| The process of amphibian limb regeneration is initiated by injury following limb loss. Cells of the mesodermal stump tissues dedifferentiate and proliferate to form an outgrowth, or blastema, the cells of which dedifferentiate to form a complete regenerate. My research began with the characterization and identification of an antigen upregulated in regenerating newt limbs which is recognized by monoclonal antibody (mAb) MT4. I characterized the MT4 antigen by both immunohistochemistry and Western blotting. By screening a blastema cDNA library, obtaining and sequencing cDNAs, and searching Genbank for homologous sequences, the MT4 antigen was identified as the extracellular matrix protein fibronectin (FN).;I next determined by in situ hybridization that both the wound epithelium and the blastema cells themselves transcribed the FN gene. In addition, by comparing FN protein extracted from early regenerates and late regenerates with newt plasma by Western blotting as well as utilizing a mAb specific for the cellular form of FN (cFN), I determined that the plasma form of FN (pfN) was the form present during early stages of regeneration and at later stages both forms were present.;Next, I asked what factors regulate FN expression during limb regeneration. Since nerves, wound epithelium, and injury are required for limbs to regenerate, I hypothesized that FN could be regulated by at least one of these factors. Since denervated and wound epithelium-deprived limbs exhibited no change in FN transcription or protein distribution, I concluded that factors released as a result of injury likely regulate FN expression. Additionally, the spatial distribution of FN transcripts was affected by retinoic acid.;Nerves supply a factor, the so-called neurotrophic factor (NTF), to the regenerating limb which acts as a mitogenic stimulus. It has been hypothesized that the NTF may be FGF-1. By Western blotting I confirmed the presence of FGF-1 protein in the regenerating limb and immunolocalized FGF-1 in the blastema, associated with the blastema cells. In addition, FGF-1 was unable to substitute for nerves in the nerve dependence to independence transition. We concluded that FGF-1 plays an important role in regeneration but that it is likely not the NTF. |
