Study of non- and mammalian inner ear hair cell regeneration by time lapse imaging
Zusammenfassung der Projektergebnisse
Non-mammalian vertebrates regenerate sensory hair cells in response to ototoxic insults. Conventional population-based RNA-sequencing analysis of cultured regenerating utricular sensory epithelia at various time points after streptomycininduced hair cell loss have previously revealed candidate genes and signaling pathways that potentially play important roles in the regenerative process. However, the order of events that initiate, execute, sustain, and terminate the different modes of hair cell regeneration remain unknown. We proposed to develop an in vitro-based system for hair cell regeneration by utilizing time lapse imaging of regenerating chicken utricle sensory epithelia after aminoglycoside induced hair cell loss. We intended to determine whether all utricle supporting cells or a smaller distinct subset of cells serve as somatic stem cells. We aimed to discriminate cellular features such as cell fates and morphological changes between the two regenerative modes: trans-differentiation and proliferation. A series of technical difficulties deterred us from pursuing our original experiments and as a result we did not succeed to i) culture the chicken utricle sensory epithelia and maintain its native structure in vitro and ii) transfect supporting cells to be able to visualize the morphological changes during regeneration after hair cell loss. Therefore, we decided to revise the experimental framework of our study and studied the two modes of regeneration on primarily a transcriptional level. For that we employed fluorescence-activated cell sorting and single-cell gene expression analysis. We also established a reliable in vivo ototoxic insult model that results in reproducible 70-95% hair cell loss in the utricle. We will also quantify mitotically active cells at various time points after ototoxic insult using EdU labeling. This will allow us to establish a timeline of symmetric and asymmetric cell divisions as well as to resolve occurrences of phenotypic conversion of supporting cells. Bioinformatics cluster analyses of expression data of 192 genes in individual cells of untreated chicken utricle sensory epithelia facilitates the classification of transcriptionally distinct cell populations including hair cells and supporting cells. In treated utricles we will identify discrete populations of ‘responding’ cells with transcriptional profiles displaying features of both, hair cell and supporting cell identity. These newly occurring groups may be grouped by their gene expression profiles for example as i) early streptomycin-responding hair cells, ii) supporting cells that respond to signals received from affected hair cells, iii) supporting cells that begin to phenotypically convert into new hair cells, iv) supporting cells that initiate asymmetric cell division, and finally v) as distinct groups of regenerated hair cells. Furthermore, we will utilize dimension-reduction algorithms to resolve cellular heterogeneity on a temporally defined scale. This will enable an in-depth characterization of the transcriptional changes that occur during. Our experiments revealed that the chicken utricle presents a powerful model to study the molecular events that control hair cell regeneration and the results may provide important insights into development of therapeutic strategies for mammalian hair cell regeneration.
Projektbezogene Publikationen (Auswahl)
- (2013) A Simple Method For Purification Of Vestibular Hair Cells And Non-Sensory Cells, And Application For Proteomic Analysis. PLoS One. 2013 Jun 4;8(6):e66026
Herget M., Scheibinger M., Guo Z., Jan TA., Adams C.M., Cheng A.G., Heller S.
