Single-Cell Profiling of the Developing Organ of Corti Identifies Etv4/5/1 as Key Regulators of Pillar Cell Identity

Abstract The mammalian auditory sensory epithelium, the organ of Corti, contains a number of unique cell types, including the inner and outer pillar cells that form the walls of the tunnel of Corti. The limited number of pillar cells and their close physical proximity to other cells within the organ of Corti has limited efforts to transcriptionally characterize their development. To identify potential regulators of pillar cell formation, we isolated cochlear duct epithelial cells at time points between embryonic day 11 and 16 for single cell RNA-sequencing. The resulting data were used to build a developmental trajectory from undifferentiated precursor cells to each of the major cell types in the organ of Corti including inner pillar cells. Bioinformatic analyses including SCENIC, TradeSeq and CellOracle identified the Etv4/5/1 transcription factors as likely regulators of inner pillar cell development. To specifically examine the role of Etv4/5/1 in pillar cell development, conditional triple mutants were generated. Results indicate defects in the formation of inner pillar cells as well as changes in other cochlear cells likely as a result of secondary interactions. Finally, we demonstrate that expression of Etv4/5/1 in pillar cells is dependent on Fgfr3 and identify downstream targets of Fgfr3/Etv signaling in inner pillar cells. These results provide significant insights regarding the specification and early development of inner pillar cells, which will have implications for understanding congenital deficits and potential applications in the development of regenerative strategies.