EXAMINATION OF FIBRONECTIN DISTRIBUTION AND ITS SOURCES IN THE REGENERATING NEWT LIMB BY IMMUNOCYTOCHEMISTRY AND IN-SITU HYBRIDIZATION

Using monoclonal antibodies (mAbs) reactive to newt limb regenerates, we hope to gain insight into the identity and function of regeneration significant molecules. mAb MT4 (matrix 4) identifies an extracellular matrix (ECM) protein that is strongly up-regulated first in the dista l stump and then in the blastema during regeneration. Within the first 24 hr after amputation the MT4 antigen is localized to an acellular s pace beneath the wound epithelium, and first appears in the basal cell s of the wound epithelium between days 5 and 7. At mid-bud blastema st ages, the MT4 antigen is homogeneously distributed as thin fibers in t he blastema ECM, and is later largely restricted to the distal tip of the blastema and the areas of cartilage condensation. After extraction and immunoblotting, the MT4 antigen was observed as three reduced spe cies of M(r) 225, 250, and 260. Taken together, the immunoblot and imm unocytochemistry results suggested that mAb MT4 recognized newt fibron ectin (FN). Sequence from a cDNA (NvFN.10) obtained by screening a new t blastema cDNA expression library with mAb MT4 conclusively identifie d the MT4 antigen as FN. To further investigate the expression of FN i n regeneration, cDNA NvFN.10 was used to construct a riboprobe and in situ hybridization was done. In the unamputated limb only a few scatte red cells expressed the FN gene. Within the first 3 days after amputat ion strong hybridization signal was observed in the basal cells of the wound epithelium. Most of the stump cells that dedifferentiated and a ccumulated beneath the wound epithelium at 7 days expressed the FN gen e, while the basal cells of the wound epithelium maintained their expr ession. At mid- and late-bud blastema stages the vast majority of the blastema cells were strongly expressing the FN gene, but the wound epi thelial cells now showed only weak FN transcription. Thus initially FN comes from the plasma. Then FN is synthesized by both the wound epith elium and mesenchyme. Finally, at blastema stages FN is produced prima rily by the mesenchyme. The expression pattern of FN throughout regene ration suggests that this glycoprotein has roles in wound epithelial a nd mesenchymal cell migration and mesenchymal cell proliferation and d ifferentiation. (C) 1995 Wiley-Liss, Inc.