Mukaigasa K et al. (2009), The keratin-related Ouroboros proteins function...

XB-ART-40482

Proc Natl Acad Sci U S A 2009 Oct 27;10643:18309-14. doi: 10.1073/pnas.0708837106.

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The keratin-related Ouroboros proteins function as immune antigens mediating tail regression in Xenopus metamorphosis.

Mukaigasa K , Hanasaki A , Maéno M , Fujii H , Hayashida S , Itoh M , Kobayashi M , Tochinai S , Hatta M , Iwabuchi K , Taira M , Onoé K , Izutsu Y .

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Tail resorption during amphibian metamorphosis has been thought to be controlled mainly by a cell-autonomous mechanism of programmed cell death triggered by thyroid hormone. However, we have proposed a role for the immune response in metamorphosis, based on the finding that syngeneic grafts of tadpole tail skin into adult Xenopus animals are rejected by T cells. To test this, we identified two tail antigen genes called ouro1 and ouro2 that encode keratin-related proteins. Recombinant Ouro1 and Ouro2 proteins generated proliferative responses in vitro in T cells isolated from naive adult Xenopus animals. These genes were expressed specifically in the tail skin at the climax of metamorphosis. Overexpression of ouro1 and ouro2 induced T-cell accumulation and precocious tail degeneration after full differentiation of adult-type T cells when overexpressed in the tail region. When the expression of ouro1 and ouro2 were knocked down, tail skin tissue remained even after metamorphosis was complete. Our findings indicate that Ouro proteins participate in the process of tail regression as immune antigens and highlight the possibility that the acquired immune system contributes not only to self-defense but also to remodeling processes in vertebrate morphogenesis.

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Species referenced: Xenopus laevis
Genes referenced: hsp70 hspa1l krt12.1 mhc1a myc myh6 ouro1 ouro2 rpl8

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	Fig. 2. ouro1 and ouro2 are expressed in the skin during metamorphosis. (A) Northern blot analysis for ouro1 expression in J strain tadpoles. Tail and trunk skin tissues were isolated from various stages of tadpoles as indicated. A representative blot is shown (Upper Left), because five independent sets of experiments showed basically the same results. Ribosomal RNA visualized by ethidium bromide as a loading control (Lower Left). Relative expression levels were calculated using the image J software (Right). (B) RT-PCR with J strain tadpoles. Tail and trunk skin tissues as indicated were analyzed for ouro1, ouro2, Xenopus adult keratin (xak-b), Xenopus larval keratin (xlk), and Xenopus rpl8 (rpl8) as an internal control. -RT, rpl8 without RT. (C) Western blot analysis for Ouro1 and Ouro2 with J strain tadpoles. Tail and trunk skin cell lysates were used. (D) WISH with albino (non-J strain) X. laevis tadpoles. ouro1 antisense probe was used for tadpoles at stage 55 (n = 7), 58 (n = 7), and 62 (n = 3). ouro1 sense probe was used as a negative control for tadpoles at stage 58 (n = 5). Positive signals in blue were reproducibly detected in the tail and trunk (stage 55) or in the tail (stages 58 and 62). Arrowheads show the boundary between the tail and trunk region. (E) The vertical section of the tadpole at stage 62 after WISH using ouro1 antisense probe. The section includes the boundary between the tail and trunk skin as indicated. Purple signals are specifically seen in the tail epidermis (n = 2). ep, epidermis.
	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 Â°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 (âˆ’) 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 1â€“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. (Fâ€“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) (Fâ€“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).
	Fig. 4. Knockdown of ouro1 and ouro2 gene expression results in retention of tail skin. (A) Antisense constructs for ouro1 and ouro2. Reverse-oriented ouro1 or ouro2 cDNA were placed after DsRed or gfp, respectively, which were driven by the hsp70 promoter. (Bâ€“E and Eâ€²) Suppression of tail regression in DsRed-anti-ouro1/gfp-anti-ouro2 DT tadpoles. Tails of DsRed-anti-ouro1/gfp-anti-ouro2 DT tadpoles (F0) on days 1â€“14 after HS at stage 58/59 are shown by bright field and fluorescence microscopy (only GFP is shown). Note: DsRed-anti-ouro1/gfp-anti-ouro2 DT animals exhibit a pronounced delay in tail regression with a folded epidermis. (Fâ€“J and Jâ€²) gfp transgenic control. HS-induced gfp transgenic tadpole (line 9, F2) show normal tail degeneration. Boxed areas in Eâ€² and Jâ€² are magnified for E and J, respectively.

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