Figure 3.

Supporting cells phagocytosis is closely correlated with hair cell death. Utricles (E17–E19) expressing β-actin-EGFP were incubated with 1 mm streptomycin and 0.1 μm TOTO-3, a fluorescent nucleic acid dye excluded by the plasmalemma of viable cells. Dual-wavelength time-lapse images were captured for EGFP (green) and TOTO-3 (blue). A, B, Two examples where supporting cells expressing β-actin-EGFP remove nonexpressing hair cells (stars) from the sensory epithelium. The position of the hair cell bundle is marked in A (arrow). A, Excision of the stereocilia bundle occurs with negligible uptake of TOTO-3 into the hair cell cytosol (−100′ to −5′) (supplemental Movie 3, available at www.jneurosci.org as supplemental material). Significant uptake of TOTO-3 is observed as supporting cells engulf the hair cell soma within a phagosome (+5′). TOTO-3 fluorescence confirms that the phagosome contains chromatin and that the hair cell confined within is dead. B, In the second example, uptake of TOTO-3 occurs with formation of the supporting cell phagosome. After the initial engulfment, TOTO-3-labeled hair cell chromatin is transported into the supporting cells' cytoplasm. Not all of the hair cell nucleus is engulfed by the β-actin-EGFP-expressing supporting cell. Fragments of TOTO-3-labeled chromatin (arrowheads) remain either in the extracellular space or within supporting cells that did not express β-actin-EGFP. C, Histogram of time delays between the onset of supporting cell phagocytosis and TOTO-3/PI uptake in hair cells (n = 35 events). The modal response was for these events to occur in the same frame, i.e., a delay of 0 min. Negative delays indicate that TOTO-3/PI uptake preceded phagosome formation and vice versa. Scale bars: 10 μm. Time is expressed in minutes, and the axis has been compressed where indicated.