Gene expression analysis

Despite advances in genetic analysis, there remains a significant delay in the elucidation of the pathophysiology of the auditory and vestibular systems as the inner ear is surrounded by bone, making it impossible to perform biopsies without causing irreversible deafness or valance defects. To overcome this difficulty, we are now performing gene expression analysis to reveal the gene expression profile of the mouse and human inner ear. As part of this study, we are using RNA-Seq analysis (short-read and long-read sequencing) to reveal spatio-temporal gene expression dynamics.

Gene expression analysis of the mouse inner ear

Tonotopy is one of the most fundamental principles in auditory function. While gradients in various morphological and physiological characteristics of the cochlea have been reported, little information is available on the gradient patterns of gene expression. In addition, while audiograms in autosomal dominant non-syndromic hearing loss can be distinctive, the underlying mechanism has not been clarified. To analyze the mechanism underlying the distinct audiograms for different genetic causes, we examined and compared gradient gene expression profiles, between the apical, middle, and basal turns of the cochlea using microarray technology. As a result,, we identified several genetic causes of deafness that gradually increase gene expression from the basal to apical turn of the cochlea (Yoshimura et al., PLoS One 2014. Figure 1)

Figure 1. Gene expression patterns found by microarray analysis and quantitative RT-PCR. Values of each gene expression are indicated as a relative value to the basal turn. The expression level of each gene measured by microarray analysis (solid lines) was comparable with the level measured by quantitative RT-PCR (dotted lines).

Laser-capture micro dissection and gene expression analysis of the mouse inner ear

To elucidate precise gene expression levels in each part of the cochlea, we performed laser-capture micro dissection in combination with next-generation sequencing analysis and determined the expression levels of all known deafness-associated genes in the organ of Corti, spiral ganglion, lateral wall, and spiral limbs (Nishio et al., Hear Res. 2017 Figure 2). The results were generally consistent with previous reports based on immuno-cytochemistry or in situ hybridization. As a notable result, the genes associated with many kinds of syndromic hearing loss (such as Clpp, Hars2, Hsd17b4, Lars2 for Perrault syndrome, Polr1c and Polr1d for Treacher-Collins syndrome, Ndp for Norrie Disease, Kal for Kallmann syndrome, Edn3 and Snai2 for Waardenburg Syndrome, Col4a3 for Alport syndrome, Sema3e for CHARGE syndrome, Col9a1 for Sticker syndrome, Cdh23, Cib2, Clrn1, Pcdh15, Ush1c, Ush2a, Whrn for Usher syndrome and Wfs1 for Wolfram syndrome) showed higher levels of expression in the spiral ganglion than in the other parts of the cochlea (Nishio et al., Hear Res. 2017 Figure 3).

Figure 2. Microscopic images of the laser-capture micro dissections of the mouse cochlea. A, D: Microscopic view before laser cutting of the organ of Corti, B, E: Microscopic view after laser cutting of the organ of Corti (including the basement membranes), C, F: Microscopic view of the post-laser captured organ of Corti.

Figure 3. Gene expression patterns obtained by LMD-NGS analysis for the organ of Corti, lateral wall, spiral limbus and spiral ganglion. Expression values of each gene were indicated as relative read numbers calculated from the total reads taken as 1M reads. The averaged gene expression level of the organ of Corti, lateral wall, spiral limbus, and spiral ganglion are shown as solid red lines.