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. 2020 Dec 30;9:e60432. doi: 10.7554/eLife.60432

Figure 3. scRNA-seq analysis of the hair cell (HC) lineage in neuromasts.

(A) Uniform manifold approximation and projection (UMAP) plot colored by Louvain clustering annotated with cell types in the HC lineage. (B) Violin plots of selected HC (red bar) and supporting cell (blue bar) marker genes in each cluster. (C) Distribution of cells expressing emx2 and atoh1a on UMAP. Clusters of precursors (#4) and nascent HCs (#3) are enlarged in bottom panels. (D) Heat map representation of selected gene expression dynamics in pseudotime. (E) A violin plot showing significantly higher read counts of emx2 in the nascent HC cluster than other clusters (p=0.0069, two tail Student's t-test, source data 1). (F) A bar graph showing a significantly higher number of emx2-positive cells in the nascent HC cluster than others (X2 = 22.44, df = 3, p<0.0001, source data 2). A post-hoc 2 × 2 chi-squared test was also performed for multiple comparisons: precursor HC cluster vs nascent HC cluster, X2 (df = 1)=15.92, p<0.0001; nascent HC cluster vs young HC cluster, X2 (df = 1)=12.73, p=0.0004; nascent HC cluster vs mature HC, X2 (df = 1)=15.08, p<0.0001. The following figure supplements are available for Figure 3—figure supplement 1. Identification and annotation of cell types from Louvain clustering.

Figure 3—source data 1. Comparison of the emx2 log-counts in nascent hair cell (HC) cluster versus other clusters.
Figure 3—source data 2. Comparison of emx2-positive and -negative cells among four hair cell (HC) clusters.

Figure 3.

Figure 3—figure supplement 1. Identification and annotation of cell types from Louvain clustering.

Figure 3—figure supplement 1.

(A) Uniform manifold approximation and projection (UMAP) plot colored by Louvain clustering showing six clusters that included rpl10-HA (RiboTag) positive and negative cells. (B) Violin plots of selected marker genes of hair cell (HC) and SC. High levels of transgene tg_rpl10_3X_HA_short transcripts were concentrated in cluster #0, 1, 3, and 4 indicating they are cells within the HC lineage. Relative lower levels of rpl10-HA transcripts were found in cluster #2 of unknown identity, and #5, which expresses a high level of a SC marker, si:dkey-205h13.2. (C) Distribution of selected marker genes in UMAP. tg_rpl10_3X_HA_short is distributed in all the clusters except cluster #2. Supporting cell markers, si:dkey-205h13.2 and klf17 positive cells are concentrated in cluster #5. Thus, clusters #2 and #5 were excluded from the reclustering. (D) UMAP plot of reclustering of #0, 1, 3, and 4 shown in (A). (E) Heatmap showing the top 30 genes expressed in each cluster after the reclustering. (F) Violin plot showing distribution of n-counts, n-genes, mitochondrial fraction, and percentage of ribosomal genes of each cluster. The lower count depth, lower transcripts, and higher fraction of mitochondrial genes in cluster #2 indicate that they are damaged HCs. High percentages of ribosomal genes in clusters #3 and #4 are features of HC precursors and nascent HCs (Lush et al., 2019).