Figure 4

Rapid externalization of phosphatidylserine (PtS) in response to neomycin treatment. Larval zebrafish (6 dpf) were incubated in Alexa 555-conjugated annexin V and neomycin was added to the water, for a final concentration of 100 μM. Fish were euthanized and fixed after 90 s, 2, 5, or 10 min of exposure to neomycin. Annexin V labeling was observed on the stereocilia bundles of most neomycin-treated fish (e.g., arrow, 90-s image, each red structure represents a single stereocilia bundle), and indicates the presence of PtS on the outer membrane surface. We observed annexin V binding as early as 90 s after the initiation of neomycin treatment (**p = 0.0045, one-way ANOVA), and the number of annexin V-labeled cells increased with longer exposure times (***p < 0.0001, one-way ANOVA). Annexin V labeling was limited to the apical (external) surface of hair cells, as confirmed by side view reconstruction of confocal stacks (arrow, “side view”). We observed no labeling on the basal hair cell surface, which is located within the epidermal barrier of the fish. We also did not observe annexin V binding on any other cells within the body of the fish, suggesting that annexin V does not become internalized. Plotted data were collected from neuromasts L4/5, for a total of 17–40 neuromasts/time point.