Project description:A major cause of human deafness and vestibular dysfunction is permanent loss of the mechanosensory hair cells of the inner ear. In non-mammalian vertebrates such as zebrafish, regeneration of missing hair cells can occur throughout life. While a comparative approach has the potential to reveal the basis of such differential regenerative ability, the degree to which the inner ears of fish and mammals share common hair and supporting cell types remains unresolved. Here we perform single-cell RNA sequencing of the zebrafish inner ear at embryonic through adult stages to catalog the diversity of hair and non-sensory supporting cells. We identify a putative progenitor population for hair and supporting cells, as well as distinct hair and supporting cell types in the maculae versus cristae. The hair and supporting cell types differ from those described for the lateral line, a distributed mechanosensory organ in zebrafish in which most studies of hair cell regeneration have been conducted. In the maculae, we identify two subtypes of hair cells that share gene expression with mammalian striolar or extrastriolar hair cells. In situ hybridization reveals that these hair cell subtypes occupy distinct spatial domains within the two major macular organs, the utricle and saccule, consistent with the reported distinct electrophysiological properties of hair cells within these domains. These findings suggest that primitive specialization of spatially distinct striolar and extrastriolar hair cells likely arose in the last common ancestor of fish and mammals. The similarities of inner ear cell type composition between fish and mammals also support using zebrafish as a relevant model for understanding inner ear-specific hair cell function and regeneration.
Project description:To understand the basic biological property of hair cells (HCs) from lower vertebrates, we examined transcriptomes of adult zebrafish HCs. GFP-labeled HCs were isolated from the utricle, saccule, and lagena, the three inner-ear sensory epithelia of a pou4f3 promoter-driven GAP-GFP line of transgenic zebrafish. 2,000 HCs and 2,000 non-sensory cells from the inner ear were individually collected by suction pipet technique. RNA sequencing was performed and the resulting sequences were mapped, analyzed, and compared. Comparisons allow us to identify enriched genes in HCs, which may underlie HC specialization.
Project description:To understand the basic biological properties of inner ear hair cells (HCs) from non-mammalian vertebrates, we examined the transcriptome of adult zebrafish auditory and vestibular HCs. GFP-labeled HCs were isolated from inner-ear sensory epithelia of a pou4f3 promoter-driven GAP-GFP line of transgenic zebrafish. One thousand HCs and 1,000 non-sensory surrounding cells (nsSCs) were separately collected for each biological replicate, using the suction pipette technique. RNA sequencing of three biological replicates for the two cell types was performed. The resulting sequenced reads were mapped. Comparisons between HCs and nsSCs allow identification of enriched genes in HCs, which may underlie HC specialization. Our dataset provides an extensive resource for understanding the molecular mechanisms underlying morphology, function, and pathology of adult zebrafish HCs. It also establishes a framework for future characterization of the genes expressed in HCs and for the study of HC evolution.
Project description:We have developed and tested the efficiency of the Tg(myo6b:GFP-2A-rpl10a-3xHA) zebrafish to specifically enrich for and evaluate the translatome of inner ear and lateral line sensory hair cells (IP) compared to the whole fish transcriptome (IN). We show through RNA-seq that HA-tagged ribosome immunoprecipitation significantly enriches for RNA transcripts of known zebrafish hair cell expressed transcripts.
Project description:Retinoblastoma gene (Rb1) is required for proper cell cycle exit in the developing mouse inner ear and its deletion in the embryo leads to proliferation of sensory progenitor cells that differentiate into hair cells and supporting cells. In the Pou4f3-Cre:Rb1 flox/flox (Rb1 cKO) inner ear, utricular hair cells differentiate and survive into adulthood whereas differentiation and survival of cochlear hair cells are impaired. To comprehensively survey the pRb pathway in the mammalian inner ear, we performed microarray analysis of Rb1 cKO cochlea and utricle. P6 or 2-month control and Rb1 cKO littermates were euthanized and the inner ear tissues were dissected. Total RNA was extracted from the pooled samples. Technical duplicates of the pooled RNA were used for microarray.
Project description:In adult mammals, hair cell loss is irreversible and may result in hearing and balance deficits. In contrast, birds can regenerate hair cells through differentiation of supporting cells and restore inner ear function, suggesting that hair cell progenitors are present in the population of supporting cells. We used microarrays to identify novel genes related to the regeneration in the chicken utricle. Supporting cell and hair cell populations of chicken utricle obtained by laser capture microdissection, following to do RNA extraction and hybridization on Affymetrix microarrays.
Project description:Retinoblastoma gene (Rb1) is required for proper cell cycle exit in the developing mouse inner ear and its deletion in the embryo leads to proliferation of sensory progenitor cells that differentiate into hair cells and supporting cells. In the Pou4f3-Cre:Rb1 flox/flox (Rb1 cKO) inner ear, utricular hair cells differentiate and survive into adulthood whereas differentiation and survival of cochlear hair cells are impaired. To comprehensively survey the pRb pathway in the mammalian inner ear, we performed microarray analysis of Rb1 cKO cochlea and utricle.