HAIR CELL-DIFFERENTIATION IN CHICK COCHLEAR EPITHELIUM AFTER AMINOGLYCOSIDE TOXICITY - IN-VIVO AND IN-VITRO OBSERVATIONS

Inner ear epithelia of mature birds regenerate hair cells after ototox ic or acoustic insult. The lack of markers that selectively label cell s in regenerating epithelia and of culture systems composed primarily of progenitor cells has hampered the identification of cellular and mo lecular interactions that regulate hair cell regeneration. In control basilar papillae, we identified two markers that selectively label hai r cells (calmodulin and TUJ1 beta tubulin antibodies) and one marker u nique for support cells (cytokeratin antibodies). Examination of regen erating epithelia demonstrated that calmodulin and beta tubulin are al so expressed in early differentiating hair cells, and cytokeratins are retained in proliferative support cells. Enzymatic and mechanical met hods were used to isolate sensory epithelia from mature chick basilar papillae, and epithelia were cultured in different conditions. In cont rol cultures, hair cells are morphologically stable for up to 6 d, bec ause calmodulin immunoreactivity and phalloidin labeling of filamentou s actin are retained. The addition of an ototoxic antibiotic to cultur es, however, causes complete hair cell loss by 2 d in vitro and genera tes cultures composed of calmodulin-negative, cytokeratin-positive sup port cells. These cells are highly proliferative for the first 2-7 d a fter plating, but stop dividing by 9 d. Calmodulin- or TUJ1-positive c ells reemerge in cultures treated with antibiotic for 5 d and maintain ed for an additional 5 d without antibiotic. A subset of calmodulin-po sitive cells was also labeled with BrdU when it was continuously prese nt in cultures, suggesting that some cells gener ated in culture begin to differentiate into hair cells.