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.

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