Department of Cell and Developmental Biology and 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 13 June 2005
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SUMMARY |
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Key words: Neural regeneration, Netrin receptor, Axon guidance, Planarian, Schmidtea mediterranea
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Introduction |
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Freshwater planarians have been classic models for studying regeneration
(Brøndsted, 1969).
Recent work has applied modern techniques to unravel the cellular and
molecular basis of the planarian's regenerative abilities
(Newmark and Sánchez Alvarado,
2002
; Saló and
Baguñà, 2002
;
Agata et al., 2003
). Planarian
plasticity is also shown by the ability of these animals to grow or de-grow
depending on culture conditions; these processes are dependent on the balance
between cell proliferation and death
(Romero and Baguñà,
1991
). Both regeneration and cell turnover in intact animals
depend on a population of stem cells known as neoblasts
(Baguñà et al.,
1989
; Newmark and
Sánchez Alvarado, 2000
), which can differentiate into all
cell types in the flatworm, including neurons.
The planarian CNS consists of two cephalic ganglia (the brain) and two
ventral nerve cords (VNCs) that run the length of the body
(Agata et al., 1998;
Cebrià et al., 2002a
).
After amputation, planarians can regenerate a complete CNS de novo within 1
week (Reuter et al., 1996
;
Cebrià et al., 2002a
).
Recent studies have shown both the complexity of this CNS at the molecular
level, as well as a high degree of evolutionary conservation between planarian
and vertebrate neural genes (Umesono et
al., 1997
; Umesono et al.,
1999
; Cebrià et al.,
2002a
; Cebrià et al.,
2002b
; Cebrià et al.,
2002c
; Pineda and Saló,
2002
; Mineta et al.,
2003
; Nakazawa et al.,
2003
). Therefore, planarians are an attractive model in which to
study regeneration and renewal of the CNS.
We report the characterization of two planarian netrins and one member of
the Deleted in Colorectal Cancer (DCC) family of netrin receptors. Netrins are
secreted molecules that act as chemoattractants or chemorepellents for guiding
axons during development (Ishii et al.,
1992; Kennedy et al.,
1994
; Serafini et al.,
1994
; Colamarino and
Tessier-Lavigne, 1995
; Harris
et al., 1996
; Mitchell et al.,
1996
; Serafini et al.,
1996
). There are two families of single-pass transmembrane
receptors for netrin: DCC and UNC5. The DCC receptors mediate the attractive
effects of netrins (Chan et al.,
1996
; Keino-Masu et al.,
1996
), whereas the UNC5-type mediate repulsion, either alone or by
association with DCC (Hong et al.,
1999
; Keleman and Dickson,
2001
). The DCC family belongs to the immunoglobulin (Ig)
superfamily and includes DCC and neogenin in vertebrates
(Fearon et al., 1990
;
Vielmetter et al., 1994
;
Keino-Masu et al., 1996
),
frazzled in Drosophila melanogaster
(Kolodziej et al., 1996
) and
UNC-40 in Caenorhabditis elegans
(Chan et al., 1996
). Recent
work has suggested possible roles for netrin and its receptors in the adult
nervous system as well as during the regeneration of some neural cells
(Madison et al., 2000
;
Petrausch et al., 2000
;
Ellezam et al., 2001
;
Manitt et al., 2001
;
Astic et al., 2002
;
Manitt and Kennedy, 2002
);
however, no functional evidence has been presented. Outside the CNS, netrin
and its receptors function in the proper morphogenesis of several tissues and
organs (Hinck, 2004
).
Here we show that a netrin receptor gene from Schmidtea mediterranea (Smed-netR) is required for the proper patterning of the cephalic ganglia and the outgrowth of the VNCs during planarian regeneration. Our results suggest that in intact and regenerating animals, Smed-netR and Smed-netrin2 might function to establish and maintain the relationship between the brain and the VNCs. Smed-netR may mediate the response to Smed-netrin2 in the CNS, and both genes also play roles in targeting the photoreceptor axons to the brain visual center. Finally, the morphological defects observed after Smed-netR and Smed-netrin2 RNAi translate into some behavioral abnormalities. These results show that planarians are a suitable model in which to characterize the function of axon guidance cues in the regeneration and maintenance of the nervous system.
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Materials and methods |
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Isolation of Smed-netR, Smed-netrin1 and netrin2 and phylogenetic analyses
Expressed sequence tag (EST) Clone H.108.3a
(Sánchez Alvarado et al.,
2002) encodes a predicted protein similar to the DCC family of
netrin receptors. In order to identify additional 5' sequence, a series
of RACE reactions was performed. To identify planarian netrin
homologs, we took advantage of the ongoing S. mediterranea Genome
Project. Netrin proteins from different organisms were used in tblastn
searches of S. mediterranea genomic sequences (NCBI Trace Archives).
Genomic clones encoding predicted proteins similar to netrins were identified
and assembled using Sequencher 4.2.2 (Gene Codes Corp.). Specific primers were
designed to amplify both putative netrin genes. RACE was then used to obtain
additional cDNA sequences.
For phylogenetic analyses, maximum likelihood trees were made with Phyml
(http://atgc.lirmm.fr/phyml/)
with WAG model of substitution and with a gamma distribution; node support was
obtained by Tree-Puzzle5.2 (1000 quartet-puzzling replicates)
(Schmidt et al., 2002).
Whole-mount immunostaining and Hoechst labeling
Immunostaining was carried out essentially as described in Sánchez
Alvarado and Newmark (Sánchez
Alvarado and Newmark, 1999). We used the following monoclonal
antibodies: VC-1, specific for photosensitive cells (kindly provided by
Kiyokazu Agata, used at 1:10,000), anti-tubulin Ab-4 (NeoMarkers, used at
1:200) to visualize the axon bundles of the VNCs and the transverse
commissures, and anti-phospho-tyrosine P-Tyr-100 (Cell Signaling Technology,
used at 1:500) to visualize the brain and the ganglia of the VNCs. Highly
cross-absorbed Alexa Fluor 488 goat anti-mouse IgG secondary antibodies
(Molecular Probes) were used at 1:400. For double immunostaining with
anti-phospho-tyrosine and VC-1, after staining with anti-phospho-tyrosine,
planarians were fixed in 4% formaldehyde for 1 hour at room temperature (RT),
washed 3x10 minutes in PBS, blocked and incubated with VC-1 labeled
using the Zenon One Mouse IgG1 Labeling Kit (Molecular Probes) for
4 hours at RT. They were then washed 3x10 minutes in PBS and re-fixed in
4% formaldehyde. Samples were labeled with 0.5 µg/ml Hoechst for nuclear
staining, mounted in Vectashield (Vector Laboratories), and observed through a
Nikon TE 2000-S inverted microscope and a CARV spinning disk confocal
(AttoBioscience). Images were collected using a CoolSnap HQ camera
(Photometrics) and Metamorph software v6.1.
Whole-mount in-situ hybridization
After fixing and bleaching the planarians as previously described
(Umesono et al., 1997),
samples were loaded into an Insitu Pro hybridization robot (Intavis) and
processed as described in Sánchez Alvarado et al.
(Sánchez Alvarado et al.,
2002
). Samples were observed through a Leica MZ125
stereomicroscope or a Nikon Eclipse TE200 inverted microscope. Images were
collected using a MicroFire digital camera (Optronics).
RNAi analyses
Double-stranded RNAs (dsRNAs) of Smed-netR, netrin1, netrin2 and
semcap-1 were synthesized by in-vitro transcription (MegaScript,
Ambion) and injected into planarians as described
(Sánchez Alvarado and Newmark,
1999). Injected planarians were amputated prepharyngeally, allowed
to regenerate, and then processed for immunostaining. For long-term
experiments, intact planarians of the same size and physiological stage were
used; injected planarians were starved for the entirety of the experiment. The
samples were re-injected 2 or 3 weeks after the first round of injections. In
all the RNAi experiments, control animals were injected with water. To analyze
the efficacy of Smed-netR RNAi, two non-overlapping fragments of
Smed-netR cDNA were used to synthesize dsRNA and as an in-situ
hybridization probe (see Fig. S1A in the supplementary material). Similarly,
two non-overlapping fragments of Smed-netrin2 cDNA were used for
dsRNA synthesis and in-situ hybridization (see Fig. S1B in the supplementary
material).
Phototaxis assay
A circle of white light (NCL 150, Volpi USA) 4.4 cm in diameter was
directed from above to the center of a Petri dish (8.5 cm in diameter);
planarians were placed at the center of the circle and the time they needed to
move toward the dark regions of the dish was measured. The assay was performed
in a dark room.
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Results |
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In intact planarians, Smed-netR was highly expressed throughout the CNS (Fig. 1B,C). Smed-netR was also expressed in the nerve ganglia of the pharynx, in cells that are likely to be the neurons of the submuscular plexus, and in the photosensitive cells. During anterior regeneration, (body pieces regenerating a new head), Smed-netR was first expressed in the blastema at day 1 (Fig. 1D). By day 2 Smed-netR was detected within two clusters of cells that correspond to the new brain primordia (Fig. 1E). As regeneration proceeded, these clusters increased in size and differentiated into the new brain, in which Smed-netR continued to be expressed (Fig. 1F-I).
During posterior regeneration (head pieces regenerating new pharynx and tail), Smed-netR was expressed within both the regeneration blastema and the new pharynx. The first signal appeared around day 3 of regeneration (Fig. 1J). At day 5, Smed-netR was highly expressed in the regenerated VNCs and throughout the newly forming pharynx (Fig. 1K). As regeneration proceeded, the expression of Smed-netR within the pharynx became restricted to the nerve ganglia (Fig. 1L).
Smed-netR is necessary for proper patterning of the CNS and peripheral nervous system during regeneration
RNA-interference (RNAi) (Fire et al.,
1998) is a powerful technique for studying gene function in
planarians: it results in gene-specific knockdowns throughout the animal,
including the CNS (Sánchez Alvarado
and Newmark, 1999
; Pineda et
al., 2000
; Cebrià et
al., 2002c
; Newmark et al.,
2003
; Reddien et al.,
2005
). Following Smed-netR RNAi, the expression of this
gene was inhibited in both the newly regenerated tissues and throughout the
uninjured region (see Fig. S1A in the supplementary material). All the
planarians injected with Smed-netR dsRNA showed consistent defects in
the patterning of the regenerated nervous system (88 dsRNA-injected samples
versus 73 control-injected samples that regenerated normal nervous
systems).
Planarians normally regenerate cephalic ganglia indistinguishable from
those observed in intact animals, with two lobes connected by a thin, anterior
commissure (Fig. 2A). Nuclear
staining showed the cellular organization of the new ganglia, with most of the
neuronal cell bodies in the periphery surrounding a central neuropil
(Oosaki and Ishii, 1965;
Morita and Best, 1965
;
Morita and Best, 1966
)
(Fig. 2B). Lateral neuronal
projections that extend from the cephalic ganglia toward the head periphery
were evident (Fig. 2C).
After Smed-netR RNAi, however, the pattern of the newly regenerated cephalic ganglia was dramatically disrupted. The two ganglia appeared shorter and wider compared with controls, and the anterior commissure that connected them was thickened significantly [Fig. 2D; 91.3±3.4 µm in dsRNA-injected samples (n=16) versus 41.6±1.2 µm in controls (n=30); mean±s.e.m.; P<0.001, t-test]. Although the morphology of these ganglia was abnormal, the cell bodies were mainly localized to the periphery (Fig. 2E). The lateral projections were thinner and shorter, or even absent, compared with controls (Fig. 2F). Neural cell bodies appeared to occupy the regions that normally contain lateral projections, giving rise to a continuous layer of cells (compare Fig. 2C,F).
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During regeneration, the remaining, uninjured tissues are remodeled to restore proper body proportions. We analyzed the effects of Smed-netR RNAi on the nervous system in the uninjured, posterior region of planarians that were regenerating a new head. The submuscular plexus consists of a network of thin nerve fibers (Fig. 3A). After Smed-netR RNAi, the fibers of the submuscular plexus appeared wider and more disorganized than controls (Fig. 3B). Ectopic nerve fibers were observed between the two VNCs (Fig. 3D) and the bundles of nerve fibers that constitute the VNCs did not appear as tightly compacted (compare Fig. 3C,D).
During posterior regeneration, planarians normally regenerate VNCs that grow into the new tail and connect to each other by a few, thin processes (Fig. 3E). By contrast, after Smed-netR RNAi, the VNCs regenerated abnormally: ectopic nerve fibers were found between them and a dense, disorganized neural meshwork was observed in the most posterior end (Fig. 3F). This phenotype is strikingly similar to that observed in the anteriorly regenerating nerve cords (compare Fig. 2J and Fig. 3F). These results indicate that during regeneration Smed-netR is required for proper patterning of the new brain and normal growth and patterning of the VNCs.
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Following head amputation, planarians regenerate a normal visual system. An
optic chiasm forms along the posterior extent of the commissure that connects
both ganglia, and the visual axons project posteriorly to target the visual
center in the brain (Fig. 4A).
After Smed-netR RNAi, all animals showed aberrant axonal projections
(Fig. 4A)
(Newmark et al., 2003). These
abnormal phenotypes could be classified into three groups: (1) those lacking
posterior axonal projections toward the brain visual center, with an anterior
ectopic projection along the midline (Fig.
4A; n=17/51); (2) those lacking posterior projections to
the visual center with no ectopic anterior projections
(Fig. 4A; n=21/51);
and (3) those with more severe disruptions
(Fig. 4A; n=13/51). In
spite of the abnormal pattern of the cephalic ganglia and the defects in
axonal targeting to the brain visual center, the projection of the visual
axons along the posterior domain of the brain commissure proceeded normally in
most cases (Fig. 4A). The
photoreceptors are normally positioned very close to the anterior end of the
brain commissure; after Smed-netR RNAi, however, the brain commissure
was wider and extended anteriorly relative to the photoreceptors
(Fig. 4A).
Photophobic response is altered after Smed-netR RNAi
We sought to determine whether the mispatterning of the regenerated CNS and
the mistargeting of the visual axons observed after Smed-netR RNAi
led to behavioral defects. Smed-netR dsRNA-treated planarians moved
normally, responded to mechanical stimuli, were able to evaginate their
pharynges in response to food and could eat (data not shown). However, the
response to light was significantly different between Smed-netR
dsRNA-treated animals and controls.
Planarians normally display negative phototaxis: they respond to light by
moving away from the source (Taliaferro,
1920; Inoue et al.,
2004
). Smed-netR RNAi knockdown animals showed a
statistically significant difference in the time required to move away from
light [48.8±3.7 seconds in controls (n=34) versus
84.8±8.7 seconds after Smed-netR RNAi (n=37);
mean±s.e.m.; P<0.005, t-test;
Fig. 4B]. To ensure that the
observed differences resulted from knocking down Smed-netR, as an
additional control we tested planarians treated with dsRNA corresponding to
Semcap-1, a gene that regulates the distribution of the transmembrane
semaphorin M-SemF (Wang et al.,
1999
). Smed-semcap-1 is expressed throughout the CNS and
in the pharynx (see Fig. S3 in the supplementary material). Smed-semcap-1 RNAi-treated planarians regenerated cephalic ganglia
connected by a significantly thinner anterior commissure [26.2±1.4
µm in dsRNA-injected samples (n=17) versus 41.6±1.2 µm
in controls (n=30); mean±s.e.m.; P<0.001,
t-test; Fig. 4B and
Fig. S3 in the supplementary material]. Also, the visual axons projected much
more posteriorly than controls (Fig.
4B); the ratio between the lengths of the visual axons and the
cephalic ganglia from the optic chiasm was 0.49±0.01 in control animals
and increased to 0.76±0.01 after Smed-semcap-1 RNAi. These
phenotypes are roughly opposite those observed after Smed-netR RNAi.
No differences in the time required to move away from light were observed
between controls and Smed-semcap-1 dsRNA-injected animals
[49.6±4.5 seconds in dsRNA-injected samples (n=18) versus
48.8±3.7 seconds in controls (n=34); mean±s.e.m.;
Fig. 4B].
The slowed photophobic response of Smed-netR dsRNA-injected
animals did not seem to be caused by slower movements. Controls and
Smed-semcap-1 dsRNA-injected animals moved away from the light
following a rather direct course. By contrast, after Smed-netR RNAi
the planarians turned more often, following much more irregular paths. These
results are similar to those described for planarians with both eyes removed
(Taliaferro, 1920), or after
RNAi knockdowns for genes expressed either within or surrounding the
photoreceptor axons (Inoue et al.,
2004
).
Smed-netR is necessary for maintaining neuronal pattern in intact planarians
To analyze the function of Smed-netR in intact adult planarians,
we carried out long-term RNAi experiments. After 6 weeks of treatment, axonal
projection length from the optic chiasm to the most posterior extent was
significantly reduced in Smed-netR dsRNA-injected planarians
[35.6±6.2 µm in dsRNA-injected samples (n=14) versus
63.4±4.9 µm in controls (n=22); mean±s.e.m.;
P<0.005, t-test] (Fig.
5A,B). This reduction ranged from no posterior growth away from
the chiasm to less drastic (but significant) shortening of visual axons. By
contrast to photoreceptor regeneration, reduction in visual axon length was
the only abnormal phenotype observed in the photoreceptors following
Smed-netR RNAi in intact planarians.
Strikingly, Smed-netR RNAi resulted in dramatic patterning defects in the CNS of intact, non-regenerating animals. Four weeks after the first round of injections, the CNS of control animals (n=8/8) retained its normal pattern, with the brain positioned on top of the VNCs (Fig. 5C-F). Nuclear staining shows the cell bodies located normally around the periphery of both the brain and the VNCs (Fig. 5D,F). By contrast, in Smed-netR dsRNA-injected planarians (n=8/8), the ventral region of the brain appeared to have expanded laterally with respect to the VNCs (Fig. 5G,I). This expansion is also evident after nuclear staining with Hoechst; the cell bodies from the ventral portion of the brain were clearly separated from the cell bodies of the VNCs (compare brackets in Fig. 5F and 5J). Also, ectopic nerve fibers appeared between the VNCs in the cephalic region, giving rise to a disorganized meshwork similar to that observed during regeneration (data not shown). In the submuscular nerve plexus, the fibers appeared wider and slightly disorganized when compared with controls (Fig. 5K,M). Outside the cephalic region, ectopic nerve fibers appeared between the VNCs, which also looked loosely organized relative to controls (compare Fig. 5L and 5N). All these defects are similar to those observed during regeneration following Smed-netR RNAi. The defects observed in the CNS of intact planarians after Smed-netR RNAi were first apparent after 2 weeks of dsRNA injection (11/17 dsRNA-injected planarians showed defects in the CNS compared with 15/15 normal controls), and could still be observed after 6 weeks (9/9 dsRNA-injected planarians showed defects in the CNS compared to 10/10 normal controls). Thus, in addition to being required for proper CNS regeneration, Smed-netR is also required to maintain the architecture of the CNS and visual system in intact planarians.
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Finally, we carried out long-term RNAi experiments to determine whether Smed-netrin1 and netrin2 play roles in maintaining photoreceptor axonal projections. After 4.5 weeks of RNAi for Smed-netrin1 and netrin2, the length of the photoreceptor projections from the chiasm to the brain visual center was shortened (Fig. 8H). The ratio between visual axon length and the length of cephalic ganglia from the optic chiasm was 0.55±0.03 in controls (n=10) and was significantly reduced after RNAi knockdowns of Smed-netrin 2 (0.35±0.01; n=13; P<0.005; t-test), Smed-netrin 1 (0.45±0.02; n=9; P<0.05; t-test) and double Smed-netrin1; netrin2 (0.32±0.03; n=9; P<0.005; t-test). Smed-netrin2 knockdowns resulted in greater reductions in visual axon length than Smed-netrin1 knockdowns (P<0.05, t-test). Together, the above results indicate that, like Smed-netR, Smed-netrin2 is required for the normal regeneration and maintenance of the planarian nervous system.
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Discussion |
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Smed-netR is also required for the patterning of the nervous system outside the cephalic region. During posterior regeneration following Smed-netR RNAi knockdowns, the VNCs did not grow normally and showed disorganization similar to that observed in anteriorly regenerating VNCs. Moreover, the uninjured VNCs were slightly disorganized the axonal bundles appeared to be less tightly associated and ectopic nerve fibers appeared between them. Similarly, the fibers that constitute the peripheral submuscular nerve plexus appeared wider and slightly disorganized. At present, however, we cannot determine whether the ectopic axonal projections derived from defasciculation, improper outgrowth of the VNCs, and/or from ectopic neurons.
During development, DCC family members play key roles in axonal growth and
guidance, as well as in neural migration
(Chan et al., 1996;
Keino-Masu et al., 1996
;
Fazeli et al., 1997
;
Deiner et al., 1997
;
Gong et al., 1999
;
Murase and Horwitz, 2002
).
However, the function of DCC genes following CNS injury and
regeneration remains unclear. Some studies have shown that in mammals
DCC is downregulated in axotomized retinal ganglion cell (RGC)
neurons (Petrausch et al.,
2000
; Ellezam et al.,
2001
) and that DCC is not upregulated in regenerating
olfactory axons (Astic et al.,
2002
). By contrast, our results indicate that in planarians
Smed-netR is upregulated within the blastema, is expressed in the
newly differentiating brain as well as the VNCs, and is essential for proper
regeneration of the CNS.
Smed-netR is required for targeting the photoreceptor axons to the brain visual center
Despite the stereotypical pattern of axonal projections displayed by
planarian visual cells, little is known about the cues that guide these
projections. The results presented here clearly indicate that silencing
Smed-netR resulted in a failure to target properly the brain visual
center during regeneration (Fig.
4) (Newmark et al.,
2003). However, other aspects of the outgrowth of the
photoreceptor axons were not affected; thus, the photoreceptor cells
associated normally with the pigmented eye-cups and sent axonal projections
posteriorly to the region where the chiasm is formed. Also, in about
two-thirds of the samples, the axons projected along the posterior domain of
the cephalic commissure, giving rise to a partially formed chiasm
(Fig. 4A). These results
suggest that different stages of the development and guidance of planarian
photoreceptor axons are governed by different guidance cues; the final step in
this process targeting the appropriate brain region seems to
require Smed-netR. A similar situation is found in vertebrates in
which different guidance cues such as Netrin and DCC
(Deiner et al., 1997
), Slit
(Plump et al., 2002
) and
Semaphorins (Oster et al.,
2003
) play roles in the projection of the retinal axons from the
retina to their targets. Smed-netR function in targeting the visual
axons suggests that axon guidance mechanisms involved in patterning the visual
system have been evolutionarily conserved.
|
Smed-netR function in intact planarians
Planarians grow and de-grow continuously, depending on culture temperature
and food availability. Growth and de-growth are the result of alterations in
the balance between cell proliferation and death
(Baguñà, 1976;
Romero and Baguñà,
1991
). Planarians are somehow able to monitor the relative sizes
of their structures and the number of cells in order to maintain body
proportions, in spite of the continuous remodeling that they are undergoing
(Oviedo et al., 2003
).
Our results show that in intact planarians Smed-netR is necessary to maintain the proper architecture of the nervous system. Within a few weeks of Smed-netR RNAi, the cephalic ganglia expanded laterally relative to the VNCs, as if the ganglia and the VNCs were no longer properly associated. Also, ectopic nerve fibers appeared between the VNCs, which appeared to be defasciculated. These phenotypes are remarkably similar to those observed during regeneration following Smed-netR RNAi. Moreover, a significant shortening of the projections of the visual axons was observed in dsRNA-injected intact planarians. By contrast, control animals starved for a few weeks retained normal patterning of the brain, VNCs and visual system. The phenotypes observed in intact planarians after Smed-netR RNAi might be explained either by disorganization of the pre-existing structures and/or by guidance defects of newly generated cells. Further experiments are required to distinguish between these two possibilities, but the fact that structural defects were detected throughout the entire brain as early as 2 weeks after dsRNA injections suggests that Smed-netR plays an important role in maintaining the organization of the mature nervous system.
Expression of netrin and netrin receptors has been
detected in the CNS of adult vertebrates
(Manitt and Kennedy, 2002);
however, their functions remain to be elucidated. Some studies suggest that
Netrin-1 may function as a short-range cue mediating cell-cell interactions in
the adult CNS (Manitt et al.,
2001
). Outside the CNS there is growing evidence that axon
guidance cues also play roles in the morphogenesis of a variety of tissues and
organs (Hinck, 2004
). Thus,
during development of the mammary gland, Netrin-1 seems to act through
Neogenin as a short-range attractant mediating proper cell adhesion, rather
than guidance (Srinivasan et al.,
2003
). Similarly, Smed-netR could mediate cell-cell
interactions in the planarian CNS in order to establish and maintain proper
connectivity between the cephalic ganglia and the VNCs, as well as to maintain
bundling of the VNCs. The requirement of Smed-netR function for
patterning the nervous system in both regenerating and intact planarians
indicates that the same mechanisms may operate during regeneration of new
tissues and the remodeling of pre-existing structures during growth and
de-growth.
Smed-netrins are required for regeneration and maintenance of the CNS
Several studies have suggested that DCC functions as a receptor for Netrins
to mediate a neuronal chemoattractant response
(Chan et al., 1996;
Keino-Masu et al., 1996
;
Kolodziej et al., 1996
;
Serafini et al., 1996
). Here,
we reported the isolation of two netrin homologs from S. mediterranea.
Smed-netrin2 RNAi led to defects in the CNS that were very similar to
those observed after Smed-netR RNAi, indicating that
Smed-netR could act as a receptor to mediate the response to
Smed-netrin2. Although RNAi for Smed-netrin1 did not result
in CNS defects during regeneration, the observation that the double
Smed-netrin1; Smed-netrin2 RNAi knockdowns resulted in more
severe visual axon phenotypes than Smed-netrin2 alone
(Table 1) suggests that both
Smed-netrins may act synergistically. RNAi experiments in
intact non-regenerating planarians support a role for Smed-netrin1 in
the maintenance of the visual axon pattern.
Our data clearly indicate that Smed-netrin 2 is required not only to regenerate a proper CNS but also to maintain neural architecture in intact animals. To our knowledge this represents the first evidence of netrin function in the adult CNS. In contrast to other animals, planarian netrins are not expressed along the midline; rather they are expressed bilaterally along each of the two VNCs. This expression pattern, together with the results of RNAi knockdowns, suggests that Smed-netrin2 probably functions to establish and maintain the structure of the CNS, rather than to guide commissural axons across the midline. The soon-to-be completed genome sequence of S. mediterranea will aid the identification and functional characterization of other axon guidance cues in planarians, and help us to understand how these remarkably plastic animals are able to regenerate and maintain their CNS.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/16/3691/DC1
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Agata, K., Soejima, Y., Kato, K., Kobayashi, C., Umesono, Y. and Watanabe, K. (1998). Structure of the planarian central nervous system (CNS) revealed by neuronal cell markers. Zool. Sci. 15,433 -440.[CrossRef]
Agata, K., Tanaka, T., Kobayashi, C., Kato, K. and Saitoh, Y. (2003). Intercalary regeneration in planarians. Dev. Dyn. 226,308 -316.[CrossRef][Medline]
Araújo, S. J. and Tear, G. (2003). Axon guidance mechanisms and molecules: lessons from invertebrates. Nat. Rev. Neurosci. 4,910 -922.[CrossRef][Medline]
Astic, L., Pellier-Monnin, V., Saucier, D., Charrier, C. and Mehlen, P. (2002). Expression of netrin-1 and netrin-1 receptor, DCC, in the rat olfactory nerve pathway during development and axonal regeneration. Neuroscience 109,643 -656.[CrossRef][Medline]
Baguñà, J. (1976). Mitosis in the intact and regenerating planarian Dugesia mediterranea n.sp. I. Mitotic studies during growth, feeding and starvation. J. Exp. Zool. 195,53 -64.[CrossRef]
Baguñà, J., Saló, E. and Auladell, C. (1989). Regeneration and pattern formation in planarians. III. Evidence that neoblasts are totipotent stem cells and the source of blastema cells. Development 107,77 -86.[Abstract]
Benazzi, M., Ballester, R., Baguñà, J. and Puccinelli, I. (1972). The fissiparous race of the planarian Dugesia lugubris S. L. Found in Barcelona (Spain) belongs to the biotype G: comparative analysis of the karyotypes. Caryologia 25,59 -68.
Brøndsted, H. V. (1969). Planarian Regeneration. London: Pergamon Press.
Cebrià, F., Nakazawa, M., Mineta, K., Ikeo, K., Gojobori, T. and Agata, K. (2002a). Dissecting planarian central nervous system regeneration by the expression of neural-specific genes. Dev. Growth Differ. 44,135 -146.[CrossRef][Medline]
Cebrià, F., Kudome, T., Nakazawa, M., Mineta, K., Ikeo, K., Gojobori, T. and Agata, K. (2002b). The expression of neural-specific genes reveals the structural and molecular complexity of the planarian central nervous system. Mech. Dev. 116,199 -204.[CrossRef][Medline]
Cebrià, F., Kobayashi, C., Umesono, Y., Nakazawa, M., Mineta, K., Ikeo, K., Gojobori, T., Itoh, M., Taira, M., Sánchez Alvarado, A. et al. (2002c). FGFR-related gene nou-darake restricts brain tissues to the head region of planarians. Nature 419,620 -624.[CrossRef][Medline]
Chan, S. S., Zheng, H., Su, M. W., Wilk, R., Killeen, M. T., Hedgecock, E. M. and Culotti, J. G. (1996). UNC-40, a C. elegans homolog of DCC (Deleted in Colorectal Cancer), is required in motile cells responding to UNC-6 netrin cues. Cell 87,187 -195.[CrossRef][Medline]
Colamarino, S. A. and Tessier-Lavigne, M. (1995). The axonal chemoattractant netrin-1 is also a chemorepellent for trochlear motor axons. Cell 81,621 -629.[CrossRef][Medline]
Deiner, M. S., Kennedy, T. E., Fazeli, A., Serafini, T., Tessier-Lavigne, M. and Sretavan, D. W. (1997). Netrin-1 and DCC mediate axon guidance locally at the optic disc: loss of function leads to optic nerve hypoplasia. Neuron 19,575 -589.[CrossRef][Medline]
Ellezam, B., Selles-Navarro, I., Manitt, C., Kennedy, T. E. and McKerracher, L. (2001). Expression of netrin-1 and its receptors DCC and UNC-5H2 after axotomy and during regeneration of adult rat retinal ganglion cells. Exp. Neurol. 168,105 -115.[CrossRef][Medline]
Fazeli, A., Dickinson, S. L., Hermiston, M. L., Tighe, R. V., Steen, R. G., Small, C. G., Stoeckli, E. T., Keino-Masu, K., Masu, M., Rayburn, H. et al. (1997). Phenotype of mice lacking functional Deleted in colorectal cancer (Dcc) gene. Nature 386,796 -804.[CrossRef][Medline]
Fearon, E. R., Cho, K. R., Nigro, J. M., Kern, S. E., Simons, J. W., Ruppert, J. M., Hamilton, S. R., Preisinger, A. C., Thomas, G., Kinzler, K. W. et al. (1990). Identification of a chromosome 18q gene that is altered in colorectal cancers. Science 247,49 -56.[Medline]
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.Nature 391,806 -811.[CrossRef][Medline]
Gong, Q., Rangarajan, R., Seeger, M. and Gaul, U.
(1999). The netrin receptor frazzled is required in the target
for establishment of retinal projections in the Drosophila visual system.
Development 126,1451
-1456.
Guan, K.-L. and Rao, Y. (2003). Signalling mechanisms mediating neuronal responses to guidance cues. Nat. Rev. Neurosci. 4,941 -956.[Medline]
Harris, R., Sabatelli, L. M. and Seeger, M. A. (1996). Guidance cues at the Drosophila CNS midline: identification and characterization of two Drosophila Netrin/UNC-6 homologs. Neuron 17,217 -228.[CrossRef][Medline]
Hinck, L. (2004). The versatile roles of "axon guidance" cues in tissue morphogenesis. Dev. Cell 7,783 -793.[CrossRef][Medline]
Hong, K., Hinck, L., Nishiyama, M., Poo, M. M., Tessier-Lavigne, M. and Stein, E. (1999). A ligand-gated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion. Cell 97,927 -941.[CrossRef][Medline]
Inoue, T., Kumamoto, H., Okamoto, K., Umesono, Y., Sakai, M., Sánchez Alvarado, A. and Agata, K. (2004). Morphological and functional recovery of the planarian photosensing system during head regeneration. Zool. Sci. 21,275 -283.[CrossRef][Medline]
Ishii, N., Wadsworth, W. G., Stern, B. D., Culotti, J. G. and Hedgecock, E. M. (1992). UNC-6, a laminin-related protein, guides cell and pioneer axon migrations in C. elegans. Neuron 9,873 -881.[CrossRef][Medline]
Keino-Masu, K., Masu, M., Hinck, L., Leonardo, E. D., Chan, S. S., Culotti, J. G. and Tessier-Lavigne, M. (1996). Deleted in Colorectal Cancer (DCC) encodes a netrin receptor. Cell 87,175 -185.[CrossRef][Medline]
Keleman, K. and Dickson, B. J. (2001). Short- and long-range repulsion by the Drosophila Unc5 netrin receptor. Neuron 32,605 -617.[CrossRef][Medline]
Kennedy, T. E., Serafini, T., de la Torre, J. R. and Tessier-Lavigne, M. (1994). Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Cell 78,425 -435.[CrossRef][Medline]
Kolodziej, P. A., Timpe, L. C., Mitchell, K. J., Fried, S. R., Goodman, C. S., Jan, L. Y. and Jan, Y. N. (1996). frazzled encodes a Drosophila member of the DCC immunoglobulin subfamily and is required for CNS and motor axon guidance. Cell 87,197 -204.[CrossRef][Medline]
Madison, R. D., Zomorodi, A. and Robinson, G. A. (2000). Netrin-1 and peripheral nerve regeneration in the adult rat. Exp. Neurol. 161,563 -570.[CrossRef][Medline]
Manitt, C. and Kennedy, T. E. (2002). Where the rubber meets the road: netrin expression and function in developing and adult nervous systems. Prog. Brain Res. 137,425 -442.[Medline]
Manitt, C., Colicos, M. A., Thompson, K. M., Rousselle, E.,
Peterson, A. C. and Kennedy, T. E. (2001). Widespread
expression of netrin-1 by neurons and oligodendrocytes in the adult mammalian
spinal cord. J. Neurosci.
21,3911
-3922.
Marchler-Bauer, A. and Bryant, S. H. (2004).
CD-Search: protein domain annotations on the fly. Nucleic Acids
Res. 32,W327
-W331.
Mineta, K., Nakazawa, M., Cebrià, F., Ikeo, K., Agata, K.
and Gojobori, T. (2003). Origin and evolutionary
process of the CNS elucidated by comparative genomics analysis of planarian
ESTs. Proc. Natl. Acad. Sci. USA
100,7666
-7671.
Mitchell, K. J., Doyle, J. L., Serafini, T., Kennedy, T. E., Tessier-Lavigne, M., Goodman, C. S. and Dickson, B. J. (1996). Genetic analysis of Netrin genes in Drosophila: Netrins guide CNS commissural axons and peripheral motor axons. Neuron 17,203 -215.[CrossRef][Medline]
Morita, M. and Best, J. B. (1965). Electron microscopic studies on planarian. II. Fine structure of the neurosecretory system in the planarian Dugesia dorotocephala. J. Ultrastruct. Res. 13,396 -408.[CrossRef][Medline]
Morita, M. and Best, J. B. (1966). Electron microscopic studies of planarian. III. Some observations on the fine structure of planarian nervous tissue. J. Exp. Zool. 161,391 -413.[CrossRef][Medline]
Murase, S. and Horwitz, A. F. (2002). Deleted
in colorectal carcinoma and differentially expressed integrins mediate the
directional migration of neural precursors in the rostral migratory stream.
J. Neurosci. 22,3568
-3579.
Nakazawa, M., Cebrià, F., Mineta, K., Ikeo, K., Agata, K.
and Gojobori, T. (2003). Search for the evolutionary
origin of a brain: planarian brain characterized by microarray.
Mol. Biol. Evol. 20,784
-791.
Newmark, P. A. and Sánchez Alvarado, A. (2000). Bromodeoxyuridine specifically labels the regenerative stem cells of planarians. Dev. Biol. 220,142 -153.[CrossRef][Medline]
Newmark, P. A. and Sánchez Alvarado, A. (2002). Not your father's planarian: a classic model enters the era of functional genomics. Nat. Rev. Genet. 3, 210-219.[CrossRef][Medline]
Newmark, P. A., Reddien, P. W., Cebrià, F. and
Sánchez Alvarado, A. (2003). Ingestion of bacterially
expressed double-stranded RNA inhibits gene expression in planarians.
Proc. Natl. Acad. Sci. USA
100,11861
-11865.
Okamoto, K., Takeuchi, K. and Agata, K. (2005). Neural projections in planarian brain revealed by fluorescent dye tracing. Zool. Sci. 22,535 -546.[CrossRef][Medline]
Oosaki, T. and Ishii. S. (1965). Observation on the ultrastructure of nerve cells in the brain of the planarian Dugesia gonocephala. Z. Zellforsch. 66,782 -793.[CrossRef][Medline]
Oster, S. F., Bodeker, M. O., He, F. and Sretavan, D. W.
(2003). Invariant Sema5A inhibition serves an ensheathing
function during optic nerve development. Development
130,775
-784.
Oviedo, N. J., Newmark, P. A. and Sánchez Alvarado, A. (2003). Allometric scaling and proportion regulation in the freshwater planarian Schmidtea mediterranea. Dev. Dyn. 226,326 -333.[CrossRef][Medline]
Petrausch, B., Jung, M., Leppert, C. A. and Stuermer, C. A. (2000). Lesion-induced regulation of netrin receptors and modification of netrin-1 expression in the retina of fish and grafted rats. Mol. Cell Neurosci. 16,350 -364.[CrossRef][Medline]
Pineda, D. and Saló, E. (2002). Planarian Gtsix3, a member of the Six/so gene family, is expressed in brain branches but not in eye cells. Mech. Dev. 119,S167 -S171.[CrossRef][Medline]
Pineda, D., Gonzalez, J., Callaerts, P., Ikeo, K., Gehring, W.
J. and Saló, E. (2000). Searching for the
prototypic eye genetic network: Sine oculis is essential for eye regeneration
in planarians. Proc. Natl. Acad. Sci. USA
97,4525
-4529.
Plump, A. S., Erskine, L., Sabatier, C., Brose, K., Epstein, C. J., Goodman, C. S., Mason, C. A. and Tessier-Lavigne, M. (2002). Slit1 and Slit2 cooperate to prevent premature midline crossing of retinal axons in the mouse visual system. Neuron 33,219 -232.[CrossRef][Medline]
Reddien, P. W., Bermange, A. L., Murfitt, K. J., Jennings, J. R. and Sánchez Alvarado, A. (2005). Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria. Dev. Cell 8, 635-649.[CrossRef]
Reuter, M., Sheiman, I. M., Gustafsson, M. K., Halton, D. W., Maule, A. G. and Shaw, C. (1996). Development of the nervous system in Dugesia tigrina during regeneration after fission and decapitation. Invert. Reprod. Dev. 29,199 -211.
Romero, R. and Baguñà, J. (1991). Quantitative cellular analysis of growth and reproduction in freshwater planarians (Turbellaria; Tricladida). I. A cellular description of the intact organism. Invert. Reprod. Dev. 19,157 -165.
Saló, E. and Baguñà, J. (2002). Regeneration in planarians and other worms: New findings, new tools, and new perspectives. J. Exp. Zool. 292,528 -539.[CrossRef][Medline]
Sánchez Alvarado, A. and Newmark, P. A.
(1999). Double-stranded RNA specifically disrupts gene expression
during planarian regeneration. Proc. Natl. Acad. Sci.
USA 96,5049
-5054.
Sánchez Alvarado, A., Newmark, P. A., Robb, S. and Juste, R. (2002). The Schmidtea mediterranea database as a molecular resource for studying platyhelminthes, stem cells, and regeneration. Development 129,5659 -5665.[CrossRef][Medline]
Schmidt, H. A., Strimmer, K., Vingron M. and von Haeseler,
A. (2002). TREE PUZZLE: maximum likelihood phylogenetic
analysis using quartets and parallel computing.
Bioinformatics 18,502
-504.
Serafini, T., Kennedy, T. E., Galko, M. J., Mirzayan, C., Jessell, T. M. and Tessier-Lavigne, M. (1994). The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell 78,409 -424.[CrossRef][Medline]
Serafini, T., Colamarino, S. A., Leonardo, E. D., Wang, H., Beddington, R., Skarnes, W. C. and Tessier-Lavigne, M. (1996). Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system. Cell 87,1001 -1014.[CrossRef][Medline]
Srinivasan, K., Strickland, P., Valdes, A., Shin, G. C. and Hinck, L. (2003). Netrin-1/neogenin interaction stabilizes multipotent progenitor cap cells during mammary gland morphogenesis. Dev. Cell 4,371 -382.[CrossRef][Medline]
Stein, E., Zou, Y., Poo, M. and Tessier-Lavigne, M.
(2001). Binding of DCC by netrin-1 to mediate axon guidance
independent of adenosine A2B receptor activation.
Science 291,1976
-1982.
Taliaferro, W. H. (1920). Reactions to light in Planaria maculata with special reference to the function and structure of eyes. J. Exp. Zool. 31,59 -116.
Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22,4673 -4680.[Abstract]
Umesono, Y., Watanabe, K. and Agata, K. (1997). A planarian orthopedia homolog is specifically expressed in the branch region of both the mature and regenerating brain. Dev. Growth Differ. 39,723 -727.[CrossRef][Medline]
Umesono, Y., Watanabe, K. and Agata, K. (1999). Distinct structural domains in the planarian brain defined by the expression of evolutionarily conserved homeobox genes. Dev. Genes Evol. 209,31 -39.[CrossRef][Medline]
Vidal-Sanz, M., Bray, G. M., Villegas-Perez, M. P., Thanos, S. and Aguayo, A. J. (1987). Axonal regeneration and synapse formation in the superior colliculus by retinal ganglion cells in the adult rat. J. Neurosci. 7,2894 -2909.[Abstract]
Vielmetter, J., Kayyem, J. F., Roman, J. M. and Dreyer, W. J. (1994). Neogenin, an avian cell surface protein expressed during terminal neuronal differentiation, is closely related to the human tumor suppressor molecule deleted in colorectal cancer. J. Cell Biol. 127,2009 -2020.[Abstract]
Wang, L. H., Kalb, R. G. and Strittmatter, S. M.
(1999). A PDZ protein regulates the distribution of the
transmembrane semaphorin, M-SemF. J. Biol. Chem.
274,14137
-14146.
|