Fig. 2: Labeling, live imaging, and identification of lens cell-derived exosomes in zebrafish embryos.
From: In vivo imaging reveals exosome-mediated intercellular communication in lens development
A Schematic diagram of the plasmid construct used for live tracking of Cd63+ exosomes in the lens. cryaa-Cd63-AcGFP pDNA served as the experimental plasmid, whereas cryaa-AcGFP pDNA served as the control. B Expression of Cd63-AcGFP in the lens of zebrafish at 3.5 dpf after the injection of cryaa-Cd63-AcGFP pDNA at the 1-cell stage. C Live confocal images of zebrafish lens at 3.5 dpf showing the expression of Cd63-AcGFP or AcGFP after plasmid injection. The left panel shows the whole lens layer scan overlay, and the right panel shows the central section of the lens. Z-scan images of the whole lens are shown in Fig. S1D. D Still image from a time-lapse video (Video S1) of the lens in a 48 hpf embryo expressing Cd63-AcGFP. Dashed box 1 highlights the area with notable movement of AcGFP-labeled exosomes in the center of the lens, with enlarged images shown in (E). Box 2 shows the AcGFP signal in the retina surrounding the lens, with enlarged images shown in (H2). Box 3 shows the AcGFP signal in the boundary area between the lens and the surrounding retina, with enlarged images shown in (H3). E Enlarged images of box 1 in (D). The dashed areas indicate two examples of regions with notable movement of AcGFP-labeled exosomes, labeled (a and b), with their enlarged images shown in (F and G), respectively. The moving exosomes are marked with white arrows. F, G Magnified views of regions (a and b) in (E), highlighting the moving exosomes indicated by white arrows. The scale bar is 2 μm. H H2 and H3 show magnified images of Box2 and Box3 from (D), respectively. H2 shows the movement of AcGFP signals in the retina surrounding the lens, whereas H3 illustrates the process of AcGFP signals moving from the lens to the surrounding retina. I Immunoelectron microscopy (IEM) image of the lens in the zebrafish expressing cryaa-Cd63-AcGFP at 3.5 dpf, showing exosome-like vesicles positive for GFP-immunogold. J Diagram of zebrafish EVs extraction. K TEM analysis of zebrafish EVs. L Nanoflow cytometry analysis revealed that nearly 72% of the GFP-positive particles were also positive for the EVs membrane dye.
