Caspase-dependent proliferation after stem cell ablation. a Schematic of a 4-day post-fertilization (dpf) zebrafish larvae. Large region denotes area of the animal where cell death and proliferation were quantified before and after cell ablation. Small region marks the area used for fixed and live imaging. b Timeline for the addition and removal of metronidazole (MTZ). c The zc1036a GAL4 enhancer trap line drives expression of fluorescently tagged nitroreductase (NTR) in a subset of p63-positive basal stem cells (scale = 100 µm, 50 µm inset). Maximum intensity projections of confocal images for activated caspase-3 (d–f) and bromodeoxyuridine (BrdU) (g–i) at different points after inducing damage (scale = 50 µm). j Quantification of active caspase-3- and BrdU-positive cells reveals a temporal relationship for the proliferative response. Mean number of positive cells from at least n = 11 individual larvae across three independent experiments per time point are plotted. NT=No treatment. k Mean number of BrdU-positive cells in individual larvae after induced apoptosis (n = 31) and in combination with treatment of the apoptosis inhibitor, NS3694 (AI) (n = 77) or caspase-3 peptide inhibitor zDEVD-fmk (zDEVD) (n = 49). Data are from three independent experiments and error bars represent sd; ****p < 0.0001. One-way analysis of variance (ANOVA) with Dunnett’s mutiple comparisons test (j, k)

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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