FIGURE 2.

Stria vascularis (SV) cell types show clear transcriptional differences at the single cell and single nucleus level. (A) Unbiased clustering was performed on both single cell RNA-Seq (scRNA-Seq) and single nucleus RNA-Seq (snRNA-Seq) datasets from the P30 mouse SV. Known cell type-specific markers were utilized to identify these agnostically determined cell clusters. Based on these known markers, clusters consisting of marginal cells, intermediate cells, basal cells, spindle/root cells, and immune cell types, including macrophages, B cells, and neutrophils, were identified. tSNE plot of P30 mouse SV scRNA-Seq dataset demonstrates clustering of SV cell types (LEFT panel). tSNE plot of P30 mouse SV snRNA-Seq dataset demonstrates clustering of SV cell types with similar identification of cell type-specific clusters (RIGHT panel). (B) Feature plots for P30 SV scRNA-Seq data demonstrate expression of known markers for 4 main SV cell types: marginal cells (Kcne1, Kcnq1), intermediate cells (Cd44, Met), basal cells (Cldn11, Tjp1), and spindle/root cells (Slc26a4). 4 main cell types are highlighted on the inset tSNE plot of the scRNA-Seq dataset. Maximum expression (normalized counts) is noted in the black outlined gray box for each gene in the lower left corner of each feature plot. (C) Grid violin plot of scRNA-Seq dataset demonstrates expression of known marker genes amongst SV cell types. Normalized counts were scaled to a range of 0–1 using min-max scaling. (D) Feature plots for P30 SV snRNA-Seq data demonstrate expression of known markers for 4 main SV cell types: marginal cells (Kcne1, Kcnq1), intermediate cells (Cd44, Met), basal cells (Cldn11, Tjp1), and spindle/root cells (Slc26a4). 4 main cell types are highlighted on the inset tSNE plot of the snRNA-Seq dataset. Maximum expression (normalized counts) is noted in the black outlined gray box for each gene in the lower left corner of each feature plot. (E), Grid violin plot of snRNA-Seq dataset demonstrates expression of known marker genes amongst SV cell types. Normalized counts were scaled to a range of 0–1 using min-max scaling. (F) Validation of known gene expression was performed using single molecule fluorescent in situ hybridization (smFISH) and fluorescence immunohistochemistry. Known marginal cell-specific transcripts (Kcne1, Kcnq1) and intermediate cell-specific transcripts (Cd44, Met) are co-localized by smFISH. Yellow dashed lines outline the SV marginal cell layer showing Kcnq1 transcript expression in marginal cells with DAPI to label nuclei. Yellow dashed lines outline the SV intermediate cell layer in the image with DAPI only. CLDN11 and ZO-1 (protein product of Tjp1) are localized to the basal cell layer with anti-CD44 and anti-KCNJ10 immunostaining to label the adjacent intermediate cell layer for contrast, respectively. Finally, Slc26a4 transcripts are localized to the spindle/root cells by smFISH. Yellow dashed lines in the image to the right with only the DAPI labeling demarcate the SV and spindle cells (SpC) and root cells (RC) in the spiral prominence (SP) are identified. DAPI (4′,6-diamidino-2-phenylindole). All scale bars 10 μm.