Functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer

Abstract

Sensory hair cells in the mammalian cochlea convert mechanical stimuli into electrical impulses that subserve audition. Loss of hair cells and their innervating neurons is the most frequent cause of hearing impairment. Atonal homologue 1 (encoded by Atoh1, also known as Math1) is a basic helix-loop-helix transcription factor required for hair-cell development, and its misexpression in vitro and in vivo generates hair-cell-like cells. Atoh1-based gene therapy to ameliorate auditory and vestibular dysfunction has been proposed. However, the biophysical properties of putative hair cells induced by Atoh1 misexpression have not been characterized. Here we show that in utero gene transfer of Atoh1 produces functional supernumerary hair cells in the mouse cochlea. The induced hair cells display stereociliary bundles, attract neuronal processes and express the ribbon synapse marker carboxy-terminal binding protein 2 (refs 12,13). Moreover, the hair cells are capable of mechanoelectrical transduction and show basolateral conductances with age-appropriate specializations. Our results demonstrate that manipulation of cell fate by transcription factor misexpression produces functional sensory cells in the postnatal mammalian cochlea. We expect that our in utero gene transfer paradigm will enable the design and validation of gene therapies to ameliorate hearing loss in mouse models of human deafness.

Figure 1. In utero gene transfer to the developing mouse inner ear

a, Expression plasmid was microinjected into the E11.5 otic vesicle. b, The vesicle was centered between the cathode (-) and anode (+) and electroporated. c, Destabilized GFP (ZsGreen) was expressed in the otic territory 24hrs post-electroporation. d, E12.5 progenitors in the medial and ventral otic epithelium expressed ZsGreen robustly. e, E18.5 Atoh1/GFP-transfected cochlea (left) immunostained for Myo7a (right). Arrowheads indicate left otocyst. Scale bars: a, 200μm; b,c, 500μm; d, 50μm; d, inset, 10μm; e, 100μm.

Figure 2. Atoh1 misexpression generates supernumerary Myo7a⁺ cells bearing stereociliary bundles

a, schematic of cell types in the organ of Corti transfected with Atoh1/enhanced GFP (filled green): CC, Claudius' cells; HeC, Hensens' cells; DC, Deiters' cells; OHC, outer hair cell; PC, pillar cell; IPC, inner phalangial cell; IHC, inner hair cell; BC, border cell. b-g, laser confocal micrographs of E18.5 GFP-transfected (b,d,f) and Atoh1/GFP-transfected (c,e,g) organs of Corti immunostained for Myo7a (red). All of the Atoh1/GFP⁺ cells in panels c,e, and g are Myo7a⁺. h,i, scanning electron micrographs of postnatal day 35, untransfected (h) and Atoh1/GFP-transfected (i) organs of Corti. Asterisks in panels h,i indicate stereociliary bundles imaged at higher magnification in panels j,k, respectively. The arrows indicate 3 cells with atypical bundles. Scale bars: b-i, 20μm; j,k, 2 μm.

Figure 3. Atoh1/GFP⁺ cells demonstrate morphological and molecular correlates of innervation and synaptogenesis

a, NeuroVue-Red placed in the cochlear nucleus labels radial fibers (RF) of spiral ganglion neurons (SGN) that project to Atoh1/GFP⁺/Myo7a⁺ cells (arrow and arrowhead). Inset shows basal regions of indicated cells. b, Neurofilament⁺ (NF⁺) processes associate with Myo7a⁺ inner and outer hair cells (apical). NF⁺ processes terminated at the base of an Atoh1/GFP⁺/Myo7a⁺ cell cluster as seen in the 90° rotation of the apical 3-dimensional reconstruction. c, Ctbp2 (red) was localized to the basolateral domain at P35. d, The Ctbp2 (red) signal in panel c that was contained within the Myo7a⁺ hair cell cytoplasm. Scale bars: a, 50 μm; a, inset, 20μm; b-d, 10μm.

Figure 4. Atoh1/GFP⁺ cells mechanotransduce and elaborate age-appropriate basolateral conductances

Biophysical response properties of control (black) and GFP⁺ (green) hair cells. Data are presented as mean ± standard deviation, and two-tailed t-tests were used for statistical comparisons. b, c, Mechanically-evoked currents from a holding potential of -84 mV elicited from the stimulus in a. d, Normalized peak current against displacement; solid line is Boltzmann fit. f, g, Current responses to the voltage-clamp stimuli in e. h, Steady-state current-voltage plot from the data in f,g; solid line is Boltzmann fit. i, The expanded current traces from the regions indicated by arrows in f,g. j, Normalized peak current against potential for inward current. k, Stimulus protocol to probe inactivation that elicited currents in l. m, Prepulse potential against normalized peak inward current; solid line is Boltzmann fit.