Secreted inhibitors drive the loss of regeneration competence in Xenopus limbs

Abstract

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.

Appendage regeneration is context dependent at the cellular level

“…Xenopus is the only commonly used model organism that has a clearly identified developmental AER and limb regeneration-associated AEC [ 31 , 44 ]. By using Xenopus and scRNA-seq, the cellular composition of the AEC and the AER tissues and their molecular similarities were found to be almost identical [ 45 ]. This suggests that the AEC is a re-deployment of the developmental AER transcriptional programme.…”

“…Recently, an epithelial cell type that is termed regeneration-organizing cells (ROCs) was found to be the cell type defining the specialized wound epidermis during Xenopus tail regeneration [ 19 ]. Afterwards, ROCs and AER cells were found to have differences in their transcriptome, single-cell morphology and cellular mechanisms [ 45 ]. Specifically, while ROCs are present in the body before tail amputation and their relocation to the amputation plane is critical for regeneration [ 19 ], AER cells have to be specified during regeneration [ 45 ] ( figure 2 ).…”

“…Afterwards, ROCs and AER cells were found to have differences in their transcriptome, single-cell morphology and cellular mechanisms [ 45 ]. Specifically, while ROCs are present in the body before tail amputation and their relocation to the amputation plane is critical for regeneration [ 19 ], AER cells have to be specified during regeneration [ 45 ] ( figure 2 ). While FGF inhibition does not significantly affect ROCs relocalization to the tail amputation plane to act as a specialized wound epidermis [ 19 ], it impairs AER cell specification during limb regeneration [ 45 ] ( figure 2 ).…”

“…Specifically, while ROCs are present in the body before tail amputation and their relocation to the amputation plane is critical for regeneration [ 19 ], AER cells have to be specified during regeneration [ 45 ] ( figure 2 ). While FGF inhibition does not significantly affect ROCs relocalization to the tail amputation plane to act as a specialized wound epidermis [ 19 ], it impairs AER cell specification during limb regeneration [ 45 ] ( figure 2 ). Although both ROCs and AER cells elicit the properties of the specialized wound epidermis, they do not compose the whole epidermal tissue at the amputation plane ( figure 2 ).…”

Distinct gene expression dynamics in developing and regenerating limbs

“…Several studies provide evidence of significant similarities in the patterning mechanisms that operate during development and regeneration, in axolotl limbs (e.g. Muneoka andBryant 1982, Roensch et al 2013;reviewed in Nacu and Tanaka 2011) and in other systems (Aztekin et al 2021, Czarkwiani et al 2021, while others point to significant differences (Fan et al 2012, Bosch et al 2010, Warner et al 2019, Khan and Crawford 2020.…”