Figure 5.
Figure 5. Whole-mount high-resolution imaging of fixed adult hearts to characterize extracellular vesicle (EV) transfer to recipient cells.A–D, Cardiomyocyte (CM)-EV transfer to endothelial cells (ECs). A, Overview maximum projection showing the surface view of an adult Tg(myl7:HRAS-mCherry); Tg(fli:EGFP) zebrafish ventricle, boxed region highlights position of B and C. B and C, Digital zoom of the boxed region in A. B, A maximum projection; C, Three-dimensional reconstruction of the same image reveal CM-EVs within ECs (CMs removed for clarity). D, Schematic summary depicts localization of CM-EVs within ECs. E–H, Limited EC-EV transfer to CMs. E, Overview maximum projection showing the surface view of an adult Tg(kdrl:mCherry-CAAX); Tg(myl7:EGFP) zebrafish ventricle, boxed region highlights position of F and G. F and G, Digital zoom of the boxed region in E. F, A maximum projection; G, A 3-dimensional reconstruction of the same image reveal EC-EVs within CMs (ECs removed for clarity). H, Schematic summary depicts localization of EC-EVs within CMs. I–L, CM-EV transfer to macrophages. I, Overview maximum projection showing the surface view of an adult Tg(myl7:HRAS-mCherry); Tg(mpeg:EGFP) zebrafish ventricle, boxed region highlights position of J and K. J and K, Digital zoom of the boxed region in I. J, A maximum projection; K, A 3-dimensional reconstruction of the same image reveal CM-EVs within macrophages (CMs removed for clarity). L, Schematic summary depicts localization of CM-EVs within macrophages. M–P, EC-EV transfer to macrophages. M, Overview maximum projection showing the surface view of an adult Tg(kdrl:mCherry-CAAX); Tg(mpeg:EGFP) zebrafish ventricle, boxed region highlights position of N and O. N and O, Digital zoom of the boxed region in M. N, A maximum projection; O, A 3-D reconstruction of the same image reveal EC-EVs within macrophages (ECs removed for clarity). P, Schematic summary depicts localization of EC-EVs within macrophages. Q and R, Volume measurements of EV containing compartments are shown as a percentage of volume measurements of the cells within the field of view from 3D image analysis. The boxed region in Q highlights the expanded view in R. Arrows indicate EVs within recipient cells. Scale bars: A, E, I, M=20 μm; B, C, F, G, J, K, N, O=5 μm.