Transcriptomes of cochlear inner and outer hair cells from adult mice

Abstract

Inner hair cells (IHCs) and outer hair cells (OHCs) are the two anatomically and functionally distinct types of mechanosensitive receptor cells in the mammalian cochlea. The molecular mechanisms defining their morphological and functional specializations are largely unclear. As a first step to uncover the underlying mechanisms, we examined the transcriptomes of IHCs and OHCs isolated from adult CBA/J mouse cochleae. One thousand IHCs and OHCs were separately collected using the suction pipette technique. RNA sequencing of IHCs and OHCs was performed and their transcriptomes were analyzed. The results were validated by comparing some IHC and OHC preferentially expressed genes between present study and published microarray-based data as well as by real-time qPCR. Antibody-based immunocytochemistry was used to validate preferential expression of SLC7A14 and DNM3 in IHCs and OHCs. These data are expected to serve as a highly valuable resource for unraveling the molecular mechanisms underlying different biological properties of IHCs and OHCs as well as to provide a road map for future characterization of genes expressed in IHCs and OHCs.

Figure 1. Study design workflow for RNA-seq and suction pipette technique for collecting isolated hair cells.

(a) Workflow of experimental design for RNA-seq transcriptomic analysis of IHCs and OHCs isolated from adult mouse cochleae. (b) Representative images (from left to right) of two isolated IHCs and two OHCs from adult mice. (c) A pick-up pipette before and after an isolated OHC was drawn into the pipette. This technique was used to individually collect isolated hair cells. Bars: 5 (b) and 10 (c) μm.

Figure 2. Reproducibility of biological replicates and RT qPCR validation of differential expression of 27 genes in IHCs and OHCs.

(a) Correlation coefficient between biological replicates from OHCs. Correlation coefficient (r) for each comparison is given in each panel. (b) Correlation coefficient between technical repeats of RNA-seq from OHC samples. (c) PCA analysis of the gene expression profiles of IHCs and OHCs compared with liver cells. Filled circles with different colors represent different biological and technical repeats for IHCs and OHCs. (d) Validation of differential expression of 26 genes between IHCs and OHCs using RT qPCR and RNA-seq. Positive values indicate higher expression of the genes in IHCs than in OHCs, while negative values indicate higher expression the genes in OHCs than in IHCs. Fold differences, based from RPKM values from RNA-seq, are all calculated in log2 base. ∆∆Ct for each gene was calculated from RT qPCR.

Figure 3. Differential expression of SLC7A14 and DNM3 in IHCs and OHCs by immunocytochemistry and confocal microscopy.

(a) Expression of SLC7A14 in IHCs in a whole mount preparation from an adult mouse cochlea. Stereocilia bundles were labelled with rhodamine-phalloidin while SLC7A14 was labelled with anti-SLC7A14 antibody. Bar: 20 μm. (b) Confocal optical sectioning from the whole mount preparation in (a). Expression of SLC7A14 (in red) is only detected in IHCs. Bar: 10 μm. (c) Top view of stereocilia bundles of hair cells. Rhodamine-phalloidin (in green) was used to label actin. Bar: 5 μm (for c, d and e). (d) Expression of DNM3 (in red) in the same view as in c. (e) A merged image of c and d. (f): Optical sectioning image of OHC stereocilia bundles under high magnification. (g) Optical sectioning image of IHC stereocilia bundles. (h) Image of vestibular hair cells. In f, g and h, antibodies against phalloidin and DNM3 were used. Expression of DNM3 was only seen in the stereocilia bundle of OHCs. Bar: 5 μm for f and g and h.

Figure 4. Comparison of differentially expressed genes in IHCs and OHCs quantified by microarray and RNA-seq techniques.

The microarray data are based on genes presented in Fig. 6 of Liu et al. (a) Log2 fold difference between IHCs and OHCs (IHCs/OHCs). (b) Log2 fold difference between OHCs and IHCs (OHCs/IHCs).