Cellular principles of Xenopus appendage regeneration

Unlike humans, some animals can regenerate their lost appendages, such as limbs and tails. To identify cellular principles driving this ability, I investigated Xenopus laevis tadpoles. Tadpoles can regrow their lost tails and limbs only at specific developmental stages, allowing comparative studies to determine principles of regeneration. I first compared tail amputation responses of regeneration-competent and regeneration- incompetent tadpoles by using single-cell transcriptomics. This approach allowed me to identify a previously uncharacterised cell population that we termed Regeneration-Organising- Cells (ROCs). Upon tail amputation, ROCs are relocated from the body to the amputation plane only in regeneration-competent tadpoles. ROCs secrete multiple growth factors for the underlying stem/progenitor cell types to proliferate and grow a new tail. Meanwhile, ROCs in regeneration-incompetent tadpoles are not able to relocalise on the amputation plane, and regeneration is stalled. Eliminating ROCs block regeneration, and grafting ROC-containing tissues enable ectopic-outgrowths. Identification of ROCs suggests that signalling centres and stem/progenitor cells in their proximity are critical requirements to build a lost appendage. Next, I found that the immune system of regeneration-competent tadpoles use reparative myeloid lineage cells that have an impact on the environmental changes, allowing ROCs to relocalise to the amputation plane. Meanwhile, in the regeneration-incompetent tadpoles, inflammatory myeloid lineage cells prevent ROCs' relocalisation. These findings suggest a regeneration-permissive environment is also required to regenerate an appendage. Finally, I focused on Xenopus limb regeneration by comparing limb amputation responses of various regeneration-competency stages by single-cell transcriptomics. Limb regeneration ability is correlated with the re-deployment of a developmental signalling centre cell type, apical-ectodermal-ridge (AER) cells. By using ex vivo regenerating limb cultures, I then demonstrated that, during tadpole development, there is an enrichment of mature chondrogenic lineage cells which express inhibitory secreted factors, including NOGGIN, blocking AER cell formation. Inhibitory factors can be overridden by the application of FGF10 which operates upstream of the effect of NOGGIN, prevents chondrogenesis, and restores regeneration. As mammals have AER in their embryonic development, revealing their connection to chondrogenesis opens new avenues for research aiming to regenerate mammalian limbs. Comparing cellular views of two appendage regeneration scenarios suggests appendage regeneration is context-dependent at the cellular level. Nonetheless, Xenopus appendage regeneration requires the presence of three main cellular principles: (1) Signalling centres (e.g. ROCs, AER cells) to instruct the growth of the surrounding cells, (2) Stem and progenitor cell types which will be the building blocks of a new appendage (e.g. existing stem/progenitor cells, injury-induced plastic cells), (3) A regeneration-permissive-environment (e.g. immune system, secreted factors).