Apical localization of Acf7a precedes hair-bundle and CP formation. Micrographs collected using confocal microscopy. A, Hair cells of a posterior lateral-line neuromast, from a zebrafish at 5 dpf, expressing Acf7a–Citrine (yellow) are labeled with phalloidin (red). B displays only the red channel of A. The CPs of the hair cells are oriented opposite to each other. Two cells (B, arrowheads) within the rosette lack significant actin labeling at their apical surfaces, indicating lack of hair bundles and CPs, but contain robust levels of Acf7a–Citrine (A). C, D, A live neuromast expressing Acf7a–Citrine and β-actin–mCherry 24 h after neomycin treatment. β-Actin–mCherry of the CP and hair bundle is red, and Acf7a–Citrine is yellow. D displays red channel only. Four hair cells have regenerated and contain both Acf7a–Citrine and β-actin–mCherry at apical regions (asterisk); however, there are cells within the rosette that contain Acf7a–Citrine and no significant amounts of β-actin–mCherry (arrowheads). E, F, At 48 h after neomycin treatment, the cells that lacked apical localization of β-actin–mCherry developed CPs with PCP, confirming their status as hair cells (arrowheads). F displays red channel only. Scale bar, 1 μm.