treatment, in the number or appearance of supporting cells (green, top and lower panels) in phoenix mutants compared to wild-type larvae. This finding suggests that, while hair cell regeneration is greatly decreased in phoenix mutant neuromasts, the number of supporting cells appear unaffected. To further support this finding, we used confocal imaging to count supporting cells in the immuno-stained larvae (Figure 4C and 4D) in wild-type (green bars) and phoenix mutants (yellow bars). We counted the supporting cells in 4 different neuromasts in the head (Figure 4C) and in two different trunk neuromasts (Figure 4D) in each larva. Cell numbers were determined for untreated or copper treated larvae at +0h, +4h, +24h and +48h after the treatment. We did not find significant differences in any of the observed stages between wild-type and mutant larvae. Another possible explanation for the observed phenotype is that mutant progenitor cells are dividing but that the newly formed precursor cells do not survive because they are hyper-sensitive or unstable after the chemical exposure. To detect dying cells in the supporting cell layer in the mutant neuromasts, we analyzed semi- thin sections (n = 2/2 wild-type and mutant larvae, respectively in Figure 4E). We reconstructed each neuromast using between 12 and 20 successive sections. We show representative examples of a wild-type (left panels) and a mutant (right panels) neuromast, untreated (Figure 4E, top panels) and 4 hours after neomycin treatment (lower panels). Before and after treatment, wild-type and mutant neuromasts were indistinguishable. As outlined in the camera lucida drawings under each section, the wild-type neuromast had three hair cells (nuclei outlined in red) and ten supporting cells visible (nuclei outlined in blue). In the mutant neuromast, three hair cells and ten supporting cells were visible. Four hours after neomycin treatment hair cells were completely destroyed in the wild-type and in the mutant neuromast (Figure 4E, lower panels). The supporting cells appeared unaffected in both wild-type and phoenix mutant neuromasts. Ten nuclei in the wild-type and ten in the mutant neuromast were visible (as outlined in the camera lucida in blue). Thus, at all observed stages, we did not notice a significantly higher cell death rate among the supporting cell population in phoenix mutant larvae. Next, we examined cell death in live larvae using acridine orange [37]. We stained larvae at +24h, +48h and +72h post treatment. Dying cells displayed strong staining as shown in two wild-type (Figure 4G left panels) and two mutant (Figure 4G right panels) neuromasts at +24h post copper treatment. We counted the acridine orange positive cells in ten head and all trunk and tail neuromasts (Figure 4F) after copper treatment in the wild-type (green line) and mutant (yellow line), or after neomycin treatment (wild-type: blue, mutant: pink). At all stages observed we did not find a significant difference. Additionally, we looked at cell death in the neuromasts, using TUNEL stainings and did not find any obvious difference in the rate of dying cells at all stages observed (data not shown). We therefore conclude that the copper and neomycin treatments do not significantly affect the number of supporting cells in either wild-type or mutant larvae. Moreover, we rule out the possibility that progenitor cells may be dying in phoenix neuromasts as we did not observe an increase in cell death rates in mutant larvae compared to wild types. Taken together these results indicate that the regenerative capacity rather than the survival of supporting cells is affected by the phoenix mutation. Supporting cell proliferation is impaired during regeneration in phoenix mutant neuromasts Since the number of supporting cells in mutant neuromasts is not unlike that of wild type neuromasts, we turned our attention to differences in cell division to explain the regeneration phenotype Phoenix Is Required for Hair Cell Regeneration observed in phoenix larvae. To test the hypothesis that the phoenix mutation is affecting proliferation of the progenitor cell popula- tion, we exposed SCM1 transgenic wild-type and mutant larvae, treated with copper or neomycin, to BrdU for 6h prior to fixing them, at +24h, +48h and +72h after chemical treatment. We subsequently double-stained the larvae with an anti-BrdU antibody (red in Figure 5A, middle and right panels) and with an anti-GFP antibody (green in left and right panels). At +24h after copper (Figure 5A) and neomycin (not shown) treatments, the BrdU-labeled transgenic wild-type (Figure 5A, top panels) and the transgenic phoenix mutant (lower panels) larvae showed a striking difference in the number of BrdU positive cells. On average, we found eight BrdU positive cells in the transgenic wild-type neuromasts, in mutant neuromasts we typically found no positive cells while at most we found two. Thus the number of BrdU positive cells in the progenitor cells population in the mutant neuromasts is drastically reduced, most likely explaining the decrease in the number of newly regenerated hair cells in phoenix mutant embryos. To obtain quantitative data to support the above conclusion and to follow the dynamics of BrdU incorporation over time, we counted BrdU positive cells after copper treatment (Figure 5B) in at least ten wild-type (green line) and ten phoenix mutant (yellow line) neuromasts per larva. BrdU was added to the medium 6h before fixation allowing us to determine rates of DNA synthesis in equivalent time-windows during the 3 days regeneration period. Since recovery to a normal number of hair cells is reached at around 72 hours after treatment, this time point represents the rate of BrdU incorporation in undamaged neuromasts, or the BrdU baseline. At +24h of recovery, we had an eight-fold difference in the number of BrdU positive cells between wild-type and mutant neuromasts. This difference was still two-fold at +48h of recovery and both returned to the base line frequency at +72h. We performed similar tests after neomycin treatment (Figure 5C, wild-type: blue line and mutant: pink line). At +24h, we found close to a two-fold difference of BrdU labeled cells, and at +48h a 33% decrease between wild-type and mutant neuromasts. Taken together, these results show that the lack of the Phoenix protein has a significant negative impact on proliferation in the regenerating neuromast, which is not due to a reduction in the number of supporting cells. Thus, phoenix has an important role in ensuring proper proliferation of the supporting cells during hair cell regeneration in neuromasts. Discussion We have isolated a mutation in a previously uncharacterized gene in zebrafish that we have named phoenix because of an observed deficiency in the regeneration of the hair cells of the lateral line. Identical phenotypes were observed in two different alleles, caused by independent retroviral integrations in the phoenix gene in different genetic backgrounds. This unambiguously links the phenotype to this particular gene, which encodes a rapidly evolving protein with no obvious homologs in the current genomic databases. The predicted Phoenix protein is a long peptide of low complexity with a predicted ATPase domain reminiscent of many cytoskeletal proteins. This would argue in favor of a structural protein, possibly interacting with the cytoskeleton and subcellular membranes. How it is linked to the ability of the supporting cells to regenerate will require further investigation. It also remains to be discovered whether mammalian cells have a similar molecule; we were unable to detect a related sequence in the mammalian genomes. Interestingly, inner ear hair cells in mammals are unable to regenerate from supporting cells postnatally and, therefore, PLoS Genetics | www.plosgenetics.org 9 April 2009 | Volume 5 | Issue 4 | e1000455