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First published online 17 October 2007
doi: 10.1242/dev.007138
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1 MIT Biology, Whitehead Institute, 9 Cambridge Center, Cambridge, MA 02138,
USA.
2 Howard Hughes Medical Institute, Department of Neurobiology and Anatomy,
University of Utah School of Medicine, 401 MREB, 20N 1900E, Salt Lake City, UT
84132, USA.
Author for correspondence (e-mail:
sanchez{at}neuro.utah.edu)
Accepted 25 August 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Asymmetry, BMP, Planaria, Regeneration, Adult homeostasis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Planarians are members of the
phylum Platyhelminthes and are currently viewed as part of the lophotrochozoan
grouping of animal phyla (Adoutte et al.,
2000). Planarians possess bilateral symmetry and are triploblastic
(Hyman, 1951). Reproduction is
accomplished either asexually through fission and regeneration or sexually as
cross-fertilizing hermaphrodites. Among the anatomical features of planarians
are a cephalized central nervous system (in addition to the cephalic ganglia,
photoreceptors and other sensory cell types are present in the head), a
musculature, a branched intestinal system for the delivery of nutrients, and a
single, centrally located, body opening (the pharynx). The ability of
planarians to regenerate entire animals from multiple types of body fragments
has been known for almost two centuries, but there is a limited understanding
of how this is achieved (Dalyell,
1814; Morgan,
1898; Randolph,
1897). Planarian regeneration involves formation of new tissues at
the wound site (blastema formation) and re-modeling of pre-existing tissue to
create an animal of proper proportions (morphallaxis)
(Morgan, 1898). Blastema
formation requires the action of a population of stem cells called neoblasts.
Because almost any type of planarian fragment can regenerate, a blastema can
form in cephalic, caudal, or lateral body regions and produce appropriate
corresponding tissue. Irregularly shaped fragments must create new tissue and
re-arrange pre-existing tissue in a coordinated manner to generate an animal
with symmetry, a complete complement of organ systems, and the proper
proportions and shape. How do wounded planarian tissues specify what to
regenerate, and what rules govern the rearrangement of body coordinates?
Recent technological developments (Newmark
and Sánchez Alvarado, 2000;
Newmark et al., 2003;
Reddien et al., 2005a;
Sánchez Alvarado and Newmark,
1999) make it now possible to begin to address these long-standing
questions.
Two genes that are predicted to encode components of a BMP signal
transduction pathway were found in a recent RNAi screen to be required for
normal blastema morphology (Reddien et
al., 2005a). Here we describe the roles of these genes, and of a
Schmidtea mediterranea BMP-like gene (smedbmp4-1), in
regeneration and homeostasis. BMPs are TGF-ß signaling ligands that
mediate a number of developmental events, such as embryonic dorsal-ventral
patterning and Drosophila imaginal disc patterning
(Diaz-Benjumea et al., 1994;
Martindale, 2005;
Padgett et al., 1987;
Spencer et al., 1982). Little
is known about the role this pathway may play in the maintenance of
differentiated structures and their regeneration after amputation. Our data
indicate that the regulated pattern of expression of a BMP-like gene in
planarians has a conserved dorsal-ventral patterning function in regeneration
and adult tissue maintenance, promotes midline patterning, and is needed for
the initiation of regeneration in cases in which bilateral symmetry is
disrupted by injury.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) served as a template for the PCR. Gene-specific primers in
nested PCR (5'-TGGACTGATTATGCGACC-3') followed by the inner primer
(5'-TGGATTTCCCCTCCGCAAAC-3') were used. The degenerate primer pool
(5'-CARGCNATGMGNCAYTGGG-3') served as the 5' primer in both
rounds. The resulting 850 bp band was T/A cloned using the pGEM-T Easy Vector
System II (Promega) and sequenced. The 5'-end of the
smedolloid-1 cDNA was isolated with 5'-RLM-RACE using the
FirstChoice RLM-RACE Kit (Ambion). The outer primer
(5'-CGACGTATGAACAACATCCG-3') followed by the inner primer
(5'-GGAATAAAGCTAAGGCACG-3') were used in the consecutive rounds of
nested PCR. The resulting 650 bp band was T/A cloned into pGEM-T Easy
(Promega) and sequenced. The smedbmp4-1 gene was isolated by PCR
amplifications from cDNA cloned into pBluescript
(Sánchez Alvarado et al.,
2002) with the primer 5'-GCACGGTTTTGGAATAGAAAGGTC-3'
and a primer that hybridizes to vector sequence. A second product used the
primer 5'-ACTGTCCTTACCCGTTATCAG-3' and a vector primer to amplify
the 3' end of the smedbmp4-1 gene. cDNAs were amplified by PCR
and cloned into the vector pDONRdT7 for RNAi using the Gateway (Invitrogen)
cloning procedure as previously described
(Reddien et al., 2005a). RNA
interference was performed by feeding bacteria expressing dsRNAs mixed with
blended liver to planarians as previously described
(Reddien et al., 2005a).
Antibody staining
Animals were fixed as previously described
(Newmark et al., 2003;
Sánchez Alvarado and Newmark,
1999). Following rehydration animals were blocked for 6 hours at
room temperature (RT) in PBTxB (PBS+0.3% Triton X-100+0.25% BSA), then
incubated overnight with 1:5000 VC-1 (kind gift of Dr K. Agata, RIKEN, Kobe,
Japan), 1:133 anti-Synaptotagmin (kind gift of Dr K. Agata), 1:400
anti-acetylated tubulin (Robb and
Sánchez Alvarado, 2002). Animals were rinsed with PBTxB,
then washed six times in PBSTxB over 6 hours, and labeled overnight with 1:400
goat anti-mouse Alexa Fluor 568 (Molecular Probes). Animals were washed as
before and mounted in Vectashield (Vector).
In situ hybridizations
Animals were fixed in Carnoy's fixative as described previously and stored
in methanol (Sánchez Alvarado et
al., 2002). Animals were rehydrated with a methanol:PBST (PBS,
0.1% Triton X-100) concentration series, pre-hybridized (50% formamide,
5x SSC pH 7.0, 100 µg/ml heparin, 0.05% Tween 20, 0.05% Triton X-100,
5 mM DTT, 1 mg/ml yeast RNA, 1x Denhardt's solution; wash Hybe) at
56°C for 2 hours, and hybridized with digoxigenin (DIG)-conjugated
riboprobes for 16 hours (pre-hybridization solution plus 5% dextran sulfate).
Animals were washed with 50% formamide, 5x SSC, 1x Denhardt's,
then transferred through a wash Hybe dilution series (75%, 50%, 25% in
2x SSC+0.1% Triton X-100), then twice with 2x SSC+0.1% Triton
X-100, then twice with 0.2x SSC+0.1% Triton X-100, and finally into MABT
(100 mM maleic acid, 150 mM NaCl, 0.1% Tween 20, pH7.5). Animals were blocked
in MABT plus 1% BSA and 10% horse serum for 1 hour at RT, and then labeled
with a sheep anti-DIG antibody (1:2000; Roche) in MABT+BSA+10% horse serum for
4 hours at RT. Animals were washed eight times with MABT and signal developed
using nitro-blue tetrazolium chloride (NBT) and
5-bromo-4-chloro-3'-indolyl phosphate p-toluidine salt (BCIP). For some
in situ hybridizations with smedolloid-1 and smedbmp4-1,
animals were killed in 10% N-acetyl cysteine for 5 minutes prior to
fixation. Some in situ hybridizations were done with the assistance of a
hybridization robot (Intavis, Köln, Germany).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Phenotypic analyses were used to group screen isolates into categories that
correspond to the sequential steps of regeneration - from wound healing and
regeneration initiation to the restoration of normal behavior. Inhibition of
two genes in this screen caused very similar abnormalities in blastema
patterning. Specifically, smedolloid-1(RNAi) and
smedsmad4-1(RNAi) animals regenerated new heads and tails that were
indented at the midline. This result suggested the possibility that these two
genes act together in planarians to regulate the midline patterning of
cephalic and caudal blastemas of these bilaterally symmetric animals.
smedsmad4-1(RNAi) animals that were challenged to go through two
rounds of regeneration following gene inhibition completely failed to form
blastemas (see below for more details). We report an analysis of the functions
of these genes in controlling positional patterning in homeostasis,
regeneration, and in the initiation of blastema formation in left-right
asymmetric fragments. Fig. 1A
defines relevant terminology for the planarian body regions and the surgical
manipulations performed.
The S. mediterranea SMEDBMP4-1 pathway
The smedolloid-1 and smedsmad4-1 genes were identified
from the cDNAs H.14.4a and H.17.9e, respectively. smedbmp4-1 was
identified by RT-PCR (see Materials and methods). smedolloid-1
encodes a protein similar to the Drosophila Tolloid protein (see Fig.
S1 in the supplementary material). Tolloid (BMP1-like) is a metalloprotease
that activates Decapentaplegic (DPP; BMP2/4-like) signaling by inhibition of
the BMP antagonist Short gastrulation (SOG; Chordin-like)
(Ferguson and Anderson, 1992;
Piccolo et al., 1997;
Shimell et al., 1991;
Wozney et al., 1988).
smedsmad4-1 encodes a protein similar to the SMAD4 protein (see Fig.
S2 in the supplementary material). SMAD4 is a co-SMAD that mediates the
signaling of multiple TGF-ß pathways
(Hahn et al., 1996;
ten Dijke and Hill, 2004). A
bmp2/4-like gene was previously identified in the planarian
Dugesia japonica and found to be expressed in the dorsal midline
(Orii et al., 1998).
smedbmp4-1 encodes a S. mediterranea protein similar to the
D. japonica BMP, Drosophila DPP, and to other BMP proteins,
including BMP2, 4, 5 and 7 proteins, and ADMP proteins (see Fig. S3 in the
supplementary material). BMP2, 4 and DPP proteins are TGF-ß signaling
ligands that mediate the dorsal-ventral patterning of embryos
(Holley and Ferguson, 1997;
Martindale, 2005;
Padgett et al., 1987;
Spencer et al., 1982). By
analogy to known functions for homologous proteins, the planarian pathway is
predicted to act as follows: SMEDOLLOID-1 inhibits BMP antagonists, promoting
the activity of SMEDBMP4-1; SMEDBMP4-1 acts as a signaling ligand, for which
signal transduction is mediated by SMEDSMAD4-1.
smedolloid-1 is expressed in scattered dorsal cells and smedbmp4-1 is expressed in dorsal midline cells (Fig. 1B,E). Significantly fewer cells on the ventral surface express smedolloid-1 than on the dorsal surface (Fig. 1C). smedbmp4-1 expression is broader in the head than in the body, expression weakens laterally, and an additional site of expression at the anterior end of the pharynx was observed (Fig. 1E). smedbmp4-1-expressing cells are dorsally localized, but subepidermal (Fig. 1F). RNAi of smedbmp4-1 and of smedolloid-1 eliminated detectable messages in in situ hybridizations (7/7 and 5/5, respectively, Fig. 1D,G). We could not detect an expression pattern for smedsmad4-1 with our in situ hybridization methods, but we detected greater than 80% knockdown of smedsmad4-1 by RNAi with quantitative RT-PCR (data not shown). Because of its dorsal and medial spatial distribution, the expression of smedbmp4-1 suggested a candidate role in midline formation and dorsal-ventral patterning.
SMEDBMP4-1 signaling controls the maintenance of adult body form
During adult planarian life, differentiated cells age and are constantly
replaced by the progeny of neoblasts
(Reddien and Sánchez Alvarado,
2004). This process not only requires neoblasts, but presumably
also the active control of body patterning even after development of adult
structures has occurred. Given the prominent role of BMP signaling in the
dorsal-ventral patterning of animal embryos
(De Robertis and Kuroda, 2004)
and the expression of BMP pathway genes in adult planarians, we sought to
determine whether homeostatic cell turnover requires BMP signaling. The C.
elegans unc-22 gene was used as a negative RNAi control for these and
other experiments presented throughout the text. This control presents animals
with dsRNA-containing bacteria, and because it lacks exact sequence identity
to any known planarian sequence fails to cause perturbation of any aspect of
planarian biology (Reddien et al.,
2005a). We continuously inhibited smedolloid-1,
smedbmp4-1 and smedsmad4-1 with RNAi and observed intact animals
over an extended period of time (Fig.
2A). Neoblasts in these animals must have been capable of basic
cell replacement because RNAi animals did not display any of the defects, such
as regression, curling and lysis within approximately 20 days, that are always
observed in irradiated animals that lack neoblasts
(Reddien et al., 2005a). By
contrast, intact smedolloid-1(RNAi), smedsmad4-1(RNAi), and
smedbmp4-1(RNAi) animals did display body plan abnormalities that
indicate a requirement for BMP signaling in the maintenance of the adult body
plan (Fig. 2A-F).
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).
Furthermore, smedsmad4-1(RNAi) and smedbmp4-1(RNAi) animals
lacked the normal pattern of dorsal cilia, instead possessing a ventral-like
distribution of cilia on their dorsal surfaces
(Fig. 2F).
smedsmad4-1(RNAi) animals could eventually flip over and glide on
their dorsal surface, indicating that the dorsal surface had in fact acquired
functional ventral-like cilia and animals had become functionally ventralized.
In other regeneration experiments, smedbmp4-1(RNAi) animals also
displayed the ability to glide on their dorsal surface (data not shown). In
short, animals largely consisted of two ventral sides. Unlike abnormal embryos
in many model systems, in which gross patterning abnormalities can lead to
embryonic lethality and preclude studies of function in later development or
adult life, gene perturbation in otherwise normal adult planarians can result
in live ventralized animals. Our RNAi experiments therefore allow the study of
late functional roles for genes that also have essential embryonic patterning
roles. Dorsal-ventral patterning throughout bilaterally symmetric animals
appears to be controlled by BMP signaling
(De Robertis and Sasai, 1996;
Martindale, 2005). Our data
indicate BMP signaling maintains the dorsal-ventral polarity of adult
planarians, even after proper dorsal-ventral patterning has been previously
set.
SMEDBMP4-1 signaling regulates midline tissue formation in symmetric regeneration
RNAi of smedolloid-1, smedsmad4-1 or smedbmp4-1 followed
by decapitation and tail removal led to the regeneration of blastemas indented
at the midline (e.g. n=62/62, 49/49, and 17/21, respectively;
Fig. 3A). Control RNAi animals
essentially never regenerated cephalic or caudal blastemas that were indented
at the midline. Because blastemas still arose after RNAi, yet showed a midline
defect, we surmised that BMP signaling is not needed for regenerative
outgrowth. In order to examine the cellular defects underlying the observed
morphological phenotypes, we examined the differentiation of distal blastema
cells by assessing expression of the D.21.6 gene. The D.21.6 gene is expressed
in cells that reside around the entire periphery of the animal
(Kato et al., 1999). D.21.6
expression was perturbed at the midline of cephalic and caudal blastemas of
RNAi animals (smedolloid-1(RNAi): 9/10 cephalic and 9/10 caudal
blastemas abnormal; smedsmad4-1(RNAi): 8/8 cephalic and 7/8 caudal
blastemas abnormal; smedbmp4-1(RNAi): 3/10 cephalic and 10/10 caudal
blastemas were abnormally dispersed in dorsal-ventral directions with some
gaps at the midline; Fig. 3B).
These data indicate indented blastemas may arise because of absence of
particular cell types.
|
).
Therefore, additional TGF-ß signaling may be affected by inhibition of
smedsmad4-1. We previously observed that cephalic blastema formation
failed in smedsmad4-1(RNAi) animals during a second round of
regeneration (Reddien et al.,
2005a). We found that smedsmad4-1(RNAi) animals can fail
to produce a blastema following a single amputation. Specifically, animals cut
17 days following the initial of four dsRNA feedings completely failed to
regenerate blastemas (Fig. 3C).
In some of these animals photoreceptors and outgrowths eventually developed in
pre-existing tissues, indicating abnormal attempts at replacing missing
tissues occurred in the absence of blastema formation
(Fig. 3C). Minimal development
of head structures appeared in the pre-existing tissue of
smedsmad4-1(RNAi) animals that were cut 23 days following initial
dsRNA exposure. Together, these results suggest that smedsmad4-1
controls a process that is necessary for blastema formation. Similar RNAi
protocols with the smedbmp4-1 and smedolloid-1 genes failed
to block blastema formation. Because SMAD4 proteins can mediate the
transduction of multiple TGF-ß signals, smedsmad4-1 may function
with additional TGF-ß proteins - separate from SMEDBMP4-1 - to mediate
blastema formation.
SMEDBMP4-1 signaling affects patterning during symmetric regeneration
BMP signaling is known to regulate the dorsal-ventral patterning of embryos
in many species (De Robertis and Sasai,
1996; Martindale,
2005) and, as described above, controls the dorsal-ventral
patterning in adult planarian tissue homeostasis. To determine the patterning
role of SMEDBMP4-1 signaling during symmetric regeneration, we assayed for the
regeneration of dorsal midline cilia. In the cephalic blastemas of
smedbmp4-1(RNAi) and smedsmad4-1(RNAi) animals, we detected
abnormal cilia (Fig. 4A).
Specifically, the normal stripe of dorsal cilia seen in control cephalic
blastemas was absent in both smedsmad4-1(RNAi) and
smedbmp4-1(RNAi) animals (10/10 and 2/4, respectively), accompanied
by ventral-like and dispersed cilia (8/10 and 3/4, respectively). These
observations suggest a dorsal-ventral patterning abnormality existed during
regeneration in the animals. In the case of smedolloid-1(RNAi)
animals, dorsal cilia were either not readily detected at all or displaced
from the midline of blastemas after 7 days of regeneration (n=6;
Fig. 4A).
We also determined patterns of photoreceptor neuron axons and the cephalic
ganglia in RNAi animals. Normal photoreceptor neurons have projections that
cross the midline and connect to the cephalic ganglia
(Fig. 4B). In
smedolloid-1(RNAi) animals, both photoreceptor neurons and the
cephalic ganglia failed to reach and cross the midline (n=11/11). By
contrast, the photoreceptor neuron axons and cephalic ganglia of
smedsmad4-1(RNAi) and smedbmp4-1(RNAi) animals crossed the
midline (11/11 and 10/10, respectively). However, the photoreceptor axons of
smedsmad4-1(RNAi) and smedbmp4-1(RNAi) animals projected in
an atypical manner dorsally and laterally (11/11 and 9/10, respectively). Only
one of 22 control RNAi animals displayed a similar dorsal projection. To
determine whether the neuronal midline crossing abnormalities of
smedolloid-1(RNAi) animals required SMEDBMP4-1 signaling, we
inhibited both smedbmp4-1 and smedolloid-1 by feeding
mixtures of bacteria expressing dsRNA for each of the two genes. Control
animals were fed mixed sets of bacteria, in which one set expressed dsRNA for
the C. elegans unc-22 gene. Animals in which smedbmp4-1 and
smedolloid-1 were both inhibited had more severely indented blastemas
than did control single gene-inhibited animals (n=28; not shown).
Whereas control smedolloid-1(RNAi) animals had midline crossing
abnormalities (8/10), doubly inhibited animals had the
smedbmp4-1(RNAi)-like appearance
(Fig. 4B, 9/9). This
observation suggests that some of the cellular abnormalities of
smedolloid-1(RNAi) animals resulted from an aberrant pattern of
SMEDBMP4-1 activity. Tolloid proteins generally function to enhance the
signaling activity of BMP2/4/DPP-like proteins by cleaving a Chordin-like BMP
antagonist (Piccolo et al.,
1997). Why photoreceptor axon patterning differences between
smedolloid-1(RNAi) and smedbmp4-1(RNAi) animals exist is
unknown, but could reflect a role for SMEDOLLOID-1 in determining the normal
pattern - as well as level - of SMEDBMP4-1 activity. It is also possible that
there exists some independent roles for these genes. Together, our
observations reveal midline and dorsal-ventral patterning abnormalities that
occur following perturbation of BMP signaling in planarian regeneration.
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).
Blastemas were scored as unpigmented outgrowths from pigmented pre-existing
tissue at the wound site. smedolloid-1(RNAi) thin fragments failed to
regenerate along the body length (n=38/43), but all produced a head
with both photoreceptors; thick fragments also failed to regenerate
(n=45/49). smedsmad4-1(RNAi) thin (n=34) and thick
(n=37) fragments failed to regenerate along the body length. RNAi of
smedbmp4-1 caused a less severe block in lateral regeneration than
did RNAi of smedolloid-1 or smedsmad4-1 and was coupled with
aberrant growths and tissue ruffling; however, animals fed dsRNA six times and
amputated parasagittally 29 days following dsRNA exposure were robustly
abnormal (n=18 thick fragments displayed minimal to no blastema
formation and n=14 thin fragments displayed folding and ruffling of
tissue along the wound site as well as minimal apparent blastema formation).
In addition, perturbation of BMP pathway genes in animals cut sagittally -
along the axis of left-right symmetry - produced animal halves that displayed
failures in blastema formation (see Fig. S4 in the supplementary
material).
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), it appears to control a necessary step in blastema
formation in bilaterally asymmetric fragments. Though RNAi may not result in
complete gene knockdown, the difference between symmetric and asymmetric
regeneration is highly penetrant and has been demonstrated with the same sets
of RNAi animals. Therefore, we suggest that asymmetric and symmetric blastema
formation have different genetic requirements.
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), appears
anew in the pre-existing tissue of asymmetric fragments between 6 hours (0/8
expressed smedbmp4-1) and 12 hours following wounding (10/10 thin
lateral fragments expressed smedbmp4-1;
Fig. 6A). These new
smedbmp4-1-expressing cells appeared initially along the wound site,
resolved to a midline pattern within 8-14 days
(Fig. 6A), and appeared similar
in size and position to the smedbmp4-1-expressing cells of an intact
animal.
To assess whether smedbmp4-1 expression results from the
production of new cells, requiring cell proliferation, we used irradiation.
Irradiation has long been known to specifically eliminate the only known
mitotically active cells in planarians, i.e. the neoblasts
(Bardeen and Baetjer, 1904;
Dubois, 1949;
Reddien et al., 2005b). We
amputated irradiated animals parasagittally and examined expression of
smedbmp4-1. We found that smedbmp4-1 expression can occur in
irradiated thin fragments, indicating expression does not require cell
proliferation. For example, we performed parasagittal amputations 7 days after
irradiation and the resultant thin fragments, lacking original
smedbmp4-1 expression, newly expressed smedbmp4-1 normally
(n=15; Fig. 6B). The
initial expression of smedbmp4-1 in these irradiated fragments failed
to resolve into a midline pattern before animal death, indicating resolution
of the smedbmp4-1 pattern requires regeneration
(Fig. 6B). Regenerating
planarian fragments not only produce new tissue at wound sites (blastema
formation), but also dramatically re-arrange pre-existing tissues to result in
a small animal with a complete complement of organ systems of appropriate
proportions. Our data suggest that new smedbmp4-1-expressing cells
are produced from a change in function of other cells. We suggest that
activity of smedbmp4-1 at these new sites changes positional
identities of existing tissues and plays an instructive role in the formation
of lateral blastemas.
smedbmp4-1 expression expands towards missing sides
Why do thick fragments lacking a side, but containing a pre-existing
midline, have blastema formation abnormalities when the smedbmp4-1
pathway is perturbed by RNAi? We found that asymmetric thick fragments
displayed changed expression of smedbmp4-1
(Fig. 6C). Specifically, the
field of cells expressing smedbmp4-1 expanded towards the missing
lateral side, such that cells expressed smedbmp4-1 more strongly in
more lateral regions than is normally observed in the wild type or in freshly
amputated animals (Fig. 6C). By
contrast, simple wounding was not sufficient to induce robust new expression
of smedbmp4-1. This was demonstrated by generating side incisions,
both perpendicular and parallel to the midline and which healed by resealing.
We detected no obvious increase in expression of smedbmp4-1 along
these wound sites (Fig. 6D).
These observations indicate that maintenance of smedbmp4-1 signaling
only at the midline requires an intact, lateral side. Furthermore, amputation
near the midline did not obviously cause an expansion on the unwounded side,
indicating expansion occurs towards missing sides only
(Fig. 6D). Given
smedbmp4-1 pathway activity is needed for thick pieces lacking a side
to regenerate, an expanded field of smedbmp4-1 expression could have
an instructive role in the regeneration of a missing side.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The process of bilaterally asymmetric regeneration can be observed after parasagittal amputations that generate a thick fragment possessing the original midline - now off-center - and a thin fragment with no original midline tissue. How do these pieces re-acquire bilateral symmetry? Our data suggest that new smedbmp4-1 expression in asymmetric fragments directs the changes to tissues that must occur in regeneration. In thin lateral pieces, for example, expression was induced within 12 hours of amputation on the side of the fragment near the old midline. This new smedbmp4-1 expression did not require new cell production. Therefore, mechanisms exist that allow a rapid change in site of expression of a signaling protein regulating planarian body patterning within existing cells. We suggest change in lateral positional identity is a needed step in asymmetric blastema formation. Changes in the site of expression of smedbmp4-1 are also involved in the regeneration of sides in thick asymmetric fragments that still possess the original midline. Specifically, lack of a side resulted in the spreading of the field of smedbmp4-1-expressing cells towards the wound. Because simple wounding did not induce smedbmp4-1 expression, we suggest that the left and right flanks of the planarian body plan normally constrain smedbmp4-1 expression. In this model, the absence of a side alleviates constraint and smedbmp4-1 expression expands. Animals may detect that they are missing a side because SMEDBMP4-1 becomes expressed at or expands towards wound sites capable of generating a regeneration blastema. There are multiple known BMP antagonists; such an antagonist could act in planarians to normally restrict smedbmp4-1 expression. Based upon data from other organisms, a Chordin-like molecule would be the best candidate, but no Chordin-like gene can, as yet, be identified in existing planarian gene and genome sequence. The ongoing planarian genome sequence project could allow exploration of such a hypothesis in the future.
BMP2/4/DPP functions in many animal embryos in dorsal-ventral patterning
(Holley et al., 1995). Our
data indicate that BMP signaling in planarians regulates dorsal-ventral
patterning of new tissues in regeneration and during the maintenance of adult
tissues. Given the current positioning of planarians in the understudied
Lophotrochozoa (Adoutte et al.,
2000), one of three main groupings of bilaterally symmetric
animals, our data also support the view of a BMP system controlling dorsal
ventral patterning throughout the Bilateria
(De Robertis and Sasai, 1996;
Martindale, 2005).
Specifically, smedbmp4-1 signaling regulated dorsal-ventral and
midline patterning during adult tissue homeostasis and regeneration. Normal
dorsal-ventral patterning is likely to be accomplished by regulated expression
of smedbmp4-1 to the dorsal midline. In the absence of planarian BMP
signaling, we observed otherwise normal, fully formed adults slowly transform
into animals with two ventral sides. This transformation of adult form did not
preclude animal viability and is a striking demonstration of the role of
signaling molecules in the maintenance of the pattern of adult tissues. Adult
life requires the ability to functionally replace damaged and aged cells.
These tasks are accomplished by stem cells and the descendants of stem cells;
approximately 10 billion cells are produced per day in the human body during
normal homeostasis (Heemels,
2000). How new cells are deployed to enter solid tissues and
maintain adult form is an understudied arena of biology. Our data indicate
that BMP signaling has an instructive role in the maintenance of dorsal
character in tissues being replaced gradually by stem cells.
Animals lacking the normal function of a planarian Tolloid-like gene
(smedolloid-1) or a BMP2/4/DPP-like gene (smedbmp4-1) were
able to regenerate anterior and posterior blastemas, symmetrically around the
midline, but these were abnormal (indented and lacking some differentiated
tissues) at the midline. These data indicate BMP-like signaling regulates
midline regeneration. Midline formation is a critical component of bilaterally
symmetric body plans (Meinhardt,
2004). The conserved localization of BMP2/4/DPP activity at the
dorsal midline suggests that universal BMP-mediated medial patterning
strategies may be deployed to accomplish a multitude of developmental tasks
across the metazoa.
Despite the similar aspects of the phenotypes associated with inhibition of the BMP pathway components studied in this report, some differences exist. For example, smedolloid-1(RNAi) animals display very severe midline patterning abnormalities (e.g. failures of axons to cross the midline), but do not display the ventralization defects observed in smedbmp4-1 and smedsmad4-1 RNAi animals. Furthermore, smedbmp4-1(RNAi) animals showed a generally weaker lateral regeneration defect than did animals in which smedolloid-1 or smedsmad4-1 were perturbed. The reasons for these differences are unknown, but could indicate independent functions for some of these genes, or redundancy with undiscovered factors. Future investigation of these genes and other BMP pathway components should help illuminate the mechanisms by which the BMP pathway components act together, or separately, in regeneration.
Planarians constantly rebuild their adult bodies during normal homeostasis
and are capable of dramatically altering pre-existing tissues during
regeneration (Morgan, 1898;
Morgan, 1900;
Morgan, 1902;
Reddien and Sánchez Alvarado,
2004). These properties of planarian regeneration have long
puzzled biologists. Robust patterning and regulatory systems must exist that
allow animal tissue to specify what to make and in what manner to re-pattern
existing tissue. Understanding these patterning and regulatory systems should
prove critical in understanding regeneration. The problems faced by a
regenerating planarian bear some similarities to those faced by a surgically
manipulated embryo. Specifically, some injured embryos and animals can restore
the pattern of the entire animal and produce what would have existed in the
removed tissue. Experimental embryology introduces these concepts as
regulative development, in which some animal embryos replace missing cells via
self-regulation. In extreme examples, one cell of the two-cell sea urchin
embryo can develop into a smaller, but normally patterned larva, and the
dorsal half of a Xenopus blastula embryo can produce an entire embryo
(Driesch, 1893;
Reversade and De Robertis,
2005; Spemann,
1924). Self-regulation of dorsal-ventral patterning, following
surgical manipulation of Xenopus embryos, involves a BMP signaling
system (Reversade and De Robertis,
2005). In planarians the ability to restore animal form can occur
from a large array of wound types and involves the production of new tissue at
wound sites acting in concert with changes in pre-existing tissue. These
processes lack mechanistic explanation. Our data identify a key role for a BMP
signaling system in lateral blastema formation. Our experiments with the
smedbmp4-1 pathway provide some of the first genetic insights into
the topics of blastema specification and restoration of form in regeneration,
and provide paradigms for future study of the genetic and cellular bases for
the tissue patterning principles of planarian regeneration.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/22/4043/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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