Ontogeny of cellular organization and LGR5 expression in porcine cochlea revealed using tissue clearing and 3D imaging

Abstract

Over 11% of the world's population experience hearing loss. Although there are promising studies to restore hearing in rodent models, the size, ontogeny, genetics, and frequency range of hearing of most rodents' cochlea do not match that of humans. The porcine cochlea can bridge this gap as it shares many anatomical, physiological, and genetic similarities with its human counterpart. Here, we provide a detailed methodology to process and image the porcine cochlea in 3D using tissue clearing and light-sheet microscopy. The resulting 3D images can be employed to compare cochleae across different ages and conditions, investigate the ontogeny of cochlear cytoarchitecture, and produce quantitative expression maps of LGR5, a marker of cochlear progenitors in mice. These data reveal that hair cell organization, inner ear morphology, cellular cartography in the organ of Corti, and spatiotemporal expression of LGR5 are dynamic over developmental stages in a pattern not previously documented.

Keywords: Biological sciences research methodologies; Cell biology; Imaging anatomy; Optical imaging.

Graphical abstract

Figure 1

Clearing, imaging, and analyses of porcine cochleae (A) A block diagram outlining the key steps of sample preparation, imaging, and computational pipeline that uses 3D visualization and analysis. The schematic illustration depicts the porcine cochlea and organ of Corti; tunnel of Corti: TC, organ of Corti: OC, inner hair cell: IHC, outer hair cell: OHC, inner border cell: IBC, inner phalangeal cell: IPHC, pillar cells: PC, Deiters cell: DC, and Hensen cell: HS. (B) A cochlea of a newborn pig before and after tissue clearing. Scale bar, 0.5 cm. (C) Maximum intensity projection (MIP) image with radial view orientation of a transgenic porcine cochlea (P120; LGR5-H2B-GFP) before and after noise reduction. Scale bar, 1000 μm. (D) The zoom-in digital section where the white boxed region in (C) resides (5 μm thick). The sample was stained for MYO7a (hair cells; magenta) and GFP (LGR5⁺ cells; green). Scale bar, 50 μm. (E) An overlay 3D frequency-cellular map. Overlaying the 3D frequency map with cellular coordinates allows comparison between different samples and ages. Inner and outer LGR5⁺ cells relate to cells on either the inner or outer side of the tunnel of Corti, respectively.

Figure 2

Developmental stages of the porcine cochlea In all the images the autofluorescence revealed the overall cochlear morphology, while the specific markers provided information about the cellular organization. (A) A radial view of the porcine cochlea duct in a day 38 embryo (LGR5-GFP staining). At this stage, the Otic Capsule has formed, and the 3 turns of the cochlea are identifiable. Scale bar, 500 μm. (B) On day 53 of the embryo (MYO7a staining), the cochlear duct lengthened. The formation of the scala vestibuli and scala tympani was incomplete. Scale bar, 500 μm; in zoom-in images, 100 μm. The sensory epithelium (SE) at this age is immature (See Figure S1). The Reissner’s membrane (RM) in the basal turn began to form. (C) On day 80 of the embryo (MYO7a staining), the cochlear duct is well developed, with the arrows pointing towards a developed scala vestibuli (SV), scala tympani (ST), and scala media (SM). The tunnel of Corti (TC) and Stria Vascularis (StV) formation is relatively mature. Scale bar, 500 μm; in zoom-in images, 100 μm. (D–F) On postnatal day 0, postnatal day 60, and postnatal day 120, the cochlea (MYO7a staining) is morphologically mature. We also observed that the height of the Tunnel of Corti increases in a basal-to-apex gradient (MYO7a) (See Figure S1). Scale bar, 500 μm; in zoom-in images, 100 μm. (G) The equivalent gestation period of the pig in comparison to the human, marmoset, and mouse is presented (Basch et al., 2016; Hosoya et al., 2021; Igarashi and Ishii, 1980; Kim et al., 2011; Locher et al., 2013; Cantos et al., 2000; Roccio and Edge, 2019). The yellow arrow denotes equivalent postnatal development in mice. Scale bar, 500 μm.

Figure 3

The organ of Corti morphology is frequency specific (A) The basilar membrane lengths of E80 (N = 4), P0 (N = 15), P60 (N = 4), and P120 (N = 11) cochleae (mean ± SD) were measured using IMARIS for both the inner and outer hair cells (IHC, OHC) spiral trajectory. (B) The basilar membrane length across different species is presented (West, 1985; Fay, 2012; Békésy et al., 1990; Greenwood, 1990; Heffner and Heffner, 1985, 1990, 1991; Heffner and Masterton, 1980; Heffner et al., 1971, 1971; Lovell and Harper, 2007; Manoussaki et al., 2008; Ryan, 1976b; Johnson et al., 2012); the pigs spiral length is the same as humans. (C) The inner and outer hair cells numbers were counted for P0 (N = 12) cochleae (mean ± SD), and their numbers are very similar to humans. (D) A comparison of hair cell organization as a function of age and estimated auditory frequency is illustrated at (1) 40 Hz, (2) 100 Hz, (3) 700 Hz, (4) 2 kHz, (5) 10 kHz, and (6) 20 kHz related to 0, 0.03, 0.2, 0.37, 0.68, and 0.82 location along the spiral, respectively, if the apex is 0 and the base is 1. At low frequencies (e.g., 40 and 100 Hz) the hair cells are disorganized compared to higher frequencies. Scale bar, 50 μm (See Figure S2 and Videos S1, S2, and S3).

Figure 4

Hair cell length measurement in 3D images (A) Digital slices (2 μm thickness) of hair cells (MYO7a staining) at 100 Hz, 2,000 Hz, and 10,000 Hz. The outer hair cells exhibit variable lengths. The images are from P0 porcine cochlea. Scale bar, 10 μm. (B) The measurement of hair cell length using 3D images. The inner and outer hair cells’ lengths are shown as mean ± SD (number of animals, N = 3, P60, and P120). Using 3D images allows us to capture the extent of the outer hair cells across multiple slices leading to less variability. (Video S4 shows how we measured the length of each hair cell across multiple z-planes. See Figure S2).

Figure 5

The organ of Corti cartography and the relative position of the supporting cells with respect to the inner and outer hair cells (A) Anti-MYO7a antibody (hair cells) and ToPro3 (nuclear stain) are used to establish the supporting cell organization in the Organ of Corti. Three representative areas in the P0 cochlea are shown at the apex (100 Hz), middle (2,000 Hz), and base (10,000 Hz). The tunnel of Corti is used to learn the position of the pillar cells. Scale bar, 30 μm. (B) Based on multiple porcine cochleae and the representative figures presented in (A), schematic models of cell organization in the organ of Corti are constructed for the apical, middle, and basal turns (See Figures S3–S5). The inner border cells (IBC), inner phalangeal cells (IPHC), inner and outer pillar cells (PCs), 1^st, 2^nd, and 3^rd Deiters’ cells (DCs), and Hensen cells (HS).

Figure 6

LGR5 expression in supporting cells has a strong dependency on cell location in the organ of Corti (A) Digital sections of porcine cochlea stained for MYO7a and GFP at P120 (LGR5-H2B-GFP). The supporting cells were assigned to the following groups: GER/ISC, IBC, IPHC, IPC, 1^st and 2^nd DC (DC12), 3^rd DC (DC3), and HS. Scale bar, 20 μm. (B) The quantification of LGR5 expression at different frequencies and in different supporting cells (number of animals, N = 4; P120). Each point in the graph represents the average relative intensity (LGR5/background) of ten individual cells for each cell type per sample and the standard deviation of all four averaged points in all four samples (mean ± SD) (See Figure S3). The cells’ relative intensity expression was measured at the apex (40 Hz and 100 Hz), middle (700 Hz and 2 kHz), and base (10 kHz and 20kHz). The mixed-effect model has been used to find significant differences between the apical and basal LGR5 expression for individual cell types. The stars indicate the level of significance. If a p-value is less than 0.05, it is represented with one star (∗), a p-value less than 0.01 is marked with two stars (∗∗), and less than 0.001 with three stars (∗∗∗). If the p-value is above 0.05, it is shown as ns. Only in the base, the DC12 group might include HCs (See Figures S4–S6 and Table. S1).

Figure 7

LGR5 expression in supporting cells changes with age (A) Digital sections of the porcine cochlea were stained for MYO7a (Hair cells) and GFP (LGR5) at the apex, middle, and base of the cochlea. The images were extracted from LGR5-H2B-GFP transgenic pigs (E80, P0, P60, and P120). Scale bar, 30 μm. (B) The quantification of LGR5 expression across ages at the apex (40 and 100 Hz), middle (700 and 2000 Hz), and base (10 and 20 kHz) of the cochlea. We have reported the log of the average LGR5 intensity of each cell type across four samples/cochleae at each age and provided the individual frequencies. For this measurement in each cochlea, ten cells from each supporting cell subset were selected and quantified per cochlea (number of cochleae, N = 4 for each age, and a total of 16 cochleae). Please note, that the GER population only exists at the E80 stage (See Figure S6 and Table. S2).