Hair Cell Differentiation in Chick Cochlear Epithelium after Aminoglycoside Toxicity:In VivoandIn VitroObservations

Abstract

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 calmodulinnegative, 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 calmodulinpositive 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; differentiationHair cells are sensory receptors for hearing, equilibrium, and motion detection. Some animals demonstrate the capacity to generate hair cells throughout their lifetime (Popper and Hoxter, 1984;Corwin, 1985; Jörgenson and Mathiessen, 1989;Roberson et al., 1992) or to initiate hair cell regeneration in the event of their loss (Corwin and Cotanche, 1988;Ryals and Rubel, 1988). The progenitors of hair cells seem to be a subset of support cells that reside adjacent to hair cells in the sensory epithelia (Girod et al., 1989;Balak et al., 1990;Raphael, 1992;Hashino and Salvi, 1993;Weisleder and Rubel, 1993;Stone and Cotanche, 1994;Tsue et al., 1994a;Roberson et al., 1996). Although mature mammals normally do not generate new hair cells, recent in vivo and in vitro studies have documented mitotic activity and immature-looking hair cells in mammalian vestibular epithelia after exposure to ototoxic drugs Warchol et al., 1993;Rubel et al., 1995), suggesting that hair cell regeneration in mammals may be inducible. The development of culture methods for mature cochlear and vestibular end organs has been initiated to identify molecules that regulate cell proliferation and differentiation in avian and mammalian hair cell epithelia. Co-culture experiments suggest that...

Supporting Cell Division Is Not Required for Regeneration of Auditory Hair Cells After Ototoxic Injury In Vitro

“…4B, D). A similar result was obtained using antibodies to class III ß-tubulin or calmodulin (data not shown), which are additional HC-selective markers (Stone et al 1996). A similar degree of HC loss was seen in cultures grown in BME/EBSS, 10% FBS, and streptomycin for a similar period (data not shown).…”

“…At the end of the culture period, the tectorial membrane was removed from the sensory epithelium using a combination of enzymatic and mechanical methods (Stone et al 1996). Tissue was fixed with 4% buffered paraformaldehyde for 30 min to 1 h at room temperature and then rinsed in PBS.…”

“…Standard indirect immunofluorescence or immunohistochemistry was used to detect proteins in wholemount preparations (see Stone et al 1996). The following primary antibodies were used: mouse antiBrdU (Becton Dickinson, Franklin Lakes, NJ, USA), rat anti-BrdU (clone BU1/75; Sera Laboratories, West Sussex, UK), rabbit antiphosphohistone H3 (antipH3; Upstate Biotechnology, Lake Placid, NY, USA), rabbit anti-myosinVI (purchased from University California San Diego, San Diego, CA, USA or from Proteus Biosciences, Ramona, CA, USA), mouse antihair-cell antigen (gift from Dr.…”

“…Secondary antibodies conjugated to biotin were purchased from Vector Laboratories (Burlingame, CA, USA). Diaminobenzidine (DAB) labeling was performed using the horseradish-peroxidase-conjugated ABC reagent from Vector Laboratories (Stone et al 1996).…”

“…DABlabeled organs were whole-mounted onto microscope slides in 90% glycerol in PBS and coverslipped. Others were processed for plastic sectioning, as described in Stone et al (1996).…”