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Amputation of the larval tail of Xenopus injures the notochord, spinal cord, muscle masses, mesenchyme, and epidermis, induces the growth and differentiation of cells in those tissues, and results in tail regeneration. A dorsal incision in the larval tail injures the same tissues and induces cell growth and differentiation, but never results in the formation of any extra appendages. The first sign of tail regeneration is the multilayered wound epidermis and Xwnt-5a expression in the distal region, neither of which is observed in the recovering region after a dorsal incision. To evaluate the role of Xwnt-5a in tail regeneration, Xwnt-5a was overexpressed in the recovering region. When an animal cap injected with Xwnt-5a mRNA was grafted into the dorsal incision, an ectopic protrusion was formed. Morphological and molecular analyses revealed that the protrusion was an ectopic larval tail, which was equivalent to the regenerating tail but different from the tail that develops from the embryonic tail bud. Lineage labeling revealed that the major differentiated structures of the ectopic tail were formed from host cells, suggesting that Xwnt-5a induced host cells to make a complete tail. The ectopic tail was not induced by Xwnt-8 or Xwnt-11, demonstrating the specificity of Xwnt-5a in this process. A pharmacological study showed that JNK signaling is required in tail regeneration. These results support the proposition that Xwnt-5a plays an instructive role in larval tail regeneration via Wnt/JNK signaling.
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18977433
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Fig. 3. Ectopic Xwnt-5a induces an ectopic tail. (A) Schematic procedure for the grafting experiment. The larval tail was grafted with an animal cap expressing Xwnt-5a. (B and C) show the same larva at different times after the operation. The fluorescent images showing the grafted cells were merged. (D and E) show the same larva grafted with a control animal cap. Recovery following the incision proceeded slowly because the animal-cap-grafted larva was maintained at low temperature (see Section 4). The well-elongated (F) and curly (H) protrusions induced by Xwnt-5a were transversally sectioned and stained with hematoxylin and eosin (G and I). The section level is indicated by the red bar in (F and H). Arrowheads in (G and I) indicate the spinal cord. (J) Muscle cells were stained in the ectopic protrusion using the monoclonal antibody 12/101 (brown). (K) An ectopic protrusion was sectioned and stained with anti-fluorescein antibody to detect the donor cells (blue). (L) An ectopic tail formed after an animal cap injected with Xwnt-5a and tetramethylrhodamine (red) was grafted onto a transgenic larva expressing EGFP. Arrowheads in (L) indicate the notochord labeled with EGFP. (M) Whole-mount in situ hybridization of the ectopic protrusion using a 5′ UTR probe to detect endogenous Xwnt-5a expression. Arrowheads in (M) indicate positive signals. (N–Q) Whole-mount (N, P and Q) and section (O) in situ hybridization for Xes1 expression. Xes1 was expressed in the wounded epidermis after amputation (N and O). Its expression at a higher level was confined to the distalepidermis (arrowheads in O). A high level of Xes1 expression was observed in the incised region grafted with the Xwnt-5a-injected animal cap (Q) but not in the control (P). The animal cap grafted within the incision was removed during the hybridization procedure. sc, spinal cord; nc, notochord; me, mesenchyme. (B–F, J, L–Q) lateral view; (H) dorsal view, anterior to the left. Scale bar in (B–F, H, J and M) 500 μm; (G, I and K) 50 μm; (L, N, P and Q) 250 μm; (O) 100 μm.
Fig. 5. Effect of JNK inhibitor on tail regeneration. (A–H) Tail-amputated tadpole was incubated in water containing 8 μM JNK inhibitor SP600125 (B, D, F and H) or in control water containing DMSO (A, C, E and G). (E and F) Sections stained with hematoxylin and eosin. (G and H) Whole-mount in situ hybridization for Xes1. Arrowheads in (A–D) show amputation planes. Arrow in (E) indicates notochord precursor cells. White arrowhead in (G) indicates positive signal for Xes1 expression. All specimens are lateral view, anterior to the left. sc, spinal cord; nc, notochord. Scale bars in (A–D, G and H) 500 μm; (E and F) 50 μm. (I) The mean length of the regenerated tails was determined on day 5 and is indicated with the standard deviation. *P value determined with Student’s t test is less than 0.000001. (J) RNA was isolated from the amputated tail on day 0, and from the tail amputated and treated with SP600125 (8 μM) or DMSO for two days, and used for RT-PCR analysis.
