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First published online 14 May 2008
doi: 10.1242/dev.021543
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UMR7009 Laboratory of Developmental Biology, CNRS and Université Pierre et Marie Curie (Paris 6), Observatoire Océanologique, F-06234 Villefranche-sur-mer, France.
* Author for correspondence (e-mail: tsuyoshi.momose{at}obs-vlfr.fr)
Accepted 21 April 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Wnt pathway, Cnidaria, Localised RNA, Axis specification
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Logan and Nusse,
2004). It is well established that maternally driven activation of
this pathway on one side of the embryo is a key component of embryonic
patterning in a variety of deuterostome species (including sea urchins,
ascidians, amphibians and fish), in which it is associated with the
development of endomesoderm and/or `organiser' fates
(Heasman, 2006;
Huelsken and Birchmeier, 2001;
Imai et al., 2000;
Logan et al., 1999;
Schier and Talbot, 2005;
Wikramanayake et al., 1998).
More recently, equivalent mechanisms have been described in the basally
diverging phylum Cnidaria (Wikramanayake
et al., 2003) on the future `oral' side of embryos, which is
derived from the animal pole of the egg. This suggests that early canonical
Wnt pathway activation already operated to specify axial polarity and/or
endomesoderm fates in a common cnidarian/bilaterian ancestor or in an even
earlier metazoan (Lee et al.,
2006; Martindale,
2005). Regionalised β-catenin stabilisation in cleavage-stage
embryos has been visualised in two phylogenetically distant cnidarians, the
anthozoan (sea anemone) Nematostella vectensis and the hydrozoan
jellyfish Clytia hemisphaerica. In Clytia, analysis of
regionally expressed genes showed that β-catenin stabilisation specifies
the oral-aboral axis of the developing embryo by defining a presumptive oral
territory (Momose and Houliston,
2007). In both these species, the animal pole of the egg marks the
future gastrulation site from which endoderm forms, and later the oral pole of
the larva (see Fig. 1A):
inhibition of the canonical Wnt pathway blocks gastrulation, whereas its
forced activation favours endoderm formation
(Wikramanayake et al., 2003;
Momose and Houliston, 2007).
Wnt signalling has also been shown to be involved in maintaining oral-aboral
polarity in cnidarian adult stages, notably in Hydra polyps, in which
Wnt ligands, as well as the intracellular regulators TCF and β-catenin,
are expressed around the hypostome (oral opening) and contribute to the `head
organiser' properties of this region along with other genes implicated in
vertebrate organiser function (Bode,
2003; Broun et al.,
2005; Hobmayer et al.,
2000).
Much effort has been spent in trying to identify localised maternal factors
that drive canonical Wnt activation in the early embryo in different
experimental species, without a clear picture emerging. In sea urchins, the
cytoplasmic regulator Dishevelled (Dsh) localises dynamically to a vegetal
cortical layer of the egg, and has been shown to mediate Wnt signalling
activation in this region of the embryos; however, its global overexpression
does not affect germ layer specification, implying the involvement of
additional localised factors that restrict its activity
(Leonard and Ettensohn, 2007;
Weitzel et al., 2004). Dsh
protein is likewise maternally localised and necessary for subsequent
patterning events in Nematostella
(Lee et al., 2007), in which
the absence of detectable maternal expression of any of the genomic Wnt
repertoire (Lee et al., 2006)
has led to the suggestion that Dsh protein might play a primary directive
role. In Xenopus embryos, in which β-catenin stabilisation is
favoured on the prospective `organiser' side of the cleavage-stage embryo,
corresponding enrichments have been reported not only for Dsh but also for
β-catenin, whereas an inhibitor of the regulatory kinase GSK3-β
moves in the appropriate direction in the fertilised egg. All of these
molecules can force organiser development upon overexpression
(Larabell et al., 1997;
Miller et al., 1999;
Weaver et al., 2003). These
various examples of localised intracellular canonical Wnt pathway regulators
in the absence of detectable localised ligand have encouraged the idea that
cytoplasmic determinants can bypass the usual extracellular phase of
signalling in the single large cell of the egg. This view was challenged by
the discovery in Xenopus of a key role for the ligand Xwnt11
(Heasman, 2006;
Tao et al., 2005).
Xwnt11 RNA is tightly localised to the vegetal cortex of the oocyte,
and becomes enriched on the future dorsal side in cleavage-stage embryos.
Depletion of the Xwnt11 transcript from oocytes prevents organiser
formation, whereas overexpression promotes it. Localised Wnt pathway
activation by Xwnt11 could contribute, at least in part, to the observed
asymmetries of Dsh and β-catenin proteins in the early embryo, by
canonical Wnt pathway-directed stabilisation and/or by recruitment to the
membrane by the Wnt-planar cell polarity (PCP) pathway
(Axelrod et al., 1998). In
ascidians, in which neither Dsh nor β-catenin protein is localised
maternally, the maternally localised RNA Wnt5a provides a good
candidate for directing vegetal-specific nuclear localisation of
β-catenin, but the consequences of morpholino-mediated inhibition of
Wnt5a translation were inconclusive (Kawai
et al., 2007).
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),
but it was not clear whether a maternal ligand was required upstream
(Lee et al., 2007). We have
now identified five embryonically expressed Wnt ligands in Clytia,
including one, CheWnt3, the maternally expressed RNA of which is tightly
localised to the animal cortex of the egg. We show using morpholino antisense
oligonucleotides that CheWnt3 is necessary for maternally driven canonical Wnt
pathway activation on the future oral side of the embryo. Morpholino- and
RNA-injection experiments further show that CheWnt3, CheFz1 and CheFz1 then
cooperate as part of a dynamic system that defines opposite territories of
oral and aboral identity during subsequent development. |
MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Momose and Houliston, 2007),
except that β-catenin-Venus localisation in early gastrula stage was
visualised using a Leica SP2 confocal microscope.
cDNA cloning
Seven Wnt sequences and an Axin sequence were identified from a
Clytia expressed sequence tag (EST) collection, and full-length cDNA
clones retrieved from the corresponding cDNA library
(Chevalier et al., 2006). A
Clytia GSK3 (CheGSK3) cDNA fragment was amplified by PCR using a set
of degenerate primers as described previously
(Hobmayer et al., 2000), and
used to retrieve a full-length CheGSK3 cDNA clone by screening a
Clytia embryonic-stage phage library.
GenBank accession numbers: CheAxin (EU374716), CheGSK3 (EU374723), CheWntX1A (EU374720), CheWntX1B (EU374722), CheWntX2 (EU374717), CheWntX3 (EU391658), CheWnt3 (EU374721), CheWnt5 (EU374718), CheWnt9 (EU374719).
Morpholino and RNA injection
The Wnt3 coding sequence was amplified by PCR from the EST-identified
Express1 clone using primers that introduced five silent mutations in the
target sequence of Wnt3-MO and was subcloned into the pRN3 vector (P. Lemaire,
Marseille). The coding sequence of CheGSK3 was amplified by PCR and cloned
into pRN3 to form pRN3-CheGSK3. The K85R mutation in the kinase domain
(Pierce and Kimelman, 1995)
was introduced into the plasmid to produce a dominant-negative (dn) form of
GSK3 (pRN3-dnGSK3) using the QuickChange Site-directed Mutagenesis Kit
(Stratagene).
mRNAs encoding Wnt3 and dnGSK3 were in vitro transcribed from the plasmids using the mMessage mMachine Kit (Ambion). The Wnt3 RNA was introduced into eggs prior to fertilisation at 0.4 µg/µL, or into single 8-cell stage blastomeres of Wnt3-MO-injected embryos. The RNA encoding dnGSK3 was injected into unfertilised eggs at 2 µg/µL. FITC-labelled dextran was co-injected with Wnt3 RNA to identify progeny of the injected blastomeres.
Antisense morpholino oligonucleotide (GeneTools) for CheWnt3 (Wnt3-MO,
5'-CCAAAACACACCAGTGTCGAGCCAT-3') was microinjected at 0.1 µM
prior to fertilisation. The in vivo target specificity of the Wnt3-MO-mediated
inhibition was confirmed by the opposite phenotype being obtained upon
injection of Wnt3 RNA, and oral pole rescue upon localised injection
of Wnt3 RNA into Wnt3-MO-injected embryos. Other morpholino oligos
and RNAs were used as described previously
(Momose and Houliston,
2007).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Of the seven Clytia Wnt genes identified, expression of five was
detected in embryonic or larval stages by in situ hybridisation: CheWnt3,
CheWnt5, CheWnt9, CheWntX1A and CheWntX2 (see Fig. S2 in the
supplementary material; see also Fig.
2I). Unlike in the anthozoan Nematostella, in which no
expression of the genomic Wnt repertoire is detected before gastrulation
(Lee et al., 2006), one
Clytia Wnt RNA, CheWnt3, was detectable in eggs and
cleavage-stage embryos (see below). Following gastrulation, the five
detectable Clytia Wnt ligand RNAs defined overlapping domains of the
oral ectoderm (see Fig. S2 in the supplementary material). There was no clear
correspondence between these domains and those described for the
Nematostella Wnts; notably, the five Clytia Wnts were
detected only in the ectoderm, whereas a subset of Nematostella Wnt
orthologues (including the CheWnt5 orthologue) is uniquely
endodermal. Furthermore, CheWnt9, expressed at the oral tip of the
Clytia larva, has no orthologue in Nematostella; indeed, the
Wnt9 subfamily previously lacked a cnidarian member
(Guder et al., 2006a;
Kusserow et al., 2005). These
findings demonstrate that the sequences and expression domains of the Wnt
genes expressed along the oral-aboral axis in cnidarian embryos have diverged
extensively within the cnidarian clade from a set of genes already present in
the eumetazoan common ancestor.
CheWnt3: a maternal RNA localised to the animal cortex
CheWnt3 RNA was the only Wnt RNA detectable in the Clytia
egg. It showed a highly localised distribution both before and after
fertilisation, with tight restriction to a cortical layer of the animal
hemisphere (Fig. 1B). This
distribution is distinct from that of two previously described localised
maternal Clytia RNAs, CheFz1 (which forms a cytoplasmic
gradient from animal to vegetal pole) and CheFz3 (localised to the
vegetal cortex) (Momose and Houliston,
2007). The maternal localisation of the CheWnt3 RNA
resembles that reported for HeWnt, a Wnt3 orthologue
identified in Hydractinia
(Plickert et al., 2006).
CheWnt3 RNA remained tightly localised to the animal cortex during
cleavage divisions, until at least the 64-cell stage. It accumulated in the
cleavage furrow at each division, becoming progressively more concentrated
around the animal pole. Thus, CheWnt3 RNA was confined to a small
patch inherited by three to four animal blastomeres at the 8-cell stage, and
about 8 cells at the 64-cell stage. By the mid-blastula stage, it was detected
as one or more small but strongly stained patches in a restricted area at the
future oral end, presumed to be maternal RNA inherited from the egg animal
pole. From this stage onwards, an additional diffuse in situ hybridisation
signal was detectable in a wider oral region, becoming progressively stronger
during blastula development. This signal was considered to correspond to the
onset of zygotic expression because of its clear distinction from that of
residual maternal RNA, the timing coinciding with the onset of transcription
of a number of other developmental genes in Clytia (our unpublished
observations). CheWnt3 expression restricted to the oral ectoderm was
maintained through gastrulation and in planula larvae
(Fig. 1B and see Fig. S2 in the
supplementary material). At the early gastrula stage, expression within this
domain appeared slightly reduced at the oral pole region, from where cell
ingression was starting.
CheWnt3 is essential for axial polarity
We tested the function of CheWnt3 by injection of a specific morpholino
antisense oligonucleotide (Wnt3-MO) into eggs prior to fertilisation to block
translation. The use of morpholinos in Clytia was validated in the
previous study of CheFz1 and CheFz3
(Momose and Houliston, 2007),
and in the current study the specificity of Wnt3-MO was confirmed by
comparison of the phenotypes with those obtained by Wnt3 RNA
overexpression, and by the reversal of Wnt3-MO phenotypes by local
introduction of the Wnt3 RNA (see below). Eggs microinjected with
Wnt3-MO underwent regular cleavage divisions but developed without
morphological polarity (Fig.
2A). They neither elongated along the oral-aboral axis, nor showed
any sign of presumptive endoderm ingression from the oral end during the
normal gastrulation period (10-18 hpf at 18°C). The developing embryos
remained completely spherical for at least 2 days. In contrast to CheFz1-MO
embryos, in which cell ingression leading to endoderm formation was
permanently blocked, Wnt3-MO-injected embryos eventually formed
morphologically normal endoderm by a multipolar `recovery' mechanism starting
at the late gastrula/early planula stage (
18-20 hpf)
(Fig. 2B,C). In addition, the
ectoderm of CheWnt3-MO-injected embryos formed highly motile cilia and embryos
swum vigorously, albeit without the normal aboral-oral directionality. This
contrasts with the defective cilia formation and orientation in
CheFz1-MO-injected embryos, a phenotype associated with disruption of PCP
(Momose and Houliston, 2007).
We conclude that the ligand CheWnt3 is required together with the Frizzled
receptors for development of the oral-aboral axis, although, unlike CheFz1, it
might not be essential in the acquisition cellular polarity.
To characterise further the role of CheWnt3 in establishing cell fate along
the oral-aboral axis, we examined the effect of CheWnt3-MO injection on
regional gene expression (Fig.
2D-H). Oral ectoderm expression of the Brachyury
orthologue CheBra was almost completely lost at the early gastrula
stage (Fig. 2D) and was no
longer detectable in planulae (Fig.
2E). Another oral marker, CheAxin, a component of the
canonical Wnt signalling pathway that is expressed in oral endoderm and
ectoderm, was also abolished in planulae by CheWnt3 knockdown
(Fig. 2F). Conversely, the
expression domain of the aboral marker FoxQ2A
(Chevalier et al., 2006)
expanded to cover nearly the entire embryo at early gastrula and planula
stages, with just a small region of low expression remaining
(Fig. 2G,H). Expression of the
four orally expressed zygotic Wnt genes (CheWntX2, CheWnt9, CheWntX1A
and CheWnt5) was also undetectable in Wnt3-Mo embryos at the planula
stage (Fig. 2I), indicating
that they too are dependent on prior expression of CheWnt3. It thus appears
unlikely that these later-expressed Wnt ligands are responsible for the
delayed endoderm formation observed in the absence of the early-expressed
ligand Wnt3.
Taken together, the morpholino experiments show that the ligand CheWnt3 is essential for the development of axial polarity in Clytia. CheWnt3 functions to promote the development of oral fates at the expense of aboral fates.
CheWnt3 is required for early canonical Wnt pathway activation
Regionalised activation of canonical Wnt signalling at as early as the
32-cell stage (3-3.5 hpf) has been demonstrated in Clytia and in
Nematostella, largely preceding morphological and cytological
manifestation of axial properties (12-15 hpf). To address whether maternal
CheWnt3 participates with CheFz1 and CheFz3 in establishing this initial
β-catenin-stabilisation domain, we monitored canonical Wnt signalling
activation using a β-catenin-Venus (modified GFP with yellow
fluorescence) fusion protein (Momose and
Houliston, 2007). At the early- and mid-blastula stages, embryos
injected with RNA encoding β-catenin-Venus alone showed a restricted
domain of stabilised nuclear and cytoplasmic β-catenin-Venus protein on
one side (Fig. 3A). This was
almost completely undetectable following co-injection of Wnt3-MO
(Fig. 3B), clearly indicating
that the ligand CheWnt3, translated from maternal RNA, is necessary for early
regionalised activation of the canonical Wnt pathway.
To determine the contribution of animally localised CheWnt3 RNA to the spatial restriction of canonical Wnt pathway activation, we injected synthetic CheWnt3 mRNA along with the RNA encoding β-catenin-Venus prior to fertilisation to achieve global expression across all regions of the embryo (Fig. 3C). The exogenous CheWnt3 did not prevent formation of a sharply demarcated β-catenin-stabilisation domain at the early blastula stage (4.5 hpf). The only discernable effect of CheWnt3 RNA at this stage was a slightly elevated nuclear β-catenin-Venus signal observed in the aboral domain of RNA-injected embryos (Fig. 3C). During subsequent development (6.5-9 hpf), however, the domain of β-catenin stabilisation spread progressively across the embryo, such that by the onset of gastrulation, nuclear β-catenin was detectable in nearly all cells (Fig. 3C). In the absence of injected Wnt3 RNA, nuclear β-catenin at this stage had become tightly restricted to the small group of inwardly migrating presumptive endoderm cells at the gastrulation site (Fig. 3A). The initial failure of injected CheWnt3 RNA to cause strong Wnt pathway activation in aboral regions is unlikely to be due to slow translation, as β-catenin-Venus protein from co-injected RNA could be detected at least 1 hour earlier.
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CheWnt3 is required for the cross-regulation between CheFz1 and CheFz3
We showed previously that strong reciprocal downregulation mechanisms
between CheFz1 and CheFz3 involving unknown additional components are
responsible for maintaining and sharpening the opposite localisation of these
RNAs by the early gastrula stage (Momose
and Houliston, 2007). Now we were able to show that CheWnt3 is an
essential component of this regulatory system. MO-mediated inhibition of
CheWnt3 translation was found to cause a dramatic increase in the levels of
both CheFz1 and CheFz3 transcripts by the early gastrula
stage (Fig. 4A), mimicking the
phenotype of double knockdown of CheFz1 and CheFz3. There was no detectable
effect on CheFz1 or CheFz3 RNA distribution at the early
blastula stage (4.5 hpf; Fig.
4A), indicating that the regulatory interactions between these
molecules operate during the blastula-early gastrula period, and probably
involve zygotic transcription.
To test the participation of CheWnt3 expression itself in the feedback system for canonical signalling regulation involving the two Frizzled receptors, we examined its response to manipulating different regulatory components (Fig. 4B). Somewhat surprisingly, neither forced global activation of the canonical Wnt pathway (by injection of an RNA encoding a dominant-negative form of the regulatory kinase GSK3-β, or by MO-mediated inhibition of CheFz3), or global β-catenin degradation (by MO-mediated inhibition of CheFz1), had a significant effect on the extent of the oral CheWnt3 expression domain, despite the severe loss-of-polarity phenotypes observed following these treatments. These results indicate that zygotic CheWnt3 transcription is not regulated principally by the canonical Wnt pathway, and imply the existence of Frizzled- and β-catenin-independent pattern regulation mechanisms operating along the oral-aboral axis of the developing embryo.
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To test more precisely the oralising capacity of CheWnt3 and to provide confirmation of the specificity of the Wnt3-MO phenotype, we injected CheWnt3 RNA into one blastomere of 8-cell stage embryos derived from Wnt3-MO-injected eggs (Fig. 6). Since the injected blastomeres were chosen randomly, they were equally likely to be derived from animal or vegetal halves. The ectopically expressed Wnt3 restored characteristic pointed oral poles to the spherical Wnt3-MO embryos in all cases (n=15), with the introduced RNA and co-injected fluorescent dextran always positioned at the oral pole. Cells descended from the Wnt3 RNA-injected blastomere showed shape changes typical of presumptive endoderm cells: apical constriction, basal migration of the nucleus and inward migration (Fig. 6C). These shape changes were also observed in neighbouring CheWnt3-RNA-negative cells (Fig. 6C, asterisks). Thus, CheWnt3 can act at short range in a paracrine manner to direct neighbouring cells to an oral fate. The resultant embryos showed limited elongation along a new axis defined by the position of RNA injection, although Wnt3-MO-containing cells distant from the RNA injection site were not affected. Thus, CheWnt3 protein is able to confer an oral/presumptive endoderm fate at any position in Wnt3-MO embryos, as well as in Wnt3-overexpressing wild-type embryos (see above). We further tested oral- and aboral-specific gene expression in Wnt3-MO embryos `rescued' by Wnt3 RNA injection at the 4-cell stage (Fig. 6D). The CheBra expression abolished by Wnt3-MO was recovered at the pointed end of RNA-injected embryo (n=15, 87%). Conversely, the expanded CheFoxQ2a expression domain became restricted to the side opposite the newly created oral pole (n=18, 78%).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Riechmann and Ephrussi, 2001).
In deuterostomes, the Wnt signalling pathway is commonly involved in
maternally directed steps of pattern formation (see Introduction), and the
discovery that cnidarian embryos share this feature suggests that it might be
evolutionarily ancient (Wikramanayake et
al., 2003). We have shown in Clytia that a Wnt ligand as
well as Frizzled family receptors are crucially involved in axis determination
via localised maternal mRNAs, and also in a dynamic regulatory system that
maintains and refines the developing oral-aboral axis.
A maternal Wnt ligand required for axial patterning in Clytia
Only one of the five Wnt ligands found to be expressed during
Clytia embryogenesis, CheWnt3, was detectable as maternal RNA.
Morpholino-mediated inhibition of CheWnt3 translation completely blocked
canonical Wnt pathway activation and the development of oral-aboral polarity.
After Xenopus Xwnt11, CheWnt3 is only the second example of a Wnt
ligand conclusively shown to be required maternally to initiate embryonic
patterning.
Our model for the initial phase of axis specification in Clytia
(Fig. 7A) is that it results
from the combined effects of the ligand CheWnt3 and two receptors, all
translated from maternal RNA. CheWnt3 protein almost certainly acts directly
as a ligand for the classic receptor CheFz1 to activate the canonical pathway
in the animal half of the early embryo. The negatively acting Frizzled,
CheFz3, plays an important role in establishing a boundary of β-catenin
stabilisation by preventing canonical pathway activation in the vegetal half
of the embryo, apparently by having a `dominant-negative' effect
(Momose and Houliston, 2007).
By contrast, although CheWnt3 RNA shows tight animal localisation
until the 64- to 128-cell stage, and is absolutely required upstream of CheFz1
for canonical Wnt pathway activation at this time, its role in the initial
spatial restriction of activation may be relatively minor. Thus, whereas
Wnt3-MO completely abolished canonical signalling, ubiquitous CheWnt3
overexpression by RNA injection into the egg had little effect on the initial
β-catenin nuclear localisation pattern. Indeed, it appears likely that
the endogenous distribution of CheWnt3 protein, although being asymmetric
across the embryo, is more widespread than that of its RNA, as β-catenin
stabilisation can be promoted throughout the early embryo following knockdown
of CheFz3 function (Momose and Houliston,
2007). A broad CheWnt3 protein distribution could be generated
from tightly localised RNA by rapid translation within the egg following
fertilisation, and from subsequent intracellular and extracellular diffusion.
Based on these considerations we propose that canonical Wnt signalling is
prevented in the future aboral half of cleavage-stage embryos by a combination
of reduced ligand availability and dampening of the cellular response by
CheFz3.
Two phases of Wnt-dependent axis formation in Clytia
In situ analyses revealed two phases of CheWnt3 expression. Maternally
deposited RNA is localised to the animal cortex and is inherited by a small
group of animal blastomeres during cleavage stages. Zygotic expression is
first detectable at the mid-blastula stage, overlapping with the maternal
expression. The transition from maternal to zygotic CheWnt3
transcription coincides with a change in its role from being a necessary but
non-directive factor in early β-catenin activation, to defining the oral
pole within a regulative oral-aboral patterning system operating in blastula
and early gastrula stages (Fig.
7B). The importance of this second role was dramatically
demonstrated by the strong oralised phenotype upon CheWnt3 RNA
injection, despite the initial refractiveness of the
β-catenin-stabilisation domain to this treatment, and by the ability of
ectopic Wnt3 mRNA injection to restore a morphologically normal oral
pole to morpholino-injected embryos. There is an interesting similarity
between the two phases of Wnt-dependent signalling in Clytia and
axial patterning in vertebrates. In vertebrates, the classic `dorsal
organiser' signalling centres responsible for anterior-posterior patterning
are also established within previously established domains of canonical Wnt
pathway activation, specified maternally in the case of amphibian and fish and
by Wnt-ligand-dependent self-organising mechanisms in mouse and chick (see
Meinhardt, 2006;
Stern et al., 2006).
Wnt-based feedback regulatory systems are well placed to account for the
well-characterised regulative abilities of hydrozoans, including the blastulae
of Clytia and Podocoryne
(Bode, 2003). Axis regulation
can be explained by the presence of a `reaction-diffusion' system
(Meinhardt, 2008), in which
the role of a locally produced activator could be filled by Wnts expressed at
the oral pole, and that of fast-diffusing inhibitors perhaps by antagonists of
the sFRP and Dickkopf families, gene sequences for which are found in the
Nematostella and Hydra genomes
(Guder et al., 2006a;
Guder et al., 2006b;
Lee et al., 2006) and in our
Clytia EST collections. Consistent with this hypothesis,
Hydra polyps rapidly upregulate the expression of Wnt3 and of
downstream activators in the `head organiser' (hypostome) region during
budding and regeneration to define oral fate
(Broun et al., 2005;
Hobmayer et al., 2000). The
axial regulatory system in the Clytia blastula remains largely
uncharacterised; however, negative regulation between CheFz1 and CheFz3 to
define mutually exclusive oral and aboral domains is clearly an important
component. Massive RNA accumulation of each receptor is observed when
translation of the other is prevented
(Momose and Houliston, 2007),
a reciprocal regulation now shown to require CheWnt3. The oral downregulation
of CheFz3 is most easily explained by negative regulation by
canonical Wnt signalling, because lithium treatment, which upregulates the
canonical pathway, clearly reduces CheFz3 RNA levels. By contrast,
aboral downregulation of CheFz1 might involve a non-canonical
mechanism, as LiCl treatment does not have a strong effect on CheFz1
RNA levels (Momose and Houliston,
2007). The dependence of CheFz1 downregulation on Wnt3
could involve direct interaction of the ligand with CheFz3 in the aboral
region, or be effected indirectly, for instance via factors secreted from the
oral region. Signalling systems other than the canonical Wnt pathway clearly
operate during oral-aboral patterning in the Clytia embryo because
zygotic Wnt3 transcription was initiated correctly when the canonical
Wnt pathway was blocked.
Evolution of axis determination
The requirement for maternal Wnt ligands in both Xenopus and
Clytia strongly suggests that the entire ligand receptor-cytoplasmic
signalling module was used to regionalise the embryo in the common ancestor of
bilatarians and cnidarians. One attractive scenario is that an intercellular
Wnt signalling system originally functioned within groups of equivalent cells
to create stable asymmetries of canonical activity. There is evidence for
systems with such properties in reconstituted cell aggregates from
Hydra, which can be accounted for theoretically using the
reaction-diffusion system evoked to explain pattern regulation
(Gierer et al., 1972;
Meinhardt, 2008;
Technau et al., 2000). An
alternative possibility is that Wnt ligand-Fz receptor signalling between the
oocyte and somatic cells in the gonad was used ancestrally in directing the
acquisition of oocyte polarity during oogenesis.
From an ancestral usage of the whole Wnt pathway, maternal concentrations
of one or several regulatory components might have been adopted during
evolution in different species to provide a reliable maternal cue. In
Clytia, at least three localised maternally coded Wnt pathway
regulators, comprising one ligand and two antagonistically acting receptors,
exhibit localised distributions. All three are required for proper axis
formation and are separately localised in the eggs and early embryos.
Additional localised regulators might also contribute: in
Hydractinia, RNA for the transcriptional regulator Tcf is localised
along with that of a Wnt3 orthologue in the egg
(Plickert et al., 2006).
Furthermore, we have found animal pole localisation of a Clytia Dsh
RNA in the egg, localisation of Dsh protein being detectable from the 8-cell
stage (T.M., unpublished). The parallel localisation of more than one maternal
factor might help to ensure a robust mechanism to establish a reliable Wnt
activity asymmetry in the face of intrinsic developmental variability and
extrinsic disturbances. In Xenopus, Dsh, GBP and β-catenin
proteins as well as Xwnt11 RNA show localised maternal distributions
(Heasman, 2006). In
Nematostella, requirement for the ligand might have been lost or
diminished, with Dsh protein localisation assuming a more important role
(Lee et al., 2007;
Lee et al., 2006). In sea
urchin, maternal Dsh concentrations also appear to be important, although
additional localised factors are required to account for vegetally restricted
β-catenin nuclear localisation
(Leonard and Ettensohn, 2007;
Logan et al., 1999).
To conclude, localised maternal Wnt ligands contributing to axial
patterning have been clearly demonstrated in Xenopus and
Clytia, and cannot be discounted in other models in which
intracellular regulators may play dominant roles to localise Wnt pathway
activation. Different determination factors might have been favoured during
evolution, depending on egg structure and reproduction pattern. The absence of
maternal Wnt3 but not of Frizzled in Hydra
(Frobius et al., 2003), whereas
both elements are present maternally in Hydractinia
(Plickert et al., 2006;
Teo et al., 2006) and
Clytia, could represent an example of ongoing developmental
flexibility within the hydrozoan clade associated with very different
strategies for early development. Currently, insufficient information is
available to support clear evolutionary scenarios, but our study suggests that
ligand-dependent Wnt signalling was already available in a eumetazoan common
ancestor to determine the body axis. A recent report of polarised zygotic Wnt
expression in a sponge embryo pushes this usage back even further in evolution
(Adamska et al., 2007). It will
be of great interest to re-examine the involvement of Wnt ligands in canonical
Wnt signalling activation and axis determination in other deuterostome,
lophotrochozoan and cnidarian models.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/12/2105/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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