Investigating cell cycle regulation and morphogenesis during axolotl limb development and regeneration

Abstract

Mammals, including humans, are severely limited in their capability for limb regeneration, only being able to regenerate amputated digit tips. Axolotl salamanders (Ambystoma mexicanum), however, are able to regenerate whole, functional limbs following amputation. For this reason, limb regeneration in the axolotl is a strong model for understanding how to elicit a more robust regenerative response in mammals. The process by which axolotls regenerate their limbs broadly occurs in three steps: wound healing, blastema formation/proliferation, and limb patterning. In this dissertation, I focus on the latter two steps. In my first chapter, I provide a brief overview of limb regeneration with focus on cell cycling and patterning during the regenerative process while relating limb regeneration to embryonic limb development. In my second chapter, I present published work from a collaboration between the Monaghan and Shefelbine Labs outlining a method for three dimensional volumetric imaging of macromolecule synthesis in whole mount tissues. Using this method, we quantify the rate of DNA synthesis in whole mount innervated and denervated limb blastemas and show that transection of the nerve supply slows DNA synthesis. In chapter three, I present work in revision where we generated a ubiquitous FUCCI (fluorescent ubiquitination-based cell cycle indicator) transgenic axolotl that reports cell cycle state in-vivo. This animal line enables discrimination of cells in G1 phase from cells in S/G2/M phases. By using this line, we were able to live image blastema formation, demonstrate the local contribution of cells to the blastema, and show that blastema cells arrest in G1 phase following limb denervation. My next two chapters focus on patterning during limb regeneration and limb development. In chapter four, I show that retinoic acid (RA) breakdown is required for positional identity during regeneration of distally amputated limbs, but not proximally amputated limbs. If this process of RA breakdown is perturbed in distal limbs, we observe duplications of proximal limb segments from distally amputated limbs. We show that this proximalization is due to molecular reprogramming of distal blastema cells and that it requires de-novo RA synthesis and RA signaling to proximalize. Limb regeneration is often described as a recapitulation of limb development. Thus, I explore patterning during limb development in chapter five. I utilize hybridization chain reaction fluorescence in-situ hybridization (HCR FISH) to provide evidence for an evolutionarily novel mechanisms of limb patterning during axolotl limb development whereby RA is produced in the distal limb bud, unlike amniotes. We speculate that this difference may contribute to the axolotl's lifelong regenerative capabilities which are absent from amniotes. In my final chapter, I outline future directions for the work presented in chapters two through five. In all, the work in this dissertation should provide a foundation for many future studies which will hopefully contribute to improving the regenerative response in humans.--Author's abstract