Identification of regenerative roadblocks via repeat deployment of limb regeneration in axolotls

We sought to determine if we could experimentally compromise the axolotl’s ability to regenerate limbs and, if so, discover the molecular changes that might underlie their inability to regenerate. We found that repeated limb amputation severely compromised axolotls’ ability to initiate limb regeneration. Using RNA-seq, we observed that a majority of differentially expressed transcripts were hyperactivated in limbs compromised by repeated amputation, suggesting that mis-regulation of these genes antagonizes regeneration. To confirm our findings, we additionally assayed the role of amphiregulin, an EGF-like ligand, which is aberrantly upregulated in compromised animals. Overall design: There are two conditions, with four biological replicates per condition. Condition 1 samples, "Multi-amp" in the sample names, are derived from axolotl limbs that have undergone six rounds of amputation-regeneration and mRNA was extracted at three days following the last amputation. Condition 2 samples, "Contol" in the sample names, are age- and size-matched sibling animals that have undergone a single amputation and mRNA was extracted at three days following this amputation.