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Fig. 5
Hair cell PCP and neuromast position govern dependence of mechanotransduction channel function on Tmc2b. a?f Confocal images of hair cells from L1, LII.1, and IO4 neuromasts of tmc2b +/? or tmc2b ?/? animals. FM1-43FX (red) uptake reveals functional channels, and phalloidin (cyan) shows hair bundle polarity. Qualitative maps (yellow) of micrographs from tmc2b ?/? mutants show that subpopulations of posterior- and ventral-facing hair bundles preferentially function (red) in L1 and LII.1, respectively; in contrast, hair cell uptake in IO4 is non-biased. g In PLL neuromasts with A?P orientations of tmc2b ?/? mutants, the vast majority (n?=?21) of FM1-43FX uptake is by hair bundles that are posterior facing (P-facing). Similarly, in the A?P-oriented neuromasts of the ALL of tmc2b ?/? (O1, O2, OP1, and MI2), the overwhelming majority of FM1-43FX uptake is by P-facing hair cells (n???5, except for O1, n?=?4). Red bars represent tmc2b ?/?, and black bars signify tmc2b +/+ and tmc2b +/?. h In the PLL neuromasts with D?V orientations of tmc2b ?/? mutants, ventral-facing (V-facing) hair cells dominate the population that uptakes the fluorophore (n?=?16). In ALL neuromasts with D?V orientations of tmc2b ?/?, the patterns of hair cells that take up FM1-43FX are complex. For IO4, there is no significant difference between the numbers of D- and V-facing hair cells that loaded with FM1-43FX. The FM1-43FX loading percentages are 82.6?±?11.1 % (n?=?5) for V-facing hair cells and 83.0?±?11.1 % (n?=?5) for D-facing hair cells in tmc2b ?/?, respectively. One-way ANOVA with Holm-Sidak?s multiple comparisons test, P? =?0.9986. D?V-oriented neuromasts MI1 and M2 have opposite PCP-related loading preferences in mutants. In MI1 and M2 of tmc2b ?/? animals, the percentages of V-facing hair cells that load with FM1-43FX are 36.1?±?3.4% (n?=?6) and 2.9?±?2.9% (n?=?5), respectively. Whereas, the percentages of D-facing hair cells from MI1 and M2 that load with FM1-43FX are 7.9?±?3.6% (n?=?6) and 44.9?±?8.3% (n?=?5), respectively (see Supplementary Table 1 for statistics). Corresponding images of MI1 and M2 displayed in Supplementary Fig. 7. Scale bar?=?5??m. Arrowheads, immature hair cells that do not take up FM1-43FX. *dying hair cell