Murine muscle stem cell response to perturbations of the neuromuscular junction are attenuated with aging
- Department of Biomedical Engineering, University of Michigan, United States
- Biointerfaces Institute, University of Michigan, United States
- Parker H. Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, United States
- School of Biological Sciences, Georgia Institute of Technology, United States
- Wallace Coulter Departmentof Biomedical Engineering, Georgia Institute of Technology, United States
- Internal Medicine-Hematology/Oncology, University of Michigan, United States
- Michigan Center for Translational Pathology, University of Michigan, United States
- Department of Pharmacology and Physiology, University of Rochester Medical Center, United States
- Department of Biomedical Engineering, University of Rochester Medical Center, United States
- Wilmot Cancer Institute, Stem Cell and Regenerative Medicine Institute, and The Rochester Aging Research Center, University of Rochester Medical Center, United States
- Departmentof Molecular Biology, Cell Biology and Biochemistry, Brown University, United States
- Center for Translational Neuroscience, Robert J. and Nancy D. Carney Institute for Brain Science and Brown Institute for Translational Science, Brown University, United States
- Department of Molecular & Integrative Physiology, University of Michigan, United States
- Department of Surgery, University of Texas Southwestern, United States
- Childrens Research Institute and Center for Mineral Metabolism, United States
- Program in Cellular and Molecular Biology, University of Michigan, United States
Figures
Figure 1
Denervation induces muscle stem cell (MuSC) actions proximal to the neuromuscular junction (NMJ).
(A) Schematic of NMJ from Pax7CreER/+-Rosa26nTnG mice, which display red fluorescent protein (RFP) in their nuclei and following administration of tamoxifen, Pax7+ MuSCs (labeled with green arrow) …
Figure 2 with 1 supplement
Aging attenuates muscle stem cell (MuSC) engraftment into synaptic myonuclei after nervous perturbation.
(A) Representative immunofluorescence image of MuSC in proximity to neuromuscular junction (NMJ) from tibialis anterior (TA) muscle of young (3 months) Pax7CreER/+-Rosa26TdTomato/+ mouse. Pax7-Pink, …
Figure 2—figure supplement 1
Muscle stem cell loss and changes in neuro-muscular junctions in aged muscle.
(A) Total number of muscle stem cells (MuSCs) along the entire length of myofibers isolated uninjured control and sciatic nerve transection (SNT) tibialis anterior (TA) muscles from young (2–3 …
Figure 3 with 2 supplements
Single cell analysis of skeletal muscle stem cells (MuSCs) in age shows a subset express transcripts associated with the neuromuscular junction.
(A) Experiment design schematic. MuSCs were fluorescent activated cell sorting (FACS) enriched from young and aged wild-type (WT) mice, then analyzed by IF, single cell mRNA sequencing (scRNA-Seq), …
Figure 3—figure supplement 1
Single-cell sequencing of sorted muscle stem cells in young and aged muscle.
(A) Representative fluorescent activated cell sorting (FACS) scatter plots showing gating of muscle stem cells (MuSCs) based on negative (Sca-1, Mac-1, CD31, CD45, Ter-119) and positive (CXCR4, …
Figure 3—figure supplement 2
Synaptic factors in aged muscle stem cells.
(A) Plots of myogenic regulatory factor and rapsyn expression in Pax7+S100b+ cells and Pax7+S100b- cells in cells enriched from young vs. aged animals. Cells were filtered into groups based on raw …
Figure 4 with 1 supplement
Muscle injury induces a continuum of muscle stem cell (MuSC) states distinct from the synaptic state induced by neurodegeneration.
(A) Schematic of experiment design, whereby tibialis anterior (TA) and extensor digitorum longus (EDL) muscles from aged (22–24 months) wild-type mice were injured via intramuscular injections of …
Figure 4—figure supplement 1
Single-cell RNA sequencing of aged muscle stem cells during muscle regeneration.
(A) Violin plot of the number of unique molecular identifiers (top) and number of genes (bottom) in each dataset. (B) Stacked bar graph showing Pax7- NMJ, Pax7+ NMJ, and muscle stem cells (MuSCs) as …
Figure 5 with 2 supplements
Neurodegeneration activates muscle stem cells (MuSCs) in a similar manner to aging and rescue of motor neurons partially reverses this activation state.
(A) Schematic of neuromuscular junction (NMJ) in young (top) and aged or Sod1-/- (bottom) muscle, whereby NMJ becomes fragmented or partially denervated. (B) Representative images of Sod-/- and …
Figure 5—figure supplement 1
Single-cell RNA sequencing of Sod1-/-, and SynTgSod1-/ muscle stem cells.
(A) Violin plots of the number of unique molecular identifiers (UMIs) (top) and number of genes (bottom) recovered in each dataset. (B) Uniform manifold approximation and projection (UMAP) overlay …
Figure 5—figure supplement 2
Validation of synaptic proteins in sorted muscle stem cells from Sod1-/- and SynTgSod1-/- muscles.
(A) Representative IF images of Pax7 and S100b expression in muscle stem cells (MuSCs) sorted from Sod1-/- and SynTgSod1-/- muscles. Scale bar indicates 100 μm for full size images and 50 μm for …
Figure 6 with 1 supplement
Mislocalization of S100β signaling from denervation promotes muscle stem cell maladaptation.
(A) Schematic of S100β-GFP mice administered sciatic nerve transection (SNT) on one limb and the other limb acts as a control (Ctrl). (B) Representative immunofluorescence z-stack images of isolated …
Figure 6—figure supplement 1
Overexpression of S100β induces muscle stem cell dysfunction as observed in aging.
(A) Representative immunofluorescence images of untransfected myoblasts (control), myoblasts transfected with an empty vector (blank sgRNA), and myoblasts transfected with an S100β sgRNA-containing …
Figure 7
Proposed model for relationship between muscle stem cells and the neuromuscular junction.
Author response image 1
Dendrogram of transcriptional similarity across models.
SOD1.KO: SOD1-/-, SOD1.Rescue: SynTgSod1-/-.
Author response image 2
(A) UMAP overlays of transcriptomes reported in Kimmel et al., Development 2020 (peach) and the datasets reported in this manuscript.
(B) Marker-gene overlays showing canonical myogenic regulatory factors as well as S100b showing S100b is confined to the UMAP cluster containing primarily cells reported in this manuscript. (C) …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | APC anti-mouse Ly-6A/E (Sca-1), clone: D7, isotype: Rat IgG2a, κ | BioLegend | BioLegend 108112; RRID:AB_313349 | FACS (1:400) |
| Antibody | APC anti-mouse CD45, clone: 30-F11, isotype: Rat IgG2b, κ | BioLegend | BioLegend 103112; RRID:AB_312977 | FACS (1:400) |
| Antibody | APC anti-mouse/human CD11b, clone: M1/70. Isotype: Rat IgG2b, κ | BioLegend | BioLegend 101212; RRID:AB_312795 | FACS (1:400) |
| Antibody | APC anti-mouse TER-119, clone: TER-119, isotype: Rat IgG2b, κ | BioLegend | BioLegend 116212; RRID:AB_313713 | FACS (1:400) |
| Antibody | PE anti-mouse/rat CD29, clone: HMβ1–1, isotype: Armenian Hamster IgG | BioLegend | BioLegend 102208; RRID:AB_312885 | FACS (1:200) |
| Antibody | Biotin Rat Anti-Mouse CD184, clone: 2B11/CXCR4 (RUO), isotype: Rat IgG2b, κ, lot # 6336587 | BD Bioscience | BD Bioscience 551968; RRID:AB_394307 | FACS (1:100) |
| Antibody | Streptavidin PE-Cyanine7, lot # 4290713 | eBioscience | eBioscience 25-4317-82; RRID:AB_10116480 | FACS (1:1000) |
| Antibody | Rabbit anti-mouse laminin 1+2, Isotype: Polyclonal IgG | Abcam | Abcam ab7463; RRID:AB_305933 | IF (1:500) |
| Antibody | Goat anti-mouse IgG1, Alexa Fluor 488 conjugate | Thermo Fisher | Thermo Fisher A21121; RRID:AB_2535764 | IF (1:500) |
| Antibody | Goat anti-rabbit H+L, Alexa Fluor 555 conjugate | Thermo Fisher | Thermo Fisher A21428; RRID:AB_141784 | IF (1:500) |
| Other | 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI), FluoroPure grade | Thermo Fisher | Thermo Fisher D21490 | IF (1:500) Nuclear stain |
| Antibody | Mouse anti-Pax7, Isotype MIgG1, kappa light chain, supernatant | Developmental Studies Hybridoma Bank | DHSB Pax7-s | IF (1:100) |
| Antibody | Rabbit polyclonal anti-S100B | Thermo Fisher | Thermo Fisher PA5-78161; RRID:AB_2736549 | IF (1:500) |
| Antibody | Mouse monoclonal anti-MyoD (G-1) | Santa Cruz Biotechnology | Santa Cruz Biotechnology Sc-377460; RRID:AB_2813894 | IF (1:200) |
| Antibody | Donkey anti-rabbit IgG (H+L), Alexa Fluor 647 | Thermo Fisher | Thermo Fisher A31573; RRID:AB_2536183 | IF (1:500) |
| Antibody | Goat anti-mouse H+L, Alexa Fluor 555 conjugate | Thermo Fisher | Thermo Fisher A28180; RRID:AB_2536164 | IF (1:250) |
| Antibody | Alexa Fluor 488 goat anti-rabbit secondary antibody | Thermo Fisher | Thermo Fisher A11034; RRID:AB_2576217 | IF (1:500) |
| Antibody | Alexa Fluor 488 conjugated α-Bungarotoxin | Thermo Fisher | Thermo Fisher B13422 | IF (1:500) |
| Antibody | Hoechst 33342 | Thermo Fisher | Thermo Fisher H3570 | IF (1:500) |
| Antibody | Goat anti-rabbit IgG (H+L), Alexa Fluor 647 | Thermo Fisher | Thermo Fisher A21245; RRID:AB_2535813 | IF (1:500) |
| Antibody | Goat anti-mouse IgG (H+L), Alexa Fluor 647 | Thermo Fisher | Thermo Fisher A21235; RRID:AB_2535804 | IF (1:500) |
| Other | Dispase II (activity ≥0.5 units/mg solid) | Sigma Aldrich | Sigma D4693-1G | Digestion of tissue to extract MuSCs |
| Other | Collagenase Type II | Thermo Fisher | Thermo Fisher 17101015 | Digestion of tissue to extract MuSCs |
| Chemical compound, drug | Propidium Iodide – 1.0 mg/mL solution in water | Thermo Fisher | Thermo Fisher P3566 | FACS (1:400) |
| Commercial assay or kit | Mouse on Mouse blocking reagent | Vector Laboratories | Vector Laboratories MKB-2213; RRID:AB_2336587 | |
| Other | Corning CellTak | Fisher Scientific | Fisher Scientific C354240 | 22.4 µg/mL Adhere MuSCs to tissue culture plastic |
| Peptide, recombinant protein | XbaI | New England Biolabs | NEB R0145S | |
| Peptide, recombinant protein | SalI | New England Biolabs | NEB R3138S | |
| Peptide, recombinant protein | Matrigel Basement Membrane Matrix, LDEV-free | Corning | Corning 356234 | |
| Commercial assay or kit | CellROX Deep Red Reagent | Thermo Fisher | Thermo Fisher C10422 | |
| Commercial assay or kit | Stellaris RNA FISH Wash Buffer A | LGC-Biosearch | LGC-Biosearch SMF-WA1-60 | |
| Commercial assay or kit | Stellaris RNA FISH Wash Buffer B | LGC-Biosearch | LGC-Biosearch SMF-WB1-20 | |
| Commercial assay or kit | Stellaris RNA FISH Hybridization Buffer | LGC-Biosearch | LGC-Biosearch SMF-HB1-10 | |
| Sequence-based reagent | Fos FISH probe | LGC-Biosearch | LGC-Biosearch VSMF-3011–5 | |
| Commercial assay or kit | Single cell 3’ Library and Gel Bead Kit v2 and v3 | 10× Genomics | 10× Genomics 120267 | |
| Commercial assay or kit | QIAprep Spin Miniprep Kit | Qiagen | Qiagen 27104 | |
| Commercial assay or kit | Satellite Cell Isolation Kit, mouse | Miltenyi Biotec | Miltenyi Biotec 130104268 | |
| Commercial assay or kit | In Situ Cell Death Detection Kit, TMR red | Sigma Aldrich | Sigma 12156792910 | |
| Biological sample (Mus musculus) | C57BL/6 mice | Charles River Labs | C57BL/6NCrl Strain 027 | Female, 3–4 months |
| Biological sample (Mus musculus) | C57BL/6 mice | NIA | C57BL/6JNia; RRID:IMSR_JAX:000664 | Female, 20–24 months |
| Biological sample (Mus musculus) | SynTgSod1-/- mice | Oklahoma Medical Research Foundation | PMID:24378874, 31470261 | Female, 10–12 months |
| Biological sample (Mus musculus) | Sod1-/- mice | Oklahoma Medical Research Foundation | PMID:9264557, 15531919 | Female, 10–12 months |
| Biological sample (Mus musculus) | Pax7CreER+/+ mice | Jackson Labs | RRID:IMSR_JAX:017763 | Male, 2–3 months |
| Biological sample (Mus musculus) | Rosa26mTmG+/+ mice | Jackson Labs | RRID:IMSR_JAX: 007676 | Female, 2–3 months |
| Biological sample (Mus musculus) | Rosa26TdTomato+/+ mice | Jackson Labs | RRID:IMSR_JAX: 007909 | Female, 2–3 months |
| Biological sample (Mus musculus) | Rosa26nTnG+/+ mice | Jackson Labs | RRID:IMSR_JAX: 023035 | Female, 2–3 months |
| Biological sample (Mus musculus) | S100b-GFP mice | Jackson Labs | RRID:IMSR_JAX: 005621 | Female |
| Biological sample (Mus musculus) | Rosa26dCas9-SunTag/+ mice | Jackson Labs | Stock # 43926-JAX | Female |
| Software, algorithm | CellRanger v2.0.0, v3.0.0 | 10× Genomics | https://support.10xgenomics.com/single-cell-gene-expression/software/downloads | |
| Software, algorithm | R v.4.0.2 | The R Foundation for Statistical Computing | RRID:SCR_001905 | |
| Software, algorithm | Bioconductor v.3.11 | Huber et al., 2015 | Nature Methods 12:115–121, PMID:25633503 | |
| Software, algorithm | DESeq2 v.1.28.1 | Love et al., 2014 | RRID:SCR_015687 | |
| Software, algorithm | scVelo v.0.2.2 | Bergen et al., 2020 | RRID:SCR_018168 | |
| Software, algorithm | ggplot2 v.3.3.2 | Wickham, 2016 | RRID:SCR_014601 | |
| Software, algorithm | EnhancedVolcano v.1.6.0 | Blighe et al., 2021 | RRID:SCR_018931 | |
| Software, algorithm | Python v3.6.5 | Python Software Foundation | RRID:SCR_008394 | |
| Software, algorithm | Seurat v.3.2.2 | Stuart et al., 2019 | RRID:SCR_007322 | |
| Software, algorithm | SeuratWrappers v0.3.0 | https://github.com/satijalab/seurat-wrappers | ||
| Software, algorithm | scCATCH | Shao et al., 2020 | https://github.com/ZJUFanLab/scCATCH | |
| Software, algorithm | LIGER v0.5.0 | Welch et al., 2019 | RRID:SCR_018100 | |
| Software, algorithm | AnnotationDbi v1.50.3 | Pagès et al., 2021 | ||
| Software, algorithm | GenomeInfoDb 1.24.2 | Arora and Morgan, 2021 | ||
| Software, algorithm | Jupyter v.4.4.0, notebook v.5.7.8 | Project Jupyter 2019 | https://jupyter.org/ | |
| Software, algorithm | dplyr v1.0.2 | Wickham, 2021 | RRID:SCR_016708 | |
| Software, algorithm | tibble v3.4.0 | Müller and Wickham, 2021 | ||
| Software, algorithm | tximport v1.16.1 | Soneson et al., 2016 | RRID:SCR_016752 | |
| Software, algorithm | Velocyto v0.17 | La Manno et al., 2018 | RRID:SCR_018167 | |
| Software, algorithm | Kallisto v0.44 | Bray et al., 2016 | RRID:SCR_016582 |
