DEV033985 Supplementary Material

Files in this Data Supplement:

• Supplemental Tables S1 - Adobe PDF
• Supplemental Tables S2 - Adobe PDF
• Supplemental Tables S3 - Adobe PDF
• 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.

• Supplemental Figure S2 -

  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.

• Supplemental Figure S3 -

  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).

• Supplemental Figure S4 -

  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).

• Supplemental Figure S5 -

  Fig. S5. Thematic model of the developmental stage-dependent regenerative ability in Xenopus tadpole tails.