Nakai Y et al. (2016), Ouro proteins are not essential to tail regress...

XB-ART-51817

Genes Cells 2016 Mar 01;213:275-86. doi: 10.1111/gtc.12337.

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Ouro proteins are not essential to tail regression during Xenopus tropicalis metamorphosis.

Nakai Y , Nakajima K , Robert J , Yaoita Y .

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Tail regression is one of the most prominent transformations observed during anuran metamorphosis. A tadpole tail that is twice as long as the tadpole trunk nearly disappears within 3 days in Xenopus tropicalis. Several years ago, it was proposed that this phenomenon is driven by an immunological rejection of larval-skin-specific antigens, Ouro proteins. We generated ouro-knockout tadpoles using the TALEN method to reexamine this immunological rejection model. Both the ouro1- and ouro2-knockout tadpoles expressed a very low level of mRNA transcribed from a targeted ouro gene, an undetectable level of Ouro protein encoded by a target gene and a scarcely detectable level of the other Ouro protein from the untargeted ouro gene in tail skin. Furthermore, congenital athymic frogs were produced by Foxn1 gene modification. Flow cytometry analysis showed that mutant frogs lacked splenic CD8(+) T cells, which play a major role in cytotoxic reaction. Furthermore, T-cell-dependent skin allograft rejection was dramatically impaired in mutant frogs. None of the knockout tadpoles showed any significant delay in the process of tail shortening during the climax of metamorphosis, which shows that Ouro proteins are not essential to tail regression at least in Xenopus tropicalis and argues against the immunological rejection model.

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Species referenced: Xenopus tropicalis
Genes referenced: foxn1 ighx krt12.1 ouro1 ouro2

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	Figure 1. Developmental expression of ouro1 and ouro2 mRNA and proteins during Xenopus tropicalis metamorphosis. (A, B) The expression levels of ouro1 mRNA (A) and ouro2 mRNA (B) in tail skin (solid line) and skinned tail (dotted line) from stage 56 to 63. Data are expressed as the means ± SEM (N = 3–6). (C, D) Western blots showing the expression levels of Ouro1 protein (C) and Ouro2 protein (D) in back and tail skin from stage 56 to 64.
	Figure 2. Characterization of the ouro1-knockout tadpole. (A) The target sites (arrowheads) of anti-ouro1 TALEN in ouro1 genomic gene and Ouro1 protein. The black boxes and the arrow indicate exons and the transcriptional direction, respectively. (B) Expression levels of ouro1 and ouro2 mRNA in the tail skin of stage 60 wild-type and ouro1-knockout tadpoles. Levels of ouro1 mRNA were determined using a pair of primers downstream and another pair of primers (5′) upstream of the TALEN target sites. Data from wild-type tadpoles are expressed as the means ± SEM (N = 6). (C, D) Expression levels of Ouro1 (C) and Ouro2 (D) proteins in the tail and back skin of stage 60 wild-type and ouro1-knockout tadpoles.
	Figure 3. Characterization of the ouro2-knockout tadpole. (A) The target sites of anti-ouro2 TALEN in ouro2 genomic gene and Ouro2 protein. The alignment is labeled as described in the legend to Fig. 2. (B) Expression levels of ouro1 and ouro2 mRNA in tail skin of stage 60 wild-type and ouro2-knockout tadpoles. Levels of ouro2 mRNA were determined using a pair of primers downstream and another pair of primers (5′) upstream of the TALEN target sites. Data from wild-type tadpoles are expressed as the means ± SEM (N = 6). (C, D) Expression levels of Ouro1 (C) and Ouro2 (D) proteins in the tail and back skin of stage 60 wild-type and ouro2-knockout tadpoles.
	Figure 4. Characterization of the Foxn1-knockout tadpoles. (A) The target sites of anti-Foxn1 TALEN in Foxn1 genomic gene and Foxn1 protein. The alignment is labeled as described in the legend to Fig. 2. (B) Photographs of heads of stage 56 wild-type and Foxn1-knockout tadpoles. Arrowheads indicate the thymus in the wild-type tadpole. There is no thymus in the Foxn1-knockout tadpole. Scale bars = 3 mm. (C) Flow cytofluorometric analysis of splenocytes from wild-type and Foxn1-knockout frogs after staining with mouse anti-CD8 monoclonal antibody (AM22 or F17) and Alexa Fluor 488 goat anti-mouse IgM antibody. (D) Transplantation of ventral skin grafts from wild-type Nigerian H (NH) and Ivory Coast (IC) strains to the backs of IC wild-type and Foxn1-knockout frogs. Note that both grafts survived on the back of Foxn1-knockout frog for more than 100 days. Scale bars = 1 mm.
	Figure 5. Tails regressed in ouro1-, ouro2- and Foxn1-knockout tadpoles similarly to wild-type tadpoles. (A) Photographs of wild-type stage 62 and 65 tadpoles. Scale bars = 5 mm. (B) The time required for wild-type tadpoles and ouro1-, ouro2- and Foxn1-knockout tadpoles to develop from stage 62 to 65. Data are expressed as the means ± SEM. wt, wild-type; in, in-frame mutation; out, out-of-frame mutation; Ldel, a large deletion.

References [+] :

Berry, The expression pattern of thyroid hormone response genes in the tadpole tail identifies multiple resorption programs. 1998, Pubmed, Xenbase