb on chromosome 6, and exon 2 harbors almost half of the TEA DNA-binding domain. The conditional mouse line Tead4lox/lox was obtained by inserting loxP sites into the introns flanking exon 2 (Fig. 1A). Heterozygous mice were established by crossing male chimeras with C57Bl/6 females. Founders were bred among themselves to establish a homozygous Tead4lox/lox line (Fig. 1F). These mice were viable and fertile, and exhibited no obvious morphological malformations or abnormal behavior.
To address the role of TEAD4 in early mouse development, an unconditional
knockout line was generated by crossing lox/lox mice with EIIa-Cre mice that
ubiquitously express Cre recombinase
(Lakso et al., 1996). This
resulted in germline transmission of the recombined allele. Heterozygous
offspring from this cross were also viable, fertile and apparently normal in
morphology and behavior. Next, Tead4 heterozygotes were mated and
their progeny genotyped. Out of a total of 367 pups, only wild-type and
heterozygous offspring, but no homozygotes were identified
(Table 2).
|
Tead4 is first expressed during preimplantation development and trophectoderm differentiation
Previous studies did not detect Tead4 mRNA in preimplantation
mouse embryos (Hamatani et al.,
2004; Kaneko et al.,
1997). To understand why TEAD4-deficient embryos arrest prior to
implantation, the relative amount of Tead4 mRNA was quantified by
real-time RT-PCR during early development. The results revealed that
Tead4 mRNA was barely detectable in unfertilized and fertilized eggs,
but increased from the 2-cell embryo through blastocyst stages with the
maximum level observed in 8-cell embryos and morulae
(Fig. 2A).
|
;
Kunath et al., 2004). In
vitro, TS cells proliferate in response to fibroblast growth factor 4 (FGF4),
and in the absence of FGF4, they differentiate into invasive trophoblast giant
(TG) cells (Cross, 2005) that
mediate the process of implantation and invasion of the embryo into the
uterine endometrium and deciduum. In contrast to TS cells, embryonic stem (ES)
cells are derived from the ICM and contribute exclusively to embryonic cell
types and to nontrophoblast-derived extra-embryonic tissues
(Smith et al., 1988;
Williams et al., 1988). In
vitro, ES cells can be induced to differentiate into embryoid bodies that
contain cells derived from all three embryonic layers. Tead4 expression was 27-fold greater in TS cells than in ES cells, and this high level of expression was maintained as TS cells differentiated into TG cells in vitro (Fig. 2B). Similarly, the relatively low level of Tead4 expression in ES cells was maintained upon their differentiation into embryoid bodies. This result was confirmed and extended using in situ hybridization technology. Tead4 mRNA was found predominantly in the extraembryonic portion of the conceptus, in particular in the dividing trophoblast cells of the ectoplacental cone (EPC) (Fig. 2C,D). Expression was also observed in extraembryonic layers of the embryo, including the chorion and giant trophoblast cells underlying the maternal part of the placenta, as well as in the maternal deciduum. Tead4 expression was not detected in the embryo proper. Taken together, these data demonstrate that Tead4 is expressed primarily in the trophectoderm-derived cell lineage during early and mid-embryonic development.
TEAD4 is required for expression of trophectoderm-specific genes
Cdx2 expression is required for the establishment of TS cells, and
CDX2-deficient embryos develop to the blastocyst stage but fail to implant due
to loss of trophectoderm cell integrity
(Strumpf et al., 2005). By
contrast, the Oct4/Pou5f1 gene is required for establishment of ES
cells, and OCT4-deficient embryos develop a blastocoel cavity but not an ICM
(Nichols et al., 1998).
Moreover, at the blastocyst stage, Cdx2 is expressed only in
trophectoderm cells, and Oct4 is expressed only in the ICM
(Niwa et al., 2005;
Strumpf et al., 2005).
To determine if TEAD4 is required for Cdx2 expression, embryos obtained from heterozygous matings were stained with antibodies against CDX2 and then genotyped. As expected, CDX2 protein was present in the trophectodermal cells of Tead4+/+ and Tead4+/- blastocysts at E3.5 (Fig. 3A). In contrast, CDX2 was not detected at E3.5 in Tead4-/- embryos, all of which exhibited an abnormal morphology characterized by poorly formed blastomeres unevenly distributed within the zona pellucida, and by the absence of a blastocoel cavity. These embryos were henceforth referred to as `abnormal morulae'. CDX2 also was absent from Tead4-/- morulae at E2.5, although these embryos were morphologically indistinguishable from Tead4+/+ and Tead4+/- embryos of the same age. In fact, CDX2 was present in only a few cells of normal 8-cell embryos or morulae recovered at E2.5 with genotypes of either Tead4+/+ or Tead4+/-, and was absent in 4-cell embryos (data not shown). Presumably, these CDX2-positive cells were destined to become trophectoderm.
Consistent with previous studies
(Strumpf et al., 2005), OCT4
protein was present only in the ICM of Tead4+/+ or
Tead4+/- blastocysts at E3.5, all of which appeared
morphologically normal (Fig.
3B). However, the E3.5 abnormal morulae, all of which were
Tead4-/- (Table
2), produced OCT4 protein in all of their blastomeres. Thus, TEAD4
was required for Cdx2, but not Oct4 gene expression. Since
Cdx2-/- embryos also express OCT4 in all of their
blastomeres (Strumpf et al.,
2005), these results further reveal that TEAD4 is required for
Cdx2 expression which normally suppresses Oct4 expression in
those cells destined to become trophectoderm
(Niwa et al., 2005).
RT-PCR was used to determine if TEAD4 was required for expression of a
battery of genes associated with preimplantation development
(Fig. 3C). Consistent with the
immunofluorescence analysis, Cdx2 mRNA was present in E3.5
blastocysts that were either Tead4+/+ or
Tead4+/-, but absent from E3.5
Tead4-/- abnormal morulae, whereas Oct4 mRNA was
present in all embryos. Moreover, E3.5 Tead4-/- abnormal
morulae expressed eomesodermin (Eomes) at a reduced level, consistent
with a requirement for TEAD4 to specify the trophectoderm lineage.
Eomes is expressed in the trophectoderm layer of blastocysts and
postimplantation extraembryonic tissue, where it acts downstream of CDX2 and
is required for trophoblast development
(Russ et al., 2000;
Strumpf et al., 2005). In the
absence of CDX2, Eomes expression is low but detectable
(Strumpf et al., 2005),
demonstrating that CDX2 is partially responsible for activating
Eomes, and consistent with the effects of TEAD4 deficiency presented
here. By contrast, expression of Fgfr2 was not affected.
Fgfr2 is normally restricted to the outer cells of compacted morulae
and highly expressed in the trophectoderm layer of blastocysts
(Haffner-Krausz et al., 1999).
Thus, TEAD4 was required for expression of some, but not all
trophectoderm-specific genes.
|
|
Compaction of 8-cell embryos into morulae, and the subsequent formation of
trophectoderm requires E-cadherin [renamed cadherin 1 (Cdh1)], a
calcium-dependent cell adhesion molecule
(Kan et al., 2007). However,
Cdh1-/- mouse embryos can develop normally until E2.5
because of maternally inherited CDH1. Thereafter, in the absence of zygotic
Cdh1 expression, defects in the embryos are detected at E3.5 and E4.5
when morulae appear with an abnormal morphology similar to those described
here for Tead4-/- embryos
(Ohsugi et al., 1997).
Therefore, to determine if TEAD4 was required for Cdh1 expression,
E3.5 embryos were stained with antibodies specific to CDH1 protein
and then genotyped. The results revealed that Tead4-/-
blastomere membranes contained similar amounts of CDH1 protein as wild-type
embryos (Fig. 4A).
Nevertheless, blastocoel cavities were clearly absent in E3.5
Tead4-/- embryos, and the well-defined adhesion boundaries
characteristic of trophectodermal polar epithelium were absent. Thus, TEAD4
was required for the formation of a blastocoel cavity, but not through
regulation of Cdh1.
|
). To test if TS cells can be derived from
Tead4-/- embryos, E3.5 embryos were isolated from
heterozygote matings, and then cultured under conditions permissive to TS cell
proliferation (Fig. 5). After
1-2 days, E3.5 blastocysts shed their zona pellucida and attached to the dish.
After 5 days, they had produced characteristic cellular outgrowths in which TG
cells were clearly evident. No difference was detected between
Tead4+/+ and Tead4+/- blastocysts. By
contrast, the abnormal morulae shed their zonae but never attached to the
dish. Neither did they produce cellular outgrowths or form blastocoel
cavities. Thus, all TS cell lines were either Tead4+/+ or
Tead4+/-; none of them were Tead4-/-
(Table 2).
The ability to derive TS cell lines has been demonstrated for early
blastocyst stage to 10-somite pair stage (E8) embryos
(Kunath et al., 2004). Since
E3.5 Tead4-/- embryos arrested development prior to
blastocoel formation, they might not have been capable of generating TS cells.
Therefore, an attempt was made to isolate TS cells from E2.5 embryos, some of
which developed blastocoel cavities and yielded TS cell outgrowths, and some
of which yielded outgrowths without forming blastocoel cavities. However, none
of the TS cell lines derived from E2.5 embryos were
Tead4-/- (Table
2). We conclude that at least one Tead4 allele is
required for establishment of TS cells.
TEAD4 is not required for establishment and differentiation of embryonic stem cells
The fact that Tead4-/- embryos failed to develop into
blastocysts, the stage from which ES cells are usually derived, together with
the fact that E3.5 Tead4-/- embryos failed to attach to
the surface of culture dishes, suggested that TEAD4 may also be required to
derive ES cells. To test this hypothesis, ES cells were derived from E2.5
embryos using a method that allows ES cell lines to be isolated from morulae
with high efficiency (Tesar,
2005). Our results revealed that Tead4 was not required
for establishment of ES cells (Fig.
6B; Table 2), since
Tead4-/- and Tead4+/+ ES cells were
morphologically indistinguishable (Fig.
6A). Moreover, Tead4-/- ES cells expressed
genes specific for pluripotent ES cells at levels comparable to those in
Tead4+/+ ES cells (Fig.
6C), including Oct4/Pou5f1, Fgf4, Nanog, Sox2 and
Rex1 (also known as Zfp42 - Mouse Genome Informatics).
Additionally, neither Tead4-/- nor wild-type cells
expressed the mesoderm marker brachyury (T), whereas it was clearly
detected in control samples (data not shown).
To test the developmental potential of Tead4-/- ES
cells in vitro, embryoid bodies were derived from both
Tead4-/- and Tead4+/+ ES cells and
examined by semi-quantitative RT-PCR for expression of differentiation
markers. Embryoid bodies are aggregates of cells derived from ES cells and
propagated under conditions that prevent them from adhering to a surface. Upon
aggregation, differentiation is initiated and the cells begin to recapitulate
embryonic development (Keller,
2005). Embryoid bodies could be generated from both wild-type and
Tead4-/- ES cells with similar ease. During
differentiation, both wild-type and mutant ES cell lines rapidly repressed
expression of pluripotency markers Rex1
(Fig. 6D) and Sox2
(data not shown) and increased expression of genes specific for endoderm
(Hnf4a, Gata6), ectoderm (Fgf5) and mesoderm (brachyury,
T) (Fig. 6D). Taken
together, these results revealed that TEAD4 was not required for ES cell
differentiation into the three primary tissue lineages.
TEAD4 is not required during postimplantation development
Tead4 is expressed in several embryonic tissues between E10.5 and
E15.5 (Jacquemin et al.,
1996). Therefore, to determine if TEAD4 is required during
postimplantation development, the conditional Tead4 allele (see
Fig. 1) was disrupted in the
embryo after implantation had occurred. Tead4 heterozygotes were
mated with Meox2-Cre+ mice that express Cre recombinase
exclusively in the epiblast beginning at 
E5.5
(Tallquist and Soriano, 2000).
The epiblast originates from the ICM and gives rise to the three germ layers
of the embryo. The Tead4+/-;Meox2-Cre+
mice were then mated to mice carrying the Tead4 conditional allele
(`lox'), producing
Tead4lox/-;Meox2-Cre+ embryos. Their
offspring were genotyped and found to include Tead4 knockouts
(Fig. 7A). These conditional
Tead4-/- mice exhibited no obvious morphological
abnormalities.
Tissue from each major organ system of the Tead4-/-;Meox2-Cre+ mice was assayed by RT-PCR for the presence of Tead4 mRNA. Tead4 mRNA was detected at varying levels in all tissues from wild-type mice except for liver and spleen (Fig. 7B). Controls included Tead4+/+ and Tead4-/- ES cells. In contrast to Tead4+/+, Tead4-/-;Meox2-Cre+ tissues did not express Tead4 mRNA. These results revealed that TEAD4 is required for establishment of the trophectoderm lineage during preimplantation development, whereas is it dispensable for survival later in development.
|
) was
similar to the deletion that eliminated exon 2 in the Tead4 gene. In
each case, the knockout allele lacked the translational start codon and a
large portion of the DNA binding domains, respectively. The next in-frame
start codon occurs in the fourth exon of each gene, downstream of the TEAD
domain. Thus, even if truncated mRNAs were translated adventitiously, the
resulting proteins would not bind DNA
(Kaneko and DePamphilis,
1998). However, whereas Tead4-/- embryos
failed to develop into blastocysts, Tead2-/- embryos
formed blastocysts at normal Mendelian frequency and were capable of
developing into viable and fertile adults
(Table 2). Of the two highly
homologous TEAD transcription factors expressed during preimplantation
development, only TEAD4 was required for mouse development prior to
implantation. |
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
) (this work). One report suggests that maternally
inherited CDX2 protein is present as early as the two-cell stage
(Deb et al., 2006), but the
efficacy of this study has been questioned
(Vogel, 2006). Thus, TEAD4
clearly acts upstream of CDX2, and given the fact that Tead4 mRNA
appears as early as the 2-cell stage, it may be directly responsible for
activating Cdx2. There are several TEAD consensus-binding sites
within the Cdx2 promoter region, including intron sequences, but it
is as yet unclear whether TEAD4 binds to them.
In contrast to Cdx2-/-, Tead4-/-
embryos did not form blastocoel cavities either in vivo or in vitro.
Therefore, TEAD4 must activate other trophectoderm-specific genes in addition
to Cdx2. Since Eomes is down regulated in both
Cdx2-/- (Strumpf et
al., 2005) and Tead4-/- embryos,
Eomes is either directly or indirectly regulated by CDX2, which in
turn, is regulated by TEAD4. Conversely, Fgfr2, which encodes the
trophectoderm receptor for FGF4 (Arman et
al., 1998; Haffner-Krausz et
al., 1999), and Cdh1, which encodes a cell adhesion
molecule required for trophectoderm formation
(Kan et al., 2007), are fully
expressed in both Cdx2-/-
(Strumpf et al., 2005) and
Tead4-/- embryos. Thus, TEAD4 does not regulate expression
of all genes involved in formation of trophectoderm but appears to trigger a
critical event early in the establishment of trophectoderm, sometime during
the 8-cell to morula transition.
|
Cdx2 
Oct4 
trophectoderm).
Moreover, changing the ratio of OCT4 to CDX2 in ES cells can determine their
fate. A high OCT4:CDX2 ratio promotes the maintenance of the totipotent ICM,
whereas a low ratio promotes differentiation into trophectoderm
(Niwa et al., 2005). Thus,
trophectoderm differentiation appears to occur in response to an increase in
the ratio of CDX2 to OCT4. We show here that TEAD4 deficiency prevented the expression of CDX2, but not OCT4. Whereas OCT4 was restricted to the ICM of wild-type and heterozygous blastocysts, it appeared in every blastomere of mutant E3.5 embryos. This is consistent with TEAD4 regulating Cdx2 by stimulating its expression in the outer blastomeres of morulae. Thus, it may be that OCT4:TEAD4 is actually the critical ratio that specifies the trophectoderm lineage. Tead4 mRNA levels are high in TS and TG cells relative to ES cells and embryoid bodies, but it remains to be determined if increasing TEAD4 levels in ES cells can mimic the ability of CDX2 to induce ES cells to behave like trophectoderm.
OCT4 drives expression of FGF4 from the ICM, and FGF4 maintains the pluripotency of cells in the polar trophectoderm. The FGF receptor protein FGFR2 is required for trophectoderm to respond to FGF4. Both Oct4 and Fgfr2 expression occur in the absence of TEAD4. Therefore, failure of Tead4-/- embryos to establish the trophectoderm layer in blastocysts and TS cells could result from the failure to express one or more genes that respond to the FGFR2 signal. Alternatively, TEAD4-CDX2 and FGF4-FGFR2 could control separate pathways that cooperate to control proper development of the trophectoderm.
During development, TEAD4 is required only for trophectoderm specification
Given the expression of Tead4 in 2-cell embryos, the possibility
was considered that Tead4 functioned as a master switch for
differentiation of all blastomeres into either trophectoderm or ICM. Our data
demonstrate that TEAD4 was required exclusively for trophectoderm
specification. Only wild-type or heterozygous TS cells could be isolated
either from E2.5 or E3.5 embryos (Table
2). By contrast, Tead4-/- ES cells could be
isolated from E2.5 morulae. These cells did not express Tead4 mRNA,
but they did differentiate into ectoderm, mesoderm and endoderm in vitro.
Wild-type ES cells isolated from E2.5 morulae have been reported to make
chimeric mice (Tesar, 2005),
and similar experiments are in progress with Tead4-/- ES
cells.
The ES cells derived from Tead4-/- embryos were similar in appearance to wild-type ES cells and expressed similar levels of pluripotency markers. Moreover, they differentiated normally in vitro, as embryoid bodies derived from wild-type and Tead4-/- cells downregulated pluripotency markers and upregulated markers of the three primary cell types with similar kinetics.
Conditional ablation of Tead4 in the postimplantation epiblast by intercrossing Tead4lox/-;Meox2-Cre+ mice confirmed these results in vivo. Unlike the full knockout, specific Tead4 inactivation in the epiblast of 5- to 7-day-old embryos resulted in viable Tead4-/- offspring despite the lack of Tead4 mRNA in all major tissues. Moreover, these mice had no obvious defects at the level of gross morphology. The fact that Tead4 is expressed in many tissues of wild-type mice certainly suggests that it plays a role either in the maintenance or in regeneration of tissues, but whatever those roles may be, they are not crucial to embryonic development. Thus, it appears Tead4 is indispensable only at the earliest stages of development and only for trophectoderm specification.
Trophectoderm specification requires functional TEAD4 protein
One concern in the analysis of any genetic mutation is that the observed
phenotype resulted from the absence of the mutated gene's function or from the
unexpected production of a dominant negative inhibitor from the remaining gene
fragment. The C-terminal half of all four mammalian TEAD proteins contains a
highly conserved transcriptional co-activator binding site for YAP65
(Vassilev et al., 2001) and
TAZ (Mahoney et al., 2005).
Translational start codons that could potentially translate this
protein-binding domain from a truncated mRNA exist at identical sites in both
the Tead4 and Tead2 knockout alleles. Therefore, one would
expect the C terminal fragment in Tead2-/- embryos to have
the same potential toxicity as the C-terminal fragment in
Tead4-/- embryos.
Three lines of evidence suggest the early preimplantation arrest of Tead4 nullizygous embryos did not result from production of a toxic C-terminal polypeptide. First, Tead4+/- mice were viable and fertile, indicating that any toxic effects that might arise from the C-terminal fragment of the deleted allele were negligible. Second, very similar deletions in two highly homologous genes, Tead2 and Tead4, did not produce the same phenotype; Tead4-/- embryos arrested development prior to formation of blastocysts, but Tead2-/- embryos did not (Table 2). In fact, most Tead2-/- embryos developed into adult mice. Finally, deletion of Tead4 in postimplantation embryos using the Meox2-Cre strategy resulted in viable Tead4-/- adults in which the Tead4 gene was ablated in all of the tissues examined, despite expression of Tead4 in postimplantation tissues. Taken together, these results strongly suggest any adventitious expression of the C-terminal protein fragment was not toxic during postimplantation development.
TEAD transcription factors are not functionally redundant
The role of TEAD4 in specifying the trophectoderm lineage appears to be
unique among TEAD family members. A very similar deletion in the closely
related Tead2 gene did not interfere with preimplantation development
or implantation, even though both genes are expressed concurrently during
preimplantation development (Kaneko et
al., 2007). This was surprising since their DNA binding and
transactivation domains are highly similar and because all four TEAD proteins
appear to bind the same transcriptional co-activators
(Mahoney et al., 2005;
Vassilev et al., 2001).
Therefore, either TEAD2 is not expressed in the same blastomeres as TEAD4, or
the two genes bind to different DNA sequences in the presence of their
transcriptional co-activator (Halder and
Carroll, 2001), or they bind to the same DNA sequence but to
different co-activators (but see above). Evidence argues against the first
possibility since Tead2 is expressed in both ICM and trophectoderm of
blastocysts at relatively equivalent levels
(Kaneko et al., 2004). What is
clear is that inactivation of the Tead2 alleles markedly increased
the risk of exencephaly, a defect in neural tube closure that occurs during
postimplantation development as early as E11.5
(Kaneko et al., 2007). As for
the remaining Tead genes, mouse embryos lacking TEAD1 fail to develop a proper
heart and die between E11 and E12 (Chen et
al., 1994), and TEAD3-deficient mice have not yet been described.
Thus, most, if not all, of the TEAD transcription factors serve at least one
nonredundant function in mammalian development. Additional roles may be
revealed in the future that are currently masked by the ability of other
members to substitute for the ablated TEAD protein. Other roles for TEAD
proteins may also exist in adult animals during regeneration of adult neural
stem cells (Ramalho-Santos et al.,
2002) or muscle (Zhao et al.,
2006). Furthermore, other defects in adult Tead2 and
Tead4 nullizygous mice may manifest themselves with age.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Arman, E., Haffner-Krausz, R., Chen, Y., Heath, J. K. and Lonai,
P. (1998). Targeted disruption of fibroblast growth factor
(FGF) receptor 2 suggests a role for FGF signaling in pregastrulation
mammalian development. Proc. Natl. Acad. Sci. USA
95,5082
-5087.
Boiani, M. and Scholer, H. R. (2005).
Regulatory networks in embryo-derived pluripotent stem cells. Nat.
Rev. Mol. Cell Biol. 6,872
-884.[CrossRef][Medline]
Chen, Z., Friedrich, G. A. and Soriano, P.
(1994). Transcriptional enhancer factor 1 disruption by a
retroviral gene trap leads to heart defects and embryonic lethality in mice.
Genes Dev. 8,2293
-2301.
Cross, J. C. (2005). How to make a placenta:
mechanisms of trophoblast cell differentiation in mice - a review.
Placenta Suppl. 26,S3
-S9.
Deb, K., Sivaguru, M., Yong, H. Y. and Roberts, R. M.
(2006). Cdx2 gene expression and trophectoderm lineage
specification in mouse embryos. Science
311,992
-996.
Deshpande, N., Chopra, A., Rangarajan, A., Shashidhara, L. S.,
Rodrigues, V. and Krishna, S. (1997). The human transcription
enhancer factor-1, TEF-1, can substitute for Drosophila scalloped during
wingblade development. J. Biol. Chem.
272,10664
-10668.
Haffner-Krausz, R., Gorivodsky, M., Chen, Y. and Lonai, P.
(1999). Expression of Fgfr2 in the early mouse embryo indicates
its involvement in preimplantation development. Mech.
Dev. 85,167
-172.[CrossRef][Medline]
Halder, G. and Carroll, S. B. (2001). Binding
of the Vestigial co-factor switches the DNA-target selectivity of the
Scalloped selector protein. Development
128,3295
-3305.
Hamatani, T., Carter, M. G., Sharov, A. A. and Ko, M. S.
(2004). Dynamics of global gene expression changes during mouse
preimplantation development. Dev. Cell
6, 117-131.[CrossRef][Medline]
Jacquemin, P., Hwang, J. J., Martial, J. A., Dolle, P. and
Davidson, I. (1996). A novel family of developmentally
regulated mammalian transcription factors containing the TEA/ATTS DNA binding
domain. J. Biol. Chem.
271,21775
-21785.
Kan, N. G., Stemmler, M. P., Junghans, D., Kanzler, B., de
Vries, W. N., Dominis, M. and Kemler, R. (2007). Gene
replacement reveals a specific role for E-cadherin in the formation of a
functional trophectoderm. Development
134, 31-41.
Kaneko, K. J. and DePamphilis, M. L. (1998).
Regulation of gene expression at the beginning of mammalian development and
the TEAD family of transcription factors. Dev. Genet.
22, 43-55.[CrossRef][Medline]
Kaneko, K. J., Cullinan, E. B., Latham, K. E. and DePamphilis,
M. L. (1997). Transcription factor mTEAD-2 is selectively
expressed at the beginning of zygotic gene expression in the mouse.
Development 124,1963
-1973.[Abstract]
Kaneko, K. J., Rein, T., Guo, Z. S., Latham, K. and DePamphilis,
M. L. (2004). DNA methylation may restrict but does not
determine differential gene expression at the Sgy/Tead2 locus during mouse
development. Mol. Cell. Biol.
24,1968
-1982.
Kaneko, K. J., Kohn, M. J., Liu, C. and DePamphilis, M. L.
(2007). Transcription factor TEAD2 is involved in neural tube
closure. Genesis (in press).
Keller, G. (2005). Embryonic stem cell
differentiation: emergence of a new era in biology and medicine.
Genes Dev. 19,1129
-1155.
Kunath, T., Strumpf, D. and Rossant, J. (2004).
Early trophoblast determination and stem cell maintenance in the mouse - a
review. Placenta Suppl.
25,S32
-S38.
Lakso, M., Pichel, J. G., Gorman, J. R., Sauer, B., Okamoto, Y.,
Lee, E., Alt, F. W. and Westphal, H. (1996). Efficient in
vivo manipulation of mouse genomic sequences at the zygote stage.
Proc. Natl. Acad. Sci. USA
93,5860
-5865.
Mahoney, W. M., Jr, Hong, J. H., Yaffe, M. B. and Farrance, I.
K. (2005). The transcriptional co-activator TAZ interacts
differentially with transcriptional enhancer factor-1 (TEF-1) family members.
Biochem. J. 388,217
-225.[CrossRef][Medline]
Nagy, A., Gertsenstein, M., Vintersten, K. and Behringer, R.
(2003). Manipulating the Mouse Embryo: A Laboratory
Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
Press.
Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H.,
Klewe-Nebenius, D., Chambers, I., Scholer, H. and Smith, A.
(1998). Formation of pluripotent stem cells in the mammalian
embryo depends on the POU transcription factor Oct4.
Cell 95,379
-391.[CrossRef][Medline]
Niwa, H., Toyooka, Y., Shimosato, D., Strumpf, D., Takahashi,
K., Yagi, R. and Rossant, J. (2005). Interaction between
Oct3/4 and Cdx2 determines trophectoderm differentiation.
Cell 123,917
-929.[CrossRef][Medline]
Nothias, J. Y., Majumder, S., Kaneko, K. J. and DePamphilis, M.
L. (1995). Regulation of gene expression at the beginning of
mammalian development. J. Biol. Chem.
270,22077
-22080.
Ohsugi, M., Larue, L., Schwarz, H. and Kemler, R.
(1997). Cell-junctional and cytoskeletal organization in mouse
blastocysts lacking E-cadherin. Dev. Biol.
185,261
-271.[CrossRef][Medline]
Ramalho-Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R. C. and
Melton, D. A. (2002). "Stemness": transcriptional
profiling of embryonic and adult stem cells. Science
298,597
-600.
Russ, A. P., Wattler, S., Colledge, W. H., Aparicio, S. A.,
Carlton, M. B., Pearce, J. J., Barton, S. C., Surani, M. A., Ryan, K., Nehls,
M. C. et al. (2000). Eomesodermin is required for mouse
trophoblast development and mesoderm formation. Nature
404, 95-99.[CrossRef][Medline]
Schultz, R. M. (2002). The molecular
foundations of the maternal to zygotic transition in the preimplantation
embryo. Hum. Reprod. Update
8, 323-331.
Smith, A. G., Heath, J. K., Donaldson, D. D., Wong, G. G.,
Moreau, J., Stahl, M. and Rogers, D. (1988). Inhibition of
pluripotential embryonic stem cell differentiation by purified polypeptides.
Nature 336,688
-690.[CrossRef][Medline]
Strumpf, D., Mao, C. A., Yamanaka, Y., Ralston, A.,
Chawengsaksophak, K., Beck, F. and Rossant, J. (2005). Cdx2
is required for correct cell fate specification and differentiation of
trophectoderm in the mouse blastocyst. Development
132,2093
-2102.
Tallquist, M. D. and Soriano, P. (2000).
Epiblast-restricted Cre expression in MORE mice: a tool to distinguish
embryonic vs. extra-embryonic gene function. Genesis
26,113
-115.[CrossRef][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.
Tesar, P. J. (2005). Derivation of
germ-line-competent embryonic stem cell lines from preblastocyst mouse
embryos. Proc. Natl. Acad. Sci. USA
102,8239
-8244.
Vassilev, A., Kaneko, K. J., Shu, H., Zhao, Y. and DePamphilis,
M. L. (2001). TEAD/TEF transcription factors utilize the
activation domain of YAP65, a Src/Yes-associated protein localized in the
cytoplasm. Genes Dev.
15,1229
-1241.
Vogel, G. (2006). Developmental biology. Fraud
investigation clouds paper on early cell fate. Science
314,1367
-1369.
Wilkinson, D. G. and Nieto, M. A. (1993).
Detection of messenger RNA by in situ hybridization to tissue sections and
whole mounts. Meth. Enzymol.
225,361
-373.[Medline]
Williams, R. L., Hilton, D. J., Pease, S., Willson, T. A.,
Stewart, C. L., Gearing, D. P., Wagner, E. F., Metcalf, D., Nicola, N. A. and
Gough, N. M. (1988). Myeloid leukaemia inhibitory factor
maintains the developmental potential of embryonic stem cells.
Nature 336,684
-687.[CrossRef][Medline]
Yasunami, M., Suzuki, K., Houtani, T., Sugimoto, T. and Ohkubo,
H. (1995). Molecular characterization of cDNA encoding a
novel protein related to transcriptional enhancer factor-1 from neural
precursor cells. J. Biol. Chem.
270,18649
-18654.
Yasunami, M., Suzuki, K. and Ohkubo, H. (1996).
A novel family of TEA domain-containing transcription factors with distinct
spatiotemporal expression patterns. Biochem. Biophys. Res.
Commun. 228,365
-370.[CrossRef][Medline]
Yockey, C. E., Smith, G., Izumo, S. and Shimizu, N.
(1996). cDNA cloning and characterization of murine
transcriptional enhancer factor-1-related protein 1, a transcription
factor that binds to the M-CAT motif. J. Biol. Chem.
271,3727
-3736.
Zhao, P., Caretti, G., Mitchell, S., McKeehan, W. L., Boskey, A.
L., Pachman, L. M., Sartorelli, V. and Hoffman, E. P. (2006).
Fgfr4 is required for effective muscle regeneration in vivo. Delineation of a
MyoD-Tead2-Fgfr4 transcriptional pathway. J. Biol.
Chem. 281,429
-438.
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:
|
|
 |
|
  P. Home, S. Ray, D. Dutta, I. Bronshteyn, M. Larson, and S. Paul GATA3 Is Selectively Expressed in the Trophectoderm of Peri-implantation Embryo and Directly Regulates Cdx2 Gene Expression J. Biol. Chem., October 16, 2009; 284(42): 28729 - 28737. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Nichols, J. Silva, M. Roode, and A. Smith Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo Development, October 1, 2009; 136(19): 3215 - 3222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. C. Douglas, C. A. VandeVoort, P. Kumar, T.-C. Chang, and T. G. Golos Trophoblast Stem Cells: Models for Investigating Trophectoderm Differentiation and Placental Development Endocr. Rev., May 1, 2009; 30(3): 228 - 240. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Rossant and P. P. L. Tam Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouse Development, March 1, 2009; 136(5): 701 - 713. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Fujimori, Y. Kurotaki, K. Komatsu, and Y.-i. Nabeshima Morphological Organization of the Mouse Preimplantation Embryo Reproductive Sciences, February 1, 2009; 16(2): 171 - 177. [Abstract] [PDF]
|
|
|
|
|
|
R. W. Tsika, C. Schramm, G. Simmer, D. P. Fitzsimons, R. L. Moss, and J. Ji Overexpression of TEAD-1 in Transgenic Mouse Striated Muscles Produces a Slower Skeletal Muscle Contractile Phenotype J. Biol. Chem., December 26, 2008; 283(52): 36154 - 36167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ota and H. Sasaki Mammalian Tead proteins regulate cell proliferation and contact inhibition as transcriptional mediators of Hippo signaling Development, December 15, 2008; 135(24): 4059 - 4069. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Cao, S. L. Pfaff, and F. H. Gage YAP regulates neural progenitor cell number via the TEA domain transcription factor Genes & Dev., December 1, 2008; 22(23): 3320 - 3334. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Ullah, M. J. Kohn, R. Yagi, L. T. Vassilev, and M. L. DePamphilis Differentiation of trophoblast stem cells into giant cells is triggered by p57/Kip2 inhibition of CDK1 activity Genes & Dev., November 1, 2008; 22(21): 3024 - 3036. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Jedrusik, D.-E. Parfitt, G. Guo, M. Skamagki, J. B. Grabarek, M. H. Johnson, P. Robson, and M. Zernicka-Goetz Role of Cdx2 and cell polarity in cell allocation and specification of trophectoderm and inner cell mass in the mouse embryo Genes & Dev., October 1, 2008; 22(19): 2692 - 2706. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Sawada, H. Kiyonari, K. Ukita, N. Nishioka, Y. Imuta, and H. Sasaki Redundant Roles of Tead1 and Tead2 in Notochord Development and the Regulation of Cell Proliferation and Survival Mol. Cell. Biol., May 15, 2008; 28(10): 3177 - 3189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yoshida MCAT Elements and the TEF-1 Family of Transcription Factors in Muscle Development and Disease Arterioscler Thromb Vasc Biol, January 1, 2008; 28(1): 8 - 17. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
