Molecular mechanisms of salamander limb regeneration

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. 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. 7