Identification of hair cell progenitors and intermitotic migration of their nuclei in the normal and regenerating avian inner ear

Abstract

Postembryonic production of sensory hair cells occurs in both normal and aminoglycoside-damaged avian inner ears. The cellular source and mechanism that results in new differentiated hair cells were investigated in the avian vestibular epithelia using three distinct cell-cycle-specific labeling methods to identify proliferating sensory epithelial cells. First, immunocytochemical detection of the proliferating cell nuclear antigen, an auxiliary protein of DNA polymerase, allowed labeling of cells in late G1, S, and early G2 phases of the cell cycle. Second, a pulse-fix tritiated thymidine autoradiographic protocol was used to identify cells in S phase of the cell cycle. Finally, Hoechst 33342, a fluorescent DNA stain, was used to identify epithelial cells in mitosis. The distribution of cells active in the cell cycle within the normal and ototoxin-damaged vestibular epithelium suggests that supporting cells within the sensory epithelia are the cellular precursors to the regenerated hair cells. Differences between the proliferation marker densities in control and damaged end organs indicate that the upregulation of mitotic activity observed after streptomycin treatment is due primarily to an increase in the number of dividing progenitor cells. The differences between the extent of ototoxic damage and the level of reparative proliferative response suggest a generalized stimulus, such as a soluble chemical factor, plays a role in initiating regeneration. Finally, after DNA replication is initiated, progenitor cell nuclei migrate from their original location close to the basement membrane to the lumenal surface, where cell division occurs. This pattern of intermitotic nuclear migration is analogous to that observed in the developing inner ear and neural epithelium.

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

“…First, progenitor cells maintain a physical connection with the basal lamina during mitosis, and postmitotic cells retain a connection with the basal lamina that is permanent in the case of support cells and transient among hair cells. This hypothesis is not consistent with studies of the chick basilar papilla during development (Katayama and Corwin, 1993) and regeneration (Raphael et al, 1994;Tsue et al, 1994a), which have shown that mitotic cells are round and have no apparent connection with the basal lamina. An alternative possibility is that support cells lose contact with the basal lamina during mitosis and daughter cells migrate to more basal positions in the epithelium after mitosis.…”

“…In inner ear epithelia, progenitor cell nuclei migrate from the basal lamina to the lumen during progression through the cell cycle (Ruben et al, 1971;Raphael, 1992;Katayama and Corwin, 1993;Stone and Cotanche, 1994;Tsue et al, 1994a). The temporal and spatial patterns of calmodulin expression in our study suggest that postmitotic cells also migrate from the lumen to a region below the hair cell layer before establishing their permanent location at the lumen.…”

“…9). This cellular profile is characteristic of support cells that are in S phase or gap 2 phase of the cycle; by the time of mitosis, support cell nuclei have migrated into the lumenal layer (Raphael, 1992;Katayama and Corwin, 1993;Stone and Cotanche, 1994;Tsue et al, 1994a). No cells were double-labeled in the damaged region at this time (Fig.…”

“…This finding is in contrast to basilar papillae at 2 hr, when the nuclei of 3 H-thymidine/BrdU-positive and calmodulin-negative cells were located primarily within the adlumenal compartment. This pattern of labeling demonstrates that support cells had progressed beyond S phase and were either differentiating into hair cells or support cells or going through a second round of cell division (Raphael, 1992;Katayama and Corwin, 1993;Stone and Cotanche, 1994;Tsue et al, 1994a). It is unlikely that support cells were poised in the first round of division at this time, because it takes ϳ6 hr for them to progress from S phase (at the time of injection) to M phase (Stone and Cotanche, 1994).…”

“…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.…”