Watanabe M et al. (2003), Ontogenic emergence and localization of larval ...

XB-ART-5617

Dev Growth Differ 2003 Feb 01;451:77-84. doi: 10.1046/j.1440-169x.2003.00676.x.

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Ontogenic emergence and localization of larval skin antigen molecule recognized by adult T cells of Xenopus laevis: Regulation by thyroid hormone during metamorphosis.

Watanabe M , Ohshima M , Morohashi M , Maéno M , Izutsu Y .

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Results from previous studies using an inbred strain of Xenopus laevis have led to the proposition that metamorphosis includes the events by which the newly differentiating adult immune system, including T lymphocytes, recognizes and eliminates larval skin cells as 'non-self'. More recently, a larval antigen targeted by adult T cells was identified as a 59 kDa protein with a specific peptide sequence. Using antisera directed against the larval antigen and the peptide, immunohistochemistry and western blotting were done to examine expression of the 59 kDa larval antigen in the skin during larval and metamorphic periods. There was no expression before Nieuwkoop and Faber stage 53. Expression was first seen at the beginning of metamorphic stage 54, when hind limbs appear, and increased thereafter, in apical and skein cells of both trunk and tail regions. In the trunk region, expression started to decrease at stage 58, until it completely disappeared at stage 62 (metamorphic climax). In the tail skin, however, expression persisted throughout the metamorphic stages. Treatment of larvae with thyroid hormone (TH) resulted in repression of expression of the 59 kDa molecule in a dose-dependent manner. Downregulation occurred earlier in the trunk than in the tail skin. These results suggest involvement in metamorphic events of an immunological mechanism: differential expression of the larval antigen in the trunk and tail skin cells due to their differing concentration of TH results in the tail, but not the trunk skin, being selectively attacked by the newly differentiating adult-type immune system.

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Species referenced: Xenopus laevis
Genes referenced: actl6a ouro1 thra
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	Fig. 1. Immunohistochemical staining of skin from larval and metamorphosing animals using anti-larval skin (LS) frog antiserum. Red signals show the larval antigens as contrasted with counterstained quinacrine (green). (A,B) Stage 47 dorsal trunk; (C,D) stage 49 tail; (E,F) stage 54 tail; (G) stage 59 tail; (H) stage 59 dorsal trunk; and (I) tail and trunk junction region at stage 63. (B), (D), and (F) are control staining reacted with normal frog sera, showing no signal in the epidermis except a faint nonspecific staining in the dermis, including muscle. Thickness of the epidermis is shown by the white lines on the right side. No difference in the staining pattern was seen between tail and trunk regions until stage 54. Differences were observed at stage 59 and at the metamorphic climax stage (I): the signal is seen only in the regressing tail region but not in the trunk epidermis, showing a clear boundary between tail and trunk regions. Arrowheads indicate typical stained skein cells localized on the basement membrane of the epidermis. Bar, 50 μ m.
	Fig. 2. Western blot analysis using anti-larval peptide (LP) rat antiserum of tail and trunk skin lysates from various developmental stages. Lysates from whole tail tissues (Ta) and whole trunk skin (Tr) from developmental stages indicated were separated by sodium dodecylsulfate (SDS)– polyacrylamide gel electrophoresis (PAGE) for detection of 59 kDa larva-specific proteins. Actin bands (42 kDa) are shown as a loading control of the tail, but not for the trunk skin including contaminating muscle tissue. (a) Shows that a 59 kDa signal is detected first at stage 50 in the tail, and increases thereafter to a maximum level at stage 59 and 62, but is not detected in the trunk at stage 62 and 66. (b) Shows that there is no difference in the expression of the 59 kDa molecule between tail and trunk regions at stage 50–57, but a decrease in the trunk occurs at stage 58 and thereafter.
	Fig. 3. Downregulation of expression in the larval antigen molecule by thyroid hormone (TH). Tadpoles at stage 49 not expressing the larval antigen molecules (a) and at stage 54 expressing the molecules (b) were treated with TH at concentrations as indicated. After 4 days of treatment, lysates form four or five whole tails were separated by sodium dodecylsulfate (SDS)– polyacrylamide gel electrophoresis (PAGE) for western blotting of the 59 kDa molecule (arrows). Actin bands at 42 kDa (arrows) are shown as a loading control. Expression of the 59 kDa molecule was completely inhibited with 1 10 –8 M TH in stage 49 tadpoles, but only partially inhibited in stage 54 tadpoles. Note that during 4 days of treatment from stage 49 onwards, control larvae developed to stage 51, and the animals from stage 54 remained at stage 54. Co, controls.
	Fig. 4. Downregulation by thyroid hormone (TH) of expression of larval antigen in the tail and trunk regions. (a) Tadpoles at stage 50/ 51 treated with TH at concentrations as indicated for 3–4 days. When the stage 50/51 larvae were treated with TH at 1 10 –8 M , the tail did not change whereas the trunk region showed morphological changes typical of spontaneous metamorphosis: the head became smaller, the width between the eyes became narrower, and the limbs developed during the course of the treatment. After 4 days of treatment with 1 10 –8 M , hind limbs developed knees, and fore limbs without fingers appeared (arrowheads). In the tadpoles treated with TH at 1 10 –9 M , the head became slightly smaller than that of controls (Co) and rudiments of hind limbs became longer. Bar, 5 mm. (b) Stage 50/51 tadpoles were treated with TH for 3–4 days, and the lysates from four or five whole tails were separated by sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) for western blotting of the 59 kDa molecule (arrows). After 3 days of treatment with 1 10 –8 M but not 1 10 –9 M TH, expression of the 59 kDa molecule was completely inhibited in the trunk but not in the tail. After 4 days of treatment with 1 10 –8 M TH, expression of the molecule was inhibited in both the tail and trunk. Ta, tail tissues; Tr, trunk skin.