Copper 2 sulphate

The ablation of hair cells and afferent terminals was achieved by the application of different concentrations of copper (II) sulphate (cat n: 451657, Sigma-Aldrich, UK), which was dissolved in plain embryonic medium E3 (in mM: 5 NaCl, 0.17 KCl, 0.33 CaCl2, 0.3 MgSO4). The different zebrafish lines (see above) at 3 dpf (early-larval) or 12 dpf (late-larval) were bath-treated with copper for 2 h at 28.5°C in the dark. Following this, the fish were washed three times in E3 and left to recover in petri dishes (<5.2 dpf) or in a non-circulating system tank (>5.2 dpf) prior to being processed for the different experiments. The use of copper was preferred to the ototoxic aminoglycoside antibiotics (e.g. neomycin and streptomycin), since under our experimental conditions streptomycin was only able to reduce the number of hair cells within a neuromast by about 70% even at very high concentrations (2 mM, Supplementary Figure 1). In addition, high concentrations of copper, but not aminoglycosides, also affected the survival of the afferent fibres and supporting cells (see Fig. 10), which allowed us to compare the time course of regeneration of different cell populations within the neuromast.

Copper (II) sulphate and Bathocuproinedisulfonic acid disodium salt (BCS) were purchased from Sigma-Aldrich (Steinheim, Germany). Copper and BCS solutions were prepared in dH₂O with stock concentrations of 10–20 mM and 200 mM, respectively. The solutions were filtered through 0.2 μM filters before usage. PD98059 was obtained from Sigma-Aldrich. A stock solution of PD98059 was prepared in DMSO.