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The results of recent studies have supported the idea that the ability to organize the formation of axes such as the anteroposterior and proximodistal axes corresponds to limb regeneration ability in Xenopus. In this study, we investigated the mechanism by which the dorsoventral (D-V) axis of regenerating Xenopus limbs is established and the relationships between D-V patterning and regenerative ability. Transplantation experiments were performed to study which epidermis or mesenchyme is responsible for the D-V patterning in regenerating limbs. Naked mesenchyme of a donor limb was rotated and implanted on a host opposite-side limb stump to make a reversed recombination about the D-V axis. The resultant regenerates had a normal-looking D-V pattern, including Lmx-1 expression, muscle pattern, and joints, in stage 52 recombinants and a reversed D-V pattern in stage 55 recombinants. Further experiments in recombination at stage 52 and stage 55 showed that the epidermal signal is responsible for producing the D-V pattern in the regenerating blastema. These results, together with the finding that Lmx-1 expression is absent in the froglet forelimbblastema, suggest that D-V axis formation is a key step in understanding the loss of regenerative ability.
FIG. 1. (A) A schematic representation showing isolation of the presumptive zeugopodmesenchyme and subsequent recombination with the host epidermis. d, dorsal; v, ventral. (B) Chimeric analysis of a recombinant limb bud. (B) Chimera was composed of stage 55 mesenchyme (X. borealis) and stage 55 epidermis (X. laevis). (C and D) Higher magnification photograph of (B). (C) Dark field. Note that the transplanted mesenchyme (shown by mottled staining of the nucleus of X. borealis) is covered by the host epidermis (shown by uniformly bright staining of the nucleus of X. laevis). Arrowheads in B show the boundary between the host and graft. (D) Bright field. Bar in (B), 30 ï°m; bar in (C and D), 5 ï°m.
FIG. 2. D-V pattern in recombinant limbs after regeneration. (A) Control experiments by sham-transplantation (not rotated) with the same-side host and donor at stage 52 (A, C, and D) and stage 55 (B). Note that limbs at both stage 52 and stage 55 correctly regenerate a D-V pattern that has accurate flexion of joints. (C) Muscle pattern of phalangeal region of a sham-transplanted limb at stage 52, stained by MF20. (D) Higher magnification photograph of (C). Ventralmuscle is much thicker than dorsal muscle (white arrowheads) at the phalangeal level. (E) Flexion of joints (E and F) and muscle pattern (G) in rotated and transplanted limbs at stage 52 (E, G, and H) and stage 55 (F, I, and J). At stage 52, both flexion of joints (E) and muscle pattern along the D-V axis (G and H) appear normal and dependent on the host epidermis. At stage 55, both flexion of joints (F) and muscle pattern (I and J) are reversed, corresponding to the D-V axis of the grafted mesenchyme. Black arrowheads show the proximal boundary between the host and the graft. Structures distal from solid lines are regenerates. Bar in (C and I) 500 ï°m; bar in (D, H, and I), 250 ï°m; bar in (J), 100 ï°m.
FIG. 3. (A) Comparison of deduced amino acid sequence of Lmx-1. (B) A phylogenetic tree, constructed by the neighbor-joining method, illustrating the relationship of Lmx-1 family members to other LIM homeodomain proteins. (C) Lmx-1 expression in developing limb buds at stage 51 (C), stage 52 (D), stage 53 (E), and stage 55 (F). Lmx-1 is expressed throughout the dorsal mesenchyme at stages 513 (C), and a weak signal is restricted to the dorsal mesenchyme of the presumptive digits region (arrowheads) at stage 55 (F). Staining in the epidermis in (F) is nonspecific background. d, dorsal; v, ventral. Bar, 100 ï°m
FIG. 4. Lmx-1 expression in regenerating hindlimb buds (A) of tadpoles and forelimbs of froglets (indicated by arrowheads). Lmx-1 expression in regenerating blastema is detected at 3 (C), 5 (E), and 7 days (G) after amputation at the presumptive ankle level of stage 52 limb buds, but in stage 55 blastemas (B, D, F, and H) Lmx-1 is detectable only at 7 days after amputation (H). Lmx-1 expression in the blastema after amputation of a froglet forelimb at wrist level (I and J) at 7 (I) and 14 days (J). Lines show the predicted amputation level. Bar, 150 ï°m.
FIG. 5.Lmx-1 expression in recombinant limbs at 7 days after transplantation. (A and D) Diagram of recombination. d, dorsal; v, ventral. (B) A stage 52 recombinant limb. The two solid lines indicate the predicted graft tissue. (C) Higher magnification of the square in (B). Note that Lmx-1 is expressed not only in the transplanted graft but also in the blastema (arrowheads) underlying the host dorsal epidermis. (E) A stage 55 recombinant limb. (F) Higher magnification photograph of (E). Lmx-1 expression (arrowheads) is detectable only within the blastema (distal to arrows) underlying the host ventralepidermis. Bar in (C), 100 um; bar in (F), 200 um.
FIG. 6. D-V pattern (flexion of joints and Lmx-1 expression) in swapped recombination between stage 52 and stage 55 limbs. In recombination with stage 52 mesenchyme and stage 55 epidermis, flexion of the joints (A) and Lmx-1 expression (C) are reversed, indicating that the D-V axis depends on grafted mesenchyme itself. When stage 55 mesenchyme is transplanted onto stage 52 host limb stump, flexion of the joints (B) and Lmx-1 expression (D) appear normal, based on the D-V axis of the host epidermis. Arrowheads in (A) and (B) indicate the proximal boundary between the host and the graft. Distal structures from the solid lines in (A) and (B) are resultant regenerates. In (C) and (D), the region between the two solid lines is the predicted graft and the arrowheads show the expression of Lmx-1 in the blastemas. d, host dorsal side; v, host ventral side. Bar in (C), 150 ï°m; bar in (D), 100 ï°m.
