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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