Abstract
Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages ‘dwell’ within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.
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Data availability
The single-cell RNA-seq dataset has been deposited at the NCBI Gene Expression Omnibus (GEO) repository under accession number GSE162979. All other data supporting the findings of this study are found within the manuscript and its Supplementary Information. Source data are provided with this paper.
Code availability
The single-cell experiment dataset was processed and analysed with programs and packages as described in the Methods. Scripts can be found at https://github.com/fjrossello/Ratnayake_et_al_2020_SC.
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Acknowledgements
We thank Monash Micro Imaging and Monash Ramaciotti Centre for Cryo-Electron Microscopy for microscopy support, Hubrecht Institute single-cell sequencing facility for sequencing service, Hubrecht Institute flow cytometry core facility and Monash FlowCore for sorting of cells using FACS and Monash Fishcore staff for technical assistance, A. Kawakami for the mmp9:eGFP and mmp9:eGFP-NTR BAC transgenic lines, F. J. Tulenko for advice on phylogenetic tree construction, H. Janovjak for advice on in vitro assays, A.-M. Tichy, E. Gerrard and A. Ruparelia for technical assistance and B. T. Kile, P. Gönczy and R. P. Currie for comments on the manuscript. P.D.N. is supported by an EMBO Long Term Fellowship (ALTF1129-2015), HFSPO Fellowship (LT001404/2017-L) and a NWO-ZonMW Veni grant (016.186.017-3). This work was supported by the National Health and Medical Research Council of Australia (APP1041885, APP1136567, APP1104190, APP1159278 to P.D.C.; APP1044754, APP1069284, APP1086020 to G.J.L.; and APP1140229, APP1176213 to M.M.M.). The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government.
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Authors and Affiliations
Contributions
D.R. (live imaging, photoconversions, immunohistochemistry, in situ hybridization, scRNA-seq sample preparation, tissue-specific loss-of-function studies, zebrafish mutant studies, drug and protein supplementation studies, RT–PCR, NAD+/NADH assays) designed and performed experiments, analysed data and co-wrote the manuscript; P.D.N. (scRNA-seq), V.C.W. (live imaging), J.L.T. (mouse experimentation (NAMPT supplementation, flow cytometry, immunohistochemistry, histology), cytokine array), L.A.G. (primary myoblast coculture), A.J.W. (C2C12 cell culture), Z.J. (binding assays, ELISAs), V.O. (correlative light and electron microscopy), S.B. (histology, immunohistochemistry, zebrafish mutant studies) and C.S. (in situ hybridization) performed experiments; A.I.I. (macrophage and neutrophil lineage-specific gene-editing system) established techniques; F.J.R. (scRNA-seq analysis) and T.B. (light-sheet cell tracking) analysed data; K.L.R., C.M., G.J.L., M.M.M. and J.B. provided reagents; P.D.C., G.J.L., J.B. and M.M.M. designed and supervised experiments and P.D.C. co-wrote the manuscript.
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P.D.C., M.M.M. and D.R. through Monash University have filed provisional patent application no. 2020901237 on the clinical use of NAMPT. All other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Dwelling macrophages establish a transient niche following muscle injury that is indispensable for the regenerative process.
a, The migratory paths of injury-responding macrophages located within a 260-μm radius from the centre of the laser ablation injury (a region encompassing 2 myotomes on either side of the injury) from Supplementary Video 1 are shown, where (0, 0) is set to the centre of the wound (see still images in Fig. 1a, b). b–d, Morphological characterization of macrophage (MΦ) subsets revealed transient macrophages presenting a more stellate phenotype (b) with lower sphericity (d; 0.4163 ± 0.1260 (mean ± s.d.)), whereas dwelling macrophages appear more rounded (c) with higher sphericity (d; 0.6639 ± 0.1296 (mean ± s.d.); a 0.248 ± 0.022 higher sphericity value than to transient macrophages). Scale bar, 20 μm. d, n = 69 macrophages per group, from n = 18 injuries carried out in 6 independent experiments). The thick black lines and thin black lines within the violin plot indicate the median and quartiles, respectively. Unpaired two-tailed t-test; t136 = 11.38, P < 0.0001. e–g″, Maximum intensity projection images of the same fish transgenic for Tg(actc1:BFP) (labelling differentiated muscle fibres; magenta; e′, f′, g′), TgBAC(pax3a:GFP) (labelling pax3a+ myogenic cells; cyan; e″, f″, g″) and Tg(mpeg1:GAL4FF/UAS:nfsb-mCherry (labelling macrophages; yellow; e‴, f‴, g‴) after a needle-stab muscle injury. Scale bar, 50 μm. h, By 2 dpi, 51.6% of the original injury-responsive macrophages remain in the wound site, and these dwelling macrophages go on to establish a transient niche that is pro-myogenic (n = 20). i–p, Establishing nitroreductase-mediated macrophage ablation parameters. The transgenic line, Tg(mpeg1:GAL4FF/UAS:nfsb-mCherry) expresses the enzyme nitroreductase specifically within macrophages, enabling the temporally controlled genetic ablation of macrophages by addition of the pro-drug Mtz. The optimum Mtz concentration was established by treating larvae with 5 and 10 mM Mtz for 24 h (starting at 4 dpf) and visualizing (i) and quantifying (j) trunk-present macrophages. Macrophage ablation of 77% was achieved at 5 dpf by 5 mM Mtz treatment (j; n = 10). Although uninjured larvae were able to tolerate 10 mM Mtz, needle-stab injury carried out at this concentration resulted in significant lethality. Therefore, all further experiments used 5 mM Mtz for macrophage ablations. k, Titrating the Mtz dosing time to specifically ablate macrophage subsets of interest. Temporal response to 5 mM Mtz was visualized by time-lapse imaging the head and tail region of larval zebrafish immediately after Mtz addition (4 dpf). Macrophage ablation initiated at 3 h after Mtz treatment. Scale bar, 500 μm. Frames were obtained from Supplementary Video 5 (n = 8). l, m, Ablation of macrophages did not alter the neutrophil response to wounding at 2 dpi (representative images (l, scale bar, 50 μm) and quantification (m); n = 12). n, Addition of Mtz at the point of injury ablated all macrophages (yellow; shown superimposed on bright-field images of the zebrafish trunk), whereas addition of Mtz at 1.75 dpi (5 dpf) selectively ablated dwelling macrophages (n = 10). Scale bar, 500 μm. The temporal sequence of neutrophil migration in response to skeletal muscle injury was assayed (o; scale bar, 50 μm) and quantified (p; n = 14 at 5–6 hpi, n = 13 at 1 dpi and n = 12 at 2 dpi). q–t, As a control experiment for nitroreductase-mediated genetic cell ablation, neutrophils—the other key cellular component of the innate immune system—were selectively ablated using the transgenic line Tg(mpx:KALTA4/UAS:nfsb-mCherry). Ablation efficiency after 24 h of 5 mM Mtz treatment starting at 4 dpf was visualized (q) and quantified at 5 dpf (r; n = 10). After needle-stab injury, larvae were soaked in 5 mM and 10 mM Mtz (4 dpf, 0 dpi) to ablate all neutrophils and 5 mM Mtz at 5 dpf (1.75 dpi) to specifically ablate late injury-present neutrophils (Mtz treatment was maintained until experimental end point at 7 dpf, 3 dpi with daily Mtz drug refreshment) and regeneration monitored by imaging for birefringence (s; scale bar, 500 μm) and quantified (t, n = 12). Neutrophil-ablated larvae showed no regeneration defects at either Mtz dose or timing, highlighting a specific requirement for macrophages in stimulating regeneration and precluding the possibility that any regeneration defect was Mtz-related. h, j, p, r, The central lines, upper and lower lines within the violin plot indicate the median, upper and lower quartiles, respectively. One-way ANOVA with Dunnett’s post hoc test for multiple comparisons. m, The black lines and grey lines within the violin plot indicate the median and quartiles, respectively. Unpaired two-tailed t-test; t122 = 1.158, P = 0.2591. t, Individual data points are shown. Two-way ANOVA with Tukey’s multiple comparison test.
Extended Data Fig. 2 Macrophage ablation does not disrupt tissue debris clearance after muscle injury.
a–f, Clearing of wound site debris is mediated by a number of cell types81, but largely attributed to phagocytic macrophages82. Absent or ineffective clearance might be one explanation for the lack of regeneration observed when macrophage populations are ablated early in the regenerative process. At 3 dpf, macrophages within the Tg(mpeg1:GAL4FF/UAS:nfsb-mCherry) transgenic line were ablated in larval zebrafish by the addition of 5 mM Mtz. Macrophage-ablated larvae were then subject to a needle-stab skeletal muscle injury at 4 dpf and the wound site assayed for clearance of damaged muscle fibres by a number of criteria. a, Fluorescently conjugated phalloidin staining to detect the actin cytoskeleton of remnant necrotic skeletal muscle fibres in the injury site. No difference is evident between control and macrophage-ablated larvae. n = 3 independent injuries per time point presented, n = 12 injuries per time point imaged. Scale bar, 50 μm. b, c, Annexin-V marking the apoptotic and phagocytic signalling process was visualized in vivo using the transgenic line Tg(ubi:secAnnexinV-mVenus) (magenta, n = 3 independent injuries per time point presented; b; scale bar, 50 μm) and quantified (c). d, e, Acidic membranous organelles and the autophagocytic process were marked by LysoTracker fluorescent vital dye (magenta), imaged (d; scale bar, 50 μm) and quantified (e). The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test (c) or unpaired two-tailed t-test; t12 = 0.3311, P = 0.7463 (e). No difference is evident between control and macrophage-ablated larvae. f, Haematoxylin and eosin-stained cross sections through needle-stab-injured myotomes demonstrates cleared wound site (black arrowheads) at 3 dpi in macrophage-ablated larvae, demonstrating the removal of dead and dying fibres evident in the wound site at 1 dpi (n = 3 larvae per group).
Extended Data Fig. 3 Dwelling macrophage–MuSC associations precede MuSC proliferation.
a–d, Macrophage and MuSC dynamics after injury were assayed using the Tg(mpeg1:GAL4FF/UAS:nfsb-mCherry);TgBAC(pax3a:GFP) (yellow, macrophages; cyan, muscle stem/progenitor cells) compound transgenic line. The pax3a:GFP reporter is particularly useful in this context as it is expressed in both the MuSC compartment and the dividing progenitor population. After laser-ablation muscle injury (white dotted line) transient macrophages migrate into the wound site (a–c). Simultaneously, activated pax3a+ myogenic stem cells from the injured and adjacent myotomes travel to line the edge of the injury site (a–c, white arrowheads). Once macrophages transition to a dwelling subtype, they initiate interactions with wound-edge-lining pax3a+ cells (d, orthogonal views). Scale bar, 50 μm. Frames were obtained from Supplementary Video 6 (n = 5). e–g, MuSCs migrate into the injury site independent of macrophage-derived signals. As macrophages and MuSCs migrate and populate the injury site simultaneously, the dependence of one on the other was assessed. e, f, Both control larvae and macrophages-ablated larvae (5 mM Mtz added at the point of injury on 4 dpf to ablate all macrophages) displayed pax3a+ stem cells in the injury site (white dotted line) at 2 dpi (white arrowheads) after needle-stab skeletal muscle injury (e; scale bar, 50 μm) and MuSCs lining the edge of the injury site (yellow asterisks) after laser-ablation muscle injury at 14 hpi (f; scale bar, 50 μm). g, Quantification of laser-ablation muscle-injury-responsive macrophages. The thick black lines and thin black lines within the violin plot indicate the median and quartiles, respectively. Not significant in unpaired two-tailed t-test; t12 = 0.1715, P = 0.8667. In the control setting (e, f, top), these cells were associated with macrophages. e, At 3 dpi after needle-stab injury, control larvae displayed regenerated cyan-fluorescence-persistent muscle fibres (red arrowheads) that arose from pax3a+ MuSCs present in the wound, a hallmark of a healing muscle injury. By contrast, although the wound site was still occupied by pax3a+ stem cells at 3 dpi in the macrophage-ablated larvae, there were no nascent pax3a+ stem cell-derived muscle fibres present, highlighting that the presence of macrophages in the wound site is not prerequisite for pax3a+ myogenic cell migration; however, it is required for those migrated cells to enter the myogenic replenishment program (n = 12). h–j, The pax3a+ myogenic cells that dwelling macrophages interact with also express the MuSC markers met (h, i) and pax7b (j). h, After needle-stab skeletal muscle injury (white dotted line) in larvae transgenic for TgBAC(pax3a:GFP) (cyan) and Tg(met:mCherry-2A-KALTA4/UAS:nfsb-mCherry) (magenta), wound-present MuSCs are pax3a+met+. These cells undergo both symmetric expansion (white open arrowheads) and asymmetric divisions (closed yellow arrowheads), giving rise to two pax3a+met+ daughter cells or a pax3a+met+ and a pax3a+met− daughter cell, respectively. The broad myogenic marker pax3a+ visualizes each of these events (n = 20, scale bar, 50 μm). i, Time-lapse imaging was used to visualize MuSC (TgBAC(pax3a:GFP)/Tg(met:mCherry-2A-KALTA4/UAS:nfsb-mCherry), cyan/yellow double-positive cells) and dwelling macrophage (Tg(mpeg1:GAL4FF/UAS:nfsb-mCherry), yellow) interactions after a laser-ablation skeletal muscle injury (white dotted line). Dwelling macrophages (white closed arrowhead) interact with pax3a+met+ MuSCs (magenta open arrowhead), after which the MuSC undergoes symmetric division to generate two pax3a+met+ progenitors. Scale bar, 30 μm. Frames were obtained from Supplementary Video 9 (n = 6). j, Dwelling macrophages (Tg(mpeg1:GFP); yellow) that are present at the wound site (white dotted line) interact with MuSCs that also express the marker pax7b (cyan; Tg(pax7b:GAL4FF/UAS:nfsb-mCherry)). In this muscle stem/progenitor cell marker gene trap line, GAL4FF is integrated into the pax7b gene (referred to as Tg(pax7b:GAL4FF)) and used to drive Tg(UAS:nfsb-mCherry). Scale bars, 50 μm (n = 9 per time point). k, Dwelling macrophages interact with wound-responsive MuSCs. Examples of three independent laser-ablation muscle injury sites showing dwelling macrophage–stem cell associations followed by stem cell divisions (arrowheads). Scale bars, 15 μm (injury 1), 30 μm (injuries 2 and 3). Frames were obtained from Supplementary Video 7. l, After MuSC division, the associated dwelling macrophage, and generated daughter cells, migrate away from each other. Dwelling macrophages interact with MuSCs located within the injury zone (white dotted line). After MuSC division (closed white, magenta, yellow and grey arrowheads highlight four independent stem cells), movements of the daughter cells (open white, magenta, yellow and grey arrowheads highlighting the daughter cells that arose from the stem cells indicated by closed arrowheads of the same colour) within the wound site are highlighted such that their relation to the dwelling macrophages can be visualized. Cell movements are tracked until dwelling macrophages localize to the vertical myosepta. Scale bar, 40 μm. Frames were obtained from Supplementary Video 10. Representative of n = 9 stem cell divisions assayed in n = 5 long-term time lapses.
Extended Data Fig. 4 Correlative light and electron microscopy analyses of the transient macrophage–stem cell niche reveals that macrophages and stem cells maintain direct heterocellular surface appositions in xyz planes.
a, Confocal microscopy of the wound site at 25 h after laser-ablation muscle injury in the compound transgenic zebrafish line, Tg(mpeg1:GAL4FF/UAS:nfsb-mCherry);TgBAC(pax3a:GFP), labelling macrophages (yellow) and pax3a+ muscle stem/progenitor cells (cyan). Scale bar, 50 μm (n = 1). b, Large STEM tile set of the same trunk region of the larva illustrated in a generated after Epon-embedding and sectioning. This dataset was used to correlate and identify the highlighted macrophage–stem cell interaction of interest (white asterisk in a, b). Dotted square marks the area that was further examined by transmission electron microscopy (TEM) in c, d. c, Region of interest encompassing a macrophage and stem cell that maintain a close interaction examined through z-depth. The cells of interest and interaction area are segmented (macrophage cytoplasm, yellow; macrophage nucleus, orange; stem cell cytoplasm, cyan; stem cell nucleus, blue; cell–cell interaction surface, magenta). d, High-resolution images of two planes through the z-depth (plane shown correlated with red dotted lines to the segmentation images) further demonstrate the close association between the two cells. Scale bar, 5 μm (n = 1).
Extended Data Fig. 5 Prox1a+Pou2f3+ macrophages (cluster 6) identified by scRNA-seq are a dwelling macrophage subset independent from Mmp9+ macrophages (cluster 2).
a, After appropriate filtering, 1,309 macrophages were analysed by scRNA-seq. These cells express antigen processing and presentation genes, confirming their macrophage character. Feature plots highlighting antigen-presenting genes expressed by the majority of cells (cd83, cd81a and cd40) as well as genes differentially expressed by individual subsets. The percentage of cells expressing a gene of interest is also recorded (maroon, information extracted from Supplementary Tables 1, 3). b, Graphical representation of UMAP scatter plot clusters. Uninjured macrophages cluster together (cluster 3). Macrophages isolated from a ‘transient’ time point (1 dpi) also predominantly cluster together (cluster 1). The remaining six clusters (0, 2, 4, 5, 6, 7) are mainly composed of macrophages isolated from ‘dwelling’ time points (2–3 dpi). c, Lineage analysis using PAGA visualizes gene expression changes leading to the definition of an mmp9+ dwelling macrophage subset along path 3-0-4-2 of the PAGA cluster and a prox1a+pou2f3+ dwelling macrophage subset along path 3-5-1-6 of the PAGA cluster. The numerical scale (right) expresses normalized gene expression from Seurat, whereas clusters (bottom) indicate subsets along the PAGA path with length being proportional to the cell numbers in each cluster. d, Violin plots show the expression pattern of mmp9 and nampta based on cluster identity and isolation time point. The percentage of cells expressing the markers at 2 dpi are documented. e–j, Violin plots (percentage of cells in cluster 6 expressing a specific marker recorded as percentage (e, h)) and antibody staining for a subset of the cluster-6 markers (Pou2f3 (f) and Prox1a (i)) after needle-stab muscle injury (white dotted line), showing a specific, late-injury-dwelling macrophage population (Supplementary Table 3 (worksheet 7)). Antibody staining against mCherry was used to identify all mpeg1+ macrophages in the Tg(mpeg1:GAL4FF/UAS:nfsb-mCherry) (yellow) line. After injury, at 1 dpi, almost no Pou2f3+mpeg1+ (f; scale bar, 50 μm) or Prox1a+mpeg1+ (i; scale bar, 50 μm) macrophages are present in the wound site; their numbers start to increase slightly at 2 dpi with the highest percentage being present at 3 dpi, identifying this subset as a late-injury-dwelling macrophage subset. g, j, Quantification (n = 10 at 2 and 3 dpi in g and n = 7, n = 11 and n = 7 at 1, 2 and 3 dpi, respectively, in j). The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. Unpaired two-tailed t-test; t17 = 4.574, P = 0.0003 (g) or one-way ANOVA with Dunnett’s post hoc test for multiple comparisons (j). k, l, Cluster-2 macrophages display a metabolic shift towards glycolysis at the gene expression level. KEGG pathway analysis of cluster-2 differentially expressed genes (Supplementary Table 3) identified genes associated with glycolysis (term: dre00010) and oxidative phosphorylation (term: dre00190) pathways. Genes associated with glycolysis were upregulated (k), whereas the majority of genes associated with oxidative phosphorylation were downregulated (l), highlighting a metabolic shift towards glycolysis in cluster-2 mmp9+ dwelling macrophages.
Extended Data Fig. 6 The intracellular enzymatic function of NAMPT is not needed for its pro-proliferative activity during skeletal muscle regeneration.
a–c, mmp9 labels a subset of wound-dwelling macrophages. a, These mmp9-expressing macrophages (magenta) associate with wound-present pax3a+ stem cells (cyan; wound site, white dotted line; n = 15). b, After laser-ablation skeletal muscle injury (white dotted line), a subset of macrophages (Tg(mpeg1:GAL4FF/UAS:nfsb-mCherry); yellow) starts expressing mmp9 (TgBAC(mmp9:eGFP); magenta) upon dwelling (yellow/magenta double-labelled cells). After injury, mpeg1+ macrophages respond to wounding. At the same time, mmp9-expressing neutrophils are found to be present in the injury (magenta only; green open arrowheads); these neutrophils have a cellular phenotype and wound site dynamics that are distinct from macrophages. The mmp9-expressing neutrophils co-express the neutrophil-specific marker mpx (data not shown; n = 16) and exit the wound site by 5.21 ± 1.27 hpi (n = 6). Once macrophages start to dwell, a subset of mpeg1-expressing macrophages also start to express mmp9 (white closed arrowheads). Frames were obtained from Supplementary Video 10 (n = 6). c, Titrating the Mtz dose enables specific ablation of mmp9-expressing immune cells in the TgBAC(mmp9:eGFP-NTR) (magenta) transgenic line. After treatment with 5 mM Mtz for 6 h (5 dpf, 1.75 dpi to 6 dpf, 2 dpi) uninjured larvae show ablation of mmp9-expressing immune cells (phenotypically distinct, stellate morphology, mmp9 high-expressing cells), whereas skin cells—which express mmp9 at significantly lower levels (phenotypically distinct, hexagonal morphology)—are resistant to ablation. After needle-stab skeletal muscle injury (4 dpf, 0 dpi), larvae treated with Mtz at 5 dpf, 1.75 dpi demonstrate an absence of mmp9 high-expressing cells, specifically in the wound site (dotted white line) (n = 12). d, e, The dose and duration of Mtz treatment used specifically ablates 77.94% of mmp9+mpeg1+ macrophages (mmp9−mpeg1+ macrophages, 6.65 ± 1.50 in Mtz-treated versus 5.47 ± 2.75 in control; mmp9+mpeg1+ macrophages, 2.18 ± 1.19 in Mtz-treated versus 9.87 ± 2.72 in control). d, Representative images, with white arrowheads identifying mmp9+mpeg1+ (magenta and yellow, respectively) macrophages within the needle-stab skeletal muscle injury zone (white dotted line). e, Quantification. The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. f–h, Ablation of mmp9+ macrophages leads to a significant reduction in muscle stem/progenitor cells present in the wound site. After Mtz treatment there is a significant reduction in Pax7+ MuSCs located in the wound site (white dotted line) (f). g, Quantification. The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. Unpaired two-tailed t-test; t41 = 6.196, P < 0.0001. This reduction is not due to a stem-cell migration deficit, as Mtz treatment is carried out after MuSC migration (MuSCs lining the wound edge in the Mtz-treated larvae is shown in f), but rather due to a MuSC proliferation deficit within the wound site (yellow dotted line) as observed by assaying EdU incorporation (h; 3 independent examples of larval injuries for both control and Mtz-treated larvae are shown; images are representative of the quantification presented in Fig. 3r). i, nampta mRNA expression is specifically upregulated in the injury site from 2 dpi onwards (black arrowhead, n = 18) in larval zebrafish. j–n, Assessing Nampt expression in larval zebrafish. Nampt antibody staining carried out in a developmental series (1, 3 and 4 dpf) confirmed a match between the distribution pattern of the protein and the previously published RNA transcript data12 and demonstrated ubiquitous expression during early development, with enrichment at the somite boundaries (white closed arrowheads) at 1–4 dpf and further enrichment in the intestine (white open arrowhead) from 3 dpf onwards (n = 10) (j). k, l, After needle-stab skeletal muscle injury, Nampt protein expression in the wound site is upregulated starting at 6 dpf, 2 dpi. l, Quantification (n = 9 control, n = 10 Mtz-treated). The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. Unpaired two-tailed t-test; t18 = 5.430, P < 0.0001. m, n, Nampt upregulation in the wound site (white dotted line) is of macrophage origin, as selectively ablating macrophages (5 mM Mtz added at the point of injury (4 dpf, 0 dpi) to Tg(mpeg1:GAL4FF/UAS:nfsb-mCherry) larvae to ablate all macrophage) leads to a significant reduction in Nampt expression in the wound site. This is further confirmed by Nampt levels being unperturbed after selective ablation of neutrophils (Tg(mpx:KALTA4/UAS:nfsb-mCherry) larvae used in neutrophil ablation). n, Quantification. The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. o, p, Increased NAMPT activity leads to elevated levels of intracellular NADH. o, NADH autofluorescence (magenta) displays a localized upregulation in dwelling macrophages (yellow), indicating that these cells are the primary source of wound-present Nampt (n = 15). Scale bar, 20 μm. p, This was quantitatively confirmed by means of a bioluminescence-based assay, which demonstrated wound-located dwelling macrophages have a 0.6781 ± 0.1650 lower NAD+/NADH ratio compared with transient macrophages. The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. Unpaired two-tailed t-test; t8 = 4.110, P = 0.0034. q, r, Inhibiting the enzymatic function of Nampt does not affect MuSC proliferation. After needle-stab skeletal muscle injury (white dotted line) TgBAC(pax3a:GFP) (cyan) larvae were treated with the small-molecule, competitive inhibitor GMX1778 from 5 dpf, 1.75 dpi untill experimental end point (6 dpf, 2.5–2.75 dpi). GMX1778 selectively inhibits the rate-limiting enzymatic function of Nampt in NAD+ biosynthesis and at the administered concentration results in a severe reduction in larval NAD+/NADH levels (Extended Data Fig. 9c). q, Inhibiting the enzymatic function of Nampt had no effect on MuSC proliferation in the injury zone, highlighting that the functionality of Nampt during stem cell proliferation is distinct to its intracellular role in energy metabolism. r, Quantification (n = 17 control, n = 13 GMX1778-treated). The central lines, upper and lower lines within the violin plot indicate the median, upper and lower quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. a–d, f, h, i–k, m, q, Scale bars, 50 μm.
Extended Data Fig. 7 NAMPT binds to the CCR5 receptor present on MuSCs and induces proliferation.
a, b, NAMPT selectively binds to CCR5. a, ELISA plates were coated with human recombinant CCR5 (hrCCR5) or BSA and further incubated with hrNAMPT(1) at increasing concentrations (0 nM to 800 nM). NAMPT molecules bound to CCR5 were detected using a biotinylated antibody. NAMPT binds to CCR5 with a KD of 172 ± 18 nM (n = 2 independent experiments performed in triplicate). The graph shows a representative binding curve from which non-specific binding to BSA was deducted. Data are mean ± s.e.m. b, NAMPT is a highly evolutionary conserved protein, with human NAMPT sharing 96% and 88.24% identity with mouse NAMPT and zebrafish Nampta proteins, respectively. As we use hrNAMPT in mouse cell line assays, we also assayed the affinity of hrNAMPT for mrCCR5 by means of a competitive ligand-binding assay with the cognate ligand, CCL4. ELISA plates were coated with mrCCR5 or BSA and incubated with mrCCL4 at increasing concentrations (0 nM to 400 nM) along with 100 nM hrNAMPT(1). hrNAMPT(1) molecules bound to CCR5 were detected using a biotinylated antibody. mrCCL4 shows an IC50 of 34.4 ± 2.2 nM (n = 2 independent experiments performed in triplicate) revealing both hrNAMPT’s strong affinity for mrCCR5 and its binding to the receptor via the same sites used by CCL4. The graph shows a combined binding curve from which non-specific binding to BSA was deducted. Data are mean ± s.e.m. c, Exogenous NAMPT supplementation enhances myoblast proliferation. In vitro assay assessing the effects of exogenously introduced factors on C2C12 myoblast proliferation. Proliferation is identified by EdU incorporation. NAMPT administration (two commercially available NAMPT sources tested, hrNAMPT(1) and hrNAMPT(2)) leads to a dose-dependent increase in myoblast proliferation. This effect is specifically mediated by the CCR5 receptor. Co-administration of NAMPT with the CCR2/CCR5 dual inhibitor CVC and CCR5-specific inhibitor MVC abolishes the pro-proliferative response of NAMPT, whereas co-administration with the CCR2 inhibitor PF-4136309 (PF4) does not hinder the stimulatory effect of NAMPT on myoblast proliferation. In agreement with this finding, the endogenous ligands of CCR5, mrCCL8 and mrCCL4, functioned to enhance C2C12 proliferation, whereas the CCR2-specific ligand mouse recombinant CCL2 did not to increase proliferative rates beyond that of the control. The pro-proliferative function of NAMPT is separate from its intracellular role in energy metabolism, as co-administering NAMPT with the NAMPT enzymatic inhibitor (GMX1778) did not affect its effect on myoblast proliferation. The central lines, upper and lower lines within the violin plot indicate the median, upper and lower quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. n = 20 control, n = 10 1.9 nM hrNAMPT(1), n = 10 9.5 nM hrNAMPT(1), n = 10 19 nM hrNAMPT(1), n = 9 1.9 nM hrNAMPT(2), n = 10 9.5 nM hrNAMPT(2), n = 10 100 nM CVC, n = 10 100 nM MVC, n = 10 100 nM PF4, n = 10 100 nM CVC + 9.5 nM hrNAMPT(1), n = 10 100 nM MVC + 9.5 nM hrNAMPT(1), n = 9 100 nM PF4 + 9.5 nM hrNAMPT(1), n = 10 9.5 nM mrCCL8, n = 10 9.5 nM mrCCL4, n = 10 9.5 nM mrCCL2, n = 10 500 nM GMX1778, n = 10 500 nM GMX1778 + 9.5 nM hrNAMPT(1). d, Cultured MAF DKO macrophages actively secrete NAMPT. ELISA-quantified NAMPT concentration in the supernatants of 16-h M-CSF-stimulated MAF DKO and Raw 264.7 macrophages (n = 1 experiment in triplicate, stimulated with 10 ng ml−1 M-CSF). The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. Unpaired two-tailed t-test; t4 = 0.3302, P = 0.7578. e, f, Cultured MAF DKO macrophages do not actively secrete the cognate ligands of CCR5, CCL3, CCL4 and CCL5. e, Representative array spots detecting CCR5 ligands expressed by MAF DKO macrophages after 16 h stimulation with M-CSF (10 ng ml−1). f, Quantification. Data are mean ± s.e.m. (n = 1 array per group performed in duplicate). g, h, PAX7+ satellite cells in mouse primary myoblast monocultures display enhanced proliferation upon CCR5 receptor signalling, mediated by either exogenous NAMPT (g, h) or CCL4 (h) supplementation. Coculturing myoblasts with macrophages stimulated satellite cell proliferation. This pro-proliferative response is abolished by administrating CVC (g) and MVC (h), whereas PF-4136309 had no negative effect (h), confirming that the pro-proliferative function of macrophages on stem cells is mediated by CCR5 signalling. In addition, coculturing myoblasts with 3T3 cells that do not naturally secrete NAMPT83 does not stimulate satellite cell proliferation, suggesting that NAMPT is the macrophage-derived pro-proliferative cue that drives stem cell proliferation. g, n = 18 control, n = 18 9.5 nM hrNAMPT(2), n = 18 100 nM CVC, n = 18 100 nM CVC + 9.5 nM hrNAMPT(2) for myoblasts, myoblast + macrophages and myoblasts + 3T3 cells); h, n = 35 control, n = 36 9.5 nM hrNAMPT(1), n = 36 100 nM MVC, n = 36 100 nM PF4, n = 55 9.5 nM mrCCL4 for myoblasts, n = 42 control, n = 36 9.5 nM hrNAMPT(1), n = 36 100 nM MVC, n = 31 100 nM PF4, n = 46 9.5 nM mrCCL4 for myoblast + macrophages and n = 36 control, n = 36 9.5 nM hrNAMPT(1), n = 36 100 nM MVC, n = 36 100 nM PF4, n = 51 9.5 nM mrCCL4 for myoblast + 3T3 cells. The black lines and grey lines within the violin plot indicate the median and quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. i–m, Inhibiting Ccr5 receptor activation does not affect injury-responsive immune cell dynamics in larval zebrafish. i, j, The predicted orthologous Ccr5 protein in zebrafish was identified by determining the appropriate best hit for the protein most similar to the human CCR5 amino acid sequence in zebrafish using BLAST. A maximum-likelihood phylogenetic tree constructed using protein sequences of CCR5 positioned the putative zebrafish Ccr5-like (Ccr5l) sequence as a homologue of mammalian CCR5 with strong bootstrap support. Bootstrap values with 500 replicates are documented below the branches. CCR7 was used as an outgroup. The accession numbers for genes included in our analysis are provided in the table (j). k, pax3a+ MuSCs express the ccr5 receptor at 2 dpi as detected by RT–PCR (n = 3). l, m, Larvae soaked in CVC or MVC displayed a marked regeneration deficit revealed by birefringence imaging (l; scale bar, 500 μm) and quantification (m; n = 20 control, n = 22 5 μM CVC, n = 14 10 μM CVC, n = 16 5 μM MVC, n = 12 10 μM MVC). Individual data points are shown. Two-way ANOVA with Tukey’s multiple comparison test. n–s, Ccr5 inhibition by CVC does not affect macrophage (Tg(mpeg1:GAL4FF/UAS:nfsb-mCherry); yellow) migration into the injury site (skeletal muscle labelled using Tg(actc1b:BFP); magenta) or the successful transition into a dwelling macrophage subtype (n; scale bar, 50 μm). o, Quantification (n = 10 per group at each time point). The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. CVC-treated macrophages appear morphologically indistinguishable from controls, with transient macrophages possessing lower sphericity values then their dwelling counterpart (p; scale bar, 20 μm). q, Quantification (n = 45 transient control, n = 42 transient 5 μM CVC, n = 50 dwelling control, n = 39 dwelling 5 μM CVC). Data are mean ± s.d. Two-way ANOVA with Tukey’s multiple comparison test. r, CVC treatment does not alter the response of neutrophils (Tg(mpx:eGFP); magenta) to needle-stab muscle injury (white dotted line). Scale bar, 50 μm. s, Quantification (n = 11 control, n = 14 5 μM CVC). The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. Unpaired two-tailed t-test; t23 = 0.5838, P = 0.5650. t–u, pax3a+ myogenic stem cell (TgBAC(pax3a:GFP); cyan) proliferation is inhibited by CVC addition as demonstrated by decreased EdU incorporation (magenta) of these cells in the wound site (white dotted line) after injury (t; scale bar, 50 μm) and quantification (u, n = 19 per group). The black lines and grey lines within the violin plot indicate the median and quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. Two-way ANOVA with Tukey’s multiple comparison test. v, CVC treatment (pre-treatment for 2 h and maintained after laser ablation skeletal muscle injury (white dotted line) until experimental end point) does not interfere with the initiation and maintenance of dwelling macrophage (white arrows)–MuSC (white arrowheads) associations in the wound site. Scale bar, 50 μm. Frames were obtained from Supplementary Video 13.
Extended Data Fig. 8 Zebrafish germline nampta and ccr5 mutants present severe skeletal muscle regeneration deficits in response to acute muscle injury.
a–j, CRISPR–Cas9 was used to target exon 2 of nampta, which resulted in a germline deletion–insertion mutation that produced an altered amino acid sequence and a subsequent premature stop codon (asterisks) (nampta p.Try61Profs*4, referred to as namptapc41). a, Sanger trace of the genotyping PCR amplicon demonstrating the effect of Cas9–gRNA-induced mutation at a DNA and amino acid sequence (AA) level in the target nampta locus (n = 3). b, Schematic representation of the nampta gene highlighting its exons, mutation site, transcript and primer-binding sites for RT–PCR. c, RT–PCR for nampt cDNA demonstrates a reduction in the level of mutant transcript, encoding a truncated protein, highlighting that it is targeted for degradation by nonsense-mediated decay. actc1b transcript levels act as a loading control. d–f, In contrast to mice, where unconditional Nampt gene knockout results in embryonic lethality84, larval zebrafish are able to survive nampta germline knockout. This is probably due to the fact that Namptb can functionally compensate and fulfil, at least in part, the enzymatic role of Nampta in the NAD+ salvage pathway12. Macrophage and stem cell dynamics are unaffected in the nampta mutant. d, Both nampta heterozygous and homozygous mutants have wound site (white dotted line) macrophage (yellow) dynamics that are comparable to wild-type siblings, with macrophages transition to a dwelling state at 2 dpi. e, Quantification (n = 12 1dpi, n = 19 2 dpi nampta, n = 23 1 dpi, n = 43 2 dpi nampta+/pc41, n = 12 for both 1 and 2 dpi namptapc41). f, Furthermore, dwelling macrophages in the heterozygous and homozygous nampta mutants go on to interact with Pax7+ MuSCs that are present in the wound site. Representative of n = 20 observations. Homozygous mutants present a severe regeneration deficit after needle-stab muscle injury as observed by birefringence imaging (g; scale bar, 500 μm) and quantified (h; n = 12 nampta, n = 13 nampta+/pc41, n = 11 namptapc41). Individual data points are shown. Two-way ANOVA with Tukey’s multiple comparison test. i, This repair deficit could be correlated to a significant proliferation deficit (EdU, white) observed within the injury site after needle-stab skeletal muscle injury (myosin heavy chain (MyHC) to visualize skeletal muscle, magenta). Proliferation within the injury zone in mutant larvae decreased to homeostatic levels observed external to the wound site, highlighting that the mutants failed to elicit the additional proliferative response needed to sustain repair. Furthermore, these observations recapitulate what is seen after ablation of dwelling and mmp9-expressing macrophages. j, Quantification (n = 23 nampta, n = 19 nampta+/pc41, n = 18 namptapc41). k–t, CRISPR–Cas9 was also used to target exon 2 of ccr5 (using two gRNAs) resulting in a deletion–insertion mutation that induced a frame shift and subsequent premature stop codon (ccr5 p.Pro24Leufs*28, referred to as ccr5pc42) (k). l, Schematic of the two exons of ccr5, along with the site of mutation (513-bp deletion and 90-bp insertion) and mRNA transcript. The primer-binding sites for RT–PCR are also documented. m, RT–PCR analysis of ccr5 cDNA demonstrates a 234-bp product, corresponding to the mutant transcript (red arrowhead). No wild-type transcript corresponding to a 657-bp product (red arrow) is present in the mutant. Both PCR fragments have been sequence-verified. The mutant transcript lacks the majority of the chemokine domain encoded by exon 2 of ccr5, and as such the mutant protein, if translated, would be non-functional as it lacks the ligand-binding site. actc1b transcript levels act as a loading control (n = 3). n–p, As for the nampta mutant described above, the ccr5 mutant presented with macrophage dynamics (representative images (n) and quantification (o; n = 8 for both 1 dpi and 2 dpi ccr5, n = 20 1 dpi, n = 23 2 dpi ccr5+/pc42, n = 8 for both 1 and 2 dpi ccr5pc42)) and macrophage–stem cell interactions (p, representative of n = 20 observations) comparable to that of their wild-type siblings. q–t, Furthermore, this mutant mirrored the phenotypic defects described above for the nampta mutant and presented with a significant skeletal muscle-repair deficit upon injury (q (scale bar, 500 μm); quantification (r; n = 11 ccr5, n = 26 ccr5+/pc42, n = 9 ccr5pc42; individual data points shown; two-way ANOVA with Tukey’s multiple comparison test)), due to a wound-site MuSC-proliferation defect (s, muscle labelled by phalloidin; quantification (t; n = 12 ccr5, n = 25 ccr5+/pc42, n = 8 ccr5pc42)). e, j, o, t, The central lines, upper and lower lines within the violin plot indicate the median, upper and lower quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. h, r, Data are mean ± s.d. Two-way ANOVA with Tukey’s multiple comparison test. d, f, i, n, p, s, Scale bars, 50 μm.
Extended Data Fig. 9 Immune-cell-specific gene editing of nampta and namptb and MuSC-specific gene editing of ccr5 in larval zebrafish.
a–c, Validating the macrophage-specific nampta gene editing strategy. a, Macrophages were isolated by FACS at 3 dpf from nampta-gRNA-injected mpeg1-cas9 larvae. DNA isolated from these cells was used to generate a PCR amplicon of the region encompassing the gRNA target site. Sanger sequencing of the amplicon confirmed the presence of sequence disruptions, starting from a few base pairs upstream of the PAM site. b, Nampt protein expression in the wound site was assessed in the nampta-gRNA-injected mpeg1-cas9 larvae after needle-stab muscle injury (white dotted line). The gene-edited larvae presented observably reduced Nampt expression (magenta) within the injury zone (n = 12). Scale bar, 50 μm. c, FACS-isolated macrophages from gene-edited larvae were assayed for Nampt functionality by measuring NAD+/NADH levels using a luminescence-based assay. Macrophages isolated from control, uninjected larvae were used to measure the baseline NAD+/NADH levels for larval zebrafish macrophages. Macrophages from larvae treated with the Nampt enzymatic inhibitor GMX1778 were used to identify the NAD+/NADH levels present in macrophages in the absence of Nampt function. Furthermore, macrophages from larvae treated with nicotinamide mononucleotide (NMN), the main product of the rate-limiting enzymatic reaction of Nampt were used to establish the maximum threshold of the sensitivity of the assays. Macrophages isolated from nampta-gRNA-injected mpeg1-cas9 larvae presented with a reduction in NAD+/NADH levels, reflective of a loss-of-function of Nampt activity within macrophages present in these larvae. This assay would also detect the residual enzymatic activity of Namptb, which would not be affected by this gene-specific targeting approach. The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. One-way ANOVA with Dunnett’s post hoc test for multiple comparisons. d–g, Macrophage (yellow) dynamics in the nampta-gene-edited larvae were assayed after wounding and demonstrated no observable deviation from the control larvae (d, n = 10). nampta-gene-edited larval macrophages, located within the injury zone (white dotted line), transitioned to a dwelling state at 2 dpi (e (scale bar, 50 μm); f, quantification (n = 16 at 1 and n = 2 dpi control, n = 11 at 1 dpi and n = 14 at 2 dpi nampta gRNA-injected)). These dwelling macrophages go on to interact with pax3a+ MuSCs present in the wound (g; scale bar, 50 μm; representative of n = 20 observations). h, Macrophage-specific nampta-knockout larvae were unable to elicit the required proliferative response in the injury zone. Scale bar, 50 μm. Representative of the quantification shown in Fig. 4f. i–l, In zebrafish, Nampta and not Namptb governs the regenerative role of Nampt in muscle regeneration. i, Lateral view of namptb expression by in situ hybridization in the wound site demonstrates constitutive expression from 1 to 3 dpi in the injury site (scale bar, 100 μm). mpeg1-cas9 larvae injected with two gRNAs targeting namptb (schematic, j) demonstrated modest skeletal muscle regenerative abilities by birefringence imaging (k; scale bar, 500 μm) after needle-stab muscle injury. l, Quantification (n = 36 control, n = 34 namptb gRNA-injected). Individual data points are shown. Two-way ANOVA with Tukey’s multiple comparison test. m–o, Macrophages, not neutrophils, are the primary and functional source of Nampta in muscle regeneration. nampta was specifically knocked down in neutrophils (using the mpx-cas9 line; schematic, m), the other key innate immune cell type present in the regenerating area. Using this approach, no regeneration deficit was observed after needle-stab muscle injury, as observed by birefringence imaging (n; scale bar, 500 μm). o, Quantification (n = 14 control, n = 40 nampta gRNA-injected). Individual data points are shown. Two-way ANOVA with Tukey’s multiple comparison test. p–s, MuSC-specific ccr5 gene editing does not affect injury-responsive (white dotted line demarcates wound site boundaries) macrophage dynamics and the transition to a dwelling phenotype at 2 dpi (p (scale bar, 50 μm); q, quantification (n = 11 at 1 dpi and n = 13 at 2 dpi control, n = 9 at 1 dpi and n = 16 at 2 dpi ccr5 gRNA-injected)). Furthermore, these ccr5-gene-edited pax7b+ MuSCs (cyan) display phenotypically wild-type interactions with dwelling macrophages (yellow) located in the injury zone (r; scale bar, 50 μm; representative of n = 20 observations). However, pax7b+ cell-specific ccr5-knockout larvae are not able to maintain the required proliferative response in wound-resident MuSCs (s; scale bar, 50 μm; representative for the quantification in Fig. 4g). f, q, The continuous lines and dotted lines within the violin plot indicate the median and quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. l, o, Data are mean ± s.d. Two-way ANOVA with Tukey’s multiple comparison test. t, Summary schematic of the role of injury-responding macrophages in modulating MuSC proliferation. Following acute muscle trauma (1), macrophages and MuSCs migrate into the injury zone (2). Around half of these injury-responsive macrophages dwell in the wound site for the duration of repair (3). A subset of the dwelling mmp9+mpeg1+ macrophages start to actively interact with pax3a+ MuSCs that are present in the wound (4). These macrophages secrete Nampta, which in turn binds to Ccr5 expressed on MuSCs (4′) and activates a signalling cascade that results in MuSC proliferation (5).
Extended Data Fig. 10 NAMPT supplementation after acute skeletal muscle injury enhances proliferation, specifically in the injury zone.
a–c, NAMPT supplementation does not alter the immune cell response to injury. Needle-stab muscle-injured larvae (white dotted line) were immediately supplemented with hrNAMPT(1) (4 dpf, 0 dpi) and their immune cell dynamics were assayed at 2 dpi. The number of macrophages (Tg(mpeg1:GAL4FF/UAS:nfsb-mCherry); yellow) and neutrophils (Tg(mpx:eGFP); cyan) were comparable to untreated controls (a; scale bar, 50 μm). b, Quantification (n = 11 for neutrophils and n = 23 for macrophages in control, n = 6 for neutrophils and n = 23 for macrophages in 57 nM NAMPT(1)). The central lines, upper and lower lines within the violin plot indicate the median, upper and lower quartiles, respectively. Two-way ANOVA with Tukey’s multiple comparison test. a, Furthermore, the autophagocytic process within the injury zone was compared by LysoTracker (magenta) and found to be similar in control and hrNAMPT(1)-supplemented larvae a. c, Quantification (n = 6 control, n = 23 57 nM hrNAMPT(1)). The central lines, upper and lower lines within the violin plot indicate the median, upper and lower quartiles, respectively. Unpaired two-tailed t-test; t27 = 1.143, P = 0.2629. d, The effect on wound site (white dotted line; skeletal muscle visualized by phalloidin staining, magenta) proliferation (EdU, white) was assayed following NAMPT and combination drug supplementation in the Tg(mpeg1:GAL4FF/UAS:nfsb-mCherry) line. Four individual examples are provided for each group and these images are representative of the quantification presented in Fig. 4h. NAMPT supplementation increases wound site proliferation in control settings and rescues the proliferative deficit that occurs after macrophage ablation. However, as NAMPT acts on the Ccr5 receptor to elicit its proliferative response, inhibiting this receptor by CVC treatment resulted in a proliferation deficit that was resistant to NAMPT-mediated rescue. Furthermore, although the CCR5 ligand, CCL8, functioned to increase proliferation in the wound site, it also increased proliferation external to the injury zone, highlighting that it lacks the wound specificity that NAMPT is able to exert. Scale bar, 50 μm. e, Proliferating satellite cells in mouse muscle injuries supplemented with NAMPT. The gating strategy to isolate the proliferating satellite cell population is shown. Cells that are CD45−, CD11b−, Ly6G−, CD31−, VCAM-1+ and PAX7+ are considered to be satellite cells. Proliferating satellite cells are additionally Ki67+. Quantification at 4 days after treatment is shown in Fig. 4m, n. f, Representative muscle regeneration cryosections stained for PAX7 (satellite cells, yellow), wheat-germ agglutinin (WGA, magenta) and nuclei (DAPI, blue) for tissues collected 6 days after treatment. Scale bar, 50 μm. g, h, Representative tissue sections stained with haematoxylin and eosin (g; scale bar, 100 μm) used to quantify centrally nucleated muscle fibres in the injury zone at 6 days after treatment (n = 6 mice per group) (h). The central lines, upper and lower lines within the violin plot indicate the median, upper and lower quartiles, respectively. Unpaired two-tailed t-test; t10 = 2.770, P = 0.0198. i, j, Immune cell profile after mouse muscle injury supplemented with NAMPT. i, Gating strategy to analyse wound-associated immune cell subsets in muscle injury. The gating strategy is shown for the neutrophil, macrophage and T cell subset panels. j, Percentages were calculated over total live cells in the collected tissue (n = 5 mice for day 6, n = 6 mice for day 8). Data are mean ± s.e.m. Two-way ANOVA with Bonferroni post hoc test for pair-wise comparisons. k, l, Angiogenesis following mouse VML injury treated with NAMPT. k, Muscle injuries were treated with NAMPT (0.5 μg) delivered in fibrin or fibrin-only control and tissues were collected at 6 days after treatment. Representative regenerated muscle cryosections were stained for CD31 (endothelial cells; yellow) and laminin (magenta). Scale bar, 50 μm. l, Quantification of the CD31+ area (n = 6 mice per group). Data are mean ± s.e.m. Unpaired two-tailed t-test; t8 = 3.275, P = 0.00113.
Supplementary information
Supplementary Table 1
Top 500 most expressed genes in all sequenced macrophages. Excel worksheet listing the most expressed genes common to all macrophages analysed. Biological process enrichment analyses on these top 500 genes describe the overall nature of the isolated and sequenced cells.
Supplementary Table 2
UMAP cluster composition. Excel worksheet documenting the composition of the 7 clusters identified by UMAP scatter plot. The macrophage frequency, isolation time point and percentage make up in relation to total number of analysed macrophages is presented.
Supplementary Table 3
Differentially expressed genes and biological enrichment analysis for macrophage subsets identified by UMAP scatter plot. Excel workbook of the differentially expressed genes for each individual cluster (8 worksheets). Biological processes enriched in each individual cluster are presented in the form of a bar graph of the top non-redundant enrichment terms and their relationships in the form of network visualisation.
Video 1
: Following injury, macrophages in the vicinity of the wound site mount an immediate wound migratory response. The video begins by showing a 4 dpf zebrafish larva’s unperturbed skeletal muscle (Tg(actc1b:GFP), magenta) with patrolling muscle resident macrophages (Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry), yellow). Three frames into the video, the larva is subject to an acute laser ablation injury targeting the ventral myotome. Following injury, a subset of macrophages near the wound site actively migrate into the injury site (see stills in Fig. 1a-b’’). Migrating macrophages project large cytoplasmic protrusions on route to the wound site. Macrophages migrate along the mid line as well as over both the dorsal and ventral myotomes. Each migrating macrophage path is shown in individually tracked colours (see stills Extended Data Fig. 1a). Maximum intensity projections of confocal z-stacks imaged at each time point. Larval view: lateral, anterior to left. Video length: 1.02 h, 4 frames/min. Video representative of n=4 observations.
Video 2
: A subset of injury-responding macrophages ‘dwell’ in the wound site for the duration of the regenerative process. The video shows two clips presented side-by-side, extracted from a single continuous 24 h confocal time-lapse movie of macrophages (Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry), yellow) responding to skeletal muscle injury (Tg(actc1b:GFP), magenta). The left clip illustrates the early macrophage response to injury from 2 to 9 hours following injury. Macrophages migrate into the wound site extending long cytoplasmic protrusions, resulting in an initial increase in the wound-located macrophages. These transient macrophages remain in the wound site up to 9.80 h (see stills in Fig. 1c). The right clip shows the late injury-present dwelling macrophages, which are composed of 50% of the original macrophages that migrated into the wound site (see stills in Fig. 1d). Dwelling macrophages remain in the wound site until injury resolution is complete (see macrophage injury residing time quantification in Fig. 1e). Maximum intensity projections of confocal z-stacks imaged at each time point. Larval view: lateral, anterior to left, laser ablation targeted to ventral myotome. Video length: left clip 7 h, right clip 6 h, 6 frames/h. Video representative of n=8 observations.
Video 3
: Transient and dwelling macrophages are phenotypically distinct. This video illustrates the morphological differences between early wound-located transient macrophages and late wound remaining, dwelling, macrophages. The video is presented in a tabular format with the left column highlighting a transient macrophage and the right a dwelling macrophage. The bottom row shows the fluorescent pre-processed image of the macrophage (Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry), yellow) while the top row shows the processed surface rendered image. The surface rendering is pseudocoloured with a sphericity scale where a perfect sphere would have a sphericity value of 1. Transient macrophages have lower sphericity values while dwelling macrophages appear more rounded having significantly higher sphericity values (see graph in Extended Data Fig 1d).
Video 4
: Macrophages that go onto dwell are initially located in close proximity to the wound site at the point of injury. Light-sheet microscopy time-lapse imaging video of a whole 4 dpf Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry)/Tg(actc1b:GFP) (grey) zebrafish larvae, documenting the macrophage response to laser ablation injury from 0.17 h post laser ablation injury until 19.04 h post injury. Retrospective tracking identifies macrophages that go on to dwell within the wound site. The video was processed such that dwelling macrophages are highlighted in yellow and their migration into the injury site (blue) can be observed over the imaged time course. The migratory paths into the injury zone of macrophages that do not adopt a dwelling phenotype (transient macrophages) are also visualised (multi-colour paths). Maximum intensity projection of confocal z-stacks imaged at each time point. Lateral view: anterior to the top, dorsal to the right. Video length: 19 h, 0.67 frames/min. Video representative of n=4 observations.
Video 5
: Timeline of nitroreductase-mediated macrophage ablation in larval zebrafish. Video consists of two clips presented one after the other, the first clip shows the head region and the second the tail region of a 4 dpf compound Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry);Tg(actc1b:GFP) (yellow macrophages, magenta skeletal muscle) larval zebrafish. 5 mM metronidazole (Mtz) is added to the Ringer’s solution in which the anesthetised larvae are maintained for the duration of imaging. Following Mtz addition, larvae are continuously imaged to visualise the temporal response of macrophage ablation (see stills in Extended Data Fig. 1k). Maximum intensity projections of confocal z-stacks imaged at each time point. Lateral view: anterior to the top, dorsal to the right. Each clip length: 13.5 h, 0.67 frames/h. Video representative of n=8 observations.
Video 6
: Once macrophages transition to a dwelling state, they actively start interacting with pax3a+ muscle stem cells present in the wound site. Video consists of two clips extracted from two time-lapse movies taken in immediate succession of the same larvae revealing macrophages (Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry), yellow) and pax3a+ muscle stem/progenitor cells (TgBAC(pax3a:GFP), cyan) responding to muscle injury (muscle injury shown in differential contrast). The first clip is taken immediately following injury and reveals that both macrophages and pax3a+ cells migrate into the injury site independently of each other. pax3a+ cells originate from the undamaged regions of the ablated myotome as well as from adjacent myotomes and migrate to line the edge of the wound site (pax3a+ cells that go onto line the wound edge are indicated by magenta arrowheads) (see stills in Extended Data Fig. 3a-c). At the beginning of the second clip (10.25 hpi) macrophages have transitioned to a dwelling state. Dwelling macrophages initiate intense and continuous interactions with pax3a+ cells lining the wound edge (see stills in Extended Data Fig. 3d). A surface rendered image of these interactions is shown to further highlight the specific nature of these associations. Maximum intensity projections of confocal z-stacks imaged at each time point. Larval view: lateral, anterior to left, laser ablation to ventral myotome. Video length: 1st clip 4 h, (1.54 frames/min), 2nd clip 3.75 h (0.5 frames/min). Video representative of n=5 observations.
Video 7
: Following dwelling macrophage interactions, associated myogenic cells undergo cell division. Video documents an example of an injury-present dwelling macrophage (Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry), yellow) interacting with a pax3a+ cell (TgBAC(pax3a:GFP), cyan) which consequently undergoes cell division (see stills in Fig. 2e). The video shows, in high resolution, the spatial control macrophages exert on specific myogenic cells present in the wound site. Maximum intensity projections of confocal z-stacks imaged at each time point. Video commences at 20 hpi. Larval view: lateral, anterior to left, laser ablation to ventral myotome. Video length: 0.65 h, 14.75 frames/min. Video representative of n=10 time-lapse videos documenting n=26 cell divisions.
Video 8
: Further documentation of dwelling macrophage associated pax3a+ cells undergoing cell division. Video showing dwelling macrophage-myogenic cell contacts leading to the associated myogenic cell undergoing cell division in 3 independent larval laser ablation injuries. Maximum intensity projections of confocal z-stacks imaged at each time point. White arrowheads point to cells undergoing division. Larval view: lateral, anterior to left. Clip 1 (see stills in Extended Data Fig. 3k, injury 1), video commences at 18.5 hpi, video length: 0.4 h, 15 frames/min. Clip 2 (see stills in Extended Data Fig. 3k, injury 2), video commences at 17 hpi, video length: 0.32 h, 7.25 frames/min. Clip 3 (see stills in Extended Data Fig. 3k, Injury 3), video commences at 26 hpi, video length: 5.91 h, 1.4 frames/min. Video’s representative of n=10 time-lapse videos documenting n=26 cell divisions.
Video 9
: Dwelling macrophages interact with pax3a+/met+ muscle stem cells in the wound site. Video documents dwelling macrophage (Tg(mpeg1:GAL4FF/UAS:NfsB-mcherry), yellow)-muscle stem cell (TgBAC(pax3a:GFP);Tg(met:mCherry-2A-KALTA4/UAS:NfsB-mcherry), cyan and yellow double labelled cells) associations within the injury site (white line demarcates boundaries of the injury, video starts 9.33 h post-laser ablation skeletal muscle injury in a 4 dpf larval zebrafish). The video is presented such that two smaller single fluorescence channel windows (cyan channel: pax3a, yellow channel: met and mpeg, stem cells and macrophages, respectively, are distinguished based on morphology and co-labelling with pax3a for muscle stem cells) are placed above the main video of the merged channels. White arrowheads highlight two pax3a+/met+ muscle stem cells that dwelling macrophages are interacting with. During the video, both of these muscle stem cells undergo symmetric cell divisions (highlighted in single fluorescent channels by asterisks). See stills in Extended Data Fig. 3i. Maximum intensity projection of confocal z-stacks imaged at each time point. Lateral view: anterior to the top, dorsal to the right. Video length: 6.34 h, 6 frames/h. Video representative of n=6 observations.
Video 10
: Following muscle stem cell division the pre division-associated dwelling macrophage migrates away from the daughter cells. Video documents the muscle stem cells (cyan, TgBAC(pax3a:GFP)) and dwelling macrophages (yellow, Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry)) response to laser ablation skeletal muscle injury from 11 hpi to 16.5 hpi in a 4 dpf zebrafish larvae. The video is presented such that two smaller single fluorescent channel windows are placed bellow the main video of the merged channel. The video captures two dwelling macrophages in the midst of each simultaneously interacting with two wound-present muscle stem cells. These interactions lead to 4 successive muscle stem cell divisions (white arrows). Following division, the dwelling macrophage on the left maintains short-lived interactions with the stem cell division-generated daughter cells prior to migrating away and taking up a myoseptal position (magenta arrowhead). Following associated-stem cell divisions, the dwelling macrophage on the right maintains cursory interactions with one pair of daughter cells, and consequently actively migrates away from the other pair of daughter cells and relocates to the vertical myosepta. See stills in Extended Data Fig. 3l. Maximum intensity projection of confocal z-stacks imaged at each time point. Lateral view: anterior to the top, dorsal to the right. Video length: 5.53 h, 15 frames/h. Video representative of n=5 observations.
Video 11
: A subset of macrophages that dwell express mmp9. Video captures the response to laser ablation skeletal muscle injury (white line demarcates boundaries of the injury) starting at 1 hpi in a 4 dpf larval zebrafish. A compound transgenic larvae is used to visualise yellow macrophages (Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry) and magenta mmp9-expressing cells (TgBAC(mmp9:EGFP). At the start of the video, mmp9+ neutrophils are seen in the wound site; these cells are morphologically distinct from macrophages and have exited the wound site prior to 4.5 hpi when mmp9 expression is initiated in a subset of mpeg+ macrophages. The video also presents a single magenta channel window of the wound site superimposed towards the top of the main video. White arrowheads identify macrophages that will go onto express mmp9. When a macrophage starts expressing mmp9, the arrowhead changes colour to magenta. This moment is also highlighted in the single channel window by a magenta asterisks. See stills in Extended Data Fig. 6b. Maximum intensity projections of confocal z-stacks imaged at each time point. Lateral view: anterior to the top, dorsal to the right. Video length: 11.83 h, 3 frames/h. Video representative of n=6 observations.
Video 12
: Dwelling macrophages express high levels of NADH. Multiphoton microscopy was used to capture the intrinsic two-photon autofluorescence of NADH (magenta) in a compound transgenic line labelling both macrophages (yellow, Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry)) and muscle stem/progenitor cells (cyan, TgBAC(pax3a:GFP)) following laser ablation skeletal muscle injury. The video captures stem/progenitor cell-associated dwelling macrophages expressing high levels of NADH. Maximum intensity projection of z-stacks imaged at each time point. Lateral view: anterior to the top, dorsal to the right. Video length: 24.5 min, 2 frames/min. Video representative of n=15 observations.
Video 13
: Inhibition of Ccr5 signalling does not affect dwelling macrophage-muscle stem cell interactions following laser ablation skeletal muscle injury. Video consists of two representative clips extracted from two independent time-lapse movies of Tg(mpeg1:GAL4FF/UAS:NfsB-mCherry)/TgBAC(pax3a:GFP) (macrophages in yellow and stem cells in cyan) compound transgenic larvae pre-treated with the Ccr5 inhibitor cenicriviroc (5 µM, pre-treated for 2 h prior to injury and maintained in the drug till experimental end point) and subject to laser ablation skeletal muscle injury (white line demarcates boundaries of the injury). The first clip is taken immediately following muscle injury and documents both macrophages and muscle stem cells migrating into the injury zone. Over time, a subset of the injury-responsive macrophages are seen to take on a more rounded dwelling macrophage phenotype and initiate interactions with wound site-located muscle stem cells (white arrow). At the beginning of the second clip (10 hpi) dwelling macrophages are actively interacting with wound-present muscle stem cells (white arrows). These interactions persist until the end of the time-lapse video (23 hpi). However, none of these interactions are seen to terminate with the associated muscle stem cell undergoing proliferation. Maximum intensity projections of confocal z-stacks imaged at each time point. Larval view: lateral, anterior to left, laser ablation to dorsal myotome. Video length: 1st clip 14 h, (2 frames/h), 2nd clip 13 h (3 frames/h). Video representative of n=11 observations.
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Ratnayake, D., Nguyen, P.D., Rossello, F.J. et al. Macrophages provide a transient muscle stem cell niche via NAMPT secretion. Nature 591, 281–287 (2021). https://doi.org/10.1038/s41586-021-03199-7
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DOI: https://doi.org/10.1038/s41586-021-03199-7
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