Differential Gene Expression Patterns Between Apical And Basal Inner Hair Cells Revealed By RNA-seq

1 ObjectiveInner hair cell（IHC）are the main sensory receptors in the mammalian cochlea and can transduce mechanical stimuli into electrical activity.In lower vertebrates,such as the turtle and bullfrog,electrical tuning displayed the hair cells frequency selectivity in synaptic exocytosis.Whether mammalian inner ear hair cells displayed similar function characteristics of intrinsic tuning is still a question worthy of discussion and research.Recent studies have shown tonotopic differences in IHC along the longitudinal cochlear axis in the synapse number and structure,membrane properties,biophysical properties of Ca²⁺channels,Ca²⁺dependence of exocytosis,and vesicle pool replenishment.Meanwhile,frequency selective hearing loss is a common phenomenon of sensorineural hearing loss caused by age,noise,ototoxic drugs,genetic defects.One of the main reasons is IHC damage,indicating a different survival ability of IHC along the cochlea.These results indicate that the cochlear IHC are also intrinsically tuned and the study on the differences of IHC gene expression patterns in different parts of the cochlea is an important basis for exploring the characteristics and mechanisms of IHC intrinsic tuning.However,IHC have not been distinguished accurately along the apex to the base axis of the cochlea to analyze the gene expression patterns.Herein,based on specific morphological features,we used pulled glass micropipettes to isolate solitary IHC from the apical and basal turns separately for RNA-seq and analyzing the gene expression patterns.Then,we validated these results by immunostaining.Our data might serve as a valuable resource for exploring the molecular mechanisms underlying different biological properties as well as the survival regulation of IHC,which is of great significance to understand the intrinsic tuning of IHC and its role in auditory signal transmission and frequency analysis.2 Methods2.1 Dissection and Isolation of apical and basal IHC from C57BL/6J miceFollowing basilar membrane dissection,collagenase digestion and mechanical isolation,apical and basal IHCs were captured by micromanipulation method.2.2 Transcriptome sequencing and data analysisReverse transcription and amplification were performed using the Smart Seq2 protocol,following IHC’s isolation.After accomplishing RNA quality control,the libraries were multiplexed and sequenced on an BGISEQ-500 platform.HISAT2 was used to transcript abundance quantification analysis and DESeq R package was used to differential expression analysis of the two groups.Then,we analyzed the expression of genes related to IHC machinery,deafness,Ca²⁺binding proteins（CaBPs）,and cell survival.2.3 Validation of differentially expressed genes by ImmunostainingWe used immunofluorescence and immunohistochemistry to detect the expression of parvalbumin-α（PVα）,oncomodulin（OM）,γ-Synuclein,and Fbx32 both in C57BL/6J mice and BALBc mice.3 Results3.1 40 IHCs from apical and basal turns were collected separately and each group prepared three replications.We found that cause of the complex structure of the cochlea,gossamer-thin membranous labyrinth,and scarcity of IHC,isolating the cells is relatively challenging and inefficient.3.2 13,908 and 12,915 transcripts were expressed in apical and basal IHC,respectively,and only 689 significant differentially expressed genes have been found.Interestingly,644/689（93.4%）differentially expressed genes（DEGs）were upregulated in the apical IHC.No differentially expressed deafness-related genes were found,a gradient of gene expression was indeed detected in Pvalb,Prkd1,Fbxo32 and Sncg,which may play putative roles in the Ca²⁺buffering and survival regulation.Both Pvalb and Fbxo32 showed a higher expression in basal IHC than apical IHC,while Prkd1 and Sncg expressed higher in apical IHC than in basal IHC.3.3 In C57BL/6J mice,labeling of PVαand Fbx32 displays a longitudinal gradient,with intense staining in basal IHC and weak in apical IHCs.γ-Synuclein displays selectively express deletion initially observed in basal IHC.These immunostaining results are consistent with RNA-seq data.However,immunofluorescence showed that OM was highly expressed in OHC but not in IHC.The results of immunohistochemistry were consistent with Immunofluorescence.Both PVαand Fbx32 showed a higher expression in basal IHC than apical IHC.However,we haven’t found any difference ofγ-Synuclein between two regions of IHC.In BALBc mice,the labeling of PVαand Fbx32 were intensely in basal IHC and weakly in apical IHC,which were consistent with those in C57BL/6J mice.However,no selectively express deletion was observed in basal IHCs ofγ-Synuclein.4 Conclusion4.1 This study established a stable method of apical and basal IHC isolation and collection from mice cochlea.Cell debris or impurities pollution and interference of the samples can be reduced by two-step collection.4.2 This study demonstrated the different gene expression profiles of IHC from different mouse cochlear turns.4.3 This study found the differential expression patterns of CaBPs,PVα,which was highly expressed in basal IHC.4.4 This study found the differential expression patterns of genes related to cell survival.Fbxo32,the negative regulatory gene,showed a higher expression in basal IHC than apical IHC,while Sncg,the protective gene,expressed higher in apical IHC than in basal IHC.4.5 This study provided the basic data of IHC gene expression along the longitudinal cochlear axis of C57BL/6J mice,which might serve as a valuable resource for exploring the molecular mechanisms underlying different biological properties as well as the survival regulation of IHC.