The keratin-related Ouroboros proteins functionas immune antigens mediating tail regressionin Xenopus metamorphosisKatsuki Mukaigasaa,1, Akira Hanasakia,1, Mitsugu Maenoa, Hiroshi Fujiia, Shin-ichiro Hayashidaa, Mari Itohb,Makoto Kobayashic, Shin Tochinaid, Masayuki Hattae, Kazuya Iwabuchif, Masanori Tairab, Kazunori Onoef,and Yumi Izutsua,2

aDepartment of Biology, Faculty of Science, Niigata University, Niigata 950-2181, Japan; bDepartment of Biological Sciences, Graduate School of Science,University of Tokyo, Tokyo 113-0033, Japan; cInstitute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8575, Japan; d Division of BiologicalSciences, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan; eFaculty of Science, Ochanomizu University, Tokyo 112-8610, Japan;and fDivision of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan

Edited by Igor B. Dawid, National Institutes of Health, Bethesda, MD, and approved September 3, 2009 (received for review September 18, 2007)

Tail resorption during amphibian metamorphosis has beenthought to be controlled mainly by a cell-autonomous mechanismof programmed cell death triggered by thyroid hormone. However,we have proposed a role for the immune response in metamor-phosis, based on the finding that syngeneic grafts of tadpole tailskin into adult Xenopus animals are rejected by T cells. To test this,we identified two tail antigen genes called ouro1 and ouro2 thatencode keratin-related proteins. Recombinant Ouro1 and Ouro2proteins generated proliferative responses in vitro in T cells iso-lated from naive adult Xenopus animals. These genes were ex-pressed specifically in the tail skin at the climax of metamorphosis.Overexpression of ouro1 and ouro2 induced T-cell accumulationand precocious tail degeneration after full differentiation of adult-type T cells when overexpressed in the tail region. When theexpression of ouro1 and ouro2 were knocked down, tail skin tissueremained even after metamorphosis was complete. Our findingsindicate that Ouro proteins participate in the process of tailregression as immune antigens and highlight the possibility thatthe acquired immune system contributes not only to self-defensebut also to remodeling processes in vertebrate morphogenesis.

amphibian � skin � cell death � T cell � remodeling

During amphibian metamorphosis, most tissues in the tadpoleundergo a complete transformation from the larva to the

adult. However, the tail tissue is unique in that it retains its larvalform until it is resorbed (1, 2). It has been hypothesized as earlyas 1916 that amphibian metamorphosis is regulated by thethyroid gland (3). Indeed, recent studies have shown that thyroidhormone (TH) plays a crucial role in tail regression and mayregulate programmed cell death in a variety of tissues in acell-autonomous manner (4, 5). However, it is also possible thatneighboring cells induce nonautonomous tissue destruction (6).

We have suggested a possible role for the immune system indegeneration of larval tail tissues during metamorphosis, basedon several lines of evidence (7–13). For instance, syngeneic graftsof larval tail skin into adult frogs are rejected and exhibit anaccelerated secondary immune response (7). Moreover, T cellsfrom adults and larvae at the metamorphic climax stage—butnot earlier—show a prominent proliferative response againstlarval tails in vitro, and tail explants undergo apoptosis in thepresence of adult Xenopus serum (8). Based on these observa-tions, we proposed that newly differentiated adult-type, nonthy-mic T cells recognize and eliminate larval cells as ‘‘non-self’’targets during metamorphosis. This model leads to the predic-tion that larval-specific antigens recognized by adult T cells areexpressed in the larval skin (9). Recently, we isolated 59- and53-kDa proteins as candidate target antigens using alloantiserumproduced by larval skin grafts in adult frogs (10). The spatio-temporal localization of these two proteins in larval tail skin (11)

is compatible with their predicted role as immune antigensinvolved in metamorphic tail regression (13). However, it isunresolved whether these proteins mediate an immune-basedmechanism of tail regression.

In this study, we isolated genes encoding 59- and 53-kDa proteins,named Ouro1 and Ouro2, respectively, and carried out gain- andloss-of-function analyses. We show that ouro1 and ouro2 arespecifically expressed in the regressing tail skin at the climax ofmetamorphosis and that recombinant Ouro proteins are recog-nized in vitro by adult T cells as foreign antigens. By analyzing thephenotypes of single- and double-transgenic (DT) tadpoles, wedemonstrate that overexpression of ouro genes results in a signif-icant acceleration of tail regression, whereas knockdown causesdelayed tail regression. Together, these data provide the evidencefor an unprecedented immune-based mechanism regulating theprocess of tissue reorganization in Xenopus metamorphosis.

ResultsOuro1 and Ouro2 Proteins Induce Adult T-Cell Proliferation. Theisolated 59- and 53-kDa larval skin proteins (10) were suffi-ciently pure to determine their partial amino acid sequences [Fig.1A, red Y marks for amino acids (AA) nos. 363–381 in Ouro1and AA nos. 307–321 in Ouro2]. Rat antibodies raised againstsynthetic peptides containing these amino acid sequences spe-cifically recognized the 59-kDa (10, 11) and 53-kDa proteins (seeFig. 2C), and these peptides elicited a T-cell response in vitrowith cells isolated from syngeneic adult frogs immunized bygrafting the larval skin (10). Oligonucleotide primers designedfor the partial amino acid sequences of the 59- and 53-kDaproteins were used to amplify corresponding cDNA fragments,which were subsequently used to clone 2,009- and 1,764-bpcDNAs (accession nos. AB299972 and AB299973, respectively).These cDNAs contained coding sequences (CDS) for 59-kDa(634 residues) and 53-kDa (500 residues) proteins, both of whichincluded a central rod domain flanked by glycine-serine-richdomains with no apparent strong homology to one another (Fig.1A). We named these genes ouro1 for the 59-kDa CDS and ouro2for the 53-kDa one, derived from the Greek word ouroboros,

Author contributions: Y.I. designed research; K.M., A.H., M.M., H.F., S.-I.H., M.I., and Y.I.performed research; A.H., M.K., S.T., M.H., K.I., M.T., K.O., and Y.I. analyzed data; and M.T.,K.O., and Y.I. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: Ouroboros1, Accession no. AB299972; Ouroboros2, Accession no.AB299973 have been deposited in the DNA Data Bank of Japan (DDBJ).

1K.M. and A.H. contributed equally to this work.

2To whom correspondence should be addressed. E-mail: izutsu@gs.niigata-u.ac.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0708837106/DCSupplemental.

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which means ‘‘one who devours his own tail.’’ These genes areputative orthologs of the hagfish thread keratin genes � and �,respectively, which have unknown function and belong to dif-ferent subgroups of the keratin superfamily (14).

To characterize the Ouro1 and Ouro2 proteins, we producedrecombinant proteins in Escherichia coli using their partialcDNA sequences (Fig. 1 A, bold lines; AA nos. 197–520 inOuro1, AA nos. 120–334 in Ouro2). Both recombinant proteinsstimulated adult T cells as strongly as larval tail tissues fromstage 57 tadpoles (Fig. 1B; see Figs. 2 A and B for the expressionof ouro genes at stage 57), indicating that Ouro proteins functionas antigens for adult immune cells.

The ouro1 and ouro2 Transcripts and Proteins Are Expressed in theMetamorphosing Tadpole Skin. To examine whether ouro1 and ouro2are expressed in the appropriate spatiotemporal pattern to beinvolved in tail regression, we performed Northern blot analysis andRT-PCR amplification for tadpole tissues. The transcripts for bothgenes were detected in the tail skin in a restricted period from stages50–62 during metamorphosis (Fig. 2 A and B). Although weakexpression levels of ouro1 and ouro2 were also observed in thetrunk, such a sharp peak in expression in the tail skin appears to beunique to the ouro genes, compared with other types of keratin,adult (xak-b) and larval (xlk) keratin (Fig. 2B).

Western blotting analyses showed that both Ouro proteinscould be detected at stage 50, and they peaked at stage 59 in thetail (Fig. 2C). Substantial expression of both proteins wasobserved at stages 54–62. Although the tail regresses dramati-cally during the metamorphic climax stage (stages 62–65),immunostaining of the tail with the Xenopus serum against thelarval antigens including Ouro1 and Ouro2 showed that expres-sions of Ouro proteins were still detected at stage 64 at a highlevel (Fig. S1), similar to stage 59 (11). Thus, even though ourotranscripts were not detected after stage 62 (see Fig. 2 A and B),

B

Adult proliferative T cells (%)0 2 4 6 8 10 12 14 16

WO

Tail

rOuro1

rOuro2

rGFP

Ouro1

Ouro2

GS central rod GS1 204 519 634AA

197-520AA

363-381AA

GS central rod GS1 154 471 500AA

120-334AA

307-321AA

A

n(exp.)

9(3)

6(2)

6(2)

5(2)

9(3)

* *

Fig. 1. Recombinant Ouro1 and Ouro2 proteins induce adult T-cell prolifera-tion. (A)SchematicpresentationofOuro1andOuro2.Bothproteinsarepredictedto contain central rod domains flanked by glycine-serine rich domains (GS). Boldlines with amino acid (AA) nos. represent sequences used for His-tagged recom-binants. Y marks with AA nos. (red) show sequences used for raising specificantisera (see Results). (B) T-cell proliferation assay. Columns indicate the percent-age of proliferating cells (mean � SD from two to three independent experi-ments) culturedwithoutastimulus (WO)orwithsyngeneic larval tail tissues (Tail),Ouro1 recombinant protein (rOuro1), Ouro2 recombinant protein (rOuro2), orGFP recombinant protein (rGFP). The ANOVA test was used to assess statisticalsignificanceamongvalues.*,P�0.01.Significantdifferencesare indicatedbytheTukey’s HSD test. n, number of assays; exp., number of experiments.

Fig. 2. ouro1 and ouro2 are expressed in the skin during metamorphosis.(A) Northern blot analysis for ouro1 expression in J strain tadpoles. Tail andtrunk skin tissues were isolated from various stages of tadpoles as indi-cated. A representative blot is shown (Upper Left), because five indepen-dent sets of experiments showed basically the same results. Ribosomal RNAvisualized by ethidium bromide as a loading control (Lower Left). Relativeexpression levels were calculated using the image J software (Right). (B)RT-PCR with J strain tadpoles. Tail and trunk skin tissues as indicated wereanalyzed for ouro1, ouro2, Xenopus adult keratin (xak-b), Xenopus larvalkeratin (xlk), and Xenopus rpl8 (rpl8) as an internal control. -RT, rpl8without RT. (C) Western blot analysis for Ouro1 and Ouro2 with J straintadpoles. Tail and trunk skin cell lysates were used. (D) WISH with albino(non-J strain) X. laevis tadpoles. ouro1 antisense probe was used fortadpoles at stage 55 (n � 7), 58 (n � 7), and 62 (n � 3). ouro1 sense probewas used as a negative control for tadpoles at stage 58 (n � 5). Positivesignals in blue were reproducibly detected in the tail and trunk (stage 55)or in the tail (stages 58 and 62). Arrowheads show the boundary betweenthe tail and trunk region. (E) The vertical section of the tadpole at stage 62after WISH using ouro1 antisense probe. The section includes the boundarybetween the tail and trunk skin as indicated. Purple signals are specificallyseen in the tail epidermis (n � 2). ep, epidermis.

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the Ouro proteins are likely to be present in the tail at a certainlevel from the prometamorphic stage (stages 54–57) to theclimax stage (see Fig. S2).

Whole mount in situ hybridization (WISH) on albino tadpoles(non-J strain) demonstrated that the expression of ouro1 beganthroughout the entire body but diminished in the trunk at stage 58,with a clear boundary between the tail and the trunk (Fig. 2D, whitearrowheads). The expression in the tail persisted until stage 62 (Fig.2D and Fig. S3 for enlargement) and was strongly reduced by stage63 (data not shown). No signal was detected with the sense probecontrol (Fig. 2D). Apparent inconsistency in expression levels atstage 62 between RT-PCR (Fig. 2B) and WISH (Fig. 2D) may havebeen caused by interindividual variability (Fig. S4) or differencesbetween the J strain and the non-J albino strain. Cross-sections ofstained tadpoles at stage 62 revealed that the expression of ouro1was mainly confined to the epidermis of the skin (Fig. 2E). Notably,T-cell accumulation was observed in the tail epidermis (Fig. S1).Taken together, the results indicate that ouro genes are expressedin the tail epidermis specifically during metamorphosis, suggestingthe possibility that Ouro proteins function to recruit T cells to thetail skin for regression.

Overexpression of ouro1 and ouro2 Enhances Tail Degeneration andT-Cell Accumulation. The potent antigenicity and expression pat-tern of the ouro transcripts and proteins, as well as T-cellaccumulation in the tail at the metamorphic climax as describedabove, support our hypothesis that the Ouro proteins functioningas tail antigens mediate an immune-based mechanism of taildegeneration. To analyze the function of the ouro genes in vivo,transgenic animals generated by nuclear transplantation (15)were used to express FLAG- or Myc-tagged GFP-Ouro fusionproteins under the control of the Xenopus heat shock (HS)protein promoter (Fig. 3A). When the tail tip of transgenictadpole was subjected to HS at stages 57–59 (Fig. 3B and Fig. S5),GFP expression was successfully induced with a clear boundarybetween HS-treated and nontreated areas (Fig. 3C). Westernblot analysis confirmed that the tagged Ouro-GFP fusion pro-teins (87 kDa for Ouro1-FLAG-GFP, 81 kDa for Ouro2-Myc-GFP) were induced by HS in transgenic tadpoles as expected(Fig. 3D). In the ouro1-gfp/ouro2-gfp DT tadpoles, both Ouroproteins were detected in the same cells as assayed by confocalmicroscopy of immunostaining with anti-FLAG and anti-Myctag antibodies (data not shown). Subsequently, F1 and F2 lines1–9 were generated with the F0 transgenic frogs (Fig. S6).

To observe the effect of ouro1 and ouro2 overexpression onthe tail before tail regression normally starts at stage 62, HS wasadministered at stages 57–59. Notably, precocious degenerationof tail tissue was observed in a GFP-positive region on days 1–4after HS when both Ouro1-GFP and Ouro2-GFP were induced(Fig. 3E). This phenomenon was observed in F0 tadpoles (TableS1) in F1 and F2 lines 1–3 with a high incidence of 72% (31/43)(Table 1, and see Table S1 for reproducibility in each line). Bycontrast, no degeneration was observed in the single transgenicouro1-gfp (0/37), ouro2-gfp (0/20), and gfp alone (0/72) tadpoles(Table 1, lines 4–9). These data suggest that a combination ofOuro1 and Ouro2 overproduction can initiate tail degeneration.

We next examined the possibility that Ouro-initiated prematuredegeneration is mediated by a T-cell immune response. For thispurpose, HS treatment was done at the premetamorphic stages50–52 before differentiation of adult-type T cells at stage 54 (13,16). As expected, precocious tail degeneration was not observed inthe HS-treated transgenic tadpoles (0/27; Table 1, lines 1–3).Furthermore, a second administration of HS at stages 57–59 topretreated tadpoles (Table S1, line 1) resulted in severe taildegeneration (7/9) with a high incidence (8/9) than the single HStreatment at stages 57–59 (see Table S1). This enhanced responseby the two rounds of HS treatment resembles a boosted immuneresponse.

Fig. 3. Precocious tail degeneration by overexpression of ouro1 and ouro2genes. (A) DNA constructs used to generate transgenic animals. Ouro proteinswere fused to the FLAG- or Myc-tag and the GFP protein. The expressionconstructs 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’ssolution at 37 °C. (C) Induction of GFP expression by HS. GFP was only detectedin a HS-treated region of the tail. The panel shows a typical case, which is thegfp 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 introducedgenes was detected in the HS-treated area (�), but not in nontreated area (�)on day 1 after HS. Arrowheads indicate the Ouro fusion proteins. Blottedproteins were stained with Coomassie Brilliant Blue (CBB). A representativeblot from two independent experiments is shown. (E) Induction of precocioustail regeneration by HS. Tails of ouro1-gfp/ouro2-gfp DT (line 2) on days 1–4after HS at stage 58/59 showed precocious degeneration (Upper). HS-inducedgfp-transgenic tadpoles (line 9) showed a normal tail (Lower). Bright field(Left) and GFP fluorescence image (Right) are paired. (F–H) Accumulation of Tcells in the HS-treated tails. Vertical frozen sections of HS-treated tails ofouro1-gfp/ouro2-gfp DT tadpole (line 1) (n � 8) (F and G) and gfp transgenictadpole (line 8) (n � 8) (H) F1 tadpoles on day 3 after HS were stained withanti-GFP antibody (green) (F–H) and anti-Xenopus T cells (red) (F and H) or withanti-Xenopus MHC class II antibody (red) (G). Dotted circles in serial sections (Fand G) indicate an assembly of T cells (F) expressing MHC class II (G). Arrow-heads, a few T cells seen in the tail epidermis of the gfp control (H).

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The precocious degenerating tails of ouro1-gfp/ouro2-gfp DTtadpoles were bloody as a result of hemolysis and/or dilation ofperipheral capillaries, whereas the tails of gfp control tadpolesappeared transparent (Fig. 3E), like the normal metamorphos-ing tadpoles. Therefore, we next analyzed T-cell accumulation indegenerating tails. Immunohistochemistry of tail tissues for aXenopus pan-T-cell marker showed an accumulation of a largenumber of T cells in the GFP-positive degenerating area (Fig.3F). The accumulated T cells did not express GFP, indicatingthat they migrated from outside the HS-treated region. No suchaccumulation was seen in gfp (Fig. 3H) or single ouro transgenictadpoles (data not shown). In addition, T cells in the HS-treatedtail of ouro1-gfp/ouro2-gfp DT tadpoles expressed MHC class II(Fig. 3G), a marker of adult-type T cells (13, 16). Thus, taildegeneration was observed at stages after adult-type T-celldifferentiation (see Fig. S2). These data suggest that T cells areprimed by Ouro antigens during metamorphosis. These data areall consistent with the idea that tail degeneration by Ouro1 andOuro2 involves T-cell-mediated immune responses.

Knockdown of ouro1 and ouro2 Gene Expression Results in Retentionof the Tadpole Tail. Next, loss-of-function analysis was performed byexpressing antisense RNA in vivo in transgenic animals usingDsRed-anti-ouro1/gfp-anti-ouro2 constructs (Fig. 4A). In this anal-ysis, we were unable to generate F1 antisense-ouro tadpoles, and itwas even difficult to raise their F0, possibly because of some toxicityfrom the leaky expression of antisense RNAs (see Table S2). Thedistal part of the transgenic tadpole tail was heat-treated in thesame manner as shown in Fig. 3B and Fig. S5. On day 1 after HS,expressions of GFP (Fig. 4B) and DsRed (data not shown) wereobserved in the HS-treated area in the antisense-DT tadpole,similar to the GFP expression as shown in Fig. 3C. RT-PCR analysisconfirmed that the antisense RNAs had been induced correctly byHS treatment (Fig. S7). Repression of the endogenous ouro2 geneupon HS was clearly shown at both the RNA and protein levels bycomparing HS and non-HS regions in the same tail of antisense-DTor single-trasngenic tadpoles (Fig. S8).

Remarkably, tail degeneration in DsRed-anti-ouro1/gfp-anti-ouro2 DT tadpoles was delayed in the HS-treated region,compared with that of the gfp control tadpoles (Fig. 4, compareC and H on day 9). Furthermore, some of HS-treated tails wereretained even after day 14 (Fig. 4E), even though control animals

had completed tail degeneration by stage 66. We defined thecriterion for a ‘‘retained tail’’ in that the tail fails to regress;mainly the epidermis remains resulting in a ‘‘folded tail’’ phe-notype (see Fig. 4 C–E). As summarized in Table 2, this retainedtail phenotype was observed in 58% (7/12) of the DsRed-anti-ouro1/gfp-anti-ouro2 DT tadpoles. Furthermore, this phenotypewas also observed in 20% (2/10) of the gfp-anti-ouro2 single-transgenic tadpoles (Table 2), suggesting that both Ouro1 andOuro2 are required for tail degeneration.

Some of the folded tails in antisense tadpoles were retained forover 2 weeks after metamorphosis had been completed. Thisretention was not caused by a transformation from larva- toadult-type skin, because the expression of adult-type keratin xak-bwas not detected in the tail (Fig. S8A). In normal metamorphosingtadpoles, the endogenous ouro1 is expressed specifically in theepidermis as shown above (see Fig. 2E). This expression pattern canexplain our observation that the epidermis persisted even thoughthe other tail tissue components completely degenerated when ourogene expression was knocked down.

DiscussionIn this paper, we have shown that the Ouro1 and Ouro2 proteinsare necessary and sufficient for the regression of the tail skin duringXenopus metamorphosis. Our data substantiate that the Ouro1 andOuro2 proteins represent the long-postulated 59- and 53-kDamolecules, respectively (9, 10), based on their antigenicity, calcu-lated molecular mass, and specific expression in the skin epidermispeaking at, and limited to, the metamorphic periods.

Immunocompetent cells undergo profound reorganizationduring metamorphosis, including the large-scale death of larvallymphocytes and the emergence of adult-type T cells (16).Precocious tail regression by overexpressed Ouro proteins de-pends on the period when adult T cells are fully differentiated,being consistent with our previous finding that the T cells fromclimax-stage, but not prometamorphic-stage animals, display aprominent proliferative response against larval tail tissues (8).Taken together, we propose an immune-based mechanism, inwhich the Ouro proteins actively function in tail regression astargets for newly differentiated adult-type immune cells duringmetamorphosis. This is in contrast to the role of innate immunityto clean up larval cells during metamorphosis, which had beengenerally believed since Weber’s original observations (17).

Table 1. Effects of overexpression of ouro genes on tail regression in transgenic tadpoles

Transgene Stage of HS Line (F)No. of HS

tadpoles (nHS)No. of GFP-expressing

tadpoles (nG) nG/nHS, %Degenerated

tails (nD) nD/nG, % P

Line 1 (F1)ouro1-gfp/ouro2-gfp 57–59 Line 2 (F2) 55 43 78 31 72 —

Line 3 (F2)

Line 1 (F1)50–52 Line 2 (F2) 38 27 71 0 0 �0.01

Line 3 (F2)

Line 4 (F1)ouro1-gfp 58/59 Line 5 (F1) 74 37 50 0 0 �0.01

Line 6 (F2)

ouro2-gfp 58/59 Line 7 (F2) 36 20 56 0 0 �0.01

gfp 57–59Line 8 (F1)

97 72 74 0 0 �0.01Line 9 (F2)

F1 andF2 lines1–9weregeneratedbymatingtheir transgenic foundersasdescribed inTableS1andFigS6.GFP-expressinganimals (nG)wereselectedfromHS-treatedspecimens (nHS) on day 1 after HS as described in Materials and Methods. The phenotype �Degenerated tails� (nD) indicates that �50% of the GFP-expressing areadisappeared by day 4 (around stages 61) as shown in Fig. 3E. The number of samples and phenotypes in the same experiment groups were combined (see Table S1 fordetails of each line). Note: HS treatment at the earlier stages (stages 50–52) for ouro1-gfp/ouro2-gfp double transgenic lines 1–3 did not initiate premature taildegeneration (see Results). P, one-way ANOVA, followed by Duncan’s multiple range test against ouro1-gfp/ouro2-gfp DT tadpoles treated with HS at stage 57–59.

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Almost a century has passed since scientific argument beganin 1916 concerning the mechanism underlying amphibian meta-morphosis, in which removal of the thyroid from tadpolesinhibited their metamorphosis (3). To date, two mechanisms

underlying induction of apoptosis in amphibian metamorphosishave been proposed (6). The first is the ‘‘suicide model,’’ in whicha cell-autonomous apoptosis pathway is triggered by TH. Thesecond is the ‘‘murder model,’’ in which death factors secretedfrom the neighboring cells kill larval cells. Our data demonstratea third mechanism, as detailed above.

We have shown that, individually, neither the Ouro1 nor Ouro2protein is sufficient for inducing precocious tail degeneration inoverexpression (Table 1) that suppression of Ouro2 alone is nec-essary for tail retention in knockdown experiments (Table 2). Thesephenomena could be explained by the formation of a complex,because it has been reported that the hagfish counterparts, threadkeratin � and �, form a stable complex in vitro (18, 19).

ouro1 and ouro2 expressions are initiated during early metamor-phosis in both the tail and trunk epidermis, but become restrictedto the tail region at the climax of metamorphosis. Although themechanisms regulating ouro gene expression remain elusive, thedown-regulation of ouro expression in the trunk may be caused byTH, because the Ouro1 protein is down-regulated in the trunk ofTH-treated tadpoles more rapidly than in the tail (11), and becauseTH is present in higher concentrations in the trunk than in the tailcells (20). Whatever the mechanism, this tail-specific expression ofouro genes is likely to function as a prepattern for tail regression,which is targeted by an immune response.

Our results demonstrate not only the role of the acquiredimmune system in self/non-self recognition, but also how adult/larva recognition contributes to the tissue remodeling process.This report shows how this discovered mechanism is involved inamphibian metamorphosis and suggests that the immune systemmay participate more generally than suspected in tissue remod-eling during vertebrate morphogenesis. Although here we re-ported on Ouro proteins in Xenopus, we have also isolated someputative orthologs from other amphibians and fish (unpublisheddata), which lead to the idea that a similar mechanism may beinvolved in the development of species other than Xenopus.

Materials and MethodsAnimals. An MHC-homozygous inbred J strain of Xenopus laevis was used formost experiments and was staged according to Nieuwkoop and Faber (21).Outbred X. laevis animals were used for preparing transgenic animals. Apartially inbred albino strain (non-J strain) of X. laevis was used for WISH. Alllarvae and adults were reared at 23 � 1 °C in dechlorinated tap water.

Cloning of ouro Genes. Degenerate primers were designed against partialpeptide sequences of 59- and 53-kDa proteins that were immunopurified fromtail skin lysates of stage 57 J strain tadpoles using a frog alloantiserum (10).PCR products were used as probes to clone full-length ouro1 and ouro2 cDNAsby screening a ZAP cDNA library of whole tails at stage 62 (a kind gift from Dr.Yoshio Yaoita). 5� RACE was performed to identify the 5�UTR of ouro1.

Fig. 4. Knockdown of ouro1 and ouro2 gene expression results in retentionof tail skin. (A) Antisense constructs for ouro1 and ouro2. Reverse-orientedouro1 or ouro2 cDNA were placed after DsRed or gfp, respectively, which weredriven by the hsp70 promoter. (B–E and E�) Suppression of tail regression inDsRed-anti-ouro1/gfp-anti-ouro2 DT tadpoles. Tails of DsRed-anti-ouro1/gfp-anti-ouro2 DT tadpoles (F0) on days 1–14 after HS at stage 58/59 are shown bybright field and fluorescence microscopy (only GFP is shown). Note: DsRed-anti-ouro1/gfp-anti-ouro2 DT animals exhibit a pronounced delay in tailregression with a folded epidermis. (F–J and J�) gfp transgenic control. HS-induced gfp transgenic tadpole (line 9, F2) show normal tail degeneration.Boxed areas in E� and J� are magnified for E and J, respectively.

Table 2. Effects of knockdown of ouro genes on tail metamorphosis in transgenic tadpoles

TransgeneStageof HS

F0 or line(F)

No. of HStadpoles (nHS)

No. ofGFP/DsRed-expressing

tadpoles (nG) nG/nHS, %Retainedtails (nR) nR/nG, % P

DsRed-anti-ouro1/gfp-anti-ouro2 58/59 F0 144 12 8 7 58 �0.01

gfp-anti-ouro2 58/59 F0 115 10 9 2 20 �0.05

gfp F0 19 6 3257–59 Line 8 (F1)

73 56 77 0 0 —Line 9 (F2)

The number of samples and phenotypes in the same experiment groups were combined (see Table S2 for details of each F0 experiment or line). F1 and F2 gfplines were generated as described in Fig. S6D. GFP-expressing animals (nG) were selected on day 1 from HS-treated tadpoles (nHS). Retained tails (nR) were countedon days 7–14 (stages 64–66). The same specimens of gfp lines 8 and 9 as in Table 1 were analyzed for tail retention. Note: the number of HS-tadpoles was reducedbecause some of tadpoles were killed for histochemical examination or died before being counted. P, one-way ANOVA, followed by Duncan’s multiple rangetest against gfp.

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T-Cell Proliferation Assay with His-Tagged Recombinant Proteins. cDNA frag-ments encoding AA197–520 of ouro1 and AA120–334 of ouro2 were cloned intothe pQE70 vector with a 6 His-tag at the C-terminal end. gfp was a gift fromQiagen GmbH. IPTG-induced recombinant proteins were obtained using the E.coli M15 strain, the QIA expressionist kit (Qiagen), a Ni-NTA column, dialyzedagainst PBS, and sterilized through a 0.22-�m filter after concentration in Cen-tricon columns (Millipore). The T-cell proliferation assay was performed as de-scribed (8, 9). Briefly, 5 107 leukocytes, including antigen presenting cells (APCs)from spleens of 1- to 2-year-old J strain adult frogs, were cocultured in 70% L15mediumwith22mm2 squarecut larval tailfins fromthesamestrainof tadpolesat stage 57 or with each His-tagged recombinant at a concentration of 35 �M in10% resin-treated FCS, from which hormones (including TH) had been excluded.Proliferation was evaluated by BrdU incorporation.

Northern Blot Analysis. Total RNA was extracted from the tail or trunk skinfrom one or five pooled J strain tadpoles at stages 48–66. The tail skin wascarefully separated from the muscle and notochord. The trunk skin wasexcised from the dorsal region of the trunk and head, including the forelimbskin. RNA (10 �g) was run on a 1% agarose formaldehyde gel, transferred ontoa membrane, and hybridized at 50 °C with 32P-labeled ouro1 cDNA probe(nucleotide nos. 1090–1497).

RT-PCR Analysis. Total RNA was extracted from the tail or trunk skin as describedabove. RNA (2 �g) was reverse-transcribed using random primers (Invitrogen).Specific primers were used for ouro1 (sense: 5�-TTT-GAT-AAC-ACG-CCC-AAA-CTG-G-3�, antisense 5�-CAT-CTT-CAC-TGC-CAA-GAG-GTC-3�) and ouro2 (sense:5�-GGC-ATT-TTC-TTT-GGG-GCG-TTC-TTT-GAC-TGC-3�, antisense: 5�-GCT-CTC-AGT-TTG-TTT-AAT-GCA-GTG-GTG-AGG-3�). PCR was performed for 22–32 cyclesas described for xlk, xak (22), and rpl8 (23). The amplified products were run on2% agarose gels and visualized by ethidium bromide.

Western Blot Analysis. Western blot analysis was performed essentially as de-scribed in ref. 11. Briefly, lysates obtained from J strain tadpole tail and trunkskins,or fromwholetissuesof transgenic tadpoles,wereelectrophoresedon10%SDS-polyacrylamide gel (50 �g total protein per lane). Immunoblots were per-formedwithratantiserumagainstsyntheticpeptidesforOuro1(10)orOuro2(seeFig. 1), mouse anti-GFP monoclonal antibody (Santa Cruz Biotechnology), rabbitanti-FLAG polyclonal antibodies (Sigma), and mouse anti-c-Myc monoclonal an-tibody (Roche Molecular Biochemicals). AP-conjugated rat, mouse, or rabbit IgGantibodies were used as secondary antibodies for color detection.

Whole Mount in Situ Hybridization. WISH was performed on albino (non-Jstrain) X. laevis tadpoles with digoxigenin-labeled probes as described in ref.24 with a few modifications. Antisense and sense probes were synthesized

from the CDS of ouro1 in the pBSIISK� plasmid vector (Stratagene) andhybridized at 60 °C for 25 h.

Immunohistochemistry on Sections. Nonfixed frozen skin tissues were embed-ded in OCT-Compound, and 4-�m sections were cut (11) and incubated withmouseanti-XenopusT-cellmonoclonalantibody(XT-1) (25),mouseanti-XenopusMHC class II monoclonal antibody (AM20) (26), or rabbit anti-GFP polyclonalantibody (Medical Biological Lab). Secondary antibody detection was performedusing Cy3 or Alexa488-conjugated mouse or rabbit IgG antibodies.

Construction of Plasmids for Transgenesis. The Xenopus HS promoter-drivenexpression plasmids pHsS1/EGFP (27) and pHsS1/DsRed were used as vectors. CDSfragmentsofouro1orouro2withFLAG-orMyc-tag, respectively,weresubclonedinto the BamHI and EcoRI sites of pHsS1/EGFP. pHsS1/EGFP was used as the gfpcontrol. Antisense constructs were made by inserting the ouro1 or ouro2 cDNA(the ouro1 cDNA including 31-bp 5�UTR and 76-bp 3�UTR or the ouro2 cDNAincluding 64-bp 5�UTR and 200-bp 3�UTR) in the reverse orientation.

Nuclear Transplantation Transgenic Technique. Transgenesis was performed asdescribed in ref. 15. Briefly, NotI-linearized plasmid was mixed with spermnuclei, and this mixture was then microinjected into unfertilized eggs. Trans-genic embryos were raised to feeding stage in 6 days in Steinberg’s solutionwith 50 �g/mL gentamicin.

HS Treatment and Screening Animals. The tip of tail was heat-shocked for 15 minat 37 °C three times, keeping them at room temperature in Steinberg’s solutionfor 15 min in between (see Fig. S5). To identify transgenic animals, expression ofGFP or DsRed in the HS area was analyzed by fluorescence microscopy on day 1after HS. Animals with high expression of GFP or DsRed were selected, andnegative-, weak-, moderate-, or mosaic-expressing animals were subsequentlyexcluded. The ouro1-gfp/ouro2-gfp DT tadpoles were confirmed by double-staining for FLAG- and Myc-tags using immunohistochemistry as describedabove. The selected animals were carefully reared individually in 200-mL glassbeakers with autoclaved Steinberg’s solution to avoid undesirable effects. Tailswere observed without anesthetizing the animals, using a fluorescent micro-scope. A picture of the tail was constructed from partially overlapped serialimages captured using a digital camera (at least 6 to 15 images for a tadpole tail).

ACKNOWLEDGMENTS. We thank M. Asashima and T. Michiue (University ofTokyo, Tokyo, Japan) for providing the transgenic vector, and Y. Yaoita(Hiroshima University, Higashi-Hiroshima, Japan) for the Xenopus cDNA li-brary. We also thank Yu-ichi G. Watanabe, K. Fujiwara, K. Yamazaki, M.Yashima, N. Sudou, and Y. Ito for technical assistance, K. Igarashi, T. Ohshima,and A. Sasaki for technical support producing transgenic tadpoles, M.Yamamoto, M. Ito, M. Ato, Y. Maeda, and M. Yamaguchi for comments, N.Funayama, I. Hamano, and Y. Okubo for their many kindnesses, and C. Katagiriand C. Hannah for critical reading.

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