Monoclonal antibody methodology will be utilized in experiments designed to understand the roles of injury, nerves, and wound epithelium in amphibian limb regeneration. The reactivity to regenerating limb tissues of 3 already available monoclonal antibodies (mAbs) will be examined. mAb 3WE reacts to adult newt skin mucus glands of the body and to wound epithelium during blastema stages. Examination of timing and distribution of 3WE reactivity led to the hypothesis that functional wound epithelium cells must come from a glandular source. To test this view, a thorough immunoflourescence analysis of 3WE reactivity to cryostat prepared tissue sections of normal and experimentally modified (denervation, skin and limb grafts, retinoid treatment) regenerates is proposed. 3WE reactivity will be examined in Amystoma larvae and adults, including during aneurogenic limb regeneration. Immunoflourescence and autoradiography will be combined to determine the origin of 3WE+ cells. Two other mAbs, which react strongly to an intracellular component of all newt blastema cells (9G1) and to an extracellular matrix antigen (4G3) during regeneration, will be analyzed similarly. mAb 9G1 reacts differentially to frog tadpole hindlimb blastema and wound epithelial cells and provides an investigative tool to compare urodele and anuran regeneration. To identify additional developmentally significant antigens, hypothesized to be present in blastema cells, nerves, and wound epithelium, mice will be immunized against blastema tissues, hybridomas will be obtained, and mAbs will be surveyed against regenerating limb tissues. Reactivity of mAbs to antigens of potential significance to the regeneration process will be further analyzed as above. The mAbs and ultimately the purified antigens provide the opportunity for comparative studies between regenerating (salamanders) and non-regenerating (frogs) species.
