SLC26A4 is known to cause auditory and vestibular disorders, including Pendred syndrome, DFNB4 and enlarged vestibular aqueduct. Recent investigations using Slc26a4-knockout (KO) mice have revealed diverse anomalies in the bony labyrinth, suggesting that abnormal otoconial formation largely underlies the balance deficits in this condition. However, a detailed understanding of how these defects diverge from normal otic capsule and otoconia development remain elusive. Here, we compared Slc26a4-KO mice with wild-type (WT) mice to clarify the pathogenic mechanism of SLC26A4-related vestibular dysfunction. First, we documented normal otic capsule and otoconial development in WT mice from birth to several weeks of age using micro-computed tomography (micro-CT). This revealed a stepwise calcification of the cochlear base, modiolus, and semicircular canals, as well as a progressive increase in otoconial volume in the utricle and saccule. By two to three weeks postnatally, WT otoconia reached functional maturity, supporting normal vestibular reflexes and stable locomotor behavior. In contrast, Slc26a4-KO mice exhibited a marked reduction or delay in otoconial growth, particularly in the saccule. The micro-CT scans also indicated enlarged vestibular aqueducts and decreased bone mineral density in parts of the labyrinth. Behavioral assays by the rotarod test showed significantly impaired balance and frequent circling behaviors in the KO mice, while vestibulo-ocular reflex analyses indicated diminished responses to tilt (otolith-based) rather than rotational (semicircular canal-based) stimuli. These findings suggest that SLC26A4 mutations disrupt the early mineralization processes of the otic capsule and otoconia, culminating in characteristic vestibular impairments that resemble those encountered in benign paroxysmal positional vertigo. Elucidating the differences between normal and aberrant progression of labyrinthine ossification and otoconial formation may aid in the development of therapeutic strategies—such as pharmacological interventions targeting ion homeostasis—to mitigate balance disorders in SLC26A4-related pathologies.
