Amphibian metamorphosis provides a model to elucidate the mechanisms underlying how vertebrates reconstitute a body plan and how the immune system develops during ontogeny. In Xenopus, T cells are expanded from the early developmental stages just after hatching. These T cells switch from larval-type in an easily tolerizable state into an adult-type having a potent immune responsiveness comparable to that of mammals. During metamorphosis, tadpoles exhibit morphological changes in skin that completely transforms from larval-type to adult-type. Only tail tissue behaves differently; it remains a larval-type tissue until it disappears at the end of metamorphosis. Thus, at metamorphic climax, four different types of cells co-exist in a tadpole body: larval tissue cells; adult tissue cells; larval immune cells; and adult immune cells. Based on the results showing that tadpole tail skin is rejected by syngeneic adult, it is proposed that the elimination of the larval tissue cells by the adult T cells that occurs during metamorphosis is immunologically mediated. Recent results indicate that the antigenic proteins expressed in the metamorphosing skin cells participate in the process of tail regression. This chapter describes how animals adjust and survive through such crises associated with large scale replacement of entire body cells.

2. INTRODUCTION

Research on the immune system of anuran amphibians has relied heavily on Xenopus laevis as a model system. Studies with this species have revealed that the immune system of larvae is immature compared to that of adults. Among a multiplicity of interesting findings in the area of the ontogeny of immunity is the ease with which allotolerance can be induced during larval life. It is thought that this allotolerance prone period, which disappears after the completion of metamorphosis, reflects a normal state of larvae that is necessary for acceptance of newly appearing adult type tissues. It is less well known, but no less interesting, that the immune system of the young postmetamorphic frog appears to play a role in eliminating larval tissues, another function that disappears soon after metamorphosis. In this paper, I summarize the ontogenesis of the immune system in Xenopus with a focus on the transition from larvae to adults. Next, I introduce studies on remodeling of skin tissue in anuran amphibians including Xenopus and our finding that the immunity excludes the larval skin remaining in the tadpole at the metamorphic climax. Finally I discuss the possibility that the acquired immune system contributes not only to self-defense but also to the remodeling processes in vertebrate morphogenesis.

3. XENOPUS IMMUNE CELLS

Electron microscopy has revealed Xenopus macrophage-like cells with phagocytotic activity in tadpole tails that are regressing at metamorphic climax (1). Immunocompetent Xenopus cells that had been noted histologically were confirmed as T cells (2, 3) and B cells (4) in the 1980's with monoclonal antibodies. These T and B cells are well distributed in the spleen (Figure 1). As shown in Figure 1, splenic B cells are found in the white pulp area (previously known as the antigen-trapping region (5)). In contrast to mammals, Xenopus splenic T cells are located in the red pulp area surrounding the white pulp (6). Most of the T cells are believed to be educated in the thymus since thymectomy, but not splenectomy, has a major effect on immune responsiveness (7). In Xenopus, the thymus anlagen arise around day 3 after fertilization at NF-stage 40 according to the Normal Table of Nieuwkoop and Faber (8). However, even after thymectomy by microcautery at the early NF developmental stage 45-47 around day 4-5 days of age, T cells still can be found (comparable to controls) in the tail tissues of tadpoles undergoing metamorphic climax (9). These T cells lack the expression of mammalian CD8 surface marker (AM22 monoclonal antibody (mAb)), which reacts with the thymus-derived cytotoxic T cell subset (10). Thus, non-thymic T cells differentiate in Xenopus; whether these cells differentiate in other major lymphopoietic organs, i. e., the liver and the mesonephros (11) is unknown.

Before the thymus develops, leukocytes that stain with XL-1 mAb against leukocyte common antigen, appear from the early embryonic stage 35/36 and are scattered in the entire tadpole body (12). These cells seem to be macrophages although whether these cells have phagocytotic activities is still controversial.

　　On the other hand, Horton et al. reported that a Xenopus-specific anti-NK cell monoclonal antibody, 1F8, reacts with a subset of non-thymus derived larval cells (13). The number of the 1F8-reactive cells in the larva in the absence of thymus was almost the same as that derived from the normal larva. This 1F8 antibody begins to detect some population of cells at the metamorphic period when the MHC class Ia is also expressed. It is likely that the IF8-reactive cells are the larval natural killer (NK) cells, which do not have cytotoxic activity against the MHC-negative cells. In contrast, the adult NK cells do (14, 15) (cf. a mAb against Xenopus MHC class I (TB17 mAb) as isolated by Harding et al., (16) and MHC class II (AM20 mAb) identified by Flajnik et al., (10)).

The immunology of Xenopus including these immune cells and their molecular markers as mentioned above has been well studied because, owing to its aquatic existence during larval and adult life and the ease of fertilization and husbandry in a laboratory environment, this species is ideal for immunologists to observe and manipulate from embryogenesis to organogenesis.

4. REMODELING OF IMMUNE CELLS DURING XENOPUS METAMORPHOSIS

To investigate whether T cells recognize foreign leukocytes derived from another individual or strain, the mixed leukocyte reaction (MLR) assay with radiolabeled-thymidine or BrdU-incorporation has been used as an established method for mammals (17, 18). The most remarkable finding using a MLR assay for Xenopus is that adult and larval immune cells are immunologically distinguishable. Specifically, when lymphocytes from cloned larval and syngeneic adult Xenopus are co-cultured, a significant and bi-directional prominent proliferative response has been reported (19, 20). However, in our experiments, when the lymphocytes from tadpoles were mixed with syngeneic adult tissues cells, proliferative responses were not observed, whereas adult lymphocytes showed prominent responses against the larval tissue cells (9). These proliferation responses seen in the MLR are severely impaired by early thymectomy, indicating that this response is generated by T cells (21).

Organ transplantation has been used to estimate how the immune cells are replaced during metamorphosis. Specifically, transplantation of triploid (3N) thymuses into diploid (2N) tadpoles showed a complete exchange of lymphocytes during metamorphosis (22) with large scale of apoptosis in the thymus medulla (23, 24). The number of lymphocytes both in the thymus (25, 26) and the spleen (26) is reduced to approximately 50-60 % at metamorphic climax stages (NF-stage 64-66) as compared to that of cells obtained from tadpoles at prometamorphosis (NF-stage 57-61). This renewal is not affected by thyroid hormone which induces metamorphic morphological changes (27). Taken together, immune cells seem to be totally replaced during metamorphosis from larval-type into adult-type. However, some larval immune cells do appear to remain after metamorphosis; this is based on the observation of a memory response to sheep red blood cells (SRBC) in postmetamorphic adults that had been immunized during larval life (28). From these results, it is difficult to reach a definitive conclusion due to the insufficiency of molecular probes that distinguish larval and adult immune cells. Another related but unresolved issue concerns the origin of stem cells that occur during the shift from a larval to an adult type of immunity. That is, cell lineages have not been characterized. Although the bone marrow (BM) is major lymphopoietic organs in mammals, in Xenopus, the BM develops after metamorphosis (29), suggesting that it is unlikely to be lymphopoietic (30, 31). Other organs i. e., spleen, liver, thymus and kidney, have been proposed as candidates, but the source and indeed, the very existence of adult stem cells has yet not been confirmed.

5. TOLERANCE INDUCED IN LARVAE BUT NOT IN ADULT XENOPUS

Allotollerance of skin grafts is easily induced in larvae but not adults (32-35). To investigate mechanisms underling this larval tolerance, MHC-identical clones (19, 36) or MHC-homozygous inbred strains, J (j/j) (37), K (k/k) (38) and F (f/f) (39) were established for applying skin transplantation techniques. LG clones were obtained from endoreduplicated Xenopus hybrid clones between a female of Xenopus laevis and a male of Xenopus gilli, so called LG. The J strain, a completely inbred strain established in Japan ("J" for the first initial of Japan) has been maintained for almost 40 years (more than 30 generations) as a closed breeding colony. A major finding from those transplantation experiments is that adult frogs can recognize minor histocompatibility (H) antigens in essentially the same way as mammals, whereas larvae cannot reject the skin from donors that differ only by minor H antigens or in certain combinations having a one MHC haplotype disparity (33-35) (Table 1). This larval allotolerance seems to depend on the graft size from the adult donor (33, 35). Relatively large grafts were always tolerated regardless of differences in the H-antigens between donors and hosts, whereas small grafts were sometimes rejected. These results suggest that the larval tolerant state is unstable or sensitive to the balance between antigens and immunocompetent cells. These studies led us to question how amphibians confront newly appearing antigens during ontogeny and how metamorphosed animals eliminate larval antigens.

6. IMMUNE SYSTEM INVOLVEMENT IN XENOPUS METAMORPHIC TAIL REGRESSION

6.1. Skin cell remodeling during metamorphosis

In anuran metamorphosis, tadpoles show morphological and functional tissue remodeling from larval- to adult-type organs (40). Extreme examples of this remodeling process are seen in the epithelial organs where the epidermal cells in larvae are completely replaced by adult cells in the corresponding tissues of the frog (41-43). Skin, the largest organ in the body, is also the largest peripheral immune organ (44). Moreover, skin has the physiological function of a barrier, so that it shows most drastic changes for adaptation from an aquatic life to a terrestrial one (43). Although Xenopus spend their entire lives in water, they, like other amphibians, also show morphological and functional changes in the skin that are adaptive to a land environment (45). The skin of Xenopus larvae contains apical cells (flat and square cells that make up the uppermost single layer of the epidermis) and skein cells (located in the inner portion of the epidermis). During metamorphosis, larval basal cells appear along the basement membrane of the epidermis; these are thought to become stem cells that reconstitute the adult epidermis. By contrast, the adult epidermis does not contain the apical cells or the skein cells, and its structure is basically comparable to the mammalian epidermis (46), which includes basal germinative cells, granular cells, spinous cells and cornified cells in the outermost layer of adult skin (47). Both larval-specific apical and skein cell are eliminated during metamorphosis. However, histological observation revealed that the skein cells persist in part of the tail skin until the very end of the metamorphosis, showing mitotic activity even though they will soon be eliminated (43).

In metamorphic climax, the most remarkable change is tail regression, as the tail behaves uniquely, retaining a larval-type tissue until it disappears (43, 48). This resorption of the tail tissue, including the skin cells, has been explained solely in terms of a cell-autonomous programmed death of tissues induced by thyroid hormone (49). We, however, have focused our attention on the possible involvement of an immunological component to the elimination of larval skin during the metamorphic tissue reorganization process (Figure 2).

6.2. Larval tail skin is rejected by the adult frog

J strain of Xenopus is a fully homozygous inbred strain, so that frogs cannot reject skin graft from each other among adults (50) (Figure 3A). However, the grafts of larval skin, including both tail and trunk. obtained from young tadpoles at the beginning of metamorphosis, are rejected by the syngeneic adults (Figure 3B and 3B'). Around day 2 to 9 after transplantation, the larval skin grafts appear to have been accepted by the hosts (Figure 3B', Day2). Around day 7-9, vascular infiltration is observed in the grafts (Figure 3D, Day9). Host and graft tissue are completely fused into a skin sheet, whereas accumulation of lymphocytes is observed only underneath the grafted larval skin grafts (Figure 3D, see cross section). Finally, grafted skin from early metamorphic tadpole is almost rejected around day 48 (Figure 3B'). The most important result from these transplantation experiments is that the tail skin obtained from tadpoles at metamorphic climax stage when the tadpole tail regresses, is still rejected, whereas the trunk skin from the same animals is not, suggesting that the trunk skin has already changed into adult-type skin (Figure 3C and 3C'). These rejections exhibit accelerated secondary responses, suggesting that adult T cells are involved in this disruption of larval skin grafts. During the same developmental period, adult-MHC class II positive T cells have already appeared in the metamorphosing tadpole body (Figure 4). Therefore, it seems that at the metamorphic climax stage, larval tail skin appears foreign to the "adult-type T cells" that emerge during metamorphosis. Notably, skin grafts obtained from tadpole tails are rejected irrespective of the metamorphic stage of the donors. The mean survival time of skin grafts from premetamorphic tadpoles is much longer than that from individuals at metamorphic climax stages. In other words, the rejection represents a tendency towards increasing rapidity as the metamorphic stage of the donor progresses up to stage 63 (50) (Figure 3C' compared to 3B'). Recently (as described below), this has been found to be correlated with the expression of tail antigen proteins recognized by the adult T cells that increase and reach the maximum level at metamorphic climax stages (51).

6.3. Larval skin cells are recognized and killed by adult T cells

Splenocytes including T cells from J strain metamorphic climax larvae and from adults, but not from early metamorphic larvae, show prominent proliferative responses indicated by BrdU incorporation when co-cultured with syngeneic larval tails in the presence of fetal bovine serum (9) (Figure 5A and Table 2). Furthermore, tail fragments co-cultured with adult splenocytes from the same strain in the presence of adult Xenopus serum, undergo apoptosis (9) (Figure 5B). Apoptosis of larval epidermal cells was confirmed by DNA ladder formation and morphological features such a as nuclei fragmentation. Although these proliferation and killing assays are not able to detect both events in the same culture due to the different requisite experimental conditions, it is suggested that the larval tail cells are recognized as foreign by the T cells of metamorphic climax tadpoles. The rate of responsiveness to the syngeneic larval tail cells is comparable to that against the semi-xenogeneic adult skin obtained from the hybrid between a female of Xenopus laevis J strain and a male of Xenopus borealis B strain (52). These proliferative responses by the adult-type T cells including adult-type antigen presenting cells (APC) are completely blocked by anti-Xenopus MHC class II mAb (52). In Xenopus, MHC class II gene and its protein are expressed not only in the T cells but also in the epidermal cells in the skin during metamorphosis (52) (Figure 4). Since MHC class II molecules are expressed only in the apical cells but not in the skein cells, this implies that in vivo the apical cells are recognized directly by the adult T cells without the APC. In vitro, however, the skein cells require the APC to be recognized. Therefore, there are likely two pathways for recognition by the adult T cells in an MHC class II-restricted manner (52). Whatever the mechanism for recognition, these results suggest that a sufficient number of adult-type T cells differentiate during the course of metamorphosis, and become able to recognize larval tail cells. The effector T cells are suggested to be non-thymic (9). These results prompted us to propose that newly differentiating adult-type immune cells recognize and eliminate larval cells as "non-self" targets and led to the prediction that larva-specific antigens recognized by adult T cells may be expressed on the larval skin cells.

6.4. Isolation process and function of larval antigens recognized by the adult T cell

To investigate larval-specific antigens, an "alloantiserum" in the J strain of adults was produced by repeated grafts of syngeneic larval tail skin (53, 54). This strategy was successful because the antibody repertoire produced in the adult exclusively recognizes larval specific antigens. Using the alloantiserum, 59 kDa and 53 kDa proteins specifically expressed in the tadpole skin cells in a coordinated fashion during metamorphosis, have been isolated as candidate target antigens (51, 53). The isolated 59 kDa and 53 kDa proteins were sufficiently pure for determining their constituent partial amino acid sequences (53). Synthetic peptides containing these amino acid sequences elicited specific rat antibodies to the same 59 kDa (51, 53) and 53 kDa larval tail molecules (unpublished data) as well as an in vitro T cell response of syngeneic adult frogs immunized with larval epidermis. The expression of the 59 kDa protein initially starts at the early metamorphic stages both in the trunk and the tail epidermis, then diminishes in the trunk, exhibiting a clear boundary between the tail and the trunk, and persists in the tail epidermis until the end of metamorphosis (51). This tail-specific expression of the protein can be explained in terms of a suppressive effect by thyroid hormone (51). As the hormone is present in higher concentration in the trunk than in the tail cells (49), the expression is retained only in the tail even at the metamorphic climax stage when the expression of those proteins are down regulated by the thyroid hormone. At the same period, T cell accumulation is observed only in the tail epidermis but not in the trunk regions (9). Thus far, these results are consistent with the involvement of 59 kDa and 53 kDa proteins in metamorphic tail regression as immune target antigens (50).

To further substantiate our immunological elimination hypothesis in ontogenetic tissue reorganization, the two genes encoding 59 kDa and 53 kDa proteins respectively were recently isolated in our laboratory, and farther analyses will address their functions. The possibility of innate immunity that cleans up larval cells in the tail tissue has been suggested (1). By contrast, our model propose that acquired immune response to specific antigen molecules selectively expressed at the metamorphic period is involved in swift elimination of larval tissues.

7. SUMMARY

The mechanism underlying amphibian metamorphosis was first considered in 1916 when it was discovered that removal of the thyroid from tadpoles inhibits their metamorphosis (55). It is currently held that amphibian metamorphosis is induced by the action of the thyroid hormone secreted from the thyroid in a cell-autonomous manner (49). Our data demonstrate that the physiological and functional replacement of the immune system is closely related to the tissue remodeling in Xenopus. Indeed, we propose that the immune system is an important contributor to the process of metamorphosis. These results demonstrate not only the role of the acquired immune system for self/non-self recognition, but also how adult/larva recognition contributes to the tissue remodeling process. Whether the immune system participates more generally in tissue reorganization during vertebrate morphogenesis and to metamorphosis in urodele as well and anuran amphibians in particular, are questions worthy of future investigation.

8. ACKNOWLEDGMENTS

I would like to thank Dr. Shin Tochinai at Hokkaido University, Dr. Carmen Hannah at Niigata University and Dr. Michihiko Ito at Kitasato University for critical reading of this manuscript. I also thank Mr. Katsuki Mukaigasa and Arito Nishikura for help in preparing the Figures.

9. REFERENCES

1. Weber R.: Ultrastructural changes in regressing tail muscles of Xenopus larvae at metamorphosis. J Cell Biol, 22, 481-487 (1964)

doi:10.1083/jcb.22.2.481

http://dx.doi.org/10.1083/jcb.22.2.481

2. Nagata S.: A cell surface marker of thymus-dependent lymphocytes in Xenopus laevis is identifiable by mouse monoclonal antibody. Eur J Immunol, 15, 837-841 (1985)

doi:10.1002/eji.1830150818

http://dx.doi.org/10.1002/eji.1830150818

3. Nagata S.: Development of T lymphocytes in Xenopus laevis: appearance of the antigen recognized by an anti-thymocyte mouse monoclonal antibody. Dev Biol, 114, 389-394 (1986)

doi:10.1016/0012-1606(86)90203-4

http://dx.doi.org/10.1016/0012-1606(86)90203-4

4. Hsu E., Du Pasquier L.: Studies on Xenopus immunoglobulins using monoclonal antibodies. Mol Immunol, 21, 257-270 (1984)

doi:10.1016/0161-5890(84)90096-8

http://dx.doi.org/10.1016/0161-5890(84)90096-8

5. Manning M. J.: Histological organization of the spleen: implications for immune functions in amphibians. Res Immunol, 142, 355-359 (1991)

doi:10.1016/0923-2494(91)90091-V

http://dx.doi.org/10.1016/0923-2494(91)90091-V

6. Obara N., Tochinai S., Katagiri C.: Splenic white pulp as a thymus-independent area in the African clawed toad, Xenopus laevis. Cell Tissue Res, 226, 327-335 (1982)

doi:10.1007/BF00218363

http://dx.doi.org/10.1007/BF00218363

7. Turner R. J.: Response of the toad, Xenopus laevis, to circulating antigens. Responses after splenectomy. J Exp Zool, 183, 35-45 (1973)

doi:10.1002/jez.1401830105

http://dx.doi.org/10.1002/jez.1401830105

8. Nieuwkoop P. D., Faber J. Normal Table of Xenopus laevis (Daudin) (North-Holland, Amsterdam) (1956)

9. Izutsu Y., Yoshizato K., Tochinai S.: Adult-type splenocytes of Xenopus induce apoptosis of histocompatible larval tail cells in vitro. Differentiation, 60, 277-286 (1996)

doi:10.1046/j.1432-0436.1996.6050277.x

http://dx.doi.org/10.1046/j.1432-0436.1996.6050277.x

10. Flajnik M. F., Ferrone S., Cohen N., Du Pasquier L.: Evolution of the MHC: antigenicity and unusual tissue distribution of Xenopus (frog) class II molecules. Mol Immunol, 27, 451-462 (1990)

doi:10.1016/0161-5890(90)90170-5

http://dx.doi.org/10.1016/0161-5890(90)90170-5

11. Manning M. J., Horton J. D.: Histogenesis of lymphoid organs in larvae of the South African clawed toad, Xenopus laevis (Daudin). J Embryol Exp Morphol, 22, 265-277 (1969)

12. Ohinata H., Tochinai S., Katagiri C.: Ontogeny and tissue distribution of leukocyte-common antigen bearing cells during early development of Xenopus laevis. Development, 107, 445-452 (1989)

13. Horton T. L., Minter R., Stewart R., Ritchie P., Watson M. D., Horton J. D.: Xenopus NK cells identified by novel monoclonal antibodies. Eur J Immunol, 30, 604-613 (2000)

doi:10.1002/1521-4141(200002)30:2<604::AID-IMMU604>3.0.CO;2-X

http://dx.doi.org/10.1002/1521-4141(200002)30:2<604::AID-IMMU604>3.0.CO;2-X

14. Horton T. L., Ritchie P., Watson M. D., Horton J. D.: NK-like activity against allogeneic tumour cells demonstrated in the spleen of control and thymectomized Xenopus. Immunol Cell Biol, 74, 365-373 (1996)

doi:10.1038/icb.1996.64

http://dx.doi.org/10.1038/icb.1996.64

15. Horton T. L., Ritchie P., Watson M. D., Horton J. D.: Natural cytotoxicity towards allogeneic tumour targets in Xenopus mediated by diverse splenocyte populations. Dev Comp Immunol, 22, 217-230 (1998)

doi:10.1016/S0145-305X(98)00003-2

http://dx.doi.org/10.1016/S0145-305X(98)00003-2

16. Harding F. A., Flajnik M. F., Cohen N.: MHC restriction of T-cell proliferative responses in Xenopus. Dev Comp Immunol, 17, 425-437 (1993)

doi:10.1016/0145-305X(93)90034-N

http://dx.doi.org/10.1016/0145-305X(93)90034-N

17. Von Boehmer H., Shortman K., Adams P.: Nature of the stimulating cell in the syngeneic and the allogeneic mixed lymphocyte reaction in mice. J Exp Med, 136, 1648-1660 (1972)

doi:10.1084/jem.136.6.1648

http://dx.doi.org/10.1084/jem.136.6.1648

18. Festenstein H.: Immunogenetic and biological aspects of in vitro lymphocyte allotransformation (MLR) in the mouse. Transplant Rev, 15, 62-88 (1973)

19. Kobel H. R., Du Pasquier L.: Strains and species of Xenopus for immunological research. Developmental Immunology North-Holland, Amsterdam299-306 (1977)

20. Du Pasquier L., Blomberg B., Bernard C. C.: Ontogeny of immunity in amphibians: changes in antibody repertoires and appearance of adult major histocompatibility antigens in Xenopus. Eur J Immunol, 9, 900-906 (1979)

doi:10.1002/eji.1830091112

http://dx.doi.org/10.1002/eji.1830091112

21. Du Pasquier L., Horton J. D.: The effect of thymectomy on the mixed leukocyte reaction and phytohemagglutinin responsiveness in the clawed toad Xenopus laevis. Immunogenetics, 3, 105-112 (1976)

doi:10.1007/BF01576943

http://dx.doi.org/10.1007/BF01576943

22. Turpen J. B., Smith P. B.: Precursor immigration and thymocyte succession during larval development and metamorphosis in Xenopus. J Immunol, 142, 41-47 (1989)

23. Ruben L. N., Ahmadi P., Johnson R. O., Buchholz D. R., Clothier R. H., Shiigi S.: Apoptosis in the thymus of developing Xenopus laevis. Dev Comp Immunol, 18, 343-352 (1994)

doi:10.1016/S0145-305X(94)90359-X

http://dx.doi.org/10.1016/S0145-305X(94)90359-X

24. Grant P., Clothier R. H., Johnson R. O., Ruben L. N.: In situ lymphocyte apoptosis in larval Xenopus laevis, the South African clawed toad. Dev Comp Immunol, 22, 449-455 (1998)

doi:10.1016/S0145-305X(97)00057-8

http://dx.doi.org/10.1016/S0145-305X(97)00057-8

25. Du Pasquier L., Weiss N.: The thymus during the ontogeny of the toad Xenopus laevis: growth, membrane-bound immunoglobulins and mixed lymphocyte reaction. Eur J Immunol, 3, 773-777 (1973)

doi:10.1002/eji.1830031207

http://dx.doi.org/10.1002/eji.1830031207

26. Rollins-Smith L. A., Parsons S. C., Cohen N.: Effects of thyroxine-driven precocious metamorphosis on maturation of adult-type allograft rejection responses in early thyroidectomized frogs. Differentiation, 37, 180-185 (1988)

doi:10.1111/j.1432-0436.1988.tb00719.x

http://dx.doi.org/10.1111/j.1432-0436.1988.tb00719.x

27. Rollins-Smith L. A., Blair P. J., Davis A. T.: Thymus ontogeny in frogs: T-cell renewal at metamorphosis. Dev Immunol, 2, 207-213 (1992)

28. Manning M. J., Al Johari G. M.: immunological memory and metamorphosis. Metamorphosis the eighth symposium of the British Society for Developmental Biology; edited by Michael Balls and Mary Bownes, Clarendon Press, Oxford, 420-434 (1985)

29. Du Pasquier L., Schwager J., Flajnik M. F.: The immune system of Xenopus. Annu Rev Immunol, 7, 251-275 (1989)

30. Hadji-Azimi I., Coosemans V., Canicatti C.: Atlas of adult Xenopus laevis hematology. Dev Comp Immunol, 11, 807-874 (1987)

doi:10.1016/0145-305X(87)90068-1

http://dx.doi.org/10.1016/0145-305X(87)90068-1

31. Green N., Cohen N.: Phylogeny of immunocompetent cells: III. Mitogen response characteristics of lymphocyte subpopulations from normal and thymectomized frogs (Xenopus laevis). Cell Immunol, 48, 59-70 (1979)

doi:10.1016/0008-8749(79)90099-6

http://dx.doi.org/10.1016/0008-8749(79)90099-6

32. Chardonnens X., Du Pasquier L.: Induction of skin allograft tolerance during metamorphosis of the toad Xenopus laevis: a possible model for studying generation of self tolerance to histocompatibility antigens. Eur J Immunol, 3, 569-573 (1973)

doi:10.1002/eji.1830030909

http://dx.doi.org/10.1002/eji.1830030909

33. DiMarzo S. J., Cohen N.: Immunogenetic aspects of in vivo allotolerance induction during the ontogeny of Xenopus laevis. Immunogenetics, 16, 103-116 (1982)

doi:10.1007/BF00364398

http://dx.doi.org/10.1007/BF00364398

34. Barlow E. H., Cohen N.: The thymus dependency of transplantation allotolerance in the metamorphosing frog Xenopus laevis. Transplantation, 35, 612-619 (1983)

doi:10.1097/00007890-198306000-00018

http://dx.doi.org/10.1097/00007890-198306000-00018

35. Nakamura T., Maeno M., Tochinai S., Katagiri C.: Tolerance induced by grafting semi-allogeneic adult skin to larval Xenopus laevis: possible involvement of specific suppressor cell activity. Differentiation, 35, 108-114 (1987)

doi:10.1111/j.1432-0436.1987.tb00157.x

http://dx.doi.org/10.1111/j.1432-0436.1987.tb00157.x

36. Kobel H. R., Du Pasquier L.: Hyperdiploid species hybrids for gene mapping in Xenopus. Nature, 279, 157-158 (1979)

doi:10.1038/279157a0

http://dx.doi.org/10.1038/279157a0

37. Tochinai S., Katagiri C.: Complete abrogation of immune response to skin allografts and rabbit erythrocytes in the early thymectomized Xenopus. Dev Growth Differ, 17, 383-394 (1975)

doi:10.1111/j.1440-169X.1975.00383.x

http://dx.doi.org/10.1111/j.1440-169X.1975.00383.x

38. Nakamura T., Kawahara H., Katagiri C. : Rapid production of a histocompatible colony of Xenopus laevis by gynogenetic procedure. . Zool Sci 2, 71-79 (1985)

39. Chardonnens X.: Tissue typing by skin grafting during metamorphosis of the toad Xenopus laevis (Daudin). Experientia, 31, 237 (1975)

doi:10.1007/BF01990725

http://dx.doi.org/10.1007/BF01990725

40. Fox H. Amphibian metamorphosis, Humana Press, Clifton, New Jersey (1983)

41. Ishizuya-Oka A., Shimozawa A.: Ultrastructural changes in the intestinal connective tissue of Xenopus laevis during metamorphosis. J Morphol, 193, 13-22 (1987)

doi:10.1002/jmor.1051930103

http://dx.doi.org/10.1002/jmor.1051930103

42. Ishizuya-Oka A., Shimozawa A.: Programmed cell death and heterolysis of larval epithelial cells by macrophage-like cells in the anuran small intestine in vivo and in vitro. J Morphol, 213, 185-195 (1992)

doi:10.1002/jmor.1052130205

http://dx.doi.org/10.1002/jmor.1052130205

43. Izutsu Y., Kaiho M, Yoshizato K.: Different distribution of epidermal basal cells in the anuran larval skin correlates with the skin's region-specific fate at metamorphosis. J Exp Zool, 267, 605-615 (1993)

doi:10.1002/jez.1402670608

http://dx.doi.org/10.1002/jez.1402670608

44. Bos J. D.: The skin as an organ of immunity. Clin Exp Immunol, 107 Suppl 1, 3-5 (1997)

45. Watanabe Y., Kobayashi H., Suzuki K., Kotani K., Yoshizato K.: New epidermal keratin genes from Xenopus laevis: hormonal and regional regulation of their expression during anuran skin metamorphosis. Biochim Biophys Acta, 1517, 339-350 (2001)

46. Yoshizato K.: Molecular mechanism and evolutional significance of epithelial-mesenchymal interactions in the body- and tail-dependent metamorphic transformation of anuran larval skin. Int Rev Cytol, 260, 213-260 (2007)

doi:10.1016/S0074-7696(06)60005-3

http://dx.doi.org/10.1016/S0074-7696(06)60005-3

47. Watanabe Y., Tanaka R., Kobayashi H., Utoh R., Suzuki K., Obara M., Yoshizato K.: Metamorphosis-dependent transcriptional regulation of xak-c, a novel Xenopus type I keratin gene. Dev Dyn, 225, 561-570 (2002)

doi:10.1002/dvdy.10196

http://dx.doi.org/10.1002/dvdy.10196

48. Izutsu Y., Tochinai S., Onoe K.: Loss of reactivity to pan-cadherin antibody in epidermal cells as a marker for metamorphic alteration of Xenopus skin. Dev Growth Differ, 42, 377-383 (2000)

doi:10.1046/j.1440-169x.2000.00527.x

http://dx.doi.org/10.1046/j.1440-169x.2000.00527.x

49. Brown D. D.: The role of deiodinases in amphibian metamorphosis. Thyroid, 15, 815-821 (2005)

doi:10.1089/thy.2005.15.815

http://dx.doi.org/10.1089/thy.2005.15.815

50. Izutsu Y., Yoshizato K.: Metamorphosis-dependent recognition of larval skin as non-self by inbred adult frogs (Xenopus laevis). J Exp Zool, 266, 163-167 (1993)

doi:10.1002/jez.1402660211

http://dx.doi.org/10.1002/jez.1402660211

51. Watanabe M., Ohshima M., Morohashi M., Maeno M., Izutsu Y.: Ontogenic emergence and localization of larval skin antigen molecule recognized by adult T cells of Xenopus laevis: Regulation by thyroid hormone during metamorphosis. Dev Growth Differ, 45, 77-84 (2003)

doi:10.1046/j.1440-169X.2003.00676.x

http://dx.doi.org/10.1046/j.1440-169X.2003.00676.x

52. Izutsu Y., Tochinai S., Iwabuchi K., Onoe K.: Larval antigen molecules recognized by adult immune cells of inbred Xenopus laevis: two pathways for recognition by adult splenic T cells. Dev Biol, 221, 365-374 (2000)

doi:10.1006/dbio.2000.9681

http://dx.doi.org/10.1006/dbio.2000.9681

53. Izutsu Y., Tochinai S., Maeno M., Iwabuchi K., Onoe K.: Larval antigen molecules recognized by adult immune cells of inbred Xenopus laevis: partial characterization and implication in metamorphosis. Dev Growth Differ, 44, 477-488 (2002)

doi:10.1046/j.1440-169X.2002.00660.x

http://dx.doi.org/10.1046/j.1440-169X.2002.00660.x

54. Izutsu Y., Maeno M.: Analyses of immune responses to ontogeny-specific antigens using an inbred strain of Xenopus laevis (J strain). Methods Mol Med, 105, 149-158 (2005)

55. Allen B. M.: The results of extirpation of the anterior lobe of the hypophysis and of the thyroid of Rana pipiens larvae. Science, 44, 755-758 (1916)

doi:10.1126/science.44.1143.755

http://dx.doi.org/10.1126/science.44.1143.755

Key Words: Xenopus, Immune Cell, Skin, Epidermal Cell, Larval Antigen, T cell, Remodeling, Keratin, Review