Fig 2: Single-cell RNA-Seq of cochlear cells reveals broad domains and identifies novel markers of auditory hair cells.(a) Heatmap representing the k-means clustering of 91 P1 cochlear epithelial cells (x axis) using the top 260 genes (y axis) identified by PCA. Gap statistics identified four cell groups, which based on known marker genes and recorded log2-transformed normalized fluorescent intensities (bottom, x-axis bars), were classified into known cell types (top, x-axis bars): NSC (grey), SC (green) and HC (red). Genes were separated into 10 k-means groups (left, y-axis bars) as specified by gap statistics, and were grouped and colour-coded by their associated enrichment with specific cell groups. Two separate k-group clusters (NSC.i–ii) were identified by less unique expression of gene clusters, including genes known to be differentially expressed in the medial and lateral non-sensory domains of the cochlea. Some cells within the NSC groups had high GFP expression, suggesting they are either from a subdomain of the organ of Corti, or non-sensory cells on the border (see Supplementary Fig. 2). (b) PCA plot of the samples and genes used in a across the first two principle components, depicted with the same cell group colours. (c) Violin plots summarizing the distribution of expression of the housekeeping gene Actb, the HC-specific genes Pou4f3, Myo7a, Pcp4 and two novel genes identified and shown to be specifically expressed in HCs, Rasd2 and Anxa4. (d–k) Antibody labelling of HC-specific proteins. Rasd2, localized to the base of stereocilia bundles and cuticular plates, as seen in axial (d), tranverse (e) and wholemount (f,g) views. Anxa4 (h,i) and Pcp4 (j,k) localized to HC membranes and soma, respectively. Scale bars, 10 μm (d–f,h–k); 3 μm (g).

Fig 3: Immunolocalization of HC type specific markers at different developmental time points. Surface and orthogonal views of the indicated markers (green) and Myo7a (magenta) at the indicated time points. At P0, Calb2 and Anxa4 are broadly expressed in nearly all HCs. In contrast, Mapt is rarely expressed in HCs while Spp1 is expressed in only a limited number. The orthogonal view indicates that Spp1 is already restricted to the neck regions of HCs. At P12, Calb2 and Anxa4 are expressed in fewer HCs while expression of Mapt and Spp1 has increased. Finally, at P64, expression of Calb2, Anxa4 and Mapt is largely restricted to Type II HCs while Spp1 is restricted to Type I HCs. Scale bar: 10 µm.

Fig 4: Cochlear hair cells and auditory function are unaffected in Anxa4–/– mice. (A) Cochlear hair cells of P7 Atoh1-GFP or Atoh1-GFP; Anxa4–/– mice were stained with anti-ANXA4 antibody (R&D Systems) and imaged using confocal microscope. Hair cells were labeled by nuclear GFP. (B) Cochlear hair cells of P60 wild type or Anxa4–/– mice were stained with anti-MYO7A antibody and imaged using confocal microscope. Shown are images taken from the middle turn. (C) Numbers of MYO7A-positive hair cells from different genotypes as indicated were calculated according to the results from (B). (D) Raw traces of ABR responses to click stimuli in 2-month-old wild type or Anxa4–/– mice. (E) The ABR thresholds for pure tone and click stimuli in 2-month-old wild type or Anxa4–/– mice. (F) The ABR thresholds for pure tone and click stimuli in 4–5-month-old wild type or Anxa4–/– mice. (G) Two-month-old wild type or Anxa4–/– mice were subjected to 2–20 kHz noise at 110 dB DSL for 2 hours, and ABR thresholds were measured pre-exposure and at different post-exposure time points as indicated. The numbers of animals used in each experiments are indicated in brackets. Scale bars, 20 µm in (A,B). ns, not significant.

Fig 5: Vestibular function is unaffected in Anxa4–/– mice. The vestibular function of 2-month-old Anxa4–/– and wild type mice (n = 4) was evaluated by performing (A) head bobbing, (B) retropulsion, (C) circling stereotyped movement, (D) swimming, (E) tail hanging reflex, and (F) rotarod test. Five-month-old Cdh23 mutant mice (V2J) (n = 3) were included as positive control. ns, not significant; ***P < 0.001.