Transcriptional Dynamics of Hair-Bundle Morphogenesis Revealed with CellTrails

Protruding 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 pseudotime 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. Ordering single cells along branching trajectories using transcriptomic data is bioinformatically challenging. Ellwanger et al. developed CellTrails and applied this tool to showcase the bifurcating sequence of gene expression as sensory hair cells develop into different subtypes that feature spatially distinct morphologies of the mechanosensitive hair bundle.