Phoenix Is Required for Hair Cell Regeneration Figure 5. Supporting cells do not divide as efficiently after hair cell damage in the phoenix mutant neuromast. (A) BrdU staining (red, in the middle and right panels) in the SCM1::GFP transgenic wild-type (top) and the SCM1::GFP transgenic phoenix mutant neuromast (bottom) of recovering larvae (n = 20/20), 24h after copper treatment. The supporting cells are counterstained with GFP antibodies (green in left and right panels). (B) Quantification of the BrdU positive cells/neuromast in wild-type (green line) and mutant (yellow line) recovering neuromasts, at +24h (n = 11/10), +48h (n = 10/6), and +72h (n = 7/6) after copper treatment. Larvae are exposed to BrdU for 6h prior to being sacrificed. (C) Quantification of the BrdU positive cells/neuromast in wild-type (blue line) and mutant (pink line) recovering larvae, at +24h (n = 22/40), +48h (n = 20/33), and +72h (n = 13/32) after neomycin exposure. – 10 microns in (A). doi:10.1371/journal.pgen.1000455.g005 exploring the molecular differences between supporting cells between mammals and other vertebrates becomes of interest when trying to understand differences in regenerative capacity. We show that the expression of the phoenix gene is strongly upregulated in the supporting cells of the lateral line and that the lateral line morphology is essentially normal in the phoenix mutant larvae at all stages observed, from 2dpf to 12dpf. However it is important to note that, because we did not assess a possible maternal contribution for this particular gene, we cannot definitively exclude a potential role for phoenix during early development in general or of the lateral line in particular. Nevertheless, our regeneration assay performed in larvae between 6 and 8dpf, clearly shows that the number of regenerated hair cells is reduced by five-fold after copper treatment and by two-fold after neomycin treatment in phoenix mutant neuromasts. Therefore, the zygotic expression of the phoenix gene is clearly crucial for proper regeneration of the hair cells in the lateral line. Observations at later stages never showed full recovery to a wild- type number of hair cells in the mutant neuromasts, demonstrating that the deficit in the regenerative process is irreversible. We have also found expression of phoenix in the neuroepithelia of the inner ear, but unfortunately regeneration of the hair cells in the ear cannot be assessed with our assay, as water-borne ototoxic compounds do not diffuse into the otic capsule. We further show that the regeneration deficiency is specific to the lateral line, as we did not see a similar effect during tail regeneration. In combination with the restricted expression of the gene in supporting cells of the lateral line, this strongly suggests that, zygotic phoenix is involved specifically in the regeneration of hair cells in the lateral line, independent of the ototoxic treatment (copper or neomycin) used. It is intriguing that while the two treatments trigger similar responses, they are significantly different in the amplitude of the elicited effect. The reason there is a nearly 2.5 times greater inability of the supporting cells to respond properly after copper compared to neomycin treatments, is not clear. However, it is to be noted that the wild-type regeneration is also significantly slower (about two times) after the copper treatments when compared to neomycin treatments, as we found and as it has been described previously [37,43]. One possibility, as has been suggested previously [44], is that there is more than one mechanism of regeneration occurring in the supporting cells. The supporting cells probably do not represent a uniform population. For instance, one type of supporting cells, the ‘‘early responders’’ may be unaffected in the case of hair cells killed with neomycin, but might be more severely affected by the copper treatment leaving them unable, either temporarily or permanently, to respond and enter division. In less mature embryos (3dpf), the concentration of copper used (10 mM) PLoS Genetics | www.plosgenetics.org 10 April 2009 | Volume 5 | Issue 4 | e1000455