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Fig. 1. Gene expression profiles during Xenopus tadpole tail regeneration. (A–X) In situ hybridization detection of fgf8 (A–D), fgf9 (E–H), fgf10 (I–L), fgf20 (M–P), wnt3a (Q–T) and wnt5a (U–X) in control tadpoles (A, E, I, M, Q, U; stage 42–43 tadpoles) and in tail regenerates 1–3 days post amputation (1 dpa: B, F, J, N, R, V; 2 dpa: C, G, K, O, S, W; 3 dpa: D, H, L, P, T, X; stage 48+ tadpoles). Arrowheads indicate amputation levels. Scale bars: 200 μm. dpa: day post amputation. (Y) RT-PCR detection of FGF, BMP and Wnt signalling components in tadpole tail immediately after amputation (D0) and 1–4 days post amputation (D1–D4). 1 mm of stump tissue was used to prepare RNA. ODC (ornithine decarboxylase) is used as loading control. (Z) Relative expression levels of genes detected by RT-PCR, based on the density of the PCR bands in (Y). The band density on D0 is the reference standard (1 arbitrary unit) in each gene group. Note that an increased in situ signal may correspond to a limited overall change in the larger tissue region used for RNA analysis.
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Fig. 4. Immunohistochemistry of axial structures in XFD transgenic and BIO treated tail regenerates. (A) The tail of this XFD transgenic tadpole failed to regenerate after daily heat shock treatment, 7 days post amputation. (B) XFD transgenic tadpole treated with BIO shows outgrowth of tail after amputation, 7 days post amputation. (C–N) Detection of notochord (C, F, I, L; with MZ15 antibody), spinal cord (D, G, J, M; with 2G9 antibody) and muscle (E, H, K, N; with 12/101 antibody) in heat shocked XFD transgenic tadpoles (C–E), wild type tadpoles treated with BIO (F–H), heat shocked XFD transgenic tadpoles treated with BIO (I–K) and wild type tadpoles (L–N). White and black arrowheads indicate amputation levels. Scale bars: 200 μm. For the experiments shown in panels I–N, tadpole stage was 42–43.
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Fig. 6. Gene expression detection in dkk1 heat shock and small molecule treated tadpole tails. (A–F) In situ hybridization detection of fgf10 (A, D), fgf20 (B, E), msx1(C, F) in tadpoles treated with DMSO alone (A–C) or 50 nM BIO (D–F). (G–L) In situ hybridization detection of fgf8 (G), fgf 9 (H), fgf10 (I), fgf20 (J), msx1 (K), msx2 (inset in panel K) and Bmp4 (L) in dkk1 transgenic tadpole tails with heat shock treatment, 3 days post amputation. (M–O) In situ hybridization detection of wnt3a (M), wnt5a (N) and Bmp4 (O) in wild type tadpole tails treated with 100 μM SU5402, 3 days post amputation. Arrowheads indicate amputation levels. Scale bar: 500 μm.
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Fig. 2. Tail regeneration in tadpoles with FGF signaling inhibited. (A, C) Inhibition of FGF signaling with SU5402 blocks tail regeneration. (A) A tail regenerate of a tadpole treated with DMSO, for 6 days after amputation. (C) A tail of a tadpole treated with 100 μM SU5402, for 6 days after amputation. (B, D) HE staining of para- sagittal sections of 3 dpa DMSO treated tadpole tail (regenerating, B) and 100 μM SU5402 treated tadpole tail (non-regenerating, D). In panel D, the notochord and neural ampulla are not elongated and there is much less blastema tissue present. (E, F) Tail regeneration in XFD transgenic tadpoles without (E) or with heat shock treatment (bright field image overlay with fluorescent image, green arrows indicate green lens). Heat shock was given 3 h before tail amputation and then every 24 h after amputation. White arrowheads indicate amputation levels in panels A, C, E and F. dpa: day post amputation. Scale bars: 200 μm in panels A and 1 mm in panels E, F.
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Fig. 5. Tail regeneration in dkk1+fgf20 double transgenic tadpoles. (A) A tadpole with both dkk1 transgene as indicated by the green lens (short white arrow) and fgf20 transgene as indicated by the green pancreas (long white arrow). Transgenic constructs used are shown in inset. (B) Tail regeneration in wild type tadpoles after 7 days' heat shock treatment (23 of 23 in total). (C) Tail regeneration in dkk1 transgenic tadpoles with heat shock treatment for 7 days. Most dkk1 transgenic tadpoles failed to regenerate (23 of 28), some regenerated partially (5 of 28, similar as shown in panel F). (D) Tail regeneration in dkk1 and fgf20 double transgenic tadpoles, with heat shock treatment for 7 days. (D) Full tail regeneration (18 of 52). (E) Partial tail regeneration (18 of 52). (F) Partial tail regeneration (8 of 52). Some dkk1 and fgf20 double transgenic tadpoles failed to regenerate (8 of 52, similar as in panel C). (G) Representative cross sections of tail regenerates in wild type tadpoles (G) and fully regenerated (H) or partially regenerated (I) tail in dkk1 and fgf20 double transgenic tadpoles. Cross sections were immunostained with 12/101 mAb and conterstained with Haematoxylin. N: notochord, s.c.: spinal cord. White arrowheads in panels B indicate amputation levels. Scale bars in panels A: 250 μm; scale bars in panels G: 100 μm.
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Fig. 3. Cell proliferation detection in beta-catenin transgenic tadpoles and tail regeneration in dkk1 transgenic and BIO treated tadpoles. (A, B) Cell proliferation detection with antibody against PCNA in tail regenerate of beta-catenin transgenic tadpoles with (B) or without (A) heat shock induction. Cross sections were prepared from tail regenerates 4 days post amputation. N: notochord; sc: spinal cord. (C) Tail regeneration in a wild type (siblings from dkk1 transgenic) tadpole with heat shock treatment, 7 days post amputation. (D) Tail regeneration in a dkk1 transgenic tadpole with heat shock treatment, 7 days post amputation. Heat shock was given 3 h before tail amputation and then every 24 h after amputation. Arrowheads in panels C, D indicate amputation levels. hs: heat shock. (E) Tail regeneration in tadpoles treated with BIO or DMSO, 4 days post amputation. White arrows indicate regenerating tail. (F) Quantification of PCNA positive cells in tail regenerates of β-catenin transgenic tadpoles, based on cell counting on cross sections of 3 individuals for each group. (G) Quantification of tail regeneration rate in DMSO or BIO treated tadpoles. Tail restoration rate is measured as described in materials and methods section. Scale bars in panels A, B: 50 μm; scale bars in panels C: 500 μm.
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Fig. 7. Expression of wnts and fgfs in wild type and in noggin transgenic tadpole tail regenerates. (A, C, E, G) Tail regenerates of noggin transgenic tadpoles with in situ hybridization detection of fgf10 (A), fgf20 (C), wnt3a (E) and wnt5a (G), 3 days post amputation, without heat shock treatment. (B, D, F, H) with heat shock treatment 3 h before amputation and every 24 h after amputation. Arrowheads indicate amputation levels. Scale bar: 500 μm.
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