Overlapping expression of tmc1/2a/2b in nascent hair cells at 1 dpf. (A) Image of the anterior region of a 1 dpf embryo. Otocyst indicated by asterisk. Expression of tmc1(B), tmc2a(C), and tmc2b(D). Merge of all three channels (E) and overlay with a DIC image (F). In merged images, fluorescent label for tmc1 transcripts is shown in blue, tmc2a in green, and tmc2b in red. (G–L) Single optical sections (0.75 μm) of overlapping expression of tmc1/2a/2b in hair cells at 1 dpf. Dotted outlines indicate the somas of single hair cells. (G–I) Lateral view of three hair cells in the anterior macula at 1 dpf with visible apical structures. (J–L) Lateral view of the cell bodies of two hair cells in the posterior macula at 1 dpf (kinocilia are not present within the same section).

Emergence of differential expression of tmc1/2a/2b at 2 dpf. (A) Image of the anterior region of a 2 dpf embryo (asterisk indicates otocyst). Lateral views with focal planes including the anterior macula (utricle; B) and posterior macula (saccule; C) are shown. (D) Maximum projection each tmc gene including a merge of all three genes expressed in a top-down view of the anterior macula (utricle; right panel). (E) Expression of tmc1/2a/2b in the posterior macula (saccule). In merged images, fluorescent label for tmc1 transcripts is shown in blue, tmc2a in green, and tmc2b in red.

Central zones of tmc2a with tmc1/2b in peripheral hair cells at 5 dpf. (A) Lateral view of the inner ear. (B) Schematic of the five end organs at this stage of development. (C) Overview of tmc1/2a/2b expression in the ear including three overlying neuromasts at the surface of the skin. (D–F) All are top-down views of each end organ. Single-channel fluorescence for each gene is depicted, along with merged images. In merged images, fluorescent label for tmc1 transcripts is shown in blue, tmc2a in green, and tmc2b in red. The presumptive striolar zone of the utricle is outlined in (D).

Differential expression in hair cell layers of the lateral crista at 5 dpf. (A) Lateral view of the lateral portion of the crista. Layers are indicated by brackets. (B–D) Single-channel fluorescence for each tmc gene. (E) Merge indicating expression of tmc2a in the upper layer and expression of all three tmc1/2 genes in the lower layer of hair cells. Dotted lines outline cell bodies. In merged image, fluorescent label for tmc1 transcripts is shown in blue, tmc2a in green, and tmc2b in red.

Differential pattern of expression of tmc1/2a/2b persists at 15 dpf. (A) Lateral view of the inner ear. (B) Overview of tmc1/2a/2b expression in the ear. (C–E) Single-channel fluorescence for each gene is shown along with merged images (right panels). In merged images, fluorescent label for tmc1 transcripts is shown in blue, tmc2a in green, and tmc2b in red.

Expression of tmc1/2a/2b and mechanotransduction components in hair cells at 40–120 hpf using scRNAseq data. (A) UMAP distributing the striolar, extrastriolar, lateral line and ampullary/cristae inner ear hair cells with expression of tmc1/2a/2b with log-normalized scRNAseq expression output. (B) Dotplot comparing presence and expression of these components in striolar vs. extrastriolar hair cells of the macular organs. (C) Diagram of the components that comprise the mechanotransduction complex. (D) UMAP of the striolar subset and clustered to tmc2a + versus tmc2a/2b + with dotplot showing the expression distribution of MET channel components for the tmc2a + (orange) compared to tmc2a/2b + (green). (E) UMAP of the extrastriolar subset and clustered to tmc2a + versus tmc1+ with dotplot showing the expression distribution of mechanotransduction channel components for the tmc2a + (blue) compared to tmc1+ (orange). In the Y-axis of the dotplot, the age of the cell is grouped in hours with the number of samples in the group labeled in parenthesis.

Expression of cib3 correlates tightly with tmc1 expression (5 dpf). In merged images, fluorescent label for cib2 transcripts is shown in green, cib3 in red, and tmc1 in blue. (A) Single channels and merged image for utricular hair cells. (B) Single channels and merged image for saccular hair cells. (C) Single channels and merged image for ampullary hair cells of the lateral crista. The presumptive striolar zone of the utricle is outlined in (A).

loxhd1a is enriched in striolar hair cells, whereas loxhd1b varies within each sensory organ (5 dpf). In merged images, fluorescent label for loxhd1a transcripts is shown in green, loxhd1b in red, and tmc1 in blue. (A) Single channels and merged image for utricular hair cells. (B) Single channels and merged image for saccular hair cells. (C) Single channels and merged image for ampullary hair cells of the lateral crista. The presumptive striolar zone of the utricle is outlined in (A).

Expression of ush1c is enriched in peripheral or extrastriolar hair cells (5 dpf). In merged images, label for ush1c transcripts is shown in red, tmc2a in green, and tmc1 in blue. (A) Single channels and merged image for utricular hair cells. (B) Single channels and merged image for saccular hair cells. (C) Single channels and merged image for ampullary hair cells of the lateral crista. The presumptive striolar zone of the utricle is outlined in (A).