Stage-dependent effects of retinoic acid on the regenerating limbs of larval Korean salamander, Hynobius leechii, at two amputation levels.
The morphological and biochemical data obtained in this series of experiments clearly confirm the previous finding about the relationship between the stage of limb regeneration and the effects of RA on duplication, i.e., the most sensitive stage for RA-induced duplication is the stage of dedifferentiation, regardless of the level of amputation. However, when the RA effects were expressed as a function of time after amputation, the upper arm regenerates clearly showed a prolonged and delayed response to RA treatment compared to the lower arm regenerates. These findings were further supported by the data on acid phosphatase activity, which may reflect the level of dedifferentiation. Furthermore, the slow increase of this enzyme activity above the control level until 8 days after RA-injection corresponds to the late appearance of limb regenerates after RA treatment. At present, we do not have any clear explanation why upper arm regenerates and lower arm regenerates show differences in the MLP's and different temporal profiles for the RA-evoked limb pattern duplications. However, the difference in the level of dedifferentiation after simple amputation and after RA-treatment suggests that RA-induced changes in the positional identity of blastema cells may require a prolonged and augmented state of dedifferentiation. In this scheme, the level and type of CRABP may play a critical role as suggested in the case of regenerating axolotl limbs (McCormick et al., 1988).