* Author for correspondence (e-mail: lepage{at}obs-vlfr.fr)
Accepted 24 November 2003
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SUMMARY |
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Key words: ERK, MAP-kinase, MEK ets, Epithelial-mesenchymal transition, Primary mesenchyme, Ingression, Micromere, Sea urchin embryo
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Introduction |
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The SMCs derive from the macromeres. They delaminate from the tip of the
archenteron throughout gastrulation by a process that also involves an
epithelial-mesenchymal transition. They contribute to a variety of mesodermal
tissues including pigment cells, blastocoelar cells, muscle fibers and to
coelomic pouches (Cameron et al.,
1991; Cameron et al.,
1987
; Gibson and Burke,
1985
; Logan and McClay,
1997
; Ruffins and Ettensohn,
1996
; Tamboline and Burke,
1992
).
In addition to their role in formation of the skeleton of the larva,
micromeres have three other important functions in intercellular signaling.
First, soon after their formation at fourth cleavage and during the two next
divisions, they send a signal to the surrounding macromeres which is required
for the specification of the endoderm
(Horstadius, 1973;
Ransick and Davidson, 1993
).
The nature of the early signal responsible for endoderm induction is not
known. The second signaling function of the micromeres occurs between the
seventh and ninth cleavage, when they are still within the vegetal plate
epithelium. During this period, micromeres emit a signal that is crucial for
induction of the SMC fate (McClay et al.,
2000
; Sweet et al.,
1999
). This signal has been identified as the Delta ligand that
activates the Notch receptor (Sherwood and
McClay, 1999
; Sweet et al.,
2002
). Finally, during gastrulation, the PMCs send an inhibitory
signal to the SMCs that prevents them from differentiating as skeletogenic
mesenchyme. When micromeres are removed at the 16 cell stage or when the PMCs
are eliminated surgically at the mesenchyme blastula stage, skeletogenic cells
nevertheless form. In the absence of PMCs, pigment cells and blastocoelar
cells adopt a skeletogenic fate by a process called conversion
(Ettensohn, 1992
;
Ettensohn and McClay,
1988
).
A large body of evidence indicates that the fate of the large micromeres is
specified autonomously. Micromeres isolated or transplanted to any ectopic
location in the embryo always differentiate on schedule into skeletogenic
cells according to their normal fate
(Angerer and Angerer, 2003;
Brandhorst and Klein, 2002
;
Davidson et al., 1998
;
Horstadius, 1973
;
Okazaki, 1975
;
Ransick and Davidson, 1993
).
The maternal determinants responsible for this autonomous differentiation are
not known. However, several zygotic genes involved in the very early steps of
specification of the micromeres have been described recently
(Croce et al., 2001
;
Davidson et al., 2002a
;
Ettensohn et al., 2003
;
Fuchikami et al., 2002
;
Kurokawa et al., 1999
) and
epistatic analyses have helped to order them in a molecular pathway
(Oliveri et al., 2002
). One of
the earliest events in this pathway is the nuclear accumulation of
ß-Catenin in the micromeres nuclei at the 32-cell stage
(Emily-Fenouil et al., 1998
;
Logan et al., 1999
).
ß-Catenin then activates the expression of a homeobox gene called
pmar1, which is to date the earliest zygotic gene expressed
specifically in this lineage. Pmar acts as a transcriptional repressor and
therefore it is inferred that its role is to allow the expression of other
zygotic specification genes by releasing the repressive action of an
ubiquitous repressor. Among the zygotic factors acting downstream of
pmar, the ets1 and alx1 transcription factors play
crucial roles. Both genes are expressed very early in the micromere lineage
and both genes are strictly required for formation of the PMCs
(Ettensohn et al., 2003
;
Kurokawa et al., 1999
). When
the function of either gene is blocked, no PMCs form and several downstream
differentiation markers are downregulated.
In this study, we show that the specification of cell fate in the micromeres and their conversion from epithelial to migratory behaviour require a functional Raf/MEK/ERK signaling pathway. Inhibition of this pathway blocks PMC ingression and arrests the program of specification of the micromeres and SMCs at an early step. We further show that Ets1 is a key target of ERK, and that ERK may be activated in a cell-autonomous manner.
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Materials and methods |
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Detection of phosphorylated ERK by immunostaining
A monoclonal antibody specific for the dually phosphorylated form of MAP
kinase (ERK1 and ERK2) (Sigma M8159) was used for immunolocalisation, with a
vectastain ABC kit (alkaline phosphatase) and the chromogenic substrates
NBT/BCIP. In control experiments, the primary antibody was omitted.
Western blotting
Protein samples (10 µg/lane) were separated by SDS-gel electrophoresis
and electrophoretically transferred to 0.2 µm nitrocellulose filters. After
blocking for 2 hours in 5% dry milk in TBST and incubation overnight with the
anti ERK antibody, bound antibodies were revealed by ECL immunodetection.
In situ hybridization
In situ hybridization was performed following a protocol adapted from
Harland (Harland, 1991) with
antisense RNA probes and staged embryos. Most of the probes used in this study
were isolated in the course of an in situ hybridization screen (T. Lepage,
unpublished). The gene markers for secondary mesenchyme were AA29 and
23F, which encode enzymes of the nucleotide metabolism. The
endodermal marker gene 42C gene encodes a methyl transferase.
ets1, ske-T, alx1, Delta, msp130, CyIa, goosecoid and
brachyury are the P. lividus homologs of these genes. All
probes were synthesized with the T7 RNA polymerase after linearization by
NotI.
RNA injection
The coding sequence of ets1 and alx1 were amplified by
PCR using the Pfu DNA polymerase and inserted at the
BamHI-XhoI sites (ets) or BamHI and
EcoRI sites (alx) of pCS2+
(Turner and Weintraub, 1994)
to generate pCS2-ets1 and pCS2 alx1. RNA encoding wild-type
Ets1, Ets1 T107D and Ets1 T107A, and Ets1 VP16 were injected at 200µg/ml.
RNA encoding Alx1 was injected at 160 µg/ml.
pCS2-DN-TCF encodes a dominant negative TCF and was made by
deleting the ß-catenin binding domain of the sea urchin TCF (C. Gache,
unpublished). The dominant negative Ras construct used was the human
Ha-Ras cDNA which carries the Asn17 mutation cloned in pSP64T and the
constitutively active Ras was the p21v-Ha-ras
(Feig and Cooper, 1988;
Whitman and Melton, 1992
).
DnRas RNA was injected at concentrations up to 2mg/ml while
p21v-Ha-ras RNA was injected at 50 to 150 µg/ml. The
dominant-negative Raf construct is a kinase defective mutant that carries an
alanine in place of serine at position 621
(Fabian et al., 1993
); DNRaf
mRNA was injected at 800 µg/ml. The zebrafish MAP kinase phosphatase
construct contains the MKP3-coding sequence clones in pCS2
(Kudoh et al., 2001
); MKP3
mRNA was used at 450 µg/ml.
Site-directed mutagenesis and construction of expression plasmids
To make pCS2 ets T107D and pCS2 ets T107A, the ACG codon
encoding threonine in position 107 of pCS2 ets was mutated to GAT or
GCG by splicing PCR using the following oligonucleotides: Ets T107D Fw,
5'-CCCCCGCCAGATCCAGGCACCAACGCT-3' and Ets T107D Rev,
5'-AGCGTTGGTGCCTGGATCTGGCGGGGG-3'; Ets T107A Fw,
5'-CCCCCGCCAGCGCCAGGCACC and Ets T107A Rev,
5'-GGTGCCTGGCGCTGGCGGGGG-3'. For pCS2 VP16 Ets
construction, the region encoding amino acids 413-490 from VP16 (accession
number HEHSV165) was PCR amplified using (VP16 sequences in capitals)
VP16-Fw-BamHI
(5'-CGCGGATCCACCATGGCCCCCCCGACCGATGTCAGC-3') and VP16-Ets-Rev
(5'-GCAGTGCATAGATGCCATCCCACCGTACTCGTCAAT-3') and spliced by PCR to
the full-length Ets sequence amplified using Ets Fw
(5'-ATGGCATCTATGCACTGCTCC-3') and Ets TGA Xho
(5'-AGGCTCGAGTCAGTCGTCATCGCGTGCACC-3').
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Results |
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|
When observed at the pluteus stage (48/60 hours), embryos that had been treated with the inhibitor from the two-cell stage onwards, had a significantly reduced number of pigment cells and mesenchymal cells (Fig. 2D,H). The archenteron was regionalized normally into hindgut, midgut and foregut, and a stomodeum opening was present on the oral ectoderm. Although spiculogenesis was completely absent, the embryos had acquired an oral-aboral polarity, as shown by the presence of a mouth, a ciliary band and the differentiation of squamous aboral ectoderm. More than 98% of the treated embryos survived and showed these defects. In the remaining 2% of the embryos, treatment with the inhibitor caused exogastrulation (not shown).
To confirm the specificity of the defects caused by the inhibitor, we overexpressed a MAP kinase phosphatase (MKP3) by RNA injection into the egg as an independent mean of inhibiting ERK function. As with U0126 treatment, microcroinjection of MKP3 mRNA in to the egg completely prevented formation of the PMCs but did not inhibit gastrulation and formation of SMCs (Fig. 2I-L). Similarly, injection of mRNA encoding a dominant-negative form of the human Raf kinase which acts upstream of MEK, suppressed formation of the PMCs (Fig. 2M-P). These results show that a conserved Raf/MEK/ERK signaling pathway plays an essential role in PMC formation.
The small GTP-binding protein Ras is an important mediator of the MAPK
pathway activated by receptor tyrosine kinases and has been implicated in
regulating cell scattering and motility induced by growth factors such as
SF/HGF (Graziani et al., 1993;
Hartmann et al., 1994
) and EGF
(Boyer et al., 1997
). We found
that overexpression of a dominant-negative form of Ras (dnRas) by RNA
injection into sea urchin eggs did not block ingression of the micromeres but
inhibited their differentiation into spicules
(Fig. 2Q-T). This result
suggests that the transient activation of ERK in the micromere lineage and
ingression of the PMCs is not dependent on Ras-mediated receptor signaling,
while differentiation of PMCs into spicules is likely to require Ras-dependent
steps. However, the dnRas construct we used was of human origin and,
therefore, the possibility remains that it does not completely block Ras
activity in the sea urchin embryo.
The MAP kinase signaling pathway is required for PMC ingression, then for skeletogenesis
In the sea urchin embryo, ablation of the micromeres at the 16-cell stage
can be compensated later by the SMCs, some of which transfate and
differentiate into skeletogenic mesenchyme
(Ettensohn, 1992;
Ettensohn and McClay, 1988
).
This phenomenon of conversion did not occur in embryos cultured in the
continuous presence of the MEK inhibitor, as they lacked spicules. ERK
activity may thus be required for the transfating of the SMCs or for
spiculogenesis. Alternatively, the PMCs may not require ERK to send the signal
that suppress transfating. To address this issue, we treated groups of embryos
for various periods of time starting at the two-cell stage. Embryos were
scored at the mesenchyme blastula and gastrula stage for the presence of
micromeres, at the late gastrula stage for the presence of cells emigrating
from the archenteron and at 48-72 hours for the presence of pigment cells and
spicules (Fig. 3). All
treatments starting before the hatching blastula stage blocked ingression of
the PMCs and produced embryos lacking skeleton
(Fig. 3A,B). However, when the
inhibitor was added at the hatching blastula stage, ingression was delayed by
about 2 hours but eventually occurred (Fig.
3C). In these embryos, the mesenchyme cells remained as individual
cells and did not coalesce into a syncytium or produce spicules. Embryos
treated with the MEK inhibitor from the late mesenchyme blastula stage or
gastrula stage onwards also lacked spicules
(Fig. 3D). When the embryos
were treated with U0126 from the two-cell stage but washed out at the
mesenchyme blastula stage, spiculogenesis was largely rescued in most embryos
(140/160) although the shape of these spicules was often abnormal
(Fig. 3E,F). We do not know if
the skeletogenic cells formed in these embryos by delayed delamination of the
PMCs or by conversion of the SMCs. When the inhibitor was washed out at the
late gastrula stage, most embryos (151/155) lacked spicules
(Fig. 3G,H). Taken together,
these observations show that there are at least two crucial requirements for
ERK activity: first between the prehatching blastula stage and the swimming
blastula stage for the epithelial mesenchymal transition of the PMCs (green
labeling in Fig. 3); and then
during gastrulation for differentiation of the PMCs into spicules (yellow
labeling in Fig. 3).
|
|
|
MEK/ERK signaling is required for full induction of the SMC fates
SMC fates (notably pigment cells and blastocoelar cells) are induced from
macromeres decendants by adjacent micromeres expressing Delta during the late
blastula stage, between the eighth and ninth cleavage
(McClay et al., 2000;
Sherwood and McClay, 1997
;
Sherwood and McClay, 1999
;
Sherwood and McClay, 2001
;
Sweet et al., 2002
;
Sweet et al., 1999
). SMC
induction therefore coincides temporally with the activation of ERK in the
micromeres.
To test the impact of MEK/ERK signaling on SMC specification, we analyzed
the expression of the P. lividus Delta gene. In agreement with
previous reports in other sea urchin species
(Oliveri et al., 2002;
Sweet et al., 2002
), we found
that Delta is first expressed in the micromeres, starting at about the
128-cell stage. As Delta expression declines in the PMCs following their
ingression, a second wave of Delta expression is initiated in the
macromeres-derived mesendodermal cells. We found that interfering with ERK
signaling significantly reduced expression of Delta in the micromeres at the
late blastula stage (Fig. 5D
and Table 1). By contrast, at
the mesenchyme blastula stage, relatively normal levels of Delta expression
were maintained in the presumptive SMCs following treatment with the inhibitor
(Fig. 5E,F).
|
U0126 treatment was found to abolish the expression of CyIa in the
putative pigment cell precursors, whereas the ectodermal expression was
unaffected. By contrast, the late expression of CyIa in presumptive
blastocoelar cells at the gastrula stage seemed largely unaffected. Both
AA29 and 23F were expressed in cells that migrated from the
archenteron tip in U0126-treated embryos, but only about half the normal
number were present at the blastula and gastrula stages
(Fig. 5P,Q,V,W and
Table 1). Moreover,
AA29 and 23F-expressing mesenchymal cells did migrate from
the tip of the archenteron during gastrulation. Taken together, these
observations suggest that the pigment cell and blastocoelar cell fates are
affected differently when the MEK/ERK pathway is blocked. These observations
are consistent with the lack of pigment cells in the U0126 embryos because
among SMC derivatives, pigment cells have the highest requirement for Delta,
whereas specification of blastocoelar cells can occur via Notch/Delta
signaling among macromeres descendants
(Sweet et al., 2002).
Finally, we confirmed that ERK signaling is not involved in the early
signaling that is required for endoderm induction, which occurs at a time when
MAPK activation was not significantly detected. Early expression of
42C, an endodermal marker expressed in presumptive hindgut and midgut
from the mesenchyme blastula stage onwards was unaffected by treatment with
U0126 (Fig. 6A-F;
Table 1). Moreover, gut
formation and patterning appeared normal when MEK/ERK signaling was inhibited
from fertilization onwards. We also confirmed that U0126 treatment did not
affect the expression of goosecoid and brachyury, two early
markers of oral /aboral polarity (Angerer
et al., 2001; Croce et al.,
2001
; Gross and McClay,
2001
), (Fig.
6G-R).
|
The sea urchin Ets transcription factor as a direct target of MEK/ERK signaling
As shown in Fig. 4,
ets1 is expressed at a relatively normal level in the presence of
U0126 while several other genes acting further downstream in the network are
strongly downregulated. This observation suggests that one target of MAP
kinase could be Ets1 itself, or a transcription factor acting early in
parallel with Ets1 such as the homeobox gene product Alx1
(Ettensohn et al., 2003).
Sequence analysis showed that the sea urchin Ets1 protein contains a PPTP
motif, and the Alx1 protein contains a PSTP conforming to the PXS/TP consensus
MAP-kinase phosphorylation site (Marshall,
1994
) (Fig. 7A,C).
In addition, we found that the sea urchin Ets1 protein sequence contains a
stretch of amino acids KTDFLSRAPPFMGD very similar to the
KEXFLXLXPXFXGD ERK2 docking site, the most critical Phe
residue being conserved (bold indicates the most conserved residues)
(Seidel and Graves, 2002
)
(Fig. 7B). To test the
importance of phosphorylation of the MAP kinase consensus site for activation
of the sea urchin Ets1 in vivo, we substituted the threonine phospho-acceptor
residue of Ets1 by an aspartic (etsT107D) or alanine
(etsT107A) residue. We also constructed a chimeric mRNA encoding the
whole Ets1 protein sequence fused to the potent transactivation domain of the
viral VP16 protein (ets-VP16). Both the introduction of a
constitutive negative charge and the fusion to a strong transactivating domain
were designed to bypass the requirement for phosphorylation. Conversely, the
conversion of the phosphoacceptor residue into alanine should produce an
inactive or weak dominant-negative form
(Brunner et al., 1994
).
|
Equivalent experiments with alx1, which also contains a consensus
MAP kinase phosphorylation site and is required for ingression and
differentiation of the micromeres
(Ettensohn et al., 2003), were
not possible because overexpression of alx1 inhibited rather than
promoted ingression of the micromeres. This suggests that dosage of
alx1 transcript is critical for its function.
Finally, we tested whether overexpression of a constitutively active form
of Ras (CA-Ras) could promote formation of primary mesenchyme cells. PMCs
formed normally in embryos injected with mRNA encoding an activated form of
Ras. However, during early gastrulation we found that most cells of the
injected embryos rounded up and detached from the hyaline layer. In large
sectors of the injected embryos, the ectoderm became very thin
(Fig. 7K). Cells continuously
detached from the epithelium while the remaining cells stretched to cover the
embryo. This broad disorganisation of the epithelium appeared very different
from the localized epithelial-mesenchymal transition caused by overexpression
of ets1, ets1-VP16, ets1-T107D
(Fig. 7) or pmar1
(Oliveri et al., 2002), and it
occurred at a later stage. This suggests that the remodeling of the ectoderm
caused by artificial activation of the pathway at the level of Ras is a
process different from the epithelial-mensenchymal transition induced by
overexpression of ets1 or pmar.
Activation of MEK/ERK in the PMCs requires the maternal TCF/ß-Catenin pathway
We tested whether activation of ERK in the micromeres was dependent on the
maternal ß-Catenin pathway, which is responsible for patterning along the
AV axis. Injection of mRNA encoding N-TCF/Lef, a dominant-negative form
of TCF/Lef which blocks the Wnt/ß-Catenin pathway, caused a typical
animalized phenotype (Fig. 8H)
and prevented activation of ERK at the hatching blastula stage as revealed by
immunostaining. (Fig. 8E).
Similarly, ERK activation in the micromeres could not be detected in embryos
treated with ZnCl2 (Fig.
8B), a classical animalizing reagent. By contrast, neither
treatment of embryos with lithium, which activates maternal Wnt/ß-Catenin
pathway by inhibiting GSK3, nor overexpression of Wnt8 (data not shown) caused
an increase in the number of cells positive for activated ERK
(Fig. 8C,F). These results
suggest that activation of ERK requires a functional Wnt/ß-Catenin
pathway but that activation of this pathway is not sufficient to activate ERK
or trigger ingression of the micromeres.
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Discussion |
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Differentiation of the endoderm and ectoderm remained largely unaffected by the inhibition of MEK signaling with U0126. The gut separated normally into three compartments and a mouth formed. Similarly, even in the absence of spicules, the ectoderm acquired a well-developed oral-aboral polarity, as indicated by the overall morphology of the larva and the normal expression of several markers. Therefore, in the sea urchin embryo, the ERK signaling pathway appears to be required mainly before gastrulation, for the formation of the mesoderm.
In vertebrates, the ERK signaling pathway has been implicated in mesoderm
induction downstream of the FGF signaling cascade
(Curran and Grainger, 2000;
Schohl and Fagotto, 2002
;
Uzgare et al., 1998
), in
patterning the embryo along the dorsoventral axis
(Furthauer et al., 1997
) and
in the induction of the neural tissue by FGF factors
(Streit et al., 2000
). As
activation of this signaling pathway during gastrulation in deuterostomes is
often associated with signaling from the FGF receptor, it is tempting to
speculate that ligands of the FGF family are responsible for activation of ERK
in the mesoderm in the sea urchin embryo. Our preliminary data using FGF
pathway inhibitors indicate that this is not the case, consistent with the
observation that injection of dnRas RNA does not block ingression.
Activation of ERK is required for maintaining specification of the micromeres and for formation of a subset of SMCs
We found that the expression of several important genes of the gene network
regulating micromere specification was severely affected by the inhibition of
MEK signaling. Expression of the homeobox gene alx1, which is
required for ingression of the PMC and skeletogenesis, was reduced at the
blastula stage and almost absent at later stages. More strikingly, the
expression of skeT, which is restricted to the large micromere
lineage from the blastula stage, and of several differentiation markers such
as msp130, was abolished. In the absence of ERK signaling, the
micromere gene expression program, which was probably well under way at the
blastula stage, was arrested, the expression of several key genes was
downregulated and ingression was inhibited. We conclude that, although ERK
activation occurs after the initial expression of early regulators such as
pmar, alx1 and ets1, ERK function is critically required for
maintenance of this specified state.
Finally, we found that blocking the MAP kinase signaling pathway diminished
the early expression of Delta in the large micromere lineage, but did not
significantly perturb the late Delta expression in the macromeres descendants.
This effect provides an explanation for the fact that pigment cells are more
affected than other SMC derivatives because pigment cells have a strict
requirement for Delta signaling from the micromeres, while blastocoelar cells
can form by Notch/Delta signaling among macromeres descendants
(Sweet et al., 2002).
The sea urchin Ets 1 is a key target of the ERK signaling pathway
A number of different transcription factor families, including the Ets
family, have been implicated in epithelial mesenchymal transitions and cell
migrations (Savagner, 2001).
In vertebrates, ets1 and ets2 are expressed in several
tissues undergoing morphogenetic changes. For example in Xenopus,
ets1 is expressed in the involuting marginal zone and in premigratory and
migratory neural crest cells. In the sea urchin embryo, maternal ets1
mRNA is ubiquitous whereas zygotic ets1 expression becomes restricted
to the precursors of the PMCs at the early blastula stage and extends to the
precursors of the SMCs at the end of gastrulation. It has been shown
previously that overexpression of ets1 induces mesenchymalization of
most cells of the blastula stage embryo whereas overexpression of a truncated
version that inhibits Ets1 function blocks PMC ingression
(Kurokawa et al., 1999
). This
led to the suggestion that Ets1 is a key regulator of epithelial-mesenchymal
transition in the normal embryo. Our results in P. lividus support
this idea. They further demonstrate that post-transcriptional modification of
this transcription factor is a crucial step for its function. We found that
the sea urchin Ets1 protein, like the vertebrate Ets1 and Ets2 and their
probable Drosophila ortholog Pointed-P2, which contains a conserved
MAP kinase phosphorylation site PXS/T upstream of the conserved N-terminal
pointed domain (PNT). Phosphorylation of this site by
Ras>Raf>MEK>MAP-kinase signaling is thought to stimulate the
transcriptional activation function of this class of Ets family members by an
unknown mechanism. We found that embryos overexpressing an activated form of
Ets (EtsT107D) are largely insensitive to the MEK inhibitor. Conversely,
mutation of the phosphoacceptor Thr107 to Ala abrogated the phenotypic
conversion from epithelial to mesenchymal induced by ets1 mRNA
overexpression. Taken together these results show that phosphorylation of Ets1
on Thr107 is a key event for its activation.
In addition to the presence of a MAP kinase consensus site, the sea urchin
Ets1 contains a sequence very similar to the ERK2 docking site of Ets1 and
Ets2. Mutation of this docking site in the vertebrate Ets1 prevents the
transcriptional activation of a promoter containing a Ras Responsive Element
(RRE) that contains Ets1-binding sites. As stressed by Seidel and Graves
(Seidel and Graves, 2002), the
presence of ERK docking sites is not widespread among Ets family members. Only
1 out of 10 of Drosophila Ets family members and two out of 25 human
Ets protein sequences contain an ERK docking site. The combined presence of an
ERK docking site and an ERK consensus phosphorylation site distinguishes a
subset of Ets proteins activated by MAP kinase signaling
(Oikawa and Yamada, 2003
). The
sea urchin Ets1, like the vertebrates Ets1/2 and Drosophila Pointed,
appears to contain both motifs.
Activation of ERK is downstream of Wnt/ß-Catenin and may be cell autonomous
The Wnt/ß-Catenin pathway has been identified as the major pathway
regulating patterning along the animal vegetal axis of the sea urchin embryo.
Accumulation of ß-Catenin in the nuclei of micromeres from the time of
their segregation up to the blastula stage is required for specification of
the PMCs. (Emily-Fenouil et al.,
1998; Huang et al.,
2000
; Logan et al.,
1999
; Vonica et al.,
2000
). We found that ERK activation depends on a functional
TCF/Lef factor and does not occur in animalized embryos. By contrast,
artificial activation of the Wnt pathway by treatment with lithium was not
sufficient to activate ERK in other cells, indicating that additional factors
present only in the region of the vegetal pole are required the spatial
regulation of ERK.
In most models where it has been studied, the epithelial-mesenchymal
transition is a non-cell-autonomous process triggered by extracellular signals
binding to their cognate receptors. Four lines of evidence suggest, however,
that in the sea urchin embryo ERK activation occurs in absence of
extracellular signal. First, we found that phosphorylation of ERK was
transiently detectable in dissociated blastomeres and the time course of
phosphorylation analyzed by western blot was not significantly different from
that in intact embryos. Second, micromere formation was not blocked by
overexpression of a dominant-negative version of Ras that mediates the
activity of most receptor tyrosine kinases. Third, activation of ERK depends
on the TCF/ß-Catenin pathway, which is cell-autonomously activated in the
sea urchin embryo. Finally, micromeres are capable of differentiating into
skeletogenic cells when cultured in a simple medium, grafted to ectopic
positions or reassociated with various tiers of blastomeres
(Horstadius, 1973;
Okazaki, 1975
).
In most other classes of echinoderms and in some sea urchin species, embryogenesis procedes without micromeres. Embryos do not undergo an epithelial mesenchymal transition at the blastula stage and therefore resemble the P. lividus embryos treated with the MEK inhibitor. It will be interesting to examine how genes like ets1 and alx1 are expressed, and if ERK is activated at the vegetal pole in these embryos.
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ACKNOWLEDGMENTS |
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REFERENCES |
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