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Volume 16, Number 19, Issue of October 1, 1996 pp. 6157-6174
Copyright ©1996 Society for Neuroscience

Hair Cell Differentiation in Chick Cochlear Epithelium after Aminoglycoside Toxicity: In Vivo and In Vitro Observations

Received Feb. 21, 1996; revised June 27, 1996; accepted July 16, 1996.

Jennifer S. Stone¹, Sharon G. Leaño², Lauren P. Baker³, and Edwin W Rubel¹

¹ Department of Otolaryngology and Head and Neck Surgery, Virginia Merrill Bloedel Hearing Research Center, University of Washington, Seattle, Washington 98195-7923, ² Mount Sinai Medical School, New York, New York 10029, and ³ Department of Pharmacology, University of Washington, Seattle, Washington 98195-7923

Inner ear epithelia of mature birds regenerate hair cells after ototoxic or acoustic insult. The lack of markers that selectively label cells in regenerating epithelia and of culture systems composed primarily of progenitor cells has hampered the identification of cellular and molecular interactions that regulate hair cell regeneration. In control basilar papillae, we identified two markers that selectively label hair cells (calmodulin and TUJ1  tubulin antibodies) and one marker unique for support cells (cytokeratin antibodies). Examination of regenerating epithelia demonstrated that calmodulin and  tubulin are also expressed in early differentiating hair cells, and cytokeratins are retained in proliferative support cells. Enzymatic and mechanical methods were used to isolate sensory epithelia from mature chick basilar papillae, and epithelia were cultured in different conditions. In control cultures, hair cells are morphologically stable for up to 6 d, because calmodulin immunoreactivity and phalloidin labeling of filamentous actin are retained. The addition of an ototoxic antibiotic to cultures, however, causes complete hair cell loss by 2 d in vitro and generates cultures composed of calmodulin-negative, cytokeratin-positive support cells. These cells are highly proliferative for the first 2-7 d after plating, but stop dividing by 9 d. Calmodulin- or TUJ1-positive cells reemerge in cultures treated with antibiotic for 5 d and maintained for an additional 5 d without antibiotic. A subset of calmodulin-positive cells was also labeled with BrdU when it was continuously present in cultures, suggesting that some cells generated in culture begin to differentiate into hair cells.

Key words: hair cells; regeneration; chick; basilar papilla; cell culture; differentiation

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