First published online 21 December 2006
doi: 10.1242/dev.001123
Development 134, 479-489 (2007)
Published by The Company of Biologists 2007
Distinct Wnt signaling pathways have opposing roles in appendage regeneration
Cristi L. Stoick-Cooper1,2,*,
Gilbert Weidinger1,*,
,
Kimberly J. Riehle3,4,
Charlotte Hubbert1,
Michael B. Major1,
Nelson Fausto4 and
Randall T. Moon1,
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.
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Fig. 1. Wnt/ß-catenin signaling is upregulated in regenerating
zebrafish tail fins. (A) Wnt/ß-catenin reporter (TOPdGFP)
activity, detected by in situ hybridization for GFP RNA (blue), is upregulated
in the blastema of regenerating fins of zebrafish homozygous for the transgene
at 48 hpa (n=5; arrowheads indicate the amputation plane). Control is
a non-amputated TOPdGFP fin. At 3 dpa (n=3) and 5 dpa (n=3),
TOPdGFP was still upregulated (not shown). (B) In situ hybridization of
control non-amputated fins (left panels), regenerating fins at 3 dpa (middle
panels), and cross-sections of fins at the same stage (right panels). The
Wnt/ß-catenin target genes axin2 and sp8 are expressed
in the distal tip of the blastemal mesenchyme and in the basal epithelial
layer of the regeneration epidermis, respectively. wnt10a is
expressed in the distal tip of the blastema. Both wnt5a (for
nomenclature, see Fig. S2 in the supplementary material) and wnt5b
are expressed in the basal epithelial layer of the regeneration epidermis and
in the distal tip of the blastema, with wnt5a extending far
proximally in the basal epithelium. (C) wnt10a expression
levels in uncut control and regenerating fins at 0 hpa (sample isolated
immediately after fin amputation), 1 hpa, 3 hpa and 6 hpa as determined by
quantitative PCR. RNA was isolated from the tips of fins of 10 wild-type fish
for each time point. Expression levels were normalized to ß-actin levels
(normalization to 18S rRNA levels produced very similar results) and
fold-induction calculated by setting the level of uncut fins to 1.
Quantitative PCR was performed four times on the same samples; error bars
represent the s.e.m.
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Fig. 3. Wnt/ß-catenin signaling regulates specification and
proliferation of the regeneration blastema. (A) Expression of
lef1, a marker for the basal epidermal layer of the regeneration
epithelium, msxb, marking the mesenchymal progenitor cells of the
blastema, and shh, expressed in basal epidermal cells (shown in thick
sections), is strongly reduced in Dkk1-overexpressing fins. lef1 is
shown at 24 hpa (n=4), msxb (n=4) and shh
(n=4) at 72 hpa. Fish were heat shocked twice daily starting shortly
before amputation. (B) Hematoxylin-stained sections of tail fin
regenerates at 48 hpa. Dkk1-overexpressing fins (right panel; n=6)
display reduced numbers of deep mesenchymal cells of the blastema. Fish were
heat shocked twice daily starting shortly before amputation. Arrowheads
indicate the plane of amputation. (C) 72 hpa regenerates stained for
BrdU (red), phosphorylated histone H3 (PH3, green) and DAPI (blue). Cell
proliferation in both the mesenchyme and epithelium is decreased in
Dkk1-overexpressing fins. Fish were heat shocked once at 66 hpa and fixed at
72 hpa. (D) Quantification of the cell proliferation defects in
Dkk1-overexpressing regenerating fins. The fraction of BrdU-positive (left)
and PH3-positive (right) cells relative to the total number of cells
(DAPI-positive) is shown in percent (n=11). Error bars represent the
s.e.m; *P=0.0495; **P=0.0025;
***P=7.076x10-6 (two-tailed).
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Fig. 4. ß-catenin-dependent and ß-catenin-independent Wnt
signaling pathways have opposing roles in zebrafish fin regeneration.
(A,D) Overexpression of Wnt8 in hsWnt8GFP transgenic fish
induces the Wnt/ß-catenin target gene axin2 in regenerating fins
6 hours after heat shock at 3 dpa (A; 3 of 4 fins), whereas overexpression of
Wnt5b in hsWnt5bGFP transgenic fish represses axin2 expression (D;
n=4). Note that staining reactions were stopped as soon as a robust
signal could be detected in most samples of one experimental group. Robust
signal was first detected in hsWnt8GFP fins (versus wild-type controls) after
a short amount of time and reactions were stopped (A), whereas robust signal
was first detected in wild-type controls (versus hsWnt5GFP fins) after a
longer staining reaction (D), thus accounting for the difference in wild-type
signal between groups. (B,E) Cell proliferation in regenerating
fins, as detected by BrdU incorporation and staining with an anti-PH3
antibody, is increased by overexpression of Wnt8 (B; n=14), and
repressed by overexpression of Wnt5b (E; n=10). Fish were heat
shocked once at 66 hpa and fixed at 72 hpa. The percentage of BrdU-positive or
PH3-positive cells relative to the total number of cells in sections of
regenerating fins is shown. Error bars represent the s.e.m.
(C,F) Whereas overexpression of Wnt8 for 10 days starting
shortly before amputation has no obvious effect on overall length of the
regenerate (C; n=16), overexpression of Wnt5b completely inhibits
regeneration (F; n=16). (B) *P=0.0579;
**P=0.0082; ***P=0.0002 (two-tailed).
(E) *P=0.0377; **P=0.0123;
***P=0.0006 (two-tailed).
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Fig. 5. Fins regenerate faster in fish heterozygous for a loss-of-function
mutation in axin1. (A) Average length of regenerating tail
fins at 7 dpa is increased in fish heterozygous for an axin1
loss-of-function mutation (mbltm013) compared with
wild-type siblings. Results of one representative experiment of three are
shown. To determine the length of the regenerate for individual fish, the
average length of the third, fourth and fifth dorsal regenerating fin ray was
calculated. n=12 wild-type fish and 9 mbl heterozygous fish;
error bars represent the s.e.m. of the average regenerate lengths;
***, P=0.0009 (one-tailed). (B) The number of fin
rays (in percent of the total number counted) is plotted against the length of
the regenerate (in 0.1 mm intervals) for wild-type (upper bar graph) and
axin1 heterozygous fish (lower bar graph). The curves represent a
fifth-order polynomial trendline. The average regenerate length is marked by
black bars at the x-axis. 148 fin rays were counted (combined results
from three experiments) in 19 wild-type fish and 94 rays were counted in 10
axin1+/- fish.
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Fig. 6. Fins regenerate faster in wnt5b mutant fish. (A)
Dorsal half of regenerating tail fins of wild-type and wnt5b
(ppt) homozygous mutant fish at 7 dpa. The amputation plane is
indicated by a dashed red line, the length of the third fin ray by red or
green bars and arrows. Note that the regenerate is longer in ppt than
in wild-type fish. (B) The average length of the regenerate of
wild-type and ppt mutant fish at 4 and 7 dpa in two independent
experiments. To determine the length of the regenerate for individual fish,
the average length of the third, fourth and fifth dorsal regenerating fin ray
was calculated. Experiment 1: n=14 wild-type, 12 ppt fish.
Experiment 2: n=12 wild-type, 11 ppt fish. Error bars
represent the s.e.m. of the average regenerate lengths. For P values,
see Table S2 in the supplementary material. Note that absolute fin lengths
cannot be compared between experiments because water temperatures and thus
regenerative speed and exact times of photography varied between
experiments.
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Fig. 7. Wnt/ß-catenin signaling regulates FGF signaling during fin
regeneration. (A) fgf20a expression as detected by
semi-quantitative RT-PCR is greatly reduced in Dkk1-overexpressing fins at 3
hpa. Wild-type and hsDkk1GFP transgenic fish were treated according to the
schematic (hs, heat shock; amp, amputation; the green line indicates
inhibition of Wnt/ß-catenin signaling), and RNA was harvested from the
tissue adjacent to the amputation plane of nine wild-type and two groups of
nine hsDkk1GFP transgenic fins. odc1 amplification serves as a
loading control. The experiment was repeated four times using two sets of
biological samples and representative results are shown. (B)
fgf20a expression is greatly reduced as detected by in situ
hybridization in hsDkk1GFP transgenic fins (5 of 6 fins) at 24 hpa as compared
with wild-type fins. (C) sprouty4 expression is greatly
reduced 6 hours after heat shock in hsDkk1GFP fins (n=3) at 72
hpa.
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Fig. 8. A model of signaling events regulating zebrafish fin regeneration.
We propose that injury of the tail fin activates as yet unknown signals that
result in upregulation of wnt10a and wnt5b. wnt5b expression
is also regulated by Wnt/ß-catenin signaling activated by wnt10a.
Wnt10a activates a ß-catenin-dependent signaling pathway that
positively regulates fgf20a expression, which has been shown to be
required for blastema formation and subsequent regeneration
(Whitehead et al., 2005 ). In
addition to its role in regulating fgf20a expression,
Wnt/ß-catenin signaling might also regulate other genes that are required
for blastema formation and proliferation (gray arrow). We propose that
wnt5b employs a ß-catenin-independent signaling pathway that
antagonizes Wnt/ß-catenin signaling. However, we cannot exclude the
possibility that such ß-catenin-independent pathways also inhibit
regeneration without impairing Wnt/ß-catenin signaling (gray arrow).
Arrows do not imply direct events.
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© The Company of Biologists Ltd 2007
TcfGFP transgenic zebrafish and heat
shocks were applied twice daily for the time periods indicated by the colored
lines. (B) Continuous suppression of Wnt target gene expression in
hs