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Disruption of Tmc1/2A/2B Genes in Zebrafish Reveals Subunit Requirements in Subtypes of Inner Ear Hair Cells

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Disruption of Tmc1/2A/2B Genes in Zebrafish Reveals Subunit Requirements in Subtypes of Inner Ear Hair Cells Research Report: Regular Manuscript Disruption of tmc1/2a/2b genes in zebrafish reveals subunit requirements in subtypes of inner ear hair cells https://doi.org/10.1523/JNEUROSCI.0163-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.0163-20.2020 Received: 15 January 2020 Revised: 14 April 2020 Accepted: 16 April 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 the authors 1 Disruption of tmc1/2a/2b genes in zebrafish reveals subunit requirements in subtypes of 2 inner ear hair cells 3 4 Running Title: Roles of tmc1/2 subunits in zebrafish inner ear 5 6 Eliot T Smith1, Itallia Pacentine2, Anna Shipman1, Matthew Hill2, and Teresa Nicolson1* 7 8 1 Department of Otolaryngology Head and Neck Surgery, Stanford School of Medicine, Stanford 9 University, Stanford, CA 94305 10 2 Oregon Hearing Research Center, Oregon Health and Science University, Portland, OR 97239 11 *Correspondence should be addressed to T. Nicolson 12 Email: [email protected] 13 14 Pages: 34 15 Figures: 10 16 Abstract: 250 words 17 Introduction: 650 words 18 Discussion: 844 words 19 20 Conflict of Interest: The authors declare no conflicts of interest. 21 22 Acknowledgements: This study was supported by funding from the NIDCD (R01 DC013572 23 and DC013531 to T.N.). 24 25 26 1 27 Abstract 28 Detection of sound and head movement requires mechanoelectrical transduction (MET) 29 channels at tips of hair-cell stereocilia. In vertebrates, the transmembrane channel-like (TMC) 30 proteins TMC1 and TMC2 fulfill critical roles in MET and substantial evidence implicates these 31 TMCs as subunits of the MET channel. To identify developmental and functional roles of this 32 Tmc subfamily in the zebrafish inner ear, we tested the effects of truncating mutations in tmc1, 33 tmc2a, and tmc2b on in vivo mechanosensation at the onset of hearing and balance, before 34 gender differentiation. We find that tmc1/2a/2b triple-mutant larvae cannot detect sound or 35 orient with respect to gravity. They lack acoustic-evoked behavioral responses (AEBR), 36 vestibular-induced eye movements (VIEM), and hair-cell activity as assessed with FM dye 37 labeling and microphonic potentials. Despite complete loss of hair-cell function, tmc triple- 38 mutant larvae retain normal gross morphology of hair bundles and proper trafficking of known 39 MET components Protocadherin 15a (Pcdh15a), Lipoma HMGIC fusion partner-like 5 (Lhfpl5), 40 and Transmembrane inner ear protein (Tmie). Transgenic, hair cell-specific expression of 41 Tmc2b-mEGFP rescues the behavioral and physiological deficits in tmc triple mutants. Results 42 from tmc single- and double- mutants evince a principle role for Tmc2a and Tmc2b in hearing 43 and balance, respectively, whereas Tmc1 has lower overall impact. Our experiments reveal that 44 in developing cristae, hair cells stratify into an upper, Tmc2a-dependent layer of teardrop 45 shaped cells and a lower, Tmc1/2b-dependent tier of gourd shaped cells. Collectively our 46 genetic evidence indicates that auditory/vestibular end organs and subsets of hair cells therein 47 rely on distinct combinations of Tmc1/2a/2b. 48 49 Significance Statement 50 We assessed the effects of tmc1/2a/2b truncation mutations on mechanoelectrical 51 transduction (MET) in the inner-ear hair cells of larval zebrafish. tmc triple mutants lacked 52 behavioral responses to sound and head movements, while further assays demonstrated no 2 53 observable mechanosensitivity in the tmc1/2a/2b triple mutant inner ear. Examination of tmc 54 double mutants revealed major contributions from Tmc2a and Tmc2b to macular function; 55 however, Tmc1 had less overall impact. FM labeling of lateral cristae in tmc double mutants 56 revealed the presence of two distinct cell types, an upper layer of teardrop shaped cells that rely 57 on Tmc2a, and a lower layer of gourd shaped cells that rely on Tmc1/2b. 58 59 Introduction 60 Central to senses of hearing and balance, hair cells rely on the mechanoelectrical 61 transduction (MET) protein complex to convert mechanical stimuli into an electrical signal. The 62 MET complex, resident at the tips of hair-cell stereocilia, consists of a cation-permeable channel 63 and associated binding partners (Corey and Hudspeth, 1979; Hudspeth, 1982; Beurg et al., 64 2009); reviewed in (Ó Maoiléidigh and Ricci, 2019). An extracellular filament, the tip-link, 65 tethers each MET channel to an adjacent, taller stereocilium, forming the MET apparatus 66 (Pickles et al., 1984; Assad et al., 1991). Upon mechanical deflection of stereocilia, increased 67 tension on tip-links opens MET channels causing depolarization. This mechanism relies on 68 concerted function of several protein subunits. 69 The mammalian MET complex comprises at least five distinct proteins: Protocadherin15 70 (PCDH15) (Kazmierczak et al., 2007), Lipoma HMGIC fusion partner-like 5 (LHFPL5) (Xiong et 71 al., 2012; Mahendrasingam et al., 2017), Transmembrane inner ear protein (TMIE) (Zhao et al., 72 2014; Pacentine and Nicolson, 2019), and Transmembrane Channel-like (TMC) proteins TMC1 73 and TMC2 (referred to as 'TMC1/2') (Kawashima et al., 2011; Pan et al., 2013; Kurima et al., 74 2015). PCDH15 forms the lower portion of the tip-link (Kazmierczak et al., 2007; Indzhykulian et 75 al., 2013) and connects with TMC1/2 via protein-protein interactions (Kazmierczak et al., 2007; 76 Indzhykulian et al., 2013; Maeda et al., 2014). LHFPL5 co-transports with PCDH15, where it 77 resides as part of the MET complex (Xiong et al., 2012). TMIE also localizes to the tips of 78 stereocilia (Zhao et al, 2014) and, in zebrafish, is required for proper localization of Tmc1/2b 3 79 (Pacentine and Nicolson, 2019). Although unclear if PCDH15, LHFPL5, or TMIE form part of 80 the pore, accumulating evidence implicates TMC1/2 as principal pore-forming subunits of MET 81 channels (Kawashima et al., 2011; Nakanishi et al., 2014; Kawashima et al., 2015; Kurima et 82 al., 2015; Chou et al., 2017; Beurg et al., 2018; Pan et al., 2018; Beurg et al., 2019; Fettiplace 83 and Nam, 2019; Goldring et al., 2019; Jia et al., 2019). Mutations in TMC1/2 affect conductance 84 and Ca2+ permeability properties of the MET channel, with at least 40 identified TMC1 alleles 85 causing human deafness (Kawashima et al., 2015). Hair cells express TMC1/2 concurrent with 86 onset of MET (Géléoc and Holt, 2003; Kawashima et al., 2011; Scheffer et al., 2015) and 87 TMC1/2 localize with other MET components at stereocilia tips (Kurima et al., 2015; 88 Mahendrasingam and Furness, 2019). Despite absence of crystallographic data, modeling 89 indicates structural similarity between TMC1/2 and Transmembrane protein 16a (TMEM16A) ion 90 channels (Pan et al., 2018) and recent evidence demonstrates TMC1/2 can form 91 mechanosensitive channels in liposomes (Jia et al., 2019). 92 Previous in vivo studies investigating MET complex components in zebrafish elucidated 93 functions of Pcdh15a (Maeda et al., 2014; Maeda et al., 2017), Lhfpl5a (Maeda et al., 2017), 94 and Tmie (Gleason et al., 2009; Pacentine and Nicolson, 2019). Zebrafish have two TMC2 95 paralogs, tmc2a/2b, due to genetic duplication in teleost fish (Maeda et al., 2014). tmc1/2a/2b 96 are expressed in the inner ear and lateral-line organ; tmc2a is present at earlier stages and 97 higher levels in the ear, whereas tmc2b is more predominantly expressed in the lateral-line 98 (Maeda et al., 2014). To date, studies of tmc disruption in zebrafish have shown that the tmc2 99 gene duplicates are required for function in the lateral-line and in the macular organs of the 100 inner ear, where mutation of both tmc2a and tmc2b abolished hair-cell activity (Chou et al., 101 2017; Chen et al., 2020). Nevertheless, the role of each tmc gene in the inner ear has not been 102 explored comprehensively with respect to hearing and balance, especially regarding functional 103 contributions in specific subtypes of hair cells. 4 104 To identify the specific roles of Tmc1/2 in inner-ear hair cells, we generated tmc1/2a/2b 105 single-, double-, and triple-mutant zebrafish lines using reverse genetic methods. We examined 106 behavioral, cellular, and physiological consequences of loss of tmc function at the onset of 107 hearing and balance, revealing differential effects on hair-cell subpopulations in the larval inner 108 ear. 109 110 Materials and Methods 111 Zebrafish Care and Use 112 We maintained zebrafish lines for all mutant alleles and transgenes in Top Long Fin 113 (TLF) and Tübingen wild-type backgrounds. Breeding stocks were housed at 28 °C and animal 114 husbandry followed standard zebrafish methods for laboratory utilization (Westerfield, 2000), as 115 approved and overseen by the Institutional Animal Care and Use Committees at both Oregon 116 Health and Sciences University and Stanford University. The experiments used zebrafish 117 larvae ≤6 days post fertilization (dpf) before gender differentiation occurs. Embryos and 118 larvae grew in E3 medium (0.33 mM CaCl2, 0.17 mM KCl, 0.33 mM MgSO4, and 5 mM NaCl) 119 incubated at 28.5 °C. When appropriate, larvae were anesthetized in E3 + 0.03% 3-amino 120 benzoic acid ethylester (MESAB, Western Chemical) to minimize pain and distress. 121 122 Generation of tmc Mutant Lines and Transgenic Lines 123 A zebrafish line heterozygous for the tmc2aEx4, -23bp allele (Figure 1a) was produced by 124 PNA Bio, Inc. using TALEN-based gene editing techniques (Bedell et al., 2012; Bedell and 125 Ekker, 2015).
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