Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK
* Author for correspondence (e-mail: j.m.w.slack{at}bath.ac.uk)
Accepted 3 March 2004
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SUMMARY |
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We produced Xenopus laevis embryos transgenic for the CMV (Simian Cytomegalovirus) promoter driving GFP (Green Fluorescent Protein) ubiquitously throughout the embryo. Single tissues were then specifically labelled by making grafts at the neurula stage from transgenic donors to unlabelled hosts. When the hosts have developed to tadpoles, they carry a region of the appropriate tissue labelled with GFP. These tails were amputated through the labelled region and the distribution of labelled cells in the regenerate was followed. We also labelled myofibres using the Cre-lox method.
The results show that the spinal cord and the notochord regenerate from the same tissue type in the stump, with no labelling of other tissues. In the case of the muscle, we show that the myofibres of the regenerate arise from satellite cells and not from the pre-existing myofibres. This shows that metaplasia between differentiated cell types does not occur, and that the process of Xenopus tail regeneration is more akin to tissue renewal in mammals than to urodele tail regeneration.
Key words: Xenopus, Tail, Regeneration, Metaplasia, Spinal cord, Notochord, Muscle, Satellite cells, Green fluorescent protein, Cre-Lox recombination
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Introduction |
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Some capacity for regeneration of missing structures is found in several
classes of vertebrates, especially the urodele amphibians, but complete tail
regeneration can also occur in anuran tadpoles. Since the 18th century
(Spallanzani, 1768) and more
recently (Morgan and Davis,
1902
; Locatelli,
1924
; Stefanelli,
1947
; Baita, 1951
;
Holtzer, 1956
;
Roguski, 1957
; Niazi, 1966;
Hauser, 1972
), the tail
regeneration of urodele and anuran amphibians has attracted interest, but the
mechanism of new tissue formation from the stump is still not yet clearly
understood. Elsewhere, we have addressed the molecular signalling processes
involved in regeneration (Beck et al.,
2003
), and in this paper we address the issue of cell lineage.
Does the regenerate originates from mature differentiated cells or from
undifferentiated `reserve' cells? If it is the former, then does each tissue
give rise to just its own cell type in the regenerate, or can it switch to
different cell types (metaplasia)? These questions are still being answered.
In the axolotl, a urodele amphibian, Echeverri and Tanaka
(Echeverri and Tanaka, 2002
)
have demonstrated neural cell plasticity during tail regeneration. During
regeneration, the ependymal or radial glial cells of the axolotl spinal cord
are able to give rise to neurons, melanocytes, muscle and chondrocytes, cell
types of both ectodermal and mesodermal origin. In previous experiments on
limb regeneration in newts it has been shown that the multi-nucleate muscle
fibres can de-differentiate. The nuclei re-enter S phase, and the
multinucleate cells break up into mononuclear cells. These enter the
undifferentiated blastema of the limb and some can later differentiate into
other tissue types, such as cartilage (Lo
et al., 1993
; Kumar et al.,
2000
). These experiments clearly show that de-differentiation of
mature differentiated cells, and metaplasia of one cell type to another, can
take place during urodele regeneration. Recently the term
`transdifferentiation' has become popular as an alternative to `metaplasia',
particularly in the context of the ability of bone marrow stem cells to
populate various other tissue types after grafting. In our own publications we
have used the term `transdifferentiation' in its original sense to refer to
direct transformation of one differentiated cell type to another with or
without cell division, and the term `metaplasia' to refer to any conversion of
one differentiated tissue type to another regardless of pathway or mechanism
(Tosh and Slack, 2002
). Given
the uncertainty about the nature of the intermediate cell states in amphibian
regeneration, we shall refer to them here as examples of `metaplasia'.
There is currently much interest in `regenerative medicine' or stimulation
of regeneration of damaged or defective human tissues (e.g.
Cavazzana-Calvo et al., 2002).
In this context it is important to establish whether metaplasia is a necessary
feature of regeneration, or whether tissue-specific regeneration can also take
place.
In the present work, we have developed a technique to label specifically
the three principal tissues of the tail by orthotopically grafting into a
non-transgenic host explants of the appropriate tissue from a transgenic
embryo expressing GFP. The resulting tadpoles then have one tissue permanently
labelled in a manner that is unaffected by subsequent changes in cell
differentiation. After amputation of the tail and its subsequent regeneration,
we are able to follow the fate of the labelled tissue cells into the
regenerate. Using this method, we demonstrate that metaplasia does not occur
during Xenopus tadpole tail regeneration. Each of the three main
tissue types the notochord, the spinal cord and the myofibres
behaves independently. The spinal cord regenerates via the formation of a
distal bulb (`ampulla') as also described by Stefanelli
(Stefanelli, 1951) in urodele
spinal cord regeneration. The notochord regenerates directly by growth at the
tip region. The muscle does not de-differentiate in the manner seen in
urodeles. Instead, the multinucleate fibres in the vicinity of the amputation
site degenerate completely and the regenerated muscle is derived from
satellite cells that contribute to a blastema-like zone around the tips of the
notochord and spinal cord.
Our results indicate that metaplasia is not a necessary component of appendage regeneration. Does this have any significance beyond the anuran tadpole? In fact, the mode of regeneration of each tissue resembles the normal mode of growth or cell turnover seen in mammals. During their evolution the mammals have lost almost all the regenerative capacity seen in urodeles and presumably possessed by urodele-like primitive vertebrates. Although many urodeles regenerate their tails throughout life, the anuran amphibians regenerate the tail only during the larval stages. This places the anurans in an intermediate position, and it may be that the regeneration that might be stimulated in mammals will be closer to the anuran than to the urodele type.
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Materials and methods |
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Specimen analysis
Tadpoles were killed with an overdose of MS222 (Sigma) and fixed overnight
in Zamboni's fixative (40 mM Na2HPO4, 120 mM
NaH2PO4, 2% PFA and 0.1% saturated picric acid), washed
in 70% ethanol overnight, dehydrated and embedded in paraffin wax. A Leitz
microtome was used to cut 6 µm serial sections. Then the sections were
dewaxed, re-hydrated and immunostained, then counterstained with Haematoxylin
and mounted in Aquatex (Merck).
Plasmid construction
The construction of pcDNA3/CMV-nucGFP2 was performed by excising
nucGFP2 from CS2/nucGFP2 (a kind gift from E. Amaya) with
BamHI and XbaI, and cloning into
BamHI/XbaI sites in pcDNA3 (Invitrogen). It was linearized
with SmaI and diluted to 500 ng/µl final concentration for
addition to the sperm nuclei.
For the specific expression of the GFP in the muscles pCarGFP was used (a kind gift from E. Amaya). It was linearized with NotI and diluted to the final concentration of 500 ng/µl for the transgenesis injection.
For the Cre-Lox system two different constructs were used together: pCarCre (i.e. Cre recombinase driven by the cardiac actin, muscle-specific, promoter) and pcDNA3/CMV-loxSTOPlox-GFP (i.e. behaves like CMV-GFP following excision of the STOP sequence). pCarCre was made by excising Cre+polyA addition sequence from pCS2Cre (a kind gift from D. Werdien) with HindIII and NotI, and cloning into pCar, after removal of GFP+polyA with the same enzymes. pCarCre was linearized with NotI and diluted to a final concentration of 500 ng/µl. The construction of pcDNA3/CMV-loxSTOPlox-GFP was performed by substituting nucGFP2 by loxP-tpA-loxP-nucGFP from pCS2/CMV-lox-tpA-lox-nucGFP (made by M. Horb). pcDNA3/CMV-nucGFP2 was cut with XbaI and blunted with Klenow (Roche), then the nucGFP2 was excised with HindIII. The loxP-tpA-loxP-nucGFP was removed with XbaI (blunted with Klenow) and HindIII. The loxP-tpA-loxP sequence came from pGK/neo-tpA-lox2 (a kind gift from P. Soriano) and the tpA has numerous stop codons to prevent translation. pcDNA3/CMV-loxSTOPlox-nucGFP was linearized with SmaI and diluted to 500 ng/µl final concentration.
Transgenesis
In order to obtain X. laevis transgenic embryos, we followed the
procedures previously described (Kroll and
Amaya, 1996; Amaya and Kroll,
1999
), except for the omission of restriction enzyme from the
reaction and the fact that sperm nuclei were frozen in aliquots in sperm
suspension buffer before use.
Graft technique
The operations were performed on neurula stage embryos (NF 13-17), in
agar-coated dishes, in NAM/2 with 10 µg/ml trypsin (Sigma, type 9) to
promote the separation of the three embryonic tissue layers. The embryos were
manually demembranated with sharp forceps, then incised using a tungsten
needle in the posterior region next to the blastopore, at the level of the
presumptive tail-forming region (Tucker
and Slack, 1995). The neural plate was lifted using a hair loop to
free access to the underlying notochord and somites or, in the case of spinal
cord labelling, to be transplanted itself. An explant of neural plate,
notochord or pre-somite plate was replaced by a similar sized one from a
CMV-GFP transgenic donor embryo. To immobilise the graft and promote
healing the operated region was covered by a small glass bridge. The bridges
are made from cover-slip fragments that are slightly bent over a microburner.
The grafted embryos were allowed to heal under the bridges, in agar-coated
dishes, for 30 minutes in NAM/2 + 20 µg/ml trypsin inhibitor (Sigma). Then
they were moved to NAM/10 + 50 µg/ml gentamicin (Sigma, sulphate salt) for
long-term culture.
BrdU injection
The tadpoles at stage 49 NF were anaesthetized in 1/3000 MS222, and
injected with 2 µl of the thymidine analog 5-Bromo-2'-deoxyuridine
(BrdU): aqueous solution of (10:1) ratio from the Cell Proliferation Kit
(Amersham). The injection was performed 24 hours before fixation.
Tail amputation
The tadpoles were anaesthetized in 1/3000 MS222, and were kept in the
anaesthetic solution for the duration of the operation (about 5 minutes). The
distal 50% of the tail was removed with a pair of iridectomy scissors (Vannas
straight small, John Weiss). The tadpoles were allowed to heal in tap water
with aeration (1 hour) and subsequently were returned to the aquarium.
Immunohistochemistry
Sections were prepared as described above. Where necessary, antigen
unmasking was performed by boiling the slides in distilled water for 3 minutes
at full power in a microwave oven. Anti-GFP polyclonal antibody from Molecular
Probes was used at a dilution of 1:100 in 2% Boehringher Blocking Buffer. The
secondary antibody (Sigma goat anti-rabbit IgG whole molecule peroxidase
conjugate) was used at a dilution of 1:100. Myofibres were stained with 12/101
mAb (Kintner and Brockes,
1984) at 1:100 dilution. Satellite cells were stained with
anti-Pax7 mAb (Developmental Studies Hybridoma Bank) at 1:500 dilution. For
both of these the second antibody (Sigma rabbit anti-mouse IgG whole molecule
peroxidase conjugate) was used at 1:100 in 2% Boehringer Blocking Buffer. All
the detection reactions were carried out using an AEC
(3-Amino-9-ethylcarbazole) staining kit (Sigma). BrdU incorporation was
revealed with anti 5-bromo-2'deoxyuridine mAb (Amersham `Cell
Proliferation Kit'), antibody provided with nuclease for DNA unmasking. The
secondary antibody was Sigma rabbit anti-mouse IgG whole molecule alkaline
phosphatase conjugate and the staining reaction was carried out using Fast Red
tablets (Sigma).
For simultaneous detection of GFP and Pax7, a fluorescence method was used. The primary antibodies were as specified above. For secondary antibodies were used a H+L IgG fragment goat anti-rabbit FITC conjugated (Vecta Lab) for GFP detection and a H+L IgG fragment rabbit anti mouse Texas Red conjugate, Vecta Lab) for Pax7 detection. The double immunostaining was performed by making first the immunoreaction against Pax7 and then that against GFP. The procedure was followed as described above, the fluorescent secondary antibodies were incubated for 3 hours at room temperature, the slides were mounted in Aquatex (Merck) and analysed with a Leica stereomicroscope equipped with UV lamp and GFP and RFP filters. Images were taken with a colour CCD camera (diagnostic instrument RT spot camera) operating with Advanced Spot RT 3.0 software. Images were processed with Adobe Photoshop 7.0.
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Results |
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Tissue-specific labelling by neurula grafts
In order to label specific tissue types, we needed a promoter that was
active, at high level, in all cell types, and that was independent of any
specific controls operating during tail regeneration. This is because the
label must persist if cells de-differentiate, and if they re-differentiate
with a different phenotype. We made transgenics using three ubiquitous
promoters to drive GFP [cytoskeletal actin, CSKA; elongation factor 1
(EF1
); and cytomegalovirus early promoter, CMV]. The comparison
suggested that the CMV promoter was best, both in terms of the level of
activity and in terms of retention of good labelling throughout the
regeneration process. This is shown in Fig.
2. The GFP used contains a nuclear localisation signal, and so
tends to concentrate in nuclei, but some fluorescence is also visible in the
cytoplasm.
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Spinal cord
At 5 days after amputation, the regenerating spinal cord was already
visible and was in continuity with that of the stump
(Fig. 5A). The blastema
surrounding the re-forming spinal cord was completely negative for the GFP
fluorescence, as was the notochord. The labelled cells expressing GFP were
localised only in the regenerating spinal cord, all other surrounding tissue
cells being completely negative. In the following days the regenerating spinal
cord kept elongating, always in continuity with that of the stump
(Fig. 5B,C). The blastema was
not involved at all in the formation of the new spinal cord. Once the stage of
complete tail regeneration was reached, the GFP was still exclusively and
specifically expressed in the spinal cord as in it had been in the original
tail.
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Origin of muscle in the regenerate
Car-Cre/Lox labelling compared to Car-GFP
Before describing the results from the grafts of presomite mesoderm, it
will be convenient to describe one other experiment that set limits to the
origin of the regenerated muscle. This is an experiment with the Cre/Lox
system, where a proportion of myofibres were permanently labelled with GFP
after an intrachromosomal recombination to remove a translational stop signal.
This experiment was conducted in founder tadpoles, not by crossing
Cre-expressing and reporter lines together. The Cre was driven by the
muscle-specific cardiac actin promoter (Car) and the reporter plasmid
contained a CMV promoter driving GFP, with a lox-STOP-lox
sequence at the beginning of the coding region (see Materials and methods).
The effect is to label permanently with GFP a proportion of the cells that
have activated the Car promoter at any time. These specimens were
compared with others that were simply transgenic for Car-GFP, which
will show label in all cells which currently have the Car promoter active.
Ten transgenic tadpoles expressing Car-Cre and CMV-lox-STOP-lox-GFP were created with a sufficient proportion of myofibres labelled (Fig. 6A,B,E,F). Unlike the Car-GFP transgenics, not all the myofibres are labelled but the proportion gradually increases with time because of new recombination events occurring in cells where Cre recombinase is expressed. Three days post-amputation, the regeneration bud contained no fluorescent cells at all for either of the two transgenic models (Fig. 6C,G). This shows that there is no de-differentiation of multinucleate fibres to mononuclear cells. In the Cre/lox transgenics, any cells dedifferentiated from green fibres would still be making GFP. In the Car-GFP transgenics, although production of GFP would cease on de-differentiation, the protein itself should persist for a few days.
After 5 days from the tail amputation a substantial number of green myofibres appeared in the regenerationed region of the Car-GFP transgenics (Fig. 6H). This indicates the normal tempo of muscle regeneration. By contrast in the Cre/lox transgenics, there were just a few isolated green fibres (Fig. 6D). We believe that the time scale and position of appearance of the few fluorescent fibres seen in the regenerating Cre/lox transgenics indicates that they are due to new recombination events and not to de- and re-differentiation of muscle cells previously expressing GFP. The absence of any short-term labelling of mononuclear cells in the regeneration bud, and the slow appearance of the labelled myofibres in the Cre/lox tadpoles suggests that the myofibres themselves contribute no cells to the regeneration bud. This is consistent with the morphological picture, which shows large scale degeneration of myofibres in the vicinity of the cut surface, and it suggests that the new myofibres must come from some other cellular source than pre-existing myofibres.
Presomite plate grafts
The most informative data relating to this question was that from the
grafts of neurula-stage presomite mesoderm (PSM), although the behaviour was
somewhat more complex than that of neural plate and notochord grafts as it
depends on the stage of the donor and on the position of origin of the graft
within the PSM. Three different types of graft were performed: PSM adjacent to
the notochord at stage 13 (early medial); PSM adjacent to the notochord at
stage 17 (late medial); and PSM from a more lateral position at stage 13
(early lateral).
In all the three cases, the labelling of the host tadpoles was exclusively in the somites and later in the myotomes (Fig. 7). However, the behaviour during tail regeneration was completely different.
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GFP expression in different muscle cell populations
In order to characterise the difference between the three different PSM
graft types in terms of cell population, the grafted tadpoles were analysed by
immunostaining against GFP (see Materials and methods). Both longitudinal and
transverse sections were used although the transverse sections proved most
informative. The nuclear localisation signal means that GFP protein is
concentrated in nuclei, although highly expressing cells show some in the
cytoplasm as well.
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Further proof of the identity of these cells as satellite cells is given by
the incorporation of BrdU. It has been demonstrated that the only cell type
proliferating in mammalian differentiated muscles are the satellite cells
(Walsh and Perlman, 1997).
Unamputated tadpoles were injected with BrdU and processed 24 hours later.
Many cells all over the tadpoles are labelled, but in the muscle the labelling
is predominantly in the flat peripheral nuclei of the type expressing Pax7
(Fig. 12A,B). Very few, if
any, of the myonuclei are labelled, although a few fibroblasts or other cell
types within the muscle are labelled.
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As there is an excellent correlation between the extent of labelling of satellite cells by the three types of graft, and the extent to which the grafts contribute to myofibres in the regenerate, we believe that the new myofibres of the regenerated tail arise from satellite cells and not by de-differentiation of pre-existing myofibres.
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Discussion |
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The results for the spinal cord and notochord are straightforward and are
consistent with the morphological appearance. Our results show that these two
tissues each regenerate as a self-contained compartment with no export or
import of cells, and without de-differentiation or metaplasia. The labelling
of spinal cord was carried out in such a way that only the ventral part was
labelled. We did not, therefore, examine the potential of the neural crest
cells that are derived from the dorsal part of the neural tube. Our results
are in marked contrast to a recent cell labelling study on axolotl tail
regeneration in which radial glial cells were shown to give rise to neurons,
melanocytes, myofibres and chondrocytes in the regenerate
(Echeverri and Tanaka, 2002).
We believe that both sets of data are correct and that they point to radically
different modes of regeneration in the urodele and anuran amphibians.
Origin of muscle in the regenerate
Our results suggest that the regenerated muscle comes from satellite cells
in the myotomes, and not from pre-existing myofibres. The evidence is as
follows. First, the original myofibres can be seen to be degenerating in the
vicinity of the cut surface. Second, myofibres labelled by the Cre-Lox
technique do not contribute to myofibres in the regenerate in the short term.
The small numbers of labelled fibres that do appear are late and consistent
with new recombination events following the new differentiation of muscle
fibres whose time course can be seen in the Car-GFP transgenics.
Ryffel et. al. (Ryffel et. al.,
2003) also used the Cre-Lox method to label myofibres, using a
protocol involving breeding, and also showed that labelled cells did not enter
the muscle of the regenerate. Third, myofibres labelled by the early medial
PSM grafts never contribute to myofibres in the regenerate. Fourth, the
tadpole myotomes contain cells, identified by Pax7 expression, with the
morphology of muscle satellite cells. Fifth, these cells are the proliferating
cells within the myotomes as shown by the BrdU incorporation. Sixth, for the
three types of PSM graft, the labelling of myofibres in the regenerate is
correlated with the number of satellite cells labelled by the graft.
The different behaviour of the PSM grafts is fully consistent with the idea
that the satellite cell precursors lie in the far lateral part of the PSM.
During somitogenesis there is a strong dorsal convergence of cells from the
ventral side of the embryo
(Pourquié, 2001). The
PSM in the early neurula is not much thicker than the ventral mesoderm, but by
the late neurula it has become markedly thicker, at the expense of the ventral
mesoderm, and segmentation has commenced at the anterior end
(Nieuwkoop and Faber, 1967
).
We believe that our early lateral grafts capture some of the satellite cell
precursors but that the late medial grafts capture more because of the
substantial migration of tissue towards the dorsal midline that occurs in this
stage interval.
Again, our result on the origin of regenerated muscle is discordant with
previous results from urodeles. In newt, limb regeneration it has clearly been
shown that the nuclei of myofibres can re-enter S phase following a nearby
injury, that the fibres can break up into viable mononuclear cells, and that
these cells can re-differentiate to form new muscle fibres and also make some
contribution to other cell types (Kintner
and Brockes, 1984; Echeverri et
al., 2001
; Echeverri et al., 2002).
Muscle satellite cells were first described by Mauro
(Mauro, 1961), who provided EM
pictures of cells from the frog very similar in appearance to the flat nucleus
cells that we find labelled by the late PSM graft. Subsequent work has all
been on birds and mammals (reviewed by
Seale and Rudnicki, 2000
),
although it is likely that amphibian satellite cells have similar properties.
Following muscle damage, satellite cells re-enter mitosis and start to express
myogenic transcription factors, and the resulting myoblasts fuse with each
other to generate new myofibres (Schultz,
1996
; Cooper, et. al.,
1999
). The formation of mammalian satellite cells, but not primary
or secondary myofibres, is now known to depend on the transcription factor
Pax7 (Seale et. al, 2000
).
They have been previously shown to originate from the somites in birds, using
quail-chick grafting (Armand et al.,
1983
), although more recent work suggests that they may arise from
the aorta (De Angelis et al.,
1999
). As the dorsal aorta is now known to be colonised by a
population of somitic angioblasts (Pardanaud et al., 2000), these findings are
not necessarily inconsistent. Our results indicate that Xenopus has
satellite cells similar to those in birds and mammals, that they express Pax7,
and that they arise from the lateral region of the PSM.
Conclusions
The regeneration of the Xenopus tadpole tail operates through
mechanisms that are completely different from those found in the appendage
regeneration of urodeles. In urodeles functional cells de-differentiate to
form a blastema. This proliferates and re-differentiates to form the
regenerate. There is a certain amount of metaplasia shown, indicating that at
least some of the blastema cells show pluripotency. In Xenopus the
spinal cord regenerates through proliferation of an apical ampulla continuous
with the ependymal layer of the more proximal spinal cord. The notochord
regenerates by proliferation at the tip. The muscle regenerates by the
multiplication, differentiation and fusion of satellite cells to form new
myofibres.
Overall, Xenopus tail regeneration seems much more akin to the
normal tissue renewal mechanisms found in mammals than to the specialised
regeneration mechanisms found in the urodeles
(Carlson, 2003). This may make
Xenopus a more useful model organism than formerly suspected for
experimental work in regenerative medicine.
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ACKNOWLEDGMENTS |
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