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A young tadpole of an anuran amphibian can completely regenerate an amputated limb, and it exhibits an ontogenetic decline in the ability to regenerate its limbs. However, whether mesenchymal or epidermal tissue is responsible for this decrease of the capacity remains unclear. Moreover, little is known about the molecular interactions between these two tissues during regeneration. The results of this study showed that fgf-10 expression in the limb mesenchymal cells clearly corresponds to the regenerative capacity and that fgf-10 and fgf-8 are synergistically reexpressed in regenerating blastemas. However, neither fgf-10 nor fgf-8 is reexpressed after amputation of a nonregenerative limb. Nevertheless, nonregenerative epidermal tissue can reexpress fgf-8 under the influence of regenerative mesenchyme, as was demonstrated by experiments using a recombinant limb composed of regenerative limbmesenchyme and nonregenerative limbepidermis. Taken together, our data demonstrate that the regenerative capacity depends on mesenchymal tissue and suggest that fgf-10 is likely to be involved in this capacity.
FIG. 1. (A) Comparison of amino acid sequence of FGF-10 (abbreviations: x, Xenopus; c, chick; m, mouse; r, rat; h, human). (B) fgf-10
expression in developing limb buds at stage 51 (B), stage 52 (C), stage 53 (D), and stage 56 (E). (F) In situ hybridization on a section of stage
52 limb bud (only distal region). (G) Higher magnification view of (F). a, anterior; p, posterior. Arrowheads show the presumptive knee level
(amputation level) of limb buds. Bars, 250 mm for (B), (C), (D), and (E), and 50 mm for (F) and (G).
Fig 2. Regenerative capacity of Xenopus limb bud. All limb buds were amputated at the presumptive knee level at stage 52 or 56. Cartilage patterns of all specimens were visualized by Alcian blue staining. (A) A complete regenerate from stage 52 limb bud. It has a complete cartilage pattern of hindlimb. (B) A sample after amputation of stage 56 limb buds. It formed no regenerate but underwent wound healing. (C) An incomplete regenerate from stage 56 limb bud. It has only a spike-shaped cartilage. (D) An incomplete regenerate from T4-treated stage 56 limb bud. It formed a spike-shaped cartilage. Arrows show amputation level. Bars, 1 mm
FIG. 3. fgf-10 and fgf-8 expression in regenerating limb buds. Limb buds were amputated at stage 52 (A–D), or stage 56 (E–L), and examined for fgf-10 (A, E, I) and fgf-8 (C, G, K) in serial sections. (B, D, F, H, J, and L) Phase-contrast photograph of (A), (C), (E), (G), (I), and (K), respectively. Arrows indicate amputation level. Arrowheads indicate thickened epidermis. d, dorsal; v, ventral. Bars, 250 um.
Figure 4. Schematic diagram illustrating experimental pro- cedure for preparing recombinant limbs. The arrow indicates the hindlimb bud of the Xenopus tadpole. To determine whether epidermis or mesenchyme controls the regenerative capacity of the Xenopus limb bud, two types of recombinant limbs were made. In experiment A, the whole stage 51–52 limb bud was excised, the epidermis was removed, and then, naked mesenchyme was grafted onto the stage 56hindlimb stump amputated at the presumptive knee level. After grafting, epidermis of stage 56 host migrated and covered the grafted mesenchyme, and therefore, recombinant limbs composed of stage 51–52 mesenchyme and stage 56epidermis (type A recom- binant) were formed. In experiment B, whole stage 56 (T4- treated) mesenchyme was grafted onto the stage 52 host hind- limb stump amputated at the presumptive knee level. After grafting, epidermis of stage 52 host covered stage 56 mesen- chyme, and therefore, recombinant limbs were composed of stage 56mesenchyme and stage 52epidermis (type B recombi- nant). 5–7 days after recombination, we amputated these two types of recombinant limbs at the presumptive knee level in order to analyze regenerative capacities. meso, mesenchyme; epi, epidermis.
FIG. 5. Development and regeneration of recombinant limbs. (A and B) Recombinant limbs composed of stage 51–52 mesenchyme and stage 56 epidermis. (C and D) Recombinants composed of stage 56 mesenchyme and stage 52 epidermis. (A and C) Allowed to develop without amputation. They formed complete cartilage patterns. (B and D) Amputated at presumptive knee level. Note that the recombinant limb in (D) regenerates only an incomplete cartilage structure like a spike, while the recombinant in (B) regenerates a complete cartilage pattern. Arrowheads indicate host–graft boundary. Black arrows, amputation level. White arrows, recombinant limb. Asterisks, extra limbs. Bars, 1 mm.
FIG. 6. Chimeric analysis of recombinant limb. (A) A recombinant limb composed of stage 51–52 mesenchyme (X. borealis) and stage 56 epidermis (X. laevis). (B and C) Higher magnification photograph of distalmost region in the same recombinant. (B) In ordinary light. (C) In fluorescent light. Note that borealis mesenchymal cells (shown by mottled staining of nucleus) were covered with laevis epidermal cells (shown by uniformly bright staining). Arrowheads and arrows indicate host–graft boundary and epidermal cell layer, respectively. Bars, 100 um.
FIG. 7. fgf-10 (A) and fgf-8 (B) expression in recombinant limb bud with stage 51–52 mesenchyme and stage 56 epidermis and fgf-10 (C) and fgf-8 (D) expression in recombinant with stage 56 mesenchyme and stage 52 epidermis 3 days after amputation. Arrowheads indicate host–graft boundary. Arrows show amputation level. Bars, 250 um.
