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Hair cell differentiation in chick cochlear epithelium after aminoglycoside toxicity: in vivo and in vitro
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 beta tubulin antibodies) and one marker unique for support cells (cytokeratin antibodies). Examination of regenerating epithelia demonstrated that calmodulin and beta 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.
Insights
Researchers identified new markers for studying avian inner ear hair cell regeneration. These markers aid in understanding how support cells proliferate and differentiate into new hair cells after injury.
Area of Science:
- Otolaryngology
- Regenerative Medicine
- Cell Biology
Background:
- Avian inner ear epithelia possess the capacity for hair cell regeneration following injury.
- Identifying specific cell markers and developing progenitor cell cultures are crucial for studying regeneration mechanisms.
Purpose of the Study:
- To identify selective markers for hair cells and support cells in regenerating avian inner ear epithelia.
- To establish culture systems for studying hair cell regeneration.
Main Methods:
- Utilized calmodulin, TUJ1 beta tubulin, and cytokeratin antibodies to label hair and support cells.
- Isolated sensory epithelia from chick basilar papillae for in vitro culture.
- Introduced ototoxic antibiotic to induce hair cell loss and observed cell proliferation and differentiation.
Main Results:
- Calmodulin and beta tubulin mark differentiating hair cells; cytokeratins mark proliferative support cells.
- Ototoxic antibiotic treatment led to complete hair cell loss and support cell proliferation in vitro.
- New hair cells expressing calmodulin or TUJ1 reemerged after antibiotic withdrawal, with some support cells differentiating into hair cells.
Conclusions:
- Established novel markers for tracking cell dynamics during avian inner ear hair cell regeneration.
- Demonstrated the potential of support cells to proliferate and differentiate into new hair cells in culture.
- Provided a foundation for further research into the molecular regulation of hair cell regeneration.