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First published online 20 February 2008
doi: 10.1242/dev.020289
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Research Report |
1 Departament de Genètica, Universitat de Barcelona, 08028 Barcelona,
Spain.
2 Centro Andaluz de Biología del Desarrollo, Universidad Pablo de
Olavide-CSIC, 41013 Sevilla, Spain.
* Author for correspondence (e-mail: tadellc{at}ub.edu)
Accepted 4 February 2008
SUMMARY
Little is known about the molecular mechanisms responsible for axis establishment during non-embryonic processes such as regeneration and homeostasis. To address this issue, we set out to analyze the role of the canonical Wnt pathway in planarians, flatworms renowned for their extraordinary morphological plasticity. Canonical Wnt signalling is an evolutionarily conserved mechanism to confer polarity during embryonic development, specifying the anteroposterior (AP) axis in most bilaterians and the dorsoventral (DV) axis in early vertebrate embryos. β-Catenin is a key element in this pathway, although it is a bifunctional protein that is also involved in cell-cell adhesion. Here, we report the characterization of two β-catenin homologs from Schmidtea mediterranea (Smed-βcatenin1/2). Loss of function of Smed-βcatenin1, but not Smed-βcatenin2, in both regenerating and intact planarians, generates radial-like hypercephalized planarians in which the AP axis disappears but the DV axis remains unaffected, representing a unique example of a striking body symmetry transformation. The radial-like hypercephalized phenotype demonstrates the requirement for Smed-βcatenin1 in AP axis re-establishment and maintenance, and supports a conserved role for canonical Wnt signalling in AP axis specification, whereas the role of β-catenin in DV axis establishment would be a vertebrate innovation. When considered alongside the protein domains present in each S. mediterranea β-catenin and the results of functional assays in Xenopus embryos demonstrating nuclear accumulation and axis induction with Smed-βcatenin1, but not Smed-βcatenin2, these data suggest that S. mediterranea β-catenins could be functionally specialized and that only Smed-βcatenin1 is involved in Wnt signalling.
Key words: β-catenin, Planarians, Anteroposterior axis, Regeneration
INTRODUCTION
Planarians show a striking morphological plasticity that becomes evident
during regeneration and normal tissue homeostasis. They are able to regenerate
a whole organism from a piece of almost any part of their body, and,
furthermore, they have the ability to grow and degrow according to culture
conditions (Morgan, 1898
;
Saló, 2006). These
properties rely on the neoblasts, multipotent stem cells present in adult
organisms that are able to differentiate into any planarian cell type
(Saló, 2006).
The canonical Wnt signalling pathway has a common role in establishing the
anteroposterior (AP) axis during development in several species, including
mouse (Marikawa, 2006), chick
(Nordstrom et al., 2002),
zebrafish (Schier and Talbot,
2005), Xenopus
(Kiecker and Niehrs, 2001),
amphioxus (Holland, 2002),
C. elegans (Huang et al.,
2007) and Platynereis
(Schneider and Bowerman,
2007). In early vertebrate embryos it is also required for
dorsoventral (DV) polarity (De Robertis and
Kuroda, 2004). In cnidarians, it specifies the oral-aboral
embryonic axis, and it also has a reported role in axial patterning during
regeneration (Lee et al.,
2006). However, in classical models of regeneration, such as fish
or amphibians, canonical Wnt signalling has only been demonstrated to be
involved in the regenerative capacity
(Kawakami et al., 2006;
Yokoyama et al., 2007).
β-Catenin is the key intracellular effector of the canonical Wnt
signalling pathway, although it is a bi-functional protein that also regulates
cell adhesion as a component of adherens junctions
(Schneider et al., 2003).
Here, we report the characterization of two β-catenin homologs in the planarian species Schmidtea mediterranea (Smed-βcatenin1 and Smed-βcatenin2). Silencing of Smed-βcatenin1 in regenerating and intact planarians induces a gradual anteriorization of the animals that finally leads to a radial-like hypercephalized phenotype, demonstrating the requirement for Smed-βcatenin1 in AP axis re-establishment and maintenance. Analysis of the protein domains of the S. mediterranea β-catenins, and functional assays using Xenopus embryos, demonstrate the involvement of Smed-βcatenin1 but not Smed-βcatenin2 in Wnt signalling, suggesting a functional specialization of S. mediterranea β-catenins.
MATERIALS AND METHODS
Organisms
The planarians used belong to an asexual race of S. mediterranea
collected from Montjuïc, Barcelona, Spain, and maintained as described
elsewhere (Molina et al.,
2007).
Identification and cloning of S. mediterranea genes
Fragments of Smed-βcatenin1 and
Smed-βcatenin2 were identified from the S.
mediterranea genomic database through a BLAST search. The corresponding
full-length transcripts were amplified by rapid amplification of cDNA ends
(RACE) using the Invitrogen GeneRacer Kit (Invitrogen). Smed-HoxD,
Smed-AbdBa and Smed-TCEN49 were identified from the S.
mediterranea genomic database using homologs from other planarian species
(Orii et al., 1999;
Garcia-Fernandez et al., 1993;
Nogi and Watanabe, 2001;
Bueno et al., 1996). Specific
primers were designed to isolate the corresponding full-length cDNA
sequences.
Accession numbers
Smed-TCEN49, EU082822; Smed-AbdBa, EU082823;
Smed-HoxD, EU082824; Smed-βcatenin1, EU082826;
Smed-βcatenin2, EU082825.
RNAi silencing
RNAi analyses were performed by feeding planarians with bacteria expressing
double-stranded RNA (dsRNA) or by dsRNA microinjection, as described by
Newmark et al. (Newmark et al.,
2003) and Sánchez Alvarado and Newmark
(Sánchez Alvarado and Newmark,
1999), respectively. The corresponding cDNA for
Smed-βcatenin1 and
Smed-βcatenin2 subcloned into the pPR242 vector was
used for feeding. Control animals were fed bacteria containing the vector
alone. When dsRNA microinjection was used,
Smed-βcatenin1 and
Smed-βcatenin2 dsRNA was synthesized by in vitro
transcription (Roche). Control animals were injected with water. For
regeneration experiments, treated planarians were amputated pre- and
postpharyngeally, and the trunk pieces allowed to regenerate. Planarians were
processed for whole-mount in situ hybridization or whole-mount immunostaining
between 3 and 30 days after amputation or last injection (intact animals).
Whole-mount in situ hybridization
Whole-mount in situ hybridization was carried out essentially as described
previously (Nogi and Levin,
2005; Umesono et al.,
1999). Digoxigenin-labelled riboprobes for
Smed-βcatenin1, Smed-βcatenin2, Smed-HoxD,
Smed-AbdBa, Smed-TCEN49, Smed-Opsin (K. Eckelt), H.10.2f
(Sánchez Alvarado et al.,
2002), cintillo
(Oviedo et al., 2003),
Smed-GluR (F. Cebrià and P. Newmark), septin
(Zayas et al., 2005) and
eye53 (Zayas et al.,
2005; Molina et al.,
2007) were synthesized using an in vitro transcription kit
(Roche).
Whole-mount immunostaining
Immunostaining was carried out essentially as described previously
(Cebrià and Newmark,
2005; Sánchez Alvarado
and Newmark, 1999). The following monoclonal antibodies were used:
anti-arrestin (VC-1) (Sakai et al.,
2000) at a 1:15,000 dilution; anti-synapsin (anti-SYNORF1,
Developmental Studies Hybridoma Bank) at 1:25; and P-Tyr-100 (Cell Signalling
Technology) at 1:500. Highly cross-absorbed Alexa Fluor 488-conjugated goat
anti-mouse IgG secondary antibody (Molecular Probes) was used at a 1:400
dilution.
Xenopus microinjection of mRNA and in situ hybridization
The entire coding regions from S. mediterranea β-catenins
genes were amplified by PCR and inserted into pCS2+
(Turner and Weintraub, 1994).
To generate GFP-tagged constructs, a DNA fragment from the 5' region of
each cDNA, which includes unique sites within the open reading frame, was
PCR-amplified. The 5' primers contained an EcoRI site to clone
the fragments in frame within the pCS2-GFP plasmid. The PCR fragments were
cloned in pGEM-T Easy vector and sequenced prior to fusion with their
corresponding cDNA. For mRNA preparation, the DNAs were linearized and
transcribed with SP6 RNA polymerases as described
(Harland and Weintraub, 1985),
with GTP cap analog (New England Biolabs). Xenopus embryos were
injected at the two-cell stage, into one blastomere at the prospective ventral
marginal region, with 500-1000 pg of each mRNA, and fixed at the tailbud
stage. Antisense RNA probes were prepared from Otx2, Krox20 and
Cad3 cDNAs and labelled with digoxigenin (Roche). Xenopus
specimens were hybridized as described
(Harland, 1991). Antibody
staining was performed as described
(Gómez-Skarmeta et al.,
2001), using the monoclonal antibody 12/101 (Developmental Studies
Hybridoma Bank, developed by J. P. Brockes) and rabbit anti-GFP (Molecular
Probes).
RESULTS AND DISCUSSION
Smed-βcatenin1 inhibition induces a gradual anteriorization of regenerating planarians
A search for β-catenin homologs in the S. mediterranea genome
database yielded two genes, which we called
Smed-βcatenin1 and
Smed-βcatenin2. In situ hybridization experiments
revealed different expression patterns for the S. mediterranea
β-catenins: Smed-βcatenin1 was expressed
ubiquitously but more strongly in the central nervous system (CNS), whereas
Smed-βcatenin2 mRNA was mostly located in the digestive
system (see Fig. S1 in the supplementary material). During the process of
regeneration, expression of both S. mediterranea β-catenin genes
was detected in anterior and posterior blastemas (see Fig. S2 in the
supplementary material). To assess their potential role in axis
re-establishment during regeneration, we carried out RNAi experiments. In situ
hybridization for each gene demonstrated that RNAi effectively silenced its
expression (see Fig. S3 in the supplementary material). Following
Smed-βcatenin1 silencing, 100% of the regenerating
trunk pieces exhibited AP polarity defects, although with varying degrees of
transformation: tailless planarians; two-headed planarians
(Fig. 1A); two-headed
planarians with ectopic eyes next to the normal anterior eyes
(Fig. 1B); two-headed
planarians with ectopic eyes next to the anterior and posterior ones
(Fig. 1C); and, the most severe
phenotype, animals displaying apparently radial symmetry with eyes all around
the periphery of their body (Fig.
1D; see also Movie 1 in the supplementary material). This last
phenotype was referred to as radial-like hypercephalized planarians. The
different phenotypes obtained correspond to the degree of severity of the
transformation, which was dependent on the time of regeneration and also on
the dose of inhibition (Fig.
1I'). After Smed-βcatenin2 silencing,
regenerating planarians did not show morphological defects or altered
expression of tissue markers (data not shown).
Several markers were used to characterize
Smed-βcatenin1-silenced animals. The planarian CNS is
composed of two anterior cephalic ganglia (CG) located at one end of two
ventral nerve cords (VNCs), which extend along the body and converge in the
tail (Fig. 1Q)
(Agata et al., 1998). From the
two dorsally located eyes, visual projections extend to the CG
(Okamoto et al., 2005). During
the first regeneration stages (3-7 days) after
Smed-βcatenin1 silencing, in situ hybridization for
opsin and cintillo revealed the ectopic differentiation of
photoreceptors and mechanoreceptors, respectively, in the posterior blastema
(Fig. 1F,J). Analysis of the
pan-neuronal marker H.10.2f revealed differentiation of ectopic CG in the
posterior blastema (Fig. 1N).
Analysis of radial-like hypercephalized planarians (after 20-30 days of
regeneration) showed that ectopic photoreceptors and mechanoreceptors expanded
from both ends of the regenerating animal to surround the planarian body
(Fig. 1H,L). Synapsin
immunostaining, which labels neuronal synapses, and in situ hybridization for
H.10.2f and Smed-GluR (which is specifically expressed in the lateral
branches of the brain), revealed that the CNS of radial-like hypercephalized
planarians appears as a thick ring from which cephalic branches ectopically
differentiate all around (Fig.
1P,T,V). Note that all photoreceptor cells appeared to be
connected through their visual axonal projections to the circular brain, as
seen with an anti-arrestin antibody (Fig.
1R). The digestive system of planarians is composed of a pharynx
located in the middle of the trunk, from which one anterior and two posterior
branches extend (Saló,
2006). The pharynx evaginates through the mouth, which is located
ventrally in the middle part of the planarian body. Staining with an
anti-P-Tyr antibody revealed that the two posterior branches converge to form
a single branch in Smed-βcatenin1-silenced planarians
(Fig. 1E'). Use of the
same marker also showed that secretory cells located in the prepharyngeal
mesenchyme of the wild-type planarian were ectopically differentiated in the
posterior part of Smed-βcatenin1-silenced planarians
(Fig. 1G').
Altogether, these results show that both neural and non-neural structures acquire anterior identity throughout the body of Smed-βcatenin1-silenced animals, demonstrating a role for Smed-βcatenin1 in AP axis re-establishment during planarian regeneration.
Trunk and tail identities are lost in Smed-βcatenin1-silenced planarians
To assess whether the differentiation of anterior tissues in central and
posterior regions was accompanied by the loss of trunk and tail identities, we
analyzed central and posterior markers in
Smed-βcatenin1-silenced planarians. Smed-HoxD
and Smed-AbdbA, central-posterior and posterior Hox genes,
respectively, were not expressed at 3 days of regeneration, the earliest stage
at which they are detected in wild-type animals
(Fig. 2A,E). Smed-HoxD
continued not to be expressed throughout regeneration
(Fig. 2C). Note that the
absence of this marker indicated the disappearance of the mouth. Expression of
Smed-TCEN49, a central marker associated with the pharynx, had almost
disappeared at the latest stage (Fig.
2G), and disappearance of the pharynx was also evident when
analyzed with other markers (Fig.
1T,V and Fig.
2I,M). These results demonstrate that the anteriorization of
Smed-βcatenin1-silenced planarians is accompanied by
the disappearance of trunk and tail identities. A possible role for Hox genes
as targets of Smed-βcatenin1 needs further
investigation.
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Taken together, these results demonstrate that in regenerating
Smed-βcatenin1-silenced animals the posterior blastema
acquires anterior identity and the trunk region is anteriorized, ultimately
generating radial-like hypercephalized animals in which the AP axis is lost
but the DV axis remains unaffected. Although the canonical Wnt signalling
pathway has a common role in establishing the AP axis during development in
several species, from cnidarians to vertebrates
(Holland, 2002;
Kiecker and Niehrs, 2001;
Nordstrom et al., 2002;
Schneider and Bowerman, 2007),
in vertebrates, at earlier developmental stages, nuclear accumulation of
β-catenin leads to dorsalization of the embryo
(De Robertis and Kuroda, 2004).
Our results support a conserved role for the canonical Wnt signal in AP axis
specification throughout evolution, and provide further confirmation that its
role in DV axis establishment is a vertebrate innovation.
In vertebrate models of regeneration, the Wnt pathway has a reported role
in cell proliferation and regenerative outgrowth, but not in axis
re-establishment (Kawakami et al.,
2006; Yokoyama et al.,
2007). Cnidarians are the only species in which this pathway has a
demonstrated axial patterning role during regeneration
(Lee et al., 2006), although a
direct functional study of cnidarian β-catenin has not been reported. Our
results, together with two recent reports also in planarians
(Gurley et al., 2008;
Petersen and Reddien, 2008),
are the first direct demonstration of the requirement of β-catenin in AP
patterning during the process of regeneration. In contrast to other models,
such as Xenopus and mouse, in which the inhibition of β-catenin
during development produces complex defects and non-viable embryos due to the
inhibition of organizer formation and the impairment of gastrulation
(Heasman et al., 2000;
Huelsken et al., 2000), the
planarian model allows in vivo analysis of mutant phenotypes for essential
developmental genes.
Smed-βcatenin1 is required for AP axis maintenance during homeostasis
To address whether Smed-βcatenin1 activity could also be required for
maintenance of the AP axis, we silenced Smed-βcatenin1
in intact planarians. At 10-17 days after
Smed-βcatenin1 silencing, ectopic eyes began to
differentiate first in the tail region and afterwards anteriorly, adjacent to
the original eyes (Fig. 3A,D).
Ectopic mechanoreceptor cells appeared in the tail and lateral regions
(Fig. 3G), and ectopic brain
differentiated along the length of the VNCs
(Fig. 3J,M). Around 30 days
after Smed-βcatenin1 silencing, radial-like
hypercephalized organisms that were indistinguishable from
Smed-βcatenin1-silenced regenerating animals were
observed. These animals showed ectopic eyes
(Fig. 3B,E) that were all
connected to the brain by their visual axons, ectopic mechanoreceptor cells
all around the periphery of the body (Fig.
3H), and ectopic branching of the CNS all around the body
(Fig. 3K,N). Analysis of
central and posterior markers revealed the disappearance of trunk and tail
identities (Fig. 3P,R). These
data demonstrate that Smed-βcatenin1 activity is required not only during
regeneration, but also during normal tissue homeostasis to maintain AP axis
polarity in planarians. Interestingly, our data show that the anteriorization
process in intact animals follows the same pattern as in regenerating trunks:
it starts mainly in the tail region and later expands to the rest of the body
(see Fig. 3T for
quantification). Further studies are required to elucidate whether this is a
direct consequence of the suppression of Smed-βcatenin1 activity or an
indirect effect due to, for example, a higher rate of cell turnover in the
tail.
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).
Accordingly, during Xenopus development, repression of Wnt signalling
is a characteristic of the head organizer
(Niehrs, 1999;
Niehrs, 2004). However, the
effect could also be explained by a mechanism in which β-catenin
regulates sister-cell asymmetry following cell division, as occurs along the
animal-vegetal axis during Platynereis and C. elegans
embryogenesis (Schneider and Bowerman,
2007; Kaletta et al.,
1997; Lin et al.,
1998). The classical janus-headed phenotype
(Morgan, 1898), in which
two-headed planarians are produced after cutting extremely thin fragments, is
a well-known model that supports the gradient hypothesis
(Child, 1911), because the
phenotype may be explained by the inability of such a small field to support a
Smed-βcatenin1 activity gradient. The analysis of Smed-βcatenin1
activity, by analysis of its subcellular localization, for instance, will help
to resolve this question.
Functional characterization of S. mediterranea β-catenins in Xenopus embryos
Sequence analysis of S. mediterranea β-catenins demonstrated
that the GSK3-binding domain, required for β-catenin degradation during
Wnt signalling, is present in Smed-βcatenin1 but not in
Smed-βcatenin2. By contrast, residues involved in cell-cell adhesion were
conserved in Smed-βcatenin2 but not in Smed-βcatenin1 (for details,
see Fig. S4 in the supplementary material). To test whether S.
mediterranea β-catenins could have undergone a functional
specialization, we assayed their ability to induce a secondary axis in
Xenopus embryos (McMahon and
Moon, 1989). A secondary axis was induced after
Smed-βcatenin1 injection but not after
Smed-βcatenin2 injection
(Fig. 4A,B,K). A GFP-tagged
construct of each S. mediterranea β-catenin was injected to
quantify the amount of translated protein in each embryo, demonstrating that
even when twice the amount of Smed-βcatenin2 was present, it could never
induce a secondary axis (Fig.
4C-H). Moreover, anti-GFP staining demonstrated that
Smed-βcatenin1, but not Smed-βcatenin2, accumulates in the nucleus
(Fig. 4I,J).
Altogether, these results demonstrate the functional conservation of
Smed-βcatenin1 in Wnt signalling, and point to a
functional specialization of S. mediterranea β-catenins, such
that Smed-βcatenin1 would be involved in signalling and Smed-bcatenin2
would be involved in the membrane cell-cell contacts. All metazoans studied to
date have a single β-catenin gene encoding a protein containing both
kinds of functional domains, involved in signaling and in cell-cell adhesion
(Schneider et al., 2003), with
the exception of C. elegans, which is the only species in which
β-catenin gene duplication and functional specialization have been
reported (Korswagen et al.,
2000). A phylogenetic analysis using β-catenin homologs from
several species demonstrates that the duplication in S. mediterranea
occurred independently from the one in C. elegans (see Fig. S5 in the
supplementary material). Molecular studies of other species belonging to
different phyla will clarify the significance of these duplications.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/7/1215/DC1
ACKNOWLEDGMENTS
We thank F. Cebrià, P. Martinez, J. Garcia-Fernandez, M. Irimia, J. Baguñà and members of the laboratory for discussion and suggestions; F. Cebrià and P. Newmark for providing H.10.2f, cintillo, Smed-GluR, septin and Eye53 clones; K. Eckelt for the Smed-opsin clone; H. Orii and K. Watanabe for providing anti-VC-1; M. Andreazzoli, A. Fainsod, N. Papalopulu and D. Turner for reagents; and I. Patten for editorial advice. This work was supported by grants from the Ministerio de Educación y Ciencia, Spain, from AGAUR (Generalitat de Catalunya, Spain), and from La Junta de Andalucía, Spain.
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