Wnt/β-catenin signaling is upregulated in regenerating zebrafish tail fins. (A) Wnt/β-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/β-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 β-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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