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Files in this Data Supplement:
Fig. S1. Wnt/β-catenin signaling is upregulated in regenerating zebrafish heart and mouse liver. (A) Wnt/β-catenin reporter (TOPdGFP) activity (detected by in situ hybridization for GFP RNA, blue) is upregulated in regenerating zebrafish hearts (n=4) adjacent to the amputation plane (arrowheads) at 3 dpa (*=blood clot). Control is a sham-operated TOPdGFP heart at 3 days post-sham. TOPdGFP is also expressed adjacent to the amputation plane at 7 dpa (n=3) and 14 dpa (n=3) (data not shown). (B) Wnt/β-catenin reporter (TOPGal) (DasGupta and Fuchs, 1999) activity (detected by staining for β-galactosidase activity, blue) is upregulated in periportal hepatocytes in regenerating mouse liver (n=8), 48 hours post-partial hepatectomy. (C) Quantification of TOPGal activity in regenerating mouse liver. β-galactosidase activity was measured in control lobes (resected lobes removed at the time of partial hepatectomy) and regenerating lobes, collected 48 hpa. Control lobes were compared to regenerating lobes harvested from the same animal. Asterisks indicate a statistically significant difference between experimental groups (n=8, P<0.01, Paired Student’s t-Test). Error bars represent the s.e.m.
Fig. S2. The previously described zebrafish wnt5 homolog pipetail (ppt) is the ortholog of wnt5b, while a newly cloned homolog is likely to represent the zebrafish ortholog of wnt5a. (A) Multiple sequence alignment of several vertebrate Wnt5 paralogs. The sequence described here and ppt are depicted in red. (B) Phylogenetic tree analysis of Wnt5 protein sequences using the PAUP software. Zebrafish Ppt clearly groups with Wnt5b paralogs of other vertebrates. The newly described sequence, together with predicted proteins from Fugu and Tetraodon, does not clearly group with Wnt5a paralogs of other vertebrates, but the resolution of the tree is too low to exclude the possibility that they belong in the Wnt5a clade. Since ppt clearly represents zebrafish wnt5b and since there is no evidence for the existence of a third wnt5 paralog in zebrafish, Fugu or Tetraodon, we suggest that the newly described sequence represents the zebrafish ortholog of wnt5a. Multiple sequence alignment of Wnt5 homologs was built using ClustalW. The non-conserved N-terminus (signal sequence) was excluded from the alignment, as were some exons from predicted Wnt5 homologs that are non-conserved. Settings used in PAUP to build the tree were: outgroup to wnt5\ciona, 10000 bootstrap replicates, neighbor joining, 50% majority rule consensus. Accession numbers: wnt5a\mouse=NP_033550, wnt5a\rat=NP_072153, wnt5a\dog=XP_541837, wnt5a\human=NP_003383, wnt5a\cow=XP_878444, wnt5a\chick=BAA75242, wnt5a\Xenopus=P31286, wnt5a\axolotl=WN5A_AMBME, wnt5b\human=NP_116031, wnt5b\chimpanzee=XP_522589, wnt5b\mouse=NP_033551, wnt5b\rat=XP_342748, wnt5b\cow=XP_584724, wnt5b\axolotl=WN5B_AMBME, wnt5c\Xenopus=WN5C_XENLA, wnt5b\fugu=ENSEMBL NEWSINFRUP00000152673, wnt5b\tetraodon=ENSEMBL GSTENT00034446001, wnt5\zebrafish\ppt=NP_571012, wnt5a\fugu=ENSEMBL NEWSINFRUP00000133995, wnt5a\tetraodon=ENSEMBL GSTENT00027963001, wnt5\zfish\NEW=DQ465921, wnt5\ciona=NP_001027951.
Fig. S3. Establishment of heat-shock inducible Dickkopf1GFP and heat-shock inducible Wnt5bGFP transgenic zebrafish lines. (A) Maps of the transgenes used. (B) Strategy used to establish and characterize transgenic fish (see Materials and methods). (C,D) Heat-shock of heterozygous hsDkk1GFP transgenic embryos at 24 hpf for 2.5 hours results in ubiquitous expression of the Dkk1GFP fusion protein. (E,F) Heat-shock of heterozygous hsDKK1GFP transgenic adult fish for 1 hour causes expression of the fusion protein in amputated tail fins. (G-I) Heat-shock of heterozygous hsDkk1GFP transgenic embryos for 2 hours during gastrulation (starting at shield stage, 6 hpf) causes severe defects in anteroposterior patterning that closely resemble those caused by knockdown of both ORFs of wnt8 using translation-blocking morpholinos. Heterozygous carriers of the transgene were crossed to wild-type fish, and the resulting clutch of embryos heat shocked at 6 hpf for 2 hours. Wild-type and transgenic embryos were identified by GFP fluorescence, sorted and photographed at 24 hpf. (J,K) Overexpression of Dkk1GFP in embryos doubly transgenic for hsDKK1GFP and the reporter TOPdGFP is sufficient to suppress β-catenin-dependent expression of the TOPdGFP transgene. Heterozygous carriers of the hsDkk1GFP transgene were crossed to homozygous carriers of the TOPdGFP transgene. The resulting clutch of embryos was heat-shocked at 9 hpf for 1 hour, embryos fixed at 12 hpf and processed for two-color in situ hybridization with an EGFP probe detecting the TOPdGFP transgene in blue and a mmGFP probe detecting the dkk1GFP transgene in brown. The pictures show the head region of flat-mounted embryos, anterior left. Expression of TOPdGFP at this stage is primarily in the tectum and hindbrain. (L,M) Overexpression of Wnt5bGFP in embryos heterozygous for the hsWnt5bGFP transgene during gastrulation causes phenotypes typical for activation of β-catenin-independent Wnt signaling pathways, namely convergent-extension defects, resulting in short embryos with compressed somites and wavy notochord. Heterozygous carriers for the hsWnt5GFP transgene were crossed to wild-type fish, the resulting embryos heat shocked at 6 hpf for 2 hours, sorted into wild-type and transgenic carriers by GFP fluorescence and photographed at 24 hpf.
Fig. S4. wnt5b expression during fin regeneration is regulated by Wnt/β-catenin signaling. Fins of hsDkk1GFP transgenic and wild-type fish were amputated, fish heat shocked once at 72 hpa and fins fixed 6 hours later. wnt5b expression is downregulated in Dkk1-expressing fins (n=10).
Fig. S5. Heat-shock induced expression of Dkk1 represses fgf20a expression during fin regeneration. Wild-type and hsDkk1GFP transgenic zebrafish were heat shocked 2 hours prior to caudal fin amputation, and every 12 hours during the course of the experiment to ensure continual Dkk1 expression. Total RNA was isolated from the distal tip of the regenerating fins of eight fish at each time point indicated and fgf20a expression levels detected by quantitative RT-PCR. fgf20a transcript levels were normalized to levels of 18S rRNA. The values plotted show fold-induction over wild type and error bars represent the confidence interval of the ratios between two repeated PCRs of the same sample, P<0.001. Note that the baseline expression of fgf20a at the time of amputation and the previously reported induction of fgf20a expression during fin regeneration are suppressed upon the expression of Dkk1.
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