1 Department of Cell and Developmental Biology, Graduate School of Biostudies,
Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
2 Department of Molecular Oncology, Graduate School of Medicine and Dentistry,
Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo
113-8519, Japan
Author for correspondence (e-mail:
L50174{at}sakura.kudpc.kyoto-u.ac.jp)
Accepted 31 January 2005
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SUMMARY |
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![]() ![]() ![]() ![]() ![]() ![]() |
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Key words: JNK, Microarray analysis, p38, Preimplantaion development, Signal transduction
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Introduction |
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The mitogen-activated protein kinase (MAPK) cascades have central roles in
diverse cellular functions (Sturgill and
Wu, 1991; Ahn et al.,
1992
; Nishida and Gotoh,
1993
; Davis, 2000
;
Ono and Han, 2000
;
Chang and Karin, 2001
;
Kyriakis and Avruch, 2001
;
Pearson et al., 2001
). The
MAPK pathways include the extracellular signal-regulated protein kinase (ERK)
pathway, the Jun N-terminal kinase (JNK) pathway and the p38 pathway. Each
member of MAPK is activated in response to various extracellular stimuli, and
controls, mainly through regulating gene expression, various biological
processes such as cell proliferation, cell differentiation, cell cycle arrest,
apoptosis, etc. As previous genetic and biochemical studies have revealed the
crucial involvement of the MAP kinase family molecules in early embryonic
development, it has been expected that they might act also in preimplantation
development. However, difficulties in manipulating preimplantation embryos
have delayed progression in this field.
Recent development of specific inhibitors of each MAPK pathway, however,
enabled us to test our hypothesis that several of the MAPK family molecules
may be involved in processes during preimplantation development. In this
study, the use of the specific inhibitors has firstly shown that inhibition of
the p38 pathway or the JNK pathway, but not the ERK pathway, leads to
abnormality in mouse preimplantation development, especially in cavity
formation. Immunostaining with anti-phospho-specific antibodies has then shown
the existence of activated forms of p38 and JNK from four-cell to blastocyst
stages. These results strongly suggest the involvement of JNK and p38 in mouse
preimplantation development. As for p38, Natale et al.
(Natale et al., 2004)
reported, just after completion of this work, that p38 MAPK activity is
required to support successful murine preimplantation development, in
agreement with our results. Our experiments with actinomycin D then show that
gene expression is essential for compaction and cavitation, in accordance with
previous reports with another inhibitor
(Khidir et al., 1995
;
Kidder and McLachlin, 1985
).
Moreover, recent microarray analyses during mouse preimplantation development
revealed global gene expression changes, which could be associated with
various signaling pathways (Hamatani et
al., 2004
; Wang et al.,
2004
). These observations prompted us to examine expression
profiles of genes that are regulated by the JNK or the p38 pathway, but not by
the ERK pathway. Our microarray analyses with Affymetrix GeneChips then show
that of over 39,000 transcripts (45,000 probe sets) analyzed, there are only
156 transcripts (161 probe sets) whose expression is enhanced or decreased
significantly (by at least twofold) by the inhibition of the JNK or the p38
pathway but is insensitive to the inhibition of the ERK pathway. Of the 156
genes, 10 genes are regulated by both the JNK and p38 pathways. These genes
include several genes known for their function in axis and pattern formation.
Targeting some of these 10 simultaneously by RNAi results in abnormality in
cavity formation. Thus, our study reveals involvement of the MAPK pathways in
mouse preimplantation development and identifies a limited number of genes
that could be crucial for this developmental process.
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Materials and methods |
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Immunofluorescence confocal microscopy
Prior to fixation, the zona pellucida was removed by treatment with acid
tyrode, and then embryos were washed twice in M16 medium. Then, the embryos
were fixed overnight in 4% paraformaldehyde in PBS at 4°C and washed in 2%
BSA in PBS. The fixed embryos were permeabilized and blocked by incubation for
1 hour in 2% BSA in PBS plus 0.01% Triton X-100 at room temperature. The
embryos were then washed in 2% BSA in PBS and incubated with anti-phospho-p38
antibody (Promega) (1:250), anti-phospho-Jun (Ser 73) antibody (Cell
Signaling) (1:500), anti-phospho-MAPKAPK-2 (Thr 334) antibody (Cell Signaling)
(1:50), anti-phospho-HSP27 (Ser 82) antibody (Cell Signaling) (1:50),
anti-Dkk-1 (H-120) antibody (Santa Cruz) (1:250), or anti-CDX1 antibody
(Bai et al., 2002) (1:250) in
2% BSA in PBS for 16 hours at 4°C. Embryos were washed three times with 2%
BSA in PBS, and incubated with anti-rabbit IgG secondary antibody in 2% BSA in
PBS for 2 hours at room temperature. After three washes with 2% BSA in PBS,
fluorescence was viewed with a BioRad confocal microscope (Radiance 2000).
Generation of microarray data
We used Affymetrix GeneChip for microarray analysis. Three independent
experiments were carried out: one using GeneChip Mouse Expression Set 430 (MOE
430) and two using GeneChip Mouse Genome 430 2.0 Array (Mouse 430 2.0)
(Affymetrix). For each microarray experiment, we collected two sets of 40
embryos from four kinds of pools: wild-type embryos, SB203580-treated embryos,
SP600125-treated embryos and U0126-treated embryos. Eight-cell stage embryos
were treated with these drugs, and blastocyst stage embryos were collected and
stored at 80°C for RNA extraction. Total RNA was isolated by
following the manufacturer's instructions (Isogen, Nippon Gene). Eighty
embryos were used for one array. All amplifications started with 100 ng
whole-embryo total RNA. Two rounds of amplifications were performed for each
replicate following the protocol `Two-Cycle Target Labeling Assays' by
Affymetrix. For each replicate, 10 µg cRNA was fragmented and hybridized
following Affymetrix instructions. The microarrays were then washed and
stained using the GeneChip fluidics station, according to the manufacturer's
instructions.
Microarray data analysis
Hybridized arrays were scanned using an Affymetrix GeneChip Scanner. We
used the GeneChip Operating Software 1.0 (Affymetrix) to analyze the data. For
clustering, the first variation was the detection call with the statistical
algorithms (Affymetrix software). The detection call indicates whether a
transcript is reliably detected (present) or not detected (absent). Genes
deemed absent in all four conditions in any one experiment were excluded from
further analysis. Second, to generate change significance and change quantity
metrics for every probe set, we used a comparison analysis (Affymetrix
software). Those genes that are increased or decreased by at least twofold
over baseline (control, wild-type embryos) with a statistical significance are
judged as increased or decreased, respectively. The data generated from the
above process were imported into GeneSpring 6.1 (Silicon Genetics, Redwood
City, CA) for making a gene list. For hierarchical clustering, the cosine
similarity method was used and the distance metric (1correlation) was
calculated (Fig. 3B). The array
data have been deposited into the Gene Expression Omnibus (GEO) database
(http://www.ncbi.nlm.nih.gov/geo/)
(series Accession Number GSE2229; sample Accession Numbers GSM40799, GSM40801,
GSM40802, GSM40803, GSM40865, GSM40866, GSM40867, GSM40868, GSM40869,
GSM40870, GSM40871, GSM40872, GSM40873, GSM40874, GSM40875 and GSM40876).
|
siRNA
Chemically synthesized 21 nucleotide siRNAs were commercially obtained
(Japan Bio-service). These RNAs were designed to form 19 bp dsRNA with 2
nucleotide deoxynucleotide overhangs at both 3' ends. The targeting
sequences of the siRNA are as follows: siJNK1-1, GAAGCUCAGCCGGCCAUUUTT;
siJNK1-2, GCGAGCCUACCGAGAACUATT; siJNK2-1, GUCGUCCUUUUCAGAACCATT; siJNK2-2,
CGCACGCAAAGAGAGCCUATT; siCdx1-1, CUACUAAUGCUGGCCUUCUTT; siCdx1-2,
CAACGCCUAGAGCUGGAAATT; siDkk1-1, CCAACGCGAUCAAGAACCUTT; siDkk1-2,
GAACCACACUGACUUCAAATT; siFoxq1-1, GCAAGGACAACUACUGGAUTT; siFoxq1-2,
AGAUCAACGAGUACCUCAUTT; siSox7-1, AAGCCGAGCUGUCGGAUGGTT; siSox7-2,
ACCUUCUUCUCGUCCUCAUTT. siRNA duplexes were generated by mixing 20 µM sense
and antisense ssRNA oligomers in the annealing buffer that has been previously
described (Elbashir et al.,
2001).
Microinjection
Microinjection was performed under inverted microscope using a mechanical
micromanipulator (Narishige). We injected about 10 pl of 20 µM siRNA
duplexes into the cytoplasm of one-cell stage embryo. The injected embryos
were cultured in M16 medium.
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Results and discussion |
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In contrast to the treatment with the ERK pathway-specific inhibitors,
treatment with SB203580, a specific inhibitor of p38 and ß MAPKs
(Cuenda et al., 1995
), caused a
severe defect in blastocyst formation. The embryos treated with SB203580 at 20
µM were not distinguishable morphologically from control embryos until the
cavitation started (Fig. 1C).
They had undergone compaction normally. The indirect immunostaining pattern
for E-cadherin, connexin-43, ZO-1 or occludin was also apparently normal (data
not shown). However, the embryos treated with SB203580 did not form a cavity,
or formed a much smaller cavity than did control embryos
(Fig. 1C). The cavity formation
was inhibited markedly even when SB203580 was added at the pre-cavitation
stage (late morula) or after initiation of cavitation
(Fig. 1C), and the cavity was
not formed or remained small. There are four isoforms in the p38 MAPK family,
p38
, ß,
and
(Ono and Han, 2000
;
Pearson et al., 2001
).
p38
and ß are thought to play redundant roles.
or
is different from
and ß in their upstream activating kinases,
downstream targets and inactivating MAPK-specific dual specificity
phosphatases (Ono and Han,
2000
; Pearson et al.,
2001
). Thus, functions of p38
and
appear to be
different from those of p38
and ß. SB203580 specifically inhibits
p38
and ß, but not p38
or
. Therefore, our results
here suggest that p38
and ß are crucially involved in cavitation
during preimplantation development. Possible roles of p38
and
in the preimplantation development, however, remain to be elucidated in the
future studies.
|
To eliminate the possibility that the sensitivity of the preimplantation stages to these drugs is merely a reflection that the cultured embryos are stressed, we performed uterine transfer experiments. Eight-cell stage embryos were treated with or without the MEK-specific inhibitor U0126, that has no effect on preimplantation development in culture (see Fig. 1), and then the blastocyst stage embryos were transferred back to 2.5-day p.c. pseudopregnant recipients. Both control embryos and U0126-treated embryos implanted and developed to term (data not shown), suggesting that our drug treatment in culture per se is not severely toxic.
Apparent phenotypes of the embryos treated with SP600125 are similar to, but not completely the same as, those treated with SB203580. Differences were seen in observations of time-dependent morphological changes of a single embryo under a stereomicroscope. Each embryo was cultured in separate dish, and was examined for its apparent phenotypic changes from the eight-cell stage when the inhibitor was added (Fig. 1D). The phenotypes of embryos treated with SB203580 can be categorized into two types: no cavity formation or a small cavity. When SB was added at eight-cell, morula or blastocyst, no cavity formation was found in about 90%, 60% or 20% of the embryos, respectively. The phenotypes of embryos treated with SP600125 can be roughly categorized into three types (see Fig. 1B): a small cavity (the first type), apparent multiple small cavities in an embryo instead of a single cavity (the second type) or a normal size cavity with abnormality in the inner-cell mass (the third type). In the third type, cells were partially detached from the inner-cell mass. As these three phenotypes are overlapping one another, exact classification was difficult. In the case of the SB203580 treatment, a much severer defect in cavitation was observed when the drug was added at the eight-cell stage. By contrast, in the case of the SP600125 treatment, marked defects in blastocyst were found even when the drug was added after initiation of cavitation. These results suggest that p38 and JNK are simultaneously, but slightly differently, involved in blastocyst formation during preimplantation development.
We then examined whether p38 and JNK are activated during mouse
preimplantation development. Activation of p38 can be assessed by using
anti-phospho-specific p38
antibody. Indirect immunofluorescence showed
that p38
began to be activated from the four-cell stage and remained
active during morula and blastocyst stages
(Fig. 2A). The phosphorylated
form of p38
was detected in dots or patches near the plasma membrane
(Fig. 2A). The activation of
p38
showed no apparent asymmetry within the cells. As confirmation of
the activation of p38, we investigated whether SB203580 inhibition of the
kinase activity of p38 abolished the staining of phospho-MAPKAPK-2 and
phospho-HSP27. MAPKAPK-2, MAP kinase-activated protein kinase 2, is a direct
target of p38 MAPK (Rouse et al.,
1994
), and HSP27 is a substrate of MAPKAP kinase 2
(Landry et al., 1992
;
Rouse et al., 1994
). Treatment
with SB203580 markedly decreased the staining intensity of phospho-MAPKAPK-2
and phospho-HSP27 (Fig. 2B),
confirming that p38 MAPK is activated during mouse preimplantation
development. The activation of JNK can be assessed by using
anti-phospho-specific antibody for Jun transcription factor. Jun is a
well-known substrate of JNK. Indirect immunofluorescent staining showed that
nuclear existence of phosphorylated Jun was detected from the four-cell stage
to the blastocyst stage (Fig.
2C). Furthermore, to verify that the staining of phospho-Jun was
caused by the activation of JNK, we examined whether treatment with SP600125,
a JNK inhibitor, abolished this staining. The result, shown in
Fig. 2D, demonstrated that the
SP600125 treatment inhibited almost completely the phosphorylation of Jun,
indicating that JNK is activated during the four-cell to the blastocyst
stages. These results taken together show that both p38
and JNK are
activated in mouse preimplantation development.
|
Phenotypes of mice carrying a single or doubly targeted deletion of the
molecules comprising the MAPK cascades have previously been reported
(Pearson et al., 2001). A
defect in cavitation during preimplantation development, however, has not been
observed in any of these mice. This may be due to redundancy of the function
of multiple isoforms of the family members. Moreover, when deletion of one
gene leads to embryonic lethality, preimplantation developmental processes may
not be examined. Thus, possible roles of members of the MAPK family in
mammalian preimplantation development have not been addressed. Our present
results have revealed crucial involvement of two subfamily members of the MAPK
family, p38 and JNK, in mouse preimplantation development. After completion of
this work, a paper by Natale et al.
(Natale et al., 2004
)
appeared, reporting requirement of p38 activity for mouse preimplantation
development. Although there are subtle differences between our experiments and
theirs, such as the period of the inhibitor treatment and the focused events
in preimplantation development, the obtained conclusions are essentially the
same.
As the MAPK signaling pathways are known to regulate gene expression in the
nucleus, we next asked whether gene transcription is required for
preimplantation development. Previous experiments with -amanitin showed
that gene expression is necessary for preimplantation development
(Khidir et al., 1995
;
Kidder and McLachlin, 1985
).
To confirm this, we examined the influence of transcription inhibitor,
actinomycin D, on preimplantation development. Treatment of eight-cell stage
embryos with actinomycin D (0.05 µg/ml or 0.5 µg/ml) completely
inhibited compaction, and embryos were unable to develop into morula
(Fig. 3A, left). When we added
actinomycin D to morula stage embryos, dose-dependent effects were observed
(Fig. 3A, right). At 0.05
µg/ml actinomycin D, the cavity formation was completely blocked in about
60% of embryos. In about 40% of embryos, the smaller cavity was formed. At 0.5
µg/ml actinomycin D, the complete inhibition of cavitation was observed in
about 85% of embryos, and the remaining embryos showed a much smaller cavity.
These results, together with the previous studies
(Khidir et al., 1995
;
Kidder and McLachlin, 1985
),
suggest that both compaction and cavity formation require de novo synthesis of
mRNAs. It is likely that the p38 and JNK signaling pathways are involved in
these processes during preimplantation development through regulation of gene
expression.
Based on the above observations, we examined expression profiles of genes
that could be regulated by the MAPK pathways during preimplantation
development. We used Affymetrix GeneChips for microarray analysis. Three
independent experiments were carried out. For each experiment, we collected
eighty embryos from four kinds of pools: wild-type embryos, SB203580-treated
embryos, SP600125-treated embryos and U0126-treated embryos. Eight-cell stage
embryos were treated with these drugs, and blastocyst stage embryos were
collected for RNA extraction, labeling and hybridization. The used array
comprised over 39,000 transcripts (45,000 probe sets) and about 60% (about
24,000 transcripts) were judged to be expressed in the wild-type sample by the
GeneChip Operating Software 1.0 determination. In any of the SB203580-treated,
SP600125-treated and U0126-treated samples, the percentages of transcripts
that were judged to be expressed were about 60%. Rather surprisingly, the
expression level of most of the transcripts was not changed by the drug
treatment. Only 1-4% of transcripts showed the sensitivity to the
inhibition of MAPK pathways; the expression level of these genes was increased
or decreased by either of SB203580, SP600125 or U0126 at least by twofold
compared with baseline (wild type). Hierarchical clustering was performed with
those transcripts that were sensitive to these drugs by GeneSpring 6.1. The
result shows that the effect of SB203580 is most similar to that of SP600125,
and the effect of U0126 is not so similar to that of SB203580 or SP600125
(Fig. 3B), as was the case for
the effect of these drugs on preimplantation development (see
Fig. 1). Thus, we extracted 156
transcripts (161 probe sets) whose changes in mRNA levels were more than
twofold in at least two out of the three independent experiments; 74
transcripts (groups 1, 2 and 3) showed an increased level of expression while
the remaining 82 transcripts (groups 4, 5 and 6) showed a decreased level of
expression by SB203580 or SP600125 treatment but not by U0126 treatment
(Fig. 3C). We classified these
156 transcripts into six groups based on the sensitivity to the drugs
(Fig. 3C, Fig. 4). In
Fig. 4 (left), the average
expression levels of genes in each group were shown. Expression levels of
genes in group 1, 2, or 3 were increased by the inhibition of the p38 or the
JNK pathway, but rather insensitive to the inhibition of the ERK pathway. By
contrast, expression levels of genes in group 4, 5, or 6 were decreased by
SB203580 or SP600125, but insensitive to U0126.
|
Representative transcripts in each group (CD24a in group 1, Map2k6 in group
2, Tcstv1 in group 3, Cdx1 in group 4, Glipr1 in group 5 and Aqp8 in group 6)
were examined for their sensitivity to each drug by RT-PCR to confirm the
microarray data (data not shown). The obtained results completely conform to
the classification (see Fig.
3C, Fig. 4).
Recently, two reports performing microarray analysis revealed that there are
two major phases of stage-specific gene activity, which precede blastocoel
formation (Hamatani et al.,
2004; Wang et al.,
2004
). In addition Hamatani et al.
(Hamatani et al., 2004
) have
classified genes in terms of their expression pattern (clusters 1 to 9). Our
rough analysis has suggested that there exists little or no specific
correlation between their clustering and our grouping (1 to 6); for example,
any of the genes in our group 4 could not be classified into any of nine
clusters. Thus, at present we are unable to characterize stage-specific
expression patterns of our 156 transcripts identified. Further studies should
identify transcriptional regulation mechanisms of these genes. In this
context, it has been shown that the expression of Dkk1 is dependent on the
Ap-1 family member Jun (Grotewold and
Ruther, 2002
). We were able to find ten possible Ap-1 sites in 1
kb upstream from the ORF of Dkk1 (data not shown). Other members of group 4
also have 5-19 possible Ap-1 sites. Moreover, several members of group 4 may
have other transcription factor binding sites, e.g. MEF and CRE. Because Ap-1,
MEF and CRE are known to be regulated by the p38 and JNK pathways, it is
possible that the expression of genes in group 4 could be regulated directly
by the p38 and JNK pathways.
Last, we performed two types of experiments to assess the possible function of several genes in group 4. First, we carried out immunofluorescent examination for Cdx1 and Dkk1 in eight-cell embryos. Both proteins were clearly detected at this stage, and their expression level was decreased by SB203580 or SP600125 treatment (Fig. 5A). This result is consistent with the p38- and JNK-dependent increase of the mRNA level of both genes. Next, siRNA experiments were performed to examine the function of Cdx1, Dkk1, Foxq1 and Sox7. siCdx1-1 and siCdx1-2 are two RNA duplexes for mouse Cdx1. siDkk1-1 and siDkk1-2 are for mouse Dkk1. siFoxq1-1 and siFoxq1-2 are for mouse Foxq1. siSox7-1 and siSox7-2 are for mouse Sox7. These RNA duplexes were injected into one-cell zygotes and 3 days later, the embryos were examined. Embryos injected with DDW developed normally and were stained by anti-Dkk1 antibody (Fig. 5B). At this stage, Dkk1 protein appeared to localize at or near plasma membrane. siRNA of individual genes had little effect on preimplantation development. However, when all these siRNAs for the four genes were injected together, about one fifth of the injected embryos showed abnormality in cavity formation (Fig. 5B). These results suggest that at least several of the group 4 genes in combination may play a role in preimplantation development.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/8/1773/DC1
* Present address: RIKEN Center for Developmental Biology, 2-2-3
Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan
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