Wang Y , Wang H , Zhang P , Zhu B , Li W , Zhao X , Yan M , Song X , Lai F , Dong J , Cui J , Guo X , Wu HJ , Li J .

32370882 National Natural Science Foundation of China (National Science Foundation of China), 32370536 National Natural Science Foundation of China (National Science Foundation of China), 82300142 National Natural Science Foundation of China (National Science Foundation of China)

	Fig. 1. Comparative transcriptome analyses across vertebrates unveiled a natural feature of inner ears from species with high regenerative capacity.a Phylogenetic tree illustrating the evolutionary relationships among human, mouse, chicken, Xenopus laevis, and zebrafish. Green gradient indicates the level of regenerative capacity. b Schematic representation of the whole inner ear in Xenopus (left), chicken (middle), and mouse (right), with the sensory epithelia indicated in purple. Xenopus has 8 epithelia, chicken 7, and mammals 6. A, anterior; D, dorsal; L, lateral. These illustrations were created with Adobe Illustrator and adapted from previous publications21,29,30. c Venn plots displaying common expressed genes between Xenopus and mouse, Xenopus and chicken, and among these three species. There are 10858 commonly expressed genes between these three species. Most of the known deafness genes from the public human deafness gene database Gen4HL (206)31 were found in the 10,858 conserved genelist. d Differential expression patterns and hierarchical clustering of conserved genes in Xenopus and mouse. Representative genes for hair cell development and regeneration, including Atoh1, Insm1 Tbx2, Bdnf, and Sox genes, are labeled in red. MM1/2 and XP1/2 are the two out of the three replicates from mouse and Xenopus, respectively. Expression levels from low to high are indicated from blue to red color in the heatmap. e GO functional analysis of genes in the group 1 and group 2 in (d), which shows high gene expression levels in Xenopus and low levels in mouse, or vice versa. Development, proliferation, and regeneration-related terms are enriched in group 1, and cell death, cell cycle arrest, and apoptosis-related terms are enriched in group 2. ap amphibian papilla; bp basilar papilla; s saccule; u utricle; l lagena; aa anterior crista; pc posterior crista; lc lateral crista; cd cochlear duct.
	Fig. 2. The single-cell atlas of Xenopus inner ear and its comparison with mouse.a Light microscopy displaying of the whole inner ear, the amphibian papilla (AP) and basilar papilla (BP) parts in Xenopus, and cochlear part of mouse inner ear. All scale bar equal to 0.5 mm. A, anterior; D, dorsal. b UMAP plots showing cell atlas of the mouse cochlea. A total of 24 cell types are labeled in different colors. c Bubble plot displaying of the most specifically expressed genes for each cell type in the mouse cochlea. d UMAP plots depicting the whole inner ear cell atlas of the Xenopus. A total of 21 cell types are identified in the Xenopus inner ear, labeled in different colors. e The most specifically expressed genes or cell markers for each cell type. f Cell atlas of AP-BP auditory part of Xenopus inner ear. A total of 19 cell types are identified in AP-BP part. g Cell markers for identification of each cell types in AP-BP atlas. Iphc/IBC inner phalangeal cell /inner border cell; ISC/OSC/CC Claudius cells and inner and outer sulcus cells; AP amphibian papilla; BP basilar papilla.
	Fig. 3. Cross-species comparison studies confirmed an exceptionally conserved neuronal cell type.a UMAP analysis and overlapping of inner ear cell atlas between Xenopus (AP-BP) and the mouse cochlea. Colors indicate the different species, Xenopus, red; mouse, blue. b Sankey plot showing the conservation or similarity between mouse cochlea and Xenopus (AP-BP) cell types. The top 5 similarity coefficients are labelled. Gray and thin lines indicate very low or almost no significant similarity between the cell types. c Exaction of UMAP plots only for the mouse cochlea neuronal type cells. Including the neuron type III, there are five neuronal cell subtypes, IA, IB, IC, II, and III. d UMAP plots for the neuronal cells in the Xenopus whole inner ear. (e) and (f) Bubble plot analysis showing the expression feature of neuronal cell type-specific expressed genes in all neuronal cell types in the mouse cochlea and the Xenopus inner ear. The blue boxed areas indicate all the neuronal cell subtypes. g Immunostaining with neuronal cell marker Tuj1 and RNA in situ detection of Nefh gene in spiral ganglion neurons of mouse cochlea. The right bottom box indicates the section direction: vertical to circular plane of cochlea; A, apical; B, basal. The white dashed lines around the Tuj1 signal indicate the spiral ganglion regions. Higher magnification of the middle area enclosed by the dashed line is shown below. h RNA in situ detection of nefh gene expression in Xenopus. SGN spiral ganglion neuron; G ganglion. AP amphibian papilla; BP basilar papilla. All scale bars equal to 100 µm.
	Fig. 4. Characterization of hair cells in Xenopus inner ear revealed their high conservation in vertebrates.a UMAP plots showing the hair cells (blue painting) in Xenopus inner ear cell atlas. b The painting of canonical hair cell marker genes otof.S, myo6.L, ush2a.L, loxhd1.S, myo16.S, and calb2.L in the UMAP plots. c Co-expression of hair cell marker genes in mouse, chicken and Xenopus, including AP-BP part in Xenopus. (d–f) Double immunostaining with anti-MYO7A antibody and phalloidin for hair cell detection in both Xenopus (d and e) and mouse cochlear (f) hair cells. DAPI, blue; Myo7a, green; Phalloidin, red. AP amphibian papilla; BP basilar papilla. All scale bars equal to 100 µm.
	Fig. 5. Characterization of the auditory hair cells in AP-BP.a UMAP plots showing the hair cells (painted in blue) in Xenopus AP-BP. b Sub-clustering of four hair cell subtypes in AP-BP hair cells. c Specific differential expressed gene markers for each hair cell subtype, including genes ptprz1.S, myo1h.L, loxhd1.L, gata2.S, and abi3bp.L. d The painting of different hair cell marker genes in (c) for each subtype in the UMAP plots. (e) and (f) RNA in situ detection and confirmation of loxhd1 and myo1h gene expression in Xenopus, respectively. Certain epithelia regions with weak or no signals are indicated by *. The RNA signals are labeled in red and DAPI in blue. AP amphibian papilla; BP basilar papilla. All scale bar equals to 100 µm.
	Fig. 6. Cross-species comparative analyses of auditory hair cells confirmed that the OHCs are a recently evolved hair cell subtype existing exclusively in mammals.a UMAP plots of mouse hair cells clearly identifying two hair cell subtypes, OHC and IHC. The expression patterns of distinct hair cell markers in UMAP plots, including IHC marker genes (Tbx2 and Slc17a8) and OHC markers (Slc26a5 and Ikzf2). See also Figures S6A, hereafter the same. b UMAP plots for the four hair cell subtypes of AP-BP. The painting of marker genes tbx2 (tbx2.L+tbx2.S, hereafter the same), slc26a5, slc17a8, and ikzf2 (See also Supplementary Fig. 6b). c UMAP plots for four hair cell subtypes of chicken (See also Supplementary Fig. 6c). d UMAP showing overlay of all hair cells from Xenopus (AP-BP) and mouse cochlea (See also Supplementary Fig. 6d). According to the mouse OHC and IHC expressing gene markers, mouse OHCs are separated away from Xenopus hair cells and mouse IHCs locate close to Xenopus hair cells. e UMAP showing overlay of inner ear hair cells from mouse cochlea, chicken, and Xenopus (AP-BP) (See also Supplementary Fig. 6e). f The expression of IHC (Tbx2 and Slc17a8) and OHC (Slc26a5 and Ikzf2) marker genes in mouse and Xenopus each inner ear cell types. In contrast to mouse, the OHC marker genes are broadly expressed in several inner ear cell types and not specifically expressed in auditory hair cells in Xenopus. The hair cells are boxed and labeled in red. AP amphibian papilla; BP basilar papilla; IHC inner hair cell; OHC outer hair cell.
	Fig. 7. The evolution of outer hair cell.a Sankey plot showing the conservation and similarity between hair cell subtypes from mouse cochlea/Xenopus (AP-BP) (left), mouse cochlea/chicken (middle), and Xenopus/chicken (right). The labeled numbers indicate the similarity coefficients between each couple of cell types. b Expression patterns of hair cell-specific genes in mouse, Xenopus, and chicken. The top differentially expressed genes between mouse IHC and OHC (IHC/OHC DEGs) were exacted for plotting the expression heatmap across species. Expression levels from low to high are indicated by blue to red color. The top mouse OHC DEGs are in blue font, and the top IHC DEGs are in orange font on the right. Clear differences of these DEGs are observed between mouse IHC and OHC. The expression feature of hair cell cluster 0 and 1 in Xenopus show certain similarity with mouse IHC. c Genomic collinearity and genome browser visualization of H3K27ac ChIP-seq data of Slc26a5 gene in Xenopus and mouse. The enrichment of H3K27ac indicates the transcriptional activities and regulatory program in Slc26a5 gene locus in two species. The dashed lines between two species indicate their perfect genome collinearity. Relatively stronger enrichment of H3K27ac is observed in Slc26a5 gene locus in Xenopus.