Search

In the regenerating limbs of Korean salamanders, Hynobius leechii, retinoic acid (RA) induces duplication of skeletal structures in the proximodistal (PD) axis and often in the transverse axes. In the present study, the stage-dependent effects of RA for the duplication of limb skeletal structures at two amputation levels, the distal stylopodium and the distal zeugopodium, were studied using larval limbs of Korean salamanders. The results showed that the mean level of proximalization (MLP) by RA treatment increased during the stages of dedifferentiation and early bud formation while the MLP declined thereafter in both amputation levels. The decline of the MLP at the later stages of regeneration was due to the high frequency of hypomorphic regeneration or blocked regeneration. When the effects of RA treatment at two amputation levels were compared, the overall trends were similar but the actual timing was delayed for 2-4 days in the proximal level of amputation. Furthermore, the peak level of proximalization was achieved earlier and the peak level remained longer in the distal stylopodial level of amputation compared to the distal zeugopodial level of amputation. Since the histological observations revealed that the dedifferentiation period was also extended up to 2-4 days in the proximal level of amputation, the acid phosphatase activity during the course of regeneration was measured to look for a quantitative relationship between the enzyme activity and the states of dedifferentiation. The results show that the level and the duration of acid phosphatase activity in the upper arm regenerates are both higher and longer than those in the lower arm regenerates. Furthermore, RA treatment caused an increase in acid phosphatase activity. Thus our results suggest that the state of dedifferentiation might be closely linked to the extent of proximalization of regenerating limbs by RA treatment.

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).