Regenerative Proliferation in Organ Cultures of the Avian Cochlea: Identification of the Initial Progenitors and Determination of the Latency of the Proliferative Response

Abstract

Sensory hair cells in the cochleae of birds are regenerated after the death of preexisting hair cells caused by acoustic overstimulation or administration of ototoxic drugs. Regeneration involves renewed proliferation of cells in an epithelium that is otherwise mitotically quiescent. To determine the identity of the first cells that proliferate in response to the death of hair cells and to measure the latency of this proliferative response, we have studied hair-cell regeneration in organ culture. Cochleae from hatchling chicks were placed in culture, and hair cells were killed individually by a laser microbeam. The culture medium was then replaced with a medium that contained a labeled DNA precursor. The treated cochleae were incubated in the labeling media for different time periods before being fixed and processed for the visualization of proliferating cells. The first cells to initiate DNA replication in response to the death of hair cells were supporting cells within the cochlear sensory epithelium. All of the labeled supporting cells were located within 200 m of the hair-cell lesions. These cells first entered S-phase ϳ16 hr after the death of hair cells. The results indicate that supporting cells are the precursors of regenerated hair cells and suggest that regenerative proliferation of supporting cells is triggered by signals that act locally within the damaged epithelium.

Spontaneous hair cell regeneration in the neonatal mouse cochlea in vivo

“…5L + cells were observed in the apical turn, where the progression of cell cycle arrest first occurs Ruben, 1967) and where HC differentiation is last observed during development (Lumpkin et al, 2003;Montcouquiol and Kelley, 2003 + SCs proliferated prior to acquiring a HC fate, consistent with mitotic HC regeneration described previously in non-mammalian species (Baird et al, 1996;Corwin and Cotanche, 1988;Jones and Corwin, 1996;Ryals and Rubel, 1988;Warchol and Corwin, 1996 (Chow et al, 2006;Weber et al, 2008 the Atoh1DTA cochleae, we traced HCs using the ROSA26 CAG-tdTomato allele, which labels 99.5±0.2% of HCs at P6 in Atoh1-CreER TM mice after tamoxifen injection at P0/P1 (Fig. 7A,B Ruben, 1967), and HC differentiation and maturation is a dynamic process that occurs over 3 perinatal weeks in mice.…”

“…Direct transdifferentiation refers to a cell fate change when neighboring supporting cells (SCs) convert into HCs without cell division. Mitotic regeneration occurs when a SC first divides and, subsequently, one or both daughter cells becomes a HC (Adler and Raphael, 1996;Baird et al, 1996;Corwin and Cotanche, 1988;Jones and Corwin, 1996;Ryals and Rubel, 1988;Warchol and Corwin, 1996).…”

Sensory hair cell development and regeneration: similarities and differences

“…A major difference between the regenerating avian cochlea and the non-regenerating mammalian counterpart is the ability of supporting cells in the former to restart proliferation upon damage (Warchol and Corwin, 1996) (Fig. 2).…”

Inhibition of Caspases Prevents Ototoxic and Ongoing Hair Cell Death

“…To assay supporting cell proliferation, we added the mitotic tracer bromodeoxyuridine (BrdU; 3 g /ml; Sigma) to cultures for the last 4 hr in vitro. Specimens were processed for BrdU immunohistochemistry by following a standard protocol (Warchol and Corwin, 1996). Fixed utricles were incubated in 90% methanol with 0.03% H 2 O 2 for 20 min, 2N HC l for 30 min, and blocking solution (PBS, 2% N HS, 1% BSA, 0.2% Triton X-100) for 20 min; then they were incubated overnight in mouse anti-BrdU monoclonal antibody (1:50; in PBS, 2% N HS, 0.1% Triton X-100) at 4°C.…”

“…It has been hypothesized that the death of hair cells triggers the proliferation of nearby supporting cells (Corwin and Cotanche, 1988;Girod et al, 1989;Raphael and Altschuler, 1992;Roberson et al, 1992;Hashino and Salvi, 1993;Stone and Cotanche, 1994;Warchol and Corwin, 1996). To investigate the relationship between ongoing cell death and the level of supporting cell proliferation in the chick vestibular organs, we inhibited hair cell death by treatment with BAF.…”