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First published online 11 February 2009
doi: 10.1242/dev.033761
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Research Report |
1 Department of Genetics and Institute of Biomedicine of the University of
Barcelona (IBUB), 08028 Barcelona, Spain.
2 German Cancer Research Center, Division of Signaling and Functional Genomics,
and University of Heidelberg/Faculty of Medicine Mannheim, Department of Cell
and Molecular Biology, 69120 Heidelberg, Germany.
* Author for correspondence (e-mail: k.bartscherer{at}dkfz.de)
Accepted 12 January 2009
SUMMARY
Planarians can regenerate a whole animal from only a small piece of their body, and have become an important model for stem cell biology. To identify regenerative processes dependent on Wnt growth factors in the planarian Schmidtea mediterranea (Smed), we analyzed RNAi phenotypes of Evi, a transmembrane protein specifically required for the secretion of Wnt ligands. We show that, during regeneration, Smed-evi loss-of-function prevents posterior identity, leading to two-headed planarians that resemble Smed-β-catenin1 RNAi animals. In addition, we observe regeneration defects of the nervous system that are not found after Smed-β-catenin1 RNAi. By systematic knockdown of all putative Smed Wnts in regenerating planarians, we identify Smed-WntP-1 and Smed-Wnt11-2 as the putative posterior organizers, and demonstrate that Smed-Wnt5 is a regulator of neuronal organization and growth. Thus, our study provides evidence that planarian Wnts are major regulators of regeneration, and that they signal through β-catenin-dependent and -independent pathways.
Key words: Anteroposterior axis, Brain patterning, Evi/Wls, Planarians, Regeneration, Wnt signaling
INTRODUCTION
Planarians have a nearly unlimited capability of renewing lost body
structures (reviewed by Saló,
2006
). Candidates for providing positional information during
planarian regeneration are gradient-forming growth factors, such as Wnts. Wnts
are secreted glycoproteins that behave as major organizers during embryonic
development in various metazoan organisms (reviewed by
Logan and Nusse, 2004).
Canonical Wnt signaling is transduced through β-catenin, and mediates
developmental processes, including axis specification (reviewed by
Grigoryan et al., 2008). Some
Wnts, such as mammalian Wnt5 (Slusarski et
al., 1997), can signal through β-catenin-independent
mechanisms, and control processes, such as cell polarity and directional
movement (Witze et al.,
2008).
Recently, β-catenin has been demonstrated to be essential in the
establishment of anteroposterior (AP) identity during regeneration of the
planarian species Schmidtea mediterranea
(Gurley et al., 2008;
Iglesias et al., 2008;
Petersen and Reddien,
2008). Smed-β-catenin1 silencing leads to a loss of
posterior identity and complete anteriorization (`radial-like hypercephalyzed'
planarians) (Iglesias et al.,
2008). However, although these studies suggest that Wnt proteins
might be essential organizers of regeneration in planarians, direct evidence
is missing.
Wnt secretion requires the transmembrane protein Evenness interrupted [Evi;
also known as Wntless (Wls) and Sprinter]
(Bänziger et al., 2006;
Bartscherer et al., 2006;
Goodman et al., 2006). In
Drosophila and Caenorhabditis elegans, Wnts are not secreted
in the absence of Evi, leading to a loss of Wnt signaling in the surrounding
tissue. Owing to the lack of non-canonical phenotypes in evi mutant
flies (Bartscherer et al.,
2006), it is currently not known whether Evi also controls
β-catenin-independent Wnt signaling.
Here, we report the effect of RNAi-mediated silencing of Smed-evi and all putative S. mediterranea Wnt genes during planarian regeneration. We show that, like Smed-β-catenin1, Smed-evi, as well as Smed-wnt11-2 and Smed-wntP-1, are required for posterior identity in regenerating animals. In addition, we demonstrate that Smed-evi RNAi causes the same β-catenin-independent defects as does Smed-wnt5 loss-of-function, suggesting that Smed-Wnt5 is a non-canonical Wnt that requires Smed-Evi for its secretion, and which acts to control neuronal growth during regeneration of the planarian nervous system.
MATERIALS AND METHODS
Animals
The planarians used belong to an asexual race of S. mediterranea,
and were maintained as described elsewhere
(Molina et al., 2007).
Identification and cloning of S. mediterranea genes
Smed-evi and the nine Smed-Wnt genes were identified from the
S. mediterranea genomic database
(http://genome.wustl.edu/)
through a BLAST search
(http://ncbi.nlm.nih.gov).
The full-length transcripts of the incomplete genes were amplified by rapid
amplification of cDNA ends (RACE) using the Invitrogen GeneRacer Kit
(Invitrogen).
Accession numbers
Smed-evi, FJ463748; Smed-wnt5, FJ463749;
Smed-wntA, FJ463750; Smed-wnt11-2, FJ463751;
Smed-wntP-4, FJ463752.
RNAi silencing
dsRNA microinjection was performed as described elsewhere
(Sánchez-Alvarado and Newmark,
1999). dsRNAs were synthesized as described
(Boutros et al., 2004). Primer
details are available upon request. Control animals were injected with water.
Injected planarians were amputated pre- and post-pharyngeally, and the head-,
trunk-, and tail-pieces were allowed to regenerate for the times
indicated.
Whole-mount in situ hybridization
Whole-mount in situ hybridization was carried out as described previously
(Nogi and Levin, 2005;
Umesono et al., 1999).
Digoxigenin-labelled riboprobes were synthesized using an in vitro
transcription kit (Roche) (Iglesias et
al., 2008). Primer details are available upon request.
Whole-mount immunostaining
Immunostaining was carried out as described previously
(Cebrià and Newmark,
2005). Antibodies used were: anti-Arrestin
(Sakai et al., 2000) at a
1:15,000 dilution, and anti-Synapsin (anti-SYNORF1, Developmental Studies
Hybridoma Bank) at 1:25.
RESULTS AND DISCUSSION
An Evi homolog is expressed in S. mediterranea
We found a single evi gene in the genome of S.
mediterranea (Fig. 1A; see
also Fig. S1 in the supplementary material). Smed-evi mRNA mainly
localized to the nervous system, the brain/cephalic ganglia (CG) and the
ventral nerve cords (VNCs), as well as to the pharynx, and to the mouth. In
addition, it was expressed in discrete cells of the posterior parenchyma
(Fig. 1B).
To analyze the expression of Smed-evi in regenerating animals, we dissected heads and tails from the trunk parts of the animals and followed their regeneration. Smed-evi was expressed in the regenerating nervous system and upregulated in the pharynx primordia, as well as in the tips of posterior wounds (blastemas; Fig. 1C), suggesting that Smed-evi might be especially important for posterior regeneration. As Evi is required for the secretion of Wnts, the Smed-evi expression pattern might indicate sites of Wnt production and release in planarians.
Smed-evi is required for posterior identity
To reveal Wnt-dependent processes in planarian regeneration, we depleted
Smed-evi by RNAi, and dissected heads and tails from the trunk parts
of the animals. Within 25 days of regeneration, all head fragments developed
an ectopic posterior head with eyes and a brain
(Fig. 2A,B). In addition, the
expression of the central-posterior Hox gene Smed-hoxD was lost
(Fig. 2C). These results
indicate that, in the absence of Smed-evi, posterior fate is
suppressed to the gain of anterior structures.
We obtained similar results for evi RNAi trunk fragments, as most
of them developed an ectopic posterior head
(Fig. 2D,E). In all animals,
the anterior head showed several ectopic eyes. Both, anterior and posterior
eyes differentiated laterally along the anteroposterior axis and were
connected by their visual axons. In most cases, they did not cross the
commissure and the optic quiasm was not formed
(Fig. 2D). In 
50% of the
Smed-evi RNAi trunks, the posterior head dissociated spontaneously
during regeneration, a process we refer to as `scission'. Tail pieces
regenerated a head with ectopic eyes (for Smed-evi RNAi phenotypes,
see Fig. S2 in the supplementary material).
The role of planarian Evi in AP polarity, but not in dorsoventral (DV)
polarity (see Fig. S3 in the supplementary material), is consistent with the
recently described Smed-β-catenin1 RNAi phenotype.
Smed-β-catenin1 RNAi causes a loss of AP polarity and complete
anteriorization of regenerating planarians
(Gurley et al., 2008;
Iglesias et al., 2008;
Petersen and Reddien, 2008)
(Fig. 2F). However, we never
observed full anteriorization after Smed-evi knockdown, which might
be due to inefficient knockdown of Smed-evi, or to a stronger
blockage of signaling after the loss of Smed-β-catenin1.
Together our results suggest that Smed-Evi is required for the release of Wnts that act through a β-catenin-dependent pathway to organize AP axis polarity during planarian regeneration.
Smed-evi is required for neuronal growth regulation during regeneration
To analyze the brains of Smed-evi RNAi animals in more detail, we
tested head fragments for Synapsin expression
(Fig. 2G-J). In wild-type
animals the CG were located dorsally above the VNCs and extended as a pair
into the regenerating tail (Fig.
2G; see Movie 1 in the supplementary material). However, in the
posterior head of Smed-evi RNAi animals, both CG grew laterally along
the VNCs. Confocal sections revealed that the posterior part of the CG
projected into the ventral region (Fig.
2G'; see Movie 2 in the supplementary material). We refer to
this phenotype as `deflected-brain phenotype'. Even though
Smed-β-catenin1 knockdown also resulted in the formation of a
posterior brain, the ectopic CG never appeared deflected from the VNCs
(Fig. 2H).
|
|
);
Smed-wntA and Smed-wnt5 encode proteins homologous to Wnts
from other planarian species (Kobayashi et
al., 2007; Marsal et al.,
2003); and Smed-wnt11-2 and Smed-wntP-4 have not
been described before. Phylogenetic analysis demonstrates that Smed-Wnt5,
Smed-Wnt11-1/2 and Smed-Wnt2-1 can be assigned to established Wnt subfamilies.
However, the other planarian Wnts appear as internal duplications in this
phylum, and cannot be classified with high certainty (see Fig. S4 in the
supplementary material).
Posterior identity requires Smed-wntP-1 and Smed-wnt11-2
Next, we set out to identify the Wnts responsible for the specification of
posterior structures during regeneration. In situ hybridization experiments
showed that Smed-wnt11-2, as well as Smed-wntP-1
(Petersen and Reddien, 2008),
was expressed in discrete cells along the most posterior midline of the S.
mediterranea body (Fig.
3A,B). In regenerating animals, Smed-wnt11-2 and
Smed-wntP-1 mRNA levels were upregulated in discrete cells of the
posterior blastemas, indicating an important role in the control of posterior
identity (Fig. 3C,D).
We tested whether posterior regeneration was affected by
Smed-wnt11-2 and Smed-wntP-1 RNAi. Silencing of either mRNA
led to a `tailless' morphology, which was characterized by a shorter and more
rounded posterior end, in which VNCs terminated shortly behind the pharynx
(Fig. 3F,G). Furthermore,
10% of regenerating trunks, and more than half of all regenerating head
fragments of Smed-wntP-1 RNAi animals resulted in two-headed
planarians (Fig. 3G), a
phenotype that was not enhanced by the simultaneous knockdown of
wnt11-2 (not shown). As Smed-β-catenin1 RNAi also
causes anteriorization of planarians (Fig.
2), Smed-Wnt11-2 and Smed-WntP-1 are likely to signal through
β-catenin to permit posterior fate during regeneration.
|
), we
detected an abnormal elongation of the new brain and visual axons in
Smed-wntA RNAi animals (see Fig. S5 in the supplementary
material). In Smed-wnt5 RNAi animals, we discovered a deflection and ventral expansion of the regenerating CG (Fig. 4D; see also Movie 5 in the supplementary material). In the regenerating tail, new Synapsin-positive tissue appeared, which was thicker than, and appeared disconnected from, the old nervous tissue (Fig. 4D). Expression analysis of Smed-glutamate receptor (gluR) mRNA showed that the new posterior neuronal tissue was not of brain identity (Fig. 4E), suggesting that it was new VNCs that grew deflected from the old ones. These phenotypes were similar to those observed after Smed-evi silencing (Fig. 2I; see also Movie 5 in the supplementary material).
Consistent with this, we found that Smed-wnt5 mRNA localized mainly to distinct cells along the CG and VNCs, and was upregulated in the regenerating nervous system and the blastemas (Fig. 4B). Together, our data suggest that Smed-Evi restricts and coordinates the growth of regenerating neuronal tissue by regulating the secretion of Smed-Wnt5.
Concluding remarks
Our study reveals several regenerative processes that are regulated by Wnts
in planarians (for a summary, see Table S1 in the supplementary material).
Using Smed-evi RNAi to block Wnt secretion, we identify AP axis
polarity and neuronal growth as being Wnt-regulated processes. Specifically,
we identify Smed-wntP-1 and Smed-wnt11-2 as being the
secreted molecules responsible for AP axis polarity. Consistent with the role
of Wnts as morphogens (reviewed by
Bartscherer and Boutros, 2008),
Smed-WntP-1 and Smed-Wnt11-2 might activate posterior fate, from their site of
production in the tail (Fig.
3), along the AP body axis. As posterior identity also depends on
Smed-β-catenin1, we propose that Smed-WntP-1 and Smed-Wnt11-2
signal through β-catenin to permit posterior fate during
regeneration.
Furthermore, we show that knockdown of Smed-wnt5 results in a
deflected-brain phenotype. Our data are consistent with the reported role of
Wnt5 in axonal growth in several organisms
(Yoshikawa et al., 2003;
Fradkin et al., 2004;
Zhang et al., 2007). Smed-Wnt5
belongs to the Wnt5 family (see Fig. S4 in the supplementary material), which
has been linked to non-canonical Wnt signal transduction
(Wong et al., 1994;
Olson and Papkoff, 1994;
Shimizu et al., 1997) and
seems to regulate cell motility rather than specification
(Moon et al., 1993;
Wallingford et al., 2001;
Witze et al., 2008). As we did
not observe any deflected-brain phenotype after Smed-β-catenin1
RNAi, we suggest that Smed-Wnt5 signals through a mechanism that is
β-catenin independent.
In planarians, Evi function is therefore required for the secretion of Wnts
that signal through β-catenin-dependent and -independent pathways
(Fig. 4F). This suggests that
Evi is an ancient factor that had been an important facilitator of Wnt
secretion before Wnts functionally diverged. Even though the same Wnts might
be able to signal through both canonical and non-canonical pathways, depending
on the receptor context (reviewed by van
Amerongen et al., 2008), we demonstrate that Wnts can have
distinct canonical or non-canonical functions in planarians. The nature and
constellation of planarian Wnt receptors remains the subject of future
studies, and might help us to understand how cells translate Wnt signals into
diverse cellular responses, both in planarians and in other organisms.
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We thank F. Cebrià for helpful advice; M. Riutort for help with phylogenetic analysis of Wnt proteins; M. Carl and A. Ragab for comments on the manuscript; B. Lang and T. Horn for bioinformatics support; F. Cebrià and P. Newmark for providing Smed-gluR, septin and eye53 clones; H. Orii and K. Watanabe for providing anti-Arrestin. K.B. thanks M. Osborn and M. Schäfer for advice. This work was supported by a Marie Curie Excellence grant from the European Commission, the German Research Foundation and by the Ministerio de Ciencia e Innovación, Spain.
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/136/6/905/DC1
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