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Evolutionarily divergent mTOR remodels translatome for tissue regeneration

Nature volume 620, pages 163–171 (2023)Cite this article

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Abstract

An outstanding mystery in biology is why some species, such as the axolotl, can regenerate tissues whereas mammals cannot1. Here, we demonstrate that rapid activation of protein synthesis is a unique feature of the injury response critical for limb regeneration in the axolotl (Ambystoma mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components that are selectively activated at the level of translation from pre-existing messenger RNAs in response to injury. By contrast, protein synthesis is not activated in response to non-regenerative digit amputation in the mouse. We identify the mTORC1 pathway as a key upstream signal that mediates tissue regeneration and translational control in the axolotl. We discover unique expansions in mTOR protein sequence among urodele amphibians. By engineering an axolotl mTOR (axmTOR) in human cells, we show that these changes create a hypersensitive kinase that allows axolotls to maintain this pathway in a highly labile state primed for rapid activation. This change renders axolotl mTOR more sensitive to nutrient sensing, and inhibition of amino acid transport is sufficient to inhibit tissue regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in a highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential.

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Fig. 1: Protein synthesis activation on injury in axolotls but not in mice.
Fig. 2: Regenerative translation targets the dormant transcriptome for rapid translational remodelling.
Fig. 3: Injury is associated with rapid activation of mTOR signalling in the axolotl.
Fig. 4: mTOR activation promotes rapid wound closure and regeneration.
Fig. 5: Urodele-specific insertions give rise to hypersensitive mTOR kinase.

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Data availability

Raw sequencing data supporting this work is available at Gene Expression Omnibus (GSE185593), all other numerical data are in the accompanying source data files and all raw western blot data are included in a fully annotated file in Supplementary Data 1. Source data are provided with this paper.

Code availability

The R code used for polysome sequencing analysis is available on GitHub at https://github.com/barnalab/regeneration.

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Acknowledgements

We thank members of the Barna laboratory for critical feedback and discussion of this work; S. Arulmani and S. Stern for preliminary contributions; A. Valdefiera, S. Ahmadi, E. Martinez, S. Jensen and D. Chu of the Stanford Veterinary Services Center for animal husbandry and veterinary support; V. Natu and J. Coller of the Stanford Functional Genomics Facility for sequencing; A. Chekholko of the Stanford Genomics Center for IT system support; S. Floor (UC Berkley) for protocol assistance; R. Voss of the Ambystoma Genetic Stock Center (University of Kentucky) for training and materials. Funding: this work was supported by New York Stem Cell Foundation grant no. NYSCF-R-I36 (M.B.), New York Stem Cell Robertson Investigator (M.B.), NIH grant no. 1R01HD086634 (M.B.), Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) grant no. 1R01HD105731-01 (M.B.), Stanford Discovery Innovation Fund in Basic Biomedical Sciences (M.B.), Canadian Institutes of Health Research postdoctoral fellowship (O.Z.), K99/R00 Pathway to Independence Award NICHD grant no. 5K99HD099787-02 (O.Z.) and National Institutes of Health Developmental Biology training grant no. 5T32GM007790-41 (H.D.R.). O.Z. is a Simon’s fellow of the Helen Hay Whitney Foundation. M.B. is a NYSCF Robertson Investigator.

Author information

Author notes

  1. Olena Zhulyn

    Present address: Developmental and Stem Cell Biology Program, SickKids Research Institute, Toronto, Ontario, Canada

Authors and Affiliations

  1. Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA

    Olena Zhulyn, Hannah D. Rosenblatt, Leila Shokat, Zijian Zhang & Maria Barna

  2. Department of Developmental Biology, Stanford University, Stanford, CA, USA

    Hannah D. Rosenblatt

  3. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA

    Shizhong Dai, Davide Ruggero & Kevan M. Shokat

  4. Department of Urology, University of California, San Francisco, San Francisco, CA, USA

    Duygu Kuzuoglu-Öztürk & Davide Ruggero

  5. Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA

    Duygu Kuzuoglu-Öztürk & Davide Ruggero

  6. Department of Chemical and Systems Biology, Stanford University, Stanford, CA, USA

    Zijian Zhang

  7. Howard Hughes Medical Institute, University of California, San Francisco, CA, USA

    Kevan M. Shokat

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Contributions

O.Z. and M.B. conceived the project and designed experiments. M.B. supervised the project. O.Z. performed amputation, sucrose gradient fractionation, polysome sequencing and data analysis, western blot and co-immunoprecipitation analysis, drug administration studies, generation of chimeric cell lines by CRISPR–Cas9, amino acid titration, immunofluorescent staining on cells and tissues, multiple sequence alignments, OPP incorporation studies and training and supervision of H.D.R. and L.S. H.D.R. designed and performed ROS detection with INK128-incorporation, APO treatment and live imaging studies, carried out immunofluorescence imaging and data analysis and acquisition in cells and tissues. L.S. optimized mTOR imaging and generated preliminary data on lysosomal localization of mTOR. S.D. performed sequence analysis and structure modelling. Z.Z. generated a pipeline for semi-automated mTOR localization analysis. D.K.-O. performed cell viability assays and polysome qPCR. D.R. supervised D.K.-O. and provided critical feedback on experimental design. K.M.S. supervised S.D. and provided critical feedback and assistance with experimental design. O.Z. and M.B. wrote the manuscript with input from all the authors.

Corresponding author

Correspondence to Maria Barna.

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Competing interests

D.R. and K.M.S. are shareholders of eFFECTOR Therapeutics and members of its scientific advisory board. K.M.S. is an inventor on a patent (РСТ/US2009/005958) covering INK128 owned by the University of California. The remaining authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Rapid epithelial migration drives wound closure.

Snapshots of wound closure in a wildtype sub-adult axolotl imaged over the course of 24 h. Scale bar is 200 px (~0.90 mm).

Extended Data Fig. 2 Protein synthesis and proliferation in the mouse digit.

a, Schematic depicts location of proximal (black arrow) or distal amputation (red arrow). b, Representative images of mouse digits at 1, 4, 6, 8 and 12 days after amputation (at least 3 animals were imaged for each treatment and time-point). c, OPP incorporation in mice after distal and proximal amputation at 1 dpa. Inset illustrates elevated OPP incorporation at the cut site. Dashed line outlines digit of interest (doi). d, Quantitation of OPP signal in the doi shows no significant changes between controls (n = 7), proximal (n = 3) or distal amputations (n = 4) at 1 dpa. e, Quantitation shows elevated OPP signal in skin near the cut site after distal amputation (signal at cut site is compared to signal at base of digit for control (n = 4 individual mice) or distal amputations (n = 4 individual mice), both normalized to total OPP across digit). f, PH3 incorporation assessed at 1 dpa. g, Quantitation of PH3+ cells per tissue area (n = 4 individual mice per treatment) reveals increased proliferation after distal amputation. h, OPP incorporation assay at 4 dpa. i, Quantitation of OPP incorporation across the digit (dashed line) for n = 4 control, n = 5 distal and n = 4 proximal amputations from individual mice. j, PH3 incorporation assessed at 4 dpa. k, Quantitation of PH3+ cells per tissue area for control, distal and proximal amputations at 4 dpa in n = 4 individual mice per treatment. Statistical analysis was performed with one-way ANOVA. P values > 0.05 were considered not significant and are denoted by n.s. Mean ± s.d. are shown in all plots. Scale bar is 1 mm in 2b; 500 μm in 2c, f, h and j. In a–j, 1 digit was amputated per mouse and each n is an independent animal.

Source data

Extended Data Fig. 3 Polysome sequencing identifies translationally regulated mRNAs.

a, Scatterplot of 8,139 transcripts in our data set. The x-axis shows (log2) fold change (FC) in the heavy polysome fractions between 0 h and 24 h post-amputation. Y-axis shows (-log10) p-values (padj) adjusted for multiple-testing using the Benjamini-Hochberg method. Blue dots indicate transcripts with padj < 0.05 and two-fold change in the heavy polysome. b, The scatterplot from panel (a) colored to emphasize that transcripts with increased reads in heavy polysome include transcripts that are upregulated at the level of transcription (orange), translation (green) or both (pink) as defined in Fig. 2b, therefore change in heavy polysome on its own does not adequately describe the provenance of actively translated transcripts. As in b, p-values (padj) were adjusted for multiple-testing using the Benjamini-Hochberg method. c, Strong negative correlation between (log2) FC in TE on y-axis and (log2) FC in the free/RNP fraction. d, Poor correlation between (log2) FC in TE on y-axis and (log2) FC in mRNA abundance between 0 h and 24 h shown on x-axis. e, Distribution of 1,995 genes with annotated roles in “signaling” and “development” across expression categories defined in Fig. 2b. f, Overlay of our data with previously published single-cell RNA-Seq analysis10 suggests that translationally regulated mRNAs (green in Fig. 2b) are enriched in the skin. g, Gene Ontology (GO) enrichment analysis of transcript subsets defined in (Fig. 2b) reveals enrichment of immune processes in the orange category, h, enrichment of cell differentiation genes in the blue category, i, enrichment of developmental processes and signaling genes in the grey category, j, enrichment of translation and metabolic processes in the translationally activated (green) category. Box shows subset of transcripts enriched in the “translation” GO category. For g–j, the adjusted P values were determined using the Benjamini and Hochberg method. k, Distribution of orthologues of established TOP/PRTE-containing mTOR-sensitive genes in our data set shows significant enrichment within translationally regulated gene set based on a two-sided binomial test, n.s. is P  > 0.05 and is not statistically significant; exact P values are shown for comparison of ΔTE, ΔmRNA and “no change” categories to “all”.

Source data

Extended Data Fig. 4 Elevated expression of translationally activated targets.

a, Immunofluorescent staining of tissue sections highlights increased expression of translational targets ANXA1 and TXN in the axolotl basal epithelium at 0 hpa and wound epithelium (WE) at 24 hpa (n = 4 individual axolotls per time-point). Scale bar is 100 μm. b, Western blot of tissue harvested from the plane of amputation shows increased expression of translational targets SELENBP1, AGR and PRDX1 at 48 hpa (tissue harvested from n = 4 individual axolotls per time-point and processed independently). Student’s t-test, two-tailed, was used to assess significance in adjacent graphs, n.s. indicates P > 0.05 and deemed not significant. Mean ± s.d. shown in all plots.

Source data

Extended Data Fig. 5 Rapid activation of mTOR signaling after amputation.

a, Limb tissue lysates were harvested at indicated time-points after amputation. Each lane contains tissue from an individual animal assessed by western blot for changes in mTOR activity with antibodies against total and phosphorylated RPS6. b, Quantitation of P-RPS6Ser235/236 and c, quantitation of P-RPS6Ser240/244 each normalized to total RPS6 and ß-actin. d, Immunofluorescent staining of P-RPS6Ser240/244 is elevated throughout limb and particularly in WE at 24 hpa shown in tiled images of whole limb (blue and orange boxes show region of WE enlarged in panel e). e, in the wound epithelium, f, skin, g, bone, h, muscle. Images in d–h are representative of tissue staining from n = 3 individual axolotls. Scale bars are all 500 μm in 5d; 100 μm in 5e, g, and h. i, Western blot of total and phosphorylated 4EBP1. j, Quantitation of P-4EBP1Thr37/46, k, P-4EBP1Thr37 and l, P-4EBP1Thr70, each normalized to total 4EBP1 and ß-actin. In b-c, j–l, statistical significance was assessed with one-way ANOVA for cumulative data from western blots shown here in a, i and in Fig. 3a, b. Complete raw blots are in Supplementary Data 1. A P value < 0.05 was deemed significant. Mean ± s.d. shown in all plots. In b-c and j, we examined tissues from n = 13 individual axolotls at 0 h and tissues from n = 6 individual axolotls per time-point at 2 h, 12 h, and 24 h (18 axolotls in total). In k-l above, we examined tissues from n = 8 individual axolotls at 0 h and tissues from n = 3 axolotls per time-point at 2 h, 12 h and 24 h. m, Schematic of injection and amputation. n, For DMSO vs. INK128 treatment comparison, P-RPS6Ser235/236 levels are quantified in o, at 0 hpa or 24 hpa. p, P-RPS6Ser240/244 is quantified at 0 hpa or 24 hpa. q, P-4EBP1Thr37/46 is quantified at 0 hpa or 24 hpa. Arrow points to P-4EBP2 band. “unt.” refers to non-injected animals. For o-q exact p-values are shown above bars. r, Translationally regulated proteins are quantified in s, green asterisks indicate significant target increase (SELENBP1: P = 0.003, TXN: P = 0.04, ANXA1: P = 0.0001) for DMSO controls at 48 vs. 0 hpa (green dash). For DMSO vs. INK128 at 48 hpa, Pvalues are > 0.05 and deemed not significant (n.s.). In a, i, n and r membranes were sequentially blotted and share ß-actin which is reproduced with accompanying targets for clarity (see Supplementary Data 1 for raw gels). N = 3 animals were examined for each condition (except DMSO and INK128 at 24 hpa shown in o, q where n = 4 each). All plots show mean +/- s.d., significance assessed with Student’s t-test, two-tailed, where * is P < 0.05, ** is P < 0.01, *** is P < 0.001, **** is P < 0.0001 and n.s. is P > 0.05 and not significant. All bands are normalized to their own ß-actin.

Source data

Extended Data Fig. 6 Apocynin and INK128 treatments inhibit regeneration.

a, Axolotls were pre-treated by immersion in tank water containing 10 μM Rapamycin for 14 h (“pre-treatment”, n = 3). Limbs were amputated at the forelimb and animals were placed in fresh tank water with a high concentration of Rapamycin (5–10 μM, n = 3) for 6 h. b, Imaging reveals poor wound closure in animals treated with high concentrations of Rapamycin at 6 hpa. c, Tissue harvested from plane of amputation exhibits reduced mTORC1 activation at 6 hpa (n = 3 with Rapamycin at 5–10 μM and n = 3 DMSO carrier). Yellow arrows point to exposed bone. d, Quantitation of phenotypes observed – 3/3 axolotls treated with high concentration of Rapamycin failed to close the wound at 6 hpa. e, Western blot and f, quantitation of P-AktSer473 normalized to Akt and ß-actin. In f, statistical significance was assessed with one-way ANOVA. Complete raw blots are in Supplementary Data 1. A P value < 0.05 was deemed significant. Mean ± s.d. shown in all plots. g, Representative images of DMSO and Apocynin (APO) treated axolotls reveal reduced blastema size (dashed line) and delayed regeneration in drug-treated animals. h, Reduction of blastema size relative to the limb area is shown. An n of 3 animals was used for each condition. Significance was assessed with Student’s t-test two-tailed, and a P value < 0.05 was deemed significant. i, Additional representative images of control limbs at 36 hpa show presence of dim and bright ROS+ cells stained with H2DCFDA reported in Fig. 4h, i in which 789 cells analyzed from n = 4 DMSO-treated axolotls; 946 cells analyzed from n = 5 INK128-treated axolotls. j, Representative images of limb regeneration in axolotls treated with DMSO or INK128 4h before amputation performed on n = 3 independent axolotls. The INK128-treated animals tracked in this analysis all belong to the 37.5% of animals with partial wound closure at 24 h post-amputation referred to in Fig. 4a, b. Mean ± s.d. shown in all plots. Scale bars are 200 px (~1.65 mm) in 6b, i; 1000 μm in 6g; 50 μm in 6j.

Source data

Extended Data Fig. 7 Protein expression in axolotls and mice.

a, Short (~1ms) and b, long exposures are provided for the blots shown in Fig. 5a to illustrate that differences in mTOR responsiveness to amputation are specific to axolotls independent of exposure time.

Extended Data Fig. 8 Altered mTOR activity in axolotls and mice.

a, Western blot depicts mTOR activity measured as phosphorylation of P-4EBP1Thr37/46 and P-RPS6Ser235/236 at 0 h in n = 3 individual mice and 2 h post-amputation in n = 3 individual mice. Each lane represents a distinct pool of digit tips harvested at given time-point. Quantitation shows ratio of phosphorylated to total protein normalized to ß-actin. P-4EBP1Thr37/46 and RPS6 were sequentially blotted on the same membrane and share ß-actin blot. 4EBP1 and P-RPS6Ser235/236 were sequentially blotted on another membrane and share ß-actin. b, Immunofluorescent staining of mouse digits at 0 and 24 hpa reveals no changes in P-RPS6Ser240/244 expression after proximal amputation performed on tissues from n = 3 individual mice. c, Schematic depicts amino acid dependent translocation of mTORC1 to the lysosome is required for pathway activation. d, Rab7 (red), mTOR (green) and nuclear stain (DAPI/blue) in axolotl tissues at 24 h post-amputation illustrate co-localization of mTOR to lysosomes, n = 2 individual axolotls. e, Immunofluorescence staining of axolotl tissues depicts punctate mTOR localization (white/grey) and nuclei (DAPI/blue) in cells of the skin, muscle and bone near the wound site at 0, 2, 12, and 24 h post-amputation (hpa). Tissues from n = 1 axolotl per time-point. Mean ± s.d. are shown in all plots. Scale bar is 500 μm in 8b; 25 μm in 8d, e. Student’s t-test, two-tailed, was used to assess significance in a, and P > 0.05 deemed not significant (n.s.).

Source data

Extended Data Fig. 9 Axolotl insertions promote lysosomal retention of mTOR.

a, Schematic depicts/Cas9 targeting strategy to introduce axolotl insert 1 in-frame into exon 20 of human mTOR in HEK 293T cells. gRNA (guide RNA site), HA (homology arm), PAM (protospacer adjacent motif) MT PAM (mutated protospacer motif). b, Schematic depicts CRISPR/Cas9 strategy to introduce axolotl insert 2 in-frame into exon 21 of human mTOR in HEK 293T cells. c, Western blot analysis illustrates steady-state expression of mTOR protein at steady state in wildtype HEK 293T cells and HEK 239TaxmTOR, n = 3 independent experiments. d, Immunofluorescence staining of mTOR (green), lysosomes (Lysotracker in magenta) and nuclei (DAPI) in wildtype and HEK 239TaxmTOR cells at steady state, upon starvation (-), and stimulation. Scale bar is 25 μm. e, Quantitation of mean mTOR intensity within lysosomes. For each cell line and condition, the lysosomal intensity is expressed relative to the mean steady state intensity for that specific cell line. Two independently derived cell lines were examined for each genotype. The following cell numbers were pooled by treatment or genotype after three independent experiments (WT at steady state from parental WT line or A3 wildtype clone (70 cells), starved (71 cells), fed (81 cells) and HEK 239TaxmTOR cells from C1 or A2 double mutant clones at steady state (103 cells), starved (75 cells), fed (102) cells. Significance assessed with one-way ANOVA (P < 1.0E-15). Exact adjusted P values (Tukey’s multiple comparison test, 95% CI) for individual multiple comparisons are shown in the figure. Mean ± s.d. are shown in all plots.

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Extended Data Fig. 10 Axolotl insertions promote increased nutrient sensitivity.

a, Representative western blot (n = 3) of amino acid (AA) titration experiment illustrates greater sensitivity of P-4EBP1Thr37/46 phosphorylation in HEK 293TaxmTOR to amino acid concentration. b, Quantitation P-4EBP1Thr37/46 level normalized to ß-actin. c, Representative western blot (n = 3) of AA titration illustrates greater sensitivity of P-RPS6Ser240/244 phosphorylation in response to change to AA levels. e, Quantitation of P-RPS6Ser235/236 level and f, P-4EBP1Thr37/46 level each normalized to ß-actin in wildtype and HEK 239TaxmTOR and HEK 239TaxmTOR cells in 2 independent experiments and normalized to the control (target protein level in wildtype HEK293T cells at steady state). Data illustrates that axolotl mTOR is better at sensing nutrient withdrawal over time. g, Graphs depict the level of a given mRNA detected by qPCR in pooled free/RNP, light or heavy polysome fractions 6 h after re-feeding of starved wildtype or HEK293TaxmTOR cells. A significant shift from the free-fraction to the heavy polysome was observed for RPL19. h, no change was observed for CIRBP (n.s.), i, a significant shift was observed for ß-actin. For g-i, the experiment was performed three times and data represent n = 3 independent replicates. Significance was assessed with two-way ANOVA and exact p-values are indicated on the graphs. A P value < 0.05 was deemed significant. Mean ± s.d. are shown in all plots.

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Supplementary information

Reporting Summary

Supplementary Data 1

Raw western blot data. This .pdf contains all fully annotated raw western blots analysed in this paper as well as any replicate blots whose data were used in the analysis. All western blots contain molecular weight annotations and ROI (as shown in figures) are indicated.

Supplementary Data 2

Translational remodelling revealed by polysome sequencing. This Excel file contains expression information for all 8,139 transcripts in our dataset. The change (Δ) in total mRNA abundance, change (Δ) in free/RNP fraction enrichment and change (Δ) in TE between 0 and 24 hpa is shown for all 8,139 mRNAs in our dataset (ALL sheet). Individual spreadsheets show subsets of data as defined and colour coded in Fig. 2b,c of the paper. Further, P values adjusted for multiple testing following the method of Benjamini–Hochberg are provided under column headings FDR ΔmRNA, FDR Δfree/RNP and FDR ΔTE.

Supplementary Data 3

GO enrichment analysis of translationally and transcriptionally regulated genes. This Excel file contains the full list of biological function categories significantly enriched within each gene set as defined in Fig. 2b,c and Extended Data Fig. 3g–j. Significance was assessed using the Benjamini–Hochberg method for multiple testing and adjusted P values are shown in the table. An adjusted P < 0.05 was deemed statistically significant, exact P values are included in the file.

Supplementary Data 4

Orthologues of established mTORC1 targets. This Excel file describes the set of 101 established mammalian mTORC1 targets and the distribution of their axolotl orthologues in our dataset. The Group column refers to the regulatory status as defined by colour groups in Fig. 2b,c of the paper.

Supplementary Data 5

Conservation of the mTORC1 pathway. This Excel file shows the percentage of amino acid identity between core components of the mTORC1 pathway in axolotls, humans and mice.

Supplementary Data 6

Full-length mTOR alignment across 50 species. This .pdf contains a multiple sequence alignment of the full-length mTOR kinase amino acid sequence across 50 species.

Supplementary Data 7

Conservation of mTOR in region of insert 1 and 2. This .pdf contains a multiple sequence alignment across more than 100 metazoan species in the region of insert 1 and insert 2 of mTOR highlighting that the former is found exclusively in amphibians and the latter is found exclusively in salamanders, including the axolotl.

Supplementary Video 1

Live imaging of wound closure in DMSO-treated controls. This is a representative video showing progression of wound closure in a DMSO-treated control animal after amputation. Still captures from this video are shown in Fig. 4c.

Supplementary Video 2

Live imaging of wound closure in INK128-treated axolotls. This is a representative video showing progression of wound closure in an INK128-treated animal after amputation. Still captures from this video are shown in Fig. 4c.

Source data

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Zhulyn, O., Rosenblatt, H.D., Shokat, L. et al. Evolutionarily divergent mTOR remodels translatome for tissue regeneration. Nature 620, 163–171 (2023). https://doi.org/10.1038/s41586-023-06365-1

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