Temporal, spatial, and morphologic features of hair cell regeneration in the avian basilar papilla.

Hair cell-selective antibodies were used in combination with the nucleotide bromode-oxyuridine (BrdU) to examine the temporal, spatial, and morphologic progression of auditory hair cell regeneration in chicks after a single gentamicin injection. New hair cells are first identifiable with an antibody to class III beta (beta) tubulin (TuJ1) by 14 hours after BrdU incorporation, but progenitor cells in S phase and M phase are TuJ1-negative. TuJ1 labeling reveals that new hair cells are first detected at 3 days after gentamicin, in the base, and the emergence and maturation of regenerating hair cells spreads apically over time. Differentiation of regenerating hair cells consists of a progressive series of morphologic changes. During early differentiation (14 hours to 1 day after BrdU), regenerating hair cells are round or fusiform and remain near the lumen, where they are generated. During intermediate differentiation (2-4 days after BrdU), regenerating hair cells resemble support cells; their somata are elongated, their nuclei are in the support cell layer, and they appear to contact both the lumenal surface and the basal lamina. The 275-kDa hair cell antigen is first expressed in regenerating hair cells during this period. During late differentiation (7 days after BrdU and later), TuJ1-positive cells acquire the globose shape of mature hair cells. Labeling with antibodies to hair cell antigen, calmodulin, and ribosomal RNA confirms this morphologic progression. Examination of sister cells born at 3 days post-gentamicin reveals that there is equal likelihood that they will assume the hair cell or support cell fate (i.e., both asymmetric and symmetric differentiation occur).