Author Summary By screening for regeneration deficient zebrafish muta- tions, we identified a zebrafish mutant line deficient in a highly specific regeneration process, the renewal of hair cells in the lateral line. Although this organ is specific to fish and amphibians, it contains essentially the same mechanosensory cells (the hair cells) that function in the ear for sound and balance detection in all vertebrates. Mammals are unusual vertebrates in that they have lost the ability to regenerate functional hair cells after damage by sound or chemical exposure. All other vertebrates retain their ability to regenerate their hair cells after damage, but this process is not well understood at the molecular level. The retroviral insertion linked to the phoenix mutation is in a new gene family class that is specifically required for the supporting cells to enter into mitosis after hair cell damage. What is particularly unusual about this mutation is that it appears not to affect the normal development and differentiation pathways, but only seems to affect the cells’ post-differentiation regeneration. functional by 4dpf [30] and as the larva grows into an adult, additional neuromasts are continually added to the embryonic pattern [31,32]. In adult fish, newly formed neuromasts are thought to originate from preexisting ones, with supporting cells budding off and migrating to their new locations [33]. Additionally, this sensory organ is known to continuously replace hair cells in larvae and adults [34]. The neuromasts are also able to replace hair cells after all existing ones have been have been destroyed [35,36]. Several waterborne agents have been used to eliminate hair cells from neuromasts, including aminoglycosides, platinum-based drugs, and metal ions. Copper is a potent ototoxic agent, killing hair cells in the lateral line, as early as 3dpf [37]. After 5dpf, the hair cells also become sensitive to antibiotics of the aminoglycoside family, in particular to neomycin, presumably coinciding with their functional maturity [38,39]. At all time points, regeneration of hair cells has been documented, mainly resulting from the division of supporting cells and subsequent differentiation into new hair cells [14,40–44]. We utilized an in vivo assay using both copper and neomycin to follow regeneration of hair cells in neuromasts of 6dpf to 8dpf larvae. Using this assay, we characterized two allelic mutant lines generated by retroviral insertion [45], which we have named phoenix (pho). Homozygous mutants did not display obvious morphological or behavioral phenotypes and developed a functional lateral line as ascertained by FM1-43 incorporation. However, when phoenix larvae were treated with either copper or neomycin, they showed a strongly reduced regeneration of the hair cells. In parallel, we demonstrated that another form of regeneration, the growth of the tail after amputation, was not affected in the phoenix mutants, arguing that the regeneration defect was specific to hair cells. We show that, the number of supporting cells was not significantly different before or after copper and neomycin treatments in neuromasts of wild-type versus phoenix mutant larvae. Furthermore, we monitored cell death over the time course of our assay in treated wild-type and mutant neuromasts and did not find significant differences in supporting cell death. Strikingly, we found that the proliferation rate in this progenitor cell population was strongly reduced in mutants. Thus, impaired proliferation is tightly correlated to the observed deficit of regenerated hair cells in the phoenix mutant. Phoenix Is Required for Hair Cell Regeneration We characterized the gene carrying the retroviral integration linked to the phenotype, and find that it is a novel gene, with no previously described homologs. Expression at 3-4dpf is upregu- lated in the supporting cells of the neuromasts. Thus, Phoenix is the first documented member of a novel gene family, which has an important role in the regeneration of hair cells in the lateral line. Results Monitoring hair cell regeneration in the lateral line of zebrafish mutant lines We have utilized an assay to monitor hair cell regeneration in the lateral line of 5dpf zebrafish larvae, using transient exposure to copper sulphate (10mM) or neomycin (200mM) dissolved in the water. Our intention was to test for defective hair cell regeneration from a collection of mutant lines previously identified in a retroviral integration screen [45]. The major advantage of using retroviral constructs, over chemicals like ENU, as a mutagenic agent is the rapid identification of the mutated gene. This allows the spatio-temporal expression of the mutated genes to be compared to the observed phenotypes, facilitating selection of the mutants of interest. We reasoned that mutant larvae which develop a normal and functional lateral line and that exhibit expression of the mutated gene in neuromasts would be good candidates to test for defects in the regeneration of the hair cells. We found such a mutant line, which we called phoenix. Homozygotes displayed no behavioral defects (response to sound or mechanical stimulation was normal, data not shown) or visible morphological abnormalities until 5dpf, when the swim bladder failed to inflate. Later, around 7 to 8 dpf, mutants display necrosis in the liver, and death ensues at approximately 14 dpf. Because the gene was expressed in the lateral line neuromasts (see below), we performed a detailed assessment of this organ in 2 to 12 dpf larvae (Figure 1). Semi-thin sections through neuromasts (Figure 1A) of phoenix mutants (lower panels, 5dpf left panel and 9dpf right panel) were indistinguishable from wild-type larvae (upper panels). Camera lucida drawings (Figure 1B) of each section show the cilia (green) of hair cells and their nuclei (red) and the nuclei of the supporting cells (blue). Likewise, ultra-thin sections observed by electron microscopy (EM) (10dpf larvae shown in Figure 1C) did not present obvious differences between wild-type (top panel) and mutant neuromasts (lower panel). Camera lucida drawings (Figure 1D) show, like in Figure 1C, the cilia (green) of hair cells and their nuclei (red) and the nuclei of the supporting cells (blue). The cytoplasm of hair cells is outlined in dark red and an apoptotic body is shown in yellow in the lower panel. Note that apoptotic bodies, as the one present in the mutant, were found at the same rate in untreated wild-type and mutant sections, being probably products of the regular turnover of the supporting cells, which has been described previously [34]. We further observed wild-type and mutant neuromasts in three different transgenic backgrounds (Figure S1). As shown in live images, these lines express GFP in all the cells of the neuromast in the cldnB::GFP line [46] (Figure S1A), in the hair cells in the pou4f3::GFP line [47] (Figure S1B) or in a subset of supporting cells in the ET20::GFP line [48] (Figure S1C). Camera lucida drawings were added to each panel, for better illustration of the live images. To test the functionality of the hair cells in wild-type and phoenix mutant larvae, we used the well- described technique of monitoring the absorption of the vital dye FM1-43 [49]. We imaged live wild-type and mutant larvae in the cldnB::GFP line background (Figure S1A, GFP in green in first and third columns, FM1-43 in red in second and third columns. We added camera lucida drawings of the merged images in the fourth column). Again, we did not find a significant difference between PLoS Genetics | www.plosgenetics.org 2 April 2009 | Volume 5 | Issue 4 | e1000455