Phoenix Is Required for Hair Cell Regeneration shown (yellow rectangle). (D) Alignment that shows the region of homology for putative phoenix genes in four other fish species. One particular stretch seems to be strongly conserved in all fish species, the short SDS- X(3)-SLF-[ILV]-TQ sequence (red box). The remaining sequence for all predicted proteins shows little to no significant identity, beyond enrichment for proline and lysine residues. (E) In situ hybridization with an antisense probe directed against the phoenix gene. At 2dpf the expression was found in all the neuromasts of the anterior and posterior lateral line. (F) Higher magnification of two trunk neuromasts (left panel) and a single neuromast (right panel), showing the typical ring-like staining restricted to the supporting cells. – 100 microns in E, 50 microns in (F, left panel) and 10 microns in (F, right panel). doi:10.1371/journal.pgen.1000455.g002 Figure 2A. The retroviral integration in hi43 (black triangle) landed in the first exon and was adjacent to the first splice donor site. To confirm that the retroviral insertion disrupted the surround- ing gene, we designed primers to perform RT-PCR on total RNA extracts of 7 dpf wild-type and mutant larvae and on the zebrafish tissue culture cell line Pac2, which also expressed the gene [51]. The expected 835 bp RT-PCR product was seen (Figure 2B, left arrow), in wild-type larvae (lane 2), and in the Pac2 cell line (lane 1 and 3), but was absent in the mutant larvae (lane 4). However a shorter 366 bp product was generated in the mutant larvae exclusively (Figure 2B, right arrow). We cloned and sequenced all of the observed PCR fragments. In pho mutants, aberrant splicing occurred, fusing exon 1 to exon 7 and causing a deletion and a frame-shift truncation of the presumptive protein product (Figure 2C). Additionally, the aberrant splicing event resulted in an arginine replacing the last serine in a predicted abATPase signature, therefore potentially abrogating the putative enzymatic activity (Figure 2C). To further prove that the deficient regeneration of the hair cells in the lateral line was caused by a mutation in the phoenix gene, we acquired a second commercially available allele (Znomics, Inc. line: ZM_00003486). This mutant line carried a retroviral insertion in the first exon (75bp downstream of the ATG) of the phoenix gene (green triangle Figure 2A). The insertion of the provirus in this position most probably led to a mutation. All of the phenotypes observed in the original hi43 allele were present in the second recovered mutation and all subsequent experiments were performed in both allelic mutant lines. We did not attempt to phenocopy the regeneration phenotype using morpholino injection, as our observations started at 6dpf. This time-point is beyond the time-window of efficacy of morpholinos, which get diluted over time after the numerous cell divisions occurring in the growing embryo/larva. Taken together, our data strongly indicate that we have correctly identified the genetic defect responsible for the observed phenotype. Phoenix is a novel gene encoding a large protein with low sequence complexity and which is strongly expressed in neuromast supporting cells Using RT-PCR, we identified several alternatively spliced variants of the pho gene (Figure 2C). We compared the different isoforms against various RefSeq databases using BLAST [52]. The best predicted homologs in other species for the pho gene were of low quality and were all predicted proteins, therefore providing little information. However, using genomic homology comparisons from the UCSC Genome Browser (http://genome.ucsc.edu/) we were able to identify the syntenic region for four other fish species: Takifugu rubripes (fugu), Tetraodon nigroviridis (tetraodon), Oryzias latipes (medaka), and Gasterosteus aculeatus (stickleback). The genes flanking phoenix in zebrafish are aspa and c11orf54 homologs. In all fish species examined, this synteny was maintained, but a synteny break occurred at this location in other vertebrate genomes. Located between aspa and c11orf54 in all four fish species was a large predicted ORF. The corresponding predicted genes (GENSCAN00000016645 in fugu, chr14.906.1 in medaka, chrVII.1390.1 in stickleback and chr7.288.1 in tetraodon) had very weak but noticeable identity with the zebrafish phoenix gene. The overall identity of the fish homologues was still not significant (the best match was fugu to tetraodon at 35% identity in a limited region, with no other pair-wise comparison showing higher than 24% identity). The main feature of all proteins was a low complexity sequence with a large number of proline and lysine residues. Using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/ index.html), we showed that the only significant identity in these predicted transcripts was in the amino-terminal end of the proteins (Fig .2D red box and 2C yellow rectangle). One small region was highly conserved in all transcripts (SDS-X(2-3)-SLF- [ILV]-TQ, red box in Figure 2D), which may represent a functional motif for this class of proteins. The high rate of divergence across the different fish species suggested that, the function of the protein can tolerate significant alterations in the primary sequence. Because these fish homologs were not identified through the usual BLAST comparisons, if homologs in other vertebrates exist, they will be difficult to find using traditional approaches. It is likely they will have to be identified through functional similarities instead of primary sequence homology. To gather more information on the phoenix gene, we analyzed the putative phoenix product, looking for protein motifs and domains, using protein structure databases such as PFAM and CDD. We identified one putative functional domain using Motif Search (http://motif.genome.jp). It contained an abATPase signature, PSVHSPPSDS (P-[SAP]-[LIV]-[DNH]-X(3)-SXS), en- coded in the first exon with the last serine overlapping the splicing site (Figure 2C). It is important to note, that evidence for the ATPase signature was not found in the other fish species, but those transcripts were computationally predicted and it is possible that there were missing exons or were incorrectly sequenced. In the largest phoenix splice form, there was a strongly predicted single membrane-spanning domain (Bioweb, Pasteur Paris, Figure 2C). Therefore, the majority of the protein appeared to be a very long, poorly structured, tail with a single potential anchorage point to a membrane. All the shorter gene products were predicted to be cytoplasmic. No secretion signal peptide was found (CBS Website, University of Denmark). We performed in situ hybridization (ISH) on embryos from 8 hours post fertilization (hpf) through 5 dpf. The expression of the phoenix gene was first detected in the anterior and posterior lateral line system (Figure 2E) beginning at 2 dpf, and was uniformly expressed in all neuromasts and still maintained in this organ at 5dpf. Phoenix mRNA was found in a ‘‘ring-like’’ structure (Figure 2F, left panel), clearly staining the supporting cells, while it was absent from the center of the neuromasts, where hair cells were located (Figure 2F, right). Additionally, we found expression in discrete areas of the inner ear, which could correspond to the neuroepithelial patches (Figure S2B). The pou4f3::GFP line allowed us to observe in live larvae the hair cells in the inner ear which seemed unaffected by the mutation in wild-type (left) and mutant (right) cristae (Figure S2A) Taken together, these observations clearly show that, we have identified a new gene with an upregulated expression in the PLoS Genetics | www.plosgenetics.org 5 April 2009 | Volume 5 | Issue 4 | e1000455