seminars in C E L L & D E V E L OP M E N T A L B I OL OG Y , Vol 10, 1999: pp.
377]383
Article No. scdb.1999.0324, available online at http:rrwww.idealibrary.com on
Molecular and cellular aspects of planarian regeneration
Kiyokazu AgataU and Kenji Watanabe
Neoblasts, which are classically defined as prospective
totipotent stem cells, supporting planarian regeneration,
now have been identified by expression of the DjvlgA gene
coding for a vasa-type ATP-dependent RNA helicase.
DjvlgA-positive cells are distributed in the mesenchymal
space from head to tail, participating in formation of organ
rudiments and the blastema during regeneration.
Interestingly, neoblasts begin to transcribe tissue-specific genes
in a position-dependent manner, while they are still in the
mesenchymal space. This is prior to migrating to the organ
rudiments or blastema, and at a time when they are not yet
morphologically distinguishable from neoblasts. We speculate
that the mRNAs transcribed in the committed cells may be
trapped in a complex with RNA helicase(s), forming a
‘chromatoid body’, and are not translated into protein until
they migrate to the rudiment. After formation of the
rudiments, these committed cells may receive a signal for
organogenesis and then start to translate these mRNAs as
well as to express pattern formation genes for organogenesis.
Key words: commitment r planarian r post-transcriptional
regulation r regeneration r stem cell
Q1999 Academic Press
Introduction
PLANARIAN REGENERATION WAS extensively studied by
Thomas Hunt Morgan approximately 100 years ago.
He wrote several long papers about planarian regen-
eration from 1898 to 1903.1 ] 3 In his paper of 1900 he
described using Drosophila as a food for planaria, and
that he had succeeded in staining planarian digestive
ducts by feeding Drosophila eyes.2 However, the his-
tory of science during the 20th century shows that it
U
From the Laboratory of Regeneration Biology, Department of
Life Science, Faculty of Science, Himeji Institute of Technology,
Hyogo 678-1297, Japan. E-mail: agata@sci.himeji-tech.ac.jp
Q1999 Academic Press
1084-9521r 99 r 040377q 07 $30.00r 0
377
is the food of planaria, rather than planaria them-
selves, that have contributed greatly to the under-
standing of complex developmental processes. In
1991 we began to apply molecular approaches to the
study of planaria, to understand the underlying
mechanisms by which cells can recognize the loss of a
part of the body and then reconstruct it. To modern-
ize planarian studies we have developed clonal strains
of planaria that can be propagated under laboratory
conditions, isolated tissue-specific molecular markers
and regulatory genes, and established a highly-sensi-
tive in situ hybridization method, as well as a method
for chimeric analysis. Here, we review the recent
findings concerning planarian regeneration using the
newly established techniques and molecular probes.
Modernization of planarian regeneration studies
To obtain a genetically homogeneous population of
planaria, we have tried to establish clonal strains.
One species of planarian that was collected in the
wild was able to adapt to laboratory conditions. It can
propagate by fission, and lives in autoclaved water on
food made from chicken livers. This clonal strain of
planarian, Dugesia japonica, was named GI since it was
derived from the Iruma River in Gifu Prefecture. GI
was established in 1991 as a clonal strain and then
extensively propagated by asexual reproduction. Fur-
thermore, in 1996, we were able to obtain eggs and
embryos from GI animals maintained at 128C in the
National Institute for Basic Biology in Okazaki. It is
very interesting that this strain can be converted to a
sexual state by changing rearing conditions.4,5
Chimeric analysis is indispensable for monitoring
cellular behavior during regeneration and also to
identify the stem cells. We have succeeded in isolat-
ing a cDNA clone ŽPH20. which stains all the cells of
GI animals, but not those of HI animals. HI is a newly
established laboratory strain of D. japonica collected
in the Iwaya valley in Hyogo Prefecture.6
Cell type-specific genes were isolated by im-
munoscreening or random sequencing of 200 head-
K. Agata and K. Watanabe

and 1000 eye-cDNA clones.7 ] 9 We are now able to
identify neurons, pharynx-muscles, body wall-muscles,
type II-mucous producing cells, and visual cells using
gene probes. Otx r otd, HOX r HOM and vasa-related
genes were isolated by PCR using degenerater
primers.4,10 ] 12 We also invested a lot of time in the
development of a highly-sensitive in situ hybridization
method. The successful method includes acid-treat-
ment of the worms before fixation and long hy-
bridization times at low probe concentration.7,11
Are the stem or undifferentiatedcells the major
source of planarian regeneration?
What type of cells participate in planarian regenera-
tion? Classical observations and experiments sug-
gested that totipotent stem cells, so called ‘neoblasts’,
may exist in the mesenchymal space through the
entire body of the worm and differentiate to supply
appropriate cells for regeneration.13,14 However, the
evidence supporting this idea is still scarce. On the
other hand, in the case of regeneration in the verte-
brates, it has been clearly demonstrated by cell cul-
ture and molecular markers that differentiated cells
can participate in regeneration through dedifferen-
tiation events,15 ] 17 and see Gardiner et al,18 this issue.
Even in planarian regeneration, it has been suggested
based on electron-microscopic observations, that the
pharynx-lumen epithelium may form by transdiffer-
entiation from intestinal epithelial cells. However, we
could not find any evidence indicating a contribution
from differentiated cells to planarian regeneration,
even though we have extensively analyzed the regen-
eration process using molecular markers and
chimeric analysis.19 On the contrary, our observa-
tions clearly indicate that the cells participating in
regeneration appear in the mesenchymal space where
the stem cells are distributed.4,19 In the case of phar-
ynx regeneration, DjMHC-A Ža pharynx-muscle-
specific gene; MHC, myosin heavy chain. expressing
cells appear in the mesenchymal space surrounding
the prospective pharynx forming region before for-
mation of a pharynx rudiment.19 These observations
suggest that the cells participating in planarian re-
generation may be mainly derived from the undif-

Figure 1. The structure of the chromatoid body and expression pattern of DjvlgA. ŽA. The
chromatoid bodies are derived from the nucleus. nu, nucleus; cb, chromatoid body; mt,
mitochondoria. Arrows indicate the chromatoid bodies passing through nuclear pore Žmodified
from ref. 20.. ŽB. DjvlgA-expressing cells are located in the mesenchymal space. ŽC. Accumulation
of DjvlgA-expressing cells are observed in the blastema and in the regenerating pharynx in
planaria regenerating from a head piece. ŽD. Expression of DjvlgA decreased drastically 10 days
after X-ray irradiation. Modified from ref 4.

378

ferentiated cells distributed in the mesenchymal
space, rather than from the dedifferentiation of dif-
ferentiated cells.
Identification of stem cells by a molecular
marker
To confirm that the cells participating in regenera-
tion may be derived from stem cells or undifferenti-
ated cells in the mesenchymal space, it is indis-
pensable to isolate a molecular marker to be able to
identify the stem cells or undifferentiated cells in the
mesenchymal space. To this end we focused on a
unique morphological feature in the cytoplasm of
neoblasts the ‘chromatoid body’. Chromatoid bodies
are round, moderately electron-dense, not sur-
rounded by membrane, and derived from the nu-
cleus ŽFigure 1A..20 The structure of the chromatoid
body is reminiscent of germline granules. It has been
demonstrated that actinomycin D-treatment collapses
the chromatoid bodies and that it shows affinity for
RNase,20,21 suggesting that the chromatoid body may
be a complex of RNAs and proteins. We expected
that the chromatoid body might contain moleculeŽs.
in common with the germline granule. An ATP-de-
pendent RNA helicase with a DEAD box motif, a
member of the VASA family, has been identified as a
major component of the germline granule in a vari-
ety of animals. We have isolated two vasa-like RNA
helicase genes from planaria, and confirmed that one
of them, DjvlgA is specifically expressed in chroma-
toid body-containing cells.4
The distribution pattern and behavior of DjvlgA-
expressing cells is very interesting. DjvlgA-expressing
cells are distributed in the mesenchymal space from
head to tail ŽFigure 1B. and the number of DjvlgA-
expressing cells is drastically reduced by X-irradiation
ŽFigure 1D.. In the process of regeneration, DjvlgA-
expressing cells accumulate in the stump and stump-
proximal region within 1 day after cutting. Extensive
accumulation of DjvlgA-expressing cells was observed
in the growing blastema and pharynx rudiment ŽFig-
ure 1C.. These DjvlgA-expressing cells of the blastema
begin to decrease 3 days after cutting, when regener-
ating brain and pharynx can be distinguished mor-
phologically. These observations closely match the
behavior of neoblasts containing chromatoid bodies.
379
Planarian regeneration
Figure 2. Cells participating in regeneration appear in the
mesenchymal space in a position-dependent manner, be-
fore migrating to the organ rudiments or blastema. ŽA.
Whole mount in situ hybridization of head pieces at 3 days
of regeneration. Neck-forming Žmiddle piece. and
pharynx-muscle-forming cells Žright piece. appear in the
mesenchymal space surrounding the brain Žleft piece. at 3
days after amputation. ŽB. Saggital section at 3.5 days of
regeneration of head piece. Arrowheads indicate that
DjMHC-A-expressing cells appear in the mesenchymal space
in the stump, not in the blastema, and participate in
pharynx-muscle formation through the base of the pharynx
rudiment. Modified from ref 19.
When and where are the stem cells committed as
regenerating cells?
The study using DjvlgA gene as a probe supports the
classical view in which the stem cell system is the
major source of cells participating in regeneration.
However, in the classical view, it has been believed
that the stem cells or undifferentiated cells derived
from the stem cells, may not become committed until
they are part of the blastema. Yet, our observations
using tissue-specific probes clearly indicates that they
are committed in a position-dependent manner in
the mesenchymal space before migrating to the or-
gan rudiments or blastema ŽFigure 2..19 This finding
is very critical for an understanding of the regenera-
tion mechanisms in planaria. Although Morgan1,2
described planarian regeneration as morphallaxis, as
K. Agata and K. Watanabe

Figures 3, 4 & 5. Legends on facing page.

380
 Planarian regeneration

observed in the regeneration process of hydra, later
scientists emphasized the formation of a blastema
and compared the planarian regeneration system to
that of the vertebrates since planaria form a blastema,
whereas hydra does not. Planaria form a blastema
which is recognized externally as a white region and
in sections as a mass of morphologically undifferen-
tiated cells. However, our observations suggest that
most of the morphologically undifferentiated cells in
the organ rudiments or blastema may have already
been committed to differentiate to appropriate cell
types. For example, the pharynx muscle-forming cells
may be committed in the stump-proximal region of
the head piece, not in the blastema nor in the phar-
ynx rudiment ŽFigure 2B.. These findings support the
idea that differentiation of the stem cells or undif-
ferentiated cells in the mesenchymal space may be
regulated in a position-dependent manner, suggest-
ing that these cells may have a molecular system to
recognize their own position within the body, possi-
bly in common with hydra. This idea is also sup-
ported by the dramatic change in HOX gene expres-
sion after cutting.12
Bueno et al,22 described the expression pattern of
MHC protein using an anti-MHC monoclonal anti-
body during pharynx regeneration. They detected
the MHC protein in the pharynx rudiment but not in
migrating cells in the mesenchymal space, suggesting
that DjMHC mRNAs in migrating cells detected by in
situ hybridization may not be translated to the pro-
tein. So, we speculated that these mRNAs may be
trapped in the chromatoid body by making a com-
plex with RNA helicaseŽs., preventing translation of
MHC mRNAs, allowing the migrating cells to main-
tain their undifferentiated state until reaching the
pharynx rudiment. The cellular events and gene ex-
pression at the early stages of regeneration are sum-
marized in the left portion of Figure 3.
Does pattern formation occur after migration of
the committed cells to the rudiment?
In the case of brain regeneration we could not detect
cells committed to a neuronal fate in the mesenchy-
mal space using neuron-specific probes.7,9 We could
not understand the reason for this, because cells
committed to pharynx muscle or mucous-producing
cells can be detected at an early stage of regenera-
tion. When we isolated Otx r otd-related genes from
planaria, we expected to be able to detect cells
committed to a neuronal fate by using such transcrip-
tion factor genes as probes, since regulatory genes
are expressed prior to differentiation genes in gen-
eral. However, expression of Otx r otd-related genes
cannot be detected until after formation of the brain
rudiment, and not in the migrating cells ŽFigure
4..10,11 Expression patterns of Otx r otd-related genes
suggested that these genes may be involved in later
pattern formation of the complex brain, but not in
the commitment of neurons. So, we concluded that
Otx r otd-related genes categorized as pattern forma-
tion genes may be transcribed after formation of the
rudiment. Transcription of other neuron-specific
gene,7,9 encoding a neuropeptide processing enzyme
or synaptotagmin, may be regulated under the con-
trol of the pattern formation genes since both pro-
teins are expressed in the same sub-population of the
neuronal cells. In the case of pharynx regeneration
the cells committed to the pharynx-muscles start to
transcribe MHC genes before formation of the phar-
ynx rudiment since MHC may be a common protein
in all subtypes of pharynx muscles. After formation of
the pharynx rudiment the muscle cells start to dif-
ferentiate into specific muscle types under the con-
trol of pattern formation genes, to form the well-
organized pharynx structure. So, we speculate that
tissue-specific genes may be categorized into two

Figure 3. A schematic drawing of the cellular and molecular events during planaria regeneration. See text for further
details.
Figure 4. Expression pattern of DjotxA, DjotxB and Djotp in normal adults and worms regenerating from middle pieces. In
regenerates at 36 h after amputation, expression is detected in the paired domains corresponding to brain rudiment at the
regenerating anterior tips of the middle pieces. By 40 h, the normal adult pattern of expression appears to be established;
DjotxA is expressed medially, DjotxB in the intermediate region and Djotp in the presumptive branch region. These positive
cells are not detected in the mesenchymal space before formation of the brain rudiment. Modified from refs 10,11.
Figure 5. Comparison of the process of projection formation by DV-reversed graft with the regeneration process. ŽA.
Summary of development of DV-reversed graft. The site where dorsal tissue Žyellow. adheres to ventral tissue Žblue. induces
formation of a blastema-like region, outgrowth Žarrows. and establishment of a DV axis. ŽB. Model for the role of DV
interaction in planaria regeneration. Dorsal and ventral tissues adhere to each other as a result of wound closure.
Subsequently, DV interaction induces blastema formation, outgrowth Žarrows. and establishment of a DV axis. A, anterior;
P, posterior; D, dorsal. V, ventral; yellow, dorsal side; blue, ventral side; dark blue, brain; gray, ventral nerve cord; red, DV
boundary. Reproduced with permission from The Company of Biologists, see ref 6.

381
K. Agata and K. Watanabe
types, common and subtype-specific genes. We pro-
pose that the common genes may be transcribed in
the process of commitment in the mesenchymal space
under the control of positional cues, and that the
subtype-specific genes may be transcribed under the
control of pattern formation genes within each or-
gan.
The cellular events and gene expression patterns at
the late stage of regeneration are summarized in the
right portion of Figure 3. We need more experimen-
tal evidence to refine the schematic drawing of the
cellular events during planarian regeneration. Espe-
cially, we lack information about proliferation capac-
ity at each step. It is a very serious problem for us that
nobody has yet succeeded in staining proliferating
cells by administration of BrdU. Transplantation ex-
periments of single isolated cells into X-irradiated
worms are essential to understand the stem cell sys-
tem in planaria.
Is regeneration triggered by DV interaction?
How do cells in the mesenchymal space recognize
the amputation and newly rearranged positional
identities? We have succeeded in showing, by trans-
plantation experiments, that an ectopic dorso-ventral
ŽDV. interaction is sufficient to trigger planarian re-
generation.6 We grafted a small piece of planarian
body back to the original location but in DV reversed
orientation. Four days after grafting, blastema-like
white regions were formed on the boundaries of the
host and the donor. The regions began to grow, and
after 1 week resulted in cup shaped projections at
both dorsal and ventral sides of the worm. In the
control experiments such blastema-like white regions
were never formed although the nerve cord was
severely damaged, suggesting that an injured nerve
cord is not sufficient for the onset of the regenera-
tion. Ectopic DV interaction appears to cause
blastema formation, outgrowth and establishment of
DV axis ŽFigure 5.. In addition, tissue- and region-
specific markers have shown that the projections may
be identical to the structure from the position of the
graft to the most anterior or posterior tip. These
results suggest that DV interaction evoked by wound
closure plays an important role in the onset of regen-
eration, and especially in blastema formation. The
blastema may have the most distal characteristics and
function as a center to send positional cues for the
rearrangement of positional identity of the undiffer-
entiated cells in the mesenchymal space.
382
Conclusions
Here we have reviewed recent progress in our labora-
tory. Both authors have studied transdifferentiation
in vertebrate regenerating system.15,23 Before starting
the study of planarian regeneration, we believed that
we could find evidence for transdifferentiation dur-
ing planarian regeneration. However, we failed to
find evidence of transdifferentiation. Our studies lead
us to conclude that the planarian regeneration sys-
tem is similar to that of early vertebrate embryos.
Planaria appear to have stem cells in the body whose
differentiation is regulated by positional cues. In the
case of the mouse embryo, ES cells can differentiate
into positionally-appropriate cells and be integrated
into a chimeric mouse if they are injected into nor-
mally developing embryos, acquiring positional cues
from their neighbouring normal embryonic cells.24
Furthermore, it has been demonstrated that ependy-
mal cells, which function as neural stem cells in adult
mouse brain can become hematopoietic stem cells
after transplantation into the bone marrow.25 These
results suggest that the cellular systems operating in
planarian regeneration may share more features in
common with the rest of the animal kingdom than
expected. We therefore anticipate that progress in
planaria research will contribute to new insights into
the cellular renewal systems of all multicellular ani-
mals.
Acknowledgements
We thank Prof. S.V. Bryant for critical reading of the
manuscript. We also thank Prof. T.S. Okada, H. Kondoh
and S. Aizawa for their encouragement in our studies. We
are grateful to all of the colleagues in our laboratory and
particularly to Kentaro Kato for his help in making figures.
These studies are supported by Special Coordination Funds
for Promoting Science and Technology to K.A., and a
Grant-in-Aid for Scientific Research on Priority Areas to
K.A. and K.W.
References
1. Morgan TH Ž1898. Experimental studies of the regeneration
of planaria maculata. Arch Entwm 7:364]397
2. Morgan TH Ž1900. Regeneration in planarians. Arch Entwm
10:58]117
3. Morgan TH Ž1903. The control of heteromorphosis in pla-
naria maculata. Arch Entwm 14:683]695
4. Shibata N, Umesono Y, Orii H, Sakurai T, Watanabe K, Agata
K Ž1999. Expression of vasa Ž vas .-related genes in germline
cells and totipotent somatic stem cells of planarians. Dev Biol
206:73]87

5. Ogawa K, Wakayama A, Kunisada T, Orii H, Watanabe K,
Agata K Ž1998. Identification of a receptor tyrosine kinase
involved in germ cell differentiation in planarians. Biochem
Biophys Res Commun 248:204]209
6. Kato K, Orii H, Watanabe K, Agata K Ž1999. The role of
dorso-ventral interaction in the onset of planarian regenera-
tion. Development 126:1031]1040
7. Agata K, Soejima Y, Kato K, Kobayashi C, Umesono Y, Watan-
abe K Ž1998. Structure of the planarian central nervous
system ŽCNS. revealed by neuronal cell markers. Zool Sci
15:433]440
8. Kobayashi C, Kobayashi S, Orii H, Watanabe K, Agata K
Ž1998. Identification of two distinct muscles in the planarian
Dugesia japonica by their expression of myosin heavy chain
genes. Zool Sci 15:855]863
9. Tazaki A, Gaudieri S, Ikeo K, Gojobori T, Watanabe K, Agata
K Ž1999. Neural network in planarian revealed by an anti-
body against planarian synaptotagmin homologue. Biochem
Biophys Res Commun in press
10. Umesono Y, Watanabe K, Agata K Ž1997. A planarian orthope-
dia homolog is specifically expressed in the branch region of
both the mature and regenerating brain. Develop Growth
Differ 39:723]727
11. Umesono Y, Watanabe K, Agata K Ž1999. Distinct structure
domains in the planarian brain defined by the expression of
evolutionarily conserved homeobox genes. Dev Genes Evol
209:18]30
12. Orii H, Kato K, Umesono Y, Agata K, Watanabe K Ž1999. The
planarian HOM r HOX homeobox genes Ž Plox . expressed
along the anterio-posterior axis. Dev Biol 210:456]486
13. Wollf E, Dubois F Ž1948. Suy la migration des cellules de
regeneration chez les planaries. Rev Suisse Zool 55:218]227
14. Baguna˜` J, Salo´ E, Auladell Ž1989. Regeneration and pattern
formation in planarians III. Evidence that neoblasts are
totipotent stem cells and the source of blastema cells. Devel-
opment 107:77]86
383
Planarian regeneration
15. Agata K, Kobayashi H, Itob Y, Mochii M, Sawada K, Eguchi G
Ž1993. Genetic characterization of the multipotent dediffer-
entiated state of pigmented epithelial cells in vitro. Develop-
ment 118:1025]1030
16. Brockes JB Ž1998. Amphibian limb regeneration: rebuilding a
complex structure. Science 276:81]87
17. Okada TS Ž1991. Transdifferentiation. Clarendon Press, Ox-
ford
18. Gardiner DM, Carlson MRJ, Roy S Ž1999. Towards a functio-
nal analysis of limb regeneration. Sem Cell Dev Biol
10:385]393 Žthis issue.
19. Kobayashi C, Watanabe K, Agata K Ž1999. The process of
pharynx regeneration in planarians. Dev Biol in press
20. Hori I Ž1982. An ultrastructural study of the chromatoid body
in planarian regenerative cells. J Electron Microsc 31:63]72
21. Auladell C, Garcia-Valero J, Baguna J Ž1993. Ultrastructural
localization of RNA in the chromatoid bodies of undifferen-
tiated cells Žneoblasts. in planarians by the RNase-gold com-
plex technique. J Morphol 216:319]326
22. Bueno D, Espinosa L, Baguna J, Romero R Ž1997. Planarian
pharynx regeneration in regenerating tail fragments moni-
tored with cell-specific monoclonal antibodies. Dev Genes
Evol 206:425]434
23. Watanabe K, Aoyama H, Tamamaki N, Sonomura T, Okada
TS, Eguchi G, Nojyo Y Ž1988. An embryonic pineal body as a
multipotent system in cell differentiation. Development
103:17]26.
24. Martin GR Ž1981. Isolation of a pluripotent cell line from
early mouse embryos cultured in medium conditioned by
teratocarcinoma stem cells. Proc Natl Acad Sci USA
78:7634]7638
25. Bjornson CR, Rietze RL, Reynolds BA, Magli MC, Vescovi AL
Ž1999. Turning brain into blood: a hematopoietic fate adopted
by adult neural stem cells in vivo. Science 22:534]537