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Xenopus laevis larvae can regenerate an exact replica of the missing part of a limb after amputation at an early limb bud stage. However, this regenerative capacity gradually decreases during metamorphosis, and a froglet is only able to regenerate hypomorphic cartilage, resulting in a spike-like structure (spike). It has been reported that the spike has tissue deformities, e.g., a muscleless structure. However, our previous study demonstrated that the muscleless feature of the spike can be improved. The existence of other kinds of tissue, such as tendon, has not been clarified. In this study, we focused on the tendon and dermis, and we isolated the scleraxis and dermo-1 genes, which are known to be marker genes for the tendon and dermis, respectively. The expressions of these genes were investigated in both the developmental and regenerating processes of a Xenopus limb. Although muscle was needed to maintain scleraxis expression, scleraxis transcription was detectable in the muscleless spike. Additionally, although grafting of matured skin, including dermal tissue, inhibited limb regeneration, the expression of dermo-1, a dermal marker gene, was detected from the early stage of the froglet blastema. These results indicate that tendon precursor cells and dermal cells exist in the regenerating froglet blastema. Our results support the idea that spike formation in postmetamorphic Xenopus limbs is epimorphic regeneration.
Figure 2. A–D: Expression of scleraxis in the developing Xenopus limb bud. A: Stage 51. A': Higher magnification of A. Scleraxis transcriptions were observed in the subectodermal region. B:The scleraxis expression domain became restricted along the developing muscle at stage 52. C: A more restricted pattern of scleraxis expression was observed at stage 53. In the proximal subectodermal region, scleraxis transcription was down-regulated. D: Visualization of muscle and scleraxis by immunostaining and in situ hybridization. Differentiating muscles were visualized by MF20 antibody (Brown). The scleraxis expression domain was restricted to the muscle extremity (inset). Arrows indicated scleraxis expression in the putative ligament. E–H: Expression of dermo-1 in the developing Xenopus limb bud. E: In the early developing Xenopus limb bud, the expression pattern of dermo-1 was similar to that of scleraxis (compare with A). E': Higher magnification of E shows dermo-1 transcription in the subectodermal region that was gradually down-regulated toward the medial region (arrow). F,G: In stages 52 and 53, subectodermal cells dominantly expressed dermo-1. The dermo-1 expression pattern in the proximal subectodermal region was more restricted (G' is compared with E', arrow). H: Stage 54. dermo-1 transcription was observed not only in the subectodermal region but also in some other regions. Scale bar = 500 μm.
Figure 3. Expressions of Scleraxis and dermo-1 in the regenerating tadpoleblastema. A: Early-stage blastema. A': Higher magnification of A. B: In the middle stage, scleraxis expression was observed in the blastema around the amputation site, including the subectodermal region. B': Higher magnification of B. C: Visualization of muscle and scleraxis expression by immunostaining and in situ hybridization. Differentiating muscles were visualized by MF20 antibody (Brown). The boxed region in C is shown in C'. The scleraxis expression domain was restricted to the muscle extremity in the regenerating limb bud. The expression pattern had recovered to a nearly normal pattern. D:Dermo-1 expression pattern in the early-stage blastema. D': At higher magnification, dermo-1 expression was observed in the early-stage blastema. Arrowheads indicate regions of expression in the blastema. E: Middle stage. Dermo-1 transcription was detected in the proximal-subectodermal region. Weaker expression was detected around the amputation site. Conspicuous dermo-1 expression in the proximal-subectodermal region was not overlapped with scleraxis expression (compare B' with E'). F: Late stage. Arrows in B' and E' indicate almost identical points. Lines indicate the amputation site.
Figure 4. Scleraxis expression in the regenerating froglet blastema was analyzed by section in situ hybridization. A,B:Scleraxis was detected in the lateral region of the early-stage blastema, and expression at this stage was not detected in the medial region. Arrowheads in A indicate signals of scleraxis transcription in the early-stage blastema. C: In situ hybridization on sections of a middle-stage blastema reveals obvious expression in blastema cells (arrowheads). Longitudinal section of a late-stage blastema shows the existence of a few scleraxis-expressing cells (D, arrowhead). Arrows in A–D indicate the signals of scleraxis transcription in cartilage-muscle junctions. Expression of Dermo-1 in the regenerating froglet blastema. Dermo-1 transcripts were detected by in situ hybridization to sections of (E,F) early-stage blastema, (G,G') middle-stage blastema, and (H,H') late-stage blastema. Arrowheads in E–H indicate hybridization signals of Xenopus dermo-1. E: In the subectodermal region, dermo-1 expression was detected from the early stage of the blastema. The boxed region in E is shown in F. G,H: Section in situ hybridization shows that dermo-1 expression continues in the subectodermal region throughout the period of regeneration. G': Higher magnification of G. H': Higher magnification of H. Scale bars in A–E,G,H = 500 μm; in F = 50 μm. Lines indicate the amputation site.
