ABSTRACT
Inner ear hair cells are characterized by the F-actin-based stereocilia that are arranged into a staircase-like pattern on the apical surface of each hair cell. The tips of shorter-row stereocilia are connected with the shafts of their neighboring taller-row stereocilia through extracellular links named tip links, which gate mechano-electrical transduction (MET) channels in hair cells. Cadherin 23 (CDH23) forms the upper part of tip links, and its cytoplasmic tail is inserted into the so-called upper tip-link density (UTLD) that contains other proteins such as harmonin. The Cdh23 gene is composed of 69 exons, and we show here that exon 68 is subjected to hair cell-specific alternative splicing. Tip-link formation is not affected in genetically modified mutant mice lacking Cdh23 exon 68. Instead, the stability of tip links is compromised in the mutants, which also suffer from progressive and noise-induced hearing loss. Moreover, we show that the cytoplasmic tail of CDH23(+68) but not CDH23(-68) cooperates with harmonin in phase separation-mediated condensate formation. In conclusion, our work provides evidence that inclusion of Cdh23 exon 68 is critical for the stability of tip links through regulating condensate formation of UTLD components.
Subject(s)
Deafness , Hearing Loss , Mice , Animals , Hearing Loss/genetics , Hearing Loss/metabolism , Hair Cells, Auditory/physiology , Deafness/genetics , Hair Cells, Auditory, Inner/metabolism , Cadherins/metabolism , Exons/geneticsABSTRACT
As the sensory receptor cells in vertebrate inner ear and lateral lines, hair cells are characterized by the hair bundle that consists of one tubulin-based kinocilium and dozens of actin-based stereocilia on the apical surface of each hair cell. Hair cell development is tightly regulated, and deficits in this process usually lead to hearing loss and/or balance dysfunctions. RNA-binding motif protein 24 (RBM24) is an RNA-binding protein that is specifically expressed in the hair cells in the inner ear. Previously, we showed that RBM24 affects hair cell development in zebrafish by regulating messenger RNA (mRNA) stability. In the present work, we further investigate the role of RBM24 in hearing and balance using conditional knockout mice. Our results show that Rbm24 knockout results in severe hearing and balance deficits. Hair cell development is significantly affected in Rbm24 knockout cochlea, as the hair bundles are poorly developed and eventually degenerated. Hair bundle disorganization is also observed in Rbm24 knockout vestibular hair cells, although to a lesser extent. Consistently, significant hair cell loss is observed in the cochlea but not vestibule. RNAseq analysis identified several genes whose mRNA stability or pre-mRNA alternative splicing is affected by Rbm24 knockout. Among them are Cdh23, Pcdh15, and Myo7a, which have been shown to play important roles in stereocilia development as well as mechano-electrical transduction. Taken together, our present work suggests that RBM24 is required for mouse hair cell development through regulating pre-mRNA alternative splicing as well as mRNA stability.
Subject(s)
Alternative Splicing , Hair Cells, Auditory , RNA Precursors , Animals , Mice , Alternative Splicing/genetics , Cadherins/genetics , Mice, Knockout , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Stability/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Zebrafish , Hair Cells, Auditory/physiologyABSTRACT
Annexin A4 (ANXA4) is a Ca2+-dependent phospholipid-binding protein that is specifically expressed in the cochlear and vestibular hair cells, but its function in the hair cells remains unknown. In the present study, we show that besides localizing on the plasma membrane, ANXA4 immunoreactivity is also localized at the tips of stereocilia in the hair cells. In order to investigate the role of ANXA4 in the hair cells, we established Anxa4 knockout mice using CRISPR/Cas9 technique. Unexpectedly, the development of both cochlear and vestibular hair cells is normal in Anxa4 knockout mice. Moreover, stereocilia morphology of Anxa4 knockout mice is normal, so is the mechano-electrical transduction (MET) function. Consistently, the auditory and vestibular functions are normal in the knockout mice. In conclusion, we show here that ANXA4 is dispensable for the development and function of hair cells, which might result from functional redundancy between ANXA4 and other annexin(s) in the hair cells.
