has been shown to eliminate post-mitotic precursors, thus requiring cell division and longer times for regeneration when compared to lower doses of the metal [37]. Additionally, it was recently shown in a screen to identify genes that modify hair cell resistance in the lateral line, that different ototoxic agents can elicit different pathways for protection and survival of hair cells in treated neuromasts [56]. It is therefore also possible that the regenerative response in supporting cells depends partially on the type of ototoxic agent applied. Namely, the trigger or the response to the trigger of regeneration in supporting cells could rely on different pathways, partially overlapping or complementing each other depending on the ototoxic chemical. Further investigation will allow us to distinguish between these possibilities. It appears that phoenix is upstream of the differential response between neomycin and copper, as the differences in the rates of response in mutant larvae remain similar to the differences seen in wild-type larvae. We showed that the death rate of supporting cells was unaltered in mutant neuromasts as observed by three different approaches (histology, acridine orange and TUNEL staining). Furthermore, making use of the MSC1 transgenic line that expresses GFP exclusively in the supporting cells, we demonstrated no loss in the number of those cells in mutant neuromasts. It is to be noted that the counting of cells in the neuromast is technically challenging. Although we did not see obvious differences, we cannot exclude a possible subtle reduction in the number of supporting cells. If a key subset of cells critical to the initiation of regeneration is missing, even a subtle difference in number might have a drastic effect on regeneration, which we cannot exclude at our level of analysis. However, it was previously shown that the regeneration process is happening in many supporting cells simultaneously, encompassing most of the neuromast [44] making this possibility unlikely. We clearly show that a significant number of supporting cells are present and that the phoenix mRNA is expressed in all supporting cells, therefore the most likely explanation is that the regenerative potential of all supporting cells is severely impaired. To assess proliferation of the progenitor cells, we follow the incorporation of BrdU during the S-phase of the cell cycle in neuromasts. The incorporation of BrdU is detected in cells that have exited the cell cycle and have differentiated. Therefore, it provides no information on the post-mitotic state of the BrdU positive cells. However, as the number of regenerated hair cells correlates closely to the number of BrdU labeled cells and as we do not see additional cell death in either wild-type or mutant neuromasts, this would suggest that the progenitor cells that actually enter S-phase proceed through the entire cell cycle and into differentiation. In the phoenix mutant larvae, this regenerative process is happening in a significantly reduced number of supporting cells. Further work is required to address the timing of the blockage occurring in the supporting cells in mutant neuromasts, but it is clearly occurring before DNA synthesis initiates. This invaluable information will help to elucidate the mechanism of action of the novel phoenix gene in the regeneration process of hair cells in the lateral line. Materials and Methods All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the appropriate committee (NHGRI protocol # G-01-3). Fish care and husbandry Fish care and husbandry were performed according to [57] in compliance with NIH guideline for animal care. The hi43 allele of Phoenix Is Required for Hair Cell Regeneration phoenix was recovered in a retroviral screen performed at MIT [58]. The genomic locus of the retroviral integration and the putative cDNA were determined as described in [45]. Genotyping was done with primers GGAGATCGACAGCGCCCTGAAG and AAACTGCTGAGGGCTGCTGGACCGCATC. The zm allele (ZM_00003486) was purchased (Znomics, Inc) and carriers were genotyped using the primers CGAGACCCCGCCGCCT- GATGTT and GACGCAGGCGCATAAAATCAGTC. The MSC1 transgenic line was a gift from B. Weinstein and carriers were identified by detecting specific expression of GFP in the lateral line. The cldnB::GFP line [46] was obtained from M. Allende, the pou4f3::GFP line [47] from H. Baier and the ET20::GFP line [48] from Vladimir Korzh. Staining with vital dyes (YoPro-1, Acridine Orange, and FM1-43) and treatment with copper or neomycin of live larvae All active agents were added to system water at 28uC. During the staining/treatment, larvae were kept in cell strainers (BD Falcon) in 6 well plates (Costar, Corning, Inc)) at a maximum of 35 larvae/well. This allowed rapid transfer of the larvae during the post-treatment rinsing. All larvae were rinsed 663 minutes with system water and then transferred into large Petri dishes with abundant fresh system water. After the various treatments, only healthy looking larvae displaying no additional morphological or behavioral defects were kept for subsequent analysis over the following 72 hours. The number of hair cells and the AO positive cells were counted in 10 head and all the trunk and tail neuromasts, in each larva. We added YoPro-1 (Molecular probes) for 15 minutes at 2mM, as described in [38], FM1-43 (Molecular probes) for 1minute at 2mM, as described [49] and Acridine orange (AO) hemi (zinc chloride, Sigma) for 5 minutes at 2mg/ml, as described [37]. Copper (Copper(II) sulfate, Sigma) was added for 2h at 10mM as described in [37] and Neomycin (sulfate, Calbiochem) for 1h at 200mM as described in [38,39]. Monitoring, counting and imaging of the lateral line in live larvae, after using the different life dyes, was done on an inverted Zeiss AXIO- VERT200M equipped with an Apotome Grid Confocal. Larvae were anesthetized with MS222 (0.005%) and mounted on a cover slip in 2% Methylcellulose (Sigma). Tail transections Five day old larvae were allowed to recover for an hour after copper or neomycin treatment. Subsequently, larvae were anesthetized with 0.005% MS-222 (Sigma) and placed on a clean glass slide and a portion of their tail was amputated with a scalpel. We cut not just the fin, but also a portion of the actual tail including the neural tube, the notochord, and the somites, to evaluate the regeneration of other tissues in addition to the fin itself. Three day old untreated larvae were similarly processed. To follow and image the tail growth over 3 to 6 days post amputation, we mounted anesthetized larvae on cover slips in 2% methylcel- lulose. Measurements were made on pictures taken from live larvae on the inverted Zeiss AXIOVERT200M, using the Axiovision software from Zeiss. The region of amputation was usually easily identifiable, as it presented healing scars, allowing a reasonably accurate measurement of the newly grown tail. BrdU treatment BrdU (Sigma) was diluted to 10mM in system water and larvae were exposed for 6h before fixation at the chosen time points (24, 48 and 72 hours post treatment) o/n with 4% formaldehyde PLoS Genetics | www.plosgenetics.org 11 April 2009 | Volume 5 | Issue 4 | e1000455