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Single-cell RNA-Seq resolves cellular complexity in sensory organs from the neonatal inner ear

ABSTRACT: In the inner ear, cochlear and vestibular sensory epithelia utilize grossly similar cell types to transduce different stimuli: sound and acceleration. Each individual sensory epithelium is composed of highly heterogeneous populations of cells based on physiological and anatomical criteria. However, limited numbers of each cell type have impeded transcriptional characterization. Here we generated transcriptomes for 301 single cells from the utricular and cochlear sensory epithelia of newborn mice to circumvent this challenge. Cluster analysis indicates distinct profiles for each of the major sensory epithelial cell types, as well as less distinct subpopulations. Asynchrony within utricles allows reconstruction of the temporal progression of cell-type specific differentiation and suggests possible plasticity among cells at the sensory-nonsensory boundary. Comparisons of cell types from utricles and cochleae demonstrate divergence between auditory and vestibular cells despite a common origin. These results provide significant insights into the developmental processes that form unique inner ear cell types.

ORGANISM(S): Mus musculus

PROVIDER: GSE71982 | GEO | 2015/10/15

SECONDARY ACCESSION(S): PRJNA292657

REPOSITORIES: GEO

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Project description:We have employed a novel approach for the identification of functionally important microRNA (miRNA)-target interactions using integrated miRNA, transcriptome and proteome profiles with advanced in silico analysis. By looking at both the transcript and protein levels of expression, a thorough coverage of miRNA regulation was obtained. Microdissected auditory and vestibular sensory epithelia were used as the model system, thus being the first time such a comparison was carried out in a neuroepithelial system. Moreover, this is one of only a few studies employing proteome screening for the identification of miRNA targets. Notably, this approach can be employed for the study of other tissues and organs. We detected the expression of 157 miRNAs in the inner ear sensory epithelia, with 53 miRNAs differentially expressed between the cochlea and vestibule. By searching for enrichment and depletion of miRNA targets in the transcript and protein datasets with a reciprocal or similar expression, respectively, as the regulatory miRNA, we identified functionally important miRNAs. Finally, the interaction between miR-135b and PSIP1-P75, a transcriptional coacitvator previously unknown in the inner ear, was identified and validated experimentally. We suggest that miR-135b may serve as a cellular effector, involved in regulating some of the differences between the cochlear and vestibular hair cells. We investigated the mRNA expression profile of the cochlear and vestibular sensory epithelia from inner ears of postnatal day 2 mice using the Affymetrix GeneChip® 430 2Mouse Genome array. Cochlear and vestibular sensory epithelia were dissected from wild type C3H mice and collected separately. The vestibular epithelia consisted of the saccule, utricle and the lateral and anterior cristae. Both the cochlear and vestibular sensory epithelia were dissected with their underlying mesenchyma. Altogether three pools, three biological replicates, of each tissue type were collected consisting of cochlear or vestibular sensory epithelia dissected from 10 to 12 inner ears.

Project description:The inner ear in mammals is derived from a simple ectodermal thickening called the otic placode. Through a series of complex morphological changes, the placode forms the mature inner ear comprising of the auditory organ (cochlea) and the vestibular/balance organs (utricle, saccule, and three semi-circular canals). The vast majority of genes known to be involved during inner ear development have been found through mutational screens or by chance. To identify genes that can serve as novel candidates required for inner ear development, and also candidate genes for uncloned human deafnesses, inner ear tissues from mouse embryos from E9 to E15 were microdissected and expression-profiled at half-day intervals. Also profiled was the non-inner ear mesenchymal tissue surrounding the inner ear tissue. Various patterns of gene expression were identified, and significant biological pathways that these genes represented were identified. Also identified were mouse genes whose human orthologs are located within uncloned non-syndromic deafness intervals, thus serving as candidates for sequence analysis. Experiment Overall Design: Inner ear tissues from E9 to E15 were microdissected at half-day intervals. E9 is the earliest stage when the otic placode is clearly visible and able to be microdissected cleanly. E15 is the stage when all the organs of the inner ear have become established, as have the sensory hair and non-sensory support cells within those organs. For each of the stages from E9 to E10, whole inner ears were profiled. For each of the stages from E10.5 to E12, the primordial cochlear and vestibular organs were profiled separately. For each of the stages from E12.5 to E15, the cochlea and the saccule were profiled separately, whereas the utricle and the three ampullae were combined and profiled together. Any given tissue from any given stage was a collection of anywhere between 4 to 17 identical tissues, and was obtained in duplicate (i.e. from different litters). Hence, a total of 58 inner ear samples were obtained. Moreover, non-inner ear tissue found in the immediate vicinity of inner ear tissue was also obtained and profiled. Specifically, all non-inner ear tissue from E9 was profiled in duplicate. Non-inner ear tissue from E9.5 to E10.5 was pooled and profiled together (in duplicate), whereas that from E11 to E15 was pooled and profiled together (also in duplicate). Therefore, a total of 6 non-inner samples were obtained.

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Single-cell RNA-Seq resolves cellular complexity in sensory organs from the neonatal inner ear

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