Fibroblast growth factors in regenerating limbs of Ambystoma: Cloning and semi-quantitative RT-PCR expression studies

Urodele amphibians (newts and salamanders) have the ability to regenerate a mputated limbs throughout their life span. Because fibroblast growth factor s (Fgfs) play important roles in developing limbs, we initiated studies to investigate these growth factors in regenerating limbs. Partial cDNAs of Fg f4, 8, and 10 were cloned from both the Mexican axolotl, Ambystoma mexicanu m, and locally collected spotted salamander, Ambystoma maculatum, two salam anders well recognized for their regenerative capabilities. cDNAs from the two Ambystoma species were virtually identical, ranging from 97-100% nucleo tide identity. Axolotl Fgf4, 8, and 10 showed nucleotide sequence identity with chick Fgf4, 8, and 10 of 79%, 83%, and 72%, respectively. RT-PCR showe d that these growth factors are expressed in regenerating axolotl limbs as well as in developing salamander larvae at the three-digit forelimb stage. Fgf8 and 10 are upregulated during regeneration and thus may be involved in distal signaling similar to that of the developing chick limb. Fgf4, howev er, was undetectable by RT-PCR in the distal tips of regenerates, suggestin g that it does not play the same role in limb regeneration that it does in limb development. We also investigated the role these Fgfs may have in the nerve-dependence of regeneration. They were expressed similarly in aneuroge nic and innervated limbs, suggesting that they are not the neurotrophic fac tors responsible for nerve-dependence. Denervation prevented Fgf8 and 10 up regulation, suggesting Fgf pathways are downstream of nerve-dependence. The se data highlight important similarities and differences in Fgf expression between limb development and limb regeneration. J. Exp. Zool. 290:529-540, 2001. (C) 2001 Wiley-Liss, Inc.