Abstract
Background: Tadpoles of the clawed frog Xenopus laevis can regenerate their tails following partial amputation, replacing the missing spinal cord, muscles, and fin. However, for a brief period of development this response becomes unstable, leading to a proportion of tadpoles that undergo wound healing rather than regenerative programme. Inspired by a growing number of links between the microbiome and human inflammatory disease, we asked how the tadpole skin microbiome and innate immunity influence the regeneration of a complex appendage. We previously showed that tadpoles raised in antibiotics such as gentamicin or penicillin/streptomycin or with reduced Toll-like receptor 4 signaling regenerated tails poorly, while adding exogenous lipopolysaccharide promoted or rescued tail regeneration.
Results: Here, we show that CRISPR/Cas9 knockdown of Toll-like receptor 2 also reduces tadpole tail regeneration. Conversely, addition of the pathogen-associated molecular pattern peptidoglycan to the medium at the time of amputation increases the likelihood of regeneration. While we have previously shown that tadpoles acquire most of their early skin microbiome from their mothers, analysis of mitochondrial haplotypes did not support a genetic maternal explanation of regenerative bias. Levels of endogenous lipopolysaccharides on tail tips were also not predictive of regenerative success, and shotgun sequencing indicated that there was no difference in bacterial loads. To see if the composition of native microbiome was associated with regenerative success, we sequenced the 16S rRNA genes of 503 tadpole tail tips from 12 sibships and mapped these to the regenerative outcome of each tadpole. While no one taxon was found to be associated with regenerative success, higher proportions of Gram-positive genera overall correlated with improved regeneration outcomes. Supporting this finding, when tadpoles were raised with the antibiotic vancomycin, to select against Gram-positive bacteria, the number of individuals undergoing tail regeneration was significantly decreased.
Conclusions: Taken together, our results suggest a previously undocumented role for Tlr2, possibly activated by peptidoglycan from Gram-positive commensal skin bacteria, in tipping the balance from wound repair to regenerative programmes in Xenopus laevis refractory stage tadpoles.