Novel cell types and developmental lineages revealed by single-cell RNA-seq analysis of the mouse crista ampullaris
- Department of Biological Structure, University of Washington, United States
- Institute for Stem Cells and Regenerative Medicine, University of Washington, United States
- Department of Biochemistry, University of Washington, United States
Figures
Figure 1
Cellular diversity in the crista ampullaris.
(a) Cristae, including the ampullae, were dissected from E16, E18, P3, and P7 mice, then dissociated for single-cell RNA-seq (scRNA-seq) analysis of cell types and gene expression. (b) UMAP of …
Figure 2 with 7 supplements
Hair cell and support cell subtypes and dynamics during development.
(a) Highlights the position of support cells and hair cells in UMAP space relative to the whole dataset (i.e. E16, E18, P3, and P7 combined). (b) Cluster analysis in support cells and hair cells. (c …
Figure 2—figure supplement 1
Oncomodulin immunofluorence in type I hair cells of the crista ampullaris.
IF for oncomodulin (green), neurofilament medium (magenta) and Sox2 (blue) in a vibratome section of P3 B6 crista. Open arrowheads indicate calyx-type synapses of type I hair cells. Closed arrowheads…
Figure 2—figure supplement 2
Localization of hair cell and support cell markers.
RNA in situ hybridization data from the Allen Developing Mouse Brain Atlas is shown for cluster-specific markers in E15.5 cristae (Isl1 is E16.5). Insets show the cluster specificity in UMAP. Note …
Figure 2—figure supplement 3
Developmental stage and pseudotime in crista epithelial cells.
(a and b) show developmental stage and pseudotime on UMAP in cells from the whole dataset, respectively. (b) Is replicated from Figure 2e for easy comparison with (a). (c) shows pseudotime in cell …
Figure 2—figure supplement 4
RNA velocity analysis in crista epithelial cells.
Arrows show the RNA velocity (i.e. the rate and direction of transcriptomic change as indicated by the ratio of unspliced:spliced transcripts La Manno et al., 2018) in UMAP of batch-corrected …
Figure 2—figure supplement 5
Localization of anti-Id1 in crista.
(a) Id1 (red) localization in a subset of Sox2+ (green) support cell nuclei in optical sections of P4 Hes5-GFP crista counterstained with Hoecsht 33342 (blue). Hi magnification images in the region …
Figure 2—figure supplement 6
Heat-shock pathway dynamics during support cell development.
(a) Shows expression of heat shock response genes relative to support cell and hair cell clusters, pseudotime and developmental time. Note that most Hsp- and Dnaj- genes increase at P7. (b) Shows …
Figure 2—figure supplement 7
Fgf pathway in the developing crista ampullaris.
(a) Shows the expression of Fgf ligands and receptors in UMAP of the crista epithelium. Maximum expression is shown at lower right of each UMAP panel. In (b), The expression of Fgf ligands, …
Figure 3 with 2 supplements
Glial cell diversity and dynamics.
(a) Highlights the position of glial cells in UMAP space relative to the whole dataset. (b) Cluster analysis in glia. (c) Expression of markers relative to cell cluster, developmental stage and …
Figure 3—figure supplement 1
Developmental stage relative to UMAP in crista glial cells.
Glial cells in the whole dataset shown in UMAP with color-coding by developmental stage.
Figure 3—figure supplement 2
Localization of glial cell markers.
RNA in situ hybridization data from the Allen Developing Mouse Brain Atlas is shown for cluster-specific markers in E15.5 cristae. Insets show the cluster specificity in UMAP. Arrowheads indicate …
Figure 4 with 1 supplement
Cellular diversity in the nonsensory ampulla epithelium.
(a) Highlights the position of the nonsensory epithelial cells of the ampulla in UMAP space relative to the whole dataset. (b) Cluster analysis in nonsensory ampulla epithelium. (c) Expression of …
Figure 4—figure supplement 1
Localization of cluster-specific markers in nonsensory ampulla epithelium.
RNA in situ hybridization data from the Allen Developing Mouse Brain Atlas is shown for cluster-specific markers in E15.5 cristae. Insets show the cluster specificity in UMAP. Arrowheads indicate …
Figure 5 with 2 supplements
Mesenchymal cell diversity and dynamics.
In (a), Labeling of endogenous IgG (white) in a vibratome section of E15.5 posterior crista demonstrates the region of loose mesenchyme (bracket) bounded by regions of dense mesenchyme proximal to …
Figure 5—figure supplement 1
Localization of cluster-specific markers in otic mesenchyme surrounding the ampulla.
Panels show RNA in situ hybridization data for cluster-specific markers in E15.5 cristae from the Allen Developing Mouse Brain Atlas. Insets show specificity in the UMAP space. Expression patterns …
Figure 5—figure supplement 2
Slc1a3-CreER activity in the anterior canal, horizontal canal and utricle.
Green shows Slc1a3-CreER activation of GFP reporter (Todd et al., 2020) in a subset of support cells and also a subset of mesenchymal cells in the cristae and utricle in an adult temporal bone …
Figure 6
Melanocyte dynamics.
(a) Arrow indicates the cluster of melanocytes in UMAP space relative to the whole dataset. (b) The relative contributions of melanocytes to the composition of the mesenchyme at the indicated …
Figure 7 with 2 supplements
Macrophage and vascular cell diversity and dynamics.
(a) Highlights the positions of macrophages and vascular cells in UMAP space relative to the whole dataset. (b) The relative contributions of cell clusters to the composition of the vasculature at …
Figure 7—figure supplement 1
Localization of macrophage markers.
RNA in situ hybridization data from the Allen Developing Mouse Brain Atlas is shown for macrophage cluster-specific markers in E15.5 B6 cristae. Insets show the cluster specificity in UMAP. Arrowhead…
Figure 7—figure supplement 2
Developmental stage relative to UMAP in ampulla macrophages.
Macrophages in the whole dataset shown in UMAP with color-coding by developmental stage.
Figure 8
Comparative analysis of P7 crista ampullaris and P7 cochlea.
(a) Shows the relatedness of scRNA-seq data from Kolla et al. for P7 Swiss Webster cochlea (left) and the P7 B6 crista ampullaris data from the present study (right) in UMAP space. Note that common …
Figure 9
Expression of genes associated with vestibular disease and dysfunction in the crista ampullaris.
The heatmap summarizes the expression of a subset of genes associated with vestibular disease and dysfunction in cell clusters of the crista ampullaris. Figure 9—source data 1 shows the full list of …
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Figure 9—source data 1
Expression of genes associated with vestibular disease and dysfunction in the crista ampullaris.
The heatmap summarizes the expression of genes associated with vestibular disease and dysfunction in cell clusters of the crista ampullaris. Column color bars indicate the major cell types and subtypes. Row color bars denote the following: IMPC, genes mutated in mice that exhibit circling (cornflour blue) and hyperactivity (teal) or both (purple) phenotypes. Disorders, gene associations with Vestibular dysfunction (forest green), susceptibility to ototoxicity (magenta), peripheral vertigo (blue) and Meniere’s disease (maroon) were compiled from MSigDB, OMIM and Malacards databases. GWAS, hits in a genome-wide association studies of motion sickness (green) and vestibular migraine (orange). Balance, genes in the GO term ‘neuromuscular process controlling balance’ (sky blue). IE morph, genes in the GO term ‘inner ear morphogenesis’ (khaki). Devo., genes in GO terms ‘stereocilium organization’ (midnight blue), ‘otolith development’ (lime green) and ‘vestibulocochlear nerve development’ (gray).
- https://cdn.elifesciences.org/articles/60108/elife-60108-fig9-data1-v2.pdf
Appendix 1—figure 1
Wnt3 expression in the E11.5 otocyst.
Panels show RNA in situ hybridization data from the Allen Developing Mouse Brain Atlas for cluster-specific markers in E15.5 B6 cristae. Note that the expression of Wnt3 coincides with Nr4a3/Nor1 …
Appendix 1—figure 2
Wnt pathway in the perinatal crista.
The expression of Wnt ligands, receptors, inhibitors and mediators is visualized by heatmap.
Videos
Video 1
Id1 immunofluorescence in P0 crista.
Video shows a series of 1 micron optical sections per second imaged in P0 B6 cristae showing anti-Sox2 (green), anti-Id1 (magenta), and phalloidin (cyan).
Video 2
Id1 immunofluorescence in P4 crista.
Video shows a series of 1 micron optical sections per second imaged in P4 Hes5-GFP cristae from a colleagues’ colony showing anti-Sox2 (white), anti-Id1 (magenta), Hes5-GFP (yellow), and Hoechst 3334…
Tables
Table 1
Antibodies used for immunofluorescence.
| Antigen | Host | Dilution | Cat. no. | Manufacturer | RRID |
|---|---|---|---|---|---|
| Atoh1 | Rabbit | 1:1000 | 21215–1-AP | Proteintech | AB_10733126 |
| Hsp70 | Rabbit | 1:1000 | PA5-28003 | Invitrogen | AB_2545479 |
| Iba1 | Rabbit | 1:1000 | 019–19741 | Wako | AB_839504 |
| Id1 | Rabbit | 1:1000 | BCH-1/37–2 | Biocheck | AB_2713996 |
| Myo7a | Rabbit | 1:1000 | 25–6790 | Proteus | AB_10015251 |
| Myo7a* | Mouse | 5 μg/ml | MYO7A 138–1 | DSHB | AB_2282417 |
| Nefm | Chick | 1:200 | ab134458 | Abcam | AB_2860025 |
| Ocm | Rabbit | 1:1000 | Omg4 | Swant | AB_10000346 |
| Pecam1 | Rat | 1:50 | 551262 | BD Pharmingen | AB_398497 |
| Sox2 | Goat | 1:200 | sc-17320 | Santa Cruz | AB_2286684 |
| Wnt3* | Goat | 1:125 | PA5-18516 | Thermo | AB_10979520 |
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*Requires antigen retrieval.
Additional files
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Supplementary file 1
Cell numbers, sequencing depth, and gene detection in the crista ampullaris dataset.
- https://cdn.elifesciences.org/articles/60108/elife-60108-supp1-v2.docx
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Supplementary file 2
RNA ISH images from the Allen Developing Mouse Brain Atlas.
- https://cdn.elifesciences.org/articles/60108/elife-60108-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/60108/elife-60108-transrepform-v2.pdf
