Advanced Insights into the Regenerative Limits, Molecular Identities, and Functional Necessity of Vestibular Hair Cell Subtypes in the Adult Mouse Vestibular System

Abstract

Vestibular hair cells are specialized mechanosensory receptors in the inner ear that detect head movements and gravitational forces to support balance, gaze stabilization, and spatial orientation (Hudspeth, 1997; Corey & Hudspeth, 1983; Gillespie & Müller, 2009). In adult mammals, these cells are vulnerable to damage from aging, ototoxic drugs, and genetic mutations, and they have limited regenerative abilities (Forge et al., 1993; 1998; Kawamoto et al., 2009; Golub et al., 2012; Bucks et al., 2017; Sayyid et al. 2019; Hicks et al., 2020; Ciani Berlingeri et al., 2022; Jauregui et al. 2024) compared to those in non-mammalian vertebrates such as birds and fish (Corwin & Cotanche, 1988; Ryals & Rubel, 1988; Ma et al., 2008). It is likely that humans also have some capacity for hair cell regeneration (Taylor et al., 2018). This limited regenerative capacity contributes to persistent vestibular dysfunction and balance impairment following hair cell loss (Agrawal et al., 2009; Hicks et al., 2020). Although a few studies have shown partial recovery of function, the newly formed hair cells do not fully compensate for the loss of vestibular function (Schlecker et al., 2011; Sayyid et al., 2019; Bremer et al., 2014). Many questions arise from these previous results. Why are only some type II hair cells regenerated? What is needed for functional regeneration? These and many other questions drive my work into the adult vestibular system. In this thesis, I investigated the molecular, functional, and regenerative properties of vestibular hair cells in adult mice. Using a conditional knockout model, I show that the transcription factor SOX2, previously implicated in sensory development (Kiernan et al., 2005; Neves et al., 2012), is required in adult vestibular supporting cells for the limited regeneration of hair cells following hair cell loss. Sox2-deficient supporting cells failed to transdifferentiate into new hair cells, resulting in markedly reduced regeneration across the utricle, saccule, and ampulla (Ciani Berlingeri et al., 2022). Although we now know Sox2 and Atoh1 (Hicks et al., 2020) are necessary for the limited number of regenerated hair cells and that these cells come from supporting cells, the lack of regeneration of all the rest of the hair cells does not occur. This led to my exploration into the different types of cells in the adult vestibular sensory epithelia. Maybe there are more cell types than we think. To define the molecular identity of vestibular hair cell subtypes, I performed single-nucleus RNA-sequencing on adult mouse utricles. This transcriptomic profiling revealed five molecularly distinct hair cell subtypes spanning the classically defined type I and type II hair cell types across different epithelial zones. Markers such as Calb2, Spp1, Ocm, Agbl1, Paqr9, and others distinguished these subtypes (Xia et al., 2025; Desai et al., 2005; Simmons et al., 2010; Dechesne et al., 1988), and expression patterns were validated through immunolabeling and fluorescent in situ hybridization. The findings expand upon previous efforts by defining hair cell heterogeneity in the adult utricle at single-cell resolution and identifying novel molecular markers specific to regional and subtype identity through analysis of thousands of adult cells (McInturff et al., 2018, Xia et al., 2025). These molecular differences could result in functional differences. Therefore, while analyzing these cells molecularly, I also investigated the functional requirements of one population of vestibular hair cells. I employed a selective genetic ablation strategy to remove peripheral type I hair cells in all vestibular organs in adult mice while preserving central type I and all type II populations using a recently generated mouse line (McGovern et al., 2022; Hartman et al., 2018). This loss caused severe and lasting deficits in balance and vestibulo-ocular reflex performance, highlighting the unique and non-redundant role of peripheral type I hair cells in dynamic vestibular function. Altogether, this work identified Sox2 as a key regulator of supporting cell driven hair cell regeneration, revealed new molecular markers and subtype distinctions in adult vestibular hair cells, as well as identified the critical role of peripheral type I hair cells in maintaining complex vestibular reflexes. These findings add to the foundation for regenerative strategies aimed at restoring vestibular function after injury or degeneration.