Jessica Whited (Online)
17.15 – 18.00
Amputation-induced systemic stem cell activation in axolotl
Regenerative abilities vary dramatically across animal lineages. Yet, how systems-level injury responses in individual species shape ultimate regenerative outcomes is not well understood. We recently discovered a body-wide stem cell activation response that occurs following limb amputation in axolotls. Here, we present evidence that this systemic activation primes appendages distant to the original injury for their own future regeneration events. Using single-cell RNAseq, we identified the repertoire of systemically-activated cells to be the same cells likely to fuel organismal growth and cell replenishment during homeostasis. We investigated the mechanism of systemic activation in axolotls and found the process requires innervation by the peripheral nervous system at distant responding sites. We used transcriptomics to identify several candidate molecular pathways for controlling systemic activation. We found inhibiting adrenergic signaling is sufficient to block systemic activation, and we found that activating adrenergic signaling can promote early phases of regeneration. Inspired by previous reports of mTOR activity being required for systemic cell cycle re-entry following injury in both highly-regenerative species and species with modest regenerative abilities, we also tested mTOR signaling in axolotl. Our studies also implicate mTOR signaling as required for axolotl systemic activation and priming following amputation. Together, these results demonstrate a direct link between systemic stem cell responses and localized regeneration of an appendage, and they also highlight roles for peripheral nerves, adrenergic signaling, and the mTOR pathway in systemic injury responses in axolotls. They also suggest a possible model whereby common initial injury responses, acting on stem cells also used for growth and homeostasis across species, are followed by differing instructions that result in regeneration versus repair.
Jesus Chimal-Monroy (Online)
Endogenous RA induces proximo-distal limb duplications during limb regeneration
During limb regeneration, cells of the distal end of the stump are released from their tissue organization and acquire a mesenchymal phenotype. These cells will proliferate to give rise to a blastema from which all limb tissues will differentiate. It is known that RA binds and activates heterodimers formed by RARs and RXRs. It is catabolized in the cytoplasm by CYP26 enzymes yielding polar metabolites that are inactive or less active than RA, consequently controlling intracellular levels of active RA. Thus, the inhibition of Cyp26 results in an increase in endogenous retinoic acid. In this study, we used a specific inhibitor to determine the role of Cyp26 during limb regeneration.
Our results showed that by inhibiting the enzyme Cyp26, PD duplication is observed as occurs after the exogenous RA treatment. Transplanting a blastema from amputated limbs treated with Inh-Cyp26 to a non-treated amputated limb cannot produce a PD duplication in the recipient limb, regenerating only the missing part of the amputated limb. Surprisingly, the Inh-Cyp26-treated donor limb, from which the blastema was removed, regenerated with a new PD duplication from the cut site. Results suggest that instructive information to induce proximal-distal duplications is not retained in blastema but in the stump. Furthermore, we observed that RAR gamma rescues normal regeneration.
Olena Zhulyn (Online)
Evolutionary divergence of mTOR signaling drives translational remodeling underlying rapid wound closure and regeneration
One of the greatest outstanding questions in biology is why some species can scarlessly heal and regenerate tissues while others cannot. Here, we demonstrate that rapid activation of protein synthesis is a unique, and previously uncharacterized, feature of the injury response critical for limb regeneration in the highly regenerative axolotl (A. mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components, which do not change in their overall mRNA abundance but are selectively activated at the level of translation from pre-existing mRNAs in response to injury. In contrast, we show that protein synthesis is not activated in response to digit amputation in the non-regenerative mouse. We further identify the mTORC1 pathway as a key upstream signal that mediates this regenerative translation response in the axolotl. Inhibition of this pathway is sufficient to suppress translation, wound closure and regeneration in the axolotl. Surprisingly, although mTOR is highly conserved across evolution, we discover novel expansions in key functional regions of mTOR kinase that are unique to highly regenerative urodele amphibians. By engineering an axolotl mTOR in human cells, we demonstrate that these expansions create a hypersensitive kinase that may allow axolotls to maintain this pathway in a highly labile state primed for rapid activation. We hypothesize that fundamental differences in the sensitivity and function of mTOR kinase may underlie metabolic differences and nutrient sensing between regenerative and non-regenerative species that are key to regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential.