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First published online 21 December 2006
doi: 10.1242/dev.001123
1 Howard Hughes Medical Institute, Department of Pharmacology, Institute for
Stem Cell and Regenerative Medicine, University of Washington School of
Medicine, Seattle, WA 98195, USA.
2 Graduate Program in Neurobiology and Behavior, University of Washington School
of Medicine, Seattle, WA 98195, USA.
3 Department of Surgery, University of Washington School of Medicine, Seattle,
WA 98195, USA.
4 Department of Pathology, University of Washington School of Medicine, Seattle,
WA 98195, USA.
Author for correspondence (e-mail:
rtmoon{at}u.washington.edu)
Accepted 21 November 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Wnt, zebrafish, regeneration, ß-catenin, dickkopf, wnt8, wnt5, pipetail, axin1, masterblind
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Brockes and Kumar, 2002;
Poss et al., 2003;
Poss et al., 2002). A dramatic
example of organ regeneration is that of amphibian limbs and fish fins, where
intricate structures consisting of multiple cell types that are patterned into
complex tissues are faithfully restored after amputation. The mechanisms that
enable lower vertebrates to re-establish such structures and the reasons why
mammals are not able to do so, are incompletely understood. Elucidation of
these mechanisms and an understanding of why regenerative capacity has
diminished in vertebrate evolution hold the potential to revolutionize
clinical medicine, with practical applications ranging from organ disease and
wound treatment to possible alternatives to prosthetics for amputees.
Whereas repair of many organs, such as the chicken retina or mouse liver,
is thought to be mediated through activation of resident stem cells or
proliferation of normally quiescent differentiated cells, respectively
(Fausto et al., 2006;
Fischer and Reh, 2001),
amphibian and fish appendages regenerate through a process termed `epimorphic
regeneration', sometimes called `true' regeneration. This occurs in three
steps: (1) wound healing and formation of the wound epidermis; (2) formation
of a regeneration blastema, a population of mesenchymal progenitor cells that
is necessary for proliferation and patterning of the regenerating limb/fin;
and (3) regenerative outgrowth and pattern reformation
(Akimenko et al., 2003;
Poss et al., 2003). Progenitor
cells of the blastema in the regenerating axolotl tail can be formed by
reprogramming and de-differentiation of differentiated cells
(Casimir et al., 1988;
Echeverri et al., 2001;
Echeverri and Tanaka, 2002;
Kintner and Brockes, 1984;
Lentz, 1969;
Lo et al., 1993). These cells
express transcriptional repressors of the msx gene family that may
help maintain a pluripotent state (Akimenko
et al., 1995; Yokoyama et al.,
2001). Recently, activation of resident muscle stem cells has been
reported in regenerating salamander limbs
(Morrison et al., 2006). Thus,
it is likely that de-differentiation and stem cell activation both contribute
to formation of the blastema. Although de-differentiation of cells has not yet
been shown to occur in regenerating structures other than amphibian limbs and
tails, the morphology, ontology and gene expression profile of the zebrafish
blastema in the regenerating tail fin suggest that zebrafish tail regeneration
occurs by similar mechanisms. A major question that remains incompletely
answered involves the identification of the extracellular signals that
regulate the formation or activation of stem cells during regeneration.
Although hedgehog signaling has been implicated in newt tail and chick retina
regeneration (Schnapp et al.,
2005; Spence et al.,
2004), and BMP signaling in newt lens and Xenopus tail
regeneration (Beck et al.,
2003; Grogg et al.,
2005), the strongest evidence to date points to FGF signaling as
an essential regulator of progenitor cell formation in limb and fin
regeneration. FGF-10 is sufficient to reactivate regeneration in
Xenopus limbs at later stages of development where limbs have lost
their regenerative capacity (Yokoyama et
al., 2001), and FGF-2-soaked beads can stimulate chick limbs,
which normally do not regenerate, to do so
(Taylor et al., 1994).
Inhibition of FGF signaling by pharmacological inhibitors or expression of a
dominant-negative FGF receptor blocks blastema formation in zebrafish caudal
fin regeneration (Lee et al.,
2005; Poss et al.,
2000b), and a mutation in zebrafish fgf20a causes an
early block in blastema formation
(Whitehead et al., 2005).
Wnt/ß-catenin signaling regulates progenitor cell fate and
proliferation during embryonic development and in adult tissue homeostasis
(Logan and Nusse, 2004;
Reya and Clevers, 2005),
raising the possibility that it is also involved in progenitor cell function
during regeneration. Several studies have documented expression of Wnt ligands
and components of the ß-catenin signaling pathway in regenerating
amphibian and fish appendages (Caubit et
al., 1997a; Caubit et al.,
1997b; Poss et al.,
2000a), and other studies have suggested that Wnt/ß-catenin
signaling is functionally involved in the proliferation of cells during
regeneration of mammalian muscle, liver and bone
(Polesskaya et al., 2003;
Sodhi et al., 2005;
Zhong et al., 2006). However,
whether Wnt/ß-catenin signaling plays an essential role in the
epimorphic, `true' regeneration of complex structures has not been tested.
Many Wnt ligands can activate ß-catenin-independent ('noncanonical')
signaling pathways (Slusarski et al.,
1997; Veeman et al.,
2003) that are well documented to regulate cell polarity and cell
migration during embryonic development
(Veeman et al., 2003).
However, other than reports which indicate that ß-catenin-independent Wnt
signaling might act to suppress tumor formation
(Dejmek et al., 2005;
Jonsson et al., 2002;
Kremenevskaja et al., 2005),
nothing is known about its role in adults. Here, we provide evidence that both
ß-catenin-dependent and -independent Wnt signaling pathways regulate
zebrafish fin regeneration.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6-12 months of age were used for all studies. Zebrafish
heart and fin amputations were performed as previously described
(Poss et al., 2000a;
Raya et al., 2004), after
which fish were returned to 28-30°C water.
Partial hepatectomy in TOPGAL mice
TOPGAL mice (a gift from E. Fuchs, Rockefeller University, NY) have been
described previously (DasGupta and Fuchs,
1999). We performed 2/3 partial hepatectomy
(Campbell et al., 2006) and
sham laparotomy on 8- to 11-week-old male TOPGAL mice in the morning after a
night of fasting. Resected lobes were collected and served as control tissue
for subsequent experiments; remnant livers were harvested 48 hours later.
ß-galactosidase activity was determined in whole liver lysates as per
manufacturer's instructions (Promega, Madison, WI), and normalized to total
protein concentration as determined by the Bradford assay (Bio-Rad, Hercules,
CA). X-Gal staining was performed on glutaraldehyde-fixed 5 µm frozen liver
sections as per manufacturer's instructions (Gold Biotechnology, St Louis,
MO).
Cloning of zebrafish wnt5a
Zebrafish genomic sequence was searched for sequences homologous to the
previously known zebrafish Wnt5 ortholog, pipetail (ppt). A
sequence distinct from ppt was identified and a partial cDNA coding
for this wnt5 paralog cloned by RT-PCR from a mixture of RNA isolated
at different stages of embryonic development. The 5' end of the cDNA was
defined by RACE and by homology to EST 052-H12-2. The very 3' end of the
open reading frame and a putative 3'UTR were predicted from genomic
sequences, but have not been experimentally verified. BLAST searches, multiple
sequence alignments of the predicted protein sequence with Wnt5 paralogs from
other species and phylogenetic analysis using the PAUP program support the
conclusion that the previously described zebrafish wnt5 paralog
ppt is the zebrafish ortholog of wnt5b, whereas the newly
cloned paralog described here is most likely to be the ortholog of
wnt5a (see Fig. S2 in the supplementary material). We thus deposited
the new sequence as wnt5a in GenBank (accession number DQ465921) and
suggested that wnt5 (pipetail; ppt) should be
renamed wnt5b, which has now been done.
In situ hybridization
Whole-mount in situ hybridization was performed on amputated fins and
hearts as described previously (Poss et
al., 2000a). For Digoxigenin-labeled probe synthesis, published
templates were used, except for wnt5a cDNA, which was cloned by
RT-PCR from RNA isolated from embryos at different stages of development. When
assaying for differences in expression, the development of the staining
reaction was monitored carefully and fins or hearts of the same comparative
groups were stopped at exactly the same time. Cryosectioning of the fins was
performed as described previously (Poss et
al., 2000b).
Heat-shock inducible transgenic zebrafish lines
The hsDkk1GFP and hsWnt5bGFP lines were established as follows (see Fig. S3
in the supplementary material). mmGPF5
(Siemering et al., 1996) was
fused to the C-terminus of zebrafish dkk1 (Genbank accession #
AB023488). Upon injection into zebrafish embryos, RNA encoding this fusion
protein was found to cause posterior truncations, and increased the size of
eyes and forebrain at similar doses as the wild-type dkk1 RNA (data
not shown). Likewise, mmGFP5 was fused to the C-terminus of zebrafish
wnt5b/pipetail (see Fig. S2 in the supplementary material for
nomenclature; Genbank accession # DRU51268). Injection of RNA coding for this
fusion protein into early zebrafish embryos caused similar gastrulation
defects as RNA coding for the wild-type Wnt5b protein, but the fusion protein
appeared to be significantly less active (data not shown). Both fusion
proteins were cloned downstream of a 1.5 kb fragment of the zebrafish hsp70-4
promoter (Halloran et al.,
2000) and upstream of the SV40 polyadenylation signal of the
vector pCS2+. An I-SceI meganuclease restriction site was inserted
5' of the transgene. Supercoiled plasmid DNA containing the transgenes
was injected together with I-SceI meganuclease
(Thermes et al., 2002) into
one-cell-stage embryos to create mosaic G0 founder fish. Founders that
transmitted a functional transgene through their germline were identified by
crossing them to wild-type fish, heat shocking the resulting F1 embryos and
screening them for GFP expression. Transgenic F1 embryos were found to be
viable when heat shocked at 24 hpf or later and therefore could be raised to
adulthood. To establish transgenic lines, identified heterozygous F1 fish were
crossed to wild-type fish and the F2 generation raised. For most experiments
on adult fish, wild-type siblings from such crosses served as controls. When
siblings could not be used, age-matched wild types served as controls.
Heat shocks for these lines and the hs
TCFGFP and hsWnt8GFP lines
were performed twice daily by transferring fish from 28-30°C water to
water preheated to 38°C with subsequent incubation in an air incubator at
39°C for 1 hour.
Tissue sectioning and histology
Hematoxylin staining and histology were performed as previously described
(Poss et al., 2002) on 20
µM cryostat sections.
BrdU incorporation and mitosis analysis
BrdU incorporation and mitosis analysis were performed as previously
described (Nechiporuk and Keating,
2002). All BrdU incorporations were performed for the final 1-2
hours of the experiment. Sections were rinsed three times in PBS, then
incubated in 2N HCl for 30 minutes at 37°C. Sections were then briefly
rinsed in PBS three times and incubated in blocking solution (1% Triton X-100
and 0.25% BSA in PBS) for at least 1 hour. Slides were incubated in mouse
anti-BrdU (1:200; Sigma, St Louis, MO) and rabbit anti-phosphorylated histone
H3 (PH3; 1:200, Upstate Biotechnology, Charlottesville, VA) antibodies
overnight at room temperature. Slides were then washed all day with multiple
changes of PBS and then incubated in secondary antibodies (goat anti-mouse
Alexa-fluor-546; goat anti-rabbit Alexa-fluor-488, Molecular Probes) for 1-2
hours at room temperature. Slides were rinsed three times in PBS (20 minutes
each) and mounted with DAPI mounting media and coverslipped. DAPI-stained
nuclei, BrdU-positive cells and PH3-positive cells were counted from 3-6
sections per fin from three fins per wild-type or transgenic sample.
n=number of blastemas counted per experiment.
Fin length measurements in axin1 and wnt5b mutant fish
Heterozygous carriers of the axin1 mutation masterblind
(mbltm013) and wild-type sibling fish were identified by
genotyping using allele-specific PCR. pptta98
(wnt5b) homozygous mutant embryos were identified by their phenotype
in an incross of heterozygous carriers. Because some homozygous embryos
survive, identified embryos could be raised to adulthood. At different times
during regeneration, fins were photographed and photographs were blinded
before analysis. The length of the regenerate (from the amputation plane to
the distal tip of the fin) at the third, fourth and fifth dorsal fin ray was
measured using IMAGE J software (NIH,
http://rsb.info.nih.gov/ij/)
and the average length of the regenerate calculated for each fish. To exclude
that variations in the position of the amputation plane might have caused
differences in regenerative speed, the exact position of the amputation plane
was measured in each fish. We found that there was no significant difference
in the position of the amputation plane between wild-type and mbl or
ppt fish.
Semi-quantitative and quantitative RT-PCR
Total RNA was extracted from zebrafish fin regenerates using TRIZOL
according to the manufacturer's protocol (Invitrogen). RNA was digested with
DNase and purified using the Qiagen RNeasy Kit. Equal amounts of total RNA
from each sample were reverse transcribed with Thermoscript reverse
transcriptase (Invitrogen) using oligo(dT) and random hexamer primers. For
semi-quantitative PCR, amplification of ornithine decarboxylase
(odc1) was used as the loading control. fgf20a (primers
5'-GCAGATTTGGTATATTGGAATTCAT-3' and
5'-CTAGAACATCCTTGTAAAGCTCAGG-3') and odc1 (primers
5'-ACTTTGACTTCGCCTTCCTG-3' and
5'-CACCTTCATGAGCTCCACCT-3') PCR products were detected on Ethidium
Bromide-stained agarose gels. Quantitative PCR was performed using a Roche
Lightcycler and the SYBR Green Labeling System. wnt10a was amplified
using primers 5'-ATTCACTCCAGGATGAGACTTCATA-3' and
5'-GTTTCTGTTGTGGGCTTTGATTAG-3'. wnt10a expression levels
were normalized to ß-actin (primers
5'-GGTATGGGACAGAAAGACAG-3' and
5'-AGAGTCCATCACGATACCAG-3') or 18S rRNA (primers
5'-CGCTATTGGAGCTGGAATTACC-3' and
5'-GAAACGGCTACCACATCCAA-3') levels. Primers for quantitative PCR
of fgf20a were 5'-CAGCTTCTCTCACGGCTTGG-3' and
5'-AAAGCTCAGGAACTCGCTCTG-3'
(Whitehead et al., 2005).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and is
highly amenable to experimental manipulation, we studied the role of Wnt
signaling in regeneration using the zebrafish fin model. Expression of Wnt
ligands and of components of the ß-catenin signaling pathway has been
reported in regenerating amphibian and fish appendages
(Caubit et al., 1997a;
Caubit et al., 1997b;
Poss et al., 2000a),
suggesting that Wnt/ß-catenin signaling is upregulated during
regeneration. The endpoint of Wnt/ß-catenin signaling is transcriptional
regulation of target genes; however, because Wnt signaling is tightly
regulated by extracellular, cytoplasmic and nuclear inhibitors, expression of
Wnt ligands does not necessarily result in activation of transcription. Thus,
to test whether the Wnt/ß-catenin pathway is functional during zebrafish
fin regeneration, we asked whether a transcriptional reporter of
Wnt/ß-catenin signaling, TOPdGFP
(Dorsky et al., 2002), is
activated in response to fin amputation in TOPdGFP transgenic zebrafish. We
found that TOPdGFP is detectable in the blastema of the regenerating fin at 2
days post-amputation (dpa) (Fig.
1A). We also found that the expression of axin2, which
has been shown to be a direct Wnt target gene in several systems
(Jho et al., 2002;
Leung et al., 2002;
Lustig et al., 2002;
Weidinger et al., 2005) and of
sp8, which is regulated by Wnt/ß-catenin signaling in fin and
limb development (Kawakami et al.,
2004), were upregulated in regenerating zebrafish tail fins
(Fig. 1B).
We then investigated which Wnt ligands might be responsible for activation
of Wnt/ß-catenin signaling during regeneration of the tail fin. We found
that wnt10a, which has been shown to activate Wnt/ß-catenin
signaling during limb development (Narita
et al., 2005), is expressed early during regeneration. Expression
of wnt10a was detected in the distal tip of the blastema
(Fig. 1B). Using quantitative
PCR, we found that expression of wnt10a was upregulated very early
during regeneration, expression being 2.3-fold higher than in uncut fins 3
hours post-amputation (hpa) and 5.3-fold higher at 6 hpa
(Fig. 1C). Thus,
wnt10a is an excellent candidate for a Wnt ligand responsible for
early activation of the ß-catenin signaling pathway during fin
regeneration. Interestingly, we found that Wnt signaling activity, as detected
by transgenic reporters, is also upregulated during zebrafish heart and mouse
liver regeneration (see Fig. S1 in the supplementary material), suggesting
that activation of Wnt/ß-catenin signaling may be a conserved feature of
regeneration.
We also tested whether Wnts that have been shown to signal via
ß-catenin-independent pathways in other systems
(Slusarski et al., 1997;
Veeman et al., 2003), are
expressed during zebrafish fin regeneration. We cloned the zebrafish ortholog
of wnt5a (see Fig. S2 in the supplementary material) and found that
its expression is induced after the blastema has formed and is maintained
throughout regeneration. We observed wnt5a expression in the basal
epithelial layer of the regeneration epidermis as well as in the distal tip of
the blastema (Fig. 1B).
wnt5b (pipetail; for nomenclature, see Fig. S2 in the
supplementary material) which, like wnt5a, has been shown to signal
via ß-catenin-independent pathways in other systems
(Westfall et al., 2003), was
also expressed in the basal epithelial layer of the epidermis, albeit only at
the very tip of the regenerate, as well as in the distal tip of the blastemal
mesenchyme (Fig. 1B). These
data suggest that ß-catenin-independent Wnt signaling pathways, activated
by Wnt5 paralogs, play a role in fin regeneration.
Wnt/ß-catenin signaling is required for fin regeneration
To test the requirement of Wnt/ß-catenin signaling for fin
regeneration we created a line of zebrafish that are transgenic for heat-shock
inducible Dickkopf1 (hsDkk1GFP; see Fig. S3 in the supplementary material), a
secreted inhibitor of Wnt/ß-catenin signaling
(Glinka et al., 1998).
Activation of the transgene during embryogenesis phenocopies the effects of
wnt8 loss-of-function (see Fig. S3G-I in the supplementary material)
and is sufficient to suppress expression of the TOPdGFP Wnt/ß-catenin
reporter in doubly transgenic embryos 3 hours after induction (see Fig. S3J-K
in the supplementary material). Heat shock induces ubiquitous expression of
the transgene (as monitored by GFP expression) in embryos and regenerating
adult tail fins (see Fig. S3C-F in the supplementary material). Thus, this
transgenic line represents an excellent tool to study the functions of
Wnt/ß-catenin signaling during late embryogenesis and in adults.
Additionally, we employed a zebrafish line transgenic for a heat-shock
inducible dominant-negative form of the transcription factor Tcf3 (T-cell
factor 3) (hsTcfGFP), which has been shown to efficiently inhibit
expression of Wnt/ß-catenin target genes
(Lewis et al., 2004). When we
heat shocked the fish 2 hours before fin amputation and continued to heat
shock twice daily for 7 days, we found that regeneration was completely
blocked in both hsDkk1GFP and hsTcfGFP transgenic fish, whereas
regeneration in heat-shocked wild-type fish was unperturbed
(Fig. 2A,B).
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Wnt/ß-catenin signaling regulates blastema formation and subsequent proliferation
To characterize the cell biological functions of Wnt/ß-catenin during
fin regeneration, we carried out assays to test for specific effects of Dkk1
overexpression on cell specification and proliferation. Heat shock of
hsDkk1GFP fish starting shortly before amputation resulted in a loss of
expression of lef1, a marker for the basal epidermis
(Poss et al., 2000a), by 24
hpa, indicating that the basal layer of the wound epidermis was not being
specified correctly (Fig. 3A).
We also found that expression of msxb, a marker for the mesenchymal
progenitor cells of the regeneration blastema
(Poss et al., 2000b), and of
shh, which is normally expressed within basal epidermal cells
(Poss et al., 2000b), was lost
by 72 hpa in Dkk1-expressing fins (Fig.
3A). Histological examination confirmed that formation of the
regeneration blastema was severely impaired in hsDkk1GFP fish, although the
wound healed properly (Fig.
3B). These data show that neither the blastema mesenchyme nor the
overlying epithelium are specified correctly following loss of
Wnt/ß-catenin signaling.
To test whether Wnt/ß-catenin signaling is required for proliferation of the blastema, we inhibited Wnt signaling by a single pulse of Dkk1 expression in regenerating fins during the outgrowth phase of regeneration at 3 dpa. We assayed for cell proliferation 6 hours after heat shock using BrdU incorporation and staining for phosphorylated histones. We observed that loss of Wnt/ß-catenin signaling lead to a reduction in proliferation of both the blastema mesenchyme and the overlying epithelium (Fig. 3C,D). Thus, Wnt/ß-catenin signaling is required for the formation and subsequent proliferation of the blastema.
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).
During embryogenesis, heat shock of these fish causes characteristic
Wnt/ß-catenin gain-of-function phenotypes
(Weidinger et al., 2005). Induction of Wnt8 during fin regeneration increased expression of the Wnt/ß-catenin target gene axin2 (Fig. 4A), showing that overexpression of Wnt8 in the fin is sufficient to augment ß-catenin signaling. Importantly, overexpression of Wnt8 at 72 hpa significantly increased proliferation of the blastema mesenchyme and overlying epithelium 6 hours after induction of the transgene, as detected by BrdU incorporation and anti-phosphorylated histone H3 antibody staining (Fig. 4B). Despite its ability to increase proliferation, overexpression of Wnt8 had no consistent effect on fin length by 10 dpa (Fig. 4C). However, the short half-life of Wnt proteins and the pulsed activation of the transgene raise the question of whether a more prolonged and consistent activation of the pathway might be sufficient to augment overall fin regeneration.
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), and
asked whether axin1+/- fins regenerate more rapidly. To
minimize effects of the genetic background, we used wild-type and
axin1+/- fish that were siblings derived from a cross of a
wild-type fish with an axin1 heterozygous carrier. We genotyped the
fish, amputated fins of 12 wild-type and 9 axin1 heterozygous mutant
fish, allowed them to regenerate for 7 days, photographed the fins, blinded
the photographs, measured the length of the regenerate (from the amputation
plane to the distal tip of the fin) in the third, fourth and fifth dorsal fin
ray in each fish and calculated the average length of the regenerate of each
fish (experiment 1). The same fish were re-amputated and remeasured twice
(experiments 2 and 3, respectively) after a 2- to 3-week recovery period. In
the third experiment, several additional fish were included.
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In summary, these findings not only indicate that increased Wnt/ß-catenin signaling results in faster regeneration, but also provide genetic evidence for the involvement of Wnt signaling in regenerative processes, which has not been previously addressed in any system.
wnt5b overexpression inhibits fin regeneration
As Wnts that can act through the Wnt/ß-catenin pathway
(wnt10a) and through ß-catenin-independent pathways (wnt5a,
wnt5b) are expressed during fin regeneration
(Fig. 1), we next tested
whether these distinct Wnt pathways might have different roles in fin
regeneration. We compared the effects of activation of Wnt/ß-catenin
signaling with those produced by activation of ß-catenin-independent Wnt
signaling. To this end, we generated a transgenic zebrafish line carrying a
heat-shock inducible Wnt5bGFP transgene (hsWnt5bGFP; see Fig. S3 in the
supplementary material). Wnt5b has been shown to activate
ß-catenin-independent signaling pathways in zebrafish embryos
(Westfall et al., 2003).
Accordingly, heat-shocked hsWnt5bGFP embryos display the characteristic
phenotypes associated with gain-of-function of ß-catenin-independent Wnt
pathways, namely defects in convergence-extension cell movements during
gastrulation and somitogenesis (see Fig. S3L,M in the supplementary
material).
Interestingly, whereas overactivation of Wnt/ß-catenin signaling enhances regeneration, overexpression of Wnt5b represses regeneration. Heat shock of hsWnt5bGFP transgenic fish for 10 days starting shortly before fin amputation completely inhibited fin regeneration (Fig. 4F). This is in marked contrast to the effects of overexpressing Wnt8, which had no obvious effect on overall fin morphology (Fig. 4C), but closely resembled the defects caused by inhibition of Wnt/ß-catenin signaling via Dkk1 overexpression (Fig. 2B). As with overexpression of Dkk1, but in contrast to Wnt8, overexpression of Wnt5b significantly reduced proliferation of the blastema mesenchyme and overlying epithelium 6 hours after induction of the transgene, as detected by BrdU incorporation and anti-phosphorylated histone H3 antibody staining (Fig. 4E). Thus, activation of Wnt5b inhibits fin regeneration.
Although it is difficult to test which signaling pathways Wnt5b activates
in the regenerating fin, the fact that it causes dramatically different
effects than Wnt8, which signals via ß-catenin, suggests that it is
likely to act through ß-catenin-independent pathways. Since Wnt5b
overexpression causes the same phenotypes as Wnt/ß-catenin
loss-of-function, and because ß-catenin-independent Wnt signaling has
been reported to be able to inhibit Wnt/ß-catenin signaling in other
systems (Weidinger and Moon,
2003), we hypothesize that Wnt5b overexpression inhibits fin
regeneration by repressing Wnt/ß-catenin signaling. In support of this
model, we found that Wnt5b overexpression abolished expression of the direct
Wnt/ß-catenin target gene axin2 6 hours after heat shock at 3
dpa (Fig. 4D).
wnt5b loss-of-function augments fin regeneration
We next tested whether endogenous wnt5b acts as an essential
modulator of fin regeneration. If non-canonical Wnt signaling activated by
wnt5b inhibits regeneration in vivo, loss of wnt5b function
in the regenerating fin might result in enhanced or faster regeneration. To
test this prediction, we made use of homozygous adult wnt5b
(pipetail) mutant fish. We amputated tail fins of wnt5b
mutant and age- and size-matched wild-type fish of the same genetic
background, measured the length of the third, fourth and fifth dorsal fin ray
at 4 and 7 dpa, and calculated the average length of the regenerate for each
fish (Fig. 6A). In two
independent sets of experiments using different fish (experiments 1 and 2), we
found that wnt5b mutants had significantly longer regenerates than
wild types at both 4 and 7 dpa (Fig.
6A,B and see Table S2 in the supplementary material). The
difference in length between wild-type and wnt5b mutant regenerates
increased between 4 and 7 dpa, showing that wnt5b mutant fins
regenerate faster (Fig. 6B).
These data provide genetic evidence that wnt5b acts as a negative
modulator of fin regeneration. Wnt5b mutant regenerating fins did not
show any obvious patterning defects or indications of tumor formation or other
signs of inappropriate growth (Fig.
6A), suggesting that wnt5b is only required to modulate
the overall rate of regeneration.
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Wnt/ß-catenin signaling regulates FGF signaling during fin regeneration
FGF signaling has been shown to be required for regeneration of amphibian
and fish appendages (Lee et al.,
2005; Poss et al.,
2000b; Yokoyama et al.,
2001) and recently fgf20a was found to be induced early
during zebrafish fin regeneration and to be required for blastema formation
(Whitehead et al., 2005).
Similarly, we observe that wnt10a is induced very early in
regenerating fins and that Wnt/ß-catenin signaling is essential for
formation of the blastema. Therefore, to gain more mechanistic insight into
the role of Wnt/ß-catenin signaling in fin regeneration, we investigated
whether Wnt/ß-catenin signaling regulates FGF signaling during
regeneration. Strikingly, we found that levels of fgf20a transcripts
are suppressed 3 hours after amputation in Dkk1-overexpressing fins
(Fig. 7A), and that
fgf20a expression is still not detectable in hsDkk1GFP fins at 24 hpa
(Fig. 7B). Quantitative PCR
revealed that induction of Dkk1 2 hours prior to amputation resulted in severe
downregulation of the baseline of fgf20a expression at the time of
amputation and in the suppression of fgf20a upregulation during the
first 48 hours of regeneration (see Fig. S5 in the supplementary material).
These findings show that Wnt/ß-catenin signaling is required for
initiation of fgf20a expression during regeneration. The fast
response and the repression of basic fgf20a levels in hsDkk1 fins
indicate that fgf20a downregulation is not an indirect consequence of
a failure of these fins to regenerate, but is likely to reflect a more direct
regulation of fgf20a expression by Wnt/ß-catenin signaling.
In addition, we observed that in fins that have been allowed to regenerate
normally for 72 hours, a single pulse of Dkk1 expression quickly results in
the repression of sprouty4, an FGF target gene
(Lee et al., 2005)
(Fig. 7C). We conclude that
Wnt/ß-catenin signaling is also required for the maintenance of FGF
signaling. These findings indicate that Wnt/ß-catenin signaling acts
upstream of FGF signaling during regeneration, placing Wnt/ß-catenin
signaling at the top of the hierarchy of signaling pathways known to be
required for epimorphic regeneration (Fig.
8).
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Imokawa et
al., 2004; Tanaka et al.,
1999). Wnt10a activates the ß-catenin signaling pathway,
which directly or indirectly activates expression of fgf20a, which in
turn activates (directly or indirectly) the events resulting in blastema
formation and thus regeneration (Whitehead
et al., 2005). Although we have not tested whether wnt10a
activates fgf20a expression directly in the regenerating fin, it is
intriguing that fgf20a has been found to be a direct target of
Wnt/ß-catenin signaling in cultured human cells
(Chamorro et al., 2005). Since
we have not tested whether Wnt/ß-catenin signaling acts solely through
fgf20a to regulate blastema formation, we cannot exclude the
possibility that ß-catenin signaling also controls regeneration in
parallel to FGF signaling (gray arrow in
Fig. 8). The same injury-activated signal(s) that regulate wnt10a expression might also activate expression of wnt5a and wnt5b and potentially other Wnt ligands that activate ß-catenin-independent signaling. We postulate that these signaling pathways modulate regeneration by negatively regulating Wnt/ß-catenin signaling. However, we cannot exclude the possibility that ß-catenin-independent Wnt signaling also represses regeneration independently of its antagonistic effect on ß-catenin signaling (gray arrow in Fig. 8). Because we find that expression of wnt5b is regulated by Wnt/ß-catenin signaling, we hypothesize that these separate Wnt pathways establish a negative feedback loop whose function might be to ensure proper levels, duration or location of ß-catenin signaling in the regenerating fin.
In addition to its role in blastema formation, FGF signaling appears to be
absolutely required for the regenerative outgrowth of the fin, as drugs that
block FGF signaling can inhibit fin regeneration during this phase
(Poss et al., 2000b). Our
experiments indicate that Wnt/ß-catenin signaling is also required for
regenerative outgrowth. However, overexpression of Dickkopf1 does not cause a
complete inhibition of outgrowth. It is possible that the expression levels of
Dkk1 are not sufficient to completely block ß-catenin signaling during
this regenerative phase. Alternatively, other signals that are partially
redundant with Wnt/ß-catenin signaling might compensate for the loss of
Wnt signaling. We have found that Wnt/ß-catenin signaling regulates FGF
signaling during regenerative outgrowth, and thus it appears likely that
ß-catenin signaling acts through FGF signaling in this phase of
regeneration as well.
Elucidation of the exact cell biological role of Wnt/ß-catenin and FGF
signaling in blastema formation awaits further experiments. Whereas
regeneration of the zebrafish tail fin occurs in similar steps to salamander
limb regeneration, blastema formation by de-differentiation of differentiated
cells has so far only been reported in salamanders. Interestingly, a recent
report has shown that resident muscle stem cells are activated during
salamander limb regeneration and that progeny of these cells take part in the
formation of the blastema (Morrison et
al., 2006). It is likely that the relative contribution of
de-differentiation and resident stem cell activation to the formation of
progenitor cells during regeneration varies between organs and organisms, with
amphibian limbs likely to represent one end of the spectrum where
de-differentiation is prominent and, at the other end, processes like
mammalian muscle or bone regeneration being driven only by activation of
resident stem cells. Whether Wnt/ß-catenin and FGF signaling regulate
de-differentiation or stem cell activation or both in blastema formation is at
present unclear. Interestingly, Wnt/ß-catenin signaling has been shown to
be important for regeneration or repair of systems that are thought to rely
largely or solely on activation of resident stem cells. Inhibition of
Wnt/ß-catenin signaling reduces proliferation of CD45+ resident stem
cells in mammalian muscle regeneration
(Polesskaya et al., 2003) and
inhibits proliferation of osteoblasts, which drive bone repair, in culture
(Zhong et al., 2006).
Wnt/ß-catenin signaling has also been reported to be active during
regeneration of deer antlers and to be required for survival of antler bone
progenitor cells in culture (Mount et al.,
2006). Very recently, Hayashi et al. have shown that
Wnt/ß-catenin signaling is necessary and sufficient for regeneration of
newt lenses in culture (Hayashi et al.,
2006). More specifically, Wnt signaling appears to regulate the
second step of regeneration in which, subsequent to proliferation of the iris
pigmented epithelium and activation of early lens genes in the whole iris,
only the dorsal iris continues to develop
(Hayashi et al., 2006). Thus,
together with our results showing that ß-catenin signaling is required
for fin regeneration and our data showing that ß-catenin signaling is
activated during mouse liver and zebrafish heart regeneration, evidence is
beginning to emerge that Wnt/ß-catenin signaling might play central roles
in many regenerative processes. However, the specific function of Wnt
signaling in the regeneration of different organs is most likely to differ.
For example, in the newt lens, Wnt signaling is only activated after the
initial phase of proliferation and gene expression and is required for the
second step of regeneration. By contrast, we have shown that Wnt signaling
regulates gene expression very early in fin regeneration and that it is
required for the early events of blastema formation.
A better understanding of the role of Wnt/ß-catenin and FGF signaling in de-differentiation and/or stem cell activation during epimorphic regeneration is hampered by the fact that our insights into signaling events that regulate epimorphic regeneration come mainly from systems such as zebrafish, where de-differentiation has not been reported. Thus, further insights into the role of these pathways awaits better characterization of the cell biological events of blastema formation in zebrafish or the development of tools that facilitate genetic and other in vivo functional studies in salamanders.
Our study not only demonstrates an important role for Wnt/ß-catenin
signaling during regeneration, but also adds to our knowledge about the
functions of ß-catenin-independent Wnt signaling in adults. In
vertebrates, it is well established that ß-catenin-independent Wnt
signaling is required for cell polarity and cell movements during
gastrulation, and has also been implicated in endoderm cell migration,
pancreas cell migration, the migration of neurons and organization of hair
cell polarity in the inner ear (Bingham et
al., 2002; Carreira-Barbosa et
al., 2003; Curtin et al.,
2003; Jessen et al.,
2002; Kim et al.,
2005; Matsui et al.,
2005; Wada et al.,
2005). It is less clear whether ß-catenin-independent Wnt
signaling plays roles in cell fate determination. Interestingly, however, it
has been shown that ß-catenin-independent Wnt signaling can inhibit
Wnt/ß-catenin signaling and thus can, indirectly at least, regulate cell
fate. For example, overexpression of ß-catenin-independent Wnt ligands in
Xenopus blocks the ability of `canonical' Wnt ligands to activate
ß-catenin signaling and to induce a secondary body axis. Genetic evidence
for the existence of such opposing roles of ß-catenin-independent Wnt
signaling on Wnt/ß-catenin signaling comes from zebrafish, where maternal
loss of wnt5b has been reported to result in ectopic ß-catenin
signaling and a consequent increase in dorsal cell fates
(Weidinger and Moon, 2003;
Westfall et al., 2003).
Furthermore, loss of wnt5a in mouse limb buds likewise results in
ectopic ß-catenin signalling, causing defective chondrocyte
differentiation (Topol et al.,
2003). We propose that ß-catenin-independent Wnt signaling,
activated by wnt5a and wnt5b, plays a similar antagonistic
role in fin regeneration. Our finding that wnt5b expression appears
to be regulated by ß-catenin signaling suggests the existence of a
negative feedback loop. Such a loop represents a mechanism for regulation of
ß-catenin signaling that, to our knowledge, has not been described
before. It will be interesting to see whether the transcriptional activation
of Wnt ligands that activate antagonistic ß-catenin-independent pathways
is a more widespread regulatory mechanism employed by organisms to keep
ß-catenin signaling in check.
Taken together, our findings add to our mechanistic insight into the regulation of regeneration by demonstrating separate and opposing roles for ß-catenin-dependent and ß-catenin-independent signaling pathways during fin regeneration. Furthermore, although regeneration of the mammalian liver and the zebrafish heart employ different cellular mechanisms than regeneration of the zebrafish fin or amphibian limbs (with only the latter two involving formation of a blastema), it is intriguing that Wnt/ß-catenin signaling is upregulated during regeneration of all three organs. Although beyond the scope of the present study, it will be very interesting to test what role Wnt signaling plays in regeneration of these organs. It is conceivable that our findings will prove to be important for the goals of regenerative medicine, as the modulation of Wnt signaling pathways might augment the regeneration of human tissues.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/3/479/DC1
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ACKNOWLEDGMENTS |
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|
Footnotes |
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Present address: Biotechnological Center, Technical University of Dresden,
Tatzberg 47, 01307 Dresden, Germany
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