NERVE DEPENDENCY OF REGENERATION - THE ROLE OF DISTAL-LESS AND FGF SIGNALING IN AMPHIBIAN LIMB REGENERATION

Dlx-3, a homolog of Drosophila Dll, has been isolated fi;om an axolotl blastema cDNA library, and its expression in developing and regenerat ing limbs characterized. The normal expression pattern, and the change s that occur during experimental treatments, indicate a correlation be tween Dlx-3 expression and the establishment of the outgrowth-permitti ng epidermis. Dlx-3 is expressed at high levels in a distal-to-proxima l gradient in the epidermis of developing limb buds, and is upregulate d in the apical ectodermal cap (AEC) during limb regeneration. Express ion is maximal at the late bud stage of regeneration, coincident with the transition from the early phase of nerve dependency to the later p hase of nerve independence. Dlx-3 expression in the epidermis is rapid ly downregulated by denervation during the nerve-dependent phase and i s unaffected by denervation during the nerve-independent phase. We inv estigated this relationship between nerves and Dlx-3 expression by imp lanting FGF-2 beads into regenerates that had been denervated at a ner ve-dependent stage. Dlx-3 expression was maintained by FGF-2 after den ervation, and regeneration progressed to completion. In addition, we d etected FGF-2 protein in the AEC and in nerves, and observed that the level of expression in both tissues decreases dramatically in response to denervation. We conclude that both limb development and regenerati on require a permissive epidermis, characterized by Dlx-3 and FGF expr ession, both of which are maintained by FGF through an autocrine loop. The transformation of the limb epidermis into a functional AEC that p roduces and responds to FGF autocatalytically, is presumed to be induc ed by FGF. Since nerves appear to be a source of this priming FGF, it is possible that a member of the FGF family of growth factors is the e lusive neurotrophic factor of limb regeneration.