Wang Y et al. (2024), Single-cell atlas comparison across vertebrates...

XB-ART-61143

Commun Biol 2024 Dec 19;71:1648. doi: 10.1038/s42003-024-07335-7.

Show Gene links Show Anatomy links

Single-cell atlas comparison across vertebrates reveals auditory cell evolution and mechanisms for hair cell regeneration.

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 .

???displayArticle.abstract???
Mammals suffer permanent hearing impairment from the loss of auditory hair cells due to their inability to regenerate. In contrast, lower vertebrates exhibit extraordinary capacity for hair cell regeneration and hearing restoration, but the mechanisms remain unclear. Here we characterize the single-cell atlas of Xenopus laevis inner ear and perform a comprehensive comparison with mouse model. An exceptionally conserved inner ear neuronal cell type is discovered. The results reveal that the outer hair cells (OHCs) exist exclusively in mammals. Importantly, our analyses reveal an orchestrated gene expression program in Xenopus, characterized by upregulation of hair cell regeneration-related genes, coupled with downregulation of proliferation inhibitory genes. These findings unveil a natural feature of regenerative capacity in Xenopus, and provide molecular and evolutionary evidences for differential regenerative capacities across vertebrates. This work offers insights from amphibians into developing strategies to solve the challenges of hair cell regeneration in humans.

???displayArticle.pubmedLink??? 39702452
???displayArticle.pmcLink??? PMC11659469
???displayArticle.link??? Commun Biol
???displayArticle.grants??? [+]

Species referenced: Xenopus laevis
Genes referenced: abi3bp atoh1 bdnf cacna1b calb2 cdkn1a cdkn1b cemip chga cilp coch dclk3 dnajc2 espn flt3 fxyd2 gata2 grik3 grm8 ikzf2 insm1 kcnk2 kcnq1 kcnq4 lmod2 loxhd1 mpz myo1h myo6 myo7a nefh nefl nefm otof pmp22 pmpcb pou4f1 pou4f3 psmc2 ptn rb1 sh2d4b slc17a8 slc26a5 sox15 srrm3 tbx2 th tjp1 tmc1 ush2a
GO keywords: regeneration

???attribute.lit??? ???displayArticles.show???

	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.
	Supplementary Fig. 1 \| Comparative transcriptome analyses across vertebrates unveiled a natural feature of inner ears from species with high regenerativecapacity. a Venn plots showing the commonly expressed genes betweenchicken/mouse and Xenopus/mouse. Most of the known deafness genes (226) fromthe human deafness gene database were present in the 12605 common expressedgenes between Xenopus and mouse. b Differential expression patterns andhierarchical clustering of common genes among mouse, chicken, and Xenopus. Theheatmap on the right lists the representative genes that are critical for inner ear development or hair cell regeneration, labelled in red. The GO functional analysis indicates a significant enrichment of inner ear development, proliferation andregeneration-related terms for group 1, which exhibits high gene expression levels inXenopus and low levels in mouse and chicken. In contrast, group 4 exhibitedlowexpression levels in Xenopus and chicken but high levels in mouse. Negativeregulation of proliferation, autophagy, and death-related terms were significantlyenriched in this group. MM1/2, GG1/2, and XP1/2 are the two out of the threereplicates from mouse, chicken, and Xenopus, respectively.
	Supplementary Fig. 2 \| The single cell atlas of Xenopus inner ear andits comparison with mouse. Heatmaps in (a) and (b) display the relative expressionlevels of the top three differentially expressed genes (y-axis) from each cluster (x-axis) of mouse and Xenopus. c UMAP plots of Fxyd2 and Kcnk2 genes in mouse single-cell atlas .
	Supplementary Fig. 3-1 \| . The comparison of the inner ear cell atlas across species highlights the neuronal type III cells as one of the most conserved types. aOverlapping of inner ear cell atlas between Xenopus whole inner ear and AP-BPpart, with different samples indicated by colors. A good overlap between themconfirmedtheir same origin and cell type similarity. b UMAP analysis and overlapping of theinner ear cell atlas between Xenopus inner ear and mouse cochlea, with different species indicated by colors. The overlap between these two species revealed their cell type conservation. c The Sankey plot illustrates the degree of conservationor similarity between inner ear cell types in mouse and Xenopus (whole inner ear). Thetop 6 similarity coefficients are labelled. Gray and thin lines indicate very lowor almost no significant similarity between the cell types. d UMAP plots demonstratedthe integration of the Xenopus (AP-BP) and mouse cell atlas. The merged cell atlas was subclustered into 26 cell types. e UMAP analysis and overlapping of inner ear neurons cells between Xenopus and mouse inner ear.
	Supplementary Fig. 3-1 \| . The comparison of the inner ear cell atlas across species highlights the neuronal type III cells as one of the most conserved types. aOverlapping of inner ear cell atlas between Xenopus whole inner ear and AP-BPpart, with different samples indicated by colors. A good overlap between themconfirmedtheir same origin and cell type similarity. b UMAP analysis and overlapping of theinner ear cell atlas between Xenopus inner ear and mouse cochlea, with different species indicated by colors. The overlap between these two species revealed their cell type conservation. c The Sankey plot illustrates the degree of conservationor similarity between inner ear cell types in mouse and Xenopus (whole inner ear). Thetop 6 similarity coefficients are labelled. Gray and thin lines indicate very lowor almost no significant similarity between the cell types. d UMAP plots demonstratedthe integration of the Xenopus (AP-BP) and mouse cell atlas. The merged cell atlas was subclustered into 26 cell types. e UMAP analysis and overlapping of inner ear neurons cells between Xenopus and mouse inner ear.
	Supplementary Fig. 3-2 \| The comparison of the inner ear cell atlas across species highlights the neuronal type III cells as one of the most conserved cell types.Characterization of ganglion neuron subtypes. a Validation of neuronal type III cells by immunostaining with marker Tuj1 and RNA in situ detection with Nefh genes in P8 mouse cochlea, higher magnification of the areas indicated by dashed box. Thedashed cycles indicate the cochlear ganglions. b Validation of neuronal type III cells by immunostaining with marker Tuj1 and RNA in situ detection with Nefmgene inP8mouse cochlea, higher magnification of the areas indicated by dashed box. indicates the Nefm- (or weak) cells in ganglions in the middle, suggesting the existence of other cell types. The Nefm-(or weak)/Tuj1+ neurons (indicated by ) in the right figurerepresent the neuronal cell type with low expression or without expression of Nefm, which is different from the newly identified type III neurons. c RNA in situ detectionof Nefm gene expression in adult mouse inner ear. d Conserved expression of nefhgene expression in Xenopus inner ear ganglion. e RNA in situ detection of cemip geneexpression in Xenopus. The boxes at the bottom left corner demonstrate the sectiondirection, A, apex; B, base. Abbreviation: SGN, spiral ganglion neuron; G, ganglion. All scale bars equal to 100 µm
	Supplementary Fig. 3-2 \| The comparison of the inner ear cell atlas across species highlights the neuronal type III cells as one of the most conserved cell types.Characterization of ganglion neuron subtypes. a Validation of neuronal type III cells by immunostaining with marker Tuj1 and RNA in situ detection with Nefh genes in P8 mouse cochlea, higher magnification of the areas indicated by dashed box. Thedashed cycles indicate the cochlear ganglions. b Validation of neuronal type III cells by immunostaining with marker Tuj1 and RNA in situ detection with Nefmgene inP8mouse cochlea, higher magnification of the areas indicated by dashed box. indicates the Nefm- (or weak) cells in ganglions in the middle, suggesting the existence of other cell types. The Nefm-(or weak)/Tuj1+ neurons (indicated by ) in the right figurerepresent the neuronal cell type with low expression or without expression of Nefm, which is different from the newly identified type III neurons. c RNA in situ detectionof Nefm gene expression in adult mouse inner ear. d Conserved expression of nefhgene expression in Xenopus inner ear ganglion. e RNA in situ detection of cemip geneexpression in Xenopus. The boxes at the bottom left corner demonstrate the sectiondirection, A, apex; B, base. Abbreviation: SGN, spiral ganglion neuron; G, ganglion. All scale bars equal to 100 µm
	Supplementary Fig. 3-3 \| The comparison of the inner ear cell atlas across specieshighlights the neuronal type III cells as one of the most conserved cell types. aThe UMAP painting of differentially expressed marker genes of mouse type I SGN(Ryr2 and Flt3) and type II SGN (Cilp). b RNA in situ detection of Flt3 geneexpression in P8 mouse inner ear. c RNA in situ detection of Cilp gene expressioninmouse inner ear. The boxes at the bottom left corner demonstrate the section direction, A, apex; B, base. d The UMAP painting of differentially expressed genes of mouseSGN type I subtypes (Ia, Ib and Ic), which is exactly consistent with previous studies and validates our snRNA-seq quality. The type Ia markers are shown in the above row; type Ib, middle row; type Ic, bottom row. All scale bars equal to 100 µm
	Supplementary Fig. 3-3 \| The comparison of the inner ear cell atlas across specieshighlights the neuronal type III cells as one of the most conserved cell types. aThe UMAP painting of differentially expressed marker genes of mouse type I SGN(Ryr2 and Flt3) and type II SGN (Cilp). b RNA in situ detection of Flt3 geneexpression in P8 mouse inner ear. c RNA in situ detection of Cilp gene expressioninmouse inner ear. The boxes at the bottom left corner demonstrate the section direction, A, apex; B, base. d The UMAP painting of differentially expressed genes of mouseSGN type I subtypes (Ia, Ib and Ic), which is exactly consistent with previous studies and validates our snRNA-seq quality. The type Ia markers are shown in the above row; type Ib, middle row; type Ic, bottom row. All scale bars equal to 100 µm
	Supplementary Fig. 4 \| Characterization of hair cells in adult Xenopus inner ear(AP-BP) revealed their high conservation in vertebrates. a Specific and conservedexpression of canonical hair cell markers in Xenopus AP-BP. The expressionof loxhd1, otof, lmod2, sh2d4b, and srrm3 genes clearly identified the auditory hair cells. b RNA in situ detection and confirmation of otof gene expression in BP, cristae andsaccule in Xenopus. c Immunostaining with myo7a (green) antibody and RNAinsitudetection of loxhd1 (red) mRNA in Xenopus BP hair cells. Abbreviation: AP, amphibian papilla; BP, basilar papilla. All scale bars equal to 100 µm.
	Supplementary Fig. 4 \| Characterization of hair cells in adult Xenopus inner ear(AP-BP) revealed their high conservation in vertebrates. a Specific and conservedexpression of canonical hair cell markers in Xenopus AP-BP. The expressionof loxhd1, otof, lmod2, sh2d4b, and srrm3 genes clearly identified the auditory hair cells. b RNA in situ detection and confirmation of otof gene expression in BP, cristae andsaccule in Xenopus. c Immunostaining with myo7a (green) antibody and RNAinsitudetection of loxhd1 (red) mRNA in Xenopus BP hair cells. Abbreviation: AP, amphibian papilla; BP, basilar papilla. All scale bars equal to 100 µm.
	Supplementary Fig. 5-1 \| Characterization of hair cell subtypes in the wholeinner ear of Xenopus. a Two cell clusters were identified as hair cells usingcanonical markers (blue and green). b Differential expressed genes for the twohair cell clusters in Xenopus whole inner ear. c Exaction and sub-clustering of Xenopus inner ear hair cell. Five hair cell subtypes were identified. d The top10 differentiallyexpressed genes for each hair cell subtype. e Specific differential expressed genemarkers for each hair cell subtype. f UMAP plots painting of the hair cell marker genes for each subtype in (e).
	Supplementary Fig. 5-2 \| Expression of hair cell marker genes in Xenopus innerear. a RNA in situ detection of hair cell marker genes gata2 (a), ush2a (b), dclk3(c), and calb2 (d) in Xenopus inner ear. These genes specifically expressed in the hair cells of AP (a, b, c and d) and cristae (d, bottom). calb2 gene also shows strong expression in inner ear ganglion neurons (as indicated by the dashed white cycle). *Represents the regions with absence or weak signals. All scale bars equal to 100µm.
	Supplementary Fig. 5-2 \| Expression of hair cell marker genes in Xenopus innerear. a RNA in situ detection of hair cell marker genes gata2 (a), ush2a (b), dclk3(c), and calb2 (d) in Xenopus inner ear. These genes specifically expressed in the hair cells of AP (a, b, c and d) and cristae (d, bottom). calb2 gene also shows strong expression in inner ear ganglion neurons (as indicated by the dashed white cycle). *Represents the regions with absence or weak signals. All scale bars equal to 100µm.
	Supplementary Fig. 6 \| Cross-species comparative analyses of auditory hair cells confirmed that the OHCs are a recently evolved hair cell subtype existingexclusively in mammals. The expression patterns of IHC marker gene (Tbx2) andOHC marker (Ikzf2) in UMAP plots from mouse (a), Xenopus (b), chicken(c), merged hair cells of mouse/Xenopus (d), and merged hair cells of mouse/Xenopus/chicken (e). f UMAP showing overlay of auditory hair cells betweenXenopus and chicken. g The UMAP painting of IHC marker genes (tbx2 and slc17a8) and OHC marker genes (slc26a5 and ikzf2) in the merged hair cells of Xenopus andchicken (f). h UMAP showing overlay of auditory hair cells between mouse andchicken. i UMAP painting of IHC marker genes (Tbx2 and Slc17a8) and OHCmarker genes (Slc26a5 and Ikzf2) in the merged hair cells of mouse and chicken (H).
	Supplementary Fig 7 \| Characterization and evolution of auditory hair cells across vertebrates. a Transcriptomic feature of all auditory hair cell subtypes inmouse, Xenopus and chicken. The top differentially expressed genes between mouse IHC and OHC (IHC/OHC DEGs) were used to plot the expression heatmap across species. The expression levels are indicated by a color gradient fromblue to red, withblue and orange font used to highlight the top mouse OHC and IHC DEGs ontheright. b Expression patterns of IHC/OHC DEGs in Tbx2- or Slc26a5-positive hair cell groups in mouse and Xenopus. c A perfect genomic collinearity of Otof and nearbygene loci and genome browser visualization of H3K27ac ChIP-seq data in Xenopus and mouse inner ear. The enrichment of H3K27ac indicates the transcriptional activities and regulatory program in Otof gene locus in two species. Abbreviation: IHC, inner hair cell; OHC, outer hair cell; DEG, differentially expressed gene; ChIP, chromatin immunoprecipitation sequencing.
	Supplementary Fig 7 \| Characterization and evolution of auditory hair cells across vertebrates. a Transcriptomic feature of all auditory hair cell subtypes inmouse, Xenopus and chicken. The top differentially expressed genes between mouse IHC and OHC (IHC/OHC DEGs) were used to plot the expression heatmap across species. The expression levels are indicated by a color gradient fromblue to red, withblue and orange font used to highlight the top mouse OHC and IHC DEGs ontheright. b Expression patterns of IHC/OHC DEGs in Tbx2- or Slc26a5-positive hair cell groups in mouse and Xenopus. c A perfect genomic collinearity of Otof and nearbygene loci and genome browser visualization of H3K27ac ChIP-seq data in Xenopus and mouse inner ear. The enrichment of H3K27ac indicates the transcriptional activities and regulatory program in Otof gene locus in two species. Abbreviation: IHC, inner hair cell; OHC, outer hair cell; DEG, differentially expressed gene; ChIP, chromatin immunoprecipitation sequencing.

	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.
	Supplementary Fig. 1 \| Comparative transcriptome analyses across vertebrates unveiled a natural feature of inner ears from species with high regenerativecapacity. a Venn plots showing the commonly expressed genes betweenchicken/mouse and Xenopus/mouse. Most of the known deafness genes (226) fromthe human deafness gene database were present in the 12605 common expressedgenes between Xenopus and mouse. b Differential expression patterns andhierarchical clustering of common genes among mouse, chicken, and Xenopus. Theheatmap on the right lists the representative genes that are critical for inner ear development or hair cell regeneration, labelled in red. The GO functional analysis indicates a significant enrichment of inner ear development, proliferation andregeneration-related terms for group 1, which exhibits high gene expression levels inXenopus and low levels in mouse and chicken. In contrast, group 4 exhibitedlowexpression levels in Xenopus and chicken but high levels in mouse. Negativeregulation of proliferation, autophagy, and death-related terms were significantlyenriched in this group. MM1/2, GG1/2, and XP1/2 are the two out of the threereplicates from mouse, chicken, and Xenopus, respectively.
	Supplementary Fig. 2 \| The single cell atlas of Xenopus inner ear andits comparison with mouse. Heatmaps in (a) and (b) display the relative expressionlevels of the top three differentially expressed genes (y-axis) from each cluster (x-axis) of mouse and Xenopus. c UMAP plots of Fxyd2 and Kcnk2 genes in mouse single-cell atlas .
	Supplementary Fig. 3-1 \| . The comparison of the inner ear cell atlas across species highlights the neuronal type III cells as one of the most conserved types. aOverlapping of inner ear cell atlas between Xenopus whole inner ear and AP-BPpart, with different samples indicated by colors. A good overlap between themconfirmedtheir same origin and cell type similarity. b UMAP analysis and overlapping of theinner ear cell atlas between Xenopus inner ear and mouse cochlea, with different species indicated by colors. The overlap between these two species revealed their cell type conservation. c The Sankey plot illustrates the degree of conservationor similarity between inner ear cell types in mouse and Xenopus (whole inner ear). Thetop 6 similarity coefficients are labelled. Gray and thin lines indicate very lowor almost no significant similarity between the cell types. d UMAP plots demonstratedthe integration of the Xenopus (AP-BP) and mouse cell atlas. The merged cell atlas was subclustered into 26 cell types. e UMAP analysis and overlapping of inner ear neurons cells between Xenopus and mouse inner ear.
	Supplementary Fig. 3-1 \| . The comparison of the inner ear cell atlas across species highlights the neuronal type III cells as one of the most conserved types. aOverlapping of inner ear cell atlas between Xenopus whole inner ear and AP-BPpart, with different samples indicated by colors. A good overlap between themconfirmedtheir same origin and cell type similarity. b UMAP analysis and overlapping of theinner ear cell atlas between Xenopus inner ear and mouse cochlea, with different species indicated by colors. The overlap between these two species revealed their cell type conservation. c The Sankey plot illustrates the degree of conservationor similarity between inner ear cell types in mouse and Xenopus (whole inner ear). Thetop 6 similarity coefficients are labelled. Gray and thin lines indicate very lowor almost no significant similarity between the cell types. d UMAP plots demonstratedthe integration of the Xenopus (AP-BP) and mouse cell atlas. The merged cell atlas was subclustered into 26 cell types. e UMAP analysis and overlapping of inner ear neurons cells between Xenopus and mouse inner ear.
	Supplementary Fig. 3-2 \| The comparison of the inner ear cell atlas across species highlights the neuronal type III cells as one of the most conserved cell types.Characterization of ganglion neuron subtypes. a Validation of neuronal type III cells by immunostaining with marker Tuj1 and RNA in situ detection with Nefh genes in P8 mouse cochlea, higher magnification of the areas indicated by dashed box. Thedashed cycles indicate the cochlear ganglions. b Validation of neuronal type III cells by immunostaining with marker Tuj1 and RNA in situ detection with Nefmgene inP8mouse cochlea, higher magnification of the areas indicated by dashed box. indicates the Nefm- (or weak) cells in ganglions in the middle, suggesting the existence of other cell types. The Nefm-(or weak)/Tuj1+ neurons (indicated by ) in the right figurerepresent the neuronal cell type with low expression or without expression of Nefm, which is different from the newly identified type III neurons. c RNA in situ detectionof Nefm gene expression in adult mouse inner ear. d Conserved expression of nefhgene expression in Xenopus inner ear ganglion. e RNA in situ detection of cemip geneexpression in Xenopus. The boxes at the bottom left corner demonstrate the sectiondirection, A, apex; B, base. Abbreviation: SGN, spiral ganglion neuron; G, ganglion. All scale bars equal to 100 µm
	Supplementary Fig. 3-2 \| The comparison of the inner ear cell atlas across species highlights the neuronal type III cells as one of the most conserved cell types.Characterization of ganglion neuron subtypes. a Validation of neuronal type III cells by immunostaining with marker Tuj1 and RNA in situ detection with Nefh genes in P8 mouse cochlea, higher magnification of the areas indicated by dashed box. Thedashed cycles indicate the cochlear ganglions. b Validation of neuronal type III cells by immunostaining with marker Tuj1 and RNA in situ detection with Nefmgene inP8mouse cochlea, higher magnification of the areas indicated by dashed box. indicates the Nefm- (or weak) cells in ganglions in the middle, suggesting the existence of other cell types. The Nefm-(or weak)/Tuj1+ neurons (indicated by ) in the right figurerepresent the neuronal cell type with low expression or without expression of Nefm, which is different from the newly identified type III neurons. c RNA in situ detectionof Nefm gene expression in adult mouse inner ear. d Conserved expression of nefhgene expression in Xenopus inner ear ganglion. e RNA in situ detection of cemip geneexpression in Xenopus. The boxes at the bottom left corner demonstrate the sectiondirection, A, apex; B, base. Abbreviation: SGN, spiral ganglion neuron; G, ganglion. All scale bars equal to 100 µm
	Supplementary Fig. 3-3 \| The comparison of the inner ear cell atlas across specieshighlights the neuronal type III cells as one of the most conserved cell types. aThe UMAP painting of differentially expressed marker genes of mouse type I SGN(Ryr2 and Flt3) and type II SGN (Cilp). b RNA in situ detection of Flt3 geneexpression in P8 mouse inner ear. c RNA in situ detection of Cilp gene expressioninmouse inner ear. The boxes at the bottom left corner demonstrate the section direction, A, apex; B, base. d The UMAP painting of differentially expressed genes of mouseSGN type I subtypes (Ia, Ib and Ic), which is exactly consistent with previous studies and validates our snRNA-seq quality. The type Ia markers are shown in the above row; type Ib, middle row; type Ic, bottom row. All scale bars equal to 100 µm
	Supplementary Fig. 3-3 \| The comparison of the inner ear cell atlas across specieshighlights the neuronal type III cells as one of the most conserved cell types. aThe UMAP painting of differentially expressed marker genes of mouse type I SGN(Ryr2 and Flt3) and type II SGN (Cilp). b RNA in situ detection of Flt3 geneexpression in P8 mouse inner ear. c RNA in situ detection of Cilp gene expressioninmouse inner ear. The boxes at the bottom left corner demonstrate the section direction, A, apex; B, base. d The UMAP painting of differentially expressed genes of mouseSGN type I subtypes (Ia, Ib and Ic), which is exactly consistent with previous studies and validates our snRNA-seq quality. The type Ia markers are shown in the above row; type Ib, middle row; type Ic, bottom row. All scale bars equal to 100 µm
	Supplementary Fig. 4 \| Characterization of hair cells in adult Xenopus inner ear(AP-BP) revealed their high conservation in vertebrates. a Specific and conservedexpression of canonical hair cell markers in Xenopus AP-BP. The expressionof loxhd1, otof, lmod2, sh2d4b, and srrm3 genes clearly identified the auditory hair cells. b RNA in situ detection and confirmation of otof gene expression in BP, cristae andsaccule in Xenopus. c Immunostaining with myo7a (green) antibody and RNAinsitudetection of loxhd1 (red) mRNA in Xenopus BP hair cells. Abbreviation: AP, amphibian papilla; BP, basilar papilla. All scale bars equal to 100 µm.
	Supplementary Fig. 4 \| Characterization of hair cells in adult Xenopus inner ear(AP-BP) revealed their high conservation in vertebrates. a Specific and conservedexpression of canonical hair cell markers in Xenopus AP-BP. The expressionof loxhd1, otof, lmod2, sh2d4b, and srrm3 genes clearly identified the auditory hair cells. b RNA in situ detection and confirmation of otof gene expression in BP, cristae andsaccule in Xenopus. c Immunostaining with myo7a (green) antibody and RNAinsitudetection of loxhd1 (red) mRNA in Xenopus BP hair cells. Abbreviation: AP, amphibian papilla; BP, basilar papilla. All scale bars equal to 100 µm.
	Supplementary Fig. 5-1 \| Characterization of hair cell subtypes in the wholeinner ear of Xenopus. a Two cell clusters were identified as hair cells usingcanonical markers (blue and green). b Differential expressed genes for the twohair cell clusters in Xenopus whole inner ear. c Exaction and sub-clustering of Xenopus inner ear hair cell. Five hair cell subtypes were identified. d The top10 differentiallyexpressed genes for each hair cell subtype. e Specific differential expressed genemarkers for each hair cell subtype. f UMAP plots painting of the hair cell marker genes for each subtype in (e).
	Supplementary Fig. 5-2 \| Expression of hair cell marker genes in Xenopus innerear. a RNA in situ detection of hair cell marker genes gata2 (a), ush2a (b), dclk3(c), and calb2 (d) in Xenopus inner ear. These genes specifically expressed in the hair cells of AP (a, b, c and d) and cristae (d, bottom). calb2 gene also shows strong expression in inner ear ganglion neurons (as indicated by the dashed white cycle). *Represents the regions with absence or weak signals. All scale bars equal to 100µm.
	Supplementary Fig. 5-2 \| Expression of hair cell marker genes in Xenopus innerear. a RNA in situ detection of hair cell marker genes gata2 (a), ush2a (b), dclk3(c), and calb2 (d) in Xenopus inner ear. These genes specifically expressed in the hair cells of AP (a, b, c and d) and cristae (d, bottom). calb2 gene also shows strong expression in inner ear ganglion neurons (as indicated by the dashed white cycle). *Represents the regions with absence or weak signals. All scale bars equal to 100µm.
	Supplementary Fig. 6 \| Cross-species comparative analyses of auditory hair cells confirmed that the OHCs are a recently evolved hair cell subtype existingexclusively in mammals. The expression patterns of IHC marker gene (Tbx2) andOHC marker (Ikzf2) in UMAP plots from mouse (a), Xenopus (b), chicken(c), merged hair cells of mouse/Xenopus (d), and merged hair cells of mouse/Xenopus/chicken (e). f UMAP showing overlay of auditory hair cells betweenXenopus and chicken. g The UMAP painting of IHC marker genes (tbx2 and slc17a8) and OHC marker genes (slc26a5 and ikzf2) in the merged hair cells of Xenopus andchicken (f). h UMAP showing overlay of auditory hair cells between mouse andchicken. i UMAP painting of IHC marker genes (Tbx2 and Slc17a8) and OHCmarker genes (Slc26a5 and Ikzf2) in the merged hair cells of mouse and chicken (H).
	Supplementary Fig 7 \| Characterization and evolution of auditory hair cells across vertebrates. a Transcriptomic feature of all auditory hair cell subtypes inmouse, Xenopus and chicken. The top differentially expressed genes between mouse IHC and OHC (IHC/OHC DEGs) were used to plot the expression heatmap across species. The expression levels are indicated by a color gradient fromblue to red, withblue and orange font used to highlight the top mouse OHC and IHC DEGs ontheright. b Expression patterns of IHC/OHC DEGs in Tbx2- or Slc26a5-positive hair cell groups in mouse and Xenopus. c A perfect genomic collinearity of Otof and nearbygene loci and genome browser visualization of H3K27ac ChIP-seq data in Xenopus and mouse inner ear. The enrichment of H3K27ac indicates the transcriptional activities and regulatory program in Otof gene locus in two species. Abbreviation: IHC, inner hair cell; OHC, outer hair cell; DEG, differentially expressed gene; ChIP, chromatin immunoprecipitation sequencing.
	Supplementary Fig 7 \| Characterization and evolution of auditory hair cells across vertebrates. a Transcriptomic feature of all auditory hair cell subtypes inmouse, Xenopus and chicken. The top differentially expressed genes between mouse IHC and OHC (IHC/OHC DEGs) were used to plot the expression heatmap across species. The expression levels are indicated by a color gradient fromblue to red, withblue and orange font used to highlight the top mouse OHC and IHC DEGs ontheright. b Expression patterns of IHC/OHC DEGs in Tbx2- or Slc26a5-positive hair cell groups in mouse and Xenopus. c A perfect genomic collinearity of Otof and nearbygene loci and genome browser visualization of H3K27ac ChIP-seq data in Xenopus and mouse inner ear. The enrichment of H3K27ac indicates the transcriptional activities and regulatory program in Otof gene locus in two species. Abbreviation: IHC, inner hair cell; OHC, outer hair cell; DEG, differentially expressed gene; ChIP, chromatin immunoprecipitation sequencing.