Transferrin is necessary and sufficient for the neural effect on growth in amphibian limb regeneration blastemas

Cell proliferation during the early phase of growth in regenerating amphibian limbs requires a permissive influence of nerves. Based on analyses of proliferative activity in denervated blastemas, it was proposed that nerves provide factors important for cells to complete the proliferative cycle rather than for mitogenesis itself. One such factor, the iron-transport protein transferrin (Tf), is abundant in regenerating peripheral nerves where it is axonally transported and released at growth cones. Using blastemas in organ culture, which have been widely used in previous investigations of the neural effect on growth, it was shown here that the growth-promoting activity of neural extract was completely removed by immuno-absorption with antiserum against Tf and restored by addition of Tf. Purified Tf or a low molecular weight ferric ionophore were as active as the neural extract in this assay, indicating that the trophic effect of Tf involves its capacity for iron delivery. Both Tf and ferric ionophore also maintained DNA synthesis in denervated blastemas in vivo. A dose-response assay indicated that purified axolotl Tf stimulates growth of cultured blastemal cells at concentrations as low as 100 ng/mL. The Tf mRNA in axolotl nervous tissue was shown by northern analysis to be similar in size to that of liver. These results are discussed together with those from previous in vitro studies of blastemal growth and support the hypothesis that cell division in the blastema depends on axonally released Tf during the early, nerve-dependent phase of limb regeneration.