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Xenopus laevis larvae gradually lose the ability to regenerate lost hindlimb structures as they progress through metamorphosis. Previous studies have suggested that this loss of regenerative capacity occurs in a proximal-to-distal fashion. We assessed the quality of overall regeneration and early bud blastema formation in order to evaluate previous explanations for this loss of regenerative ability in Xenopus. We further examined the extent to which epidermis, basement membrane, dermis, cartilage, bone, periosteum, and accumulated mesenchyme within the blastema are involved in the decline of regenerative abilities during mid-metamorphic stages of development. Each tissue was scored based on its contributions to the regeneration blastema, in accordance with previously reported blastemal descriptions. Tadpoles amputated at the ankle and tarsal-metatarsal joints scored objectively higher within the overall regeneration and blastema quality rating systems. Both joint sites met more criteria associated with regeneration-capable blastemas than tadpoles amputated through the middle of the tarsus, especially at later stages of metamorphosis. The three amputation sites studied began to vary in their ability to regenerate skeletal elements and to generate productive blastemas during the same stages at which we initially observed ossification of the tarsus. These results suggest that the decline of Xenopus hindlimb regeneration does not occur in a strictly proximal-to-distal fashion but rather is dependent at later stages on the state of ossification of the structure through which amputation occurs. Our morphological and cellular observations reveal specific times and places during Xenopus hindlimb development at which further investigations into tissue-specific molecular events during early regeneration should be focused.
Fig. 1. Presence of bone and cartilage within the ankle region. These whole-mount specimens were stained with alizarin red, showing ossified bone, and alcian blue, showing cartilage. Stages of development are labeled. Solid arrowheads in each figure display the diaphysis (or pre- sumptive center) of the principal tarsal bone, while open arrowheads depict the ankle and tarsal-metatarsal joints. The top of each panel corresponds to the distal end of the limb. Inset for stage 55 depicts an alizarin red-only stained limb, which showed greater sensitivity for bone detection than the doubly-stained limb. This was the only stage at which a marked difference in sensitivity was found between the singly- and doubly-stained limbs. Note that ossification becomes evident at stage 55 (inset). Stages 55 and 57 are displayed at 10ﰄ, stages 54, 55 (inset), 58 and 59 at 20x and stage 56 at 40x
Fig. 4. Blastemal characteristics. A: Selected histological features of a high quality blastema, generated by a Xenopus stage 54 hindlimb amputation through the tarsus after 7 days recovery time. B: Selected histological features of a poor quality blastema, generated by a stage L57 tibia-fibula amputation after 10 days. The plane of amputation is denoted by a transverse hatched gray line through the blastema in each panel. Both panels are shown at 100x. Black boxes indicate the locations magnified (400x) in the inset of each panel. See Experimental Procedures and Table 1 for a description of the displayed characteristics.
Fig. 6. Average blastemas within each stage and amputation site. Each panel represents a hematoxylin-and-eosin-stained histological preparation of a blastema which was rated at the average for its stage and amputation site. Panels in the left-hand column represent ankle joint (AJ) regenerate blastemas, those in the center column represent mid- principal tarsal bone (T) blastemas, and those in the right-hand column represent tarsal-metatarsal joint (TMTJ) blastemas. The numerical rat- ings for these samples are shown in Table 2, with the illustrated samples italicized in Table 2. Each row represents animals of the same stage at the time of amputation. Blastemas formed by amputees at stages 54, 55, and 56 do not demonstrate gross qualitative differences from one an- other, as is shown by the graph in Figure 4. However, after stage 56 the T blastemas rapidly begin to display the poor blastema qualities of dermis, skin glands, cut bone surface with periosteal cartilage evident in the blastema (first observed in E57T, arrow), and development of a fibrocellular pad covering the cut bone. The joint amputees, while show- ing an age-dependent decline in blastema quality, maintain high-quality characteristics such as mesenchymal accumulation, thickened AEC and appedskeletal elements at later developmental stages. AJ panels at stages 54-L57 are shown at 100x; stages 58 and 59 at 50x; T panels at stages 54, 55, and L57 are shown at 100x; stages 56, E57, 58 and 59 at 50x;; TMTJ panels at stages 54, 55, and E57-59 are shown at 50x; stage 56 at 100x.
Fig. 3. Average overall regeneration ratings. A: With increasing age, overall regeneration capacity is lost. However, the two joint amputation sites result in demonstrably better overall regeneration success than the mid-bone site until complete failure of identifiable outgrowth occurs at stage 58. The magnitude of error shown in the stage 54 regenerates is due to the difficulty in ascertaining precise amputation sites in these small, largely undifferentiated limbs. B: Selected examples of joint and bone regeneration sites are shown for stages 55, 57, and 59. In these examples, the trends illustrated in A are demonstrated: for stages 55 and 57, the TMTJ site is the most productive, followed by the AJ site, then the T site. (The stage 57 TMTJ sample was collected prior to complete structure formation, however, four metatarsal sites were observed, which resulted in a higher score than the stage 57 AJ sample.) The stage 59 samples show heteromorphic cartilaginous spikes. Arrowheads denote the original sites of amputation, and asterisks label the center of the tarsus in each panel. All samples are shown at 10x.
