Molecular mechanisms of salamander limb regeneration

Abstract: Salamanders, like newts and axolotls, stand out among adult vertebrates for their outstanding capacity to regenerate whole body parts and restore complex structures upon injury. One of the best-known examples is their ability to fully regenerate a functional limb. Despite the important progress in the field, our understanding of the molecular cues that control limb regeneration is still limited. In this thesis, I focus on the mechanisms by which skeletal muscle stimulates limb regeneration. Skeletal muscle is particularly interesting because, in newts, it contributes to limb regeneration by dedifferentiation. This unique process is characterized by fragmentation of the multinucleated myofiber and subsequent cell cycle reentry by the derived mononucleate progeny. In Paper I, we sequenced and edited the ~20 Gigabases genome of the Iberian ribbed newt Pleurodeles waltl, a commonly used species for regeneration studies in salamanders. Using CRISPR/Cas9 technology we perturbed two key transcription factors (Pax3 and Pax7) that are involved in skeletal muscle development and regeneration in vertebrates. We found that contrary to mammals, in which Pax7 expression by skeletal muscle stem cells is indispensable for regeneration, muscle regeneration was not altered when Pax7 gene was mutated in newts. Moreover, we observed that embryonic stem cell-specific microRNAs (mir-93b and mir-427), as well as Harbinger DNA transposons carrying the Myb-like proto-oncogene have expanded dramatically in the Pleurodeles waltl genome and are co-expressed during limb regeneration. This study provides a foundation for comparative genomic studies that could improve our understanding of the uneven distribution of regenerative capacities among vertebrates. In Paper II, we identified a microRNA, miR-10b-5p, which is highly abundant in muscle tissue across species and downregulated during early limb regeneration in newts. In contrast, miR-10b-5p displayed the opposite regulation in mammalian cultured myotubes, when these were induced to dedifferentiate. To investigate a possible function of miR-10b-5p in newt limb regeneration, we overexpressed it by mimic injection. We found that such manipulation of miR-10b-5p levels during the initial stages of regeneration slowed down the regeneration process. Moreover, we observed that overexpression of miR-10b-5p decreased the number of cycling cells and counteracted blastema growth. The identification of miR-10b-5p targets will be an important task for future studies. In Paper III, we showed that blood clotting proteases cleaved and activated bloodderived bone morphogenetic proteins (BMPs) to promote BMP signaling-dependent cell cycle re-entry by myofiber progeny. In particular, we found that protease-activated BMP4/7 heterodimers which were present in serum, strongly induced myotube cell cycle re-entry, with protease cleavage yielding a 30-fold potency increase of BMP4/7 compared with canonical BMP4/7. Additionally, we observed that inhibition of BMP signaling, via muscle-specific dominant-negative receptor expression, reduced cell cycle re-entry in vitro and in vivo. Furthermore, in vivo inhibition of serine protease activity depressed cell cycle re-entry, which in turn could be rescued by cleavedmimic BMP. This work provides a new molecular mechanism for the reversal of the differentiated state in muscle. In Paper IV, we carried out a comparative analysis of centrosome dynamics in mouse and newt muscle cells. We showed, through a detailed characterization of different centrosome components, that centrosomes were gradually disassembled during muscle differentiation in mammals. We also provided new insights into the underlying mechanisms and variations in gene expression during that inactivation process. On the other hand, we found that salamanders retained several centrosome components even in mature myofibers. Moreover, we observed that not only the centrosomes were maintained in salamander muscle, but they also appeared to be active as microtubule organizing centers. This study has elucidated fundamental differences between vertebrates at cellular level, which might help us to understand why species differ in their ability to produce regenerative progenitor cells.