Insights into the mechanisms and limitations of axolotl limb regeneration

Abstract

Axolotl salamanders are powerful models for understanding how limb regeneration can be achieved. However, if even highly regenerative organisms such as axolotls have finite regenerative capabilities, then understanding these limitations will have important implications for addressing these issues in mammals. This dissertation’s goal was to determine whether a limit exists to axolotl limb regeneration and to identify genes that are functionally important for this process. I discovered that repeated amputation hinders axolotls’ ability to initiate limb regeneration and that Amphiregulin, an EGF-like ligand, is aberrantly upregulated in compromised limbs. Furthermore, amphiregulin is expressed by the early wound epidermis after limb amputation, and mis-expression of this factor disrupts the initiation of regeneration. These findings suggest that axolotl limb regeneration cannot be redeployed indefinitely and that dysregulated wound healing may obstruct axolotls’ ability to repeatedly mount a regenerative response. In a related study, I challenged axolotls to repeatedly regrow limb buds in order to determine if there is a limited time window for axolotl limb development. Similar to repeated full limb amputation, an increasing number of limbs failed to form following an increasing number of bud removals. Interestingly, the limbs that were able to develop after prolonged repeated bud removal were permanently miniaturized. Additional experimentation using this model will further elucidate how limb and body size are coupled, an essential feature of successful regeneration and critical for future regenerative therapies. Though salamander limb regeneration has been appreciated and studied for hundreds of years, a lack of genomic and transcriptomic sequences has impeded investigation of the molecular mechanisms that drive this phenomenon. To overcome this hurdle, I was part of a collaboration that constructed one of the most comprehensive axolotl transcriptomes to date and discovered many genes whose expression is highly enriched in blastemas. Through functional experimentation, we identified both cirbp and kazald1 among these candidates as being required for proper limb regeneration. In summary, my work uncovered previously unknown limits to axolotl limb regeneration and established novel functional roles for several genes in this system. Further exploration of the insights presented in this dissertation has high potential to inform future regenerative medicine.