In the damaged avian inner ear regeneration occurs when supporting cells are triggered to proliferate after the loss of nearby hair cells. In order to study the signalling events that regulate and stimulate the activation of proliferation in hair cell epithelia we have adapted an epithelial culture preparation using thermolysin-treated utricular epithelia. Embryonic day 21 pre-hatch chicks were killed humanely and the utricles isolated. Utricles were treated with thermolysin for 45 min, facilitating the removal of the epithelial sheet from the underlying basement membrane for subsequent culture (Warchol, 2002). A nitrogen laser based ablation system (MicropointTM, Photonic Instruments, USA) attached to the epifluorescence port of the microscope allowed precise control of the timing and magnitude of the epithelial damage. 5-Bromo-2Ô-deoxyuridine (BrdU), which is incorporated into the DNA of cells during s-phase, was used to assess the induction of proliferative activity. Epithelia were subjected to a 2 h pulse of BrdU (3 µg ml-1) prior to fixation and labelled using BrdU antibodies. BrdU pulses were applied between 6 and 24 h after laser ablation. A minimum of four epithelia was used for each time-point. BrdU-positive nuclei were counted within a 120 µm radius circle centred over the ablation site. The number of s-phase cells varied appreciably over the 6-26 h period analysed. Two temporally distinct increases of 301 ± 72 % (n = 4) and 438 ± 153 % (n = 6, mean ± S.E.M.) over control levels were seen at 12 and 24 h, respectively. In addition to investigating the temporal distribution of s-phase entry, we were able to bin BrdU-positive cells spatially using co-ordinate data calculated with Metamorph imaging software (UIC, USA). We used concentric circles of radius 60 and 120 µm to form two binning regions (inner and outer) of 0-60 and 60-120 µm. S-phase entry in the outer region was elevated by 295 ± 218 % (n = 4) and 330 ± 120 % (n = 6) over control levels at 12 and 24 h, respectively. In the inner region s-phase entry was not significantly elevated over controls at 12 h, but was seen to markedly increase after 24 h by 1323 ± 504 % (n = 6) over controls.
The temporal data suggest that laser ablation triggers two waves of s-phase entry occurring 12 and 24 h after damage. In cells proximal to the lesion site s-phase entry is delayed when compared with more distal cells. We tentatively suggest that at early time points the primary function of cells surrounding the lesion is repair of the wound, and it is this activity which delays s-phase entry in those cells.
This work was supported by The Wellcome Trust, The Royal Society and a Medical Research Council Studentship to J.E.B.