LAUSR

ABSTRACT The utricle of the inner ear, a vestibular sensory structure that mediates perception of linear acceleration, is comprised of two morphologically and physiologically distinct types of mechanosensory hair cells, referred to as Type Is and Type IIs. While these cell types are easily discriminated in an adult utricle, understanding their development has been hampered by a lack of molecular markers that can be used to identify each cell type prior to maturity. Therefore, we collected single hair cells at three different ages and used single cell RNAseq to characterize the transcriptomes of those cells. Analysis of differential gene expression identified Spp1 as a specific marker for Type I hair cells and Mapt and Anxa4 as specific markers for Type II hair cells. Antibody labeling confirmed the specificity of these markers which were then used to examine the temporal and spatial development of utricular hair cells. While Type I hair cells develop in a gradient that extends across the utricle from posterior-medial to anterior-lateral, Type II hair cells initially develop in the central striolar region and then extend uniformly towards the periphery. Finally, by combining these markers with genetic fate mapping, we demonstrate that over 98% of all Type I hair cells develop prior to birth while over 98% of Type II hair cells develop post-natally. These results are consistent with previous findings suggesting that Type I hair cells develop first and refute the hypothesis that Type II hair cells represent a transitional form between immature and Type I hair cells. Summary: Single cell RNAseq was used to identify new markers for specific hair cell in the mammalian utricle. With these new markers, the developmental timing of specific types of hair cells was examined.