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Agata1999 PDF
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Article No. scdb.1999.0324, available online at http:rrwww.idealibrary.com on
Neoblasts, which are classically defined as prospective is the food of planaria, rather than planaria them-
totipotent stem cells, supporting planarian regeneration, selves, that have contributed greatly to the under-
now have been identified by expression of the DjvlgA gene standing of complex developmental processes. In
coding for a vasa-type ATP-dependent RNA helicase. 1991 we began to apply molecular approaches to the
DjvlgA-positive cells are distributed in the mesenchymal study of planaria, to understand the underlying
space from head to tail, participating in formation of organ mechanisms by which cells can recognize the loss of a
rudiments and the blastema during regeneration. part of the body and then reconstruct it. To modern-
Interestingly, neoblasts begin to transcribe tissue-specific genes ize planarian studies we have developed clonal strains
in a position-dependent manner, while they are still in the of planaria that can be propagated under laboratory
mesenchymal space. This is prior to migrating to the organ conditions, isolated tissue-specific molecular markers
rudiments or blastema, and at a time when they are not yet and regulatory genes, and established a highly-sensi-
morphologically distinguishable from neoblasts. We speculate tive in situ hybridization method, as well as a method
that the mRNAs transcribed in the committed cells may be for chimeric analysis. Here, we review the recent
trapped in a complex with RNA helicase(s), forming a findings concerning planarian regeneration using the
‘chromatoid body’, and are not translated into protein until newly established techniques and molecular probes.
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 Modernization of planarian regeneration studies
well as to express pattern formation genes for organogenesis.
To obtain a genetically homogeneous population of
Key words: commitment r planarian r post-transcriptional planaria, we have tried to establish clonal strains.
regulation r regeneration r stem cell One species of planarian that was collected in the
Q1999 Academic Press 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
Introduction was established in 1991 as a clonal strain and then
extensively propagated by asexual reproduction. Fur-
PLANARIAN REGENERATION WAS extensively studied by thermore, in 1996, we were able to obtain eggs and
Thomas Hunt Morgan approximately 100 years ago. embryos from GI animals maintained at 128C in the
He wrote several long papers about planarian regen- National Institute for Basic Biology in Okazaki. It is
eration from 1898 to 1903.1 ] 3 In his paper of 1900 he very interesting that this strain can be converted to a
described using Drosophila as a food for planaria, and sexual state by changing rearing conditions.4,5
that he had succeeded in staining planarian digestive Chimeric analysis is indispensable for monitoring
ducts by feeding Drosophila eyes.2 However, the his- cellular behavior during regeneration and also to
tory of science during the 20th century shows that it identify the stem cells. We have succeeded in isolat-
ing a cDNA clone ŽPH20. which stains all the cells of
U
GI animals, but not those of HI animals. HI is a newly
From the Laboratory of Regeneration Biology, Department of established laboratory strain of D. japonica collected
Life Science, Faculty of Science, Himeji Institute of Technology,
Hyogo 678-1297, Japan. E-mail: agata@sci.himeji-tech.ac.jp in the Iwaya valley in Hyogo Prefecture.6
Q1999 Academic Press Cell type-specific genes were isolated by im-
1084-9521r 99 r 040377q 07 $30.00r 0 munoscreening or random sequencing of 200 head-
377
K. Agata and K. Watanabe
and 1000 eye-cDNA clones.7 ] 9 We are now able to brates, it has been clearly demonstrated by cell cul-
identify neurons, pharynx-muscles, body wall-muscles, ture and molecular markers that differentiated cells
type II-mucous producing cells, and visual cells using can participate in regeneration through dedifferen-
gene probes. Otx r otd, HOX r HOM and vasa-related tiation events,15 ] 17 and see Gardiner et al,18 this issue.
genes were isolated by PCR using degenerater Even in planarian regeneration, it has been suggested
primers.4,10 ] 12 We also invested a lot of time in the based on electron-microscopic observations, that the
development of a highly-sensitive in situ hybridization pharynx-lumen epithelium may form by transdiffer-
method. The successful method includes acid-treat- entiation from intestinal epithelial cells. However, we
ment of the worms before fixation and long hy- could not find any evidence indicating a contribution
bridization times at low probe concentration.7,11 from differentiated cells to planarian regeneration,
even though we have extensively analyzed the regen-
eration process using molecular markers and
Are the stem or undifferentiatedcells the major chimeric analysis.19 On the contrary, our observa-
source of planarian regeneration? tions clearly indicate that the cells participating in
regeneration appear in the mesenchymal space where
What type of cells participate in planarian regenera- the stem cells are distributed.4,19 In the case of phar-
tion? Classical observations and experiments sug- ynx regeneration, DjMHC-A Ža pharynx-muscle-
gested that totipotent stem cells, so called ‘neoblasts’, specific gene; MHC, myosin heavy chain. expressing
may exist in the mesenchymal space through the cells appear in the mesenchymal space surrounding
entire body of the worm and differentiate to supply the prospective pharynx forming region before for-
appropriate cells for regeneration.13,14 However, the mation of a pharynx rudiment.19 These observations
evidence supporting this idea is still scarce. On the suggest that the cells participating in planarian re-
other hand, in the case of regeneration in the verte- 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.
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Planarian regeneration
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K. Agata and K. Watanabe
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Planarian regeneration
observed in the regeneration process of hydra, later Does pattern formation occur after migration of
scientists emphasized the formation of a blastema the committed cells to the rudiment?
and compared the planarian regeneration system to
that of the vertebrates since planaria form a blastema, In the case of brain regeneration we could not detect
whereas hydra does not. Planaria form a blastema cells committed to a neuronal fate in the mesenchy-
which is recognized externally as a white region and mal space using neuron-specific probes.7,9 We could
in sections as a mass of morphologically undifferen- not understand the reason for this, because cells
tiated cells. However, our observations suggest that committed to pharynx muscle or mucous-producing
most of the morphologically undifferentiated cells in cells can be detected at an early stage of regenera-
the organ rudiments or blastema may have already tion. When we isolated Otx r otd-related genes from
been committed to differentiate to appropriate cell planaria, we expected to be able to detect cells
types. For example, the pharynx muscle-forming cells committed to a neuronal fate by using such transcrip-
may be committed in the stump-proximal region of tion factor genes as probes, since regulatory genes
the head piece, not in the blastema nor in the phar- are expressed prior to differentiation genes in gen-
ynx rudiment ŽFigure 2B.. These findings support the eral. However, expression of Otx r otd-related genes
idea that differentiation of the stem cells or undif- cannot be detected until after formation of the brain
ferentiated cells in the mesenchymal space may be rudiment, and not in the migrating cells ŽFigure
regulated in a position-dependent manner, suggest- 4..10,11 Expression patterns of Otx r otd-related genes
ing that these cells may have a molecular system to suggested that these genes may be involved in later
recognize their own position within the body, possi- pattern formation of the complex brain, but not in
bly in common with hydra. This idea is also sup- the commitment of neurons. So, we concluded that
ported by the dramatic change in HOX gene expres- Otx r otd-related genes categorized as pattern forma-
sion after cutting.12 tion genes may be transcribed after formation of the
Bueno et al,22 described the expression pattern of rudiment. Transcription of other neuron-specific
MHC protein using an anti-MHC monoclonal anti- gene,7,9 encoding a neuropeptide processing enzyme
body during pharynx regeneration. They detected or synaptotagmin, may be regulated under the con-
the MHC protein in the pharynx rudiment but not in trol of the pattern formation genes since both pro-
migrating cells in the mesenchymal space, suggesting teins are expressed in the same sub-population of the
that DjMHC mRNAs in migrating cells detected by in neuronal cells. In the case of pharynx regeneration
situ hybridization may not be translated to the pro- the cells committed to the pharynx-muscles start to
tein. So, we speculated that these mRNAs may be transcribe MHC genes before formation of the phar-
trapped in the chromatoid body by making a com- ynx rudiment since MHC may be a common protein
plex with RNA helicaseŽs., preventing translation of in all subtypes of pharynx muscles. After formation of
MHC mRNAs, allowing the migrating cells to main- the pharynx rudiment the muscle cells start to dif-
tain their undifferentiated state until reaching the ferentiate into specific muscle types under the con-
pharynx rudiment. The cellular events and gene ex- trol of pattern formation genes, to form the well-
pression at the early stages of regeneration are sum- organized pharynx structure. So, we speculate that
marized in the left portion of Figure 3. 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.
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K. Agata and K. Watanabe
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Planarian regeneration
5. Ogawa K, Wakayama A, Kunisada T, Orii H, Watanabe K, 15. Agata K, Kobayashi H, Itob Y, Mochii M, Sawada K, Eguchi G
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