Variable dependence on Tmc2b for mechanotransduction in posterior neuromast hair cells. a Extracellular recordings of microphonic potentials measured from posterior neuromasts with A-P orientations of 6-dpf zebrafish larvae are displayed. The responses in tmc2b −/− are often absent, bottom trace (n = 9/15); however, several responses (n = 6/15) are highly asymmetric, with weakened amplitude for one direction of stimulus and no response for the other direction, penultimate trace. Deflection anteriorly (A) or posteriorly (P). b, c Qualitative confocal images of hair cells labeled with FM1-43FX (red), to assess mechanotransduction function, and parvalbumin 3 (cyan)50, as a counter label, to visualize mature hair cells. b A posterior neuromast from a tmc2b +/− animal demonstrates co-labeling of all hair cells. c In a tmc2b −/− animal, a posterior neuromast contains hair cells that label with FM1-43FX and a subset that do not (label:no label; 2:5). Orange n, hair cells labeled with parvalbumin 3 antiserum. Blue n, hair cells that load with FM1-43FX. Green n, hair cells that do not load. Scale bar = 5 μm. d, e Percentages of hair cells within PLL neuromasts that label with 4-Di-2-ASP. d In neuromasts of tmc2b +/+ and tmc2b +/− animals, most hair cells take up fluorophore. Approximately 35% of hair cells in each PLL neuromast of tmc2b −/− fish take up 4-Di-2-ASP. In contrast, in cdh23 aj64a/aj64a mutants, all hair cells take up dye, though at much lower quantities than wild-type animals (see f and Supplementary Fig. 4b). **** Kruskal–Wallis test P value < 0.0001. n values ≥ 20. e In mutants, A–P-facing and D–V-facing posterior neuromasts have similar percentages of hair cells that gather fluorophore, ~38 and 32%, respectively. ****Student’s t-test P value < 0.0001. n values ≥ 23. f Mean fluorescence intensity of 4-Di-2-ASP uptake of the brightest cell per neuromast. n values ≥ 17. Kruskal–Wallis test ****P < 0.0001, ***P = 0.001, **P = 0.0215
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