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: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.
Project description:Capping protein controls stereocilia length and width during hair bundle development. To determine what other proteins are involved in capping protein regulation, we carried out immunoaffinity purifications targeted at either CAPZA or CAPZB2. The starting material for immunopurification was crude stereocilia membranes isolated from mouse inner ear.
Project description:The inner ear sensory epithelia contain mechanosensitive hair cells and supporting cells. Both cell types arise from SOX2-expressing prosensory cells, but the mechanisms underlying the diversification of these cell lineages remain unclear. To determine the transcriptional trajectory of prosensory cells, we established a SOX2-2A-ntdTomato human ES cell line using CRISPR/Cas9, and performed single-cell RNA-sequencing analysis with SOX2-positive cells isolated from inner ear organoids at various time points between differentiation days 20 and 60. Our pseudotime analysis suggests that hair cells arise primarily from supporting cells, rather than bi-fated transitional cells in organoids. Moreover, ion channel- and ion transporter-related gene sets were enriched in supporting cells vs. prosensory cells, whereas Wnt signaling-related gene sets were enriched in hair cells vs. supporting cells. These findings provide valuable insights into how prosensory cells give rise to hair cells and supporting cells during human inner ear development, and may provide a clue to promote hair cell regeneration from resident supporting cells in individuals with hearing loss and balance disorders.
Project description:Hair cells of the inner ear are essential for hearing and balance. As a consequence, pathogenic variants in genes specifically expressed in hair cells often cause hereditary deafness. Hair cells are few in number and not easily isolated from the adjacent supporting cells, so the biochemistry and molecular biology of hair cells can be difficult to study. To study gene expression in hair cells, we developed a protocol for hair cell isolation by FACS sorting. With nearly pure hair cells and surrounding cells, from cochlea and utricle and from embryonic day 16 to postnatal day 7, we performed a comprehensive cell-type-specific RNA-Seq study of gene expression during mouse inner ear development. Expression profiling revealed new hair-cell genes with distinct expression patterns: some are specific for vestibular hair cells, others for cochlear hair cells, and some are expressed just before or after maturation of mechanosensitivity. We found that many of the known hereditary deafness genes are much more highly expressed in hair cells than surrounding cells, suggesting that genes preferentially expressed in hair cells are good candidates for unknown deafness genes.
Project description:Inner ear organoids recapitulate development and are intended to generate cell types of the otic lineage for applications such as basic science research and cell replacement strategies. Here, we use single-cell sequencing to study the cellular heterogeneity of late-stage mouse inner ear organoid sensory epithelia, which we validated by comparison with data sets of the mouse cochlea and vestibular epithelia. We resolved supporting cell sub-types, cochlear like hair cells, and vestibular Type I and Type II like hair cells. While cochlear like hair cells aligned best with an outer hair cell trajectory, vestibular like hair cells followed developmental trajectories similar to in vivo programs branching into Type II and then Type I extrastriolar hair cells. These results highlight the transcriptional accuracy of the organoid developmental program but will also inform future strategies to improve synaptic connectivity and regional specification.
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.
Project description:The inner ear utilizes sensory hair cells as mechano-electric transducers for sensing sound and balance. In mammals, these hair cells lack the capacity for regeneration. Unlike mammals, hair cells from non-mammalian vertebrates, such as birds, can be regenerated throughout the life of the organism making them a useful model for studying inner ear genetics pathways. The zinc finger transcription factor GATA3 is required for inner ear development and mutations cause sensory neural deafness in humans. In the avian cochlea GATA3 is expressed throughout the sensory epithelia; however, expression is limited to the striola of the utricle. The striola corresponds to an abrupt change in morphologically distinct hair cell types and a 180° shift in hair cell orientation. We used 3 complimentary approaches to identify potential downstream targets of GATA3 in the avian utricle. Specifically we used microarray expression profiling of GATA3 knockdown by siRNA and GATA3 over-expression treatments as well as direct comparisons of GATA3 expressing cells from the striola and non GATA3 expressing cells from the extra-striola. To identify genes that are co-expressed with GATA3 at the striola reversal zone, we compared gene expression in cells micro-dissected from the sensory epithelia of the chick utricle striola to cells from the surrounding extra-striola. There are 2 biological samples and experiments include technical replicates as well as dye-switches for a total of 8 microarrays.