Coarse mapping of single-cell transcriptomes for zebrafish cells enriched for the ear.

A. Cartoon schematic of ear anatomy that highlights tissue and cell types in the adult zebrafish ear. B. Cartoon schematic of ear developmental events queried by our experiments. C. Brightfield images of representative developmental stages examined. D. Cartoon schematic of experimental workflow. E. UMAP graph of cells clustered by transcriptomes. F. UMAP graph with presumptive ear and neuromast cells in red. G. Heat map of normalized expression indicating clusters that have cells expressing known markers of the ear and neuromast. (Scale bars in C, D 50 μm)

Identifying ear and neuromast cell states by unsupervised clustering of transcriptomes.

A. UMAP plot of clustered ear and neoromast cells based on single-cell transcriptomes. B. Heat maps of transcript counts for expression of nueromast and ear markers overlayed on UMAP graph from A. C. Multiplexed in situ hybridization analysis of ear and neuromast markers. D. Hierarchical organization of clusters form A. with heat map histogram of markers for each cluster. E. Examples of genes enriched in expression in neuromast hair cells relative to otic vesicle hair cells. F. Examples of genes enriched in expression in otic vesicle hair cells relative to neuromast hair cells. G. Examples of genes differentially enrich between otic vesicle hair cells and neuromast hair cells that appear to either be paralogs or perform homologous roles. H. Color-coding of developmental stage on UMAP plot from A. (Scale bars in C 50 μm)

Fine analysis for single-cell transcriptomes of hair cell paths in the neuromast and the otic vesicle.

A. Cartoon schematics of a 3 day old zebrafish ear, with otic vesicle hair cells highlighted in blue, and a cartoon of neuromasts with hair cells highlighted in green next to a PAGA organized graph of hair cells. B. Coarse-grained PAGA graph of hair cells. C. Color-coding of developmental stage on PAGA graph. D. Heat-map of transcript counts on PAGA graph with markers representative of different clusters. E. Collapsed heat-map histogram of normalized expression that compares gene expression in cells along the PAGA path of neuromast hair cells with calculated distance along the path. F. Collapsed heat-map histogram that compares gene expression in cells along the PAGA path of otic vesicle hair cells with calculated distance along the path. G, H. Multiplexed fluorescent in situ analysis of otic vesicle genes of interest from the PAGA graph. (Scale bar 50 μm, same for G,H)

Fine analysis for single-cell transcriptomes of semicircular canal cells.

A. Cartoon schematic of a 3 day old zebrafish ear, with canal cells highlighted in magenta, next to PAGA organized graph of SCC cells with more “mature” SCC cells colored magenta. B. Coarse-grained PAGA graph of SCC cells. C. Color-coding of developmental stage on PAGA graph. D. Heat-map of transcript counts on PAGA graph with markers representative of different clusters. E. Collapsed heat-map histogram that compares gene expression in cells along the PAGA path with calculated distance along the path. F. Multiplexed fluorescent in situ analysis of genes of interest from the PAGA graph. (Scale bar 50 μm)

Fine analysis for single-cell transcriptomes of endolymphatic duct and sac cells.

A. Cartoon schematic of a 3 day old zebrafish ear, with the endolymphatic duct and sac highlighted in orange next to PAGA organized graph of dorsal otic vesicle and EDS cells with more “mature” EDS cells colored orange. B. Coarse-grained PAGA graph of EDS cells. C. Color-coding of developmental stage on PAGA graph from A. D. Heat-map of PAGA graph with markers representative of different clusters. E. Collapsed heat-map histogram that compares gene expression in cells along the PAGA path with calculated distance along the path. F. A sagittal perspective of multiplexed fluorescent in situ analysis of EDS genes expressed at the queried developmental stages. G. A transverse perspective of multiplexed fluorescent in situ analysis of duct and sac genes expressed at 72 hpf. H. Quantification of the in situs represented in G. (Scale bars 10 μm)

Fine analysis for single-cell transcriptomes of periotic mesenchyme cells.

A. Cartoon schematic of a 3 day old zebrafish ear, with the periotic mesenchyme highlighted in green next to PAGA organized graph of periotic mesenchyme cells with more “mature” mesenchyme cells colored green. B. Coarse-grained PAGA graph of mesenchyme cells. C. Color-coding of developmental stage on PAGA graph from A. D. Heat-map of transcription counts on PAGA graphs with markers representative of different clusters. E. Collapsed heat-map histogram that compares gene expression in cells along the PAGA path with calculated distance along the path. F. Multiplexed fluorescent in situ analysis of periotic mesenchyme genes expressed at the queried developmental stages. (Scale bar 50 μm)

Comparison of wild-type vs lmx1bb mutant.

A. 3-d rendering of confocal images of the wild-type and lmx1bb mutant ear at 3 dpf. B. A sagittal perspective of multiplexed fluorescent in situ analysis of epcam expression at 3 dpf. C. Maximum intensity projection of multiplexed fluorescent in situ analysis of foxi1 and epcam 3 dpf. D. Quantification of in situs represented in C. (Scale bar 50 μm in A, 10 μm in C).