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Figure 1.
Real-time RT–PCR of xNP1-transcripts in tail tissues of the ‘refractory’ and ‘post-refractory regeneration’ period tadpoles on days 0, 3, and 5 after amputation. Real time RT–PCR was performed using total RNA extracted from the tail stumps (2-mm wide tail tissues) of the (a) ‘refractory’ and (b) ‘post-refractory regeneration’ period tadpoles on days 0, 3, and 5 after amputation. As a control, we used total RNA extracted from tissues of the same tail parts: for example, 2-mm wide tissues at the distal one-third of the intact tails from tadpoles at the same developmental stages, on days 3 and 5 after the tails of the experimental group were amputated. The shaded and open bars indicate amputated tail stumps and intact tails, respectively. The numbers of individuals used for the experiments were: 16–24 × 3–4 lots and 2–5 × 2–4 lots for ‘refractory’ and ‘post-refractory regeneration’ periods, respectively. The relative amounts of xNP1 transcripts determined by real-time RT–PCR were obtained by taking the value at day 0 as 1 for each (a) ‘refractory’ period and (b) ‘post-refractory regeneration’ period after normalization relative to elongation factor 1α (EF1α) transcript levels. The bars indicate standard errors. Asterisks indicate statistically significant differences: P < 0.05 using the Tukey-Kramer method. image, Intact tails; image, Amputated tail stumps.
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Figure 2.
Real-time RT–PCR of xNP1-transcripts in tail tissues of the ‘pre-refractory regeneration’, ‘refractory’, and ‘post-refractory regeneration’ period tadpoles within 48 h after amputation. Real time RT–PCR was performed using total RNA extracted from the tail stumps (2-mm wide tail tissues) of the (a) ‘pre-refractory regeneration’, (b) ‘refractory’, and (c) ‘post-refractory regeneration’ period tadpoles at 0, 5, 10, 15, 24 and 48 h after amputation. The numbers of individuals used for experiments were: 15–16 × 4 lots, 20 × 4 lots, and 5 × 4 lots for ‘pre-refractory regeneration’, ‘refractory’, and ‘post-refractory regeneration’ periods, respectively. The relative amounts of xNP1 transcripts determined by real-time RT–PCR were obtained by taking the value at 0 h after amputation as 1 for each (a) ‘pre-refractory regeneration’ period, (b) ‘refractory’ period and (c) ‘post-refractory regeneration’ period after normalization relative to elongation factor 1α (EF1α) transcript levels. The bars indicate standard errors. Single and double asterisks indicate statistically significant differences: P < 0.05 and P < 0.01 using the Tukey–Kramer method.
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Figure 3.
In situ hybridization of xNP1-transcripts in regenerating tails of the ‘post-refractory regeneration’ period tadpoles within 10 days after amputation. Whole mount in situ hybridization was performed using the regenerating tails of ‘post-refractory regeneration’ period tadpoles on (a and g) day 0, (b and h) days 3, (c and i) days 5, (d, j, f and l) days 7, and (e and k) days 10 after amputation, with (a–e) antisense or (g–k) sense DIG-labeled xNP1 probe. Arrowheads in (b–e) indicate signals. Yellow dash lines in (b–e) indicate amputation plane. As positive and negative controls for hybridization, the regenerating tadpole tails on day 7 after amputation were hybridized with (f) antisense or (l) sense DIG-labeled EF1α probe. xNP1 expression in the regenerating tail on day 5 after amputation was also examined by section in situ hybridization using (m–o) antisense or (p) sense DIG-labeled xNP1 probes. (n and o) are magnified views of boxed areas in (m). We also performed in situ hybridization on 10, 24 and 48 h after amputation (q–y). (r and s), (u and v), (x and y) are magnified views of boxed areas in (q, t and w), respectively. The orientation of the specimen is; left, anterior; right, posterior; top, dorsal; and bottom, ventral. The bars in (m, p, q, t and w) indicate 0.5 mm. Arrowheads in (n, o, r, s, u, v, x and y) indicate signals. ep, epidermis; me, mesenchyme; nc, notochord; sc, spinal cord.
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Figure 4.
In situ hybridization of xNP1 transcripts in wound tail stumps of the ‘refractory’ period tadpoles after amputation. Whole mount in situ hybridization was performed using amputated tail stumps of the ‘refractory’ period tadpoles on (a and d) day 0, (b and e) days 3, and (c and f) days 11 after amputation, with (a–c) antisense or (d–f) sense DIG-labeled xNP1 probe. xNP1 expression in the amputated tail stumps on 3 days after amputation was also examined by section in situ hybridization using (h) antisense or (i) sense DIG-labeled xNP1 probes. We also performed in situ hybridization on 10, 24 and 48 h after amputation (i–q). (j and k), (m and n), (p and q) are magnified views of boxed areas in (i, l and o), respectively. The bars indicate (a–f) 1 mm and (g, h, i, l and o) 0.1 mm, respectively. The orientation of the specimen in each panel was the same as in Figure 3. Arrowheads in each panel indicate signals. ep, epidermis; me, mesenchyme; nc, notochord; sc, spinal cord.
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Figure 5.
Immunoblotting of xNP1 in the regenerating tails of the ‘post-refractory regeneration’ period tadpoles. Immunoblotting was performed using homogenates of regenerating tails of the ‘post-refractory regeneration’ period tadpoles on days 0, 1, 2, 3, 8, 20, and 23 after amputation, and the anti-xNP1 antibody. The positions of bands for xNP1 are indicated with an arrowhead. The numbers at the right of panels indicate positions of molecular weight markers in kDa.
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Figure 6.
Expression analysis by immunostaining of xNP1 in the amputated tail stumps and regenerating tails of the ‘refractory’ and ‘post-refractory regeneration’ period tadpoles. Immunostaining was performed using amputated tail stumps of the refractory’ period tadpoles on (a) day 0 and (b and c) days 2 after amputation, and regenerating tails of the ‘post-refractory regeneration’ period tadpoles on (d) day 0 and (e–g) days 3 after amputation, with the anti-xNP1 antibody (a, b, d–f) or the same concentration of normal IgG as negative controls (c and g), respectively. The yellow lines indicate outlines of the sampled tails. Panel (f) indicates a magnified view of the boxed area in (e). The arrowhead in panel (f) indicates regenerating notochord. We also performed immunostaining on sectioned tails of (h–j) the ‘refractory’ period and (k–m) the ‘post-refractory regeneration’ period tadpoles at 10, 24 and 48 h after amputation. (h–m) Upper left, immunofluorescence images of xNP1 staining; Upper right, merged images of xNP1 staining (red) and Hoechst 33342 staining (blue). the position of the tissues is indicated with colored and dashed lines. sc (light blue), spinal cord; nc (yellow), notochord; ms (gray), mesenchyme; ep (green), epidermis; Lower left, magnified view of xNP1 staining of the boxed area in upper right; Lower right, magnified view of merged images of the boxed area. Arrowheads in (h–m) indicate signals. The bars indicate (a–c) 1 mm, (d, e and g) 0.5 mm, and (h–m) 0.2 mm, respectively.
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Figure 7.
Expression analysis by in situ hybridization of xNP1 in tailbud embryos. In situ hybridization was performed using tailbud embryos (stage 33/34) with (a, c–e) antisense or (b and f) sense DIG-labeled xNP1 probe. (c and d) are magnified views of the head and tail regions of (a). In situ hybridization using transverse sections are also performed using (e) antisense and (f) sense xNP1 probes. Approximate position of the sections is indicated by dashed lines in (a and b), respectively. The arrowhead in panel (e) indicates the signal at spinal cord. The bars in (e and f) indicate 50 μm. nc, notochord; op, optic vesicle; ot, otic vesicle; sc, spinal cord; so, somite; tg, trigeminal placode.
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