Sensory transduction is required for normal development and maturation of cochlear inner hair cell synapses
- Speech and Hearing Bioscience & Technology Program, Division of Medical Sciences, Harvard University, United States
- Department of Otolaryngology, Boston Children’s Hospital and Harvard Medical School, United States
- Department of Neurology, Boston Children’s Hospital and Harvard Medical School, United States
Figures
Figure 1
CtBP2+ puncta counts in Tmc1Δ/Δ;Tmc2Δ/Δ mice lacking sensory transduction are similar to those in wild-type (WT) mice at postnatal day 2 (P2).
(A) Representative 3D projections of confocal z-stacks of P2 WT and Tmc1Δ/Δ;Tmc2Δ/Δ (B) inner hair cells (IHCs) from the 16 kHz region. Scale bar: 7 µm. The tissue was immunostained with …
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Figure 1—source data 1
CtBP2+ puncta counts in Tmc1Δ/Δ;Tmc2Δ/Δ mice lacking sensory transduction are similar to those in wild-type (WT) mice at postnatal day 2 (P2).
- https://cdn.elifesciences.org/articles/69433/elife-69433-fig1-data1-v2.xlsx
Figure 2
Synapse counts are elevated at postnatal day 7 (P7) and reduced at P28 in Tmc1Δ/Δ;Tmc2Δ/Δ mice relative to wild-type (WT) mice.
(A–B) Representative image of P7 WT and Tmc1Δ/Δ;Tmc2Δ/Δ inner hair cells (IHCs) from 16 kHz region immunostained for anti-Myosin7a (gray), anti-CtBP2 (red), and anti-GluA2 (green). Higher …
Figure 3
Synapse counts were elevated at postnatal day 7 (P7) in the absence of both Tmc1 and Tmc2 and diminished at P28 in the absence of Tmc1 and Tmc2 or Tmc1 alone.
(A–C) The mean number of synapses/inner hair cell (IHC) was calculated for each frequency region. Data from wild-type (WT) (black), Tmc1Δ/Δ;Tmc2Δ/Δ (red), Tmc1Δ/Δ (blue), and Tmc2Δ/Δ (dark yellow) …
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Figure 3—source data 1
Synapse counts were elevated at postnatal day 7 (P7) in the absence of both Tmc1 and Tmc2 and diminished at P28 in the absence of Tmc1 and Tmc2 or Tmc1 alone.
- https://cdn.elifesciences.org/articles/69433/elife-69433-fig3-data1-v2.xlsx
Figure 4
Loss of sensory transduction, not of Tmc1 and Tmc2 specifically, results in the synaptic differences observed at postnatal day 7 (P7) and P28.
(A–C) The mean number of synapses/inner hair cell (IHC) at each frequency region. Data from wild-type (WT) (black), Tmc1Δ/Δ;Tmc2Δ/Δ (red), Tmc1Δ/Δ (blue), Tmiesr (purple), and Tmc1Bth (gold) groups …
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Figure 4—source data 1
Loss of sensory transduction, not of Tmc1 and Tmc2 specifically, results in the synaptic differences observed at postnatal day 7 (P7) and P28.
- https://cdn.elifesciences.org/articles/69433/elife-69433-fig4-data1-v2.xlsx
Figure 5
AAV9-PHP.B-Tmc1 restores ABR thresholds in Tmc1Δ/Δ mice.
(A) Representative ABR waveforms recorded at postnatal day 28 (P28) using 16 kHz tone bursts at sound pressure levels of increasing 5 dB increments. Waveforms from uninjected Tmc1Δ/Δ mouse (left), …
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Figure 5—source data 1
AAV9-PHP.
B-Tmc1 restores ABR thresholds in Tmc1Δ/Δ mice.
- https://cdn.elifesciences.org/articles/69433/elife-69433-fig5-data1-v2.xlsx
Figure 6
AAV9-PHP.B-Tmc1 preserves synapse counts and ribbon volume distributions in Tmc1Δ/Δ mice.
(A) The mean number of synapses/inner hair cell (IHC) was counted at each frequency region from postnatal day 28 (P28) wild-type (WT) (black), Tmc1Δ/Δ (blue), and injected Tmc1Δ/Δ (green) mice. WT …
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Figure 6—source data 1
AAV9-PHP.
B-Tmc1 preserves synapse counts and ribbon volume distributions in Tmc1Δ/Δ mice.
- https://cdn.elifesciences.org/articles/69433/elife-69433-fig6-data1-v2.xlsx
Figure 7
Developmental changes in inner hair cell (IHC) synapses in genetic models of sensory transduction dysfunction.
(A) Number of synapses/IHC from the 32 kHz region of wild-type (WT), Tmc1Δ/Δ;Tmc2Δ/Δ, Tmc1Δ/Δ, and Tmc2Δ/Δ cochleas as a function of age. (B) Schematic diagram summarizing changes in IHC synapses …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | C57B/L6-Cdh23753A>G | Derived from Lentz et al., 2010 | C57BL6 | Lentz et al. Dev. Neurobiol (2010) |
| Genetic reagent (Mus musculus) | 019146 - B6.129-Tmc1tm1.1Ajg/J | Available from Jackson Lab, obtained initially from Dr A Griffith (NIH/NIDCD) | Tmc1 Targeted (Reporter, Null/Knockout) | Kurima et al. Nat Genet. (2002) |
| Genetic reagent (Mus musculus) | 019147 - B6.129-Tmc2tm1.1Ajg/J | Available from Jackson Lab, obtained initially from Dr A Griffith (NIH/NIDCD) | Tmc2 Targeted (Reporter, Null/Knockout) | Kawashima et al. J Clin Invest (2011) |
| Genetic reagent (Mus musculus) | 003929 - BXA4/Pgn-Tmiesr-J/J | Available from Jax C57BL/6-Tmiesr (‘spinner’) mice | Spontaneous mutation in Tmie | Stock No. 000543Mitchem et al. Hum Mol Genet. (2002) |
| Genetic reagent (Mus musculus) | Tmc1Bth | Tmc1Bth/Bth mice were obtained from M Hrabé de Angelis and H Fuchs, Institute of Experimental Genetics, Neuherberg, Germany | Point mutation at residue 412 (M412K) | Vreugde et al. Nat Genet. (2002) |
| Antibody | Anti-CtBP2 (Mouse IgG1 monoclonal) | BD Transduction Laboratories | Cat #: 612044 | Primary antibody, IF (1:200) |
| Antibody | Anti-GluA2 (Mouse IgG2a monoclonal) | Millipore Sigma | Cat #: MABN1189 | Primary antibody, IF (1:2000) |
| Antibody | Anti-MyosinVIIA (Rabbit polyclonal) | Proteus Biosciences | Cat #: 25–6790 | Primary antibody, IF (1:200) |
| Antibody | Anti-Rabbit Alexa Fluor 647 (Donkey polyclonal) | Thermo Fisher Scientific | Cat #: A-31573 | Secondary antibody, IF (1:200) |
| Antibody | Anti-Mouse IgG2a Alexa Fluor 488 (Goat polyclonal) | Thermo Fisher Scientific | Cat #: A-21131 | Secondary antibody, IF (1:1000) |
| Antibody | Anti-Mouse IgG1 Alexa Fluor 546 (Goat polyclonal) | Thermo Fisher Scientific | Cat #: A-21123 | Secondary antibody, IF (1:1000) |
| Other | Vectashield Antifade | Vector Laboratories | Cat #: H-1000–10 | Mounting medium |
| Other | AAV9-PHP.B- CMV-Tmc1e × 1 | Wu et al., 2021 | Nucleic acid, Titer: 3.9 E + 13 gc/mL | |
| Software, algorithm | Imaris | https://imaris.oxinst.com/ | ||
| Software, algorithm | ImageJ software | http://imagej.nih.gov/ij/ | ||
| Software, algorithm | Eaton-Peabody Laboratories Cochlear Function Test Suite | https://www.masseyeandear.org/research/otolaryngology/eaton-peabody-laboratories/engineering-core |
Additional files
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Supplementary file 1
CtBP2+ puncta (mean ± SEM) per inner hair cell (IHC) in postnatal day 2 (P2) wild-type (WT) and Tmc1Δ/Δ;Tmc2Δ/Δ mice.
- https://cdn.elifesciences.org/articles/69433/elife-69433-supp1-v2.xlsx
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Supplementary file 2
Group comparisons of synapse counts at P7 and P14.
(A) Group comparisons of postnatal day 7 (P7) synapse counts per inner hair cell (IHC) (mean difference, 95% CI) in wild-type (WT), Tmc1Δ/Δ;Tmc2Δ/Δ, Tmc1Δ/Δ, and Tmc2Δ/Δ mice. *p < 0.05, **p < 0.01, ***p < 0.001, non-significant (ns) > 0.05.
(B) Group comparisons of postnatal day 14 (P14) synapse counts per inner hair cell (IHC) (mean difference, 95% CI) in wild-type (WT), Tmc1Δ/Δ;Tmc2Δ/Δ, Tmc1Δ/Δ, and Tmc2Δ/Δ mice. *p < 0.05, **p < 0.01, ***p < 0.001, non-significant (ns) > 0.05. (C) Group comparisons of postnatal day 28 (P28) synapse counts per inner hair cell (IHC) (mean difference, 95% CI) in wild-type (WT), Tmc1Δ/Δ;Tmc2Δ/Δ, Tmc1Δ/Δ, and Tmc2Δ/Δ mice. *p < 0.05, **p < 0.01, ***p < 0.001, non-significant (ns) > 0.05.
- https://cdn.elifesciences.org/articles/69433/elife-69433-supp2-v2.xlsx
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Supplementary file 3
ANOVA analyses as a function of genotype, age and frequency.
(A) Three-way and two-way ANOVA analyses of genotypes (wild-type [WT], Tmc1Δ/Δ;Tmc2Δ/Δ, Tmc1Δ/Δ, Tmc2Δ/Δ), timepoints (P7, P14, P28), and frequencies (8, 11.3, 16, 22.6, 32 kHz) in Figure 3A–C.
(B) Three-way and two-way ANOVA analyses of genotypes (wild-type [WT], Tmc1Δ/Δ;Tmc2Δ/Δ, Tmc1Δ/Δ, Tmiesr, Tmc1Bth), timepoints (P7, P28), and frequencies (8, 11.3, 16, 22.6, 32 kHz) in Figure 4A–C.(C) Two-way ANOVA analyses of genotypes (wild-type [WT], injected Tmc1Δ/Δ, Tmc1Δ/Δ), and frequencies (8, 11.3, 16, 22.6, 32 kHz) in Figure 6A.
- https://cdn.elifesciences.org/articles/69433/elife-69433-supp3-v2.xlsx
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Supplementary file 4
Group comparisons at P7 and P28 for WT, Tmc1Δ/Δ;Tmc2Δ/Δ, and Tmiesrmice.
(A) Group comparisons of postnatal day 7 (P7) synapse counts per inner hair cell (IHC) (mean difference, 95% CI) in wild-type (WT), Tmc1Δ/Δ;Tmc2Δ/Δ, and Tmiesr mice. *p < 0.05, **p < 0.01, ***p < 0.001, non-significant (ns) > 0.05.
(B) Group comparisons of postnatal day 28 (P28) synapse counts per inner hair cell (IHC) (mean difference, 95% CI) in wild-type (WT), Tmc1Δ/Δ;Tmc2Δ/Δ, and Tmiesr mice. *p < 0.05, **p < 0.01, ***p < 0.001, non-significant (ns) > 0.05. (C) Group comparisons of postnatal day 28 (P28) synapse counts per inner hair cell (IHC) (mean difference, 95% CI) in wild-type (WT), Tmc1Δ/Δ, and Tmc1Bth mice. *p < 0.05, **p < 0.01, ***p < 0.001, non-significant (ns) > 0.05.
- https://cdn.elifesciences.org/articles/69433/elife-69433-supp4-v2.xlsx
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Supplementary file 5
ABR thresholds (mean ± SD) in postnatal day 28 (P28) wild-type (WT) (n = 8) and injected Tmc1Δ/Δ (n = 12) mice.
- https://cdn.elifesciences.org/articles/69433/elife-69433-supp5-v2.xlsx
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Supplementary file 6
Group comparisons for synapse counts and synapse volumes at P28.
(A) Group comparisons of postnatal day 28 (P28) synapse counts per inner hair cell (IHC) (mean difference, 95% CI) in wild-type (WT), Tmc1Δ/Δ, and injected Tmc1Δ/Δ mice. *p < 0.05, **p < 0.01, ***p < 0.001, non-significant (ns) > 0.05. (B) Normalized distribution of ribbon volumes (median± SD) in postnatal day 28 (P28) wild-type (WT), uninjected Tmc1Δ/Δ, and injected Tmc1Δ/Δ mice. (C) Statistical comparison of standard deviations of normalized ribbon volumes in postnatal day 28 (P28) wild-type (WT), uninjected Tmc1Δ/Δ, and injected Tmc1Δ/Δ mice. See (B) for SD values.
- https://cdn.elifesciences.org/articles/69433/elife-69433-supp6-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/69433/elife-69433-transrepform1-v2.pdf
