Developmental transcriptomic profiling of non-sensory vestibular epithelium reveals ionic contribution to endolymph formation

Abstract

In the inner ear, various ion channels have reported and play a key role in signal transduction, their malfunction could lead to hearing and vestibular dysfunction. Ion channels contribute to formation of endolymph. Endolymph is formed from embryonic day (E) 14.5 of developing murine inner ear. Endolymph secretion leads to lumen formation in developing inner ear. Although it was suggested that osmotic gradient created by changes of ionic composition plays a role in the endolymph formation, still exact molecular mechanism of endolymph formation is unknown. We aimed to investigate the mechanism of endolymph formation in the developing inner ear. We collected non-sensory epithelium of vestibule at the age of E16.5, E18.5, and Postnatal day (P) 5, and classified the collected specimens according to the presence of dark cells as follows: non-sensory epithelium of utricle and common crus that contained dark cells and semicircular canals that did not contain dark cells. RNA sequencing was performed with those samples to analyze the changes in ion channels according to the development. After selecting candidate genes for endolymph formation, we measured endolymphatic volume changes using confocal 3D live imaging with the application of candidate ion channel inhibitors for functional study. The localization of the candidate ion channels was examined by immunohistochemistry We selected 1613 differentially expressed genes (DEGs) by RNA sequencing analysis (P <0.001 and |Fold Change|<2 at least in one comparison). The genes in each sample formed close clusters according to the cell types and development period. Most genes were related to ion activities such as ion transport, and membrane transport. Four major ions potentially involved in the endolymph formation with high probability were sodium, chloride, calcium, and potassium ions. A functional study using 3D volume change with the application of chloride-free solution and amiloride at E16.5 showed blockage of endolymphatic fluid secretion. The calcium-free solution did not show any decrease in secretion rate. Potassium ions only worked at P5, confirmed by XE991 treatment. During inner ear development, sodium and chloride ions, but not potassium ions, are strongly associated with endolymphatic fluid secretion. These findings may help elucidate the mechanism of inner ear formation as well as congenital hearing loss and vestibular disorders.