Absence of a specialized wound epidermis is hypothesized to block limb regeneration in higher vertebrates. However, the factors preventing its formation in regeneration-incompetent animals are poorly understood. To characterize the endogenous molecular and cellular regulators of specialized wound epidermis formation in Xenopus laevis tadpoles, and the loss of their regeneration-competency during development, we used single-cell transcriptomics and ex vivo regenerating limb cultures. Transcriptomic analysis revealed that the specialized wound epidermis is not a novel cell state, but a re-deployment of the apical-ectodermal-ridge (AER) program underlying limb development. Enrichment of secreted inhibitory factors, including Noggin, a morphogen expressed in developing cartilage/bone progenitor cells, are identified as key inhibitors of AER cell formation in regeneration-incompetent tadpoles. These factors can be overridden by Fgf10, which operates upstream of Noggin and blocks chondrogenesis. These results indicate that manipulation of the extracellular environment and/or chondrogenesis may provide a strategy to restore regeneration potential in higher vertebrates.
We thank Katarzyna Kania and the Cambridge Institute Genomics Core for their support with this work on the 10X-Genomics and sequencing library preparations. The transgenic testes used in this study was obtained from the European Xenopus Resource Centre, curated with funding from the Wellcome Trust/BBSRC, and maintained by the University of Portsmouth, School of Biological Sciences. We thank R. Jones-Green for excellent animal care. We thank H. Ma for use of her stereomicroscope, and E. Rawlins for use of her Zeiss Axiolmager compound microscope. We thank B. Steventon, E. Rawlins, and members of the Marioni and Simons labs for general discussion about the project. We thank R. Butler for assistance in image analysis.
C.A. is funded by University of Cambridge and Cambridge Trust. J.J. and J.B.G. are funded by a grant from the Wellcome Trust (101050/Z/13/Z). T.W.H., J.C.M., and B.D.S. are funded as part of a Wellcome Strategic Award to study cell fate decisions (105031/D/14/Z). T.W.H. is also supported by an EMBO Long-Term Fellowship (ALTF 606-2018). B.D.S. also acknowledges funding from the Royal Society E.P. Abraham Research Professorship (RP\R1\180165) and Wellcome Trust (098357/Z/12/Z). J.C.M. acknowledges core funding from the European Molecular Biology Laboratory and Cancer Research UK (A17197). This work is funded by a grant from the Wellcome Trust (101050/Z/13/Z), Molecular Research Council (MR/P000479/1), and supported by the Gurdon Institute core grant from Cancer Research UK (C6946/A14492) and the Wellcome Trust (092096/Z/10/Z).
- Limb regeneration
- Ex vivo limbs