Loss of V-ATPase does not alter proliferation but induces hair cell death in mutant neuromasts. (A,B) A 1-h pulse with BrdU results in a similar number of BrdU-positive cells in wild-type and atp6v1f−/− neuromasts at 2 dpf and 3 dpf. Representative images show BrdU-labeled cells (orange) and DAPI-stained neuromast nuclei (blue). Arrowheads denote pyknotic nuclei. Dashed line circles indicate the neuromast boundary. (B) Quantification of the number of BrdU-labeled cells per neuromast at 2 dpf and 3 dpf. (C,D) The number of pyknotic nuclei increases from 2 dpf through 4 dpf in both atp6v1f−/− (C) and atp6v1h−/− (D) neuromasts. (E) Snapshots from live time-lapse imaging of wild-type and atp6v1f−/− hair cells marked by Tg(myo6b:tdtomato) expression. The arrowhead follows a single hair cell in the atp6v1f−/− neuromast over time as it dies. Fluorescent tdTomato protein accumulates into aggregates that are reminiscent of LC3b-GFP aggregates (see Fig. 3). Wild-type hair cells remained healthy. Scale bars: 5 µm. n=number of embryos. ***P=0.0009 and ****P<0.0001 by unpaired Student's t-test with Welch's correction. ns, not significant.
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