RESUMEN
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.
Asunto(s)
Extremidades/fisiología , Metaloproteinasas de la Matriz/metabolismo , Regeneración/fisiología , Salamandridae/fisiología , Secuencia de Aminoácidos/genética , Animales , Cartílago/enzimología , Clonación Molecular , Tejido Conectivo/enzimología , Hibridación in Situ , Isoenzimas/genética , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 10 de la Matriz , Metaloproteinasa 3 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Salamandridae/genéticaRESUMEN
The expression of two regeneration-associated antigens in the blastemas of normal and retinoid-treated regenerating limbs of axolotl (Ambystoma mexicanum) was examined. One antigen, 55C12, which was similar to tenascin in expression pattern and molecular weight profile, was weakly expressed in the perichondrium and tendon of normal limbs. In the regenerating limbs, the amount of 55C12 antigen increased near the amputation site within 7 days and almost all cells of the blastema mesenchyme came to be positive to the antigen at 20 days, although those of epidermis and most stump tissues were negative. When the regenerating limbs were treated with Am80, a synthetic retinoid, which induced proximo-distal duplication, the expression of 55C12 antigen in the blastema became weak temporarily and was reactivated in the anterior region of the blastema. This expression pattern suggests that the duplicated limb is formed by the preferential growth of this 55C12-positive anterior blastema region. The other antigen, 117C1, was faintly expressed in the epidermis, dermis, muscle, perichondrium and cartilage of normal limbs, and intensely expressed in the blastema mesenchyme and wound epidermis. The Am80 treatment, however, induced no changes in the expression pattern of 117C1. These results suggest that these antigens may distinguish two different regions of the blastema in normal regeneration and retinoid-induced duplication.