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Frog metamorphosis induced by thyroid hormone (TH) involves not only cell proliferation and differentiation in reconstituted organs such as limbs, but also apoptotic cell death in degenerated organs such as tails. However, the molecular mechanisms directing the TH-dependent cell fate determination remain unclear. We have previously identified from newts an RNA-binding protein (nRBP) acting as the regulator governing survival and death in germ cells during spermatogenesis. To investigate the molecular events leading the tail resorption during metamorphosis, we analyzed the expression, the functional role in apoptosis, and the regulation of xCIRP2, a frog homolog of nRBP, in tails of Xenopus laevis tadpoles. At the prometamorphic stage, xCIRP2 protein is expressed in fibroblast, epidermal, nerve, and muscular cells and localized in their cytoplasm. When spontaneous metamorphosis progressed, the level of xCIRP2 mRNA remained unchanged but the amount of the protein decreased. In organ cultures of tails at the prometamorphic stage, xCIRP2 protein decreased before their lengths shortened during TH-dependent metamorphosis. The inhibition of calpain or proteasome attenuated the TH-induced decrease of xCIRP2 protein in tails, impairing their regression. These results suggest that xCIRP2 protein is downregulated through calpain- and proteasome-mediated proteolysis in response to TH at the onset of metamorphosis, inducing apoptosis in tails and thereby degenerating them.
Fig. 1.
Spatial expression of xCIRP2 protein in tadpole tails. Crude protein extracts and cross sections of 5-μm-thickness were prepared from tails of stage 56 and analyzed by Western blotting (A) and immunohistochemistry (B–I) for xCIRP2, respectively. Four independent experiments were conducted with three different tails. Since similar results were found, representations of them are shown. (A) β-Actin was used as the loading control; oocytes were used as a positive control for xCIRP2 expression. (B–I) The same sections were incubated with a polyclonal rabbit antibody to xCIRP2 (B, E, and H) and counterstained with PI (C, F, and I); the serial sections were incubated with normal rabbit immunoglobulins (NRI) (D and G); scale bar, 50 μm. (H and I) Enlarged figures of some cells in the same section; scale bar, 10 μm. e, Epidermis; g, ganglia; m, muscle fibers; s, spinal cord; sef, subepithelial fibroblasts.
Fig. 2.
Temporal expression of xCIRP2 mRNA and protein in tadpole tails during spontaneous metamorphosis. Tails were taken from tadpoles at stage 56, 59, and 61 and divided into two fragments of 10–15 mm in length (parts 1 and 2) (A). The fragments were then separately analyzed by RT-PCR (B) and Western blotting (C and D) for xCIRP2. Four independent experiments were conducted with three different tails for each stage. Since similar results were found, representations of them are shown. (A) A scale bar indicates 10 mm. (B) Total RNA extracted was subjected to the reverse transcription reaction without (−) or with (+) reverse transcriptase (RT); EF-1α was used as the internal control; oocytes were used as a positive control for xCIRP2 expression. (C) β-Actin was used as the loading control. (D) Results are presented as a percentage (means ± S.E.M.) of the xCIRP2 protein expression in tails of tadpoles at stage 59 and 61, relative to that in tails at stage 56, from four independent experiments (C); N.D., not detected; ∗∗p < 0.01.
Fig. 3.
Temporal expression of xCIRP2 mRNA and protein in tadpole tails during T3-induced metamorphosis. Tails derived from tadpoles of stage 56 were cultured for the indicated days without (0, control) or with the indicated doses of T3. The tails were then analyzed by RT-PCR (A) and Western blotting (B and C) for xCIRP2 and by measurement of their lengths (D). Four independent experiments were conducted with three different tails for each T3 dose in each culture day. Since similar results were found, representations of them are shown. (A) EF-1α was used as the internal control; oocytes were used as a positive control for xCIRP2 expression. (B) β-Actin was used as the loading control. (C) Results are presented as a percentage (means ± S.E.M.) of the xCIRP2 protein expression in tails cultured under each condition, relative to the control cultured for 1 day, from four independent experiments (B); ∗∗p < 0.01. (D) Results are presented as a percentage (means ± S.E.M.) of the lengths of tails cultured under each condition, relative to the control cultured for 1 day without T3, from four independent experiments; ∗p < 0.05.
Fig. 4.
Dose–responses of the xCIRP2 protein expression in tails and their lengths to T3. Tails derived from tadpoles of stage 56 were cultured for 2 days without (0, control) or with the indicated doses of T3. The tails were then analyzed by Western blotting for xCIRP2 (A and B) and by measurement of their lengths (C). Four independent experiments were conducted with three different tails for each T3 dose. Since similar results were found, representations of them are shown. (A) β-Actin was used as the loading control; oocytes were used as a positive control for xCIRP2 expression. (B) Results are presented as a percentage (means ± S.E.M.) of the xCIRP2 protein expression in tails cultured with each dose of T3, relative to the control, from four independent experiments (A); ∗∗p < 0.01. (C) Results are presented as a percentage (means ± S.E.M.) of the lengths of tails cultured with each dose of T3, relative to the control, from four independent experiments; ∗p < 0.05.
Fig. 5.
Dependency of decreased xCIRP2 protein levels and degenerated lengths in tails on T3. Tails derived from tadpoles of stage 56 were cultured for 2 days without (−) or with (+) 10−7 M T3 in the absence (0) or presence of the indicated doses of TBBPA. The tails were then analyzed by Western blotting for xCIRP2 (A and B) and by measurement of their lengths (C). Four independent experiments were conducted with three different tails for each T3 dose. Since similar results were found, representations of them are shown. (A) β-Actin was used as the loading control; oocytes were used as a positive control for xCIRP2 expression. (B) Results are presented as a percentage (means ± S.E.M.) of the xCIRP2 protein expression in tails cultured under each condition, relative to those cultured without T3 in the absence of TBBPA (control), from four independent experiments (A); ∗∗p < 0.01. (C) Results are presented as a percentage (means ± S.E.M.) of the lengths of tails cultured under each condition, relative to those of tails cultured without T3 in the absence of TBBPA (control), from four independent experiments; ∗p < 0.05.
Fig. 6.
Contribution of proteolysis systems to the xCIRP2 downregulation and the tail degeneration in tails. Tails derived from tadpoles of stage 56 were cultured for 2 days without (−) or with (+) 10−7 M T3 in the absence (−) or presence (+) of Z-VAD-fmk (A–C), MG132 (MG), or calpeptin (Calp) (D–F). The tails were then analyzed by Western blotting for xCIRP2 (A, B, D, and E) and by measurement of their lengths (C and F). Four independent experiments were conducted with three different tails for each treatment. Since similar results were found, representations of them are shown. (A and D) β-Actin was used as the loading control; oocytes were used as a positive control for xCIRP2 expression. (B and E) Results are presented as a percentage (means ± S.E.M.) of the xCIRP2 protein expression in tails cultured under each condition, relative to those cultured without T3 in the absence of inhibitors (control), from four independent experiments; ∗∗p < 0.01. (C and F) Results are presented as a percentage (means ± S.E.M.) of the lengths of tails cultured under each condition, relative to those of tails cultured without T3 in the absence of inhibitors (control), from four independent experiments; ∗p < 0.05.
