Chapman PA et al. (2022), Manipulating the microbiome alters regenerative...

XB-ART-59048

Wound Repair Regen 2022 Nov 01;306:636-651. doi: 10.1111/wrr.13003.

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Manipulating the microbiome alters regenerative outcomes in Xenopus laevis tadpoles via lipopolysaccharide signalling.

Chapman PA , Gilbert CB , Devine TJ , Hudson DT , Ward J , Morgan XC , Beck CW .

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Xenopus laevis tadpoles can regenerate functional tails, containing the spinal cord, notochord, muscle, fin, blood vessels and nerves, except for a brief refractory period at around 1 week of age. At this stage, amputation of the tadpole's tail may either result in scarless wound healing or the activation of a regeneration programme, which replaces the lost tissues. We recently demonstrated a link between bacterial lipopolysaccharides and successful tail regeneration in refractory stage tadpoles and proposed that this could result from lipopolysaccharides binding to Toll-like receptor 4 (TLR4). Here, we have used 16S rRNA sequencing to show that the tadpole skin microbiome is highly variable between sibships and that the community can be altered by raising embryos in the antibiotic gentamicin. Six Gram-negative genera, including Delftia and Chryseobacterium, were over-represented in tadpoles that underwent tail regeneration. Lipopolysaccharides purified from a commensal Chryseobacterium spp. XDS4, an exogenous Delftia spp. or Escherichia coli, could significantly increase the number of antibiotic-raised tadpoles that attempted regeneration. Conversely, the quality of regeneration was impaired in native-raised tadpoles exposed to the antagonistic lipopolysaccharide of Rhodobacter sphaeroides. Editing TLR4 using CRISPR/Cas9 also reduced regeneration quality, but not quantity, at the level of the cohort. However, we found that the editing level of individual tadpoles was a poor predictor of regenerative outcome. In conclusion, our results suggest that variable regeneration in refractory stage tadpoles depends at least in part on the skin microbiome and lipopolysaccharide signalling, but that signalling via TLR4 cannot account for all of this effect.

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Species referenced: Xenopus laevis
Genes referenced: cda mrc1
GO keywords: lipopolysaccharide binding [+]

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	Figure 1. The tadpole tail skin microbiome varies between sibships and can be altered dramatically by raising tadpoles in antibiotics. (A) Schematic of the experimental design. Three sibships of 4-cell embryos were randomly assigned to gentamicin-treated and control groups. Tail samples for microbiome analysis were obtained at stage 46 from two replicate cohorts of 24 tadpoles for each treatment and sibship. Tadpoles were scored for regeneration 7 days after tail amputation. (B) Regeneration data from three tadpole sibships. Each point represents the percentage of tadpoles regenerating any tissue at all, is the sum of full, partial good and partial bad tadpoles and is a replicate Petri dish with sample size of 24 tadpoles per dish, with the exception of controls for Sibship B where N = 22 as two died in each before they could be scored for regeneration. Unpaired t-tests, p < .05, p < .01. (B0) Stacked categorical graphs comparing regeneration phenotypes for each sibship. Linear-by-Linear association test, ***p < .0001. (C) Principal coordinates analysis (PCoA) ordination plot of tadpole tail samples with >1500 reads, calculated based on Bray–Curtis distance. (D) Pie charts showing the percentage of Gram-negative versus Gram-positive annotated reads for each sibship when raised with or without gentamicin. (E) Relative abundance of the 10 most abundant bacterial classes in tadpole tail skin, stratified by sibship and treatment status. Raw data can be found in Supporting information S1 [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 2. Genus-level interactions between sibship, antibiotic treatment and regeneration. (A) The relative abundance of genera within the five most abundant bacterial classes in treated and untreated sibships, highlighting the three most abundant genera in each. Read counts were rarefied to 1500 reads. (B) Violin plots show log-transformed relative abundance (y-axis) of six genera positively associated with regeneration (q < 0.01, Benjamini–Hochberg false discovery correction) stratified by gentamicin status (colour). Raw data can be found in the Supporting information S1 [Color figure can be viewed at wileyonlinelibrary.com]

	Figure 4. LPS from the commensal bacterium Chryseobacterium spp. XDS4 rescues regeneration in stage 46 tadpoles raised in the antibiotic gentamicin (gent). (A) Timeline of treatments. (B–D) Data from three sibships of tadpoles. Each point represents the percentage of tadpoles regenerating any tissue at all, is the sum of full, partial good and partial bad tadpoles and is a replicate Petri dish with sample size of 38-48 (A), 35-43 (B) or 23-43 tadpoles per dish. (C) One-way ANOVA with Tukey post hoc comparisons of all means. p < .05, p < .01, p < .001, **p < .0001. (A0–C0) are stacked categorical graphs of the same tadpoles, showing the proportion of each phenotype by dish. Compact letter display has been used to indicate statistical significance; each treatment is assigned a letter, with treatments within the same letter group having no statistically significant difference from each other. Extended Cochran–Armitage test, followed by post hoc pairwise ordinal independence test with Benjamini–Hochberg correction. Raw data can be found in the Supporting information S1 [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 5. LPS from an exogenous Delftia spp. rescues regeneration in stage 46 tadpoles raised in the antibiotic gentamicin (gent). Timeline of treatments as for Figure 4A. (A–C) represent data from three sibships of tadpoles. Each point represents the percentage of tadpoles regenerating any tissue at all, is the sum of full, partial good and partial bad tadpoles and is a replicate Petri dish with sample size of 32-65 (A), 23-60 (B) or 40-65 tadpoles per dish (C). One-way ANOVA with Tukey post hoc comparisons of all means. p < .05, p < .01, p < .001, **p < .0001. (A0– C0) are stacked categorical graphs of the same tadpoles, showing the proportion of each phenotype by dish. Compact letter display has been used to indicate statistical significance; each treatment is assigned a letter, with treatments within the same letter group having no statistically significant difference from each other. Extended Cochran–Armitage test, followed by post hoc pairwise ordinal independence test with Benjamini–Hochberg correction. Raw data can be found in the Supporting information S1 [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 6. Rhodobacter sphaeroides LPS, a TLR4 antagonist, can significantly reduce regeneration quality, but not quantity. (A) Timeline of treatments. Exposure of the cut tail stump to agonistic LPS should enhance regeneration in antibiotic-raised tadpoles, as in Figures 2–4, and antagonistic LPS (RS-LPS) is expected to reduce regeneration in naturally raised tadpoles. (B and C) Scatterplots where each point represents the percentage of tadpoles regenerating any tissue at all, is the sum of full, partial good and partial bad tadpoles and is a replicate Petri dish with sample size of N = 15 (B) or 11-17 tadpoles per dish (C). (B) 50 μg/ml ultrapure RS-LPS (Invivogen) treatment versus controls. Unpaired t-test showed no significant difference between groups. (C) Post-amputation treatment with three concentrations of antagonistic-extracted RS-LPS was compared to control tadpoles and gentamicin-treated tadpoles with or without E. coli LPS rescue. One-way ANOVA with Tukey post hoc comparisons of all means showed no significant differences between groups. (B0 and C0) are stacked categorical graphs of the same tadpoles, showing the proportion of each phenotype by dish. Compact letter display has been used to indicate statistical significance; each treatment is assigned a letter, with treatments within the same letter group having no statistically significant difference from each other. Extended Cochran– Armitage test, followed by post hoc pairwise ordinal independence test with Benjamini–Hochberg correction. *p < .05. Raw data can be found in the Supporting information S1 [Color figure can be viewed at wileyonlinelibrary.com]

References [+] :

Adams, H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration. 2007, Pubmed, Xenbase

	Figure 1. The tadpole tail skin microbiome varies between sibships and can be altered dramatically by raising tadpoles in antibiotics. (A) Schematic of the experimental design. Three sibships of 4-cell embryos were randomly assigned to gentamicin-treated and control groups. Tail samples for microbiome analysis were obtained at stage 46 from two replicate cohorts of 24 tadpoles for each treatment and sibship. Tadpoles were scored for regeneration 7 days after tail amputation. (B) Regeneration data from three tadpole sibships. Each point represents the percentage of tadpoles regenerating any tissue at all, is the sum of full, partial good and partial bad tadpoles and is a replicate Petri dish with sample size of 24 tadpoles per dish, with the exception of controls for Sibship B where N = 22 as two died in each before they could be scored for regeneration. Unpaired t-tests, p < .05, p < .01. (B0) Stacked categorical graphs comparing regeneration phenotypes for each sibship. Linear-by-Linear association test, ***p < .0001. (C) Principal coordinates analysis (PCoA) ordination plot of tadpole tail samples with >1500 reads, calculated based on Bray–Curtis distance. (D) Pie charts showing the percentage of Gram-negative versus Gram-positive annotated reads for each sibship when raised with or without gentamicin. (E) Relative abundance of the 10 most abundant bacterial classes in tadpole tail skin, stratified by sibship and treatment status. Raw data can be found in Supporting information S1 [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 2. Genus-level interactions between sibship, antibiotic treatment and regeneration. (A) The relative abundance of genera within the five most abundant bacterial classes in treated and untreated sibships, highlighting the three most abundant genera in each. Read counts were rarefied to 1500 reads. (B) Violin plots show log-transformed relative abundance (y-axis) of six genera positively associated with regeneration (q < 0.01, Benjamini–Hochberg false discovery correction) stratified by gentamicin status (colour). Raw data can be found in the Supporting information S1 [Color figure can be viewed at wileyonlinelibrary.com]

	Figure 4. LPS from the commensal bacterium Chryseobacterium spp. XDS4 rescues regeneration in stage 46 tadpoles raised in the antibiotic gentamicin (gent). (A) Timeline of treatments. (B–D) Data from three sibships of tadpoles. Each point represents the percentage of tadpoles regenerating any tissue at all, is the sum of full, partial good and partial bad tadpoles and is a replicate Petri dish with sample size of 38-48 (A), 35-43 (B) or 23-43 tadpoles per dish. (C) One-way ANOVA with Tukey post hoc comparisons of all means. p < .05, p < .01, p < .001, **p < .0001. (A0–C0) are stacked categorical graphs of the same tadpoles, showing the proportion of each phenotype by dish. Compact letter display has been used to indicate statistical significance; each treatment is assigned a letter, with treatments within the same letter group having no statistically significant difference from each other. Extended Cochran–Armitage test, followed by post hoc pairwise ordinal independence test with Benjamini–Hochberg correction. Raw data can be found in the Supporting information S1 [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 5. LPS from an exogenous Delftia spp. rescues regeneration in stage 46 tadpoles raised in the antibiotic gentamicin (gent). Timeline of treatments as for Figure 4A. (A–C) represent data from three sibships of tadpoles. Each point represents the percentage of tadpoles regenerating any tissue at all, is the sum of full, partial good and partial bad tadpoles and is a replicate Petri dish with sample size of 32-65 (A), 23-60 (B) or 40-65 tadpoles per dish (C). One-way ANOVA with Tukey post hoc comparisons of all means. p < .05, p < .01, p < .001, **p < .0001. (A0– C0) are stacked categorical graphs of the same tadpoles, showing the proportion of each phenotype by dish. Compact letter display has been used to indicate statistical significance; each treatment is assigned a letter, with treatments within the same letter group having no statistically significant difference from each other. Extended Cochran–Armitage test, followed by post hoc pairwise ordinal independence test with Benjamini–Hochberg correction. Raw data can be found in the Supporting information S1 [Color figure can be viewed at wileyonlinelibrary.com]
	Figure 6. Rhodobacter sphaeroides LPS, a TLR4 antagonist, can significantly reduce regeneration quality, but not quantity. (A) Timeline of treatments. Exposure of the cut tail stump to agonistic LPS should enhance regeneration in antibiotic-raised tadpoles, as in Figures 2–4, and antagonistic LPS (RS-LPS) is expected to reduce regeneration in naturally raised tadpoles. (B and C) Scatterplots where each point represents the percentage of tadpoles regenerating any tissue at all, is the sum of full, partial good and partial bad tadpoles and is a replicate Petri dish with sample size of N = 15 (B) or 11-17 tadpoles per dish (C). (B) 50 μg/ml ultrapure RS-LPS (Invivogen) treatment versus controls. Unpaired t-test showed no significant difference between groups. (C) Post-amputation treatment with three concentrations of antagonistic-extracted RS-LPS was compared to control tadpoles and gentamicin-treated tadpoles with or without E. coli LPS rescue. One-way ANOVA with Tukey post hoc comparisons of all means showed no significant differences between groups. (B0 and C0) are stacked categorical graphs of the same tadpoles, showing the proportion of each phenotype by dish. Compact letter display has been used to indicate statistical significance; each treatment is assigned a letter, with treatments within the same letter group having no statistically significant difference from each other. Extended Cochran– Armitage test, followed by post hoc pairwise ordinal independence test with Benjamini–Hochberg correction. *p < .05. Raw data can be found in the Supporting information S1 [Color figure can be viewed at wileyonlinelibrary.com]