1 Max Planck Institute for Molecular Cell Biology and Genetics,
Pfotenhauerstrasse 108, 01307 Dresden, Germany
2 Curis Incorporated, 61 Moulton Street, Cambridge, MA 02138, USA
* Author for correspondence (e-mail: tanaka{at}mpi-cbg.de)
Accepted 12 May 2005
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SUMMARY |
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Key words: Axolotl, Regeneration, Sonic hedgehog, Cyclopamine, Blastema, Sox9, Pax7
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Introduction |
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While the molecular mechanisms underlying spinal cord regeneration are
poorly understood, the patterning of the developing neural tube into distinct
DV progenitor domains has been molecularly characterized in recent years
(reviewed by Bronner-Fraser and Fraser,
1997; Ericson et al.,
1997a
; Tanabe and Jessell,
1996
). The neural tube is subdivided into distinct domains, as
defined by a series of homeodomain and paired box-containing transcription
factors, with the dorsalmost domain defined by Msx1 and 2
expression, dorsolateral cells by Pax7, and lateral domains by
Pax6, while Nkx6.1 and Nkx2.2 define increasingly
ventral domains. The size and placement of these domains is controlled by
several morphogens. Sonic hedgehog (Shh), a cholesterol-modified extracellular
signaling factor expressed in the notochord and the floor plate, induces
ventral neural tube cell types in a concentration-dependent manner
(Briscoe et al., 1999
;
Ericson et al., 1997a
;
Ericson et al., 1997b
;
Litingtung and Chiang, 2000
;
Roelink et al., 1995
). The Shh
gradient in the neural tube is antagonized by dorsally secreted bone
morphogenetic proteins (Bmps) from the epidermal ectoderm and the dorsal roof
plate cells of the neural tube (Liem et
al., 1995
), which specify a subset of interneurons in the dorsal
neural tube (Lee et al., 2000
;
Liem et al., 1997
). Whereas
Bmp4 and Bmp7 activate the expression of Msx1, Pax7 and Pax6
in the dorsal and lateral neural tube, Shh has a concentration-dependent
inhibitory effect on the expression of these markers
(Goulding et al., 1993
;
Liem et al., 1995
;
Timmer et al., 2002
). Low
concentrations of Shh block Msx1 and Pax7 expression but can
elevate Pax6 expression in lateral neural tube cells. High
concentrations of Shh, however, inhibit Pax6 expression in floor
plate cells of the neural tube (Ericson et
al., 1997b
). Thus, during embryogenesis, the notochord ventrally
and the ectoderm dorsally impose DV patterning on the neural tube through
extracellular signaling.
During development, the action of Shh and Bmps is not restricted to
patterning the neural tube. These morphogens also play important roles in
controlling cell proliferation, patterning and cell-type specification of
somite-derived cells such as the sclerotome, resulting in a coordinated
patterning of the neural tube and its surrounding mesodermal structures.
Shh mutant mice lack vertebral columns and ribs, demonstrating that
Shh signaling from the notochord and ventral neural tube is crucial for
sclerotome development (Chiang et al.,
1996). More specifically, Shh induces the expression of
sclerotomal markers such as Pax1 and Sox9
(Fan and Tessier-Lavigne,
1994
; Marcelle et al.,
1999
; Murtaugh et al.,
1999
; Zeng et al.,
2002
), which are essential for sclerotome development and
cartilage formation (Bi et al.,
1999
; Peters et al.,
1999
). Similarly, Shh regulates myogenic precursors by positively
regulating Myf5 expression
(Gustafsson et al., 2002
). In
addition to sclerotomal and myogenic markers, Shh induces proliferation of the
somitic mesoderm (Fan et al.,
1995
; Marcelle et al.,
1999
). Shh also negatively regulates its own signaling by
upregulation of its own binding receptor Patched1
(Goodrich et al., 1996
). Taken
as a whole, this information indicates that Shh signaling plays diverse roles
in the somite, namely proliferation, patterning and negative feedback. The
interplay of all three may help define the shape and size of the developing
sclerotome-derived skeletal components.
We wanted to investigate the molecular identity of the DV patterning information in the axolotl spinal cord, and how the spinal cord communicates it to the regenerating spinal cord, and subsequently to the surrounding blastema tissue. In order to identify the molecular basis of the DV patterning information in the axolotl spinal cord, we asked whether these well-described markers were present in the mature and/or regenerating spinal cord. Here we demonstrate that Shh, Pax6, Pax7 and Msx1 are expressed in their respective domains in the mature axolotl spinal cord as well as in the ependymal tube. This represents the first time that the molecular basis of DV patterning information in the mature axolotl tissue has been defined. Patched1 expression further indicates that hedgehog signaling occurs both within the spinal cord, and in surrounding blastema cells. By blocking hedgehog signaling through the drug cyclopamine, we show that it is required not only for DV patterning of the spinal cord, but also for overall tail regeneration. Specifically, the proliferation of blastema cells and Sox9 expression in the ventral blastema is dependent on hedgehog signaling. Therefore the induction of cartilage by the spinal cord during tail regeneration is mediated at least in part through hedgehog.
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Materials and methods |
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In situ hybridization on axolotl tail cryosections and sequences of probes used
Axolotl tail tissue was fixed in 4% fresh paraformaldehyde (PFA) overnight
at 4°C, washed in PBS, equilibrated in 30% sucrose and embedded in
tissue-tek (O.C.T. compound, Sakura). Cryosections 16 µm thick were mounted
on Superfrost adhesive slides and dried at room temperature (RT) for several
hours. The sections were quickly washed in PBS and treated with hybridization
denaturation mix (2% SDS, 100 mmol/l DTT in 1 x PBS) for 20 minutes at
RT. After three washes in PBS/0.1% Tween, the sections were digested with
Proteinase K (2-10 µg/ml) for 5 minutes and post-fixed directly afterward
with PFA for 10 minutes at RT. Slides were washed in PBS/Tween and incubated
at RT for 15 minutes in triethanolamine with 0.25% acetic anhydride. After
several washes in PBS/Tween, slides were prehybridized in hybridization buffer
(50% formamide, 5 x SSC, 5 x Denhardts, 750 µg/ml yeast RNA)
for 1 hour at 68°C, and then hybridized overnight at 68°C with 500
ng/ml DIG-labeled probe in hybridization solution. Slides were washed twice an
hour at 68°C in post-hybridization solution (50% formamide, 2 x SSC,
0.1% Tween) and then 3 x 10 minutes at RT in maleic acid buffer (100
mmol/l maleic acid pH 7.5, 150 mmol/l NaCl, 0.1% Tween). Sections were blocked
in maleic acid buffer plus 10% goat serum for 1 hour at RT and then incubated
overnight at 4°C in blocking buffer plus alkaline phosphatase conjugated
anti-DIG antibody (diluted 1:2000). Slides were washed 2 x 5 minutes in
maleic acid buffer and 2 x 20 minutes in alkaline phosphatase buffer
(100 mmol/l Tris pH 9.5, 50 mmol/l MgCl2, 100 mmol/l NaCl, 0.1%
Tween). Each slide was overlaid with filtered NBT-BCIP (Sigma) for 1-2 days at
RT. The staining reaction was stopped with PBS/Tween and the slides mounted in
90% glycerol.
Sense and antisense probes for in situ hybridizations were prepared from
the axolotl Shh sequence (CO786463), Msx1 sequence
(AY525844), Pax6 sequence (CO784109), Ptc1 sequence
(AY887138) and Sox9 sequence (AY894689). Shh and
Pax6 sequences were derived from EST sequences
(Habermann et al., 2004),
while the Msx1, Ptc1 and Sox9 sequences were obtained by
RT-PCR from total embryonic RNA using degenerate primers (primer sequences and
PCR conditions available upon request).
Pax7 antibody staining on axolotl tail cryosections
Axolotl tails were fixed in 4% fresh paraformaldehyde (PFA) overnight at
4°C, washed in PBS, equilibrated in 30% sucrose and frozen in tissue-tek
(O.C.T. compound, Sakura). Cross-sections of the tail 16 µm thick were
processed for immunohistochemistry with the anti-Pax7 mAB (Pax7, Developmental
Studies Hybridoma Bank, Iowa, USA). A Cy5-labeled secondary antibody (Dianova,
Hamburg, Germany,
http://www.dianova.com)
was used at 1:200 dilution. Nuclear stainings were done with 1 µg/ml of
Hoechst. To calculate the percentage of Pax7-positive cells in the blastema,
between 704 and 1128 blastema cells were counted in total per regenerate.
Cyclopamine and agonist treatment
Cyclopamine was purchased from Toronto Research Chemicals. Two hedgehog
agonists in the same chemical class as described
(Frank-Kamenetsky et al.,
2002), but with different EC50 values, were obtained from the
Curis Corp
(http://www.curis.com/),
and tested. Both gave identical results with respect to their EC50
concentrations. Hh-Ag1.9 is available from the Curis Corp. Unless indicated
otherwise, larval axolotls were exposed to cyclopamine and the hedgehog
agonist directly after tail or limb amputation. Cyclopamine-treated axolotls
were kept in 20 ml water plus 600 nmol/l cyclopamine (diluted from 5 mmol/l
stock solution in ethanol). Agonist-treated axolotls were kept in 20 ml water
plus 4, 40, 100, 300 nmol/l agonist (diluted from 400 µmol/l stock solution
in DMSO). Control animals were kept in 20 ml water, or 20 ml water plus
0.0125% ethanol, or 20 ml water plus the agonist-equivalent amount of DMSO, or
20 ml water plus 600 nmol/l tomatidine (Toronto Research Chemicals).
Cumulative BrdU labeling and anti-BrdU antibody staining
Axolotl tails were amputated and treated with 600 nmol/l cyclopamine or
equivalent amounts of ethanol. Animals were injected intra-peritoneally with
10 mg of BrdU (in a volume of 10 ml) every 8 hours starting 3 days
post-amputation (dpa). Tails were fixed in 4% freshly made PFA 48 and 72 hours
after the initial BrdU injection. Cryosections 16 µm thick were prepared
and processed for antibody staining with mouse monoclonal anti-BrdU antibody
directly coupled to rhodamine (Tanaka et
al., 1997). Nuclear staining was performed using 1 µg/ml
Hoechst. The percentages of BrdU-positive ependymal cell nuclei, and
BrdU-positive blastema cell nuclei ventral to the ependymal tubes were
calculated. The graphs in Fig.
6 represent the mean percentage of BrdU-positive cells of 2-4
regenerates. Between 76 and 255 cells in total were counted in ependymal tube
and blastema per regenerate.
Spinal cord removal from the axolotl tail
Axolotls were anesthetized and the tail was sliced open from the dorsal
side until the level of the spinal cord. The spinal cord was removed over the
length of several segments and the tail was allowed to heal for several days.
Mock operated axolotl tails were opened until the level of the spinal cord and
allowed to heal without removal of the spinal cord. Amputation was performed a
few days after the operation.
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Results |
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Our gene expression analysis indicates that the putative progenitor cells in the mature axolotl spinal cord show an embryonic pattern of DV neural tube markers. In addition, the same DV pattern is present in the ependymal tube throughout axolotl tail regeneration.
Hedgehog signaling is required for overall tail regeneration
Shh is a potent morphogen patterning the ventral half of the spinal cord,
which leads to the correct spatial organization of interneurons and
motoneurons during development (reviewed by
Litingtung and Chiang, 2000;
Marti and Bovolenta, 2002
).
Because Shh was expressed in the mature and regenerating axolotl
spinal cord, we wanted to assess its function in the establishment of the DV
identity of the regenerating tail. An interesting question for us was whether
interfering with the DV pattern in the regenerating spinal cord would have an
effect on the overall DV organization of the regenerating tail: for example,
on the position of cartilage formation. Furthermore, we wanted to examine
whether Shh is necessary for ependymal cell proliferation, as it has been
shown that Shh can act as a mitogen on neural progenitor cells, both in vitro
and in vivo (Bambakidis et al.,
2003
; Lai et al.,
2003
; Machold et al.,
2003
; Palma et al.,
2005
).
In order to inhibit the Shh signaling pathway during tail regeneration, we
turned to the widely used chemical inhibitor cyclopamine, which blocks
hedgehog signaling by antagonizing the hedgehog receptor Smoothened
(Chen et al., 2002;
Taipale et al., 2000
). The
drug can be easily administered through the axolotl water. Interestingly, we
found that in the presence of cyclopamine overall axolotl tail regeneration
was strongly inhibited (Fig.
2A-G). Wound healing and fin formation occurred normally, and the
ependymal tube grew to a limited extent, but a proper blastema did not grow
(compare Fig. 2A-C with D-F).
The rate of ependymal tube growth was substantially lower than control
regenerates (Fig. 2G). In terms
of the blastema phenotype, few blastema cells had accumulated in
cyclopamine-treated regenerates 4 days post-amputation (dpa) in comparison
with the control (compare Fig. 2A with
D). The effect became more evident at later stages of
regeneration, when even up to 14 dpa neither cartilage nor muscle
differentiation took place in cyclopamine-treated regenerates (compare
Fig. 2B,C with E,F). Cartilage
and muscle started to differentiate at 6 and 10 dpa, respectively, in control
regenerates (not shown). After 8 days of cyclopamine treatment, the initial
outgrowth of the ependymal tube stopped, and the tube slowly regressed over
the following days (Fig. 2G).
The inhibitory effect of cyclopamine on tail regeneration could be observed at
concentrations ranging from 600 nmol/l to 6 µmol/l, while the same
concentrations of tomatidine, a closely related compound to cyclopamine that
does not interfere with Shh signaling, did not have this effect
(Fig. 2G). As the various
concentrations of cyclopamine tested all yielded very similar results, only
the lowest concentration (600 nmol/l) was used for the experiments reported
here.
Experimental evidence that cyclopamine exerts a specific inhibition of the
hedgehog signaling pathway during tail regeneration was the ability to rescue
the phenotype with a hedgehog-pathway agonist. When we added a hedgehog
agonist in the same chemical class described in Frank-Kamenetsky et al.
(Frank-Kamenetsky et al.,
2002) (see Materials and methods) together with cyclopamine, a
tail with normal cartilage and muscle patterning regenerated
(Fig. 2H-K).
The inhibition of tail regeneration by cyclopamine and the rescue of this phenotype with a hedgehog-pathway agonist strongly suggest that hedgehog signaling is required for overall tail regeneration.
Hedgehog signaling is necessary for the correct establishment of DV progenitor domains in the ependymal tube during tail regeneration
When we examined the ependymal tube in cyclopamine-treated regenerates for
DV patterning defects we observed expansion of the dorsal spinal cord markers
Pax7 and Msx1 into more ventral regions (compare
Fig. 3A,D with
Fig. 1G,I). In cyclopamine and
agonist-treated regenerates, both the Msx1 and Pax7
expression domains were restored, demonstrating the rescue of the cyclopamine
effect (compare Fig. 3B,E with
Fig. 1G,I). Treatment of
regenerating tails with the agonist alone did not have any overall
morphological effects, although the regenerating tails might have been
slightly bigger. We did, however, observe an effect of the agonist alone on DV
patterning markers in the spinal cord. Even low concentrations (4 nmol/l) of
agonist abolished Pax7 expression from the dorsal spinal cord
(Fig. 3C). By contrast,
Pax7-positive cells in the surrounding lateral blastema tissue, which
presumably represent muscle progenitors, persisted in the presence of agonist
(Fig. 3C).
We conclude from these results that hedgehog signaling is required for the correct establishment of DV progenitor domains in the regenerating axolotl spinal cord.
Patched1 is expressed in the ependymal tube as well as in the blastema
As the strongest effect of inhibiting hedgehog signaling during tail
regeneration was a reduced tail blastema, we wanted to know whether blastema
cells directly receive the hedgehog signal. We therefore examined the
expression of the hedgehog binding receptor Patched1 (Ptc1)
in tail regenerates by in situ hybridization. In normal regenerates
Ptc1 was expressed in ventral and lateral spinal cord cells, and in
the blastema cells surrounding the ventral spinal cord
(Fig. 4B,C; note the absence of
staining in the epidermis). Ptc1 itself is a target gene of the
hedgehog signaling pathway that is upregulated where Shh signaling occurs
(Goodrich et al., 1996).
Agonist-treated regenerating tails showed increased Ptc1 expression:
most or all of the ependymal cells and also most of the blastema cells
expressed Ptc1 (Fig.
4E,F). Together these data indicate that blastema cells receive
the hedgehog signal directly. Although it is not known if other hedgehog
family members are also expressed during regeneration, the expression of
Ptc1 in the ependymal tube and the surrounding blastema tissue is
consistent with the regenerating spinal cord as the primary source of hedgehog
signal.
Hedgehog signaling is required for Sox9 expression in the tail blastema
Knowing that Shh signaling can occur from the ependymal tube to the
surrounding blastema cells, we wanted to investigate whether Shh is required
for patterning the blastema tissue. During development, Shh induces the
expression of the early cartilage marker Sox9 in the sclerotome
(Tavella et al., 2004;
Zeng et al., 2002
). We
examined whether cartilage progenitors in the early blastema express
Sox9, and whether this expression is controlled by hedgehog
signaling. We found that Sox9 was expressed in a defined area of the
blastema ventral to the spinal cord from 4 dpa onward (data not shown), which
is 2 days before obvious cartilage differentiation. By contrast, Sox9
expression was not detectable in cyclopamine-treated regenerates 6 dpa
(Fig. 5A,B), while
agonist-treated regenerates showed an increased expression domain of
Sox9, and occasional dorsal blastema cells expressing the gene
(Fig. 5C, arrows point to
Sox9-positive cells). Despite this expanded expression of
Sox9 in the agonist-treated sample, no overt cartilage
differentiation was observed in the dorsal blastema, and the ventral cartilage
rod appeared normal.
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Hedgehog signaling controls blastema cell proliferation rather than ependymal cell proliferation
The overall morphology of cyclopamine-treated regenerates indicated that
the fin was normal, but the size of the blastema was severely reduced (compare
Fig. 2A with 2D). On
cross-sections we observed that cyclopamine-treated regenerates had a smaller
width compared with controls (compare Fig.
3A with Fig. 1G and
Fig. 3B, and
Fig. 5A with 5B).
We examined whether the reduction of the blastema was due to apoptosis or a block in cell division. TUNEL staining of cyclopamine and control samples were indistinguishable, suggesting that massive apoptosis did not account for the blastema defect (data not shown). To examine cell proliferation, we performed cumulative BrdU labeling for 48 and 72 hours, starting 3 dpa. The percentage of BrdU-positive ependymal cells and BrdU-positive ventral blastema cells was calculated in control and cyclopamine-treated regenerates at 48 hours after the initial injection. We observed a different effect of cyclopamine on proliferation of ependymal cells versus ventral blastema cells (compare Fig. 6A with 6B). Whereas cyclopamine treatment had only a minor effect on the fraction of proliferating ependymal cells (from 99 to 86%; Fig. 6A), it had a strong, statistically significant inhibitory effect on the fraction of proliferating ventral blastema cells, from 95 to 56% (Fig. 6B). This inhibitory effect was stable over time, as we observed the same decrease of BrdU incorporation at 72 hours, indicating that all proliferating cells had incorporated BrdU. We conclude that hedgehog signaling controls the proliferation of approximately 40% of ventral tail blastema cells. This number could be an underestimate, because we could have inadvertently included some fin cells (that regenerate normally) in the analysis.
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Discussion |
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In terms of establishing the various DV progenitor cell domains within the
spinal cord, a relatively detailed understanding has been gained in embryonic
studies, and we assume that the same signaling networks are implemented during
regeneration. In particular, Briscoe et al.
(Briscoe et al., 2000) have
suggested a model to explain how Shh signaling from the floor plate could
result in the establishment of distinct neural progenitor domains along the DV
axis of the neural tube. Graded Shh signaling results in the definition of two
distinct types of molecular domains. The expression of so-called class I
homeodomain proteins such as Pax7, Irx3, Dbx1, Dbx2 and Pax6 (found in
dorsolateral regions) are repressed by Shh signaling, while expression of
class II homeodomain proteins including Nkx6.1 and Nkx2.2 are activated by Shh
signals. Cross-repressive interactions between class I and class II
homeodomain proteins, such as those between Pax6 and Nkx2.2
(Briscoe et al., 2000
),
establishes, refines and stabilizes the progenitor cell domains. Although a
specific class II protein that represses Pax7 has not been identified yet,
presumably additional class II proteins may exist
(Briscoe et al., 2000
).
Therefore, in our case the ventral expansion of Pax7 in cyclopamine-treated
regenerates is probably due both to an increase in Pax7 expression stemming
from reduced hedgehog signaling, and a decrease in the level of class II
proteins that require hedgehog signaling for their expression and that act by
restricting Pax7 expression to a dorsal domain. Conversely, reduction of Pax7
in ependymal tubes of hedgehog-agonist-treated regenerates might be due to
both an increase in hedgehog signals and in the level of class II proteins
that subsequently repress Pax7 in the dorsal tube.
During development, Bmps in the dorsal ectoderm and roof plate are crucial
morphogens for DV neural tube patterning. We surmise that Bmp4 and Wnt3a are
expressed in the dorsal axolotl spinal cord. Although we could detect Bmp4 and
Wnt3a in tail blastema RNA by RT-PCR, attempts to localize Bmp4 and Wnt3a by
in situ hybridization or phospho-Smad1 immunohistochemistry have so far been
unsuccessful. The presence of Msx1, a known downstream target of Bmp4
(Liem et al., 1995;
Timmer et al., 2002
), in the
axolotl dorsal spinal cord suggests the presence of Bmp signaling within the
spinal cord.
The role of hedgehog signaling in patterning the tail blastema
In addition to the role of hedgehog signaling in patterning the
regenerating spinal cord, we have demonstrated that hedgehog is also required
for patterning the surrounding blastema tissue. The early cartilage marker
Sox9 was not expressed in cyclopamine-treated animals. We favor the idea that
this reflects a requirement of hedgehog to induce Sox9 expression rather than
complete absence of Sox9-expressing cells in the blastema, for several
reasons. First, during development, Shh signaling from the notochord and
neural tube induces Pax1, Pax9 and Sox9 in the sclerotome, the precursors for
cartilage (Fan and Tessier-Lavigne,
1994; Marcelle et al.,
1999
; Murtaugh et al.,
1999
; Tavella et al.,
2004
; Zeng et al.,
2002
). In the blastema, the location of Sox9 expression with
respect to the regenerating spinal cord is distinct from that during
development, as it appears ventral to the ependymal tube rather than in
lateral sclerotomal cells. Although regeneration does not proceed through a
morphologically distinct somite, it is very likely, however, that the
molecular signaling pathway leading to cartilage formation in the two contexts
are the same. Second, the hedgehog agonist could induce ectopic Sox9
expression in dorsal regions of the blastema. The fact that only isolated
dorsal blastema cells expressed Sox9, rather than massive formation of
cartilage throughout the blastema in agonist-treated regenerates, is probably
due to the inhibitory role of molecules such as Bmps in the dorsal regenerate
that would antagonize the agonist effect.
The role of hedgehog signaling in blastema cell proliferation
A striking aspect of our results is the profound dependence of tail
regeneration on hedgehog signaling. BrdU labeling indicated at least a 40%
reduction in cycling blastema cells. This result probably represents an
underestimate, because it is difficult to distinguish the cycling fin cells
from the blastema cells due to lack of a blastema cell marker. We favor the
idea that sonic hedgehog is a direct mitogen for blastema cells, as the
patched receptor is expressed in the blastema, although it is possible that
hedgehog expression in the regenerate may be necessary for the expression of a
blastema cell mitogen. For example, in the limb sonic hedgehog expression
upregulates Fgf4 in the apical ectodermal ridge to promote limb bud outgrowth
(Laufer et al., 1994;
Niswander et al., 1994
). We
have tested the role of signaling through the Fgfr1 in tail regeneration and
found that it cannot account for the effect of hedgehog. While chemical
inhibition of Fgfr1 signaling during tail regeneration via the pharmacological
inhibitor SU5402 initially slowed down regeneration slightly, the regenerated
tails showed no other phenotype and grew to a normal length (data not shown)
a phenotype quite distinct from hedgehog inhibition. It appears,
however, that hedgehog is not the sole factor required for blastema cell
proliferation, as the hedgehog agonist could not rescue the blastema growth
defect produced by spinal cord removal.
Concluding remarks
Our study also has implications for understanding the origin and fate of
blastema cells. As Shh is already expressed in the mature spinal cord, the
tail blastema cells receive signals that direct them to specific cell fates as
soon as they are born. A naive blastema cell may therefore be extremely
difficult to detect. Although the ventral blastema cells behave similarly to
sclerotome, it is not clear if an early blastema cell that responds to the Shh
signal is equivalent to an early somite cell, a presomitic mesoderm cell, or
is a completely distinctive cell type. It is possible, for example, that the
blastema cell has more fates available to it than a typical sclerotomal cell.
Furthermore, it is unclear if the Sox9-expressing blastema cells derive solely
from sclerotomal derivatives in the mature tissue, or whether different tissue
types can contribute blastema cells that are induced to express Sox9.
Echeverri and Tanaka (Echeverri and
Tanaka, 2002) showed that cells can migrate from the spinal cord
and contribute to cartilage during tail regeneration, indicating that
cartilage precursors have diverse origins. Specific labeling of different cell
types in the mature tissue and long-term lineage tracing will be required to
fully address this issue.
It is noteworthy that the role of hedgehog signaling during axolotl limb regeneration is clearly different from its role in tail regeneration. In the limb blastema, Shh controlled AP digit formation and did not severely inhibit blastema outgrowth or cartilage formation. This indicates that blastema cells probably have region-specific identities that allow them to respond to inductive cues in distinct ways. Presumably this region-specific identity is maintained in the mature tissue and inherited by blastema cells, although it is possible that such identity is positively reinforced during regeneration, and that in certain cases, this identity could be reversed.
The maintenance of patterning information in the mature tissue may be a
central feature of regenerative ability. Adult mouse and chick spinal cord
tissue does not maintain the markers examined here, and this may represent a
block for regeneration (Fu et al.,
2003; Yamamoto et al.,
2001
). Interestingly, injury of the mouse spinal cord did result
in the appearance of Pax7-positive cells in the parenchyma of the dorsal horn
(Yamamoto et al., 2001
). These
Pax7-positive cells co-stained with nestin, indicating that they may have the
capacity to act as progenitor cells. Such observations suggest the possibility
that mammals harbor a latent capacity to re-induce important aspects of cell
patterning after injury. The comparison of patterning marker expression in
spinal cord progenitor cells (and other tissues) in different species may be
an important dimension of understanding the regenerative ability.
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ACKNOWLEDGMENTS |
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