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First published online October 12, 2006
doi: 10.1242/10.1242/dev.02603
UMR 7009 CNRS, Université de Pierre et Marie Curie (Paris 6), Observatoire Oceanologique, 06230 Villefranche-sur-Mer, France.
Author for correspondence (e-mail:
lepage{at}obs-vlfr.fr)
Accepted 30 August 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: NLK, Delta, TCF/Lef, ERK, MAP kinase, TAK1, Germ layers, Mesoderm, Endoderm, Sea urchin embryo
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Rottinger et al.,
2004). The SMCs derive from the sister cells of the micromeres
called macromeres, which also give rise to most of the endoderm
(Cameron et al., 1991;
Ruffins and Ettensohn, 1993).
This population of mesodermal precursors gives rise to four cell types: the
pigment cells, the blastocoelar cells, the coelomic pouch precursors and the
circumoesophageal muscles.
Fate mapping experiments and gene expression studies have shown that at
blastula stage precursors of the SMCs are present in a ring of cells within
the vegetal plate, the centre of which is occupied by the PMCs
(Ruffins and Ettensohn, 1996).
Indeed, embryological studies have revealed that during early development the
SMCs require a signal from the micromeres to be specified. Removal of the
micromeres result in embryos devoid of SMCs, while recombining micromeres with
blastomeres derived from the animal pole is sufficient to induce formation of
SMCs (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). Overactivation of the Notch pathway causes overproduction
of SMCs, while inhibition of Notch signalling prevents specification of the
SMCs (Sherwood and McClay,
1999; Sweet et al.,
2002). Delta is first expressed in the micromere progeny starting
at early blastula stage, then, after ingression of the PMCs, a second wave of
Delta expression is initiated in the SMC precursors. Further, experiments
using chimeric embryos have established that the first expression of Delta in
the micromere progeny is essential for specification of the pigment cells and
blastocoelar cells, while its later expression in the SMC territory at
mesenchyme blastula stage is important for specification of the blastocoelar
cells and muscle cells and for refining the position of the ectoderm-endoderm
boundary (Sherwood and McClay,
2001).
Another signalling pathway crucial for specification of the mesoderm is the
Wnt/ß-catenin pathway (Emily-Fenouil
et al., 1998; Logan et al.,
1999; Wikramanayake et al.,
1998). Starting at the 16-cell stage, ß-catenin is
translocated into the nuclei of micromeres and macromeres, where, associated
with the transcription factor TCF, it activates endomesodermal genes
(Huang et al., 2000;
Vonica et al., 2000).
Expression of Delta in the micromeres also requires nuclear translocation of
ß-catenin (McClay, 2000).
Up to late blastula stage, a high level of ß-catenin is detected in the
nuclei of mesodermal precursors (PMCs and SMCs). However, after hatching,
ß-catenin is progressively downregulated in these cells and by early
mesenchyme blastula stage it is no longer detected in the nuclei of SMCs
(Logan et al., 1999).
One mechanism allowing the downregulation of TCF-ß-catenin signalling
has emerged from recent work in Caenorhabditis elegans and
vertebrates. This mechanism relies on a crosstalk between the canonical Wnt
pathway and a derived MAP kinase pathway involving the MAP kinase-related
proteins Nemo Like Kinase (NLK) and TAK-1
(Behrens, 2000;
Thorpe and Moon, 2004). In
C. elegans, NLK regulates the segregation of the endodermal (E) and
mesodermal (MS) precursors at the eight-cell stage through the asymmetric
nuclear localization of POP-1, the C. elegans homologue of TCF
(Bowerman and Shelton, 1999;
Meneghini et al., 1999;
Rocheleau et al., 1997;
Rocheleau et al., 1999;
Thorpe et al., 1997).
Epistasis analysis and biochemical experiments have demonstrated that TAK-1
activates NLK, which in turn phosphorylates TCF and promotes its export from
the nucleus (Lo et al., 2004;
Rocheleau et al., 1999). The
role of this MAP kinase pathway as a negative regulator of TCF activity seems
to be conserved in vertebrates, as illustrated by its ability to suppress the
axis-inducing activity of ß-catenin in Xenopus
(Ishitani et al., 1999).
Biochemical studies have demonstrated that vertebrate NLK binds directly to
the TCF/ß-catenin complex and phosphorylates two conserved residues on
TCF, inhibiting its DNA-binding activity
(Ishitani et al., 2003b).
In this study, we analyse the role of NLK during sea urchin development. We found that nlk is expressed in a pattern strikingly similar to that of Delta, that expression of nlk in the mesodermal lineage is regulated by Notch/Delta signalling, and that nlk and Delta strongly synergize during mesoderm formation. Furthermore, we provide evidence that the Delta-induced expression of nlk serves as a mechanism to inhibit TCF in the mesodermal lineages and propose that downregulation of TCF in these cells is required for the segregation of the mesoderm from the endomesoderm.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Lepage and Gache, 1990).
Treatments with U0126 were performed as described previously
(Rottinger et al., 2004).
Lithium was used at 30 mmol/l and dexamethasone at 10 µmol/l.
Northern blot
Total RNA was extracted from embryos at various stages by the method of
Cathala (Cathala, 1983). RNA
samples (10 µg per lane) were electrophoresed through a formaldehyde
agarose gel, transferred to a Nylon membrane, and hybridized with probes
labelled by random priming corresponding to the 3'UTR or to the coding
sequence of the nlk transcript. Hybridization was carried out using
standard methods (Sambrook et al.,
1989). Both probes gave similar results.
Detection of phosphorylated ERK
For immunolocalization, embryos were fixed in methanol and following
rehydration were incubated with a monoclonal antibody specific for the dually
phosphorylated form of MAP kinase (ERK1 and ERK2) (Sigma M8159). An anti-mouse
secondary antibody conjugated to alkaline phosphatase and the chromogenic
substrates NBT/BCIP were used for detection.
Constructs, RNA and morpholino injections
The coding sequence of nlk was amplified by PCR using the Pfx DNA
polymerase (Invitrogen) and inserted at the ClaI-XbaI sites
of pCS2 + (Turner and Weintraub,
1994). RNA encoding a catalytically inactive version of NLK (NLK
K78M) was obtained by replacing, within the ATP-binding site, a conserved
lysine residue by a methionine using PCR. RNA encoding wild-type NLK or NLK
K78M were injected at 900 µg/ml.
pCS2-DN-TCF encodes a dominant negative TCF and was made by
deleting the ß-catenin-binding domain of the P. lividus TCF
(C.G., unpublished). The pCS2-TCF-VP16-GR was constructed by using as
starting plasmid the pCS2 ENR-Tbx2/3-GR
(Horb and Thomsen, 1999). The
fragment encoding ENR-Tbx was removed by digestion and replaced by an
N-terminally deleted TCF fused to VP16 (C.G., unpublished)
(Triezenberg et al., 1988).
pCS2-TCF-VP16-GR was used at 400 µg/ml. PCS2Lv Delta was
constructed by amplifying the coding sequence of Lytechinus
variegatus Delta (Sweet et al.,
2002) and cloning it in pCS2. To construct the dominant negative
Delta, we used the P. lividus Delta sequence. Using PCR, we
introduced a stop codon at amino acid 626, resulting in a truncation of the
region located after the transmembrane domain. RNAs encoding Delta or the
dominant negative Delta were used at 400 µg/ml and 200 µg/ml,
respectively.
In the experiments to test for a synergistic effect, nlk and Delta were co-injected at 450 µg/ml and 200 µg/ml, respectively. The following morpholino oligonucleotides were used at 0.9 mmol/l: 5'-CGAGATCCACAAACAGCCATATCAC-3' (NLK ATG); 5'-TCGGAGGCAGACCAGCAGCGAGAAA-3' (NLK 5'UTR); 5'-GATTCAAGGCGAGCCATTTTGGATG-3' (TAK-1).
All the experiments were repeated two or three times, and for each experiment 100-150 embryos were observed. Only representative phenotypes (present in 80-90% of the injected embryos) are shown.
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.L.,
unpublished). The bhmt (betaine-homocysteine S-methyltransferase),
bpnt (PAP-inositol 1-4 phosphatase), papss
(3'-phosphoadenosine 5'-phosphosulfate synthase). ets1, ske-T,
alx1, Delta, goosecoid and brachyury are the P. lividus
homologues of these genes. The accession numbers of the mRNA sequences used in
this study are as follows: nlk, AY442297; TCF, AM179826;
Delta, DQ536193; tak1, DQ531771; gcm, DQ666827;
alx1, DQ536192; bhmt, DQ531773; bpnt, DQ531772;
papss, DQ531774.
TOP FLASH assays
Reporter gene assays were performed as described by Vonica et al.
(Vonica et al., 2000).
Briefly, the linearized TOP FLASH reporter plasmid was injected at 5 µg/ml
together with carrier DNA at 50 µg/ml. Embryos injected with the TOP FLASH
reporter plasmid alone, with nlk mRNA, or that were treated with
lithium were collected after hatching and lysed in 70 µl of lysis buffer
(Promega) for 30 minutes at room temperature. After centrifugation, the
supernatant was stored at -20°C. To perform the luciferase assay, 60 µl
of supernatant was mixed to 150 µl of assay buffer (Promega) and the
luminescence was measured on a Glomax luminometer (Promega). About 200 embryos
were used for each experiment and the experiments were repeated three
times.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Hyodo-Miura et al., 2002;
Ishitani et al., 1999;
Thorpe and Moon, 2004), to
Drosophila Nemo (Choi and Benzer,
1994) and to C. elegans LIT-1
(Meneghini et al., 1999;
Rocheleau et al., 1999). An
alignment of these NLK protein sequences shows that conservation is very high
in the central kinase domain and the C-terminal region, while the N-terminal
part is not conserved (see Fig. S1 in the supplementary material). Northern blot analysis revealed the presence of a single 7.6 kb transcript expressed maternally and throughout cleavage. The level of this mRNA declines at the blastula stage then increases strongly during gastrulation, probably as a consequence of zygotic transcription (Fig. 1A). After gastrulation, expression of nlk decreases abruptly, a low level of mRNA is still detected at the prism stage, but expression is barely detectable at the pluteus stage.
nlk belongs to the Delta synexpression group
During development, nlk is expressed in a pattern that resembles
that of Delta, the Notch ligand. This similarity is first apparent at the
early blastula stage when, after an initial phase of ubiquitous expression
(Fig. 1B-D), nlk
transcripts become restricted to a ring of about 30 cells around the vegetal
pole that correspond to the precursors of the PMCs
(Fig. 1E-G). Delta is also
strongly expressed in the PMCs at this stage
(Fig. 1R,S)
(Sweet et al., 2002). As for
Delta, nlk expression remains elevated in the PMCs until they start
to ingress into the blastocoele but is no longer detected in these cells after
ingression is completed. At mesenchyme blastula stage, nlk and Delta
are both expressed in a domain located at the centre of the flattened vegetal
plate that, according to the fate map, corresponds to the presumptive SMCs
(Fig. 1H,I)
(Ruffins and Ettensohn,
1993).
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Overexpression of nlk or Delta causes the same phenotypes
To gain insight into the role of NLK, we overexpressed it by mRNA
microinjection into unfertilized eggs. Overexpression of nlk severely
perturbed patterning of the embryo (Fig.
3). The first visible defects were observed at the gastrula stage.
While in control embryos the gut had elongated and the PMCs had formed
bilateral clusters on each side of the oral ectoderm, in the injected embryos,
elongation of the archenteron was strongly delayed, the PMCs remained
disorganized, and no visible sign of oral aboral polarity was apparent
(Fig. 3A, part a). A second
remarkable phenotype was observed following overexpression of nlk
(although with high variability, from 10 to 90%, depending on the batch of
embryos). This phenotype was characterized by massive EMT, which started at
the vegetal pole and propagated to the rest of the embryo
(Fig. 3A, part d). This wave of
EMT resulted in the extrusion of an excess of mesenchymal cells at the vegetal
pole. These mesenchymal cells later accumulated a red pigment, and therefore
probably corresponded to pigment cells.
When the control embryos reached the pluteus stage, about half of the nlk-injected embryos had gastrulated but displayed a short and wide archenteron that remained straight (Fig. 3A, part f). Frequently, this archenteron was surrounded by multiple abnormal spicules, indicating that ectodermal patterning was perturbed and oral-aboral polarity was disrupted (Fig. 3A, part g). The other half of nlk-overexpressing embryos exogastrulated and also appeared radialized (Fig. 3A, part h). Their ectoderm consisted of a small hollow sphere containing abnormal spicules, and their digestive tract appeared enlarged. In addition, all the injected embryos were strongly pigmented, suggesting that overexpression of nlk had caused production of an excess of pigment cells.
This range of phenotypes, which comprises extrusion of pigment cells,
radialization, exogastrulation and overdevelopment of the endomesoderm, is
very reminiscent of the phenotypes caused by overexpression of an activated
form of Notch (Sherwood and McClay,
1999) or of Delta (Sweet et
al., 2002) (Fig.
3A, parts i-l). These observations provide further evidence that
NLK function is linked to the Notch/Delta signalling pathway.
nlk overexpression expands the mesoderm territory
To characterize the NLK overexpression phenotype we examined the expression
of several genes expressed in the main embryonic territories. We first
analysed the expression of the hatching enzyme gene (HE), which is a
marker of the presumptive ectoderm (Lepage
et al., 1992), and of brachyury, which is expressed
dynamically at the ectoderm-endoderm boundary
(Croce et al., 2001;
Gross and McClay, 2001). In
control embryos, the territory expressing HE covers about two-thirds
of the blastula (Fig. 3B, part
a). Overexpression of NLK caused this territory to shrink to one-third of the
embryo, indicating that the boundary between the ectoderm and endomesoderm had
been shifted toward the animal pole (Fig.
3B, part g). In agreement with this conclusion, the territory
expressing brachyury was dramatically displaced toward the animal
pole following overexpression of nlk
(Fig. 3B, parts b,h).
Conversely, the expression of goosecoid in the presumptive oral
ectoderm was abolished in these embryos, consistent with the suppression of
oral-aboral polarity and the absence of a stomodeum caused by ectopic
nlk mRNA (Fig. 3B,
parts c,i).
To examine the effects of NLK overexpression on mesoderm and endoderm, we analysed the expression of several novel markers that are specifically expressed in the these territories (see Materials and methods). bhmt is expressed in the presumptive midgut and hindgut, while bpnt and papss are expressed in the presumptive SMC territory, starting at mesenchyme blastula stage. In most of the nlk-injected embryos, the ring of cells expressing the endodermal marker bhmt was not particularly expanded but rather shifted toward the animal pole, suggesting that in these embryos, NLK had not caused formation of an excess of endoderm but had shifted the ectoderm-endoderm boundary (Fig. 3B, parts d,j). By contrast, expression of the mesodermal markers bpnt (Fig. 3B, parts e,f,k,l) and papss (data not shown) was dramatically expanded in nlk-injected embryos. Instead of a ring of cells surrounding the vegetal pole, the territory expressing these genes covered most of the vegetal plate and extended up to the equatorial region of the embryo. In addition, we confirmed that overexpression of Delta caused the same effects on mesodermal and endodermal markers as overexpression of nlk (data not shown; see Fig. 5).
On the basis of this molecular analysis, we conclude that overexpression of nlk, like overexpression of Delta, causes primarily an expansion of the mesoderm territory and a shift in the endoderm territory toward the animal pole, with a concomitant reduction of the ectodermal territory.
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).
This observation raised the possibility that ectopic expression of
nlk and/or Delta could activate ERK, which in turn would be
responsible for part of the observed phenotypes. In control embryos at
blastula stage, activation of ERK is restricted to the precursors of the PMCs
(Fig. 5U)
(Fernandez-Serra et al., 2004;
Rottinger et al., 2004).
However, following overexpression of nlk or Delta, ERK activation
could be detected in an enlarged area of the vegetal pole that encompassed the
region where the SMCs normally form (Fig.
5V,W). Most strikingly, in the embryos overexpressing nlk
and Delta, a strong activation of ERK was detected in all cells, foreshadowing
the massive conversion into mesenchymal cells observed at later stages
(Fig. 5X).
Partial redundancy during mesoderm formation between the MEK/ERK and the TAK1/NLK pathways
To determine the function of NLK, we used three different strategies. First
we constructed a kinase dead version of NLK (K78M), equivalent to the mouse
NLK (K155M), which was shown to be unable to block TCF binding to DNA and to
function as a dominant negative form
(Ishitani et al., 1999).
Second, we designed two antisense morpholino oligonucleotides directed against
nlk transcripts, one directed against the translational start and the
other further upstream in the 5'UTR. Finally, using a different
morpholino oligonucleotide, we attempted to block the translation of the mRNA
encoding TAK1, the kinase that is thought to be responsible for activating
NLK. Embryos microinjected with the antisense morpholino oligonucleotides
against nlk and/or tak1 transcripts or with mRNA encoding
the kinase dead mutant of NLK, always developed normally, even when very high
concentrations (1.5 mmol/l oligonucleotide and 1.5 mg/ml RNA) of these
reagents were used. A possible explanation for the absence of effect of these
reagents is that the activity of another factor may compensate for the loss of
NLK activity in these embryos. Because NLK is highly related to ERK, and
because ectopic activation of the MEK/ERK pathway causes the same phenotypes
as the overexpression of nlk, the kinase ERK itself appears a good
candidate for this redundant factor. To test this idea, we injected
nlk morpholinos (Mo-nlk) and treated half the embryos with
the MEK inhibitor U0126 to block ERK activation
(Fig. 6). Untreated
Mo-nlk-injected embryos developed normally, and the control embryos
treated with U0126 developed without PMCs and very few SMCs, as shown
previously (Fernandez-Serra et al.,
2004; Rottinger et al.,
2004). A surprising phenotype was observed following the double
knockdown of NLK and ERK. All these embryos failed to hatch and gastrulated
within the fertilization envelope, and their archenteron was very smooth,
suggesting that SMCs had failed to form
(Fig. 6D). In Mo-nlk-
injected embryos, the expression of gcm, which marks the pigment cell
territory (Ransick et al.,
2002), papss (Fig.
6I,O) and Delta (data not shown) was normal, while in the embryos
treated with U0126, the expression of these genes was reduced but clearly
detectable (Fig. 6H,N),
consistent with previous studies
(Fernandez-Serra et al., 2004).
By contrast, in the U0126-treated Mo-nlk-injected embryos, no
expression of papss, gcm or Delta could be detected, confirming that
specification of the SMCs had failed (Fig.
6J,P and data not shown). The same effects were observed after
simultaneous inhibition of tak1 mRNA translation and MEK activity,
including inhibition of hatching and complete loss of expression of gcm,
papss and Delta, consistent with the position of TAK1 upstream of NLK in
other systems. These results demonstrate that the TAK1/NLK pathway is partly
redundant with the MEK/ERK pathway during mesoderm formation in the sea urchin
embryo. They also show that while nlk overexpression results in
formation of an excess of mesoderm, knocking down its function causes a loss
of mesoderm, consistent with a positive requirement for NLK in mesoderm
formation.
Overexpression of NLK downregulates TCF
Taken together, the results presented so far indicate that, in the sea
urchin embryo, NLK is a target of Notch/Delta signalling and functions
together with ERK to promote formation of the mesoderm. However, previous
studies carried out on different organisms have shown that NLK, by
phosphorylating TCF and suppressing its DNA-binding activity, acts primarily
as an antagonist of the Wnt pathway. To examine whether overexpression of
nlk influences the transcriptional activity of TCF in the sea urchin
embryo, we performed a TCF reporter gene assay using the TOP FLASH construct
that contains multiple copies of a TCF-binding site upstream of the
luciferease coding sequence. This plasmid was injected into the eggs, either
alone or together with nlk mRNA, and stimulation of the Wnt pathway
was achieved by continuous treatment with lithium following fertilization. The
embryos were collected at the blastula stage and luciferase activity was
measured. When the embryos were injected with the reporter gene and treated
with lithium, a dramatic increase in the activation of the reporter gene was
observed compared with controls (Fig.
7B). When nlk was co-injected with the TOP FLASH plasmid,
it completely prevented the lithium-induced activation of the reporter gene.
Consistent with these results, we found that the sea urchin TCF sequence
contains a conserved SPGTP phosphorylation site within the central region of
the protein, which has been shown to be phosphorylated by NLK
(Fig. 7A)
(Ishitani et al., 2003b), and
that an NLK-GFP fusion protein localizes within the nucleus in the sea urchin
embryo (see Fig. S2 in the supplementary material).
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Gastrulation and maintenance of the endomesoderm gene regulatory network requires downregulation of TCF after hatching
The spatial expression pattern of nlk together with the results
from our functional analysis suggest that during normal development the role
of this kinase is to downregulate TCF in the mesodermal precursors at blastula
stages. If downregulating TCF before gastrulation is important for mesoderm
formation, then maintaining high activity of TCF would be predicted to
interfere with differentiation of PMCs and SMCs. To test this prediction, we
used a conditional, constitutively active version of TCF fused to the
transcriptional activator domain of the viral VP16 protein and to the
hormone-binding domain of the mouse glucocorticoid receptor (GR). Chimeric RNA
encoding this construct was injected in the egg, and the nuclear uptake of the
fusion protein was triggered at different stages by addition of dexamethasone
(Fig. 9A). In the absence of
dexamethasone, the embryos injected with this RNA developed normally. As
expected, when dexamethasone was added immediately after fertilization, the
injected embryos developed into extremely vegetalized larvae that expressed
bhmt up to the animal pole (Fig.
9C,G). Treatments with dexamethasone started at the early blastula
stage also caused a strong vegetalization, indicating that activation of the
Wnt pathway to a sufficiently high level can still change the fates of the
animal blastomeres towards endoderm after the cleavage period (data not
shown). When dexamethasone was added around the time of hatching, a marked
decrease in the degree of vegetalization was observed
(Fig. 9D). The embryos formed
primary and secondary mesenchymal cells, gastrulated and formed pigment cells.
In these embryos, the endodermal marker bhmt was overexpressed but
the SMC marker papss was expressed at normal levels
(Fig. 9H,Q). Finally, when
dexamethasone was added at the early mesenchyme blastula stage, the stage at
which zygotic expression of nlk peaks, a very different phenotype was
observed (Fig. 9E). Activation
of TCF at this stage instantaneously blocked migration of the PMCs and
prevented invagination of the archenteron. These embryos later formed a
rudimentary invagination and very few mesenchymal cells that remained
unpigmented. Molecular analysis revealed that expression of the endodermal
marker bhmt, of the PMC marker skeT and of the SMC marker
papss was strongly inhibited 12 hours after activation of the fusion
protein (Fig. 9I,N,R). These
results show that a high level of TCF activity at the mesenchyme blastula
stage severely interferes with development of the endomesoderm. These results
are therefore consistent with the idea that NLK, expressed in response to
Delta, functions as a negative feedback inhibitor of TCF/ß-catenin
signalling to allow segregation of the mesoderm
(Fig. 10).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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NLK and the maternal Wnt/ß-catenin pathway
Intriguingly, NLK was first characterized in Drosophila as a gene
required for planar polarity in the eye, a process that requires Delta/Notch
signalling between photoreceptors R3 and R4 downstream of frizzled
(Choi and Benzer, 1994). Mutant
alleles of Drosophila nlk were also isolated in the course of a
genetic screen for dominant modifiers of the activated Notch phenotype
(Verheyen et al., 1996).
However, a number of studies carried out in C. elegans and in
vertebrates have subsequently shown that NLK primarily acts as a modulator of
Wnt signalling (Ishitani et al.,
2003a; Ishitani et al.,
2003b; Ishitani et al.,
1999; Meneghini et al.,
1999; Thorpe and Moon,
2004). In line with these data, sequence analysis confirmed that
the sea urchin TCF contains a conserved putative MAP kinase phosphorylation
site in a region that has been shown to be phosphorylated by NLK. Further, the
TOP FLASH assays clearly showed that, like in vertebrates, NLK inhibits TCF.
Taken together, these results strongly suggest that the role of NLK as a
kinase that phosphorylates TCF and downregulates its activity is conserved in
the sea urchin. However, while in C. elegans and in vertebrates NLK
appears to act predominantly downstream or in parallel of the Wnt pathway, in
the sea urchin, the function of this kinase appears to have been recruited
downstream of the Notch/Delta signalling pathway.
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). So
why does overexpression of nlk in zebrafish or sea urchin embryos not
interfere with maternal Wnt/ß-catenin signalling? It should be kept in
mind that NLK is a MAP kinase that requires phosphorylation by an upstream MAP
kinase cascade to be activated and therefore that it is the activity of the
kinase that is important and not solely its presence. Thus, a possible
explanation for the lack of effect of overexpressed NLK on the maternal
TCF-ß-catenin pathway is that the activity of NLK is regulated spatially
or temporally. If this were the case, then overexpression of nlk
would not necessarily be predicted to interfere with the early activity of
TCF-ß-catenin but may only affect late patterning of the embryo, which is
what we observe.
Partial redundancy between NLK and ERK
We have shown previously that the strong activation of the MAP kinase ERK
that occurs transiently before gastrulation is largely restricted to the
presumptive PMC territory and that inhibition of this kinase suppresses
formation of the skeletogenic mesenchyme but only partially affects the
secondary mesenchymal cells (Rottinger et
al., 2004). The finding that another MAP kinase is expressed
specifically and at high levels in the SMC precursors raised the possibility
that NLK in the SMCs was playing a role similar to that played by ERK in the
PMCs. Surprisingly, inhibition of NLK function using dominant negative
approaches or with morpholino oligonucleotides failed to demonstrate a
requirement for this kinase in either the PMCs or SMCs. Based on the fact that
NLK and ERK are both members of the MAP kinase family, which recognize and
phosphorylate the same PXS/TP motifs, we then hypothesized that these two
kinases may play redundant roles in the SMCs. Indeed, when the function of
both kinases was inhibited, the SMCs failed to form, revealing the redundant
roles of these kinases. Interestingly, these embryos also failed to hatch.
This phenotype is reminiscent of the phenotype resulting from inhibition of
the transcription factor Ets4, which regulates the hatching enzyme gene
(Wei et al., 1999) and is a
potential target of MAP kinase (Rottinger
et al., 2004). This observation thus suggests that in addition to
its role in the endomesoderm, NLK may cooperate with ERK to regulate the
activity of ectodermal transcription factors such as Ets4.
The idea that the activities of ERK and NLK are partially redundant is
further supported by the phenotypes observed following activation of ERK.
Using an activated form of MEK, Fernandez-Serra et al.
(Fernandez-Serra et al., 2004)
have shown that activation of the MAP kinase pathway causes overproduction of
SMCs, which extrude from the vegetal pole, i.e. the nlk
overexpression phenotype. Because, NLK probably acts by phosphorylating TCF,
and because the specificities of ERK and NLK are largely overlapping, it would
be tempting to hypothesize that the phenotypes caused by overactivation of ERK
result from ERK phosphorylating TCF and downregulating its activity. However,
preliminary experiments performed to test this hypothesis indicate that ERK is
not able to downregulate TCF and therefore that ERK and NLK may promote
mesoderm formation by different mechanisms (T.L., unpublished). Therefore, the
relationships between NLK and ERK and between ERK and TCF are still
enigmatic.
Downregulation of the transcriptional activity of TCF and segregation of the mesoderm from the endomesoderm
In the sea urchin embryo, the endodermal and mesodermal precursors
originate from a common endomesodermal territory, which is initially specified
by activation of a maternal TCF/ß-catenin pathway at the vegetal pole. It
is thought that activation of this pathway is sustained during cleavage by the
expression of the Wnt8 ligand in this area. However, starting at the
hatching blastula stage, Wnt8 expression is progressively
downregulated in the endomesodermal territory and shifted towards the animal
pole so that at the mesenchyme blastula stage, wnt8 expression is
restricted to the ectoderm (Wikramanayake
et al., 2004) (E.R., J.C., G.L., L.B., C.G. and T.L.,
unpublished). Similarly, Logan et al.
(Logan et al., 1999) have
shown that up to hatching ß-catenin is present in the nucleus in the
precursors of the mesoderm and endoderm but that after hatching the level of
nuclear ß-catenin is downregulated in the precursors of the mesoderm
(PMCs and SMCs). Therefore, while the role of the Wnt/ß-catenin pathway
is crucial during the early phase of development to specify the endomesoderm,
the spatial expression pattern of Wnt8 and the pattern of nuclear
localization of ß-catenin suggest that this pathway has to be
downregulated in these cell types after hatching. The results presented in
this study agree with this idea. NLK has been shown to inhibit the
transcriptional activity of TCF and is expressed at the right place and at the
right time to participate to that downregulation at the premise of
gastrulation. Further, maintaining a high level of activity of TCF at the
beginning of gastrulation strongly interfered with the programme of
differentiation of the mesodermal cells.
Finally, the role of NLK in segregation of the mesoderm and endoderm in the
sea urchin is highly reminiscent of the role of this kinase in C.
elegans. In both species, segregation of the mesoderm and endoderm relies
on the TAK-1/NLK pathway to mediate inductive interactions between
blastomeres. However, while in C. elegans NLK appears to act
downstream or in parallel of Wnt signalling to promote endoderm development,
in the sea urchin NLK appears to act both downstream of Wnt signalling and in
cooperation with Delta signalling to promote mesoderm specification. Our
findings provide new insights to understand how the primary germ layers of the
sea urchin embryo are established and how the mesodermal and endodermal
precursors segregate from a bipotential endomesodermal territory, a process
that occurs during development of many organisms
(Rodaway and Patient,
2001).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/21/4341/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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