supporting cells of the lateral line in zebrafish. Based on the various predictions, we speculate that Phoenix is most likely a structural protein, potentially carrying an enzymatic ATPase activity at its N-terminus. Impaired regeneration of hair cells in the lateral line of phoenix mutant larvae Previous reports have demonstrated that copper [37] and neomycin, an antibiotic of the aminoglycoside family, effectively kill hair cells in the lateral line [38,39,41]. We treated 5dpf larvae with copper sulphate (10mM) for 2 hours or with neomycin (200mM) for 1 hour. After rinsing the larvae, we stained with Yo- Pro1, a vital dye that specifically accumulates in hair cells and binds irreversibly to DNA, allowing easy visualization of the hair cells in live larvae [38]. Untreated control wild-type (Figure 3A, left top panels) and phoenix mutant (Figure 3A, left lower panels) larvae at 5dpf showed a bright uniform staining of the hair cells, which adopt the shape of a rosette. Most hair cells were ablated immediately after the copper treatment (+0h) in all observed wild- type (top panel) and phoenix mutant (lower panel) larvae. At most, one remaining hair cell could be seen, but its irregular shape indicating that it was probably a dying cell (Figure 3A, second column). We concluded that we could efficiently destroy the lateral line hair cells with this treatment. Likewise, 4h after exposure to neomycin, we found that most hair cells were absent (data not shown). We next monitored, over the following three days, the reappearance of hair cells in wild-type and mutant larvae after both treatments (copper shown in Figure 3A, neomycin not shown). At one day post treatment (+24h, third column), we found an average of 6 stained hair cells in the wild-type (top panel), but at most one in the mutant (bottom panel) neuromasts. At two days post treatment (+48h, fourth column), we found on average 8 stained hair cells in the wild-type (top panel), but at most two in the mutant (bottom panel) neuromasts. Three days post treatment (+72h, fifth column), we found averages of 12 stained hair cells in the wild-type (top panel) vs. at most three in the mutant (bottom panel) neuromasts. Therefore we conclude that the regeneration process is severely impaired in phoenix mutant larvae. We further quantified the treatments, counting Yo-Pro1 positive hair cells in 10 head and all trunk and tail neuromasts in wild-type and mutant larvae, after both copper and neomycin treatments and at all three time points during the recovery period. To factor in subtle developmental delays that we might have missed in the mutant, we counted untreated larvae at comparable stages. Numbers are presented as a percentage, 100% being the total number of hair cells present in untreated wild-type and mutant larvae at comparable stages. After copper treatment (Figure 3B) at all three time points, we found close to a five-fold difference in the number of Yo-Pro1 positive cells in wild-type when compared to phoenix neuromasts. After neomycin treatment (Figure 3C) we found nearly a two-fold difference at all stages. We conclude that in the absence of the phoenix product, neuromasts are not able to efficiently regenerate the destroyed hair cells. The regeneration defect in phoenix is specific to the lateral line To determine if phoenix’s role in regeneration was specific to the lateral line, or represented a general inability to regenerate damaged tissues, we tested larval tail regeneration. We transected tails of 3dpf untreated wild-type (top panel) and mutant (lower panel) larvae and followed them to 9dpf (Figure 3D left panels). As described previously, the tails of wild-type larvae first closed the injured tips of the notochord and neural tube, followed by fin fold Phoenix Is Required for Hair Cell Regeneration outgrowth [53]. Likewise, in mutant larvae, we observed a complete regeneration of the tail (Figure 3D, left lower panel). Furthermore, to exclude the fact that the treatments with the ototoxic agents could be interfering with the tail regeneration, we sectioned tails, immediately after copper (middle panels) or neomycin treatment at 5dpf (right panels). We monitored tail regeneration at 8dpf for copper and 9dpf for neomycin. While tail regrowth did not reach completion in wild-type or in mutant larvae, we saw no differences in the growth rate between wild-type and mutant larvae with either treatment. To further exclude more subtle differences, we quantified the tail growth after copper exposure (Figure 3E top graph) in 8dpf wild-type (green bar) and mutant (yellow bar) larvae and did not find a significant difference. Similarly, after neomycin treatment (Figure 3E, bottom panel), the growth of the tail in 9dpf wild-type (blue bar) and phoenix (pink bar) larvae was identical. Thus, we conclude that the phoenix mutation affects hair cell but not tail regeneration, indicating a defect specific to the lateral line. Taken together, these observations strongly suggest that phoenix has an important role in the regeneration process of hair cells in the lateral line. The number of supporting cells is not affected by copper and neomycin treatments in mutant neuromasts The differences in regeneration capacity of hair cells in phoenix versus wild-type larvae could be due to a difference in susceptibility of the progenitor cells to the ototoxic agents used in our assays. There is substantial evidence pointing to supporting cells as the source of new hair cells in neuromast regeneration. The new hair cells are likely to arise from a population of dividing progenitor cells [42–44], however it is not clear if all or only a subset of the supporting cells represent cells capable of forming new hair cells [42–44]. In order to visualize all the supporting cells (including all the cells capable of initiating regeneration), we took advantage of a transgenic zebrafish line generated by an enhancer trap event, which expresses GFP in all supporting cells of the neuromast (MB/SB unpublished). We named this transgenic line SCM1 (Supporting Cell Marker 1). It was crossed into the phoenix mutant background in order to obtain mutant transgenic individuals. We evaluated the effect of either copper or neomycin treatment on the number of supporting cells in wild-type and mutant treated larvae after copper or neomycin exposure. We confirmed that there were no significant differences in the appearance of the supporting cells in the mutant transgenic larvae by performing the regeneration assay, followed by immuno- staining against GFP to detect supporting cells and anti-Myosin VI antibody to detect hair cells. In figure 4A, we present a schematic drawing, of the stainings in a transverse view (top) and a dorsal view (bottom) of a neuromast, as seen in both untreated wild-type and mutant neuromasts. We found centrally located hair cells (red), surrounded by the supporting cells (green) that form cytoplasmic furrows around hair cells, as described previously [54,55]. In the control untreated wild-type (Figure 4B, fist column) and phoenix mutant (second column) neuromasts, hair cells were present and centrally located as visualized by anti-myosin VI antibody staining (red, middle and lower panels). The supporting cells, as expected, surrounded them as visualized with an anti-GFP antibody staining (green, top and lower panels). After copper (shown in Figure 4B) and neomycin (not shown) treatments, we could see a clear difference between wild-type and mutant neuromasts in the number of hair cells (red, middle and lower panels) at all stages of recovery, +24h (columns 3 and 4), +48h (columns 5 and 6) and +72h (not shown). Importantly, we did not see an obvious difference at any of the observed stages post- PLoS Genetics | www.plosgenetics.org 6 April 2009 | Volume 5 | Issue 4 | e1000455