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First published online 10 May 2006
doi: 10.1242/dev.02397
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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 7 April 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Pax7, Pax7EnR, Muscle satellite cells, Regeneration, Xenopus laevis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Much of the tissue mass of the regenerated tail consists of
striated muscle, but the origin of this muscle has been a matter of some
uncertainty and controversy. In this paper, we show that an adult stem cell:
the muscle satellite cell, already known to be able to replace fibres in
damaged muscle, is also capable of building a new mass of tissue in an
extended regenerated appendage.
In principle, three possibilities have been proposed for the origin of
regenerated muscle: de-differentiation of myofibres, muscle satellite cells
and side population (SP) cells. During regeneration of the limbs of urodele
amphibians (newts and salamanders), it has been well documented that the
striated muscle fibres can de-differentiate
(Namenwirth, 1974;
Lo et al., 1993;
Kumar et al., 2000;
Echeverri et al., 2001). In
this process, the nuclei of the myofibres re-enter S-phase and the fibres
break apart to become mononuclear cells. These cells then participate in the
regeneration blastema, proliferate and eventually become re-differentiated,
mostly as new myofibres, but also as some other tissue types. In postnatal
mammals, there is no regeneration of appendages such as limbs or tails.
However, striated muscle does have some ability to repair itself following
tissue damage. In this process, the new fibres are generally considered to be
derived from muscle satellite cells: a population of small mononuclear cells
located beneath the basement membrane of the myofibres
(Seale and Rudnicki, 2000).
Muscle satellite cells can be considered as a type of adult stem cell as they
are able both to reproduce themselves and to produce myoblasts, which can
differentiate to form new myofibres. However, this idea has been challenged
because in skeletal muscle, there also exists another kind of stem cell called
side population (SP) cells that can be recognized by a DNA-binding Hoechst
(33342) dye (Gussoni et al.,
1999; Jackson et al.,
1999; Seale et al.,
2001). Transplanted SP cells have also been shown to be able to
adopt the myogenic lineage and to participate in muscle repair.
Previous studies of Xenopus tail regeneration showed that there
was no de-differentiation of the myofibres
(Ryffel et al., 2003;
Gargioli and Slack, 2004). The
fibres near the cut surface simply degenerate and die. This suggested a
different mode of muscle regeneration from that found in urodeles. It was
conjectured, on the basis of the labelling patterns from different types of
graft, that satellite cells might be the precursors of the striated muscle of
the regenerate (Gargioli and Slack,
2004). In the present paper, we show that this is the case. We
show that, as in mammals, Pax7 is expressed in muscle satellite cells and that
many satellite cells, but not myonuclei, are active in DNA replication. We
show that a domain swap inhibitor, Pax7EnR, can antagonise the biological
activity of Pax7. We then use an inducible pax7EnR transgene to
deplete the tail of satellite cells and show that this reduces or inhibits the
regeneration of new muscle.
Muscle satellite cells were actually first discovered in frogs, visualised
by electron microscopy as mononucleated cells wedged between the basement
membrane and the plasma membrane (Mauro,
1961). Later, muscle satellite cells were also found in skeletal
muscles of mammals and birds, and their role in muscle repair has been studied
recently (Chargé and Rudnicki,
2004). Upon muscle injury, quiescent satellite cells residing in
the damaged myofibres are rapidly activated to re-enter the cell cycle. The
upregulation of Myf5 and MyoD, two myogenic regulatory factors, confers on
them a myoblast identity. After proliferation, most of the cells differentiate
and fuse to generate new myofibres
(Cornelison and Wold, 1997;
Cooper et al., 1999). A
minority of satellite cells are retained in the sub-laminar location to
replenish the pool of cells for subsequent muscle repair
(Chargé and Rudnicki,
2004).
An important gene expressed in satellite cells is pax7
(Seale et al., 2000;
Halevy et al., 2004). It is a
member of the Pax (paired box) family of transcription factors, which play
important roles in cell fate, early embryonic patterning and organogenesis
(Mansouri et al., 1996;
Mansouri et al., 1999;
Ziman et al., 2001a;
Lamey et al., 2004). Pax7 has
three conserved protein domains, a DNA-binding domain called the paired
domain, a paired-type homeodomain and an octapeptide
(Jostes et al., 1990). Early
studies of the pax7 gene mainly focused on its biological function in
the central nervous system because of its abundant expression there
(Mansouri et al., 1996;
Kawakami et al., 1997;
Ziman et al., 2001a). Its
activity in muscle satellite cells has been established more recently
(Seale et al., 2000). It has
been found that Pax7 is only expressed in quiescent and newly activated muscle
satellite cells. Upon myogenic differentiation, it is rapidly downregulated.
The Pax7-/- mice contain reduced number of muscle
satellite cells and they are progressively lost during postnatal growth
(Oustanina et al., 2004;
Kuang et al., 2006;
Relaix et al., 2006). These
findings revealed an important feature of satellite cells in their capacity
for self renewal and the role of Pax7 in this context
(Zammit et al., 2004;
Collins et al., 2005;
Montarras et al., 2005).
Our results presented here show that the satellite cells are dividing during normal tail growth and, most importantly, that they are responsible for forming the muscle masses of the regenerated tail. This means that the cellular processes of regeneration in the anuran tadpole is much more akin to the tissue repair events of mammals than to the de-differentiation/re-differentiation process found in urodeles.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Embryos were dejelled with 2% cysteine (Sigma) (pH 7.8) and then cultured in
0.1xNAM. From stage 46, they were transferred to recirculating aquarium
and fed on tadpole diet (Blades Biological, Redbridge, UK).
Plasmid construction
Xenopus pax7 (Xpax7) gene was isolated from cDNA of stage
25 embryos by RT-PCR. The primers were designed according to the sequence
submitted by the laboratory of Dr Richard Harland (NCBI:AF725267): sense
primer, 5'-CAA CTT GTG AGG ACT CTT CTA GGC T-3'; antisense primer,
5'-TTT TCA CCA AGT GGC AGA CAT-3'. The pax7 DNA fragment
obtained by RT-PCR was ligated into pGEM-T easy vector (Promega) to
generate pax7-pGEMT, which was then sequenced. The full-length
Xenopus pax7 gene was excised from pax7-pGEMT plasmid by
SpeI and ApaI (Promega), and cloned into SpeI and
ApaI sites of pSL1180 vector (Amershan). This plasmid is
called pax7-pSL1180 and used for subsequent cloning.
To generate the construct for RNA injection, the full pax7 sequence was isolated from pax7-pSL1180 with HindIII and EcoRI (Promega) and cloned into the same sites of pcDNA3 vector (Invitrogen). Capped Pax7 RNA was then transcribed in vitro with T7 RNA polymerase (Promega) after linearization with Tth111I (Promega).
The dominant-negative form of pax7 was made by the following steps. The N-terminal region of the Xenopus pax7 gene (1-245 amino acids) was excised from pax7-pSL1180 with HindIII and SmaI (Promega), and was then cloned into HindIII and ClaI (blunt filled) sites of ENR-N-pCS2+ vector (kind gift of Dan Kessler). This pax7EnR plasmid is designed to produce a fusion protein, which includes the pax7 DNA-binding domain and Drosophila engrailed repressor domain. For RNA injection, the sequence of pax7EnR was cloned into pcDNA3 with HindIII and XbaI (Promega) to generate pax7EnR-pcDNA3. This plasmid was then linearized with SmaI and transcribed in vitro with T7 RNA polymerase.
A pax7EnR plasmid suitable for transgenesis was made by excising the sequence of pax7EnR from pax7EnR-pcDNA3 with HindIII and XbaI (blunt filled), and then cloning into HindIII and SmaI sites of HGEM. The pax7EnR-HGEM was linearized with XmnI before use in transgenesis.
Transgenic Xenopus tadpoles
Transgenic Xenopus laevis tadpoles were made as previously
described (Amaya and Kroll,
1999), except for the omission of restriction enzyme from the
reaction. The transgenics were sorted out by GFP expression in the lens at
stage 42.
Microinjection
For Pax7 overexpression, 500 pg pax7 and 80 pg
gfp mRNA were injected into one side of dorsal animal hemisphere at
the four-cell stage. For pax7EnR injection, 200 pg pax7EnR,
together with 80 pg gfp mRNA, was injected into one side of dorsal
animal hemisphere at the four-cell stage. For rescue experiments, 500 pg
pax7, 200 pg pax7EnR and 80 pg gfp mRNA were
co-injected.
BrdU injection
The tadpoles at stage 49 were anaesthetized in 0.02% MS222, and injected
with 2 µl of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU)
labelling reagent from the Cell Proliferation Kit (Amersham). The injection
was performed 24 hours before fixation.
In situ hybridization and immunohistochemistry
Whole-mount in situ hybridization was performed according to the standard
protocol (Harland, 1991). The
antisense pax7 probe was designed to hybridize specifically with the
C-terminal region of Pax7. The pax7-pGEMT was linearized
with SmaI and transcribed with T7 RNA polymerase. For
morphology or immunohistochemistry, embryos or tadpoles were fixed in
Zamboni's fixative (40 mM NaH2PO4, 120 mM
Na2HPO4, 2% PFA, 0.1% saturated picric acid) overnight
at 4°C. Myofibres were stained with 12/101 monoclonal antibody
(Kintner and Brockes, 1984) at
1:100 dilution of medium. The secondary antibody was horse anti-mouse IgG
whole molecular alkaline phosphatase-conjugated (Vector Labs) at 1:1000
dilution. The colour was developed with the BM purple reagent (Roche).
Activated satellite cells were stained with MyoD monoclonal antibody (kind gift of John Gurdon) at 1:4 dilution. The basement membrane of myofibres was stained with laminin (Sigma) at 1:100 dilution. Their secondary antibodies were Texas Red-conjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Vector Labs), respectively. The monoclonal PCNA antibody (Dako Cytomation) was used at 1:500 dilution. Muscle satellite cells were stained with anti-Pax7 monoclonal antibody (Developmental Studies Hybridoma Bank, University of Iowa) at 1:300 dilution. Antigen retrieval was performed for Pax7 antibody staining by boiling the slides in citrate buffer (Vector Labs) for 5 minutes in the microwave oven. For vibratome sections, the secondary antibody was horse biotinylated anti-mouse IgG (Vector Labs) at 1:500 dilution. Then the sections were incubated in ABC reagent (Dako Cytomation), followed by colour development using a DAB kit (Vector Labs). For paraffin sections, Pax7 antibody staining was either used with the ABC method as described above, or with a fluorescence method. In the latter case, the secondary antibody is Texas Red-conjugated anti-mouse IgG (Vector Labs). The slides were counterstained in 0.5% Methyl Green solution (Fluka) and mounted in Depex (BDH) or Gel mounting medium (Biomeda) before observation under the microscope.
Electron microscopy
The tadpoles at stage 49 were fixed in Zamboni's fixative containing 0.5%
glutaraldehyde overnight at 4°C, washed in PBS and embedded in 5% low
melting agarose (Sigma). Transverse sections (100 µm) were cut with a Leica
vibratome and then immunostained as described above. Following this, the
vibratome sections were washed in PBS, and post-fixed in osmium tetroxide (1%
w/v) in 0.1 M sodium cacodylate buffer (pH 7.6) for 1 hour. The post-fixed
sections were dehydrated in a series of ethanols and embedded in Epon resin
(TAAB). The polymerized resin blocks were trimmed and transversely sectioned
with a Reichert Ultracut-E ultramicrotome (Leica, Wein, Austria). Sections
(100 nm) were collected onto copper grids, some being stained with uranyl
acetate and lead citrate, and examined under a JEOL JEM1200EX transmission
electron microscope (JEOL, Tokyo, Japan). Owing to the narrow spacing between
each section, for the morphometric studies a nucleus within myofibers was
counted only once in each resin block.
Tail amputation, heat shock and satellite cell counting
The Xenopus laevis tadpoles were anaesthetized in 1/3000 MS222 and
kept in the anaesthetic solution during the operation. For the heat-shock
experiments, tadpoles were placed into warmed water at 34°C for 30 minutes
3 hours before tail amputation and again each day during tail regeneration.
Initially, the distal 50% length of the tail was removed. Twelve or 14 days
later, when the tail regenerated to its full length, the distal 75% of the
regenerated tail was amputated again. The tadpoles were allowed to recover
from anaesthesia in tap water before returning to aquarium tanks.
Muscle satellite cell counting was based on the Pax7 antibody labelling on tissue cross-sections of regenerated tails. Cells were counted on a series of sections comprising the 50 µm length of the tail that is nearest to the first amputation site or near to the second tail amputation site, as the regenerated muscle near the tail tip is too small for any quantification. Ten tails of similar size in each group were examined and statistical analysis was performed using Student's t-test.
RT-PCR
Ten regenerating tails of each experimental group were collected for RNA
isolation. Total RNA was prepared using Trizol and reverse transcribed into
cDNA with Superscript III system (Invitrogen). Primers used for
pax7EnR are: sense, 5'-GCTCTGTCCCCTCAGGTTTAGT-3';
antisense, 5'-GGTGGTGTGCGTCTGATTGTG-3'. Primers for pax7
are: sense, 5'-TCAATAATGGTCTCTCCCCGC-3'; antisense,
5'-TTGCCAGGTAATCAACAGCGG-3'. Primers for pax6 are: sense,
5'-GCA ACC TGG CGA GCG ATA AGC-3'; antisense, 5'-CCT GCC GTC
TCT GGT TCC GTA GTT-3'.
TUNEL assay
For apoptosis detection, paraffin tissue sections were prepared as
described above. The TUNEL assay was applied on 7 µm section with the in
situ cell death detection kit (Roche) as instructed, followed by colour
development with Fast Red (tablets from Sigma). For detection of apoptosis in
Pax7-expressing cells, Pax7 immunohistochemistry was carried out first,
developed with DAB, and then the apoptosis labelling reaction performed and
visualized with a GFP filter set. For detection of apoptosis in
MyoD-expressing cells, MyoD immunohistochemistry was performed first, and then
followed by the apoptosis labelling reaction. Apoptotic cells were counted in
three series of sagittal sections of 3 days regenerated tails.
Photography and microscopy
GFP was observed in live tadpoles under anaesthesia as described above,
using a Leica Fluo III fluorescent dissecting microscope with a GFP2 filter
set. Stained sections were visualized with a Leica DMRB microscope. Images
were captured using a SPOT RT camera (Diagnostic instruments) and processed
with Photoshop software (Adobe).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At later tadpole stages, in situ probes are unable to penetrate the tadpole skin. Therefore, we used anti-Pax7 monoclonal antibody to detect expression of Pax7 protein on paraffin sections. Consistent with our in situ results, the immunohistochemistry study shows that high levels of Pax7 expression are present in forebrain (data not shown), midbrain (Fig. 2A), hindbrain (Fig. 2B) and dorsal spinal cord (Fig. 2C). Moreover, Pax7 is expressed in the eye muscle and pituitary gland (Fig. 2A,D). The cells with Pax7 positive signals in the tadpole trunk and tail muscle are flat, peripheral and squeezed beneath the basement membrane, as revealed by laminin antibody staining (Fig. 2E). On the basis of their position, these cells are mostly likely to be muscle satellite cells.
In summary, the in situ hybridization and immunohistochemistry studies in Xenopus laevis demonstrate that Xenopus pax7, like its homologues in other species, is strongly expressed in the central nervous system and muscle. This expression pattern continues to adult stage, as detected by RT-PCR (data not shown). Apart from that, Xenopus Pax7 is also expressed in pituitary gland (Fig. 1I and Fig. 2A) and adult testis (data not shown).
Pax7 is a reliable marker of muscle satellite cell in the tadpole tail
The expression study of Pax7 suggested that the cells squeezed between
myofibres with positive Pax7 immunostaining signals may be Xenopus
muscle satellite cells. To investigate this, we performed immunoelectron
microscopy (IEM). Muscle satellite cells are located in dentations between the
basement membrane and plasma membrane of myofibres, while the myonucleus lies
within the plasma membrane of myofibres
(Mauro, 1961). These
morphological criteria enable us to distinguish between the nuclei of
satellite cells and the myonuclei by transmission electron microscopy.
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). To find whether these are, in fact, satellite cells, we
injected BrdU into stage 49 tadpoles and fixed the tadpole tails 1 day after
injection. They were processed for immuno-electron microscopy, using antibody
to BrdU. We found that 60% of satellite cell nuclei are labelled with
anti-BrdU (Fig. 3I,
Table 1). By contrast, almost
all the myonuclei examined from four tails are negative for BrdU
(Fig. 3J,
Table 1). Only one myonucleus
was detected with a faint BrdU antibody signal. This is perhaps due to DNA
repair synthesis, or to recent incorporation of a satellite cell in the fibre.
As satellite cells are multiplying and myonuclei are not, it seems highly
likely that the satellite cells are the source of the new fibres, or
contribute to fibre expansion during growth, as they do in mammals
(Moss and Leblond, 1971).
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). To test
whether Pax7 plays a role in this process, we performed RT-PCR assays with
regenerating tails 3 days post amputation. As shown in
Fig. 4A, the pax7 mRNA
level is increased in the regenerating tails, compared with that in tails
without amputation. We consider that this is due to an increase in number of
Pax7-positive cells. It is not possible to make a precise quantitative
comparison of cell numbers because the structure of mature tail muscle and
regeneration bud are so different. However, at this stage, there is a
considerable amount of cell proliferation in the regeneration bud, indicated
by PCNA antibody staining (Fig.
4B), and many Pax7-positive cells are found free of the muscle
lying close to the amputation level in the blastema
(Fig. 4C), which is the
undifferentiated region of the regeneration bud
(Gargioli and Slack, 2004).
Pax7EnR is a dominant negative form of Pax7
To further investigate the function of Pax7 in muscle satellite cells
during Xenopus tail regeneration, we generated a domain-swapped
construct pax7EnR, in which the C-terminal region of Xenopus
Pax7 was replaced with the transcriptional repression domain of
Drosophila Engrailed (Han and
Manley, 1993).
This method has been used many times to generate transcription factor domain swaps, but we felt it important to confirm that it really had the predicted biological activity: namely that it can inhibit normal Pax7 function. To do this, we overexpressed Pax7EnR in the brain region, which is rich in endogenous expression of Pax7. We injected 200 pg pax7EnR, together with 80 pg gfp mRNA, into the left side of the dorsal animal hemisphere of four-cell stage embryos. The injected neurulae show defects in the anterior neural fold on the left (Fig. 5A) and later on in the developing left eye (Fig. 5C and Table 2). These defects become obvious at advanced stages. The left eye is absent or smaller, while the right eye has fully developed (Fig. 5C,D,F). The GFP fluorescence on the injected side indicates that it still has a small lens underneath the epidermis (inset in Fig. 5C). To test the specificity of Pax7EnR, we co-injected the pax7EnR RNA together with 500 pg wild-type pax7 RNA. As shown in Fig. 5B,E and Table 2, the pax7 RNA is able to rescue these eyes back to approximately normal size.
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We are not claiming that Pax7 has a normal function in Xenopus eye development, but the phenomena described here provide us with a useful bioassay. The experiments show that Pax7 promotes eye development, Pax7EnR represses it and sufficient Pax7 can restore eye development in the presence of Pax7EnR. This proves that Pax7EnR is able to inhibit the function of Pax7 and we can therefore use Pax7EnR as a dominant-negative form of Pax7 to investigate the function of Pax7 during muscle regeneration.
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). This means
that the gene is inactive through development and activated only when the
temperature is raised. We performed RT-PCR analysis on four groups of tadpole
tails. They are: (1) wild-type tails regenerated for 7 days without heat
shock; (2) pax7EnR transgenic tails regenerated for 7 days without
heat shock; (3) wild-type tails regenerated for 7 days with one heat shock
each day after amputation; and (4) pax7EnR transgenic tails
regenerated for 7 days with one heat shock each day after amputation. As shown
in Fig. 6A, pax7EnR is
induced only in the case of pax7EnR transgenic tails with heat shock,
and no leaking expression of pax7EnR is detected in tails without
heat shock. These results also show that the endogenous pax7 mRNA is
reduced in the regenerating tails with Pax7EnR expression. By contrast, there
is no effect on pax6 mRNA, which is also present in the regenerating
tail (Fig. 6A).
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Pax7EnR causes cell death during muscle regeneration
The reduction of the number of muscle satellite cells in pax7EnR
transgenic tails might arise from a reduction in the normal proliferation or
from promotion of cell death of satellite cells during muscle regeneration. We
examined cell proliferation by PCNA antibody staining in 3 day regenerating
tails of wild-type and Pax7EnR transgenic tadpoles and this showed no
obvious difference (data not shown).
Then, we performed TUNEL staining to test for apoptosis in the regenerating tails. In the case of pax7EnR transgenic tails, most apoptotic cells are present in the blastema region, the same location that muscle satellite cells are found freed from the myofibres (Fig. 7A-C). In the wild-type tails, the number of apoptotic cells is smaller than that in the pax7EnR transgenic tails (Fig. 7E). Moreover, in many clusters of cells, possibly indicating small clones, those with low level of Pax7 expression are often entering apoptosis (red arrows in Fig. 7D), although those with high level of Pax7 expression are not. Given that the heat shock promoter is expected to have a similar activity in all cells, it may be that the cells with lower content of pax7 are more vulnerable to the effect of the pax7EnR. Quantification of cell death shows that cell death in mesenchyme and blastema of pax7EnR transgenic tails is more severe than that in wild-type tails, whereas cell death in the regenerating spinal cord and notochord is unaffected (Fig. 7E).
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) to
label the activated satellite cells
(Zammit et al., 2004) and then
performed a TUNEL assay. The cell quantification result demonstrates that over
60% of MyoD-labelled cells in the mesenchyme and blastema region of
pax7EnR transgenic tails are undergoing apoptosis, whereas only 25%
of MyoD-positive cells undergo apoptosis in wild-type tails. This result
confirms a protective role of Pax7 in cell survival, something that has also
been noticed in mammalian studies (Kuang
et al., 2006; Relaix et al.,
2006). In other words, Pax7EnR reduces the number of satellite
cells not by reducing their proliferation rate but by increasing their rate of
cell death.
Muscle regeneration is inhibited in pax7EnR transgenics
The above results show that the number of satellite cells in regenerating
tails is sharply decreased when pax7EnR is activated by heat shock.
So we predict that less muscle would form in the regenerating pax7EnR
transgenic tails compared with wild type, if muscle satellite cells are the
sole source of muscle regeneration. However, after 14 days heat-shock
treatment, when muscle in wild-type tail has terminally differentiated, we did
not find a significant reduction of muscle in the transgenic pax7EnR
tails. As shown in Fig. 8B,C,
the staining strength of 12/101, a marker of muscle differentiation, is
similar in pax7EnR and in wild-type tails.
However, we know that the first regenerate is depleted in satellite cells and this raises the question of whether it can regenerate yet more muscle following a second amputation. To investigate this, we followed the procedure as depicted in Fig. 8A. The Pax7EnR expression is induced with a daily heat shock throughout the regeneration period. The first regenerate is allowed to grow for 14 days and then the distal 75% of the primary regenerated tail is re-amputated and allowed to regenerate for another 14 days. As shown in Fig. 6F, the number of satellite cells in heated pax7EnR transgenics is about half that in wild types at this amputation level. After the second regeneration, we performed 12/101 immunostaining on both regenerates. As shown in Fig. 8B,D,G, the muscle in both first and second regenerates of wild-type tails was fully formed. By contrast, for the case of pax7EnR heat shocked tadpoles, 61% of the second tail regenerates showed substantially reduced differentiated muscle, and another 11% had no muscle at all (Fig. 8E,F, Table 3). In these individuals the spinal cord and the notochord do regenerate fully in the second regenerate, so even though Pax7 is expressed in some spinal cord cells it is presumably not necessary for regeneration (Fig. 8H). This experiment clearly shows that the regeneration of muscle depends on the presence of a population of satellite cells in the amputated tail.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Kawakami et al., 1997). During
embryonic myogenesis, the chevron pattern observed in Xenopus somites
is very similar to the Pax7 expression pattern in the E12.5 mouse
myotome (Horst et al., 2006).
Expression of pax7 is also observed in a subset of muscle cells, with
the position and appearance of satellite cells
(Fig. 2). This similar
expression pattern suggests a conserved involvement of pax7 in
embryonic myogenesis and specification of muscle progenitor cells
(Halevy et al., 2004;
Seale et al., 2004;
Gros et al., 2005;
Kassar-Duchossoy et al., 2005;
Relaix et al., 2005).
However, there exist minor differences of pax7 expression profile
between species. pax7 expression is found in the melanocyte lineage
in chick and quail, but not in the melanocyte lineage in Xenopus
tadpoles, mouse and rat (Lacosta et al.,
2005). Expression of pax7 in head mesenchyme is also
different in Xenopus and mouse. As shown in
Fig. 1H,M,N, pax7
transcripts appear earlier and stronger than that in the segmented somites,
while in mouse, pax7 expression in the head mesenchyme is not
detectable until E11.5 (Horst et al.,
2006).
The presence of pax7 transcripts in the pronephros of the
Xenopus embryo is interesting and its function during organogenesis
is currently under investigation. Our preliminary result suggests that pax7
also plays an important role in the growing pronephros, probably synergizing
with Pax2 and Pax8 by acting as an anti-apoptotic factor (Y.C., G.L. and
J.M.W.S., unpublished) (Relaix et al.,
2006).
Pax7 in eye development
Our testing of the function of pax7 and pax7EnR
constructs by injecting them into the dorsal side of four-cell stage embryos
resulted in a very surprising phenotype: the induction of a well-formed extra
eye. This phenotype is reproducible, despite the low frequency of occurrence
(10% of cases). In an elegant manner, this overexpression phenotype is the
`opposite' of the Pax7EnR phenotype, which involves reduction of the normal
eye. The eye-reduction phenotypes of pax7EnR can be rescued by
co-injection of pax7. An overexpression phenotype does not, of
course, prove that the gene in question is involved in the normal development
of the affected structure. In this case, it is probably not directly involved
as Pax7 is not normally expressed in the eye itself. However, it is expressed
in the region immediately anterior to the eye and so the overexpression
effects may indicate an indirect function for Pax7 in eye development
involving signals from the surrounding tissues.
Consistent with this, pax7 is expressed in chick optic tectum and
mouse superior colliculus, one of the major visual targets in brain
(Nomura et al., 1998;
Ziman et al., 2001b;
Thomas et al., 2004;
Thompson et al., 2004). When
misexpressed, Pax7 can induce ectopic formation of superior collicular tissue,
with characteristic laminae innervated by retinal ganglion cell axons
(Matsunaga et al., 2001;
Thompson et al., 2004).
Interestingly, among the tadpoles with an extra eye, we very often observed
formation of an extra tube-like structure protruding from the midbrain
(asterisks in Fig. 5J).
Detection of a series of transcription factors related to eye field
specification show that some of the transcription factors, such as Rx1 and
Tll, are downregulated by pax7EnR injection (data not shown). Thus,
these findings do support a role of Pax7 in the development and maturation of
the vertebrate visual system, even if it is indirect. It is also possible that
the overexpression experiments are interfering in some way with pax6,
well known as a master controlling gene in eye development
(Gehring and Ikeo, 1999).
However, Fig. 6A does not
indicate any effect on pax6, at least at the level of mRNA
content.
Pax7 as a satellite cell marker
In this report, we show that Pax7 is a reliable satellite cell marker by
combining nuclear Pax7 expression with morphological observations of satellite
cells by electron microscopy. We need a good molecular marker, because it is
impossible reliably to identify a satellite cell under light microscope by
morphology alone. As shown in Fig.
3 and our statistical analysis, the immuno-electron microscope
result proves that Pax7 is a very good satellite cell marker. The observation
that 88% of morphologically identified satellite cells express Pax7 would
allow us to pick up most of satellite cells in a pool of cell population. The
minority of negative cells are presumably those that have commenced
differentiation to myoblasts. Despite the occasional expression of Pax7 in
other cell types, expression of Pax7 in myonuclei is never found, providing a
very clear discrimination between the satellite cells and myofibres
themselves. The presence of Pax7 in some Schwann cells may possibly be
associated with their ability to re-enter the cell cycle for peripheral nerve
regeneration (Hall, 2005).
Several other molecules, including neural cell adhesion molecule (NCAM),
Met and M-cadherin, have been used to identify satellite cells in various
experimental approaches (for a review, see
Holterman and Rudnicki, 2005),
so we also checked the expression of these candidates in Xenopus. In
our hands, the efficiency of these markers to recognize satellite cells in
Xenopus tadpoles is not as good as for Pax7. We observed
significantly more Pax7-positive cells in tadpole muscle tissues than cells
positive for other markers (data not shown) and so consider Pax7 as the most
reliable satellite cell marker in Xenopus.
Cell origin of muscle regeneration in Xenopus tadpoles
Our results support our previous conjecture that satellite cells residing
in the tail muscle fibres are the origin of muscle regeneration in
Xenopus tadpoles (Gargioli and
Slack, 2004). First, additional expression of pax7 in the
regenerating tail suggests that satellite cells are activated to divide after
tail amputation. In regenerating tails 3 days post amputation, a large number
of Pax7-positive cells appears in the region where muscle fibres are
undergoing degeneration, and many Pax7-positive cells are visible in the
growing blastema (Fig. 4).
Second, our experiments show that inhibition of Pax7 action by Pax7EnR causes
apoptosis of muscle satellite cells, and reduces the number of satellite cells
in regenerating tails. The tails that are depleted of satellite cells do still
regenerate, but most regenerates contain little or no muscle
(Fig. 8;
Table 3). Thus, our result
demonstrates that muscle regeneration in Xenopus tails is similar to
muscle repair in mammals, where it has been generally accepted that satellite
cells are adult muscle precursor cells and contribute to repair of damaged
muscle (Wagers and Conboy,
2005). The actual requirement for Pax7 function must be in the
survival of the proliferating satellite cells rather than in their
differentiation into muscle, as the ability to form a first regenerate is not
significantly impaired. This is consistent with the observed loss of Pax7
expression on differentiation (Seale and
Rudnicki, 2000).
Recent studies have demonstrated that Pax7 has an important role in cell
survival of adult muscle satellite cells
(Kuang et al., 2006;
Relaix et al., 2006). Using
activated caspase 3 as a marker, Relaix et al. showed that apoptosis occurs in
activated satellite cells in the skeletal muscle of the pax7 mutant
(Relaix et al., 2006).
Consistent with this, our detection of cell death with TUNEL assay suggest
that cells with low level of Pax7 tend to undergo apoptosis, while those with
high level of Pax7 expression can be protected from dying. In the regenerating
tails of pax7EnR tadpoles, the number of cells undergoing apoptosis
is increased (Fig. 7) and this
ultimately results in the failure of regeneration of muscles in
pax7EnR transgenic tails.
It was found that Pax3, which is expressed both in quiescent and activated
muscle satellite cells in mammals, has partially redundant function with Pax7
in satellite cells (Relaix et al.,
2005; Relaix et al.,
2006). As we do not obtain 100% suppression of muscle regeneration
with pax7EnR, it is possible that there is a redundant component
present such as Pax3, but it is also possible that we do not obtain 100%
inhibition of pax7 function.
Our experiments do not support the view that myofibres regenerate from
resident or circulating stem cells of the side population (SP) type
(Chargé and Rudnicki,
2004). As discussed above, these stem cells should be available to
reconstitute the muscle fibres in pax7EnR tadpoles but do not do so.
Our results also differ from the previous findings on limb regeneration in
newts and axolotl, in which it has been well documented that
de-differentiation and re-differentiation of multinucleate muscle fibres
occurs (Lo et al., 1993;
Kumar et al., 2000;
Echeverri et al., 2001;
Echeverri and Tanaka, 2002). We
see no particular reason to doubt the results from the urodele species but
consider that the mode of Xenopus tail muscle regeneration to be much
closer to the regeneration of damaged muscle in mammals. Interestingly, a
recent paper on newt regeneration has also highlighted a role for satellite
cells (Morrison et al.,
2006).
Together, our experiments support the idea that Pax7 is required for the
survival of the proliferating satellite cells in muscle and hence the
maintenance of the satellite cell pool during regeneration
(Kuang et al., 2006;
Relaix et al., 2006). This
satellite cell pool is the origin of the regenerated muscle in the
Xenopus tadpole tail.
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ACKNOWLEDGMENTS |
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REFERENCES |
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