Directed axonal growth towards axolotl limb blastemas in vitro

Limb amputation in urodele amphibia is followed by formation of a blastema, which subsequently develops into a complete limb with normal pattern of innervation. In this study, we investigated the effects of axolotl limb blastemas on axonal growth in gels of collagen and extracellular matrix (matrigel). When peripheral nerves with attached dorsal root ganglia were cultured in collagen gels together with blastemas, axonal outgrowth was markedly increased compared with control preparations. Blastemas contain fibroblast growth factors, and may also contain neurotrophic factors such as nerve growth factor, brain-derived neurotrophic factor, neurotrophin 3, neurotrophin 4, glial cell line-derived neurotrophic factor and hepatocyte growth factor/scatter factor, since these factors are expressed in developing limbs in other vertebrates. In collagen gels the neurotrophins and glial cell line-derived neurotrophic factor stimulated axonal growth, but outgrowing axons were shorter than in co-cultures with blastemas. The tyrosine kinase inhibitor K252a blocked the stimulatory effects of the neurotrophins on axonal growth but had relatively little effect on axonal growth in co-cultures with blastemas. In experiments in which peripheral nerves, with attached dorsal root ganglia, were cultured in matrigel, axons grew towards blastemas over distances of about 1mm. Directed axonal growth even occurred in these co-cultures after addition of high concentrations of all the above neurotrophic factors, suggesting that blastemas may release a different factor which stimulates axonal growth. The results indicate that during early stages of limb regeneration in amphibia, factor(s) are released which are capable of attracting the growth of peripheral nerves and may play an important role in the development of innervation of regenerated limbs. The identity of the factor(s) remains to be determined.