Extent of ossification at the amputation plane is correlated with the decline of blastema formation and regeneration in Xenopus laevis hindlimbs.

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.