(Electron Microscopy Sciences) in 16 PBS (Quality Biological, Inc.). Double staining was done as described [40,44], with a fluorochrome labeled mouse monoclonal antibody against BrdU (Molecular probes) and a fluorochrome labeled rabbit polyclonal antibody against GFP (Abcam). Larvae were mounted on slides in Aquapolymount (Polyscience, Inc) and at least 10 neuromasts/ larva were counted. The imaging was done on an upright confocal microscope (Zeiss AXIOVERT) Immunofluorescence on larvae Untreated and copper treated larvae were fixed o/n with 4% formaldehyde (Electron Microscopy Sciences) in 16 PBS (Quality Biological, Inc.), at various stages of interest and subsequently stored in 100% methanol. After progressive rehydration (25%, 50% 75% and 100% PTW (PBS16, 0.001% Tween and 0.001% DMSO)), larvae were treated with acetone for 7mn at 220uC. Subsequently, we rehydrated and rinsed them 365mn in PTW. Next we digested them with 1mg/ml collagenase (Sigma) in PTB (PTW + 10% goat serum +10% BSA) for 35mn. After 565mn rinses with PBT, we pre-incubated the larvae 4 hours in PBT. Larvae were incubated o/n with the polyclonal rabbit primary antibody (1/200) against Myosin VI (Proteus Biosciences, Inc) and a fluorescently labeled monoclonal mouse primary antibody (1/ 200) against GFP (Abcam). The next day we rinsed the larvae 6610 mn in PTW and preincubated them again for 4 hours in PBT. The fluorescently labeled secondary anti-rabbit antibody (1/ 500) was added o/n. The next day 6610 mn rinses were performed. Larvae were mounted on slides in Aquapolymount (Polyscience, Inc) for imaging on an upright confocal microscope (Zeiss AXIOVERT). Semi-thin and ultra thin sections for TEM on zebrafish larvae Larvae were fixed overnight at in 2.5% glutaraldehyde (Sigma) and 4% paraformaldehyde prepared from paraformaldehyde (Sigma) in 0.1M sodium cacodylate buffer (Sigma). Larvae were then rinsed and post-fixed 1h at room temperature in reduced osmium (1:1 mixture of 2% aqueous potassium ferrocyanide) as described previously [59]. After post-fixation the cells were dehydrated in ethanol and processed for Epon (Sigma) embedding. Semi-thin sections (300 nm) were cut and collected on a glass slide, and subsequently stained using toluidine blue (Sigma). The analysis and imaging were done on an inverted Zeiss Axio- vert200M. Ultra thin sections (80 nm) were cut on a Reichter-E ultramicrotome, collected on copper grids and stained with lead citrate (Sigma) for 2 min. Sections were then examined with a CM 10 Philips electron microscope at 80kV. Whole mount in situ hybridization Performed as described previously [60]. We designed an antisense probe, using the following primers TCAACTGATG- TATTTCCTGGGC and GTTTTGCTCCACTATCT- GACCTTT. The probe was hybridized at 62uC. Larvae were mounted on slides in Aquapolymount (Polyscience, Inc) for imaging on an upright confocal microscope (Zeiss AXIOVERT). Isolation of the BAC containing the phoenix gene and bioinformatic analysis We screened the zebrafish BAC library (Chori 211, BacPac consortium) with a probe generated from total RNA (5 dpf larva), with the primers AGATCTTGAGATTGCCGAATGT and CATCTCTCTCACCTTCTTCAGTGAC. The BAC was sequenced, by shotgun assembly and brought to finished quality Phoenix Is Required for Hair Cell Regeneration (#1 error per 50kb) at the National Intramural Sequencing Center (NISC). Sequences were aligned to the zebrafish genome (UCSC Genome Browser http://genome.ucsc.edu/) and syn- tenic regions identified using genomic chain comparisons in Takifugu rubripes (fugu), Tetraodon nigroviridis (tetraodon), Oryzias latipes (medaka), and Gasterosteus aculeatus (stickleback). Genescan or N-SCAN [50] predicted transcripts between the two flanking genes were identified and compared using ClustalW [61] (http://www.ebi.ac.uk/Tools/clustalw2/index.html). The BLO- SUM protein matrix [62] was used with a window of 7 and gap penalty of 2. RT-PCR, sequencing, and cDNA identification We prepared total RNA extracts from embryos and Pac2 cells (which is an embryonic zebrafish cell line described in [51] in Trizol (Invitrogen). For the RT-PCR, we retro-transcribed specific cDNA using superscript II Reverse transcriptase (Invitrogen), following the manufacturer’s protocol. For the cloning of all transcripts shown in Figure 2B and 2C, we used the same set of 2 nested forward primers CCATATCAAAACAGAGCTGTGC- TAC and GTCAAGGCAGAGTAAGCAAGTGACACTG, both located in the 59UTR, respectively starting 142bp and 121bp upstream of the start codon. For the cloning of transcripts 2, 3 and 6, they were respectively coupled with the reverse primers GTTCTGTTTTCTTTTCCTTGTCAACGCC and CCTTCTCTCCCTGATAAAGTCTGGCAACC both found in the 59end of exon 7. For the cloning of the transcript 1, we coupled them respectively with the reverse primers CATTCATTCATT- CATTCATTTTCCT and TTTCCTTTTGCTTAGTCCCT- TATTT both found in the 39end of exon 2. For the cloning of the transcript 4 we coupled them respectively with the reverse primers TTTTACATCTACAGAATCGTGAAAAA and AATTGTGATTCTCATTTTAGCCAGA, both found in the 39 end of exon 6. For the cloning of the transcript 5 we coupled them respectively with the reverse primers TTTTTCTTTCATAGC- GAACACAAAG and TTTAAACTGTCAGCTCTTGATGC, both found in 39end of exon 5. To verify the quality of the Total RNA from different stages, which we used for the RT-PCR, we amplified a 159bp fragment of b-actin using the following primers, GACCCAGACATCAGGGAGTGATGG and AGGTGT- GATGCCAGATCTTCTCCAGT. All sub-cloning for subse- quent sequencing were done using the TOPO TA cloning Kit (Invitrogen), following the manufacturer protocol. Sequencing was performed by ACGT, Inc. The analysis of the sequences was done using the Sequencher (Gene Codes Corp.) program, with the genomic sequence of the phoenix gene (as previously isolated in the BAC) as a reference. Statistical analysis At all time points and in all graphs, the p values for wild-type versus mutant larvae were calculated using a Student’s T test (two tailed) with two samples of equal variance and considered significant when p was ,0.05. Error bars in the different graphs represent the standard deviation, or standard error, depending on the number of samples (n) analyzed. Image reconstruction Stacks of images collected on the inverted microscope were treated with the Axiovision software from Zeiss. Stacks of images collected on the confocal microscope were treated with the LSM software from Zeiss. All images or stack of images were exported as tiff files, which were subsequently processed with Photoshop. PLoS Genetics | www.plosgenetics.org 12 April 2009 | Volume 5 | Issue 4 | e1000455