Fig 1.
TRP channel expression in cochlear and vestibular hair cells.
(a) Schematic of the sorted cells, redrawn from Scheffer et al., 2015 [25]. Hair cells (green) and surrounding cells (purple) were collected from the cochlea (dark colors) and utricle (light colors) at ages E16, P0, P4, and P7. (b) Normalized RNA-seq read counts in hair cells and surrounding cells (colors as in a). Data are shown for 31 of the 33 members of the TRP channel family in mouse. Mcoln2 and Trpc4 showed negligible counts in all samples. Data from www.shield.hms.harvard.edu [25].
Fig 2.
Trpm2 is not required for hair cell mechanotransduction.
(a) FM1-43 accumulation by IHCs and OHCs from control Trpm2fll+:Gfi1-Cre+ mice (top) and deleted Trpm2fl/fl:Gfi1-Cre+ mice (bottom). FM1-43 accumulation by hair cells was similar in control and Trpm2-deleted hair cells. Age: P5+2div. Scale bar = 20 μm. (b) A family of OHC transduction current recordings (top traces) in response to stereocilia bundle deflections (bottom traces) in WT (left) and Trpm2fl/fl:Gfi1-Cre+ (right) mice. (c) Average peak transduction current (red traces in b) for control mice (WT and Trpm2fl/+:Gfi1-Cre+) and Trpm2fl/fl:Gfi1-Cre+ mice. d, ABR thresholds in response to pure tone stimuli. Trpm2fl/fl:Gfi1-Cre+ mice show normal hearing. Data are mean ± sem; n as indicated.
Fig 3.
Neither Pkd2 nor Pkd2l1 is required for hair cell mechanotransduction.
(a) In situ hybridization in cochlear sections. (Left) no label is evident with a control sense probe. (Middle) no specific label is evident in the Pkd2l1 knockout. (Right) In situ hybridization with an antisense probe shows label of inner hair cells (arrowhead), outer hair cells (arrows) and inner sulcus cells (asterisks) in the organ of Corti. Scale bar = 50 μm; age P2. (b) ABR thresholds in response to pure tone stimuli. Pkd2l1-/- mice show normal hearing at age P31-P37. (c) Pkd2-/-:Atoh1-Cre+/-mice show normal hearing at age 4~6 weeks. Data are mean ± sem; n as indicated.
Fig 4.
Mice lacking single or multiple PKD genes show normal stereocilia bundle morphology and hearing function.
(a-d) SEM images of postnatal 4~6 weeks organ of Corti hair cells at low magnification in WT and PKD-deficient mice. Scale bar = 10 μm. (e-l) OHC bundles at high magnification. Scale bar = 1 μm. (m) ABR thresholds in response to pure tone stimuli in Pkd2 and Pkd2l1 single and double knockouts. (n) Pkd2l1 and Pkd1l3 single and double knockouts. (o) Pkd2l2 knockouts. No functional deficit was observed in any combination tested. Data shown as mean ± sem.
Fig 5.
Triple PKD knockout (Pkd2fl/fl:Atoh1-Cre+:Pkd2l1-/-:Pkd2l2-/-) mice show normal mechanotransduction and normal hearing in ABR measurements.
(a) FM1-43 accumulation is similar in IHCs and OHCs from P6 Pkd2fl/fl:Atoh1-Cre-:Pkd2l1-/-:Pkd2l2-/- controls lacking Cre (left) and Pkd2fl/fl:Atoh1-Cre+:Pkd2l1-/-:Pkd2l2-/- knockout mice (right), suggesting normal transduction. Scale bar = 20 μm. (b) A family of OHC transduction currents (top traces) in response to stereocilia bundle deflections (bottom traces) in WT (left) and Pkd2fl/fl:Atoh1-Cre+:Pkd2l1-/-:Pkd2l2-/- knockout (right) mice. (c) Average peak transduction currents (red traces in b) for WT mice (blue; n = 5) and triple knockout (red bar, n = 6). (d) ABR threshold measurements in response to pure tone stimuli in Pkd2fl/fl:Atoh1-Cre+:Pkd2l1-/-:Pkd2l2-/- knockout mice indicate normal hearing. Data are mean ± sem.
Table 1.
Primers used for genotyping and validation of gene deletion.