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The regeneration of the amputated tail of Xenopus laevis larvae is an excellent model system for regeneration research. The wound left by the amputated tail is covered with epidermis within 24 h. Then, the cell number increases near the amputation plane at the notochord, spinal cord and muscle regions. An apparently complete tail with notochord, muscle and spinal cord is regenerated within two weeks. To reveal whether the molecular mechanism underlying the tail regeneration is the same as that in embryonic tail development, the gene expression patterns of the embryonic tail bud and the regenerating tail were compared by in situ hybridization and reverse transcription-polymerase chain reaction. Most genes analyzed were expressed at similar levels in both tissues, whereas two bone morphogenetic protein (BMP)-antagonists, chordin and noggin, were detected only in the embryonic tail bud. The regenerating tail also lacked expression of Xshh in the floor plate and expression of Xdelta-1 in the spinal cord and presomitic mesoderm. These results show that there are some differences in gene expression between the two processes. Furthermore, when the tail of Xenopus larvae is amputated, the regenerating tail has a gene expression pattern similar to the distal portion of the larval tail rather than the embryonic tail bud, suggesting that the cut larval tail does not make a new embryonic tail bud, but rather a new larval tail tip for regeneration.
Fig. 1. Morphological and immunological analysis of the tail regeneration. Transverse section of the normal tail of the stage 48larva was stained with hematoxylin and eosin (A). The amputated tail was observed with Nomarski optics at days 0 (B), 1 (C), 2 (D), and 3 (E) after amputation. Sagittal sections of the regenerating tail at days 1 (F), 2 (G), and 3 (H) after amputation, and frontal section at day 3 (I), were stained with hematoxylin and eosin. Muscle cells were detected on days 4 (J) and 5 (K) by staining with the monoclonal antibody 12/101, and neural cells on day 2 (L) and day 4 (M) with the monoclonal antibody Xen1. The amputation plane is indicated by two arrowheads. NC, notochord; SC, spinal cord; M, muscle mass; NS, notochord sheath; TV, terminal vesicle of the regenerating spinal cord. Bar, 50 μm.
Fig.2. Whole mount in situ hybridization of embryos and regenerating tails. Expression of the indicated genes was detected in stage 30 and stage 36 embryos and in regenerating tails on days 2 and 4 of regeneration. Arrow- heads indicate positive signals with relatively weak intensity. Magnification common to each stage. Bar, 100 μm.
Fig. 3. Reverse transcriptionâpolymerase chain reaction (RTâ PCR) analysis of gene expression in developing and regener- ating tails. Gene expression was analyzed by the RT-PCR method in regenerating tails on days 0, 2 and 4 of regeneration, in the tail buds of stage 30 embryos, and in the tail tip regions of stage 48 larvae. Specific primer pairs and PCR conditions are indicated in Table 1.
Fig. 4. In situ hybridization of sections. The expression of Xshh, Xbra-3, and Xwnt-5a was analyzed by in situ hybridization using sectioned samples. Transverse sections of stage 30 (A) and stage 42 (B) tail regions, and of the regenerating larval tail on day 4 (C), were stained with Xshh probe. Sagittal sections of stage 30 (D) and stage 36 (E) tail regions, and of the regenerating tail on day 2(F), were stained with Xbra-3 probe. Sagittal section of stage 30 (G) and stage 36 (H) tail regions and regenerating tail on day 4 (I) were stained with Xwnt-5a probe. Broken line indicates neural tube. Arrowheads in (A) and (B) show Xshh expression in the floor plate. Arrowheads in (D) and (E) show the posterior expression of Xbra-3. Bars, 20 μm (C), 50 μm (A,B,H,F), 100 μm (D,G,E,I).
