Hearing and balance rely on small sensory hair cells that reside in the inner ear. To explore dynamic changes in the abundant proteins present in differentiating hair cells, we used nanoliter-scale shotgun mass spectrometry of single cells, each ~1 picoliter, from utricles of embryonic day 15 chickens. We identified unique constellations of proteins or protein groups from presumptive hair cells and from progenitor cells. The single-cell proteomes enabled the de novo reconstruction of a developmental trajectory using protein expression levels, revealing proteins that greatly increased in expression during differentiation of hair cells (e.g., OCM, CRABP1, GPX2, AK1, GSTO1) and those that decreased during differentiation (e.g., TMSB4X, AGR3). Complementary single-cell transcriptome profiling showed corresponding changes in mRNA during maturation of hair cells. Single-cell proteomics data thus can be mined to reveal features of cellular development that may be missed with transcriptomics.

SUMMARYProtruding from the apical surface of inner ear sensory cells, hair bundles carry out mechanotransduction. Bundle growth involves sequential and overlapping cellular processes, which are concealed within gene expression profiles of individual cells. To dissect such processes, we developed CellTrails, a tool for uncovering, analyzing, and visualizing single-cell gene-expression dynamics. Utilizing quantitative gene-expression data for key bundle proteins from single cells of the developing chick utricle, we reconstructed de novo a bifurcating trajectory that spanned from progenitor cells to mature striolar and extrastriolar hair cells. Extraction and alignment of developmental trails and association of pseudo-time with bundle length measurements linked expression dynamics of individual genes with bundle growth stages. Differential trail analysis revealed high-resolution dynamics of transcripts that control striolar and extrastriolar bundle development, including those that encode proteins that regulate [Ca2+]i or mediate crosslinking and lengthening of actin filaments.

Highlights d Single-cell transcriptomic characterization of the avian cochlear sensory epithelium d Distinct hair cell and supporting cell types d Tonotopic gene expression dynamics of hair cells d Resource of cell-type-specific gene expression in the mature avian cochlea

Highlights d Efficient culture conditions for the generation of inner ear organoids d Single-cell RNA-seq resource of all principal neonatal cochlear cell groups d Greater epithelial ridge cells display robust potential to generate otic organoids d Organoid formation is synergistically enhanced by direct cellcell contact

Highlights d In vivo model to study cochlear hair cell death d Cochlear hair cells die rapidly and synchronously d Single-cell RNA-sequencing reveals two stages of hair cell demise d Major types of dying cochlear hair cells express different sets of genes