fm1-43 and Hearing-Loss

The cellular diversity of the inner ear has presented a technical challenge in obtaining molecular insight into its development and function. The application of technological advancements in cell type-specific expression enable clinicians and researchers to leap forward from classic genetics to obtaining mechanistic understanding of congenital and acquired hearing loss. This understanding is essential for development of therapeutics to prevent and reverse diseases of the inner ear, including hearing loss. The objective of this study is to describe and compare the available tools for cell type-specific analysis of the ear, as a means to support decision making in study design.. Three major approaches for cell type-specific analysis of the ear including fluorescence-activated cell sorting (FACS), ribosomal and RNA pulldown techniques, and single cell RNA-seq (scRNA-seq) are compared and contrasted using both published and original data.. We demonstrate the strength and weaknesses of these approaches leading to the inevitable conclusion that to maximize the utility of these approaches, it is important to match the experimental approach with the tissue of origin, cell type of interest, and the biological question. Often, a combined approach (eg, cell sorting and scRNA-seq or expression analysis using 2 separate approaches) is required. Finally, new tools for visualization and analysis of complex expression data, such as the gEAR platform (umgear.org), collate cell type-specific gene expression from the ear field and provide unprecedented access to both clinicians and researchers.. N/A Laryngoscope, 131:S1-S16, 2021.

Topics: Animals; Decision Making; Ear, Inner; Flow Cytometry; Fluorescent Dyes; Gene Expression; Gene Expression Profiling; Hearing Loss; Humans; Mice; Mice, Transgenic; Organ of Corti; Pyridinium Compounds; Quaternary Ammonium Compounds; Ribosomes; RNA; Sequence Analysis, RNA; Single-Cell Analysis; Tight Junctions

In hair cells of the inner ear, sound or head movement increases tension in fine filaments termed tip links, which in turn convey force to mechanosensitive ion channels to open them. Tip links are formed by a tetramer of two cadherin proteins: protocadherin 15 (PCDH15) and cadherin 23 (CDH23), which have 11 and 27 extracellular cadherin (EC) repeats, respectively. Mutations in either protein cause inner ear disorders in mice and humans. We showed recently that these two cadherins bind tip-to-tip in a "handshake" mode that involves the EC1 and EC2 repeats of both proteins. However, a paucity of appropriate animal models has slowed our understanding both of the interaction and of how mutations of residues within the predicted interface compromise tip link integrity. Here, we present noddy, a new mouse model for hereditary deafness. Identified in a forward genetic screen, noddy homozygotes lack inner ear function. Mapping and sequencing showed that noddy mutant mice harbor an isoleucine-to-asparagine (I108N) mutation in the EC1 repeat of PCDH15. Residue I108 interacts with CDH23 EC2 in the handshake and its mutation impairs the interaction in vitro. The noddy mutation allowed us to determine the consequences of blocking the handshake in vivo: tip link formation and bundle morphology are disrupted, and mechanotransduction channels fail to remain open at rest. These results offer new insights into the interaction between PCDH15 and CDH23 and help explain the etiology of human deafness linked to mutations in the tip-link interface.

Topics: Age Factors; Animals; Animals, Newborn; Cadherin Related Proteins; Cadherins; Calcium; Cells, Cultured; Electroencephalography; Ethylnitrosourea; Evoked Potentials, Auditory, Brain Stem; Extracellular Matrix; Gene Expression Regulation; Genotype; Hair Cells, Auditory; Hearing Loss; Labyrinth Diseases; Mechanotransduction, Cellular; Mice; Mice, Transgenic; Microscopy, Atomic Force; Mutagens; Mutation, Missense; Phenotype; Polymorphism, Single Nucleotide; Protein Binding; Protein Precursors; Pyridinium Compounds; Quaternary Ammonium Compounds