Unique expression patterns of matrix metalloproteinases in regenerating newt limbs

The process of regeneration of urodele limbs includes a drastic remodeling of extracellular matrices (ECMs) that is induced by matrix metalloproteinases (MMPs) and is thought to be one of the triggers of the regeneration. We studied this remodeling in limbs of Japanese newt, Cynops pyrrhogaster, by using five genes of newt MMPs (nMMPs) as probes: nMMP9, nMMP3/10-a, nMMP3/10-b, and nMMP13 that had been characterized previously, and nMMPe that was newly cloned in the present study. nMMPe was 502 amino acid residues long and showed a low homology to other known vertebrate MMPs. Reverse transcriptase-polymerase chain reactions analysis localized the transcript of nMMPe in the apical epidermal cap (AEC) and the non-blastemal wound epidermis but not in the blastemal mesenchyme or the normal epidermis. Northern blot analysis localized the transcripts of nMMP9, nMMP3/10-a, and nMMP13 in the bone of regenerating limbs, whereas those of nMMP3/10-b in AEC. mRNA in situ hybridization experiments identified the nMMP-expressing cells. nMMP9 gene was strongly expressed in chondrocytes of the cartilage of epiphysis. Of interest, basal cells of AEC, but not those of the normal skin, expressed nMMP3/10-b intensely. Immunohistochemical analysis showed that the nMMP9 proteins synthesized by chondrocytes were secreted and distributed widely in the basement membrane of bone and ECMs of the amputation plane. These nMMPs characterized in the present study might cooperatively work to remodel ECMs of regenerating limbs.