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Fig. 1. Expression analysis of genes identified by differential display during the refractory and post-refractory periods. (A-C) cathelicidin-like (A), CXCR2 (B) and RIN3 (C)expression analysis. The relative amounts of transcripts determined by qRT-PCR were obtained by taking the value at 0 hpa as 1 for each refractory period(open circle) and post-refractory regeneration period (solid circle) after normalization relative to elongation factor 1α(EF1α) transcipt levels. (D-G) In situ hybridization for cathelicidin-like (D,E) and CXCR2 (F,G) using the sagittal sections of wound stumps during the post-refractory regeneration period 10 hpa. (E,G) Magnified views of the boxed area in D and F, respectively. Leukocyte-like cells expressing the genes are indicated with arrowheads. ms,muscle; nc, notochord; sc, spinal cord; we, wound epidermis. Scale bars: 300μm in D,F; 30 μm in E,G.
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Fig. 2. Expression profiles of immune-related genes in wound stumps differ between the refractory and pre-/post-refractory regeneration periods.(A-I) CXCLb (A), CXCLe (B), CCLf (C), CXCLh (D), CCLb (E), CCL5L1 (F), CCL5L2(G), MHC IIa (H) and FOXP3 (I) expression analysis. The relative amounts of transcripts determined by qRT-PCR were obtained by taking the value at 0 hpa as 1 for each of the pre-refractory (broken line and open triangle), refractory (gray line and open circle) and post-refractory regeneration (black line and solid circle) periods. Transcript levels were first normalized relative to EF-1α transcript levels.
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Fig. 3. Immunosuppressant treatment or injection of antisense MOs for PU.1 restores the regenerative ability during the refractory period. (A-D) Vehicle control (A,C) and Celastrol-treated tadpoles(B,D) during the refractory period, observed 7 dpa. Arrowheads indicate the amputation plane. (E) Coronal section of a regenerated tail of a Celastrol-treated tadpole. df, dorsal fin; me, mesenchyme; my, myotome; nc,notochord; sc, spinal cord; vf, ventral fin. Scale bar: 50 μm. (F)Frequency of regenerative ability (see key) of control and immunosuppressant-treated tadpoles (n=24 in each experiment). The numbers of tadpoles classified in each experiment are presented in Table S1 in the supplementary material. *P<0.003 usingχ 2 test (the number of dead tadpoles was excluded). (G)A noninjected tadpole with no apparent regeneration. (H) A PU.1-MO1-injected tadpole with a completely regenerated tail. (I) A 5mis PU.1-MO1-injected tadpole with no apparent regeneration.(J,K) Frequency of regenerative ability of noninjected,PU.1-MO1-injected and 5mis PU.1-MO1-injected (J), and PU.1-MO2-injected and 5mis PU.1-MO2-injected (K) tadpoles. The number of tadpoles classified in each experiment is presented in Table S2 in the supplementary material. *P<0.05, †P<1×10-12 usingχ 2 tests against experimental (PU.1-MO1- or PU.1-MO2-injected)tadpoles. (L,M) The relative amounts of CD45transcripts determined by qRT-PCR were obtained by taking the value of the noninjected group as 100%. Transcript levels were first normalized relative to EF-1α transcript levels. *P<0.01,Student's t-test.
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Fig. 4. The decrease in regenerative ability correlates with the T cell population development. (A) Frequency of regenerative ability (see key) of 4 to 9 dpf tadpoles (n=25 in each experiment). The numbers of tadpoles classified in each experiment are presented in Table S3 in the supplementary material. (B-D) TCRα (B), TCRγ (C) and CD3γ /δ (D)expression analysis. The relative amounts of transcripts obtained by qRT-PCR were determined by taking the value at 4 dpf as 1, after normalization with ornithine decarboxylase 1.
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Supplemental Figure S1
Fig. S1. The wound epidermis completely covers the amputated tail plane in most of the tadpoles within 8 to 12 hours post amputation. (A) Amputated tadpole tail at 0 hours post amputation (hpa) during the post-refractory regeneration period. The amputation plane was rough, as no wound epidermis formed. (B) Amputated tail at 12 hpa. The amputation plane was covered by wound epidermis (arrow). (C) Follow-up observation of covering of wound stump by the wound epidermis. Wound stumps were observed every 2 hours after amputation and the number of tadpoles with complete wound epidermis coverage was counted.
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Fig. S2. Proliferating cells appear in the tail wound stump within 18 to 24 hpa. (A,B) Immunostaining using anti-proliferating cell nuclear antigen antibody (purple) and counterstaining of the nuclei (green) of tadpole tails 6 (A) and 24 (B) hpa during the post-refractory regeneration period. Proliferating cells (white) thought to form the blastema appeared near the amputation plane (circled with dashed line). (C) Number of tadpoles in which proliferating cells were observed.
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Fig. S3. FK506 antagonizes elevation of immune-related gene expression in amputated Xenopus tadpole tails during the refractory period. (A-E) We examined the effect of FK506 on the expression of five genes that were upregulated during the refractory period: CXCLh, CCLb, CCL5L1, CCL5L2 and MHC class II genes (Fig. 2D-H). In all cases, FK506 treatment decreased the expression of these genes: in particular, CCLb expression was significantly repressed at 48 hpa. (F) Furthermore, we cloned a X. laevis homolog of FASL (GenBank: AB435242), a major effector molecule that is utilized by natural killer cells and activated T cells to destroy target cells in mammals, and found that its expression was also significantly downregulated in FK506-treated groups. The relative amounts of transcripts obtained by qRT-PCR were determined by taking the value at 0 hpa as 1, after normalization using the EF-1α transcripts. Open square and dashed line, dimethyl sulfoxide-treated control groups; solid square and black line, FK506-treated groups. *P<0.05, **P<0.001 (Student�s t-test).
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Fig. S4. MO injection has negative effects on regenerative ability. In our MO experiments, MO injection itself seemed to have some negative effects on regenerative ability. For example, in this experiment (experiment 2 using PU.1-MO2), in which the experimental condition was the same as experiment 1 using PU.1-MO2, as presented in Fig. 3K, tadpoles injected with PU.1-MO2 showed significantly higher regenerative ability than tadpoles injected with 5mis PU.1-MO2 (P<0.001, χ2 test). Noninjected tadpoles, however, also showed significantly higher regenerative ability than tadpoles injected with 5mis PU.1-MO2 (P<0.001). The number of tadpoles classified in this experiment is presented in Table S2 in the supplementary material. A similar phenomenon was also observed in experiments 1 and 2 using PU.1-MO1 (Fig. 3J) and experiment 1 using PU.1-MO2 (Fig. 3K), although the extent varied among experiments. In this experiment (experiment 2 using PU.1-MO2), the regenerative ability of PU.1-MO2-injected and noninjected tadpoles was not significantly different (P>0.05), possibly due to a counteraction of the positive effect of PU.1-MO2 by a depletion of normal leukocytes and nonspecific negative effects of the MO itself. The negative effect of the MO injection on regenerative ability was not related to the inhibition of PU.1 because CD45 expression differed significantly between PU.1-MO1/MO2-injected groups and noninjected groups, whereas there was no significant difference between 5mis PU.1-MO1/MO2-injected and noninjected groups (Fig. 3M,L).
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Fig. S5. Thematic model of the developmental stage-dependent regenerative ability in Xenopus tadpole tails.
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