Apoptosis-induced stem cell division requires Wnt8a on the surface of ESABs (epithelial stem cell-derived apoptotic bodies). a–c Time-lapse confocal images of an epithelial stem cell producing an apoptotic body containing Wnt8a-GFP (scale = 5 µm), see Supplementary Movie 4. d Quantitation of green fluorescent protein (GFP) fluorescence in ESABs after overexpression of Wnt8a by heat-shock induction. e Schematic of strategy to isolate ESABs by differential centrifugation. f–h Whole cells (scale = 10 µm) and ESABs (scale = 5 µm) isolated by differential centrifugation exhibit mCherry fluorescence (magenta) and are appropriate size and morphology. i Quantification of the number of mCherry-positive ESABs after purification (magenta), compared to extracellular vesicles isolated from zebrafish larvae under homeostatic conditions (gray), using flow cytometry. j Size distribution of apoptotic bodies compared to size match bead controls (1 µm and 3 µm boxes, FSC = forward scatter, SSC = side scatter, see gating strategy in Supplementary Figure 6 a-b). k–m Transmission electron micrographs of Wnt8a immunogold labeling on whole-mount purified apoptotic bodies (scale = 500 nm). n The mean number of Wnt8a foci on individual ESABs from uninjected (n = 21) and Wnt8a CRISPR (clustered regularly interspaced short palindromic repeats)-injected (n = 10) animals. Data are from three independent experiments and error bars represent sd; ****p < 0.0001, **p < 0.001. Unpaired two-tailed t-test (d, n)
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