Project description:Hearing loss is most commonly caused by the destruction of mechanosensory hair cells in the ear. This condition is usually permanent: Despite the presence of putative hair-cell progenitors in the cochlea, hair cells are not naturally replenished in adult mammals. Unlike those of the mammalian ear, the progenitor cells of nonmammalian vertebrates can regenerate hair cells through- out life. The basis of this difference remains largely unexplored but may lie in molecular dissimilarities that affect how progenitors respond to hair-cell death. We analyzed gene expression in hair-cell progenitors of the lateral-line system. We developed a transgenic line of zebrafish called alpl:mCherry that expresses a red fluorescent protein in the presumptive hair-cell progenitors known as mantle cells. Fluorescence-activated cell sorting from the skins of transgenic larvae, followed by microarray-based expression analysis, revealed a constellation of transcripts that are specifically enriched in these cells versus hair cells and non-fluorescent skin cells. Gene expression analysis after hair-cell ablation uncovered a cohort of genes that are differentially regulated early in regeneration, suggesting possible roles in the response of progen- itors to hair-cell death. These results provide a resource for studying hair-cell regeneration and the biology of sensory progenitor cells.
Project description:Loss of sensory hair cells leads to deafness and balance deficiencies. In contrast to mammalian hair cells, zebrafish ear and lateral line hair cells regenerate from poorly characterized, proliferating support cells. Equally ill-defined is the gene regulatory network underlying the progression of support cells to cycling hair cell progenitors and differentiated hair cells. We used single-cell RNA-Sequencing (scRNA-Seq) of lateral line sensory organs and uncovered five different support cell types, including quiescent and activated stem cells. In silico ordering of support cells along a developmental trajectory identified cells that self-renew and new groups of genes required for hair cell differentiation. scRNA-Seq analyses of fgf3 mutants, in which hair cell regeneration is increased demonstrates that Fgf and Notch signaling inhibit proliferation of support cells in parallel by inhibiting Wnt signaling. Our scRNA-Seq analyses set the foundation for mechanistic studies of sensory organ regeneration and is crucial for identifying factors to trigger hair cell production in mammals.
Project description:Hearing loss is most commonly caused by the destruction of mechanosensory hair cells in the ear. This condition is usually permanent: Despite the presence of putative hair-cell progenitors in the cochlea, hair cells are not naturally replenished in adult mammals. Unlike those of the mammalian ear, the progenitor cells of nonmammalian vertebrates can regenerate hair cells through- out life. The basis of this difference remains largely unexplored but may lie in molecular dissimilarities that affect how progenitors respond to hair-cell death. We analyzed gene expression in hair-cell progenitors of the lateral-line system. We developed a transgenic line of zebrafish called alpl:mCherry that expresses a red fluorescent protein in the presumptive hair-cell progenitors known as mantle cells. Fluorescence-activated cell sorting from the skins of transgenic larvae, followed by microarray-based expression analysis, revealed a constellation of transcripts that are specifically enriched in these cells versus hair cells and non-fluorescent skin cells. Gene expression analysis after hair-cell ablation uncovered a cohort of genes that are differentially regulated early in regeneration, suggesting possible roles in the response of progen- itors to hair-cell death. These results provide a resource for studying hair-cell regeneration and the biology of sensory progenitor cells. Two sets of analyses were performed. The first compared baseline expression levels in four sorted cell types from two different transgenic lines of zebrafish, alpl:mCherry;pou4f3:GFP and alpl:mCherry:ET20. alpl:mCherry;pou4f3:GFP larvae express GFP in hair cells and mCherry in mantle cells. alpl:mCherry:ET20 larvae express GFP in most mantle cells and mChery in all mantle cells. The four cell types compared were GFP+ hair cells, mCherry+ mantle cells, mCherry+/GFP+ mantle cells from alpl:mCherry:ET20 larvae, and non-fluorescent (NF) cells. Two hair-cell, five mCherry+ mantle cell, four mCherry+/GFP+ mantle cell, and six NF cell samples were analyzed. This excludes a few samples that were discardced based on failure to cluster by principal component analysis. A second analysis, separately imported, compared gene expression in mCherry+ mantle cells and NF cells at four time points following chemical ablation of hair cells with copper sulfate. These were one, three, five, and eleven hours after treatment (hpCu). Untreated samples served as controls. For mCherry+ cells, five untreated and four each of 1 hpCu, 3 hpCu, 5 hpCu and 11 hpCu were analyzed. For NF cells, six untreated, seven 1 hpCu, and four each of 3 hpCu, 5 hpCu and 11 hpCu samples were analyzed. Untreated mCherry+ and NF cells were shared between both analyses.
Project description:Deafness due to the terminal loss of inner ear hair cells is one of the most common sensory diseases. However, non-mammalian animals (e.g. birds, amphibian and fish) regenerate damaged hair cells. In order to better understand the reasons underpinning such regeneration disparities in vertebrates, we set out to define the changes in gene expression associated with the regeneration of hair cells in the zebrafish lateral line at high resolution. We performed RNA-Seq analyses on regenerating support cells purified by fluorescence activated cell sorting (FACS). The zebrafish lateral line provides an experimentally accessible system to define the complex signaling events triggered by injury and regeneration, because these cells can be acutely killed by exposure to neomycin, after which they regenerate rapidly. Lateral line hair cells are located in the center of a mechanosensory organ known as the neuromast and are surrounded by inner support cells and an outer ring of mantle cells. Tg(sqET20) larvae express GFP strongly in mantle cells and to a lesser degree in inner support cells. We isolated GFP positive and GFP negative cells from 5 days post fertilization (dpf) Tg(sqET20) larvae at 1, 3 and 5 hours post neomycin treatment, as well as from a non-treated control. Transgenic zebrafish Tg(sqET20) larvae at 5 days post fertilization were exposed to neomycin, dissociated, and FACS sorted into GFP positive and GFP negative populations at 1, 3, and 5 hours following treatment, along with a mock treated 1 hr control. The experiment was performed in triplicate, for a total of 24 samples.