A TREE linked to mdka directs epidermal gene expression. (A) The mdkaE20-fos:EGFP enhancer reporter construct. The 1310 bp regulatory sequence (blue arrow) was placed upstream of a 96 bp c-fos minimal promoter (yellow arrow) to drive fluorescent EGFP expression (green arrow). (B) mdkaE20:EGFP larvae express EGFP in brain (br), notochord (n), lens (l) and fin fold (ff). High-magnification view of the larval tail includes the end of the notochord and caudal fin fold (B′). Scale bars: 500 μm. (C) mdkaE20:EGFP fish do not express appreciable amounts of EGFP in uninjured caudal fins. Scale bar: 300 μm. (D-F′) Longitudinal sections of 4 dpa fins indicating expression of mdka RNA in the mesenchyme (asterisks) and epidermis (arrowheads) via in situ hybridization in wild-type (D) and mdkaE20Δ/Δ (E) fin sections. Similar patterns were observed for EGFP immunofluorescence in the epidermis (strong) and mesenchyme (weak) of mdkaE20:EGFP regenerates (F,F′). Scale bars: 100 μm. (G-J) mdkaE20:EGFP reporter fish display clear injury-induced EGFP at 1 dpa, with expression detectable beyond 7 dpa and fading by 21 dpa. Arrowheads indicate amputation plane. Scale bars: 300 μm. (K) Fin regeneration in mdkaΔ/Δ animals is slightly but measurably delayed compared with wild-type fins at 4 dpa (7.3%), 7 dpa (7.9%) and 14 dpa (4.4%) (n=14). (L,M) qPCR indicating that mdka mRNA levels are similar in mdkaE20Δ/Δ and wild-type fins in expression (L), and in expression relative to respective 0 dpa mdkaE20Δ/Δ or wild type (M). 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=6). (N) Fin regeneration in mdkaE20Δ/Δ fish is similar to wild type (n=14). Paired two-tailed t-tests were performed to indicate significance. Data are mean±s.d.
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