Fig. 3: Development of OHCs.

a tSNE plot for HCs isolated at P1. Both IHCs and OHCs can be clustered into immature and more mature cell groups based on gene expression. Arrow indicates trajectory of pseudotime (see c, d). b Dot plot showing differentially expressed genes between IHCs and OHCs. IHCs exclusively express several genes, including Fgf8, Atp2a3, Cabp2, Tbx2, and Shtn1. In contrast, a more limited number of genes are exclusively expressed in OHCs, including Calca, Serpina1c, Veph1, Cacng2, Strip2, and Msln1. This may reflect the delay in OHC maturation relative to IHC. c The P1 OHCs were analyzed using Monocle to generate a pseudotime trajectory. Analysis of gene expression profiles along pseudotime identified four different developmental phases. d Feature plots for a representative gene or genes from each of the four OHC developmental phases. Expression of Tmc1 was limited to the most mature IHCs and OHCs (purple circles) e Left-hand panel, tSNE plot based on identification of transcriptional regulons using SCENIC. The HC and SC clusters are indicated by red (HC) and blue (SC) circles. Right-hand panel, feature plots for representative HC-specific regulons (blue indicates high expression). Lhx3, Atoh1, Pou4f3, and Barhl1 are known HC-specific transcription factors (TFs). Brf2 and Usf2 have not been previously localized to hair cells but are present in HCs from previous bulk RNAseq experiments (see text). f Left-hand panel, expanded regulon tSNE for the HCs. Right-hand panel, two additional TFs, Bdp1 and Arnt1 are localized to HC subsets. HC hair cells, IPhC inner phalangeal cells/border cells, IPC inner pillar cells, OPC outer pillar cells, DC1/2 Deiters’ cells rows 1 and 2, DC3, Deiters’ cells row 3, HeC Hensen’s cells, CC/OSC Claudius cells/outer sulcus cells, IdC interdental cells, ISC inner sulcus cells, KO Kölliker’s organ cells, L.KO lateral Kölliker’s organ cells, M.KO medial Kölliker’s organ cells, OC90 OC90⁺ cells.