• Animal cells and systems
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• v.14 no.2
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• pp.73-81
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• 2010

In this study, monoclonal antibodies against lysosomal acid phosphatase (LAP) of a salamander, Hynobius leechii, were used to determine the spatial and temporal expression of the LAP in the regenerating limbs. The Western blot and immunohistochemical analysis in the limb regeneration revealed that LAP was highly expressed at the dedifferentiation stage, especially in the wound epidermis and dedifferentiating limb tissues such as muscle and cartilage. With RA treatment, the LAP expression became upregulated in terms of both level and duration in the wound epidermis, blastemal cell and dedifferentiating limb tissues. In addition, in situ activity staining of LAP showed a similar result to that of immunohistochemistry. Thus, the activity profile of LAP activity coincides well with the expression profile of LAP during the dedifferentiation period. Furthermore, to examine the effects of lysosomal enzymes including LAP on salamander limb regeneration, lysosome extract was microinjected into limb regenerates. Interestingly, when the lysosome extract was microinjected into limb regenerates with a low dose of RA($50\;{\mu}g/g$ body wt.), skeletal pattern duplication occurred frequently in the proximodistal and transverse axes. Therefore, lysosomal enzymes might cause the regenerative environment and RA plays dual roles in the modification of positional value as well as evocation of extensive dedifferentiation for pattern duplication. In conclusion, these results support the hypothesis that dedifferentiation is a crucial event in the process of limb regeneration and RA-evoked pattern duplication, and lysosomal enzymes may play important role(s) in this process.

• The Korean Journal of Zoology
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• v.39 no.4
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• pp.426-436
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• 1996

In previous studies, we have shown that lysosomal add phosphatase (LAP) activity increases at the dedifferentiation stage in the regenerating larval limbs of salamander, Hynobius leechii. Monoclonal antibodies against LAP were generated to determIne the spatial and temporal distribution of the protein In the regenerates.A total of 22 monoclonal antihodies recognizIng different epftopes of the protein were obtained, of which five strongly stained the regenerating limb by imunohistochemistry. in LAP immunohistochemical examination, LAP showed distribution coincident with the state of dedifferentiation, both spatially and temporally, in the limb regenerates. When unfractioned protein of regenerating salamander limbs were separated by gel electrophoresis and immunoblotted, the antibodies recognized a single protein band of 53 kl)a, which comigrates with a monomerlc subunit of IAR Using the anti-IAP antibodIes as probe, we investigated the cross-reactivities of LAPs from other sources. The immunoreadive bands on Western blots appeared to be the same In molecular mass-53 kl)a in axoloti and Xenopus, but no protein band was detected in mouse, Drosophila, or C. elegans.These results show that the antibodies generated in this study spedfically recognize Hynoblus leeclili IAp and that IAPs may be highiy conserved among amphibians. Furthermore, the distdbution of the protein is consistent with a role for LAP in the dedifferentiation process of limb regeneration.