Single-Cell Transcriptomic Profiling of the Zebrafish Inner Ear Reveals Molecularly Distinct Hair Cell and Supporting Cell Subtypes

Abstract

A major cause of human deafness and vestibular dysfunction is permanent loss of the mechanosensory hair cells of the inner ear. In non-mammalian vertebrates such as zebrafish, regeneration of missing hair cells can occur throughout life. While a comparative approach has the potential to reveal the basis of such differential regenerative ability, the degree to which the inner ears of fish and mammals share common hair cells and supporting cell types remains unresolved. Here we perform single-cell RNA sequencing of the zebrafish inner ear at embryonic through adult stages to catalog the diversity of hair cell and non-sensory supporting cells. We identify a putative progenitor population for hair cells and supporting cells, as well as distinct hair cells and supporting cell types in the maculae versus cristae. The hair cell and supporting cell types differ from those described for the lateral line system, a distributed mechanosensory organ in zebrafish in which most studies of hair cell regeneration have been conducted. In the maculae, we identify two subtypes of hair cells that share gene expression with mammalian striolar or extrastriolar hair cells. In situ hybridization reveals that these hair cell subtypes occupy distinct spatial domains within the two major macular organs, the utricle and saccule, consistent with the reported distinct electrophysiological properties of hair cells within these domains. These findings suggest that primitive specialization of spatially distinct striolar and extrastriolar hair cells likely arose in the last common ancestor of fish and mammals. The similarities of inner ear cell type composition between fish and mammals also support using zebrafish as a relevant model for understanding inner ear-specific hair cell function and regeneration.

Differential expression of mechanotransduction complex genes in auditory/vestibular hair cells in zebrafish

“…We identified 136 hair cells as lateral line hair cells while for the inner ear, we identified 165 striolar hair cells and 78 extrastriolar hair cells ( Figure 6A ). We used previously identified markers for these distributions: cabp2b and pvalb9 for striolar hair cells and zgc:153395 ( skor2 ) and cabp1b for extrastriolar hair cells ( Shi et al, 2022 ). Based on these classifications, the single cell data correlate well with the mRNA in situ analysis, showing that tmc transcripts describe unique subpopulation of cells in the stages of development.…”

“…After 5 dpf, the patterns no longer vary. Our previous study using scRNAseq data from whole zebrafish indicated that distinct hair cell types emerge in macular organs during larval stages ( Shi et al, 2022 ). We leveraged this data set to further explore the expression of other mechanotransduction components within tmc subtypes.…”