Spatial transcriptomics reveals metabolic changes underly age-dependent declines in digit regeneration
- Department of Orthopaedics, Johns Hopkins University, United States
- Department of Surgery, Tulane School of Medicine, United States
- Department of Mathematics, Tulane University, United States
- Department of Biological & Agricultural Engineering, Louisiana State University, United States
- Center for Molecular Medicine, Maine Medical Center Research Institute, United States
- Department of Orthopaedic Surgery, Louisiana State University Health Sciences Center, United States
Figures
Figure 1 with 3 supplements
Transcriptional profiling of the blastema using spatial transcriptomics.
(A) Representative spatial gene expression of epithelial (Krt14), bone (Bglap2), blastema (Pdgfra, Mest), and hematopoietic cell (CD45, encoded by Ptprc) transcripts. (B) Manual segmentation of …
Figure 1—figure supplement 1
Overview of spatial transcriptomics approach.
(A) Fresh frozen samples are sectioned onto slides, subjected to a modified hematoxylin and eosin (H&E) and imaged. Optimization of enzymatic permeabilization, visualized through incorporation of …
Figure 1—figure supplement 2
Exclusion of cell cycling cells identified in fibroblast cluster 8 from Johnson et al., 2020, improves labeling proficiency of the spatial blastema footprint.
Module scoring of the blastema signature derived from all fibroblast-like cell populations from Johnson et al. mapped to spatial data (left), or single-cell RNA sequencing (scRNAseq) data derived …
Figure 1—figure supplement 3
Comparison of blastema gene signatures in single-cell RNA sequencing (scRNAseq) data sets.
UMAP (left) and module scoring of blastema signature genes from Supplementary file 4 mapped to the (A) (Storer et al., 2020) or (B) (Johnson et al., 2020) data sets.
Figure 2
Aged mice show impaired regeneration in the digit tip amputation model.
(A) Radiographic imaging from day 0 to day 42 (D0 to D42) in young and aged mice following amputation of the distal P3. (B) Masson’s trichrome staining of young and aged mice. (C) Micro-computed …
Figure 3 with 1 supplement
Spatial transcriptomics reveals altered cellular metabolism in aged mice.
(A) Pathway analysis of blastema genes showing differential expression between the young and aged blastema. Number at end of bar denotes the number of significantly regulated genes assigned to each …
Figure 3—figure supplement 1
SpatialTime analysis of the young and aged blastema reveals regional specific changes in pathway activation between young and aged mice.
(A) Assignment of SpatialTime values to spatial spots within the blastema from the proximal to distal ends using the residual bone stump as reference. (B) Module scoring for activation of pathways …
Figure 4 with 1 supplement
Aged blastemas have increased bioenergetics.
(A) Module scoring for cell metabolism pathways within young and aged blastemas. Combined is the cumulative module scoring values of glycolysis and oxidative phosphorylation (OxPhos). Values below …
Figure 4—figure supplement 1
Metabolic pathway-specific changes within the blastema of young and aged mice.
Spatial and violin plots of module scoring for indicated metabolic pathway in young and aged blastemas. *p<0.05, **p<0.01.
Figure 5
Increased intracellular hypoxia and vascularization in aged mice.
(A) Spatial module scoring of hypoxia-related transcripts. (B) Hypoxyprobe staining in young and aged blastema. Scale bar, 100 µm. Dotted lines indicate the P3 cortical bone stump. (C) Spatial …
Figure 6
Treatment with oxaloacetate (OAA) enhances regeneration in aged mice.
(A) Seahorse Mito Stress Test (oxygen consumption rate, OCR; extracellular acidification rate, ECAR) and Glycolytic Rate Assay (glycolygic proton efflux rate, GlycoPER) of dissected blastema from …
Figure 7
Oxaloacetate (OAA) treatment promotes proliferation and WNT signaling.
(A) Aged mice treated daily with OAA from D10 were sacrificed at D21 and subjected to spatial transcriptomics and the regenerated digit divided into fibroblast (fibro) and bone areas. (B) S- and …
Author response image 1
No notable shift in osteogenic transcript expression is observed by spatial transcriptomics.
Relative expression of osteogenic genes in the blastema of young and aged mice 10 days post amputation. This osteoscore is comprised of the following genes: Alpl, Bglap, Bglap2, Col1a1, Col1a2, …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | CD1 | Charles River Laboratories | Strain 022 | Female and male, 6–22 months of age RRID:IMSR_CRL:022 |
| Chemical compound, drug | Oxaloacetate | Sigma | 07753 | Oxaloacetic acid |
| Other | Z-Fix | Anatech | 5701ZF | Fixative |
| Other | Decal I | Surgipath | Decalcifier | |
| Other | Blocking solution | Thermo | 27515 | Blocking solution |
| Commercial assay, kit | Masson’s Trichrome Kit | Poly Scientific | K037 | Stain kit |
| Commercial assay, kit | Tyramide signal amplification | Invitrogen | T20924 | |
| Commercial assay, kit | Hypoxyprobe Plus Kit | Hypoxyprobe | HP2 | Stain kit |
| Commercial assay, kit | Mito Stress Test Kit | Agilent Seahorse | 103015-100 | |
| Commercial assay, kit | Glycolytic Rate Assay Kit | Agilent Seahorse | 103344-100 | |
| Software, algorithm | GEN5IPRIME V3.05.11 | Biotek | ||
| Software, algorithm | GraphPad Prism 9 | GraphPad | ||
| Software, algorithm | CTAn | Bruker | ||
| Software, algorithm | NRECON | Bruker | ||
| Software, algorithm | CTVox | Bruker | ||
| Antibody | (Rabbit Monoclonal) Anti-CD31 antibody | Abcam | ab182981 | (1:100) |
| Antibody | (Mouse Monoclonal) Anti-PCNA antibody | Abcam | ab29 | RRID:AB_303394 (1:200) |
| Software, algorithm | CellRanger | Version 6 | 10× Genomics | |
| Software, algorithm | Seurat | Version 3 | R package | Stuart et al., 2019 |
| Software, algorithm | ggpubr | Version 0.4.0 | R package | Kassambara, STHDA July 2016 |
Additional files
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Supplementary file 1
Differentially expressed genes (DEGs) preferentially expressed in the blastema.
- https://cdn.elifesciences.org/articles/71542/elife-71542-supp1-v1.csv
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Supplementary file 2
Differentially expressed genes (DEGs) preferentially expressed in the boundary.
- https://cdn.elifesciences.org/articles/71542/elife-71542-supp2-v1.csv
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Supplementary file 3
Differentially expressed genes (DEGs) preferentially expressed in the remaining digit.
- https://cdn.elifesciences.org/articles/71542/elife-71542-supp3-v1.csv
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Supplementary file 4
Blastema fingerprint genes identified from published single-cell RNA sequencing (scRNAseq) and spatial transcriptomics.
- https://cdn.elifesciences.org/articles/71542/elife-71542-supp4-v1.csv
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Supplementary file 5
Blastema differentially expressed genes (DEGs) differentially expressed between young and aged mice.
- https://cdn.elifesciences.org/articles/71542/elife-71542-supp5-v1.csv
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Supplementary file 6
Spatial differentially expressed genes (DEGs) differentially expressed between control and oxaloacetate (OAA)-treated mice.
- https://cdn.elifesciences.org/articles/71542/elife-71542-supp6-v1.csv
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Transparent reporting form
- https://cdn.elifesciences.org/articles/71542/elife-71542-transrepform1-v1.pdf
