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. 2011 Feb 8;108(6):2355-60.
doi: 10.1073/pnas.1016646108. Epub 2011 Jan 18.

miR-96 regulates the progression of differentiation in mammalian cochlear inner and outer hair cells

Stephanie Kuhn  1 , Stuart L Johnson, David N Furness, Jing Chen, Neil Ingham, Jennifer M Hilton, Georg Steffes, Morag A Lewis, Valeria Zampini, Carole M Hackney, Sergio Masetto, Matthew C Holley, Karen P Steel, Walter Marcotti
Affiliations

miR-96 regulates the progression of differentiation in mammalian cochlear inner and outer hair cells

Stephanie Kuhn et al. Proc Natl Acad Sci U S A. .

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs able to regulate a broad range of protein-coding genes involved in many biological processes. miR-96 is a sensory organ-specific miRNA expressed in the mammalian cochlea during development. Mutations in miR-96 cause nonsyndromic progressive hearing loss in humans and mice. The mouse mutant diminuendo has a single base change in the seed region of the Mir96 gene leading to widespread changes in the expression of many genes. We have used this mutant to explore the role of miR-96 in the maturation of the auditory organ. We found that the physiological development of mutant sensory hair cells is arrested at around the day of birth, before their biophysical differentiation into inner and outer hair cells. Moreover, maturation of the hair cell stereocilia bundle and remodelling of auditory nerve connections within the cochlea fail to occur in miR-96 mutants. We conclude that miR-96 regulates the progression of the physiological and morphological differentiation of cochlear hair cells and, as such, coordinates one of the most distinctive functional refinements of the mammalian auditory system.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Hair cell morphology and cochlear physiology in diminuendo mice. (A and B) SEM from apical coil IHCs (Upper) and OHCs (Lower) from P4+/+ and Dmdo/Dmdo cochleae. The hair bundle structure in Dmdo/Dmdo hair cells is more immature than that in controls. (Scale bar: 3 μm.) (C and D) Cm of IHCs (C: 1 ≤ n ≤ 11, P1–P30) and OHCs (D: 1 ≤ n ≤ 8, P1–P18) increases with age in control but not Dmdo/Dmdo mice. Dashed gray lines in C and D give an indication of the range of expected normal growth in WT mice (25, 26). (E) Immature Dmdo/Dmdo OHCs (P4) are significantly shorter than control cells. +/+: 270 OHCs, 9 cochleae; Dmdo/+: 240 OHCs, 8 cochleae; Dmdo/Dmdo: 329 OHCs, 11 cochleae. (F) Mean ABR thresholds (mean ± SD) are raised in P15 Dmdo/+ (n = 9) compared with age-matched +/+ mice (n = 8).
Fig. 2.
Fig. 2.
Potassium currents and voltage responses in IHCs from diminuendo mice. (A and B) Examples of K+ currents recorded from +/+ (black) and Dmdo/Dmdo (red) adult IHCs (P22), respectively. Currents were elicited by depolarizing voltage steps in 10-mV nominal increments from the holding potential of −64 mV to the various test potentials shown by some of the traces. IK (25); IK,f, (49); IK,n (25, 50); IK1 (27); ISK2 (28). (C) K+ currents characteristic of adult IHCs are smaller in Dmdo/+ cells (blue) and absent in Dmdo/Dmdo cells. The size of the isolated IK,n in adult Dmdo/+ IHCs could not be accurately assessed because it was contaminated by the persistence of the immature-type current (27). Voltage responses in control (D, induced by depolarizing current injections) and homozygous mutant (E, spontaneous Ca2+-dependent action potentials) adult IHCs. Development of the outward K+ current was measured at 0 mV (F, IK) and that of the inward rectifier K+ current was measured at −124 mV (G, IK1) in all three genotypes. Dashed gray lines give an indication of the range of expected normal growth in WT mice (25, 27). The vertical dashed line in G indicates that the size of IK1 is normally rapidly down-regulated from P12 (27).
Fig. 3.
Fig. 3.
Exocytotic Ca2+ dependence and synaptic morphology in diminuendo IHCs. (A and B) ICa and ΔCm in adult control (+/+, P21) and homozygous mutant (Dmdo/Dmdo, P15) IHCs. Recordings were obtained in response to 100-ms voltage steps from −81 mV in 10-mV increments. Only maximal responses are shown in A. (C and D) Maximal peak ICa and ΔCm values, respectively, from immature and adult control and mutant IHCs. (E) Adult mutant IHCs showed a steeper intrinsic Ca2+ dependence of exocytosis. (Right) Synaptic transfer curves obtained by plotting ΔCm against the corresponding ICa between −71 mV and −11 mV (31). Fits are according to Eq. 1 (SI Materials and Methods). (Left) Average ΔCm traces obtained from all IHCs investigated. (F and G) Transmission electron microscopy showing the cross-sectional profiles of presynaptic dense bodies (arrows) from a control IHC and a Dmdo/Dmdo adult IHC, respectively. aff, afferent endings. (Scale bar: 100 nm.) (H and I) Immunostaining of neurofilament showing a more irregular wiring pattern of fibers below adult Dmdo/Dmdo IHCs compared with that observed in controls. TC, tunnel of Corti. (Scale bar: 50 μm.)
Fig. 4.
Fig. 4.
Efferent modulation of diminuendo IHCs. (A–C) Membrane currents recorded from immature (A: Dmdo/Dmdo, red) and adult (B: +/+, black; C: Dmdo/Dmdo, red) IHCs before and during superfusion of ACh (green traces). The instantaneous current elicited by ACh is indicated by arrows. (D) Developmental change in the steady-state slope conductance measured near −84 mV in the presence of 100 μM ACh. (E) Transmission electron microscopy showing the synaptic region of an adult control IHC with efferent (eff) endings contacting radial afferent fibers (aff + arrowhead). Pre- and postsynaptic membrane densities are visible between efferents and afferents. (F) Afferent and efferent terminals onto adult Dmdo/Dmdo IHCs. (Inset) High-magnification view of the efferent terminal (*) shows vesicles and pre- and postsynaptic densities. (G) Efferent ending from another adult Dmdo/Dmdo IHC showing a large number of vesicles. (Scale bars: E, G, and Inset in F, 0.5 μm; F, 1 μm.)
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