The fgf20aE48 enhancer directs epidermal expression in regenerating fins. (A) The fgf20aE48-fos:EGFP enhancer reporter construct. The 954 bp candidate TREE (blue arrow) was placed upstream of a 96 bp c-fos minimal promoter (yellow arrow) to direct fluorescent EGFP expression (green arrow). (B,C) Larval expression of HGn21A:EGFP and fgf20aE48:EGFP enhancer reporter lines. At 5 days post fertilization (dpf), HGn21A:EGFP is detectable in brain (br), trunk muscle (mus) and other structures (B), while fgf20aE48:EGFP is present in brain in this reporter line. A high magnification view of the larval head is shown for each sample (B′,C′). Scale bars: 500 μm. (D-G′) Longitudinal sections of 4 dpa fins showing the wound epidermal expression of fgf20a (arrowheads) by in situ hybridization in wild-type fins (D) and fgf20aE48Δ/Δ fins (E), immunofluorescence detection of EGFP in HGn21A:EGFP fins (F,F′), and immunofluorescence detection of EGFP in fgf20aE48:EGFP fins (G,G′). Scale bars: 100 μm. (H-K) Expression of HGn21A:EGFP in the distal edge of an uninjured caudal fin (H,I); similar fluorescence is not detectable in fgf20aE48:EGFP fish (J,K). Autofluorescent pigment cells are present at the tips of dorsal fin rays. The distal edge is indicated by an arrowhead. Scale bars: 300 μm. (L-N) HGn21A:EGFP fins show expression in the wound epidermis at multiple stages following injury and persisting throughout regeneration at the leading edge. Arrowheads indicate the amputation plane. Scale bars: 300 μm. (O-Q) fgf20aE48:EGFP reporter regenerates display EGFP expression at 4 dpa but not at other assessed timepoints. Scale bars: 300 μm. (R,S) qPCR analysis of fgf20a transcript levels in fgf20aE48Δ/Δ and wild-type clutchmate (WT) fins. Each biological replicate pool of three fins each was performed in technical triplicate, and paired two-tailed t-tests were performed to indicate significance. (n=8). (T) Lengths of regenerates are slightly reduced at 4 dpa (5.5%), 7 dpa (6.0%) and 14 dpa (7.0%) in fgf20aE48Δ/Δ fins compared with those of wild-type siblings (n=8). Paired two-tailed t-tests were performed to indicate significance. Data are mean±s.d.
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