Auditory hair cells are mechanoreceptors in the inner ear that transduce sound waves into neural signals. In humans, hair cell loss is irreversible and leads to profound, permanent hearing deficits. In contrast, mature birds regenerate hair cells in the chicken auditory epithelium (basilar papilla, or BP). Despite this ability, little is known about the identity, cell lineage, or molecular regulation of avian hair cell progenitors.
Four Specific Aims are proposed to address these issues.
In Aim I, the dynamics of progenitor cell division are examined using in vivo and in vitro approaches. First, using two nucleotide analogs, I will determine if progenitor cells divide more than once after a single lesion and after two lesions separated by a recovery period. Second, I will perform cell lineage analysis in primary BP cultures to assess the number of hair cell progenitors that have stem-cell like behavior (i.e., form colonies containing hair cells) versus those that divide only once or twice prior to forming hair cells and/or supporting cells. These experiments will help define the types of progenitor cells present in the mature avian BP, and if the presence of stem cells is confirmed, they will provide a foundation for experiments in Aim II.
In Aim II, two methods are proposed to generate purified cultures of hair cell progenitors: clonal expansion and viral-mediated selection of cells with mitotic potential. Progenitor cells derived by each method are examined for 1) their response to known regulators of supporting cell division and 2) their ability to regenerate cells with hair-cell and supporting-cell phenotypes. Experiments in Aim III will test if systematic variations in morphology exist among quiescent and dividing supporting cells in the intact BP. Otocysts are infected with a retrovirus encoding GFP in ovo, and chicks are allowed to mature to post-hatch. GFP-labeled supporting cells in the BP of control chicks and of chicks exposed to gentamicin are compared to determine if 1) distinct morphological subsets exist among quiescent supporting cells and 2) if dividing cells exhibit morphological features that distinguish them from growth arrested cells.
In Aim I V, I will test the function of a potential regulator of progenitor cell cycling - the homeobox-like transcription factor, cProxl - in cultures enriched for hair cell progenitors. Cultured cells are infected with a retrovirus encoding full-length or dominant-negative Proxl, and the effects of cProxl misexpression or inhibition on cell division are assessed. I hypothesize that cProx 1 activity is necessary and sufficient to stimulate cells to withdraw from the cell cycle. I hope the information generated in these experiments will be useful toward promoting hair cell regeneration in mammalian species.

Agency
National Institute of Health (NIH)
Institute
National Institute on Deafness and Other Communication Disorders (NIDCD)
Type
Research Project (R01)
Project #
5R01DC003696-08
Application #
6935344
Study Section
Special Emphasis Panel (ZRG1-IFCN-6 (01))
Program Officer
Freeman, Nancy
Project Start
1998-04-01
Project End
2007-03-31
Budget Start
2005-09-01
Budget End
2007-03-31
Support Year
8
Fiscal Year
2005
Total Cost
$241,423
Indirect Cost
Name
University of Washington
Department
Otolaryngology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Stone, Jennifer S; Wisner, Serena R; Bucks, Stephanie A et al. (2018) Characterization of Adult Vestibular Organs in 11 CreER Mouse Lines. J Assoc Res Otolaryngol 19:381-399
Lewis, Rebecca M; Keller, Jesse J; Wan, Liangcai et al. (2018) Bone morphogenetic protein 4 antagonizes hair cell regeneration in the avian auditory epithelium. Hear Res 364:1-11
Warchol, Mark E; Stone, Jennifer; Barton, Matthew et al. (2017) ADAM10 and ?-secretase regulate sensory regeneration in the avian vestibular organs. Dev Biol 428:39-51
Pujol, Rémy; Pickett, Sarah B; Nguyen, Tot Bui et al. (2014) Large basolateral processes on type II hair cells are novel processing units in mammalian vestibular organs. J Comp Neurol 522:3141-59
Chonko, Kurt T; Jahan, Israt; Stone, Jennifer et al. (2013) Atoh1 directs hair cell differentiation and survival in the late embryonic mouse inner ear. Dev Biol 381:401-10
White, Patricia M; Stone, Jennifer S; Groves, Andrew K et al. (2012) EGFR signaling is required for regenerative proliferation in the cochlea: conservation in birds and mammals. Dev Biol 363:191-200
Lewis, Rebecca M; Hume, Clifford R; Stone, Jennifer S (2012) Atoh1 expression and function during auditory hair cell regeneration in post-hatch chickens. Hear Res 289:74-85
Golub, Justin S; Tong, Ling; Ngyuen, Tot B et al. (2012) Hair cell replacement in adult mouse utricles after targeted ablation of hair cells with diphtheria toxin. J Neurosci 32:15093-105
Lin, Vincent; Golub, Justin S; Nguyen, Tot Bui et al. (2011) Inhibition of Notch activity promotes nonmitotic regeneration of hair cells in the adult mouse utricles. J Neurosci 31:15329-39
Shang, Jialin; Cafaro, Jon; Nehmer, Rachel et al. (2010) Supporting cell division is not required for regeneration of auditory hair cells after ototoxic injury in vitro. J Assoc Res Otolaryngol 11:203-22

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