Studies in amphibian limb development and regeneration

Many experiments have previously been performed to investigate the phenomenon of amphibian limb regeneration. However, these past experiments have not attempted to apply rigorous statistical methods to analyze specific whole limb and regeneration blastema events. The most interesting of these events are those that occur during the developmental stages when the anuran Xenopus laevis is experiencing the most abrupt losses in the ability to regenerate hindlimbs after amputation. We have dealt successfully with the issue of variability in regeneration data by eliminating developmentally abnormal tadpoles from experimental pools and ensuring that sample sizes were large for statistically assessing normal baseline regeneration performance. We demonstrated that the degree of regeneration capability of the tadpole hindlimb was highly correlated to the amount of ossification that was present in the skeletal elements at the plane of amputation. We then employed baseline regeneration data in two experiments designed to improve ossified amputation plane hindlimb regeneration: re-amputation through the original amputation plane 7 days after the first amputation, and in vivo electroporation with an expression plasmid containing the patterning and survival gene Sonic Hedgehog. We found that both treatments significantly improved older tadpole hindlimb regeneration capability, as assessed by scoring systems we developed for whole limb regenerates and for regeneration blastemas. In other experiments, we investigated the mechanisms that determine digit identity in mammals and birds to see if those mechanisms also were those used by limb regeneration-capable species: young Xenopus laevis, and the axolotl, Ambystoma mexicanum. We found that digit identity determination is indeed conserved across these species, and that the capability to regenerate limbs and digits or to condense the digits in opposite sequence from that in most tetrapods does not influence digit identity specification. We also extensively reviewed the subjects of amphibian development and regeneration, and we concluded that it is necessary to employ both the older embryological manipulation techniques and the newer molecular approaches to investigate the related events that take place in amphibian limb development and regeneration.