Jacques Boutet de Monvel

Myosin-VIIA (MYO7A) is an unconventional myosin responsible for syndromic (Usher 1B) or nonsyndromic forms of deafness in humans when mutated. In the cochlea, MYO7A is expressed in hair cells, where it is believed to act as the motor protein tensioning the mechanoelectrical transducer (MET) channels, thus setting their resting open probability ( P _o ). However, direct evidence for this unique role for an unconventional myosin in mature hair cells is lacking. Here, we show that MYO7A has a distinct role in hair cells, being crucial for the structural integrity of hair bundles. Postnatal deletion of Myo7a leads to 87 to 96% reduction in MYO7A from hair cells by postnatal day 20 (P20), without affecting hearing function. During the following week, mice showed progressive decline in both hearing function and MET current amplitude in hair cells without affecting the resting P _o and calcium sensitivity of the MET channel. Hair-bundle stiffness was normal at P20 but halved at P30, despite it having a normal staircase morphology and tip links. The reduction of MYO7A in the stereocilia (>87%) increased their vulnerability to sound-induced damage, with significantly more hearing loss and hair bundle deterioration than in control mice. RNA-sequencing identified a downregulation of several stereociliary genes in the Myo7a -deficient cochlea, indicating the presence of indirect compensatory mechanisms. This study reveals that mature hair cells seem to use a MYO7A-independent mechanism to maintain the resting P _o of the MET channels. Instead, MYO7A is essential for maintaining the structural and functional integrity of the hair bundles.

In vivo gene delivery to tissues using adeno-associated vector (AAVs) has revolutionized the field of gene therapy. Yet, while sensorineural hearing loss is one of the most common sensory disorders worldwide, gene therapy applied to the human inner ear is still in its infancy. Recent advances in the development recombinant AAVs have significantly improved their cell tropism and transduction efficiency across diverse inner ear cell types to a level that renders this tool valuable for conditionally manipulating gene expression in the context of developmental biology studies of the mouse inner ear. Here, we describe a protocol for in utero micro-injection of AAVs into the embryonic inner ear, using the AAV-PHP.eB and AAV-DJ serotypes that respectively target the sensory hair cells and the supporting cells of the auditory sensory epithelium. We also aimed to standardize procedures for imaging acquisition and image analysis to foster research reproducibility and allow accurate comparisons between studies. We find that AAV-PHP.eB and AAV-DJ provide efficient and reliable tools for conditional gene expression targeting cochlear sensory and supporting cells in the mouse inner ear, from late embryonic stages on.

SUMMARY: Our understanding of how otoferlin, the major calcium sensor in inner hair cells (IHCs) synaptic transmission, contributes to the overall dynamics of synaptic vesicle (SV) trafficking remains limited. To address this, we generated a knock-in mouse model expressing an otoferlin-GFP protein, where GFP was fused to its C-terminal transmembrane domain. Similar to the wild type protein, the GFP-tagged otoferlin showed normal expression and was associated with IHC SV. Surprisingly, while the heterozygote Otof ^+/GFP mice exhibited a normal hearing function, homozygote Otof ^GFP/GFP mice were profoundly deaf attributed to severe reduction in SV exocytosis. Fluorescence recovery after photobleaching revealed a markedly increased mobile fraction of the otof-GFP-associated SV in Otof ^GFP/GFP IHCs. Correspondingly, 3D-electron tomographic of the ribbon synapses indicated a reduced density of SV attached to the ribbon active zone. Collectively, these results indicate that otoferlin requires a free intravesicular C-terminal end for normal SV docking and fusion.

Efficient tools allowing the extraction of 2D surfaces from 3D-microscopy data are essential for studies aiming to decipher the complex cellular choreography through which epithelium morphogenesis takes place during development. Most existing methods allow for the extraction of a single and smooth manifold of sufficiently high signal intensity and contrast, and usually fail when the surface of interest has a rough topography or when its localization is hampered by other surrounding structures of higher contrast. Multiple surface segmentation entails laborious manual annotations of the various surfaces separately. As automating this task is critical in studies involving tissue-tissue or tissue-matrix interaction, we developed the Zellige software, which allows the extraction of a non-prescribed number of surfaces of varying inclination, contrast, and texture from a 3D image. The tool requires the adjustment of a small set of control parameters, for which we provide an intuitive interface implemented as a Fiji plugin. As a proof of principle of the versatility of Zellige, we demonstrate its performance and robustness on synthetic images and on four different types of biological samples, covering a wide range of biological contexts.

Using adequate DNA barcodes is essential to unambiguously identify each DNA library within a multiplexed set of libraries sequenced using next-generation sequencers. We introduce DNABarcodeCompatibility, an R-package that allows one to design single or dual-barcoding multiplex experiments by imposing desired constraints on the barcodes (including sequencer chemistry, barcode pairwise minimal distance and nucleotide content), while optimizing barcode frequency usage, thereby allowing one to both facilitate the demultiplexing step and spare expensive library-preparation kits. The package comes with a user-friendly interface and a web app developed in Java and Shiny (https://dnabarcodecompatibility.pasteur.fr), respectively, with the aim to help bridge the expertise of core facilities with the experimental needs of non-experienced users. DNABarcodeCompatibility can be easily extended to fulfil specific project needs. The source codes of the R-package and its user interfaces are publicly available along with documentation at [https://github.com/comoto-pasteur-fr] under the GPL-2 licence. Supplementary data are available at Bioinformatics online.

Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C₂-domain, Ca²⁺-binding transmembrane protein of synaptic vesicles. To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice (Otof ^{Ala515,Ala517/Ala515,Ala517}) with lower Ca²⁺-binding affinity of the C₂C domain. The IHC ribbon synapse structure, synaptic Ca²⁺ currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca²⁺ sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca²⁺ concentration, by varying Ca²⁺ influx through voltage-gated Ca²⁺-channels or Ca²⁺ uncaging. Otoferlin thus functions as a Ca²⁺ sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone.

Significance: Hearing and balance impairments are major concerns and a serious burden for public health, but still lack an effective curative therapy. We assessed inner ear functions in a mouse model of Usher syndrome type 1, a developmental disorder characterized by profound congenital deafness and balance deficit, after local gene therapy. Viral transfer of the wild-type cDNA to the inner ear of the mutant mice shortly after birth resulted in a partial restoration of hearing and a long-lasting, almost complete, removal of the balance defect. The present results provide a basis for future clinical trials in humans.

Many genetic forms of congenital deafness affect the sound reception antenna of cochlear sensory cells, the hair bundle. The resulting sensory deprivation jeopardizes auditory cortex (AC) maturation. Early prosthetic intervention should revive this process. Nevertheless, this view assumes that no intrinsic AC deficits coexist with the cochlear ones, a possibility as yet unexplored. We show here that many GABAergic interneurons, from their generation in the medial ganglionic eminence up to their settlement in the AC, express two cadherin-related (cdhr) proteins, cdhr23 and cdhr15, that form the hair bundle tip links gating the mechanoelectrical transduction channels. Mutant mice lacking either protein showed a major decrease in the number of parvalbumin interneurons specifically in the AC, and displayed audiogenic reflex seizures. Cdhr15- and Cdhr23-expressing interneuron precursors in Cdhr23^−/− and Cdhr15^−/− mouse embryos, respectively, failed to enter the embryonic cortex and were scattered throughout the subpallium, consistent with the cell polarity abnormalities we observed in vitro. In the absence of adhesion G protein-coupled receptor V1 (adgrv1), another hair bundle link protein, the entry of Cdhr23- and Cdhr15-expressing interneuron precursors into the embryonic cortex was also impaired. Our results demonstrate that a population of newborn interneurons is endowed with specific cdhr proteins necessary for these cells to reach the developing AC. We suggest that an “early adhesion code” targets populations of interneuron precursors to restricted neocortical regions belonging to the same functional area. These findings open up new perspectives for auditory rehabilitation and cortical therapies in patients. Significance In early-onset genetic forms of deafness, deficits of the auditory sensory organ are sufficient to account for the hearing impairment. However, the possibility that intrinsic deficits of the auditory cortex (AC) coexist with the peripheral deficits is still unexplored. We show, in rodents and primates, that the cadherin-related proteins cdhr23 and cdhr15 are expressed by many interneuron precursors targeted specifically to the AC. A deficiency of either protein results in the failure of these interneuron precursors to enter the embryonic cortex and in abnormally small numbers of parvalbumin interneurons in the AC only. These findings should lead to an improvement of hearing rehabilitation strategies in patients and open up new genetic approaches for studying AC development and function.