The mutant medaka library was generated and screened as schematically outlined in Figure 1. Founder fish were repeatedly mutagenized with ENU, crossed with wild-type females, and the progeny were used to establish a permanent cryopreserved resource of 5,771 F1 males (Table 1). To get an indication about the induced mutation frequency, we performed a specific locus test using the albino mutant 4. The appearance of a white-eyed embryo at a rate of 1 in 272 (Table 1) is in line with previously observed frequencies 4, suggesting that the mutagenesis was very effective.

The mutant library was screened for genes involved in tumor biology (p53, and Blm, encoding Bloom helicase), neurodegeneration (Parkin, encoding ubiquitin ligase), aging (Sirt1, encoding deacetylase), and miRNA metabolism (Dcr-1, encoding Dicer). Although a variety of mutation discovery technologies have been established for targeted retrieval of induced mutations 11121314, we chose to use dideoxy resequencing of PCR-amplified target sequences for routine mutation discovery 15, as this technology is robust and can be automated very well at both the experimental and data interpretation levels 16. Most importantly, it provides highly informative data about the exact location and nature of the mutation.

We screened the complete library for 10 different amplicons covering 20 exons in 5 different genes (Table 2). In total, about 22 Mbp were screened and 64 independent mutations were identified (Table 3). The average ENU-induced mutation frequency for the library was found to be 1 mutation per 345,000 bp, similar to what was found for reverse genetic screens in zebrafish 12. We retrieved highly likely loss-of-function mutations for four out of five genes screened by the identification of four nonsense and two splice site mutations. Although a full loss-of-function has to be demonstrated for each mutant individually, we refer to these mutants as knockouts in this paper. Furthermore, 38 missense mutations were found in the different genes (Tables 2 and 3), some of which could potentially result in a partial or complete loss-of-function or gain-of-function phenotype.

All nonsense and splice site mutants were recovered from the frozen sperm archive by in vitro fertilization (Table 4). A very high fertilization rate of more than 90% was consistently obtained following standard in vitro fertilization procedures, with only 7% to 33% of the fertilized eggs failing to develop and hatch. Genotyping tail fin tissue from a portion of F2 offspring revealed that the ratio of wild-type fish to mutant heterozygotes was about one-to-one, as expected (data not shown).

We identified seven induced mutations in the medaka p53 gene 17, including three missense mutations, one splice site, and two nonsense mutations (Figure 2). The p53^E241X allele is a G to T substitution that results in the alteration of Glu241 to a stop codon, whereas the p53^Y186X allele is a T to A substitution that alters Tyr186 to a stop codon. Both were presumed to result in a truncated protein that terminates prematurely in the midst of a DNA-binding domain. These proteins retain the amino-terminal transactivation domain but lack the nuclear localization signal and tetramerization domain required for full activity. Furthermore, no alternative splicing variants involving these mutation-containing exons are known in any species, indicating that these nonsense mutations are most likely to result in a null phenotype. All three missense mutations are at highly conserved residues within the DNA-binding region, but more detailed characterization will be needed to conclude anything about their effect on protein function.

Impaired target gene induction upon DNA damage is one of the phenotypes that is expected in a p53 knockout animal 18. p53^E241X/E241X embryos were γ-irradiated and the induction of p21, Mdm2 and Bax genes was examined by RT-PCR. As expected, no increase of these target genes was observed in p53^E241X/E241X homozygous fish, while control fish clearly showed upregulation of p21 and Mdm2 transcription level in response to ionizing radiation (IR), (Figure 3a). Interestingly, the basal level of the p53 transcript was decreased in p53^E241X/E241X fish. This could be due to nonsense-mediated decay 19 of mutant RNA, a phenomenon that is frequently observed in ENU-induced nonsense mutants (E Cuppen, unpublished observations), although an autoregulatory mechanism cannot be excluded. The same results were obtained for the second nonsense allele (p53^Y186X/Y186X; data not shown). Next, we investigated whether IR-induced apoptosis was affected in p53^E241X/E241X mutants. Primary cell cultures were derived from wild-type and p53^E241X/E241X fish, γ-irradiated, and observed by time-lapse video microscopy for apoptosis. While 13.2% (15 out of 142 cells counted) of p53^+/+ cells underwent apoptosis, none of the p53^E241X/E241X cells (0 out of 121 cells) showed fragmentation of the nucleus (Figure 3b). These results are consistent with a complete loss-of-function phenotype of p53 in these medaka mutants.

To monitor for spontaneous tumorigenesis, p53 knockout (p53^E241X/E241X, n = 21), heterozygote (p53^+/E241X, n = 26), and wild-type (p53^+/+, n = 10) littermates were raised to adulthood to monitor for spontaneous tumorigenesis. Only a single p53^+/+ fish died within 10 months after birth with no obvious signs of cancer (Figure 4). Heterozygous fish developed some tumors during the course of observation (two out of the five fish that died during the first ten months had clear tumors), but the mortality rate was relatively low. In contrast, a dramatic tumor predisposition was observed in the homozygotes, with the first incidence of tumorigenesis observed already at 2.5 months of age. The frequency of tumor formation increased after 5 months of age, resulting in a median lifespan of 228 days. All homozygous fish died within 10 months and 11 out of the 21 animals had clear tumors. The real tumor rate is most likely higher, as a significant part of the dead fish could unfortunately not be examined properly, due to rapid decomposition. It should be mentioned that at least 2 out of the 21 p53^E241X/E241X fish died without any macroscopic signs of tumors. The p53^Y186X/Y186X fish developed tumors as well but at a lower rate compared to the p53^E241X/E241X mutant. The median lifespan was also slightly increased (311 days), but was still much shorter than for wild-type fish (Figure 4). The difference in tumorigenesis between the two different nonsense alleles is not clear at this moment. We cannot exclude the possibility that co-segregating ENU mutations affect the predisposition to develop tumors in the p53^E241X background. The analysis of heteroallelic p53^E241X/Y186X fish and/or analysis of further outcrossed lines should resolve this issue.

Stereoscopic as well as histological characterization of tumor-bearing p53^E241X mutant fish revealed a wide variety of tumor types in kidney, eye, brain, intestine, gill, thymus and testis (Figures 5 and 6). In one case, where kidney is the primary origin, lymphoid cells spread throughout the interstitial space, destroying the normal architecture of renal tubules and glomeruli (Figure 5). This is consistent with the observation that the teleost kidney is developmentally a mesonephros, which is the site for hematopoiesis in adult fish and is thought to function analogously to the bone marrow in mammals 20. Considering a very low natural occurrence of tumors in young medaka (<0.01%) and the propensity of medaka to liver tumors 21, the diversity in tumor types and the high incidence of tumors observed in p53-deficient fish implicate that the p53 knockout medaka are highly susceptible to spontaneous tumorigenesis compared to their p53-proficient littermates, even though the number of fish examined in this study was relatively small.

In p53-deficient zebrafish, peripheral nerve sheath tumors were found to predominate 22. The difference in tumor spectrum may be caused by the type of mutation introduced in the genome, namely a missense mutation at a conserved residue in zebrafish versus a nonsense mutation in medaka, or by the presence of organism-specific secondary genes that are differentially involved in tumor susceptibility. This tissue specific tumor development in different species is of great interest as this phenomenon is also found in mammals: in Li-Fraumeni syndrome patients, caused by mutations in the human p53 gene, breast cancer and sarcomas are most common, whereas p53 knockout mice develop T cell lymphomas 2324. Such differences strengthen the need for parallel studies in multiple model organisms.

We identified a nonsense mutation that results in a truncated Parkin protein at Tyr314, eliminating the inbetween RING domain (IBR) and the second RING domain (RING2), which are critical for its ubiquitin ligase activity 25. Interestingly, a similar mutation, which results in Parkin protein truncation at Glu311, has been found in a human juvenile parkinsonism patient 26. For the Blm gene, the premature stop codon was introduced at position Glu497, which removes the entire critical helicase domain. Again, a similar 515 amino acid-long truncated protein has been reported in a human disease case that results from a 1 bp insertion prior to the helicase domain 27. It should be noted that the complete knockout of the Blm gene results in embryonic lethality in mice 28, while Blm mutant medaka fish are viable, similar to human. We expect that the medaka mutants of the Parkinsonism and Bloom syndrome genes may serve as valuable disease models, and are currently characterizing their phenotypes in detail.

Kyoto-Cab, a substrain of Cab, was mutagenized as described previously with slight modifications 4. Males (102; G0) were treated weekly with 3 mM ENU (Sigma-Aldrich, St. Louis, MO USA) in 10 mM sodium phosphate buffer (pH 6.3) at 26°C for 1 h. After the third treatment with ENU, the G0 were crossed with wild-type females to monitor the recovery of fecundity. A month after the last ENU treatment, crosses with wild-type females were set up and fertilized eggs were left to develop to full term, resulting in the mutant F1 library (only males were kept). The number of offspring produced from a single mutagenized male founder varied from 1 to 239, presumably reflecting variability in ENU-induced damage to the testis. Ten mutagenized male founders were crossed with albino fish (Heino) to monitor the mutagenesis efficiency using a single locus test.

The sperm from each F1 medaka was cryopreserved as described in Section 3.3.1 of the medaka protocols book 39. The sperm was suspended in 60 μl of freezing medium (10% dimethylformamide in fetal calf serum) and was divided into 6 glass capillaries. The amount of sperm held in each capillary was enough to fertilize more than 100 eggs.

After removing the testis, fish were cut in two halves and kept at -80°C until DNA was extracted. The tail side of the fish was incubated overnight at 55°C in 500 μl of lysis buffer containing 10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0), 120 mM sodium chloride, 12 mM sodium citrate (pH7.0), 1% SDS, and 200 μg/ml of proteinase K (Sigma-Aldrich). The lysate was phenol extracted and precipitated with isopropanol. The DNA pellet was dissolved in 1 ml of TE (10 mM Tris pH 7.5, 1 mM EDTA, pH8.0). The concentration was adjusted to 40 ng/μl, aliquoted into 96 deep well plates, and stored at -20°C.

For p53, both the cDNA and genomic sequence available (NCBI accession AF212997) have been published 1737. For Blm, Sirt1 and Parkin the medaka cDNA sequences were determined using a combination of RT-PCR and rapid amplification of cDNA ends (RACE). Overall, the similarity of the encoded medaka proteins to their human counterparts was 80%, 80% and 90%, respectively, and the identity was 42%, 50% and 56%, respectively. The cDNA sequences were used to retrieve the genomic sequence from the draft medaka genome assembly 8. For Dcr-1, the zebrafish protein sequence was used in combination with gene prediction algorithms to retrieve the genomic sequences required for amplicon design. For all genes, only a single ortholog of the human gene was identified in the medaka draft genome. All genes were imported in the laboratory information management system for the identification of mutations by sequencing or TILLING: LIMSTILL 40 to facilitate amplicon design for screening and mutation annotation. Oligonucleotides for the amplification of exons of interest by nested PCR were designed in LIMSTILL. Details on oligonucleotides used can be obtained from the authors upon request. For universal processing at the sequencing stage, the primers for the second PCR reaction contained M13 tails. The sequence of the universal tails and M13 oligonucleotides used for sequencing were: M13-Forward, TGTAAAACGACGGCCAGT; and M13-Reverse, AGGAAACAGCTATGACCAT.

Genomic DNA stocks were diluted 25-fold and gridded out as 5 μl aliquots into 384 well plates. The first PCR (PCR1) was carried out using a touchdown thermocycling program (92°C for 60 s; 12 cycles of 92°C for 20 s, 65°C for 20 s with a decrement of 0.6°C per cycle, 72°C for 30 s; followed by 20 cycles of 92°C for 20 s, 58°C for 20 s and 72°C for 30 s; 72°C for 180 s; GeneAmp9700, Applied Biosystems, Foster City, CA, USA). This reaction contained 5 μl genomic DNA, 0.2 μM forward primer and 0.2 μM reverse primer, 400 μM of each dNTP, 25 mM tricine, 7.0% (w/v) glycerol, 1.6% (w/v) DMSO, 2 mM MgCl₂, 85 mM ammonium acetate pH 8.7 and 0.2 U Taq polymerase in a total volume of 10 μl. After thermocycling, the PCR1 reactions were diluted with 25 μl water, mixed by pipetting, and 1 μl was used as template for the second round of PCR. The second PCR (PCR2) was done using a standard thermocycling program (92°C for 60 s; 30 cycles of 92°C for 20 s, 58°C for 20 s and 72°C for 30 s; 72°C for 180 s; GeneAmp9700, Applied Biosystems). PCR2 mixes contained 1 μl diluted PCR1 template, 0.1 μM forward primer, 0.1 μM reverse primer, 100 μM of each dNTP, 25 mM tricine, 7.0% (w/v) glycerol, 1.6% (w/v) DMSO, 2 mM MgCl₂, 85 mM ammonium acetate pH 8.7 and 0.1 U Taq polymerase in a total volume of 5 μl. Several samples of each amplicon were tested on a 1% agarose gel containing ethidium bromide for the presence of the proper PCR fragment.

PCR2 products were diluted with 20 μl water and 1 μl was directly used as template for the sequencing reactions. Sequencing reactions, containing 0.12 μl BigDYE (v3.1; Applied Biosystems), 1.88 μl 2.5× dilution buffer (Applied Biosystems) and 0.4 μM universal M13 primer in a total volume of 5 μl, were performed using cycling conditions recommended by the manufacturer (40 cycles of 92°C for 10 s, 50°C for 5 s and 60°C for 120 s). Sequencing products were purified by ethanol precipitation in the presence of 40 mM sodium acetate and analyzed on a 96-capillary 3730XL DNA analyzer (Applied Biosystems), using the standard RapidSeq protocol on a 36 cm array. Sequences were analyzed for the presence of heterozygous mutations using PolyPhred 41 and manual inspection of the mutated positions. Every candidate mutation was verified by an independent PCR and resequencing read. Nucleotide variations that were present in more than two F1 fish were considered to be single nucleotide polymorphisms (SNPs) and, therefore, excluded from further analysis, while mutations found in only two animals were included, as examination of the breeding records revealed that, in most cases, these originated from the same mutagenized parent and are thus most likely to be derived from the same spermatogonial stem cell. These mutations are counted as a single mutation.

About 100 unfertilized eggs were squeezed out from the wild-type cab females. A single glass capillary containing 10 μl of sperm from the F1 fish identified in the screening was removed from the liquid nitrogen and thawed by placing at ambient temperature. Immediately after the sperm was thawed, the content was pushed out in balanced salt solution (BSS; 0.65% sodium chloride, 0.04% potassium chloride, 0.02% magnesium sulfate heptahydrate, 0.02% calcium chloride dihydrate, 0.00005% phenol red, 0.01% sodium hydrogen carbonate, pH 7.3) and incubated with the eggs for 20 minutes with occasional pipetting. The eggs that were not fertilized were removed 3 h later, and BSS was replaced with 0.03% Red Sea salt water. The eggs were incubated at 28°C until they hatched. The quality of thawed sperm and the fertilization rate was checked under the microscope. Only a single cryopreserved straw (out of six straws frozen in total) was needed for successful recovery of each mutation of interest. For each in vitro fertilization, between 66 and 88 fish were obtained that developed to adulthood. Progeny were genotyped by sequencing and heterozygous fish carrying the mutation of interest were incrossed to obtain homozygous fish.

For most of the studies, the p53^E241X allele was used. F2 p53^+/E241X heterozygous fish resulting from the in vitro fertilization were incrossed to produce F3 progeny of all genotypes. Four days post-fertilization, F3 embryos were irradiated with 20 Gy of ionizing radiation using ¹³⁷Cs (0.02 Gy/s, Gammacell 40, Atomic Energy of Canada Limited Industrial Products, Ontario). Six hours later, the embryos were frozen in liquid nitrogen and RNA was extracted by Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction. The embryos were genotyped by PCR and resequencing of the simultaneously extracted genomic DNA. cDNA was synthesized from each genotype using SuperScript III (Invitrogen). The mRNA expression levels were determined by PCR reactions (94°C for 1 minute; predetermined cycles of 94°C for 30 s, 55°C for 20 s, 72°C for 30 s). The numbers of cycles used were: β-actin, 15; Mdm2, 24; and p53, p21 and Bax, 26. Details on oligonucleotides used can be obtained from the authors upon request.

Primary cell cultures derived from p53^E241X/E241X and p53^+/+ embryos were obtained as described previously 42. Cells (1.5 × 10⁵) were inoculated in a 35 mm dish and irradiated with 10 Gy of γ-rays. The cells were monitored for fragmentation using a IX81 inverted microscope (Olympus, Tokyo, Japan) controlled by IPLab software (BD Biosciences, Rockville, MD, USA) from zero to eight hours after γ-irradiation.

After tumors were observed under the stereomicroscope, fish were fixed in 4% paraformaldehyde for 24 hours and embedded in paraffin. Tissue sections were stained by standard hematoxylin-eosin staining. Photographs of the slides were obtained by a VC4500G digital camera (Omron, Kyoto, Japan) mounted on an ECLIPSE E800 microscope (Nikon, Tokyo, Japan).