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First published online 24 January 2007
doi: 10.1242/dev.02794
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Research Report |
Department of Cell and Developmental Biology, Neuroscience Program, University of Illinois at Urbana-Champaign, B107 Chemical and Life Sciences Laboratory, 601 South Goodwin Avenue, Urbana, IL 61801, USA.
Author for correspondence (e-mail:
pnewmark{at}life.uiuc.edu)
Accepted 15 December 2006
SUMMARY
The process by which the proper pattern is restored to newly formed tissues during metazoan regeneration remains an open question. Here, we provide evidence that the nervous system plays a role in regulating morphogenesis during anterior regeneration in the planarian Schmidtea mediterranea. RNA interference (RNAi) knockdown of a planarian ortholog of the axon-guidance receptor roundabout (robo) leads to unexpected phenotypes during anterior regeneration, including the development of a supernumerary pharynx (the feeding organ of the animal) and the production of ectopic, dorsal outgrowths with cephalic identity. We show that Smed-roboA RNAi knockdown disrupts nervous system structure during cephalic regeneration: the newly regenerated brain and ventral nerve cords do not re-establish proper connections. These neural defects precede, and are correlated with, the development of ectopic structures. We propose that, in the absence of proper connectivity between the cephalic ganglia and the ventral nerve cords, neurally derived signals promote the differentiation of pharyngeal and cephalic structures. Together with previous studies on regeneration in annelids and amphibians, these results suggest a conserved role of the nervous system in pattern formation during blastema-based regeneration.
Key words: Neural regeneration, Planarian, Axon guidance, ROBO, Schmidtea mediterranea
INTRODUCTION
Freshwater planarians possess amazing regenerative abilities: when cut
transversely, the anterior-facing wound regenerates a new head, whereas the
posterior-facing wound regenerates a new tail
(Newmark and Sánchez Alvarado,
2002
; Agata, 2003;
Reddien and Sánchez Alvarado,
2004). After amputation, stem cells called neoblasts proliferate
to produce the regeneration blastema in which the missing structures will
regenerate (Newmark and Sánchez
Alvarado, 2002; Reddien and
Sánchez Alvarado, 2004). Previous studies suggest that
differentiated cells convey the positional information required for proper
morphogenesis in planarians (Saló
and Baguñà, 1985;
Kato et al., 2001) and that
epithelialmesenchymal interactions
(Chandebois, 1980), as well as
gap junctional communication (Nogi and
Levin, 2005), may be important for defining anterior versus
posterior regeneration. Recent large-scale RNA interference (RNAi) analysis
identified hundreds of genes required for proper regeneration
(Reddien et al., 2005);
however, the exact mechanisms governing morphogenesis of the planarian
regenerate are still largely unknown.
In the initial days of anterior regeneration, primordia of the cephalic
ganglia form within the blastema; these cephalic ganglia must then
re-establish proper connections with the ventral nerve cords and with each
other (Cebrià et al.,
2002; Cebrià and
Newmark, 2005) to produce a functional central nervous system
(CNS). To understand the mechanisms underlying the regeneration of the
planarian CNS, we have begun to identify planarian orthologs of genes required
for proper axon guidance (Cebrià and
Newmark, 2005). Here, we report the isolation and functional
characterization of a roundabout gene from the planarian
Schmidtea mediterranea (Smed-roboA). ROBO proteins
are evolutionarily conserved transmembrane receptors of the immunoglobulin
superfamily that bind secreted molecules of the SLIT family
(Brose et al., 1999;
Kidd et al., 1999); together,
they play important roles in guiding axons to their proper targets
(Araujo and Tear, 2003;
Inatani, 2005). RNAi knockdown
of Smed-roboA results in the unexpected production of a supernumerary
pharynx and ectopic cephalic outgrowth during anterior regeneration. We show
that the development of these ectopic structures correlates with improper
connectivity between the newly regenerated cephalic ganglia and the ventral
nerve cords. These results suggest that the nervous system is a source of
signal(s) required for proper morphogenesis during planarian regeneration.
MATERIALS AND METHODS
Organisms
A clonal line of the diploid, asexual strain of Schmidtea
mediterranea was used (Sánchez
Alvarado et al., 2002). Planarians were maintained as previously
described (Cebrià and Newmark,
2005) and starved for at least 1 week before use.
Isolation of Smed-robo homologues
S. mediterranea genomic sequences encoding predicted proteins
similar to ROBO were retrieved from the NCBI Trace Archives and assembled
using Sequencher 4.2.2 (Gene Codes Corporation). In total, two different genes
encoding predicted ROBO proteins were identified; both were amplified from a
planarian cDNA library (Zayas et al.,
2005) and RACE was used to obtain additional cDNA sequences.
GenBank accession numbers for SmedroboA and Smed-roboB are
DQ336174 and DQ336175, respectively.
Whole-mount in situ hybridization and immunostaining
Planarians were processed in an Intavis InsituPro hybridization robot
(Sánchez Alvarado et al.,
2002) and imaged as described previously
(Cebrià and Newmark,
2005). Immunostaining was performed as described
(Cebrià and Newmark,
2005) using: anti-tubulin Ab-4 (NeoMarkers, 1:200) to label axon
bundles of the ventral nerve cords (VNCs), transverse commissures and lateral
processes; anti-phospho-tyrosine P-Tyr-100 (Cell Signaling Technology, 1:500)
to visualize the brain, VNC ganglia, gut and pharynx; and VC-1 to label
photosensitive cells (Umesono et al.,
1999). Highly crossabsorbed Alexa Fluor 488 goat anti-mouse IgG
secondary antibodies (Invitrogen) were used at 1:400. Samples were mounted in
Vectashield (Vector Laboratories), imaged with a CARV spinning disc confocal
microscope and deconvolved using AutoDeblur 9.3 (AutoQuant Imaging, Inc.).
|
; Cebrià
and Newmark, 2005). Injected planarians were amputated
pre-pharyngeally, allowed to regenerate and processed for in situ
hybridization or immunostaining. At 15 days after the first round of
injections, some regenerating animals were reinjected on 3 consecutive days
and re-amputated. Control animals were injected with water or GFP dsRNA. No
defects were observed in intact or regenerating animals after
Smed-roboB RNAi knockdown. Smed-roboA;
Smed-roboB double knockdowns showed the same phenotypes as
SmedroboA single knockdown; thus, we limit this report to
Smed-roboA. RESULTS AND DISCUSSION
Smed-roboA is required for the proper guidance of regenerating photoreceptor axons
We identified planarian robo homologues in genomic sequences from S.
mediterranea (see Materials and methods). Similar to other robo-family
members, Smed-roboA encodes five conserved immunoglobulin repeats and
three fibronectin type III repeats in the extracellular domain
(Fig. 1A). Smed-roboA
mRNA is expressed in the CNS and pharyngeal nerve ganglia in intact planarians
(Fig. 1B), as well as in the
regenerating CNS (Fig. 1C-E)
and pharynx (Fig. 1G).
To analyze the function of Smed-roboA, we performed RNAi
(Sánchez Alvarado and Newmark,
1999). A reduction of endogenous transcript after
Smed-roboA RNAi was confirmed by in situ hybridization
(Fig. 1F,G,I,J). Specific
inhibition was observed both within the newly regenerated tissues and within
the pre-existing nervous system (Fig.
1I,J); the expression of Smed-roboB was unaffected
(Fig. 1H,K). Conversely,
Smed-roboB RNAi did not affect levels of the Smed-roboA
transcript (data not shown). No abnormal phenotypes were observed in
Smed-roboA knockdowns in intact animals (5 weeks after RNAi, two sets
of three injections; n=10).
We monitored the effects of Smed-roboA RNAi on photoreceptor
regeneration. Planarian photoreceptors consist of two cell types: pigment cup
cells and photosensitive cells that reside outside of the pigment-cup opening.
The photosensitive cells extend axons posteriorly in a stereotypical pattern;
some axons project ipsilaterally, whereas others project contralaterally,
forming an optic chiasm that extends to the brain
(Okamoto et al., 2005)
(Fig. 1L). Smed-roboA
RNAi regenerates showed a variety of visual system defects
(Fig. 1M-O): the most common
phenotypes (39/47 vs 1/43 in controls; see Table S1 in the supplementary
material) were ectopic projections that resulted in loops
(Fig. 1M,N); in some specimens
(8/47 vs 0/43 in controls), the visual axons projected to the most-anterior
portion of the brain without crossing the midline
(Fig. 1O, arrowheads). Thus,
Smed-roboA is required for the proper guidance of visual axons during
regeneration.
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Ectopic pharynges (Fig. 2A,B, magenta asterisks) developed with reversed anteroposterior polarity, as revealed by the opening of the pharyngeal lumen (Fig. 2A, magenta arrow) towards the anterior of the animal. This alteration of polarity could also be observed in the gut. In triclad planarians, the digestive system consists of three main gut branches connected to the central pharynx: one branch grows anteriorly along the midline, ending at the level of the photoreceptors; the other two branches grow posteriorly, lateral to the pharynx and dorsal to the ventral nerve cords, extending through the tail. In three Smed-roboA RNAi animals, two gut branches developed lateral to the ectopic pharynx (Fig. 2B), as they would in post-pharyngeal regions. The ectopic gut branches were connected to the main digestive tract of the animal (Fig. 2B, arrows). None of the animals developed an ectopic mouth opening (the planarian pharynx protrudes through the mouth to ingest food), so it is unlikely that the ectopic pharynges were functional.
Furthermore, approximately 13% (9/67) of Smed-roboA RNAi anterior
regenerates produced an ectopic, dorsal outgrowth between the newly
regenerated cephalic region and the pre-existing pharynx
(Fig. 3A-E). All samples that
produced an ectopic outgrowth also developed an ectopic pharynx. These
outgrowths were first visible as small, unpigmented regions within the
uninjured tissues close to the wound; they became more evident after 2 weeks
of regeneration (Fig. 3A,
arrowhead). In 7/9 cases, these outgrowths appeared lateral to the midline
(Fig. 3B). The cephalic
identity of these outgrowths was demonstrated both morphologically and by
using molecular markers for various head-specific cell types: photosensitive
cells (Fig. 3C), pigment cups
of the photoreceptors (Fig. 3D,
arrow), sensory cells (Fig. 3D,
purple labeling) and brain-specific cells
(Fig. 3E) differentiated within
these outgrowths. Even in the absence of obvious outgrowths, ectopic
Smed-netrin1-expressing cells were detected
(Fig. 3F, arrowheads); these
cells are normally confined to two narrow rows along the medial cephalic
ganglia and ventral nerve cords
(Cebrià and Newmark,
2005). In addition, a ventral-specific marker was detected
dorsally (Fig. 3G,H). A gene
similar to anosmin-1, implicated in Kallman's syndrome in humans
(Franco et al., 1991;
Legouis et al., 1991), is
expressed both in a subset of cells of the CNS and ventrally, in cells beneath
the ventral musculature (Fig.
3I; data not shown). No anosmin-1-positive cells were
detected in dorsal views of control planarians
(Fig. 3G,J). However, in 8 out
of 14 Smed-roboA RNAi animals, ectopic anosmin-1-expressing
cells were detected dorsally (Fig.
3H), confined to the region between the newly regenerated head and
the pharynx (Fig. 3L). Using a
clone with weak similarity to septin (DN302617) as a marker for
dorsal mesenchymal cells, we did not observe a concomitant displacement of
these cells ventrally (data not shown). Thus, it appears that the polarity of
the tissues in which ectopic structures form is only partially altered.
|
). By
contrast, after Smed-roboA RNAi, the regenerated cephalic ganglia
were mis-positioned relative to the nerve cords
(Fig. 4D,F,H,J). The ganglia
were displaced laterally, resulting in a curvature in the VNCs as they
reconnected to the regenerated ganglia
(Fig. 4F). In the majority of
Smed-roboA RNAi knockdown animals (29/51; 57%), a more dramatic
dissociation between the regenerated cephalic ganglia and VNCs was observed
(Fig. 4H,J). In these cases,
the newly regenerated cephalic ganglia regenerated lateral to the VNCs; the
VNCs appeared truncated and failed to extend to the anterior-most portion of
the brain. These defects were observed as early as day 5 of regeneration
(Fig. 4H), well before the
appearance of ectopic structures. Despite these defects in the organization of
the regenerated CNS, no obvious behavioral defects were observed. An ectopic pharynx was observed in approximately 90% (26/29) of the Smed-roboA RNAi animals that showed a clear disconnection between the brain and the VNCs. By contrast, an ectopic pharynx was observed in only 36% (8/22) of the knockdown animals in which these misconnections were not apparent with the markers that we used. Posterior regeneration (head pieces regenerating new pharynx and posterior regions) proceeded normally in Smed-roboA RNAi knockdown animals: the VNCs grew properly into the new posterior regions and no ectopic pharynges or outgrowths developed (data not shown).
The above results suggest that the nervous system may play an important
role in patterning the newly formed anterior tissues during planarian
regeneration. Following Smed-roboA RNAi, the VNCs and the newly
formed cephalic ganglia are not connected properly. Almost all of the
planarians with these neural defects produced ectopic pharynges and cephalic
outgrowths. The CNS-specific expression of Smed-roboA during
regeneration (Fig. 1C-E) and
the observation that the improper connection of the VNCs and ganglia preceded
ectopic tissue growth (Fig. 4H)
suggest that the development of ectopic structures results from primary
defects in the regenerated nervous system. A recent high-throughput RNAi
screen in planarians reported that the knockdown of clone H.68.4a, which has
low similarity to slit genes, resulted in the development of ventral cephalic
outgrowths (Reddien et al.,
2005). Further analyses should clarify the extent of similarities
between the defects observed after H.68.4a and Smed-roboA RNAi, and
whether or not these two genes could encode a ligand-receptor pair.
In planarians, ectopic outgrowths have also been observed following grafts
in which dorsal and ventral tissues are juxtaposed
(Santos, 1931;
Kato et al., 1999). Likewise,
the juxtaposition of anterior and posterior tissues leads to the development
of ectopic pharynges (Kobayashi et al.,
1999). Thus, discontinuities in anteroposterior or dorsoventral
positional values can lead to changes in cell proliferation (resulting in
tissue outgrowth) and differentiation (producing new tissues and organs). We
suggest that the improper connection between the cephalic ganglia and ventral
nerve cords in Smed-roboA-knockdown animals mimics the effects of
transplantation or amputation, in which the cephalic ganglia and VNCs are
separated. In the absence of this connection, we infer that neurally derived
signals are sent to the surrounding tissues; these signals could alter
positional identities and trigger the production of an ectopic pharynx and a
cephalic outgrowth. This idea is consistent with previous observations showing
an increase of neurosecretory granules following amputation
(Sauzin-Monnot, 1972) and the
stimulation of mitogenic activity in planarians by substance P and substance K
(Baguñà et al.,
1989).
Such a role for the planarian nervous system may reflect an evolutionarily
conserved function in pattern formation during regeneration; thus, in
annelids, deflected cut ends of anteriorly facing nerve cords can give rise to
ectopic heads, whereas deflected cut ends of posteriorly facing nerve cords
induce the differentiation of ectopic tails
(Kiortsis and Moraitou, 1965).
In urodele amphibians, the dependence of limb regeneration on the nervous
system has been clearly shown (Singer and
Craven, 1948; Singer,
1952); deviation of the caudal spinal cord can induce
supernumerary tails (Kiortsis and Droin,
1961) and nerve deviation to a skin wound can induce ectopic limbs
(Egar, 1988;
Endo et al., 2004). Further
analyses, combining the tools of functional genomics now available for
studying planarians (Newmark and
Sánchez Alvarado, 2002;
Reddien et al., 2005), will
help us to identify the signals that serve to re-establish proper pattern
during planarian regeneration.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/5/833/DC1
ACKNOWLEDGMENTS
We would like to thank Gene Robinson, Huey Hing, Dave Forsthoefel, Ricardo Zayas and Tingxia Guo for helpful comments on the manuscript; Alejandro Sánchez Alvarado for providing asexual EST clones; and Kiyokazu Agata for providing VC-1. Planarian genomic-sequence data were generated by the Washington University Genome Sequencing Center in St Louis. F.C. was supported by a Long-Term Fellowship from EMBO and the programme Beatriu de Pinós of Generalitat de Catalunya; he thanks Emili Saló for support. This work was supported by NIH grant R01 HD43403 and NSF CAREER Award IBN-0237825 to P.A.N. P.A.N. was a Damon Runyon Scholar supported by the Damon Runyon Cancer Research Foundation (DRS 33-03).
Footnotes
* Present address: Departament de Genètica, Universitat de Barcelona,
Av. Diagonal 645, 08028 Barcelona, Spain 
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