Phoenix Is Required for Hair Cell Regeneration Figure 1. The development of the neuromasts in the lateral line is normal in the phoenix mutant larvae. (A) Semi-thin sections showing wild-type (top panels) and mutant neuromasts (bottom panels) in 5dpf (left panels) and 9dpf (right panels) larvae. (B) Camera lucida drawing for each section, highlighting the hair cells nuclei (red) and their cilia (green) and the supporting cells nuclei (blue). (C) Ultra-thin sections viewed by Electron Microscopy (EM) of a wild-type (top panel) and a mutant (lower panel) neuromast in 10dpf larvae. The hair cells stain darker than the supporting cells. (D) Camera lucida drawings of the EM sections, highlighting the hair cell nuclei (red), cell bodies (dark red), and cilia (green). The nuclei of the supporting cells are highlighted (blue). One apoptotic body was visible in (yellow). – 5 microns in (A) and 1 micron in (C). doi:10.1371/journal.pgen.1000455.g001 the absorption of the dye in wild-type (top) and mutant (bottom) hair cells, nor did we see any significant observable structural differences. Therefore, although the gene is expressed in the lateral line, its initial development appears unaffected in mutant larvae. Mutations in the phoenix gene are linked to the regeneration phenotype The first allele (hi43) of the phoenix mutant line was generated by retroviral mutagenesis as previously reported [45]. The genomic location of the retroviral integration was isolated and used to genotype the offspring to maintain the mutant line through numerous generations [45]. We identified a BAC spanning the genomic locus from a zebrafish BAC library and sequenced it in its entirety. Using GENSCAN [50], we predicted the genomic structure of phoenix (Figure 2A). The gene spanned ,16.5 kb with seven exons, the final predicted exon being unusually long as it was comprised of 7758bp with the entire exon consisting of an open reading frame. The size of the exons and introns is indicated in PLoS Genetics | www.plosgenetics.org 3 April 2009 | Volume 5 | Issue 4 | e1000455