Two populations of morphologically distinct hair cells in the central thickness of the lateral crista of larval zebrafish. (A,B) Transient (A) and stable (B) transgenic expression of Cerulean fluorescent protein in the lateral crista reveals two morphologically distinct hair cells: short (arrows) and tall (arrowheads). The brackets mark the neck regions of tall hair cells. Scale bar = 5 μm. (C) Left panel, schematic of a dorsal view of a 7-dpf zebrafish ear, illustrating the five mechanosensory patches: the anterior crista (ac), lateral crista (lc), posterior crista (pc), anterior macula (am), and posterior macula (pm). Middle panel, enlarged view schematizing the hair cell arrangement in the lateral crista highlighted in yellow in the left panel. Each yellow circle represents a single hair cell, and the black dot within represents the position of the kinocilium (fonticulus). The red rectangle marks the section from which the hair cells are imaged and characterized. A, anterior; P, posterior; M, medial; L, lateral. Right panel, schematic of the two populations of hair cells (short and tall) in this section of the lateral crista based on imaging results. (D) The distribution of somata lengths of the lateral crista hair cells from 7-dpf zebrafish. n = 4. Pink bars represent short hair cells. Blue bars represent tall hair cells. (E) The somata lengths (mean ± SD) of the two groups of hair cells shown in panel (D). ****P < 0.0001, unpaired t-test.

Tall and short hair cells of the lateral crista display biases for Tmc proteins in mechanotransduction. (A) Representative confocal images of 4-Di-2-ASP (orange) dye uptake assays showing differential dependencies of tall and short hair cells on Tmc proteins in the lateral cristae of 7-dpf larvae. Solid lines outline tall hair cells with 4-Di-2-ASP uptake in a wild-type sibling, a tmc2a^cwr3 single mutant, and a tmc2b^cwr2tmc2a^cwr3 double mutant. In each wild-type tall hair cell, there is an accumulation of dye near the cell’s base. The dashed lines outline tall hair cells with diminished 4-Di-2-ASP uptake in the tmc1^cwr5 single mutant and the tmc1^cwr4 single mutant or short hair cells with no or little 4-Di-2-ASP uptake in the tmc2b^cwr2tmc2a^cwr3 double mutant and the tmc2a^cwr3 single mutant. Graph of lengths of hair cells that have diminished 4-Di-2-ASP uptake. Each data point represents a single cell’s length and associated genotype. Cells associated with each data point are shown in confocal images with dashed outlines. (B) Representative confocal images of FM 4–64 (red) uptake assays for a heterozygous sibling control (tmc2b^cwr2/+tmc2a^cwr3/+, B’), a tmc1^cwr4 single mutant (B”), a tmc2b^cwr2tmc2a^cwr3 double mutant (B”’), and a tmc2a^cwr3 single mutant (B””), each at 7-dpf. The lateral crista hair cells express GCaMP6s-CAAX (green), making each cell apparent. The red or white asterisks mark the short hair cells with or without FM4-64 loading, respectively. Red, yellow, or white arrowheads mark tall hair cells with normal, reduced, or no apparent FM4-64 dye loading, respectively. Red asterisk in panel (B”’) marks a short hair cell erratic that permits FM4-64 entry in the absence of Tmc2a and Tmc2b. (C–E) Graphs of normalized hair cell fluorescence intensity ratios (I_{Tall HCs (hair cells)}/I_{Short HCs}, C) and the percentages of hair cells that loaded with FM4-64 (n_{loaded HCs}/n_{total HCs}, D,E). (C) Two-tailed unpaired student’s t-test, ****P < 0.0001. (D) One-way ANOVA, P = 0.6076. (E) Two-tailed unpaired student’s t-test, ****P < 0.0001, **P = 0.0017. For the tmc1^cwr4 single mutant, n_{Tall HCs} = 23, n_crista = 4. For the tmc2b^cwr2tmc2a^cwr3 double mutant, n_{Tall HCs} = 15, n_{short HCs} = 28, n_crista = 3. For the tmc2a^cwr3 single mutant, n_{Tall HCs} = 21, n_{short HCs} = 30, n_crista = 3. For the controls, n_{Tall HCs} = 27, n_{short HCs} = 40, n_crista = 4. (F) 4-Di-2-ASP (left, live) and FM1-43FX (right, fixed with hair bundles labeled by phalloidin) uptake assays demonstrate that mechanotransduction of lateral crista hair cells is absent in the tmc2b^cwr8tmc1^cwr4tmc2a^cwr6 triple mutant, regardless of hair cell subtype. Control, tmc2b^cwr8/+tmc1^cwr4/+tmc2a^cwr6/+. (A,B,F) Scale bar = 5 μm. (G) Model of Tmc dependencies in the central thickness of the lateral crista.

Map of hair bundle polarities in the posterior macula of a 7-dpf larva. (A) Confocal image of a single posterior macula labeled with phalloidin. Circumscribed regions contain groups of hair cells with particular hair bundle polarities: yellow – anterior, blue – posterior, red – ventral, and green – dorsal. (B) Enlarged view of a hair bundle in square in panel (A). The hair bundles angle of deviation from the A-P axis is depicted. (C) Posterior macula hair bundle polarities and associated hair cell numbers. Each colored line length represents the number of hair cells with that orientation in each group from panel (A). Each line orientation is the mean deviation from the A-P axis ± SD. (D) Map of hair bundle polarities in the posterior macula from panel (A). Each color circle represents an individual hair cell, and the black dot within represents the position of the kinocilium, which reflects bundle polarity. Lines of polarity reversal are shown in the bulbus (blue dashed line) and narrow region (yellow dashed line) for D-V-facing and A-P-facing hair cells, respectively. Axes of best sensitivity are depicted by gray arrows.

Hair cells of the posterior macula are differentially dependent on Tmc proteins. (A) Representative images of FM1-43FX (green) uptake in posterior maculae of wild-type and mutant larvae at 7 dpf. Hair bundles are labeled with phalloidin (red). In a wild-type macula, passage of FM1-43FX is observed in all hair cells. In the tmc2b^cwr2 tmc2a^cwr3 double mutant, FM1-43FX uptake is limited to two symmetrical stripes of hair cells located toward the posterior of the macula at the dorsal and ventral poles (arrowheads). In contrast, the tmc2a^cwr7 single mutant or tmc1^cwr4 tmc2a^cwr7 double mutant hair cells do not reveal a pattern similar to the tmc2b^cwr2 tmc2a^cwr3 double mutant. A, anterior; D, dorsal. Scale bar = 10 μm. Similar patterns of saccular uptake were observed for five larvae for each mutant type and control. (B) (Left) Map of hair cell positions and hair bundle polarities in relation to Tmc1 use within the posterior macula based on the tmc2b^cwr2 tmc2a^cwr3 double mutant. Each color circle (red or blue) represents an individual hair cell, and the black dot within represents the position of the kinocilium, which reflects bundle polarity. Double-headed arrow indicates axis of best sensitivity preserved in the tmc2b^cwr2 tmc2a^cwr3 double mutant. (Right) Schematized zebrafish head depicting just one axis of best sensitivity (dorsal-ventral) able to elicit a startle response.