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EMBO reports 4, 10, 964–968 (2003)
doi:10.1038/sj.embor.embor939
AOP Published online: 19 September 2003
An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development
Marc K. Saba-El-Leil1, 5, Francis D.J. Vella2, 4, 5, Bertrand Vernay2, 4, Laure Voisin1, Lan Chen2, 4, Nathalie Labrecque2, 3, Siew-Lan Ang2, 4 & Sylvain Meloche1, 4
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1 Institut de Recherches Cliniques de
Montréal and Department of Pharmacology, Université de
Montréal, 110 Pine Avenue West,
Montréal, Quebec, Canada H2W
1R7
2 Institut de Génétique et de
Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 1 rue Laurent
Fries, BP 163, F-67404 Illkirch cedex,
France
3 Centre de Recherche Guy-Bernier, Hôpital
Maisonneuve-Rosemont, Université de Montréal, 5415 Boulevard
Assomption, Montréal, Quebec,
Canada H1T 2M4
4 Present address: Division of Developmental
Neurobiology, National Institute for Medical Research, Mill Hill,
London NW7 1AA, UK
5 These authors contributed equally to this work
To whom correspondence should be addressed
Sylvain Meloche Tel: +514 987 5783; Fax: +514 987 5536;
melochs@ircm.qc.ca
Received 11 April 2003; Accepted 13 August 2003; Published online 19 September 2003.
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Abstract
The closely related mitogen-activated protein kinase isoforms
extracellular signal-regulated kinase 1 (ERK1) and ERK2 have been implicated in
the control of cell proliferation, differentiation and survival. However, the
specific in vivo functions of the two ERK isoforms remain to be
analysed. Here, we show that disruption of the Erk2 locus leads to
embryonic lethality early in mouse development after the implantation stage.
Erk2 mutant embryos fail to form the ectoplacental cone and
extra-embryonic ectoderm, which give rise to mature trophoblast derivatives in
the fetus. Analysis of chimeric embryos showed that Erk2 functions in a
cell-autonomous manner during the development of extra-embryonic cell lineages.
We also found that both Erk2 and Erk1 are widely expressed
throughout early-stage embryos. The inability of Erk1 to compensate for
Erk2 function suggests a specific function for Erk2 in normal
trophoblast development in the mouse, probably in regulating the proliferation
of polar trophectoderm cells.
EMBO reports 4, 10, 964–968 (2003)
doi:10.1038/sj.embor.embor939
AOP Published online: 19 September 2003
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Introduction
Mitogen-activated protein kinase (MAPK) pathways are evolutionarily
conserved signalling modules through which cells transduce extracellular
signals into intracellular responses (Lewis et al.,
1998; Widmann et al., 1999;
Pearson et al., 2001). The prototypical
MAPK pathway is the extracellular signal-regulated kinase (ERK)1/2 pathway. Two
related MAPK isoforms, known as ERK1 (p44mapk) and ERK2
(p42mapk), which share 84% amino-acid identity, have been
identified in mammals (Boulton et al., 1990,
1991). The two proteins are ubiquitously expressed
in cell lines and tissues, although their relative abundance is variable. ERK1
and ERK2 are activated by almost all mitogenic factors, differentiation
stimuli, cytokines, and by other cellular perturbations. In fibroblasts, the
two isozymes are coordinately regulated in response to serum (Meloche, 1995).
Although much still remains to be learned about the physiological
functions of ERK1 and ERK2, several lines of evidence have implicated these
enzymes in the control of cell proliferation and differentiation (Lewis et al., 1998; Whitmarsh
& Davis, 2000). The ERK1/2 MAPK pathway has an important function
in the regulation of cell-cycle re-entry and G1-phase progression (Pagès
et al., 1993; Lavoie et al.,
1996; Roovers & Assoian, 2000).
Moreover, evidence suggests that this signalling pathway influences cell growth
by modulating the rate of protein and pyrimidine-nucleotide biosynthesis
(Servant et al., 1996; Graves et al., 2000). Activation of the ERK1/2
pathway also provides protection against apoptosis in several cell types when
they are challenged by cellular stresses or chemotherapeutic drugs (Ballif & Blenis, 2001). However, many questions remain
unanswered about the in vivo functions of ERK1 and ERK2 in normal
development and growth. In particular, the functional specificity and
redundancy of the two MAPK isoforms remain to be analysed by genetic
approaches. Pagès et al. generated mice with a null mutation in
the Erk1 gene (Pagès et al.,
1999). Erk1-/- mice are viable,
fertile and of normal size. However, the proliferation and maturation of
thymocytes are affected in these mice, despite the expression and activation of
Erk2.
We have used a gene-targeting approach to investigate the specific
function of Erk2 in development. We found that mouse embryos lacking
Erk2 die early during embryogenesis due to a defect in trophoblast
development. Mutant embryos fail to form the ectoplacental cone and
extra-embryonic ectoderm, which derive from the polar trophectoderm.
Importantly, we show that Erk1 is widely expressed in normal and mutant
embryos. The lack of compensation of Erk2 deficiency by Erk1 suggests that Erk2
has a specific function in the developing embryo.
Results and Discussion
To explore the function of Erk2 in development, we have inactivated
the Erk2 gene by homologous recombination in embryonic stem (ES) cells
(Fig. 1A). Targeted disruption of the Erk2 gene
was confirmed by Southern analysis of genomic DNA (Fig.
1B) and immunoblot analysis of total protein (Fig.
1C) isolated from whole embryos. A significant percentage of embryos,
probably corresponding to heterozygotes, expressed half the amount of Erk2
protein (Fig. 1C). No fragment of Erk2 was detected by
immunoblot analysis using an amino-terminus-specific antibody, confirming that
the targeting strategy resulted in a null allele. When Erk2
heterozygotes from a hybrid 129/Sv  C57Bl/6 background were
intercrossed, no homozygous mutants were recovered from a progeny of 128
(Table 1), indicating that absence of Erk2 leads
to embryonic lethality. Notably, Erk2+/- pups
represented 50% of the total offspring, deviating from the expected normal
mendelian frequency (p < 0.001). This indicates that some
heterozygous animals also died during embryonic development. By contrast, in a
CD1 background, the same cross (n = 394) produced 61.7% heterozygotes,
which is close to the normal mendelian frequency. The cause of death of
Erk2+/- animals in the 129/Sv C57Bl/6
background remains to be investigated. In contrast to the heterozygotes, the
phenotype of homozygous embryos was identical in the two genetic
backgrounds.
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Figure 1
Targeted disruption of the mouse Erk2 gene. (A) The
targeting vector (top), the wild-type Erk2 locus (middle) and the mutant
Erk2 locus (bottom) are shown. Black boxes represent Erk2 exons
(Ex2–Ex5) and the grey box represents the neor
(neomycin resistance gene)–poly(A) cassette. The arrowheads indicate the
positions and orientations of PCR primers that were used for genotyping
analysis. The 3' external probe used for Southern analysis is shown as
blue bars. KpnI restriction sites (K) are indicated. (B) Southern
blot analysis of wild-type and heterozygous embryonic stem cell DNA. DNA
samples were digested with KpnI and hybridized with the 3'
external probe. The positions and sizes of wild-type and mutant fragments are
indicated. (C) Immunoblot analysis of whole extracts from embryos
obtained from Erk2 intercrosses. Top panel, blotting using the
 1cp44 antibody, which recognizes both Erk1 and Erk2 isoforms. Lower
panel, control blot using anti- -actin. (D) Gross morphology of
wild-type, heterozygous and homozygous Erk2 mutant embryos at embryonic
day 6.5. Scale bar, 100  m. Erk2, extracellular signal-regulated
kinase 2.
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Table 1
Genotype of progeny from Erk2 intercrosses
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We next analysed timed pregnancies of Erk2 heterozygous matings
to determine the stage of embryonic development at which lethality occurred.
Embryos collected from embryonic days E6.5–E7.5 were found to have a
normal mendelian frequency (Table 1). However, at E8.5,
severely resorbed mutant embryos were found, suggesting that the embryos die
before this stage. Earlier, at E6.5 and E7.5,
Erk2-/- embryos were significantly smaller
than their wild-type or heterozygous littermates (Fig.
1D; and data not shown). Mutant embryos at E6.5 showed an abnormal
morphology, being oval in shape, with no obvious proximodistal and
anteroposterior polarities, which can be easily distinguished in wild-type
embryos at this stage (Fig. 1D). Analysis of blastocyst
outgrowth showed no difference between genotypes, suggesting that Erk2 is not
required for pre-implantation development (data not shown).
Histological analysis was performed to examine defects at the tissue
and cellular levels in early post-implantation embryos. At E5.5, wild-type
embryos had developed into egg cylinders with organized embryonic and
extra-embryonic structures and a proximal–distal polarity that was marked
by the ectoplacental cone (Fig. 2A). Mutant embryos were
oval in shape, with inner and outer epithelia, and lacked identifiable
ectoplacental cones (Fig. 2B). At E6.5, the inner layers
of mutant embryos looked disorganized (Fig. 2D), instead
of appearing as single layers of pseudostratified epithelia as in wild-type
embryos at the early-streak stage of gastrulation (Fig.
2C). The outer epithelial layer, known as the visceral endoderm, was
formed in mutant embryos, but visceral endoderm cells accumulated in the
proximal and distal regions of the E5.5 and E6.5 embryos, respectively (red
arrows in Fig. 2B,D). Parietal endoderm cells were also
seen along the uterine walls, suggesting that this differentiation step of the
primitive endoderm had occurred in mutant embryos (black arrow in
Fig. 2B). In addition, primary giant cells were
identified (black arrow in Fig. 2D), indicating that
further differentiation of trophectoderm cells takes place in the mutants.
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Figure 2
Impairment of extra-embryonic ectoderm development in
Erk2-/- embryos. (A–D)
Histological sections of control (A,C) and mutant embryos
(B,D) at early post-implantation stages (embryonic days (E) 5.5
and 6.5) show the absence of ectoplacental cones and reduced size of the mutant
embryo. (E–T) Marker gene expression in control
(E–L) and mutant (M–T) embryos by
whole-mount in situ hybridization followed by section analyses reveal
that the extra-embryonic ectoderm domains of Pem (n = 2)
(I,J,Q,R) and eomesodermin (Eomes; n
= 2) (K,L,S,T) are not expressed in the mutant
embryos, whereas the embryonic domains of Oct4 (n = 2)
(E,F,M,N) and Otx2 (n = 2)
(G,H,O,P) are still present.
(A–D) are shown at the same magnification, as are
(E–L) and (M–T). Scale bars, 100 m.
Erk2, extracellular signal-regulated kinase 2.
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The phenotype of Erk2 mutant embryos was characterized further
by examining the expression of appropriate marker genes for specific tissues in
embryos at E6.5. Expression of Oct4 and Otx2 is usually
restricted to all distal ectoderm or epiblast cells (Ang
et al., 1994; Scholer et al.,
1990), and is not detectable in the proximal extra-embryonic ectoderm
of wild-type embryos at this stage (Fig. 2E,F and
Fig. 2G,H, respectively). However, all cells of the inner
layer expressed these genes in Erk2-deficient embryos (Fig.
2M,N and Fig. 2O,P, respectively), suggesting that
the epiblast, but not the extra-embryonic ectoderm, was formed in these
mutants. This result was confirmed by the absence of expression of two markers
of the extra-embryonic ectoderm, Pem (Lin et
al., 1994) and eomesodermin (Eomes; Russ et al., 2000), in mutant embryos (Fig. 2Q,R and Fig. 2S,T, respectively).
Pem and Otx2 were expressed in the outer layer of wild-type as
well as mutant embryos (Fig. 2R and Fig.
2P, respectively), indicating that the visceral endoderm is specified to
some extent in these mutants. The histological and molecular data indicate that
inactivation of Erk2 leads to a specific loss of the extra-embryonic
ectoderm and ectoplacental cone, whereas other tissues are formed in the mutant
embryos. As the extra-embryonic ectoderm and ectoplacental cone are usually
derived from the polar trophectoderm, which corresponds to the subset of
trophectoderm cells that are in contact with the inner cell mass (ICM) in the
blastocyst, our results strongly suggest that the morphological defects in
Erk2 mutants may result from a primary defect at the level of the polar
trophectoderm.
Recent molecular and genetic findings indicate that fibroblast growth
factor 4 (Fgf4) is the main signal derived from the ICM that is required for
the proliferation of polar trophectoderm cells. Direct evidence for a role of
Fgf4 in promoting trophoblast proliferation comes from the treatment of
wild-type and Oct4 mutant blastocysts with Fgf4, which increases
trophectoderm cell numbers (Nichols et al.,
1998), and the ability to generate trophoblast stem (TS) cell lines
from blastocysts and extra-embryonic ectoderm in the presence of Fgf4 and
fibroblast-conditioned medium (Tanaka et al.,
1998). Fgf receptor 2 (Fgfr2), which is strongly expressed in the
proliferating trophoblast, is a likely candidate receptor for receiving the
Fgf4 signal. This model is consistent with genetic studies that show that Fgf4-
and Fgfr2-deficient embryos show similar phenotypes at the peri-implantation
stage. As ERK1/2 MAPKs are important downstream effectors of FGF receptors
(Ornitz & Itoh, 2001), it is tempting to
speculate that Erk2 functions downstream of Fgfr2 and is required cell
autonomously in polar trophectoderm cells to transduce the proliferative signal
of Fgf4.
To distinguish specific functions of Erk2 in embryonic versus
extra-embryonic tissues, we generated Erk2-/-
<-> +/+ chimeric embryos with wild-type extra-embryonic
tissues and >95% Erk2-/- epiblast cells by
injecting Erk2-/- ES cells into Rosa26
lacZ-expressing heterozygous embryos (Beddington &
Robertson, 1989). The Erk2-/- ES
cells were isolated from a heterozygous intercross (see
supplementary information online).
The resulting chimaeras (n = 5) developed ectoplacental cones and
extra-embryonic ectoderm abutting the embryonic ectoderm (Fig.
3A,B; and data not shown), indicating that the failure of these tissues
to develop correctly in Erk2-/- mutant
embryos is not because of a function of Erk2 in the epiblast. This
result suggests a specific function for Erk2 in extra-embryonic tissues
in the development of the extra-embryonic ectoderm.
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Figure 3
Erk2 is required in extra-embryonic tissues for the development of
the extra-embryonic ectoderm and the ectoplacental cone. Chimeric embryos at
embryonic day (E) 6.5 stained for -galactosidase, and the corresponding
sagittal sections, are shown. (A,B) Chimeric embryo with
wild-type extra-embryonic tissues (blue) and a predominantly
Erk2-/- epiblast (white), showing rescue of
extra-embryonic ectoderm (arrow in (B)) and ectoplacental cone
(arrowhead in (B)) development. (C,D) By contrast, a
chimeric embryo with Erk2-/- extra-embryonic
tissues (white) and a predominanly wild-type (blue) epiblast phenocopies
Erk2-/- embryos. In the latter chimaera, a
significant number of wild-type cells also contributed to the visceral endoderm
(arrows in (D)). Magnifications are the same in (A,B) and
in (C,D). Scale bars, 100 m. Erk2, extracellular
signal-regulated kinase 2.
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We also generated reciprocal +/+ <->
Erk2-/- chimeric embryos, with
Erk2-/- extra-embryonic tissues and
predominantly wild-type epiblast cells by injecting LacZ-positive
wild-type ES cells into embryos collected from
Erk2+/- intercrosses. These chimaeras (n =
3), with a contribution of 50–90% from wild-type ES-derived cells,
morphologically phenocopied the Erk2-/-
mutant embryos. Histological sections of these chimaeras revealed the absence
of extra-embryonic ectoderm and ectoplacental cones (Fig.
3C,D; and data not shown). In addition, cells accumulated in the distal
and/or proximal regions of the visceral endoderm in the chimaeras, as seen in
Erk2-/- embryos, and the epiblast cells
failed to develop into a columnar epithelium. The presence of a high
contribution of normal ES-derived cells in embryonic tissues of
Erk2-/- <-> +/+ chimeric
embryos did not rescue the development of the extra-embryonic tissues.
Altogether, the data obtained from the chimeric studies show that Erk2
functions autonomously in extra-embryonic tissues, probably in the polar
trophectoderm, for the development of the ectoplacental cone and the
extra-embryonic ectoderm. The visceral endoderm and epiblast defects observed
in Erk2-/- mutant embryos and
+/+ <-> Erk2-/- chimeric
embryos may be indirect and will require further investigation.
Disruption of the Erk1 gene did not result in any developmental
phenotype (Pagès et al., 1999),
indicating that Erk1 is dispensable during embryonic development or,
alternatively, that Erk2 can compensate for the loss of Erk1. A possible
explanation for the embryonic lethality seen on inactivation of the Erk2
gene is that Erk1 is not expressed in early-stage embryos, and thus cannot
functionally rescue the loss of Erk2. To determine whether Erk1 and
Erk2 differ in the timing of their expression during early embryonic
development, we performed radioactive in situ hybridization on sagittal
sections of wild-type and mutant embryos. Both Erk2 and Erk1 were
found to be widely expressed throughout the whole embryo (Fig.
4A,F and Fig. 4C,H, respectively).
Immunohistochemical analysis of E5.5 embryos using a phosphospecific antibody
indicated that Erk1 and Erk2 are activated in the extra-embryonic ectoderm,
ectoplacental cone and trophectoderm giant cells (see
supplementary information online).
We also found that Erk1 and Erk2 proteins are expressed and phosphorylated in
blastocyst-derived TS cells (data not shown). As Erk1 is still expressed
in Erk2 mutants (Fig. 4E,J; n = 2), the
lack of compensation for the loss of Erk2 by Erk1 strongly
suggests that Erk2 has a specific function in the developing embryo.
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Figure 4
Gene expression patterns for Erk1 and Erk2 at E6.5.
Brightfield (A–E) and corresponding darkfield images
(F–J) are shown. Erk2
(A,B,F,G) and Erk1
(C–E,H–J) are ubiquitously expressed
throughout the whole embryo in control embryos ((A,F) and
(C,H), respectively) at E6.5, as assessed by 35S
radioactive in situ hybridization. Erk1 is expressed in mutant
embryos (E,J). 3' UTR sense and antisense probes were used.
Magnifications are the same in
(A–D,F–I) and (E,J).
Scale bars, 100 m. Erk2, extracellular signal-regulated kinase
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Our results show that Erk2 has an essential function in embryonic
development in the generation of extra-embryonic ectoderm, which gives rise to
the fetal part of the placenta and the secondary trophoblast giant cells.
Hence, Erk2 is required for mouse trophoblast development, probably in
signalling that is required for the proliferation of TS cells in the polar
trophectoderm. This hypothesis is supported by the observation that TS cell
lines cannot be generated in the presence of specific inhibitors of the
upstream kinases MAPK/ERK kinase 1 (MEK1) and MEK2 (Rossant,
2001). Interestingly, the activation of ERK1/2 was recently suggested
to be dispensable for the proliferation of ES cells, but might instead have a
more important function in their differentiation (Burdon
et al., 1999). In support of this idea, we found no
significant change in the proliferative rate of
Erk2-/- epiblast cells in mutant embryos when
compared to wild-type littermates at E6.5 (see
supplementary information online).
All these findings indicate that the early cell lineages in the conceptus
respond differently to ERK1/2 signalling. TS cells and ES cells might therefore
be good in vitro model systems for examining how specific cellular
responses to the ERK1/2 MAPK pathway are generated.
Methods
Gene disruption.
The targeting vector was constructed by inserting a
PGK–neor cassette into exon 3 of Erk2, which
contains protein kinase subdomains V and VI. The linearized vector was
electroporated into the 129-derived ES cell line D3. The presence of the
targeted allele of Erk2 was determined by
restriction-fragment-length-polymorphism analysis of genomic DNA extracted from
ES cells using an external 3' probe corresponding to a 1.5-kb XbaI
fragment containing exon 4. The presence of a single neor
cassette was confirmed by Southern blot analysis using a
neor gene fragment as a probe (data not shown). Cells
heterozygous at the Erk2 locus (Erk2+/-)
were used to generate chimeric mice by injection into blastocyst-stage embryos.
Chimeric males were bred with C57Bl/6 females to produce F1
heterozygotes. Germline transmission was confirmed by Southern blot analysis.
F1 heterozygous males and females were mated to produce homozygous
mutant animals. DNA was extracted from tail biopsies obtained three weeks after
birth. Genotypes were determined by Southern blot hybridization using an
external 3' probe or by PCR (see supplementary information online).
Generation of chimeric embryos.
The Erk2-/- ES cell line B1 was
generated by the in vitro culture of blastocysts (Hogan et al., 1994) isolated from Erk2
heterozygous intercross matings. Cytogenetic analysis of the B1 line was
performed by the Banque de Cellules Leucémique du Québec and
showed a normal karyotype. The morulae-stage (E2.5) embryos used to generate
chimaeras were obtained from intercrosses of ROSA26/+ and CD1 mice.
These embryos were injected with approximately ten
Erk2-/- ES cells from the B1 line and ten
LacZ-positive ES cells from the Rosa26.1 line (Varlet et
al., 1997), respectively, and were subsequently reimplanted into
pseudopregnant females. Chimeric embryos were harvested at E6.5 and E7.5 and
processed for -galactosidase staining as described previously (Hogan et al., 1994).
Immunoblot analysis.
Protein extracts were prepared by homogenization of embryos or cells
in lysis buffer. Equal amounts of protein were resolved by
SDS–polyacrylamide gel electrophoresis and transferred to nylon membrane.
The membrane was blocked in TBS, 0.1% Tween 20, 4% non-fat dried milk, and
probed with antibody 1cp44 (1:5000 dilution), which recognizes Erk1 and
Erk2 isoforms (Meloche, 1995). To control for
protein loading, the blot was stripped and reprobed with anti--actin
(1:10,000 dilution). We also used an Erk2 N-terminal-specific polyclonal
antibody (AB3055; Chemicon) to confirm the absence of expression of N-terminal
fragments of Erk2.
Supplementary
information is available at EMBO reports online
(http://www.emboreports.org).
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Acknowledgements
We thank A. Dierich for the culture of ES cells, H. Scholer and A.
Russ for providing Oct4 and eomesodermin probes, E. Robertson for providing
Rosa26.1 ES cells, and J. Rossant for critical reading of the manuscript and
for the phospho-ERK1/2 immunostaining protocol. We acknowledge C. Benoist and
D. Mathis for their contribution during the early phase of this work. This work
was supported by grants from the Cancer Research Society and Canadian
Institutes of Health Research (CIHR) to S.M., by grants from the European
Community Biotech Programme and Association pour la Recherche Contre le Cancer,
and by funds from the INSERM, the Centre National de la Recherche Scientifique
and the Hôpital Universitaire de Strasbourg to S.-L.A. S.M. is an
Investigator of the CIHR.
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