Fig. 7

Epi site 3 contains important sequences for ectodermal reporter expression in embryonic fin folds. (A, B) unmutated Tg(2Pand1:eGFP) transient expression at 2 d.p.f. The rate of GFP positive embryos is indicated (A), and higher magnification of the median fin fold of representative fish is shown (B). (C) Four different mutations were tested within the Epi fragment for ectodermal enhancer activity to identify candidate ectodermal enhancer sites. Mutation locations are colour coded, and the mutation details are described on the right. Only embryos with visible ectodermal expression were counted as “GFP positive”. (D-O) GFP expression observed with the mutated constructs. Epi mutations 1 (D,E), 2 (F,G), and 4 (J,K) display similar rates of GFP positive embryos and levels of ectodermal expression in the median fin fold compared to unmutated Tg(2Pand1:eGFP) injections. Epi mutation 3 (H, I) yields a decreased rate of GFP positive embryos (50.9%), and lower levels of ectodermal expression in the median fin fold (indicated by yellow arrowheads) (I). Epi mutations 1 and 3 tested in combination (L, M) yield no synergistic effects. (N-O) Epi 3 mutation was tested in the Tg(epi+β-globin:eGFP) construct and displayed a drastic decrease in GFP positive embryos (6.25%), and a nearly complete loss of ectodermal reporter expression (indicated by the white arrowhead) (O). Scale bars: 200 µm in A, D, F, H, J, L, N; 20 µm in B, E, G, I, K, M, O.