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Developmental Biology 2
A MAPK pathway is involved in the control of mitosis after
fertilization of the sea urchin egg
Wen Ling Zhanga, Philippe Huitorelb, Rainer Glassa, Montserra Fernandez-Serrad,
Maria I. Arnoned, Sandrine Chiria, Andre Picardc, Brigitte Ciapaa,e,*
aUMR 7622 CNRS, Universite Paris 6, 9 Quai St Bernard, Bat C, 5e etage, case 24, 75252 Paris cedex 05, FrancebUMR 7009 CNRS, Universite Paris 6, Station Zoologique, Observatoire Oceanologique, 06234 Villefranche/Mer cedex, France
cUMR 7628 CNRS, Observatoire Oceanologique de Banyuls, Universite Paris 6/CNRS/INSU, BP 44, F-66651, Banyuls-sur-mer cedex, FrancedStazione Zoologica A. Dohrn, 80121, Italy
eGDR N- 2688
Received for publication 26 January 2005, revised 26 January 2005, accepted 12 March 2005
Available online 19 April 2005
Abstract
Activation and role of mitogen-activated protein (MAP) kinase (MAPK) during mitosis are still matters of controversy in early embryos.
We report here that an ERK-like protein is present and highly phosphorylated in unfertilized sea urchin eggs. This MAPK becomes
dephosphorylated after fertilization and a small pool of it is transiently reactivated during mitosis. The phosphorylated ERK-like protein is
localized to the nuclear region and then to the mitotic poles and the mitotic spindle. Treatment of eggs after fertilization with two different
MEK inhibitors, PD 98059 and U0126, at low concentrations capable to selectively induce dephosphorylation of this ERK-like protein, or
expression of a dominant-negative MEK1/2, perturbed mitotic progression. Our results suggest that an ERK-like cascade is part of a control
mechanism that regulates mitotic spindle formation and the attachment of chromosomes to the spindle during the first mitosis of the sea
urchin embryo.
D 2005 Elsevier Inc. All rights reserved.
Keywords: MAP kinase; Egg; Sea urchin; ERK; Mitosis; Fertilization; Mitotic spindle
Introduction
Activation of mitogen-activated protein kinase (MAPK)
is generally triggered after exit from a G0 or G2 quiescent
state. In somatic cells, stimulation by various extracellular
signals that regulate morphology, proliferation, differentia-
tion or survival often leads to the activation of at least one
member of the MAPK family, for instance the extracellular
signal-regulated kinases (ERK1/2) (reviews by Peysonnaux
and Eychene, 2001; Volmat and Pouyssegur, 2001). More
than 10 years ago, it was shown that microinjection of the
proto-oncogene mos, a MAPK kinase kinase (Sagata et al.,
0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ydbio.2005.03.008
* Corresponding author.
E-mail address: [email protected] (B. Ciapa).
1989), or of thiophosphorylated MAP kinase (Haccard et
al., 1993) into one blastomere of a two-cell Xenopus embryo
induced cell cycle arrest. This suggested that a MAPK
pathway could also regulate the cell cycle in early embryos.
It is well accepted that an ERK cascade, implying mos,
acts at different steps during maturation and regulates
meiosis in oocytes of various species (Abrieu et al., 2001;
Karaiskou et al., 2001). This pathway has been implicated in
regulating the meiotic spindle itself (Lefebvre et al., 2002;
Verlhac et al., 1996). However, whether MAPK is stimu-
lated and plays a role during the following mitotic cycles in
living embryos still remains a matter of controversy. In
intact Xenopus oocytes, Gotoh et al. (1991) observed that a
42/44-kDa protein, which phosphorylated preferentially
MBP or MAP2 (microtubule associated protein) rather than
histone H1, was activated at the time of first mitosis.
82 (2005) 192 – 206
YDBIO-01932; No. of pages: 15; 4C: 6, 7, 12
W.L. Zhang et al. / Developmental Biology 282 (2005) 192–206 193
Activation of MAPK at time of mitosis was indeed
measured in Xenopus cycling extracts (Guadagno and
Ferrell, 1998), but since the reports by Ferrell et al.
(1991), it was admitted for many years that this activity
decreased at exit of meiosis II and was not further
reactivated in early Xenopus embryos (Tunquist and Maller,
2003). This dogma was broken very recently by Yue and
Ferrell (2004) who detected, in dividing Xenopus embryos,
activation of p42 in MAPK that could be due to low levels
of Mos remaining after fertilization. In clam oocytes,
fertilization that occurs during meiosis I triggers a decrease
in MAPK activity, and no further reactivation of MAPK
during the following cell cycles has been reported so far
(Shibuya et al., 1992). We then set about this work to clarify
the situation in sea urchin eggs, where meiosis is completed
prior to fertilization, and since activation of MAPK activity
during the first mitosis following fertilization has been
detected (Chiri et al., 1998; Philipova and Whitaker, 1998)
or not (Kumano et al., 2001). We show in the present report
that an ERK-like protein, highly phosphorylated in the sea
urchin unfertilized egg, is dephosphorylated soon after
fertilization. We found that a small proportion of the
ERK-like protein is transiently rephosphorylated at the time
of mitosis, and that this pool of phosphorylated ERK is
localized to the nuclear region and then to the mitotic
apparatus.
In various types of somatic cells, several data have
shown the association of ERK1/2 and/or of upstream
partners such as MEK or RISK with the mitotic spindle
(Cha and Shapiro, 2001; Shapiro et al., 1998; Wang et al.,
1997; Willard and Crouch, 2001; Zecevic et al., 1998). It
has also been suggested that a MAPK pathway plays a role
in the spindle checkpoint, a mechanism that delays anaphase
onset in eukaryotic somatic cells when all chromosomes are
not aligned or under proper tension and that is expressed as
a negative signal produced by the Mad2/Bub1 machinery
active at unattached kinetochores (Hardwick, 1998; Hoyt,
2001; Sadler and do Carmo Avides, 2000). This hypothesis
came from results obtained in somatic cells showing that
active MAPK associates with kinetochores (Shapiro et al.,
1998; Zecevic et al., 1998) and that this checkpoint does not
operate properly after inhibition of the MAPK pathway with
PD 98059 (Cross and Smythe, 1998), anti-MAPK anti-
bodies (Takenaka et al., 1997) or injection of XCL100, a
MAPK phosphatase (Wang et al., 1997). In cycling Xenopus
extracts, various data indicate that MAP kinase is required
for the spindle assembly checkpoint (Chen, 2004; Chung
and Chen, 2003; Minshull et al., 1994; Takenaka et al.,
1997). However, it is accepted that such a mechanism
depends on the nucleus/cytoplasm ratio, ruling out its
existence in early dividing eggs where the ratio nucleus/
cytoplasm is too low. The question was then whether the
phosphorylated ERK that we detected at time of mitosis in
sea urchin early embryos could regulate mitotic events such
as the formation of the mitotic spindle. We found that
treatment of fertilized eggs with two different MEK
inhibitors, PD 98059 and U0126, at low concentrations that
selectively trigger dephosphorylation of the ERK-like
protein, or expression of a dominant-negative MEK1/2,
induced alterations in the formation of the mitotic spindle
and of the attachment of chromosomes to the spindle during
the first mitosis of the sea urchin embryo. Our results
strongly suggest that an ERK-like protein is an important
regulator of mitosis and might be part of a spindle
checkpoint mechanism in the early sea urchin embryo.
Materials and methods
Handling of gametes
Gametes collected from the sea urchin Paracentrotus
lividus were prepared and fertilized in artificial sea water
(ASW, Reef Crystals Instant Ocean) as described in our
previous papers (Chiri et al., 1998; De Nadai et al., 1998).
Eggs were treated or not before or after fertilization with
PD 98059 (Calbiochem, 15 mM stock in DMSO), U0124 or
U0126 (Calbiochem, 15 mM stock in DMSO).
Western blot analysis
Samples of eggs were prepared as previously described
(Chiri et al., 1998; De Nadai et al., 1998). In all experi-
ments, 10 Ag of proteins was separated by 12.5% SDS–
PAGE and transferred on PVDF membranes. After transfer,
the membrane was rinsed in distilled water for 5 min, dried
in air for 15 min in order to fix proteins and then incubated
for one h in blocking buffer (10 mM Tris–Cl, pH 7.4, 150
mM NaCl, 1 mM EDTA, 0.1% Tween-20, 3% BSA). The
membrane was incubated with primary antibody for 2 h at
room temperature and then washed three times for 5 min in
washing buffer (10 mM Tris–Cl, pH 7.4, 150 mM NaCl,
0.5% NP40). The membrane was incubated with secondary
antibody for 1 h, rinsed three times for 5 min in washing
buffer, and proteins were revealed by enhanced chemo-
luminescence (ECL, Amersham).
Whenever necessary, band intensities were quantified by
scanning radiographic films followed by densitometry
analysis using Adobe Photoshop.
All the antibodies were diluted in blocking buffer.
Primary antibodies and dilutions were as following: mouse
anti-phospho-MAPK42/44 (Thr202/Tyr204, Cell Signal-
ling) at 1/2000, rabbit anti-phospho-MEK1/2 (ser217/221,
Cell Signalling, 9121) at 1/1000, rabbit anti-phospho-cdc2
(Tyr15, Cell Signalling) at 1/500 and rabbit anti-cdk1/cdc2
(PSTAIR, UBI) at 1/1000. The anti-mouse HRP-conjugated
goat IgG and the anti-rabbit HRP-conjugated goat IgG
secondary antibodies (ICN) were diluted 1/1000 and 1/
10,000, respectively.
In order to verify that the anti-phospho-MAPK42/44
antibody recognized only the active form of a MAPK-like
protein, samples of unfertilized eggs were treated by
W.L. Zhang et al. / Developmental Biology 282 (2005) 192–206194
alkaline phosphatase before SDS–PAGE analysis as follow-
ing. A dry pellet of eggs (Chiri et al., 1998) containing 250
Ag proteins was incubated for 2 h at 37-C in 65 Albicarbonate buffer (100 mM NaHCO3, pH 10.5) containing
84 U/ml alkaline phosphatase. Incubation was stopped by
adding 65 Al denaturation buffer (Chiri et al., 1998) and
boiling for 10 min at 95-C. 10 Ag of proteins was then
separated on SDS–PAGE and analyzed by Western blot.
Cytochemistry
Eggs were fertilized in the presence of 1 mM ATAZ (3-
amino-1,2,4-triazole, Sigma) to prevent hardening of the
fertilization membrane. 2 min after fertilization, eggs were
diluted 10 times in normal filtered sea water (NFSW) and
quickly filtered several times through a 85-Am filter to
remove fertilization membranes. Decanted eggs were rinsed
twice in NFSW and cultured in NFSW, treated or not with
PD 98059 or U0126 and taken for fixation and labeling at
various times post-fertilization.
Fig. 1. Identification of a MEK/ERK-like pathway. (A) Detection of an ERK-like
MAPK42/44 antibody (arrow). Phosphorylation of the 42-kDa ERK-like protein
extracts with alkaline phosphatase (+AP) or after treatment of intact unfertilized
phosphorylation after fertilization. Samples were separated on two equivalent gels
transferred and blotted in parallel and in the same solutions. A very strong signa
become undetectable 20 min after fertilization. (C) Reactivation of the ERK-like p
detected around 60 min, time corresponding to the first mitosis in both experime
second experiment. (D) Time course of ERK-like and MEK activities. For ERK-
seven different experiments, indicated by seven different black or white symbols,
obtained in the next panel (E) were quantified after scanning and densitometry, and
of signal intensity measured in unfertilized eggs and that was arbitrarily taken a
fertilization analyzed by SDS–PAGE and Western blot analysis using an anti-ph
upper panel and later times on lower panel) that were run and then transferred and
were loaded on both gels to ascertain that both gels could be compared.
At a given time point, an aliquot of eggs (typically 1 ml)
was quickly decanted twice in an Eppendorf tube, and
NFSW was replaced by Ca2+-free artificial sea water
(CaFSW: 484 mM NaCl, 10 mM KCl, 27 MgCl2, 29 mM
MgSO4, 2.4 mM NaHCO3, pH 8.2) just before fixation.
Actin filaments and chromatin were labeled using a
method described by Mabuchi (1994) that was modified as
follows. Embryos were fixed for 0.5–1 h at room temper-
ature with 5% (v/v) formaldehyde in F-buffer (0.1 M KCl, 2
mM MgCl2, 1 mM EGTA, 10 mM MOPS buffer, pH 7.4).
They were then incubated for 30 min in F-buffer containing
5% formalin, 0.8 M glucose and 0.2% NP40. Eggs were
then washed with F-buffer containing 0.8 M glycerol
(glycerol-F-buffer) and stuck on 0.1% polylysine or prot-
amine-treated slide. Eggs were incubated for 30 min in
blocking buffer (1% BSA in PBS–0.1% Triton X-100
(PBS-T)), and then stained for 3 min with 0.3 AMrhodamine-conjugated phalloidin (Rh-ph, Sigma) and 0.5
Ag/ml Hoechst 33258 dissolved in PBS-T containing 0.1
mM h-mercaptoethanol in order to label actin filaments and
protein by Western blot after SDS–PAGE analysis using the anti-phospho-
detected in unfertilized control eggs (C) was lost after treatment of egg
eggs with 1 AM PD 98059 for 20 min (+PD). (B) Decrease of ERK-like
(early times in left panel and later times on right panel) that were cast, run,
l observed in unfertilized eggs decreased progressively after fertilization to
rotein at time of mitosis. Two different experiments are shown. A band was
nts, and at 92 min, time corresponding to the second mitosis shown in the
like activity, signals obtained after immunoblots obtained as in above from
were quantified after scanning and densitometry. For MEK activity, bands
results are shown in a dotted line. All values were calculated as percentages
s 100 in each experiment. (E) Changes in phosphorylation of MEK after
ospho-MEK antibody. Samples were separated on two gels (early times in
blotted at the same time. Unfertilized samples (time 0) and times 50 and 51
W.L. Zhang et al. / Developmental Biology 282 (2005) 192–206 195
chromosomes, respectively. Eggs were finally washed three
times in PBS-T and mounted in Immuno-Mount (Shandon).
For microtubule labeling (De Nadai et al., 1998), fertilized
eggs deprived of fertilization membrane were taken at
different times after sperm addition and fixed for 1 h at room
temperature with 2% (v/v) formaldehyde, 20 mM PIPES, 5
mM EGTA, 0.5 mM MgSO4 and 0.1% Triton X-100. Eggs
were then rinsed in TBS-T, stuck on 0.1% polylysine coated
slide, incubated for 1 h in blocking buffer and then overnight
with the monoclonal rat anti-tubulin antibody YL1/2
(Chemicon) diluted 1/1000 in blocking buffer containing
1% donkey serum. After three rinses in TBS-T, eggs were
incubated for 1 h with FITC- or TRITC-conjugated-donkey-
anti-rat IgG 1/250 (Jackson ImmunoResearch), rinsed again
three times and treated for 2 min with 0.5 Ag/ml Hoechst
33258. The eggs were finally rinsed three times in PBS-Tand
mounted between slide and coverslip in a homemade
mounting medium containing an anti-bleach reagent.
A similar protocol was followed for anti-phospho-
MAPK42/44 labeling, except that 20 mM beta-glycero-
phosphate, 2 mM Sodium Ortho-Vanadate and 4 mM NaF
were added in the fixation buffer.
For conventional fluorescence microscopy, all micro-
tubule and actin filaments observations were performed on a
Nikon Eclipse TE300, using a �20 plan fluor or a �40 S
Fig. 2. Effect of U0126 and PD 98059 on ERK-like phosphorylation. Western blot
44 antibody. (A) Effect of increasing concentrations of U0126 in unfertilized eggs
Effect of 1 or 10 AM U0124 or U0126 on ERK-like phosphorylation. Unfertilized
U0126 on ERK-like reactivation during mitosis. Eggs were treated or not (contro
fertilization. (D) Effect of increasing concentrations of PD 98059 in unfertilized egg
PD 98059 in fertilized eggs analyzed 6 min after fertilization or at time of mitos
fluor Nikon objectives. Pictures were taken on Kodak Gold
100 Asa films that were developed and then scanned using
an Epson Perfection 2400 scanner. Final images were
treated using Adobe Photoshop. Confocal microscopy was
done on a Leica Laser Scanning Confocal Microscope (SP2)
using a �63 lens. 1024 � 1024 images were captured using
the Leica Lite Software and edited with Adobe Photoshop.
H1 kinase activity
H1 kinase activity was measured on egg extracts
following the same protocol as that described previously
(Chiri et al., 1998; De Nadai et al., 1998). Histone H1
(Sigma type III-S) was used as substrate.
Video microscopy
In order to estimate the timing of mitosis, fertilized eggs
treated or not with U0126 or PD 98059 were observed under
bright-field illumination on an inverted microscope Leica
DM IRB and images taken every 30 s. Eggs were maintained
at 18-C under a slight and constant flow of refrigerated ASW.
The time from nuclear envelope breakdown (NEB) to
beginning of cytokinesis was referred as the duration of
mitosis.
analysis was performed after SDS–PAGE using the anti-phospho-MAPK42/
after different treatment duration. The inhibitor was added at time zero. (B)
eggs were treated during 25 or 60 min with the drug. (C) Effect of 1 AMl) 30 min after fertilization with U0126 and arrested at various times after
s treated for 30 min with the drug. (E) Effect of increasing concentrations of
is (62 min). The inhibitor was added prior to sperm addition.
W.L. Zhang et al. / Developmental Biology 282 (2005) 192–206196
MEK constructs, mRNA preparation and microinjection
We used a Mekneg expression plasmid containing the
entire coding sequence of the K96M human MEK mutant
(Mansour et al., 1994). The plasmid was constructed, and
mRNA purified and injected in the presence of 0.12 M KCl
as previously described (Fernandez-Serra et al., 2004).
Fig. 3. Localization of phospho-MAPK during mitosis by confocal imaging. (A)
(b), later prophase (c), metaphase (d) or telophase (e). Eggs were double labeled w
an anti-tubulin antibody (aV1, bV1, cV1, dV1, eV1) or an anti-phospho-MAPK42/44 (
unfertilized egg (aV2), very weak at early prophase (bV2), then localized in the nucleaLabeling disappeared before telophase (eV2). Scale bar: 50 Am. (B) Effect of U0126
eggs (b and c) were treated at 20 min post-fertilization and observed at metaphase
or with the anti-phospho-MAPK42/44 (aV, bV, cV).
Results
A MEK/ERK pathway is transiently activated at time of
mitosis
In order to detect changes after fertilization in the active
form of MAPK, we used a commercial monoclonal
Control eggs. Eggs were observed before fertilization (a), at early prophase
ith Hoechst 33258 (a1, a2, b1, b2, c1, c2, c3, d1, d2, e1, e2) and with either
aV2, bV2, cV2, cV3, dV2, eV2). PhosphoMAPK42/44 labeling was intense in the
r region (cV2), to centrosomes (cV3) and finally to the spindle at mitosis (dV2).. Unfertilized eggs (a) were treated by 5 AMU0126 for 30 min, and fertilized
(b) or at telophase (c), respectively. Eggs were labeled with Hoechst (a, b, c)
Fig. 3 (continued).
W.L. Zhang et al. / Developmental Biology 282 (2005) 192–206 197
antibody raised against the dual phosphorylated form of
human ERK1 and ERK2. On a Western blot of sea urchin
extracts, it labeled a single band of 42 kDa that likely
corresponds to the phosphorylated form of an ERK-like
protein, since this signal was lost after treatment of the egg
extracts either by alkaline phosphatase or by PD 98059, an
inhibitor of MEK (Fig. 1A). The high level of this
phosphorylated protein contained in the unfertilized egg
progressively decreased after fertilization to become unde-
tectable 20 min after fertilization (Figs. 1B and C). A slight
increase in the amount of the phosphorylated form of this
ERK-like protein occurred at the time of first and second
mitosis (Fig. 1C). Results obtained from several experi-
ments (Fig. 1D) establish that the level of transient
phosphorylation of ERK at mitosis could reach in some
experiments 20% of the level observed in the unfertilized
egg, the mean increase T SEM of 18 values obtained
between 55 and 68 min was 10.0 T 1.9%.
We investigated whether the activity of MEK, the
MAPK kinase that is the ERK1/2 activator in most
systems, was subject to the same variations after fertiliza-
tion as that of the ERK-like protein described above. The
anti-phospho-MEK antibody raised against the dual phos-
phorylated form of a mammalian MEK1/2 recognized a
single band migrating around 45 kDa (Fig. 1E). This
MEK-like protein was progressively dephosphorylated
after fertilization to become poorly phosphorylated 20
min after fertilization (Fig. 1E). An increase of this signal
was then observed 50 min after fertilization, that remained
elevated during mitosis and then decreased again after
cleavage (Fig. 1E). The quantification of the MEK signal
(Fig. 1E) shows that these variations correspond to those
observed for the ERK-like protein (Fig. 1D).
Effect of MEK inhibitors on ERK activation during the first
cell cycle
In order to analyze the role of the MEK-MAPK cascade
during mitosis, we used two different MEK inhibitors,
U0126 which inhibits both the active and the inactive forms
of MEK1/2, and PD 98059 which only inhibits activation of
inactive MEK (Davies et al., 2000). We determined the
concentration necessary to cancel ERK-like phosphoryla-
tion. 1 AM U0126 was sufficient to rapidly turn this activity
off in unfertilized eggs (Fig. 2A) and during mitosis (Fig.
2C). The inactive analog U0124 did not trigger dephos-
phorylation of the ERK-like protein, even when used at 10
AM, suggesting that the effects observed with U0126 are
specific (Fig. 2B). 20-min treatment with 0.5 AM PD 98059
could also abolish this activity in unfertilized eggs (Fig.
2D). However, 15 AM (Fig. 2E) of PD 98059, depending on
the batch of eggs, was necessary to obtain a complete loss of
the activity in fertilized eggs.
Localized activation of ERK in the fertilized sea urchin egg
We then checked whether ERK activation by phosphor-
ylation occurred homogeneously throughout the sea urchin
embryo. Eggs were fixed at different times following
fertilization and immunostained using the anti-phospho-
MAPK42/44 antibody (Fig. 3A). Unfertilized eggs (Fig.
3Aa) were highly fluorescent, the cytoplasmic labeling
appearing granular and the intranuclear space brightly
stained (Fig. 3AaV2).30 min after fertilization, eggs reached prophase (Figs.
3Ab and Ac). Control eggs started to polymerize micro-
tubules (Figs. 3AbV1 and cV1) and to condense chromatin in
the nucleus (Figs. 3Ab1, b2, c1, c2 and c3). The anti-
phospho-MAPK signal was highly reduced at early pro-
phase (Fig. 3AbV2) compared to that observed in unfertilized
eggs. At later prophase (Fig. 3Ac), the cytoplasmic signal
appeared less granular and faint, while a higher labeling
accumulated close to nuclei (Fig. 3AcV2a and h) and then in
the vicinity of the centrosomes (Figs. 3AcV2h and AcV3g).Control eggs were at metaphase 40 min after fertilization,
showing alignment of condensed chromosomes (Figs. 3Ad1
and d2) on the bipolar mitotic spindle (Fig. 3AdV1). Thecentral spindle and asters were labeled by the anti-phospho-
MAPK antibody, whereas the periphery of the central
spindle, which also contains a high density of microtubules
in sea urchin embryos, was negative (Fig. 3AdV2). The
cytoplasm surrounding the spindle at that stage still
contained a low but significant granular staining.
Finally, control eggs reached the telophase stage 60 min
after fertilization (Fig. 3Ae). A very low and diffuse
Fig. 4. Time course during the first two cell cycles of MPF activity after
fertilization of eggs treated with PD 98059 or U0126. The inhibitors were
added 30 min after fertilization. In the inset is shown a Western blot
analysis performed after SDS–PAGE using the anti-phospho-Tyr cdc2
antibody of eggs arrested at time of mitosis and treated or not (control) with
15 AM PD 98059.
W.L. Zhang et al. / Developmental Biology 282 (2005) 192–206198
staining was obtained with the anti-phospho-MAPK anti-
body (Fig. 3eV2).Unfertilized eggs treated for 30 min by U0126 showed a
much reduced and diffuse fluorescent signal throughout the
eggs even though some faintly stained granules could still be
seen (Fig. 3BaV). Only a very low and diffuse phospho-
MAPK signal was seen in eggs treated by U0126 and
observed 30 min (Fig. 3BbV) or 60 min after fertilization (Fig.
3BcV), times when eggs were at prophase (Fig. 3Bb) and
telophase (Fig. 3Bc), respectively. Similar results were
obtained using U0126 or PD 98059 (not shown). It is
important to note that the intensity of general staining
obtained in control eggs with the anti-phospho-MAPK
antibody followed that obtained after Western blotting using
this same antibody (Figs. 1B and C), that is, high in
unfertilized eggs, very low at prophase, slightly increased
during mitosis and undetectable at telophase. Taken together,
our results show that ERK is transiently activated during sea
urchin egg mitosis and that this activation mostly takes place
at the spindle poles during prophase and prometaphase.
Effect of MEK inhibition on MPF activation
We analyzed whether the MEK inhibitors affected MPF
activity. The MPF activity due to cdc2/cyclin B is often
referred to as histone H1 kinase, since MPF shows a
pronounced activity toward this substrate and is the main
source of histone H1 kinase activity in eggs during mitosis
(Meijer et al., 1989). We observed that H1 kinase activity
during the first two mitosis was slightly increased when
eggs were treated with 15 AM PD 98059 or with 5 AMU0126, concentrations that inhibited the ERK-like activity
(Fig. 4). This increase in H1 activity corresponded to a
greater decrease in the tyrosine phosphorylation status of
cdc2 during mitosis (Fig. 4 inset), a decrease in phosphor-
ylation of cdc2 corresponding to an increase in MPF activity
(Ohi and Gould, 1999).
Role of MEK/ERK on mitotic spindle formation and cell
division
The role of MAPK cascade during the first two division
cycles was tested by treating eggs 30 min after sperm
addition with concentrations of U0126 or PD 98059 that
induce ERK-like dephosphorylation (Fig. 2). Similar
altered morphologies were observed after both treatments.
Compared to controls, treated embryos showed abnormal
metaphase plates or incomplete cytokinesis, and formed
two or three daughter cells of different sizes or with altered
shapes (Fig. 5). Proportions of altered embryos are
reported in Table 1. The proportion of altered embryos
increased with the concentration of PD 98059. A very high
proportion of embryos were abnormal after treatment with
15 AM PD 98059; almost all eggs divided (Fig. 5A) and
formed morula, although with very altered morphologies,
that finally died before reaching the gastrula stage (not
shown). Treatment with 1 AM U0126 induced abnormal
divisions (Fig. 5B) or mitotic arrest in a number of eggs
(Table 1). Increasing U0126 concentration to 5 AM led to
the increase in the proportion of non-divided eggs (Fig. 5B
and Table 1). Video microscopy experiments allowed us to
determine the timing of the first cell cycle. We observed
that 15 AM PD 98059 or 5 AM U0126 significantly
delayed entry in mitosis but slightly accelerated exit of
mitosis (Table 2).
The abnormal divisions described above led us to
investigate whether U0126 and PD 98059 altered the
formation of the cleavage furrow. Examples of alterations
are shown in Fig. 6. We observed that treated eggs
normally formed a contractile ring of actin filaments
between two sets of chromosomes that were either not as
widely separated (Fig. 6B) as in the control experiment
(Fig. 6A) or not separated at all (Figs. 6C and D). This
probably led to the situation where chromosomes remained
close enough to the cleavage plane and did not separate
properly in a number of cases. Several furrows were also
observed in a number of treated eggs that formed three or
four sets of chromosomes. Those eggs with multiple
furrows seemed to form lobes or cells of different sizes
(Figs. 6E–G). No clear difference in the timing of
formation of the actin ring was observed, suggesting that
these alterations were not due to an accelerated or
premature formation of the cleavage furrow.
We tested whether the smaller gap between the two sets
of chromosomes seen at the time of furrow formation in PD
98159 or U0126 treated eggs (Fig. 6) was due to an altered
mitotic spindle. Eggs were treated in the presence of these
inhibitors 30 min after fertilization and fixed for micro-
tubule and chromatin staining at the normal time of
anaphase in control eggs. In those conditions, we observed
the same percentage of treated eggs as that reported above
showing chromosomes that were not equally distributed in
two sets and several types of aberrant mitotic spindles (Fig.
Fig. 5. Effect of increasing concentrations of PD 98059 (A) or U0126 (B) on the first two mitotic cycles. Each inhibitor was added 30 min after fertilization.
Embryos treated with PD 98059 were observed at different times after fertilization and those treated with U0126 were observed 90 min after fertilization.
U0126-treated eggs or eggs treated with 2.5 AM 98059 PD 98059 showing altered shapes are shown with a star. Almost all eggs treated with 15 AM PD 98059
in this shot had altered morphologies. No alteration was observed when eggs were treated with the inactive analog U0124 (not shown).
W.L. Zhang et al. / Developmental Biology 282 (2005) 192–206 199
7). Some eggs showed two distinct sets of chromosomes,
but one of the two sets appeared separated (Figs. 7B1 and
C1), which could lead to the formation of a tripolar spindle
(Fig. 7C2). Some other eggs contained four sets of
chromosomes (Fig. 7D1), separated on a tetrapolar mitotic
spindle (Fig. 7D2). Finally, a number of eggs showed one or
a few chromosomes lagging out of the normal sets of
chromosomes (Figs. 7D1, E1, F1 and G1). In some cases,
bipolar spindles were formed but were made either of
unequal half spindles and with a poorly defined main axis,
suggesting a loose connection between half spindles (Figs.
7E2 and F2), or with an extra lobe containing extra
microtubules (Fig. 7G2). Similar morphological alterations
were obtained with U0126 and PD 98059, at a higher
frequency in PD 98059-treated eggs, and no alteration was
observed when eggs were treated with the inactive analog
U0124 (not shown) compared to control eggs (Fig. 7A).
Higher resolution micrographs, obtained after confocal
imaging, of some alterations seen after fertilization in
U0126 treated eggs, are shown in Fig. 8. Two very altered
Table 1
Effect of two different concentrations of PD 98059 or U0126 on division
PD 98059 U0126
2.5 AM,
n = 6
15 AM,
n = 7
1 AM,
n = 5
5 AM,
n = 6
Abnormal divisions (%) 15 T 2 67 T 7 22 T 4 21 T 3
Non-divided eggs (%) 3 T 2 3 T 2 6 T 2 13 T 3
Mortality (%) / / / 4 T 2
Total (%) 18 68 28 38
Eggs were treated with MEK inhibitors 30 min after fertilization and
observed under light microscopy. Results are mean T SEM; 15–57 eggs
were counted in each experiment, n representing the number of
experiments.
Table 2
W.L. Zhang et al. / Developmental Biology 282 (2005) 192–206200
metaphase are shown in Figs. 8a and b. In these eggs,
chromosomes are condensed and well individualized (Figs.
8a and b), but microtubules are severely disorganized (Figs.
8aV and bV). Eggs observed at telophase (Fig. 8c) often
showed decondensed chromosomes that were dispersed in
the cytoplasm (Figs. 3Bc and Figs. 8c), which was never
seen in control eggs. All these results show that ERK
inactivation induced various anomalies such as mitotic
spindle defects or lagging chromosomes and lead to the
conclusion that a MEK/ERK pathway is involved in mitotic
spindle formation and function.
In order to confirm the defects caused by PD 98059 and
U0126, we overexpressed an inactive form of MEK
(MEKneg) that acts as a dominant-negative protein (Mansour
et al., 1994). The high degree of sequence homology
between human and sea urchin MEK (around 90% in the
catalytic domain, Rizzo and Arnone, unpublished) sug-
gested that these mutants could be used to activate or to
block the MEK/ERK pathway in sea urchin, and expression
of MEKneg indeed induces dephosphorylation of ERK-like
in the sea urchin embryo (Fernandez-Serra et al., 2004).
Injection of MEKneg mRNA just before fertilization induced
aberrant first and second divisions in a very high percentage
of eggs and inhibition of mitosis in a few eggs, in
comparison with control non-injected eggs or eggs injected
with KCl buffer only (Figs. 9A and B). Similar alterations to
those observed after PD 98059 or U0126 treatment were
observed, such as incomplete cytokinesis, formation of
daughter cells of different sizes or with altered shapes (Fig.
9A). Altogether, these results strongly suggest that an MEK/
ERK-like pathway controls the first mitotic divisions in sea
urchin.
Effect of U0126 and PD 98059 on the timing of mitosis entryControl
n = 14
5 AM U0126
n = 6
15 AM PD 98059
n = 5
Mean time to NEB
(minutes after fertilization)
45 T 2 52 T 4 51 T 4
Duration of mitosis (min) 30 T 1 26 T 2 27 T 2
Eggs were treated with MEK inhibitors 30 min after fertilization and
observed under video microscopy. Results are mean T SEM. n represents
the number of eggs. A Student’s t test shows that all values obtained in
U0126 or PD 98059-treated eggs are significantly different ( P < 0.01) from
those of control eggs.
Discussion
The data provided here in sea urchin embryos show that a
MEK/ERK-like cascade is transiently activated at the time
of mitosis. Moreover, we show that the activated ERK pool
is located close to the nucleus then to the mitotic spindle.
The specific inhibition of this cascade leads to mitotic
defects that strongly implicates a MEK-ERK cascade in the
formation of the mitotic spindle and in the attachment of
chromosomes during the early embryo mitotic cell cycles.
The unfertilized sea urchin egg contains a highly
phosphorylated ERK-like protein
The fact that unfertilized eggs contain a highly phos-
phorylated ERK-like protein that was gradually dephos-
phorylated during the first minutes following fertilization
(this paper and Carroll et al., 2000; Chiri et al., 1998;
Kumano et al., 2001) fits with the idea that maintaining
MAPK activity at a high level in the unfertilized egg blocks
DNA synthesis. Indeed, a decrease in this activity with
MEK inhibitors leads to progression into S phase in starfish
oocytes (Abrieu et al., 1997; Fisher et al., 1998; Tachibana
et al., 1997) and in the sea urchin eggs of S. purpuratus
(Carroll et al., 2000). In contrast, Philipova and Whitaker
(1998) observed a low MAPK activity in unfertilized L.
pictus eggs that was followed by an increase after
fertilization. These authors may not have detected MAPK
contained in the nucleus (Carroll et al., 2000) or in the
cortex (Walker et al., 1997) of sea urchin eggs since they
followed a protocol described by Shibuya et al. (1992) and
used soluble fractions that may lack nuclei and insoluble
cytoskeleton elements for Western blots and immunopreci-
pitations. This could also be explained if these authors have
detected another MAPK in their study. As a matter of fact,
these authors reported very recently that a ERK1-like
MAPK, which should then be different from the ERK that
we detect here, was required for S phase onset in sea urchin
embryos (Philipova et al., 2005).
A small pool of ERK-like protein is activated during mitosis
Only a minor fraction of total ERK-like activity is
transiently activated at time of mitosis and is located in close
vicinity to the mitotic spindle in the sea urchin egg of P.
lividus (this paper) and in some other cell types (Cha and
Shapiro, 2001; Shapiro et al., 1998; Wang et al., 1997;
Willard and Crouch, 2001; Zecevic et al., 1998). This could
explain why MAPK activity has not been detected at the time
of mitosis in oocytes of clam (Shibuya et al., 1992) or even
other species of sea urchin (Carroll et al., 2000; Kumano et
Fig. 6. Effect U0126 or PD 98059 on chromosomes separation and actin ring formation. 5 AM U0126 or 15 AM PD 98059 was added 30 min after fertilization
and eggs were taken 30 min later for fixation and labeling as described in Materials and methods. Chromosomes (upper picture) and actin (lower picture) were
labeled with Hoechst and rhodamine phalloidin, respectively. (A) An anaphase in control eggs. (B–G) Different phenotypes that were commonly observed in
U0126 (B, C, E) or PD 98059 (D, F, G) treated eggs. Scale bar: 100 Am.
W.L. Zhang et al. / Developmental Biology 282 (2005) 192–206 201
al., 2001). The changes in levels ofMAPK activity just before
or during mitosis could be a way to control exit from mitosis
during the first cell cycles of the early embryo, at least in sea
urchin, starfish and Xenopus. We observed that treatment of
fertilized eggs with 20 AMwortmannin, which leads to arrest
in prometaphase-like state (De Nadai et al., 1998), inhibited
ERK-like dephosphorylation normally seen at exit of mitosis
(results not shown and Chiri et al., 1998). This sustained
MAPK phosphorylation can then be correlated with arrest at
mitosis during the first embryonic cell cycle in sea urchin as
reported inXenopus (Chau and Shibuya, 1998; Haccard et al.,
1993; MacNicol et al., 1995; Sagata et al., 1989) and starfish
(Abrieu et al., 1997).
Specificity of PD 98059 or U0126
We believe that PD 98059 or U0126, used at concen-
trations as low as 1 AM and never exceeding 15 AM or 5 AM,
respectively, inhibited specifically the MEK/ERK-like path-
way while U0124 had no effect, considering the data
reported by Davies et al. (2000). These concentrations are
in the same range as those used by Carroll et al. (2000), and
very low if compared to the 50-AM U0126 used in somatic
cells (Horne and Guadagno, 2003) or the 200-AM PD 98059
used in Xenopus extracts (Guadagno and Ferrell, 1998). The
fact that only a very low percentage of cells was blocked in
mitosis by PD 98059 or U0126 corroborates the data
reported by Kumano et al. (2001). Increasing the concen-
trations of PD 98059 or U0126 at levels as high as 50 AMcould indeed lead either to eggs arrested at pseudo-
metaphase showing one or a few unaligned chromosomes
and an unusually small mitotic spindle, or to divided eggs
showing alterations very similar to our present observations
(Pesando et al., 1998, 1999). Indeed, 100 AM U0126 was
necessary to inhibit DNA synthesis in sea urchin embryos
(Philipova et al., 2005). However, such high concentrations
of PD 98059 or U0126 might act on other MAPK cascades
or act non-specifically on other proteins that may have an
MBP kinase activity, since several myc or MBP kinases can
be detected in the sea urchin egg and are activated during
mitosis (Chiri et al., 1998; Walker et al., 1997). Finally, we
observed that dominant-negative MEK human constructs
had a greater effect than the inhibitors, comparing the
percentage of altered eggs. This could be due to the fact that
MEK inhibitors were added 30 min after fertilization to a
population of eggs, where diffusion in eggs, access to MEK,
and sensitivity to the drugs may differ between eggs. These
limiting factors cannot be taken into account in dominant-
negative MEK experiments, where all eggs were injected
before fertilization. However, we cannot rule out non-
specific effects of human proteins in the sea urchin
embryo.
Fig. 7. Effect of PD 98059 or U0126 on mitotic spindle formation and chromosome separation. A1 to A6 show control eggs that were taken at time of early
anaphase (1,2), late anaphase (3,4) and early telophase (5,6), labeled with Hoechst (1,3,5) or with an anti-tubulin antibody (2,4,6), and observed with a light
microscope. Eggs were treated 30 min after fertilization with either 10 AM PD 98059 (B, F, G) or with 5 AM U0126 (C, D, E) and taken at different times of
mitosis for labeling with Hoechst (1) or with anti-tubulin antibody (2). Scale bar: 50 Am.
W.L. Zhang et al. / Developmental Biology 282 (2005) 192–206202
Fig. 8. Examples of alterations in U0126 treated eggs observed by confocal imaging. 5 AM U0126 was added 20 min after fertilization and eggs taken for
fixation at metaphase (a/aV and b/bV) or at telophase (c/cV). Eggs were labeled with Hoechst (a, b, c) or with an anti-tubulin antibody (aV, bV and cV) and observed
by confocal imaging. Very altered spindles were seen at metaphase (aV and bV), while the two daughter cells observed at telophase showed lots of decondensed
and dispersed chromosomes (c). Scale bar: 50 Am.
Fig. 9. Effect of injection of Mekneg on first mitosis. Embryos are observed 2 or 3 h after injection with Mekneg or not (control eggs non-injected or injected
with KCl only). (A) Different types of altered divisions seen in eggs injected with MEK mutants. (B) The percentage of altered embryos. 27, 43 and 56
embryos were counted after 2 h in control embryos, embryos injected with KCl buffer, or injected with Mekneg, respectively. 22, 21 and 62 embryos were
counted after 3 h in control embryos, embryos injected with KCl or injected with Mekneg, respectively.
W.L. Zhang et al. / Developmental Biology 282 (2005) 192–206 203
W.L. Zhang et al. / Developmental Biology 282 (2005) 192–206204
Role of ERK-like in mitotic events
The association of the phosphorylated ERK-like with
the centrosomes and the mitotic spindle in the early sea
urchin embryo corroborates various data obtained in
somatic cells (Shapiro et al., 1998; Wang et al., 1997;
Willard and Crouch, 2001). The different defects induced
by the MEK inhibitors, such as poorly defined mitotic
axis, anarchic microtubule organization within the spin-
dle, extra arrays of microtubules and poor chromosome
separation, suggest that an ERK-like cascade acts at
different levels of mitotic spindle formation. By being
associated with the cortex (Walker et al., 1997), this
ERK-like could regulate the mitotic spindle–cortex
interactions important for spindle orientation and ana-
phase B. This could explain why we frequently observed
spindles that were not properly centered and formation of
two daughter cells of different sizes in eggs treated by
MEK inhibitors. Moreover, daughter cells might contain
an abnormal number of chromosomes, since a few
chromosomes were not properly aligned on the metaphase
plate in many cases, which could lead to the delay of cell
division that accumulated as the embryos developed and
to asynchronous divisions within embryos, giving rise to
stages three or five (Fig. 5). The occurrence of multipolar
spindles (Fig. 6) also contributed to the formation of
three or four cells of unequal sizes and of unequal
content in chromosomes. The association of phosphory-
lated ERK-like with the centrosomes suggests a role of
this MAPK in the regulation of centrosome activity.
MEK inhibitors could induce centrosomes/spindle poles
to split, which would explain the formation of tri- or
tetrapolar spindles in these conditions. Finally, almost all
eggs divided, even though the number of actin rings was
variable and a function of the number of microtubule
asters (spindle poles or extra microtubule arrays). These
results suggest that the ERK-like activity is not involved
in the induction of the cleavage furrow or of the actin
ring formation. Other proteins that also have an MBP
kinase activity and have been identified in the cortex of
the sea urchin egg (Walker et al., 1997) could be
involved in the triggering of cleavage furrow formation
(Shuster and Burgess, 2002a,b).
Relations MPF/MAPK during mitosis
All these results strongly suggest that an ERK-like
contributes to regulate mitotic spindle structure and
function in the early embryo similarly to somatic cells.
These effects cannot be due to inhibition of MPF activity
normally stimulated during mitosis, since treatment with
U0126 or PD 98059 did not inhibit MPF but on the
contrary led to a slight increase in this activity during
mitosis. This is in agreement with the results obtained by
Murakami et al. (1999) and Walter et al. (2000) who
showed that the MAPK pathway regulates cdc2 phos-
phorylation and thus MPF activation through Wee1. The
activation of ERK-like seems then to be necessary to
ensure normal progression through mitosis in the early
embryo, but not essential to entry or exit mitosis. This
fits with results obtained in Xenopus extracts where
MAPK activity is not needed to enter or exit mitosis but
is required for normal mitotic progression (Guadagno and
Ferrell, 1998; Takenaka et al., 1997). Finally, similar
alteration of the length of mitosis and no arrest of mitosis
were observed in tissue culture cells where M phase ERK
activity was blocked (Roberts et al., 2002).
Is an ERK-like part of a spindle checkpoint mechanism in
the early sea urchin embryo?
Our results strongly suggest that an ERK-like cascade
is part of a mechanism that controls the formation of the
mitotic spindle and the attachment of chromosomes to the
mitotic spindle during the first mitosis of the sea urchin
embryo. In somatic cells and in cycling Xenopus extracts,
various data show a role of a MEK/ERK pathway in the
spindle checkpoint mechanism (review by Maller et al.,
2002). Several points argue for the existence of a similar
mechanism in the early sea urchin embryo. Firstly,
phosphorylated ERK-like associates with the centrosomes
and the spindle at mitosis. Secondly, alterations of the
mitotic spindle and chromosomes attachment in PD
98059 or U0126-treated eggs that we report here look
like those observed in somatic cells lacking the mitotic
checkpoint proteins Mad2 (Dobles et al., 2000) or Bub1
(Taylor and McKeon, 1997). Preliminary Western blotting
analysis using anti-Bub1 and -Bub3 polyclonal antibodies
showed that these proteins are present in P. lividus
embryos (unpublished results), and it would be interesting
to investigate whether these proteins are active during
cleavage stages or not, and whether other checkpoint
proteins are expressed in the early sea urchin embryo.
Thirdly, only a minority of mitoses were abnormal when
ERK-like was inhibited, which indicates that this MAPK
might only be required in case of mitosis defects, as
opposed to being an intrinsic part of mitosis. However,
the existence itself of a spindle checkpoint in the sea
urchin early embryo has been questioned. Mitosis is
prolonged when the zygotes are induced to form
monopolar spindles or when the spindle is cut into two
half spindles (Sluder and Begg, 1983), which is in favor
of this hypothesis. On the contrary, unattached or
misoriented chromosomes do not induce arrest of division
(Rieder et al., 1995; Sluder et al., 1994), and duration of
second mitosis is not altered in embryos containing
tripolar or tetrapolar spindles (Sluder et al., 1997), which
rather suggests that no spindle checkpoint was functional.
One possibility is that an ERK-like is part of a
mechanism that is not a ‘‘spindle checkpoint’’ stricto
sensus, since it is not able to stop division as in somatic
cells in case of a single misoriented chromosome or other
W.L. Zhang et al. / Developmental Biology 282 (2005) 192–206 205
spindle defects, and some component of this checkpoint
could be either absent or inactive. Another possibility is
that a real spindle checkpoint mechanism exists in the
early sea urchin embryo, but the inhibitory activity
produced by an unattached kinetochore either remains
local or diffuses in a large cytoplasmic volume containing
large asters and a high microtubule density, which would
render inefficient this activity. This is in agreement with
the hypothesis where the spindle checkpoint is regulated
by a mechanism that depends on the nucleo-cytoplasmic
ratio (Maller et al., 2002), ratio that then allows an active
mechanism in somatic cells or in Xenopus egg extracts
supplemented with a high concentration of nuclei per
microliter of cytoplasm (Guadagno and Ferrell, 1998;
Minshull et al., 1994; Takenaka et al., 1997). It would be
interesting to investigate whether the inhibition of the
mos/MEK/ERK pathway leads in dividing Xenopus
embryos to the same alterations of mitotic events than
those we report here. However, the dependency on the
nucleo-cytoplasmic ratio of the spindle checkpoint mech-
anism in Xenopus embryos was questioned a few years
ago by Clute and Masui (1997).
Acknowledgments
This work was supported by the Association pour la
Recherche sur le Cancer (ARC), the CNRS, The University
Pierre et Marie Curie and the French Ministere de la
Recherche. S. Chiri acknowledges receipt of an ARC
fellowship, and W.L. Zhangh a Ministere de la Recherche
and a KC Wong fellowships. We thank C. Sunkel for the
anti-Bub1-Bub3 antibodies and G. Pruliere for the Bub1 and
Bub3 experiment. We dedicate this work to our dearest
friend and colleague Andre Picard, who was a strong
supporter of the sea urchin model in France.
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