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First published online 3 August 2006
doi: 10.1242/dev.02526
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Centre de recherche en cancérologie de l'Université Laval, Centre Hospitalier Universitaire de Québec, L'Hôtel-Dieu de Québec, Québec, QC G1R 2J6, Canada.
* Author for correspondence (e-mail: jean.charron{at}crhdq.ulaval.ca)
Accepted 28 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: MAP2K1, Map kinase cascade, Conditional deletion, Placenta, Labyrinthine morphogenesis, Syncytiotrophoblast, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Multiple MAPK pathways have been described in vertebrates,
which include the extracellular signal-regulated kinase (ERK), the p38 kinase
and the Jun NH2-terminal kinase (Cano and
Mahadevan, 1995; Seger and
Krebs, 1995; Zanke et al.,
1996). The classical pathway, which appears to be the major one in
growth factor signaling, involves the extracellular signal-regulated kinases
ERK1 and ERK2 (renamed MAPK3 and MAPK1, respectively) and the ERK kinases
(MAP2K1 and MAP2K2 also known as MEK1 and MEK2), and it is known as the
ERK/MAPK pathway. In this cascade, MAP2Ks are dual specificity kinases that
activate MAPK1 and MAPK3 upon agonist binding to receptors
(Crews et al., 1992). The
ERK/MAPK pathway is implicated in cell fate determination in
Caenorhabditis elegans, Drosophila and Xenopus laevis
(Hsu and Perrimon, 1994;
Kornfeld et al., 1995;
Umbhauer et al., 1995;
Wu et al., 1995). In mammals,
this pathway is proposed to regulate cell growth and differentiation
(Johnson and Vaillancourt,
1994).
Although two different MAP2K proteins are present in the ERK/MAPK cascade
in mammals, a single Map2k gene fulfills this role in
Caenorhabditis elegans, Drosophila and Xenopus laevis.
Sequence analysis revealed that the murine MAP2K1 is more related to the
Xenopus laevis MAP2K1 (X-MEK) than to the mouse MAP2K2
(Brott et al., 1993;
Crews et al., 1992;
Russell et al., 1995). Two
regions of MAP2K1 show reduced homology with MAP2K2: (1) the N terminus (33%
identical/66% similar), which has been shown in X-MAP2K1 to be involved in the
interaction of MAP2K1 with MAPKs; and (2) a part of the MAP2K specific
sequence (MSS; 21% identical/36% similar), which is shared by MAP2K1 proteins
from different species (Fukuda et al.,
1997; Papin et al.,
1996; Xu et al.,
1997). The MSS domain of MAP2K1 contains a PAK phosphorylation
site important for its function (Coles and
Shaw, 2002). It is also involved in the interaction with the Raf
family members (Catling et al.,
1995; Dang et al.,
1998; Eblen et al.,
2002; Nantel et al.,
1998; Papin et al.,
1996). The protein sequence differences observed between MAP2K1
and MAP2K2 suggest that they have most probably diverged to achieve unique
functions in mammals.
The differential role of MAP2K1 and MAP2K2 in signal transduction during
mouse development has been revealed by the characterization of mutant mouse
lines in which the Map2k1 or Map2k2 gene has been disrupted
(Bélanger et al., 2003;
Giroux et al., 1999). The null
mutation of the Map2k1 gene results in a recessive lethal phenotype,
the mutant embryos dying at 10.5 days of gestation because of an abnormal
development of the labyrinthine region of the placenta.
Map2k1-/- embryos appear morphologically normal, and
vasculogenesis and angiogenesis seem to take place normally, as evidenced by
the presence of intersomitic vessels and capillaries in the head region.
However, the Map2k1-/- placenta presents a marked
reduction of vascularization in the labyrinth. Map2k2-/-
mice showed no obvious phenotype, suggesting compensatory effects by
Map2k1 (Bélanger et al.,
2003; Giroux et al.,
1999). By contrast, the Map2k1-/- phenotype is
observed in the presence of normal Map2k2 expression levels in the
placenta, providing genetic evidence that Map2k2 is unable to make up
for the absence of Map2k1 (Giroux
et al., 1999). Altogether, these observations indicate that
Map2k1 and Map2k2 genes accomplish specific functions in
mammals.
The placenta is a highly vascularized organ, which allows fetal-maternal
exchanges during gestation. It is composed of a vascular network and stroma
coming from the embryonic mesoderm, and of trophoblast cells that arise from
the extra-embryonic tissue and differentiate to achieve specialized functions
(Coan et al., 2005;
Cross, 2000;
Rossant and Cross, 2001;
Simmons and Cross, 2005). In
mice, placenta formation initiates around E8.5 with the fusion of the chorion
with the allantois, which will give rise to the labyrinthine region. As the
labyrinth develops, it becomes highly folded and branched, generating an
important surface area required for nutrient and gas exchanges. A large part
of the labyrinth is composed of one layer of mononuclear trophoblasts and two
layers of syncytium (syncytiotrophoblasts) that separate the maternal blood
space from the fetal blood vessels. Contribution of the ERK/MAPK pathway to
placental development has been highlighted by the characterization of mouse
lines carrying mutations in molecules involved in this signaling cascade.
Growth factors (HGF), growth factor receptors (MET, FGFR2 and PDFGR) and
components of the ERK/MAPK cascade (GRB2, GAB, SOS1, RAF1, MAP2K1 and MAPK1)
have been reported to present defects in the development of the placenta
labyrinthine region (reviewed by Rossant
and Cross, 2001). Hence, signaling via the ERK/MAPK pathway is
essential for placental development.
To dissect the physiological role of MAP2K1 in placental development, we have used the Map2k1 mutant mouse line previously generated to fully characterize the placental phenotype. Map2k1-deficient placenta exhibited a reduced proliferation combined to an augmented apoptosis of labyrinth trophoblasts. Moreover, the activation of the ERK/MAPK cascade is dramatically diminished in Map2k1 mutant placenta extracts indicating the requirement of Map2k1 function to transduce signals to the MAPKs, MAPK1 and MAPK3. In wild-type placenta, MAPK activation is mainly observed in the cell layers lining the maternal sinuses. Despite the absence of MAPK1 and MAPK3 activation, the Map2k1 mutation does not affect the determination and the differentiation of the syncytiotrophoblasts. However, the Map2k1-deficient syncytiotrophoblasts are unable to invade the placental labyrinth thereby suggesting a role for the ERK/MAPK cascade in this process. Rescue of the Map2k1 mutation in the extra-embryonic structures allows normal development of Map2k1-/- embryos and production of live-born animals. Altogether, these observations indicate that MAP2K1 acts cell autonomously in the extra-embryonic ectoderm to support the development of the labyrinthine region.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). All
experiments were performed according to the guidelines of the Canadian Council
on Animal Care and approved by the institutional animal care committee.
Tissue collection
Embryonic age was estimated by considering the morning of the day of the
vaginal plug as E0.5. Specimens were collected and processed for paraffin
inclusion or cryosection. For paraffin inclusion, the specimens were fixed
overnight in 4% paraformaldehyde at 4°C. Serial sections of 4 µm were
deparaffinized, rehydrated and either stained with Hematoxylin and Eosin
according to standard procedures, or submitted to immunohistological analyses
described below. Frozen sections were prepared from specimens fixed overnight
in 8% paraformaldehyde, equilibrated in a 30% sucrose solution in 0.1 M
phosphate buffer at pH 7 and then embedded in OCT. Serial sections of 8 µm
were mounted on polylysine-treated slides for CD31 and phospho-p38
immunodetection.
Proliferation, apoptosis and immunohistochemical analyses
Proliferation rate was assessed by immunodetection of phospho-histone H3, a
mitotic marker (pH3; Upstate Biotechnology)
(Aubin et al., 2002). Apoptotic
cells were detected by terminal transferase (TdT) DNA end labeling
(Giroux and Charron, 1998).
Rabbit polyclonal antibodies against: phospho-p38 MAPK, phospho-MAP2K1/MAP2K2
and phospho-MAPK1/MAPK3 (Cell Signaling Technology) were used at a 1/50
dilution. The placental vascular network was revealed with anti-CD31 antibody
used at a 1/50 dilution (PECAM; Pharmingen International). Antigen retrieval
was performed under pressure in a microwave for 2 minutes in 10 mM sodium
citrate buffer. Non-specific binding was blocked by incubation with 10% normal
goat serum for 1 hour at room temperature. The Vectastain HRP ABC Reagent
(Vector Laboratories) was used for detection and the sections were
counterstained with Hematoxylin.
Six specimens were analyzed per genotype. The most representative fields
were presented in the figures. Bright-field and dark-field illuminations were
photographed on a DM RB microscope (Leica) using a QImaging CCD camera (QICAM)
and the Openlab software (Improvision). The photos were processed using Adobe
Photoshop CS program. For proliferation and apoptosis studies, ratio of
positively stained cells on the total cell number was evaluated for a minimum
of five random areas. Repeated measures for the linear mixed model were
performed to assess the difference between genotypes at all stages studied
when genotype is considered the fixed effect and while area is the random
effect. The procedure PROC MIXED from the SAS System was used
(Littell et al., 1998).
Western blot analysis
Protein extracts were prepared as previously described
(Bélanger et al., 2003).
Total protein lysates (20 µg) were resolved on a denaturing 10% SDS-PAGE
and probed with rabbit polyclonal anti-MAP2K1, polyclonal anti-MAP2K2 and
polyclonal anti-MAPK1 antibodies. Mobility shift assays to resolve
phospho-MAP2K1 from phospho-MAP2K2 and the phosphorylated and
non-phosphorylated forms of MAPK1 and MAPK3 were performed as described
(Bélanger et al., 2003).
The phospho-specific antibodies for MAPK1/MAPK3 and MAP2K1/MAP2K2 were the
same as those used for immunohistochemistry.
In situ hybridization, ß-galactosidase staining and alkaline phosphatase assays
Radioactive in situ hybridization on tissue sections was previously
described (Giroux and Charron,
1998). The following murine fragments were used as templates for
synthesizing [35S]UTP-labeled riboprobes: a 1.5 kb cDNA fragment
for the Vegf 120 isoform and a 1.5 kb cDNA Gcm1 fragment.
ß-Galactosidase staining was performed on whole
Map2k1+/- and Map2k1-/- placentas
(Giroux et al., 1999). Stained
specimens were embedded in paraffin wax and sectioned (7 µm) for
photography. Alkaline phosphatase activity was assayed by incubating
rehydrated E10.5 Map2k1+/+ and
Map2k1-/- placenta sections with BM substrate (Boehringer
Mannheim).
Generation of tetraploid-aggregation chimeras
Tetraploid embryos of wild-type B6CBAF1 were prepared by electrofusion and
aggregated with diploid Map2k1+/- embryos or
Map2k1-/- ES cells as described
(Nagy et al., 1993). The
resulting aggregates, which had reached the blastocyst stage, were transferred
into E2.5 pseudopregnant females, and the embryos were dissected at E11.5 or
E13.5.
Generation of the Map2k1 conditional allele
The targeting vector was made by using a 12.4 kb genomic fragment isolated
from a 129/Sv mouse strain-derived genomic library. It encompasses exons 2 to
4 of the Map2k1 gene, which was fused to the herpes simplex
virus-thymidine kinase cassette for selection against random integration. To
disrupt the Map2k1 sequences, two loxP sequences were
inserted into the BamHI sites flanking the third exon of
Map2k1.A neo cassette flanked by loxP sites was
inserted in the third intron (Fig.
7). Deletion of the Map2k1 third exon should result in an
out-of-frame transcript, creating a Map2k1-null allele. Correctly
targeted ES clones were injected into MF1 blastocysts to generate chimeras as
described (Bélanger et al.,
2003). The chimeras were bred with 129/SvEv mice to transmit the
targeted Map2k1 allele (Map2k1flox-neo). To
generate the Map2k1 conditional allele
(Map2k1floxed), we used the EIIa-Cre deleter
mouse line that can produce mosaic mice with partial excision of loxP
flanked sequences (Lakso et al.,
1996). Specimens were genotyped by Southern analysis of a
StuI digestion using a Map2k1 genomic probe
(Fig. 7). The Map2k1
endogenous allele generates a 2.0 kb fragment, whereas the
Map2k1floxed and Map2k1
alleles
produce bands at 2.4 kb and 4.2 kb, respectively.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
However, the labyrinthine region of the Map2k1 mutant placenta was
reduced in size, a phenotype already obvious at E9.5 1 day before the death of
the Map2k1 mutant embryos (Fig.
1A) (Giroux et al.,
1999). Given the well-documented role of the ERK/MAPK cascade in
cellular proliferation and survival (Huynh
et al., 2003; Pages et al.,
1993; von Gise et al.,
2001; Wu et al.,
2004; Yu et al.,
2004), we hypothesized that the disruption of Map2k1
might affect labyrinthine expansion by limiting cell proliferation and/or
survival. We first performed immunostaining using phospho-histone H3 antibody
to detect mitotic cells in E8.5 to E10.5 placentas
(Fig. 1B). In wild-type
placentas, the proliferation rate in the labyrinthine region was elevated
resulting in extensive labyrinthine development
(Fig. 1B,D)
(Hemberger and Cross, 2001).
By contrast, the proliferation rate was halved in
Map2k1-/- placenta at E9.5 and E10.5 (P<0.01;
Fig. 1D). The decreased
proliferation observed in E8.5 Map2k1-/- labyrinth was not
statistically significant (P>0.09;
Fig. 1D). In parallel, TUNEL
assays were performed to determine if apoptosis contributes to the reduced
growth of the Map2k1-/- labyrinthine region. Very few
apoptotic cells were detected in E8.5 to E10.5 wild-type specimens
(Fig. 1C,E). Conversely,
apoptosis was increased by fivefold in E8.5 and E9.5
Map2k1-/- specimens and by tenfold at E10.5 when
Map2k1-/- embryos were dying and in resorption
(Fig. 1E). Therefore, perturbed
cell proliferation and apoptosis underlay the reduced expansion of the
labyrinthine region in Map2k1-/- placenta.
Loss of ERK/MAPK activation in Map2k1-/- placenta
Both Map2k1 and Map2k2 genes are ubiquitously expressed
in the embryo and strongly in the labyrinthine region
(Giroux et al., 1999). To
define whether the proliferation defect observed in
Map2k1-/- placenta was due to perturbed ERK/MAPK
signaling, we performed western blot analyses with embryonic and placental
whole protein extracts from E9.5 and E10.5 wild-type and
Map2k1-/- specimens using phospho-specific MAPK1/MAPK3 and
phospho-specific MAP2K1/MAP2K2 antibodies. The phosphorylation of MAPKs and
MAP2Ks at specific residues of the activation loop is usually a good read-out
of their state of activation (Gopalbhai et
al., 2003). The absence of the MAP2K1 protein and the level of
proteins loaded on the gel were controlled with antibodies against MAP2K1 and
MAPK1, respectively (Fig. 2A).
In E9.5 Map2k1-/- embryonic extracts, lack of MAP2K1 did
not seem to affect MAPK1 and MAPK3 phosphorylation (pMAPK1/pMAPK3), while in
placenta extracts, the mutation caused a diminution of the pMAPK1 and pMAPK3
levels (Fig. 2A). This decrease
was more dramatic in E10.5 Map2k1-/- placentas, despite
the presence of MAP2K2 phosphorylation (pMAP2K2;
Fig. 2B). Similarly, an
important reduction in pMAPK1 and pMAPK3 levels was detected in
Map2k1-/- embryos. In Map2k2-/-
specimens, the levels of pMAPK1, pMAPK3 and pMAP2K1 proteins were unchanged
compared with those of wild-type specimens
(Fig. 2B). These results
suggested that although MAP2K2 was phosphorylated and activated in
Map2k1-/- placentas, it could not activate the ERK/MAPK
pathway and promote cellular proliferation and survival in the placenta. A
likely explanation for the difference in pMAPK1 and pMAPK3 levels between E9.5
and E10.5 embryos could be that E10.5 Map2k1-/- embryos
were dying and in the process of resorption.
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One day later, pMAP2K1/pMAP2K2 positive signal was seen in the labyrinthine region, the cells lining the maternal sinuses, the allantoic cells and, in addition, in the endothelium lining the embryonic blood vessels in wild-type and Map2k2-/- placentas (Fig. 3B; see Fig. S1 in the supplementary material). In Map2k1-/- specimens, the highest staining was restricted to the cells of the allantois and at the chorioallantoic interface (Fig. 3B). Interestingly, despite the widespread MAP2K1 and MAP2K2 activation, pMAPK1/pMAPK3 staining was mainly detected in the cells lining the maternal sinuses, while no signal was observed in the endothelium of the embryonic blood vessels (Fig. 3B). As observed at E9.5, pMAPK1/pMAPK3 staining was restricted to the allantoic cells in E10.5 Map2k1-/- placentas.
Altogether, these results indicated that the ERK/MAPK pathway was strongly activated in the cell population lining the maternal sinuses of the placental labyrinth. In Map2k1-/- placentas, even though MAP2K2 activation was observed at the chorioallantoic interface, phosphorylation of MAPK1 and MAPK3 proteins was greatly reduced, reinforcing the notion of the specific requirement for MAP2K1 in the activation of the ERK/MAPK cascade.
Normal VEGF angiogenic signaling in Map2k1-/- placentas
We have previously shown that the fetal vascular endothelial cells are
excluded from the chorion in E9.5 Map2k1-/- placentas,
suggesting that placenta angiogenesis could be defective
(Giroux et al., 1999).
However, the data presented in Fig.
3 demonstrated that the ERK/MAPK pathway was not strongly
activated in the embryonic vascular endothelial cells of the labyrinthine
region in wild-type placentas when compared with the activation observed in
the cells lining the maternal sinuses, indicating that the ERK/MAPK cascade
may not play a direct role in labyrinthine angiogenesis
(Fig. 3). The p38/MAPK
signaling pathway was shown to be implicated in angiogenesis and activated in
response to VEGF (Mudgett et al.,
2000; Rousseau et al.,
1997). Therefore, we decided to investigate the activation status
of the p38/MAPK cascade in Map2k1 mutant placentas.
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;
Conrad et al., 2000;
Fan et al., 2005). To assess if the increased Vegf expression and the p38/MAPK activation coincide with the domain of action of MAP2K1, we took advantage of the lacZ cassette of the ROSAß-geo promoter trap vector used to generate the Map2k1 mutant allele to visualize X-Gal staining and localize Map2k1 expression in E10.5 Map2k1+/- and Map2k1-/- placentas. In Map2k1+/- placentas, a widespread X-Gal staining was observed in the labyrinth region, whereas in Map2k1-/- specimens, X-Gal stained cells were confined to the chorioallantoic plate, forming a barrier between the embryonic and the maternal blood cells (Fig. 4G,H). Thus, in Map2k1-/- placentas, the p38/MAPK cascade was activated in the embryonic vascular endothelial cells blocked at the chorioallantoic plate, indicating a capacity to generate an angiogenic response to the hypoxic stress, the latter most probably resulting from poor blood exchanges between the embryo and the mother caused by hypovascularization. The defective angiogenesis observed in Map2k1-/- placentas may therefore be secondary to a morphogenic problem.
Normal determination and differentiation of Map2k1-/- syncytiotrophoblasts
The vascularization of the labyrinth is initiated when the allantois fuses
with the chorion around E8.5. At this stage, complex morphogenic and
vascularization processes will generate the labyrinth. There is increasing
evidence to indicate that the trophoblast epithelium contributes actively to
the chorioallantoic branching (Rossant and
Cross, 2001). A subgroup of trophoblasts in the chorionic plate
starts to express Gcm1, an early marker of the syncytiotrophobasts,
at the site of allantoic mesoderm evagination before the chorioallantoic
fusion, suggesting that the morphogenesis of the labyrinth proceeds in
response to instructive signals from the allantois and involved the
syncytiotrophoblast cell line. This will then allow the vascularization of the
labyrinth by the embryonic blood vessels arising from the allantois
(Rossant and Cross, 2001).
Syncytiotrophoblasts and mononuclear trophoblasts coat the maternal sinuses to
isolate the fetal blood circulation from the maternal one
(Coan et al., 2005;
Simmons and Cross, 2005). As
MAPK1 and MAPK3 are strongly activated in the cells lining the maternal
sinuses (Fig. 3), we
investigated whether the lack of pMAPK1 and pMAPK3, as well as the
vascularization defect observed in Map2k1-/- placentas,
were due to the misspecification of syncytiotrophoblasts.
We first performed in situ hybridization experiments with a Gcm1
probe used as a marker for syncytiotrophoblast precursors on E8.5, E9.5 and
E10.5 wild-type and Map2k1-/- placentas. As previously
reported, Gcm1 was expressed in cell clusters in E8.5 wild-type
chorions (Stecca et al.,
2002), and a similar expression profile was detected in
Map2k1-/- chorions
(Fig. 5A,B). In E9.5 and E10.5
wild-type placentas, Gcm1-positive cells started to invade the
labyrinth (Fig. 5C,E), while in
Map2k1-/- placentas, Gcm1-positive cells were
blocked at the chorioallantoic junction
(Fig. 5D,F). In order to
determine whether the syncytiotrophoblast precursors of
Map2k1-/- placentas were able to differentiate into
syncytiotrophoblasts, we performed alkaline phosphatase assays on E10.5
wild-type and Map2k1-/- specimens as differentiated
syncytiotrophoblasts lining the maternal sinuses express endogenous alkaline
phosphatase activity (Matsubara et al.,
1993; Wu et al.,
2003). As expected, we observed a strong alkaline phosphatase
activity around maternal sinuses in wild-type placentas
(Fig. 5G,I). In
Map2k1-/- specimens, alkaline phosphatase activity was
detected in patches and followed a similar pattern to the Gcm1 signal
(Fig. 5H,J). These results
strongly suggested that the Gcm1-positive cells were correctly
determined at the right moment in the Map2k1-/- placentas.
They were also able to differentiate, as demonstrated by the alkaline
phosphatase activity. However, they were unable to invade the chorion,
allowing the formation of the vascular network of the labyrinth. Thus, the
ERK/MAPK signaling via MAP2K1 is not likely to be required for
syncytiotrophoblast differentiation but appears to be necessary for the
chorioallantoic branching morphogenesis by the syncytiotrophoblasts.
Tetraploid rescue of Map2k1-deficient embryos
Our results suggest that the activation of the ERK/MAPK cascade is greatly
reduced in the Map2k1-/- syncytiotrophoblasts that
originate from extra-embryonic tissues. By contrast, the p38/MAPK pathway was
normally activated in Map2k1-/- vascular endothelial cells
derived from the embryo. These data suggested that the
Map2k1-/- placental phenotype has an extra-embryonic
origin that affects the labyrinthine morphogenesis. The vascularization defect
observed might be a consequence of abnormal chorioallantoic branching. To
define in which structure, embryonic or extra-embryonic, MAP2K1 was playing an
essential role, we performed tetraploid rescue experiments.
|
). ES cells have essentially the reciprocal developmental
potential compared with tetraploid cells in chimeras. They can participate to
all inner cell mass derivatives but not to trophectoderm or primitive endoderm
lineages. Based on these characteristics, aggregation of
Map2k1-/- ES cells to wild-type tetraploid embryos should
lead to the identification of the tissue responsible for the placental
abnormality. The combination of Map2k1-/- ES cells and
tetraploid wild-type embryos allowed the survival of
Map2k1-/- embryos up to E11.5 and E13.5 (four and one
chimeric embryos, respectively; Fig.
6C,E). Gross morphology and histological staining of the chimeric
placentas revealed that the labyrinth developed normally
(Fig. 6H,J,M,O,R,T). Anti-CD31
staining showed that in Map2k1-/- tetraploid-rescued
embryos, fetal blood vessels migrated into the labyrinth and intermingled with
the maternal sinuses similarly to those observed in wild-type placenta
(Fig. 6U-W). The
Map2k1-/- tetraploid chimeras obtained at E11.5 were well
developed, whereas the gross morphology and the histology of the chimera
obtained at E13.5 suggested that the rescue was incomplete. The E13.5 chimera
was slightly underdeveloped and presented signs of hemorrhage
(Fig. 6E). Moreover, even
though the vascularization of the labyrinth was normal, no fetal blood cell
was detected in the blood vessels conversely to those seen in
Map2k1+/- tetraploid chimeras
(Fig. 6S,T). These observations
raised the possibility that the tetraploid-aggregation experiments rescued
partially the Map2k1-/- mutant phenotype. Alternatively,
as one Map2k1-/- ES cell clone was tested in the
tetraploid rescue experiments, we cannot exclude a clonal effect. Altogether, these data indicated that Map2k1 might be dispensable for the normal development of the embryo, and that MAP2K1 is required in extra-embryonic-derived structures like the trophoblasts for the normal development of the labyrinthine region.
|
). To do
so, Sox2Cre;Map2k1+/ males were bred with
Map2k1+/floxed females. With this breeding, normal
Mendelian ratio of live-born Map2k1/ animals
was obtained (Table 1;
Fig. 7B). To verify that the
Map2k1/ mice were devoid of MAP2K1 protein,
western blot analyses of protein extracts from different organs of
Map2k1+/+ and Map2k1/
adult mice were performed. No MAP2K1 protein was detected in
Map2k1/ organs, demonstrating that the
Map2k1 allele is a null allele
(Fig. 7C). Both
Map2k1/ female and male mice were fertile
and when they were intercrossed, they generated litters that died at E10.5,
exhibiting the same phenotype as the one observed in the
Map2k1-/- embryos (Fig.
8A). Altogether, these results confirmed the primordial role of
Map2k1 in placenta development, whereas, in the embryo, it can
probably be replaced by its homologue, Map2k2.
|
/ embryos;
Brunet et al., 1995;
Zhang and Liu, 2002). In E9.5
Map2k1-/- embryos, the cascade was normally activated,
whereas 1 day later, no pMAPK1- and pMAPK3-specific bands were detected in
Map2k1-/- embryos, despite the presence of activated
MAP2K2 (Fig. 2B). These data
suggested that the inability of MAP2K2 to activate the ERK/MAPK pathway at
E10.5 was due to the fact that the Map2k1-/- embryos were
dying from underdevelopment of the placenta. To address this issue directly,
we analyzed the phosphorylation status of MAPK1 and MAPK3 in E10.5
Map2k1/ embryos and their corresponding
Map2k1floxed/ placentas obtained from the breeding
of Sox2Cre;Map2k1+/ males with
Map2k1+/floxed females. Western blot analyses revealed no
decrease in pMAPK1 and pMAPK3 in Map2k1/
embryos, and in the corresponding placental extracts
(Fig. 8B). Therefore, in E10.5
conditionally rescued Map2k1/ embryos,
MAP2K2 can activate MAPK1 and MAPK3. Thus, in absence of a normal placenta,
E10.5 Map2k1-/- embryos were dying and hence could not
achieve ERK/MAPK activation.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). By
contrast, the absence of phenotype in Map2k2 mutants suggests that
Map2k2 is dispensable, as Map2k1 can compensate for the lack
of Map2k2 gene function
(Bélanger et al., 2003).
We have previously shown that both Map2k1 and Map2k2 genes
are widely expressed in the placenta
(Giroux et al., 1999).
Immunostaining of the active forms of MAP2K1 and MAP2K2, as well as their
downstream effectors (MAPK1 and MAPK3), revealed that, although MAP2K1 and
MAP2K2 can be activated in several cell types of the placenta, their action is
limited to specific lineages. For example, detectable levels of pMAPK1 and
pMAPK3 were observed in the cells lining the maternal sinuses, the allantoic
cells and some labyrinthine trophoblasts of wild-type placentas. In
Map2k1-/- placentas, pMAPK1 and pMAPK3 were restricted to
the allantoic cells at the chorioallantoic interface, suggesting that the
activation of the ERK/MAPK cascade in the labyrinth may specifically require
Map2k1. It has recently been shown that the catalytic activity of
activated MAP2K1 and MAP2K2 proteins can be repressed by the phosphorylation
of Ser212 (Gopalbhai et al.,
2003), suggesting that the phosphorylation of MAP2K2 at
Ser212 may account for the lack of MAPK1/MAPK3 phosphorylation in
the labyrinth. Alternatively, it is also possible that the levels of activated
MAPK1 and MAPK3 sufficient for MAP2K1 function in the labyrinth trophoblasts
are under the detection levels.
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;
Pages et al., 1999;
Saba-El-Leil et al., 2003).
Together, these results support a model in which placenta development requires
that the transduction of specific signals transits via distinct protein kinase
isoforms such as MAPK1 and MAP2K1 through the ERK/MAPK cascade. The presence
of various isoforms at different levels of the pathway reflects the complexity
of the controls required for the regulation of diverse mammalian cellular
processes.
Using specific MAP2K1 and MAP2K2 inhibitors, the ERK/MAPK cascade was shown
to be required for in vitro trophoblast stem (TS) cell establishment and
maintenance (Rossant, 2001).
In agreement with this, a strong activation of MAPK1 and MAPK3 was observed in
regions of the extra-embryonic ectoderm known for the presence of FGF
signaling and TS cell niches (Corson et
al., 2003; Tanaka et al.,
1998). However, the lack of phenotype in
Map2k2-/- mutants strongly indicates Map2k1 as a
key player in TS cell establishment and growth. The role of Map2k1 in
TS cell proliferation and maintenance is further supported by the fact that we
were able to generate TS cells from wild-type and
Map2k2-/- embryos but not from
Map2k1-/- blastocysts (V.B., M.G. and J.C., unpublished;
data not shown). Even though the placenta growth is perturbed in
Map2k1-/- specimens
(Fig. 1), the different
trophoblastic cell lineages derived from the TS cells (including trophoblast
giant cells, spongiotrophoblasts, syncytiotrophoblasts and other trophoblasts
from the labyrinth) were present in Map2k1-/- placentas
(Fig. 5)
(Giroux et al., 1999). These
results suggest that even if the ERK/MAPK cascade is required in vitro for TS
establishment and maintenance, other factors independent of MAP2K1 may
contribute in vivo to these processes, allowing the partial development of the
Map2k1-/- placenta. In the absence of Map2k1
function, proliferation and survival of the trophoblasts in the chorion and
the labyrinthine region were impaired, leading to the underdevelopment of the
labyrinth and ultimately to the death of the embryo.
The characterization of the activation of the ERK/MAPK cascade in
Map2k1-/- embryos and placentas indicates that MAPK1 and
MAPK3 are normally phosphorylated in E9.5 Map2k1-/-
embryos, whereas in their placenta, activation of the ERK/MAPK cascade is
reduced. At E10.5, activation of the ERK/MAPK cascade is almost completely
abolished in both Map2k1-/- embryos and placentas.
However, in Map2k1/-rescued embryos,
activation of MAPK1 and MAPK3 is comparable with that seen in wild-type
specimens (Fig. 8B), indicating
that MAPK1 and MAPK3 phosphorylation in the embryo does not require MAP2K1
protein. The absence of ERK/MAPK activation in E10.5
Map2k1-/- conceptuses
(Fig. 2) was most probably due
to the fact that these embryos were dying and undergoing resorption.
The importance of the ERK/MAPK cascade in placental development has been
suggested by the identification of placental defects in mouse mutants for
several intermediates of the ERK/MAPK cascade, as well as in mutants affecting
other MAPK pathways such as the p38/MAPK cascade
(Rossant and Cross, 2001;
Watson and Cross, 2005). For
example, the Map3k3 and Mapk14 mutants (known as
Mekk3 and p38
, respectively) present a reduced
labyrinthine layer. The development of embryonic, but not of maternal, blood
vessels in Map3k3-/- labyrinth is dramatically impaired,
suggesting a role for the p38/MAPK pathway in the vascularization of the
placenta by angiogenesis (Adams et al.,
2000; Mudgett et al.,
2000; Tamura et al.,
2000; Yang et al.,
2000). In the case of Mapk14 mutants, placenta
development can be rescued in tetraploid-aggregation experiments, suggesting
that the vascularization defect associated with this mutation is non-cell
autonomous. We showed that the p38/MAPK cascade was strongly activated in the
endothelial cells lining the embryonic blood vessels in wild-type placentas.
In Map2k1-/- specimens, the p38/MAPK cascade was still
activated in the endothelial cells blocked at the chorioallantoic interface.
This incapacity of endothelial cells as well as syncytiotrophoblasts to invade
the labyrinthine region may reflect the abnormal morphogenesis of the
labyrinth in absence of Map2k1 function. As the p38/MAPK cascade is
activated in absence of Map2k1 function, this suggests that both
pathways act independently during placenta formation, with p38 being involved
in angiogenesis and MAP2K1 in labyrinthine morphogenesis.
Altogether, our results indicate that the Map2k1 gene is required
in the extra-embryonic ectoderm for the morphogenesis of the labyrinth leading
to its vascularization by the embryonic blood vessels derived from the
allantois but it is not essential for syncytiotrophoblast cell determination
and differentiation. The cell migration deficiency observed in
Map2k1-/- mouse embryonic fibroblasts might reflect the
lack of migration of the syncytiotrophoblasts
(Giroux et al., 1999).
However, we cannot rule out the possibility that the syncytiotrophoblast
defect might be due to the undergrowth of the labyrinth. Indeed, the lack of a
sufficient number of labyrinthine trophoblasts in
Map2k1-/- placenta might explain why the
syncytiotrophoblasts are stalled at the chorioallantoic interface. The
specific deletion of Map2k1 in the syncytiotrophoblasts should allow
the definition of its role in syncytiotrophoblast cell differentiation and/or
function.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/17/3429/DC1
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ACKNOWLEDGMENTS |
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