Supplementary Materials

Asymmetric division of clonal muscle stem cells coordinates muscle regeneration in vivo

David B. Gurevich, Phong Dang Nguyen, Ashley L. Siegel, Ophelia V. Ehrlich, Carmen Sonntag, Jennifer M. N. Phan, Silke Berger, Dhanushika Ratnayake, Lucy Hersey, Joachim Berger, Heather Verkade, Thomas E. Hall, Peter D. Currie

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Figs. S1 to S10
  • Captions for Movies S1 to S10
  • References

Images, Video, and Other Other Media

Movie S1
A subset of myf5:GFP cells are activated in response to muscle injury. Continuous confocal time-lapse imaging of a 6dpf zebrafish larvae, two days after needle stick injury. The injured larvae is transgenic for both 10 Tg(actc1b:mcherry), which marks differentiated fibres (red) and Tg(myf5:GFP), which marks potential muscle stem and progenitor cells (green). Individual frames are 30 minutes apart covering a 5 hour duration. A subset of cells are activated to form hyper-polarized cells that move into the wound site, with some cells being activated and the others remaining unactivated. Images are maximum projections of a confocal z-stack, lateral view, anterior to the left.
Movie S2
Tissue contraction plays little role in the initial phase of muscle regeneration. Single confocal section images taken at the same depth in the wound at 30 min intervals during the regenerative process. This analysis reveals over the 450 min time frame (t=minutes) that there is no passive closure of the wound. In fact, the loss of muscle fibres over this time frame increases the physical size of the wound. Continuous time lapse is of a 6dpf larvae, 2 days after needle stick injury, transgenic for Tg(myf5:GFP) (green) and Tg(acta1b:mCherry) (red).
Movie S3
Myf5 positive cells enter the wound from different depths. Three movies of the same injured larvae captured at distinct phases of the regenerative process. Movies consist of confocal images that encompass the entire injury zone captured in z of a Tg(myf5:GFP) (green) and Tg(acta1b:mCherry) (red), 6dpf transgenic larvae, 2 days after needle stick injury. myf5:GFP positive cells can be seen in the majority of individual images of the z-stack movies at the three distinct time points shown.
Movie S4
In vivo imaging of muscle regeneration. Continuous confocal time-lapse imaging of three distinct 4 dpf larvae double transgenic for Tg(myf5:GFP) (green) and Tg(actc1:mCherry) (red), post laser ablation injury. These fish were subsequently continuously time-lapsed with individual frames 30 minutes apart covering a 48 hour period. Four specific phases are evident as also illustrated in Figure 6. 1. "Migration and contact", where stem cells move to the wound and contact dying fibres. 2. "Proliferation and activation", where cells undergo a proliferative response to generate the differentiation competent compartment. 3. "Bipolar extension and interaction", where activated myf5-positive cells polarize and interact. 4. "Resident fibre capture and differentiation", where differentiated uninjured muscle fibres extend filopodial–like membrane protrusions, which adhere and "lasso" the activated cells that are guided to generate new muscle adjacent to these fibres. Images are maximum projections of confocal z-stacks, lateral view, anterior to the left, of a dorsal hypaxial myotome injury. The first injury time-lapse is also illustrated in Figure 1H. Representative movies selected from a total of n=8 individually examined injured fish.
Movie S5
Myogenin mutant zebrafish are blocked at the differentiation phase of muscle regeneration. Continuous confocal time-lapse imaging of three distinct 4dpf homozygous myogfh265 mutant larvae, double transgenic for Tg(myf5:GFP) (green) and Tg(actc1:mCherry) (red), post laser ablation injury. These fish were subsequently continuously time-lapsed with individual frames 60 minutes apart covering a 35 hour period. This analysis revealed that myf5 expressing cells in myogfh265 mutant fish did migrate effectively to the injury site with a slight delay compared to their wildtype counterparts. These myf5 cells subsequently proliferated and attempted to form bipolar shape, but instead fragmented and died within the wound site rather than appropriately differentiating. Images are maximum projections of confocal z-stacks, lateral view, anterior to the left, of a dorsal hypaxial myotome injury. The first injury time-lapse is also illustrated in Figure 2A. Representative movies selected from a total of n=7 individually examined injured fish.
Movie S6
cmet-positive cells activate during muscle regeneration. Continuous confocal time lapse imaging of 4dpf larvae immediately post injury transgenic for Tg(cmet:mCherry-T2A-KalTA4) (red) and Tg(pax7a:GFP) (green), also imaged in the brightfield channel. A single cmet high/pax7 low cell (arrow) maintains cmet expression through several rounds of division that generate pax7 high cells, which elongate and differentiate. Imaging is a 21 hours time-lapse at 60 minutes per frame. Images are single confocal sections, lateral view, anterior to the left, of a dorsal hypaxial myotome injury, as illustrated in Figure 5E.
Movie S7
cmet-positive cells undergo asymmetric divisions. Four different examples of asymmetric divisions in larvae that have undergone either laser ablation injury (Injury 1 and 2) or needle stick injury (Injury 3 and 4) are presented. Laser injury allows the higher resolution imaging evident in the first two movies. Arrows mark cell division and the generation of cmet low/pax7 high progenitor cells. In each case the cmet high/pax7 low stem cell is maintained, indicated by arrowheads.
Movie S8
Uninjured musclebow larvae. 4dpf uninjured larvae transgenic for Tg(msgn1:CreERT2) and Tg(ubi:zebrabow), to which tamoxifen addition results in clonal labeling of all somite derived cells. All muscle fibres express 13 distinct zebrabow rearrangements. Animated confocal z-stack virtually sectioned in sagittal and transverse view imaged immediately prior to needle stick injury. Identical larvae as illustrated in Figure 4.
Movie S9
Injured musclebow larvae exhibit clonal muscle regeneration. The same larvae as in Supplementary Movie S8 at 7dpf, three days after injury. Regenerating fibres exhibit the same spectral properties, indicating they arose from a common progenitor and are clonally related. Animated confocal z-stack virtually sectioned in sagittal and transverse view imaged post needle stick injury. Identical larvae as illustrated in Figure 4.
Movie S10
Pre injury and post injury in toto imaging of musclebow larvae. 12 individual injuries documented prior to and after needle injury. For each larvae there are paired views of 4dpf uninjured larvae immediately prior to needle stick injury and the same larvae at 7dpf, three days after injury, transgenic for Tg(msgn1:CreERT2) and Tg(ubi:zebrabow), to which tamoxifen addition results in clonal labeling of all somite derived cells. All muscle fibres express distinct zebrabow rearrangements. Animated confocal z-stack virtually sectioned in sagittal and transverse view.