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Develop. Growth Differ. (2007) 49, 155–161
doi: 10.1111/j.1440-169x.2007.00912.x
Review
Blackwell Publishing Asia
Tail regeneration in the Xenopus tadpole
Makoto Mochii,* Yuka Taniguchi and Isshin Shikata
Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori Akou, Hyogo
678-1297, Japan
The tail of the Xenopus tadpole contains major axial structures, including a spinal cord, notochord and
myotomes, and regenerates within 2 weeks following amputation. The tail regeneration in Xenopus can
provide insights into the molecular basis of the regeneration mechanism. The regenerated tail has some
differences from the normal tail, including an immature spinal cord and incomplete segmentation of the
muscle masses. Lineage analyses have suggested that the tail tissues are reconstructed with lineagerestricted stem cells derived from their own tissues in clear contrast to urodele regeneration, in which
multipotent blastema cells derived from differentiated cells play a major role. Comprehensive gene expression
analyses resulted in the identification of a panel of genes involved in sequential steps of the regeneration.
Manipulation of genes’ activities suggested that the tail regeneration is regulated through several major
signaling pathways.
Key words: axolotl, regeneration, tail, Xenopus.
Introduction
Amphibians have long been used in the study of tail
regeneration (Goss 1969; Ferretti 2001). Urodeles
regenerate their tails throughout their lives, but
anurans do so only in a limited period, the tadpole
stage, as the anuran tail is a transient appendage
and is not present throughout an anuran’s life. Much
research has been performed using newts and salamanders, but the tail regeneration of Xenopus larva
is now considered to be a useful model system for
analyzing the molecular mechanism underlying the
appendage regeneration. The tail of the Xenopus
tadpole is transparent and suitable for whole mount
observation at the cellular level. It regenerates within
2 weeks following amputation. Molecular studies using
Xenopus have been facilitated by recent advances
in genetic resources and techniques, including
expressed sequence tags (ESTs), microarray and
transgenesis. Analyses of cell lineage and molecular
pathways using transgenic tadpoles have been
*Author to whom all correspondence should be addressed.
Email: [email protected]
Received 27 November 2006; revised 12 December 2006;
accepted 13 December 2006.
© 2007 The Authors
Journal compilation © 2007 Japanese Society of
Developmental Biologists
reviewed in detail (Slack et al. 2004). This review
briefly summarizes the results of the recent research
on Xenopus tail regeneration in comparison to tail
generation in axolotls (Ambystoma mexicanum), another
amphibian model used in recent molecular analyses
of tail regeneration.
Overview of Xenopus tail regeneration
A notochord is a major axial structure located in the
center of the larval tail (Fig. 1A). It consists of vacuolated cells surrounded by a collagenous sheath. A
spinal cord and a pair of sensory ganglions are
located at the dorsal side of the notochord. Bilateral
muscle masses cover the central notochord and the
spinal cord. Dorsal and ventral fins are located at
the midline of the tail. Major veins and arteries are
in the mesenchymal space. The Xenopus larva
regenerates most of its tail after amputation during
its larval life until metamorphosis, with an exception
in the refractory period between stages 45 and 47,
at which the ability to regenerate the tail is lost in
some populations of tadpoles (Beck et al. 2003).
Reproducible tail regenerations were obtained within
2 weeks using tadpoles at stages 48–50 (Sugiura
et al. 2004).
The surface of the amputated stump is covered
with an epidermis within 24 h after half of the tail is
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fore is not the same as the normal tail, at least in terms
of the patterns of the spinal cord and the muscle mass.
A typical feature of urodele regeneration is the formation of the blastema, which contains undifferentiated uniform cells with the potential to differentiate
into multiple types of cells. The regenerating Xenopus
tail shows no obvious structural equivalent to the
urodele blastema, and there is no evidence to show
the presence of multipotent cells in the regenerating
Xenopus tail, as described below. This is the most
critical difference between Xenopus and axolotl tail
regeneration.
Cell lineage is not changed during Xenopus
tail regeneration
Fig. 1. Morphology of normal and regenerating tail of Xenopus
tadpole. (A) Transverse section of a stage 50 tadpole tail was
stained with hematoxylin and eosin. (B, C) Sagittal sections of
the regenerating tail at days 2 and 3 after amputation. The pair
of arrowheads indicates the amputation plane. Arrows in (B)
indicate cell masses of the notochord precursor. The dorsal side
is up and the anterior side is to the left in (B) and (C). N,
notochord; SC, spinal cord; M, muscle mass. Scale bar, 50 µm.
removed. The most distal epidermis thickens to form
a multicell layer (Sugiura et al. 2004), which may be
equivalent to the apical ectodermal cap reported in
urodele limb regeneration. Notochord precursor cells
accumulate to create a compact cell mass adjacent
to the edge of the amputated notochord sheath
(Fig. 1B). The cells proliferate and align along the
proximal-to-distal direction to make an immature
notochord (Fig. 1C), which continues to elongate in
the posterior direction. The regenerating notochord
increases its diameter in the proximal region where
the cells finally differentiate into vacuolated cells. The
axolotl larva also contains a notochord in its tail but
differs greatly from Xenopus after tail amputation, as
the regenerated axolotl tail contains cartilage rather
than notochord (Echeverri et al. 2001). The amputated spinal cord creates a terminal vesicle or bulb
at its proximal end and then elongates posteriorly
along the growing notochord. The regenerating
spinal cord shows a simple tube without an apparent
dorso-ventral pattern and is sometimes known as an
ependymal tube in contrast to the complete regeneration of the spinal cord in urodeles. Myogenic cells
accumulate in a mesenchymal region of the regenerating tail and differentiate into myofibers, which are
not aligned with the regular segments observed in
the normal tail. The regenerated Xenopus tail there-
Cell lineage analyses during Xenopus tail regeneration showed a clear contrast to the results obtained
for the axolotl. The blastema cells in urodeles are
derived from differentiated cells and redifferentiate
into a variety of cells, including muscle, cartilage and
dermis (Lo et al. 1993; Kumar et al. 2000). Multinucleate
myofibers in the axolotl tail become mononucleate
cells through fragmentation, proliferate rapidly and
contribute a significant population of the blastema
cells during the regeneration (Echeverri et al. 2001).
In contrast to the case of the axolotl, Ryffel et al. (2003)
showed that non-muscle cells were never derived
from differentiated muscle cells in the regenerating
Xenopus tail. The researchers labeled differentiated
muscle cells and their descendants by making a
transgenic animal in which Cre recombinase driven
by a muscle-specific promoter induced a recombination of loxP sites and the inheritable expression of
a downstream green fluorescent protein (GFP) gene.
If labeled cells had been found in differentiated tissues other than muscles after amputation, it would
have meant that differentiated muscle cells could transdifferentiate into other types of cells. However, no
labeled cells were found in such differentiated tissues.
Gargioli and Slack (2004) labeled myotome by grafting a piece of presomitic mesoderm from a transgenic embryo expressing GFP into a non-transgenic
embryo, and showed that myofibers in the larval tail
degenerated after amputation and did not contribute
to any tissues in the regenerated tail. The same result
was obtained after our experiment in which myofibers
were labeled by electroporation-mediated gene transfer
(Fig. 2).
A mononucleate muscle satellite cell expresses
PAX7 and is a myogenic stem cell in mammals. After
stimulation by injury or exercise the satellite cells proliferate and differentiate into myofibers (Collins 2006).
Xenopus satellite cells also express PAX7, and
© 2007 The Authors
Journal compilation © 2007 Japanese Society of Developmental Biologists
Tail regeneration in Xenopus
157
PAX7-expressing cells increase in number after tail
amputation (Chen et al. 2006; Fig. 3). Gargioli and Slack
(2004) suggested from the grafting experiment that
the satellite cell is the source of regenerated myofibers
after tail amputation. Forced expression of a dominantnegative form of Pax7 resulted in incomplete regenerations with few or no muscle cells, suggesting that
the Pax7 plays an essential role in the muscle regeneration of Xenopus larva (Chen et al. 2006). These
results showed that Xenopus larvae regenerate tail
muscles through the proliferation and differentiation
of the lineage-restricted stem cells, the muscle satellite
cells, but not through the dedifferentiation of myofibers.
A lineage switch from neural cells to other cell types
was reported in axolotl tail regeneration (Echeverri &
Tanaka 2002). When radial glia cells in the spinal
cord were labeled with GFP by electroporation and
traced after tail amputation, GFP fluorescence was
found in a variety of cell types, including neurons,
glias, melanocytes and muscle and cartilage cells.
It is plausible that cells in the Xenopus spinal cord
neither change their lineage nor exit from the spinal
cord during tail regeneration, because GFP-labeled
cells were only detected in the regenerated spinal
cord when spinal cord cells were labeled by grafting
or electroporation (Slack et al. 2004; Fig. 2).
Tracing the notochord cells labeled with GFP by
grafting or electroporation showed that notochord cells
in the regenerated tail are derived from the pre-existing
notochord (Slack et al. 2004; Fig. 2). The above results
show that muscle, spinal cord and notochord are
reconstructed in the regenerating tail with cells derived
from their own type of tissues, suggesting that Xenopus
tail regeneration is performed through the proliferation
and differentiation of lineage-restricted stem cells rather
than through trans-differentiation from differentiated cells.
Gene expression analyses in Xenopus tail
regeneration
Gene expression analyses were performed in several
laboratories to elucidate the molecular basis of Xenopus
Fig. 2. Lineage analyses during the tail regeneration. Arrows
in (A) indicate green fluorescent protein (GFP)-labeled
notochord cells before amputation. (B) The same tadpole
shown in (A) at day 4 after amputation. All of the GFP-labeled
cells were found in the regenerating notochord. Arrows in (C)
indicate three muscle fibers labeled with GFP. (D) The same
tadpole as shown in (C) at day 5 after amputation. Two labeled
cells were not changed while the other cell degenerated and
was not found. (E, F) Spinal cord cells were electroporated after
amputation and observed at day 2 (C) and day 7 (D). All the
labeled cells were found in the regenerating spinal cord. The
pair of arrowheads indicates the amputation plane. The dorsal
side is up and the anterior side is to the left. Scale bar, 250 µm.
The solution containing the expression plasmid pCAX-AFP
(1 µg/µL) that coded for a mutant form of GFP (Inouye et al.
1997) was injected into the muscle, notochord or spinal cord
region of a stage 48 tadpole anesthetized in MS222. The
injected tadpole was covered with 0.1 × MMR-saturated papers
and electroporated with an electroporator (ECM 830, BTX) by
applying 10 pulses (30 V, 5 ms) according to the method of
Momose et al. (1999). After confirmation of the GFP expression
using a fluorescence microscope the tail was amputated at the
plane indicated by a white line.
© 2007 The Authors
Journal compilation © 2007 Japanese Society of Developmental Biologists
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M. Mochii et al.
Fig. 3. PAX7 expression during
tail regeneration. A frozen crosssection of the tail muscle region
was stained with anti-laminin
antibody (A), Hoechst 33342 (B)
and anti-PAX7 antibody (C). (D)
Merged image of (A) and (C). The
subpopulations of muscle nuclei
were labeled with ani-PAX7. (E)
Higher magnification view of a
section immunostained for laminin
(green) and PAX7 (magenta). A
PAX7-labeled cell is situated underneath the myofiber basement
membrane visualized with antilaminin antibody, suggesting that
it is the muscle satellite cell. (F, G)
Horizontal sections of amputated
tail at days 1 (F) and 3 (G) were
stained with anti-PAX7 antibody.
The PAX7-positive cells increase
in number during the regeneration.
The dorsal side is up in (A, B, C,
D, E) and the anterior side is to
the left in (F, G). A white line in (F,
G) indicates the amputation
plane. Scale bar, 50 µm in (D, F,
G) and 25 µm in (E). A stage 48
tadpole was fixed in 4% paraformaldehyde, infiltrated in 18% sucrose, embedded in Tissue-Tek OCT compound (Sakura) and frozen
in n-hexane cooled with liquid nitrogen. A cryosection was re-fixed in 4% paraformaldehyde and treated with a mixture of antibodies,
containing a monoclonal anti-PAX7 antibody (DSHB, Iowa; Kawakami et al. 1997) and a rabbit anti-laminin antibody (Sigma). The
signals were detected with Cy3- and Alexa488-conjugated secondary antibodies (molecular probes).
tail regeneration (Beck et al. 2003; Christen et al.
2003; Ishino et al. 2003; Sugiura et al. 2004; Tazaki
et al. 2005). Most genes analyzed are expressed
both in the developing tail bud of the embryo and in
the regenerating larval tail, suggesting the presence
of common genetic programs regulating both types
of tail formations. Conversely, it is obvious that the
mechanism underlying tail regeneration is not
identical to that of tail development. Inflammation
response and wound healing are specific events in
tail regeneration. The regenerated tail is morphologically different from the intact tail, as described above.
Neural and mesodermal cells are continuously produced
from undifferentiated cells in the embryonic tailbud,
as shown by morphological observation and the
expression of organizer-specific bone morphogenetic
protein (BMP)-antagonists, which induce neural cells
(Gont et al. 1993; Beck & Slack 1998), while neither
neural nor mesodermal cells are produced from
undifferentiated cells in the tail regeneration process
(Sugiura et al. 2004). The gene expression profile in
the regenerating tail is similar to that in the normal tail
tip region of the larva rather than that in the embryonic
tailbud, suggesting that Xenopus tail regeneration
occurs through the reconstruction and growth of the
larval tail tip rather than through reconstruction of the
embryonic tailbud. The above idea is also supported
by the following observation. The notochord, spinal cord
and muscle tissues continue to grow posteriorly in
the most distal region of the normal tail with morphological similarity to the tissues in the regenerating tail.
Comprehensive analysis of gene expression has
been performed using a cDNA array technique to
identify genes whose expression was up- or downregulated during tail regeneration (Tazaki et al. 2005).
The identified genes were categorized into three
groups, namely; early responding, late responding
and downregulated gene groups, according to their
expression timings during the regeneration. The early
responding group includes a large number of genes
related to the inflammation response and wound healing, while the late-responding group includes genes
related to cell proliferation and differentiation. The downregulated genes are reactivated at a much later stage
of the regeneration and represent the final differentiation of cells, especially muscle differentiation. The
temporal expression profile is therefore well correlated
to morphologically identified events during the tail
regeneration. Additionally, the array analysis resulted
in the identification of 20 unclassified or novel genes
© 2007 The Authors
Journal compilation © 2007 Japanese Society of Developmental Biologists
Tail regeneration in Xenopus
whose expression was upregulated during the tail
regeneration. It is necessary to reveal the functions
of the genes in both embryogenesis and regeneration
in order to obtain a complete understanding of the
molecular mechanism underlying the tail regeneration.
Molecular signals required for the tail
regeneration
Nerve-dependent regeneration is well documented in
the limbs of both urodeles and anurans, and in the tails
of urodeles (Goldfarb 1909; Dinsmore & Mescher 1998).
Studies of the nerve-derived soluble factors have
resulted in the identification of neurotrophic factors
or growth factors. Ablation of the spinal cord from
the urodele tail inhibits the regeneration completely
(Holtzer 1956). Conversely, the tail regeneration of
the anuran larva is believed to be independent of
the spinal cord. It was reported that the destruction
of the Xenopus spinal cord caused little or no effect
on the tail regeneration (Rogusuki 1954), while the
exact role of the spinal cord in Xenopus tail regeneration remained to be elucidated by ablating the
spinal cord completely. We recently developed a
simple operation method to remove the spinal cord
completely from a part of the tadpole tail and showed
that the spinal cord is required for the proper growth
and differentiation of notochord during tail regeneration
(Taniguchi et al. in preparation). This method should
be useful for identifying and characterizing the soluble
factors derived from the Xenopus spinal cord.
Fibroblast growth factors (FGFs) were expressed
in the urodele spinal cord and were suggested to be
required for the tail regeneration (Zhang et al. 2000).
But SU5402, an inhibitor of FGF signaling, does not
have significant effects on either axolotl tail regeneration (Schnapp et al. 2005) or Xenopus tail regeneration (data not shown), suggesting that FGFs are not
essential for tail regeneration in either species. Sonic
hedgehog (shh) is expressed in the ventral spinal
cord of the axolotl. Treatment with cyclopamin, an
inhibitor of hedgehog signaling, suppressed the axolotl
tail regeneration completely, while treatment with a
hedgehog agonist could not rescue the defect caused
by the spinal cord-ablation (Schnapp et al. 2005). Shh
therefore seems to be a necessary but not sufficient
factor secreted from the spinal cord in the axolotl. The
expression pattern of Xenopus shh (Xshh) in regenerating tail is quite different from that in axolotl. Xshh is
not expressed in the regenerating spinal cord but is,
rather, expressed abundantly in the regenerating notochord (Sugiura et al. 2004). Xshh is therefore not the
spinal cord-derived factor discussed above, while its role
in Xenopus tail regeneration should be determined.
159
Beck et al. (2003) reported that Xenopus tail regeneration requires signals of BMP and Notch. These
researchers made transgenic tadpoles harboring hsp
70 promoter-regulated constructs in order to activate
BMP or Notch signaling. When the expression of
the transgene was induced by heat shock at the
regeneration-deficient refractory stage, tail regeneration was restored. Conversely, the forced expression
of inhibitors of BMP or Notch signaling suppressed the
tail regeneration at the regenerative stage. A temporal
analysis using a stable transgenic line harboring hsp
promoter-driven noggin showed that BMP signaling
is not required for the early phase of the tail regeneration, including wound healing and the early morphological changes of the notochord and spinal cord,
but is required for the growth of the regenerating bud
through the activation of a target gene, Msx1.
Expression of several Wnts and a wnt inhibitor is
increased during Xenopus tail regeneration (Sugiura
et al. 2004; Tazaki et al. 2005; Sugiura et al., unpubl.
data). Wnts activate the canonical beta-catenin and/
or the non-canonical signals according to a combination of ligands and receptors. The role of the canonical
Wnt signal in cell proliferation and cell differentiation
may be essential in tail regeneration, as its role has
been well documented in other experimental systems.
Xwnt-5a is expressed in the distal part of the regenerating tail and probably activates non-canonical
signals. We recently found that forced expression
of Xwnt-5a affects the proximo-distal pattern in the
regenerating tissue (Sugiura et al., unpubl. data).
Gene manipulation in tail regeneration
research
Genetic manipulation is a powerful and direct approach
to analyzing the functions of genes in the regeneration process. Transgenesis using a sperm nucleus is
now a popular method of manipulating the Xenopus
genome (Kroll & Amaya 1996), while an inducible
promoter is sometimes required in regeneration
research because ectopic expression of a transgene
in embryonic stages often results in severe developmental defects. The promoter of hsp 70 is very useful, as the expression of a downstream gene can be
induced by a simple heat shock treatment (Beck
et al. 2003; Beck et al. 2006; Chen et al. 2006), while
repeated heat shock treatment, which may seriously
damage amputated tadpoles, is sometimes required
to obtain the desired effect. The use of other types of
inducible promoters and/or cell type-specific promoters
may overcome this problem.
Gene transfer by electroporation is another way of
expressing exogenous genes at larval stages and
© 2007 The Authors
Journal compilation © 2007 Japanese Society of Developmental Biologists
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M. Mochii et al.
is available to manipulate cells in the spinal cord,
notochord and epidermis as mentioned above (see
Fig. 2). The condition of electroporation, however, should
be modified to increase the efficiency of the gene
transfer, because only a small population of cells is
labeled using a standard method. The injection of
morpholino antisense-oligo into blastomeres is a very
popular and effective method to knock down endogenous genes during early development, but could
not be applied at the larval stage by a simple injection method. Delivering morpholino oligo by electroporation was reported to reduce the expression of
Msx1 and Pax7 during tail regeneration in the axolotl
(Schnapp & Tanaka 2005). The same strategy may
be effective in Xenopus. Improving the manipulation
methods and genetic resources, including promoters
and transgenic strains, will facilitate regeneration
studies using Xenopus.
Conclusions
The most significant difference between Xenopus
and axolotl tail regeneration is in the plasticity of the
cells that contribute to regenerates. Mononucleate
cells derived from differentiated myofibers contribute
the blastema cells, and cells derived from the spinal
cord differentiate into non-neural cells in the axolotl.
In Xenopus, however, the major components of the
tail are reconstructed with cells derived from the
same types of tissues, probably through the proliferation and differentiation of lineage-restricted stem cells.
Repair or regeneration through the activation of stem
cells is well known in mammals. Proliferation and differentiation of the muscle satellite cells expressing
PAX7 are observed in both Xenopus tail regeneration
and in mammalian muscle regeneration. The Xenopus
regeneration is still essentially different from mammalian tissue repair, even if they share a common cellular
mechanism, as Xenopus larvae regenerate an
entire appendage but mammals do not. Comparing
the molecular aspects of regeneration in anurans,
urodeles and mammals will elucidate the common and
species-specific molecular mechanisms in these
animals, thus facilitating our understanding of vertebrate regeneration.
Acknowledgements
This work was supported in part by a Grant-in-Aid
for Scientific Research from JSPS to M. M. We thank
Drs N. Ueno, K. Agata, C. Kobayashi, Y. Momose,
A. Tazaki, T. Sugiura, K. Watanabe, H. Orii and other
members of our laboratory for their technical advice
and helpful discussions.
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Journal compilation © 2007 Japanese Society of Developmental Biologists