Fig. 3.

Precocious tail degeneration by overexpression of ouro1 and ouro2 genes. (A) DNA constructs used to generate transgenic animals. Ouro proteins were fused to the FLAG- or Myc-tag and the GFP protein. The expression constructs are under the control of the HS promoter hsp70. (B) HS treatment. The distal part of the tadpole tail was heat-treated by immersion in Steinberg's solution at 37 °C. (C) Induction of GFP expression by HS. GFP was only detected in a HS-treated region of the tail. The panel shows a typical case, which is the gfp F₂ transgenic line (see Fig. S6, line 9) tadpole on day 1 after HS treatment. (D) Western blot analysis of induced Ouro fusion proteins. ouro1-gfp/ouro2-gfp DT F₂ tadpoles (see Fig. S6, line 2) were used. Expression of both introduced genes was detected in the HS-treated area (+), but not in nontreated area (−) on day 1 after HS. Arrowheads indicate the Ouro fusion proteins. Blotted proteins were stained with Coomassie Brilliant Blue (CBB). A representative blot from two independent experiments is shown. (E) Induction of precocious tail regeneration by HS. Tails of ouro1-gfp/ouro2-gfp DT (line 2) on days 1–4 after HS at stage 58/59 showed precocious degeneration (Upper). HS-induced gfp-transgenic tadpoles (line 9) showed a normal tail (Lower). Bright field (Left) and GFP fluorescence image (Right) are paired. (F–H) Accumulation of T cells in the HS-treated tails. Vertical frozen sections of HS-treated tails of ouro1-gfp/ouro2-gfp DT tadpole (line 1) (n = 8) (F and G) and gfp transgenic tadpole (line 8) (n = 8) (H) F₁ tadpoles on day 3 after HS were stained with anti-GFP antibody (green) (F–H) and anti-Xenopus T cells (red) (F and H) or with anti-Xenopus MHC class II antibody (red) (G). Dotted circles in serial sections (F and G) indicate an assembly of T cells (F) expressing MHC class II (G). Arrowheads, a few T cells seen in the tail epidermis of the gfp control (H).