Disruption oftmc1/2a/2bGenes in Zebrafish Reveals Subunit Requirements in Subtypes of Inner Ear Hair Cells

Abstract

Detection of sound and head movement requires mechanoelectrical transduction (MET) channels at tips of hair-cell stereocilia. In vertebrates, the transmembrane channel-like (TMC) proteins TMC1 and TMC2 fulfill critical roles in MET, and substantial evidence implicates these TMCs as subunits of the MET channel. To identify developmental and functional roles of this Tmc subfamily in the zebrafish inner ear, we tested the effects of truncating mutations in tmc1, tmc2a, and tmc2b on in vivo mechanosensation at the onset of hearing and balance, before gender differentiation. We find that tmc1/2a/2b triple-mutant larvae cannot detect sound or orient with respect to gravity. They lack acoustic-evoked behavioral responses, vestibularinduced eye movements, and hair-cell activity as assessed with FM dye labeling and microphonic potentials. Despite complete loss of hair-cell function, tmc triple-mutant larvae retain normal gross morphology of hair bundles and proper trafficking of known MET components Protocadherin 15a (Pcdh15a), Lipoma HMGIC fusion partner-like 5 (Lhfpl5), and Transmembrane inner ear protein (Tmie). Transgenic, hair cell-specific expression of Tmc2b-mEGFP rescues the behavioral and physiological deficits in tmc triple mutants. Results from tmc single and double mutants evince a principle role for Tmc2a and Tmc2b in hearing and balance, respectively, whereas Tmc1 has lower overall impact. Our experiments reveal that, in developing cristae, hair cells stratify into an upper, Tmc2a-dependent layer of teardrop-shaped cells and a lower, Tmc1/2b-dependent tier of gourd-shaped cells. Collectively, our genetic evidence indicates that auditory/vestibular end organs and subsets of hair cells therein rely on distinct combinations of Tmc1/2a/2b.

Tmc Reliance Is Biased by the Hair Cell Subtype and Position Within the Ear

“…A potential explanation for the discrepancy between our findings and those of Smith et al (2020) could be genetic in origin. Smith et al (2020) used a zebrafish strain with a mutation in exon 3 of the tmc1 gene. In our work, the tmc1 cwr4 and tmc1 cwr5 mutations are on exon 7 and exon 5, respectively.…”

“…Because these outcomes disagree with recent findings by Smith et al (2020) , who stated that Tmc1 and Tmc2b are individually sufficient for MET function in tall hair cells and did not note a difference in uptake of FM dye in their tmc1 mutant, we verified our findings using a second tmc1 mutant strain, tmc1 cwr4 ( Supplementary Tables S1 , S2 ; Chen et al, 2020 ). In the tmc1 cwr4 mutant, tall hair cells in the central thickness of the lateral crista displayed a substantial reduction in uptake after 4-Di-2-ASP presentation ( Figure 2A ).…”

“…It is possible that exon skipping plays a role ( Anderson et al, 2017 ), and exon 3 of tmc1 is not as critical for the protein’s function in tall hair cells. Alternatively, Smith et al (2020) may be imaging another region of the crista than the present study where hair cell Tmc dependencies are different. Nevertheless, tall hair cells in the central thickness of the lateral crista depend heavily on Tmc1 ( Figure 2G ).…”

“…One, hair cell mechanotransduction in this fish strain is dependent on just one Tmc protein, Tmc1, eliminating the chances of complex Tmc interactions that could complicate the interpretation of results. Two, the hair cells that still function are in the posterior macula, but functional hair cells have not been mapped with high resolution ( Chen et al, 2020 ; Smith et al, 2020 ). Three, this mutant can hear.…”

“…The soma of each of these epithelial cells presents a mechanosensitive hair bundle at its apical surface. Consisting of an array of stereocilia of graded lengths, the hair bundle is asymmetrical and is most sensitive along a single axis of best sensitivity (Shotwell et al, 1981). Both hearing and balance depend on hair cell heterogeneity (Eatock and Hurley, 2003;Kandel, 2013).…”