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Identification of radiation-sensitive mutants in the Medaka, Oryzias latipes
Mechanisms of Development 121 (2004) 895–902 www.elsevier.com/locate/modo
Research paper
Identification of radiation-sensitive mutants in the Medaka, Oryzias latipes Kouichi Aizawaa, Hiroshi Mitania,*, Nozomi Kogurea, Atsuko Shimadab, Yukihiro Hirosec, Takao Sasadod, Chikako Morinagad, Akihito Yasuokae, Hiroki Yodaf, Tomomi Watanabeg, Norimasa Iwanamih,i, Sanae Kunimatsuh, Masakazu Osakadaj, Hiroshi Suwad, Katsutoshi Niwad, Tomonori Deguchif, Thorsten Hennrichd, Takeshi Todoj, Akihiro Shimaa, Hisato Kondohd,f, Makoto Furutani-Seikid a
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa-no-ha 5-1-5, Kashiwa, Chiba 277-8572, Japan b Department of Biological Sciences, The University of Tokyo, Tokyo, Japan c Graduate School of Biostudies, Kyoto University, Kyoto, Japan d Kondoh Differentiation Signaling Project, JST, Sakyo-ku Kyoto, Japan e Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan f Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan g Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan h Division of Experimental Immunology, Institute for Genome Research, The University of Tokushima, Tokushima, Japan i Research Center for Allergy and Immunology, RIKEN, Saitama, Japan j Radiation Biology Center, Kyoto University, Kyoto, Japan Received 26 December 2003; received in revised form 5 March 2004; accepted 3 April 2004
Abstract We screened populations of N-ethyl-N-nitrosourea (ENU)-mutagenized Medaka, (Oryzias latipes) for radiation-sensitive mutants to investigate the mechanism of genome stability induced by ionizing radiation in developing embryos. F3 embryos derived from male founders that were homozygous for induced the mutations were irradiated with g-rays at the organogenesis stage (48 hpf) at a dose that did not cause malformation in wild-type embryos. We screened 2130 F2 pairs and identified three types of mutants with high incidence of radiationinduced curly tailed (ric) malformations using a low dose of irradiation. The homozygous strain from one of these mutants, ric1, which is highly fertile and easy to breed, was established and characterized related to g-irradiation response. The ric1 strain also showed higher incidence of malformation and lower hatchability compared to the wild-type CAB strain after g-irradiation at the morula and pre-early gastrula stages. We found that the decrease in hatching success after g-irradiation, depends on the maternal genotype at the ric1 locus. Terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling assays showed a high frequency of apoptosis in the ric1 embryos immediately after g-irradiation at the pre-early gastrula stage but apoptotic cells were not observed before midblastula transition (MBT). The neutral comet assay revealed that the ric1 mutant has a defect in the rapid repair of DNA double-strand breaks induced by g-rays. These results suggest that RIC1 is involved in the DNA double strand break repair in embryos from morula to organogenesis stages, and unrepaired DNA double strand breaks in ric1 trigger apoptosis after MBT. These results support the use of the ric1 strain for investigating various biological consequences of DNA double strand breaks in vivo and for sensitive monitoring of genotoxicity related to low dose radiation. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Medaka; Oryzias latipes; Development; Mutagenesis; Radiation; ric; Apoptosis; Repair; Malformation; Double strand break
1. Introduction * Corresponding author. Tel./fax: þ 81-471-36-3670. E-mail address: [email protected] (H. Mitani). 0925-4773/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mod.2004.04.002
Exposure to ionizing radiation at embryonic stages may predispose organisms to a high risk of developing diseases
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(Carlson and Desnick, 1979), and harmful mutations in germ cells can be transmitted to descendants (Bakker et al., 1987). Russell and Russell (1957) reported that lethality and malformations in mouse embryos induced by ionizing irradiation was highly dependent on the developmental stage. The preimplantation stage was highly sensitive to lethal irradiation effects, whereas very little increase in malformation frequency was due to irradiation during the preimplantation period. Most experimental data on the effects of radiation on developing embryos have been obtained from studies on the mouse or rat in utero because these species have high rates of reproduction. However, our knowledge of the DNA repair and cell cycle regulation in utero is limited. For example, it is not known which DNA repair pathway provides the greatest protection against malformation or lethality, or how the apoptosis pathway is activated in embryos before and after MBT. The small fish species, Medaka (Oryzias latipes), is a useful experimental animal because transparent embryos (. 20 embryos/female) that develop outside of the mother’s body are obtained every day thereby allowing the cellular effects of radiation—such as cell death—to be easily be observed during all stages of embryogenesis. The availability of inbred strains also enables qualitative and quantitative analysis of the effects of radiation using uniform genetic backgrounds. There are many reports on hatchability and developmental abnormalities of Medaka embryos irradiated at early developmental stages and in F1 embryos obtained by mating irradiated males with non-irradiated females (Hyodo-Taguchi et al., 1973; Ishikawa and Hyodo-Taguchi, 1997). Shima and Shimada (1991) reported extensive data for the Medaka specific-locus test system, which can detect the germ line mutagenesis after exposure to environmental mutagens, including ionizing radiation. Mutant Medaka strains that are sensitive to ionizing radiation will thus enable us to examine the molecular functions of genes responsible for genomic stability in early embryos. Prospective genetic screening is an ideal approach to search for functions of genes responsible for genome stability in vivo. N-ethyl-N-nitrosourea (ENU), which predominantly induces point mutations (Harbach et al., 1992), has been used for producing and isolating mutations that produce developmental anomalies during embryonic development in the mouse, zebrafish and Medaka (Hrabe de Angelis et al., 2000; Haffter et al., 1996; Loosli et al., 2000). Here, we report the isolation of three mutations associated with a high incidence of radiation-induced malformations of the tail. One of the three mutants, ric1, exhibited a defect in rejoining of DNA double-strand breaks (DSBs) at the pre-early gastrula stage. We also found that the maternal genotype at the ric1 locus determines hatchability after g-irradiation, even at the organogenesis stage, which is a developmental stage at high risk of radiation-induced malformation in vertebrates.
2. Results 2.1. Design of screening by g-ray irradiation The dose of g-ray irradiation used for screening was determined by examining lethality rates in wild-type CAB strain embryos irradiated at the organogenesis stage (stage 25). We selected this stage because irradiation at the corresponding stage in mouse induces high incidences of both abnormalities and neonatal death (Russell and Russell, 1957). After irradiating with 25 Gy (see Section 4) at the organogenesis stage, lethality in wild-type CAB strain embryos was about 80% (data not shown). After irradiation with lower doses (10.2 Gy), lethality rates were less than 10%. 2.2. Isolation of three mutants associated with a high incidence of radiation-induced curly tail malformation The first screening for radiation-sensitive mutants was carried out by irradiating F3 embryos of the mutagenized male founders with g-rays (9.1 Gy) at Radiation Biology Center, Kyoto University, at 48 h post-fertilization (hpf), which corresponds to the organogenesis stage of mouse. Morphological abnormalities were examined at 5 or 6 days post-fertilization (dpf). After screening 2130 F2 pairs, we identified 352 F2 pairs whose F3 embryos showed severe malformations, mainly related to tail or eye development. The typical malformation phenotypes (Fig. 1) of these candidate mutants were roughly similar to those frequently observed in the wild-type CAB strain after severe irradiation of g-rays (data not shown). We then examined whether the F3 embryos display malformations without g-irradiation. For the 122 F2 pairs whose non-irradiated F3 embryos developed normally, we investigated whether there were significant differences in lethality between F3 embryos irradiated or not irradiated with 8.4 Gy to select pairs showing higher sensitivity to g-irradiation. Three pairs were selected, whose progenies showed high and stable sensitivity in terms of induced severe malformations such as curly tail malformations and were also easy to maintain as homozygotes for each of the mutations. Complementation analyses from crosses of different homozygous strains demonstrated that these three strains have mutation at three different genetic loci (Table 1). Therefore, these strains were named ric (radiation-induced curly tailed) 1, ric2 and ric3, respectively. 2.3. Homozygous ric1 embryos show radiation sensitivity at various developmental stages A strain homozygous for ric1, which is highly fertile and easy to breed, was established and characterized related to response to g-irradiation at various developmental stages. We examined hatchabilities and malformation rates of the embryos irradiated at the morula (stage 8), pre-early gastrula (stage 12) and organogenesis
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Fig. 1. Typical morphogenetic abnormalities of the ric1 homozygotes after. g-irradiation (a) A wild-type (CAB strain) embryo at 2 days post-fertilization (dpf); (b) A wild-type embryo at 5 dpf; (c) A wild-type embryo at 5 dpf irradiated with 9.1 Gy of g-rays at 48 h after fertilization; (d) A ric1 homozygote embryo at 2 dpf; (e) ric1 homozygote embryos at 5 dpf; (f)–(k) Examples of ric1 embryos at 5 dpf irradiated with 9.1 Gy of g-rays at 48 h after fertilization showing (f) curly tail; (g) bent tail; (h) spherical eyes; (i) protruding lens; (j) enlarged ear vesicle; (k) cross-eyes and (l) red eyes. More than 50% of malformed embryos had curly tails.
stages (stage 25) (Table 2). After irradiation with 1.0 Gy at the radiation sensitive morula stage, although no significant increase of embryonic abnormality was observed for CAB embryos (hatchability, 90.4%, malformation rate, 0%), the ric1 embryos exhibited reduced hatchability (23.8%) and elevated malformation rates (54.3%). The embryos homozygous for ric1 irradiated with 5.1 Gy at the pre-early gastrula stage, showed reduced hatchability (36.1 vs. 94.2%) and an elevated malformation rate (71.5 vs. 4.7%) compared with wildtype embryos. Similarly, after irradiation with 10.2 Gy at the organogenesis stage, hatchability was decreased (4.1%) and the malformation rate increased (70.9%) in the ric1 homozygous embryos (Fig. 2). Malformations of the tail (curly and bent tails) were typically observed, with curly tails being the most frequent ric1 malformation (Table 3). These results suggest that the ric1 homozygous embryo is sensitive to radiation beginning at the molura and extending through the organogenesis stage. We also investigated the radiation sensitivity of embryos homozygous for ric2 and ric3 mutations. After irradiating ric2 homozygous embryos with 10.2 Gy at the organogenesis stage, we observed a reduction in hatchability (4.9%) and an increase in malformation rate (53.5%). Similar results were found for the embryos homozygous for ric3 (hatchability 6.3%; malformation rate 72.9%, Table 2).
Table 1 Hatchability and malformation rate of embryos irradiated at the organogenesis stage Genotype
Dose Number of Malformation Hatchability (Gy) examined rate (%) (%) embryos
(þ /þ ) £ (þ /þ )
0 10.2 0 10.2 0 10.2 0 10.2 0 10.2 0 10.2 0 10.2 0 10.2 0 10.2 0 10.2
(ric1/þ) £ (ric1/þ) (ric1/ric1) £ (ric1/ric1) (ric2/þ) £ (ric2/þ) (ric2/ric2) £ (ric2/ric2) (ric3/þ) £ (ric3/þ) (ric3/ric3) £ (ric3/ric3) (ric1/ric1) £ (ric2/ric2) (ric2/ric2) £ (ric3/ric3) (ric3/ric3) £ (ric1/ric1)
86 348 76 106 72 247 53 54 153 144 91 89 47 48 48 176 53 139 61 170
0 0.6 0 20.8 2.8 70.9 0 11.2 4.6 53.5 0 16.9 4.3 72.9 6.3 21.5 5.7 30.9 6.6 12.9
97.7 90.2 97.4 67.9 94.5 4.1 98.1 70.4 97.4 4.9 98.9 76.4 93.6 6.3 95.8 51.1 94.3 64.0 95.1 50.6
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Table 2 Hatchability and malformation rate of embryos irradiated at early embryonic stage and organogenesis stage
2.4. The maternal genotype in ric1 affects on hatchability after g-irradiation
Irradiated stage
Strain Dose Number of Malformation Hatchability (Gy) examined rate (%) (%) embryos
Morula stage (stage 8)
CAB (WT)
We next examined the hatchability of embryos obtained by the four types of crosses: C (þ /þ ) £ F (þ /þ ), C (þ /þ ) £ F (ric1/ric1), C (ric1/ric1) £ F (þ /þ ) and C (ric1/ric1) £ F (ric1/ric1). To examine whether the maternal genotype in ric1 could modify the radiosensitivity of embryos before MBT. After 2 Gy g-irradiation at stage 8, the hatchabilities of wild-type and the ric1 homozygous embryos were 66.7 and 0%, respectively. The hatchability of the embryos produced from wild-type eggs fertilized with sperm from the males homozygous for ric1 was 65.7%, which is similar to that of wild-type embryos, whereas the hatchability of the embryos obtained from the reverse cross was significantly lower (31.5%). We found that maternal genotype in ric1 locus could modify the hatchability after 10.2 Gy g-irradiation at stage 25, the organogenesis stage.
ric1
Pre-early gastrula CAB stage (stage 12) (WT) ric1
Organogenesis stageb (stage 25)
CAB (WT)
ric1 ric2 ric3 a b
0
104
0
94.2
1.0 0 1.0
396 84 320
0 3.6 54.3a
90.4 84.5 23.8
84
0
98.8
380 74 288
4.7 8.1 71.5a
94.2 81.1 36.1
0
86
0
97.7
10.2 0 10.2 0 10.2 0 10.2
348 72 247 153 144 47 48
0.6 2.8 70.9 4.6 53.5 4.3 72.9
90.2 94.5 4.1 97.4 4.9 93.6 6.3
0 5.1 0 5.1
Low malformation rate of curly tail. These results are entered in Table 1.
2.5. g-Irradiation induces apoptosis as an immediate response in ric1 homozygous embryos To examine the immediate responses of the ric1 homozygous embryos to g-irradiation, we carried out the terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick end-labeling (TUNEL) assay on whole mount embryos. Radiation-induced apoptotic cells are shown as TUNEL positive cells. No TUNEL-positive cells were detected in embryos irradiated at the morula stage. We therefore selected the pre-early gastrula stage for the TUNEL assay, and used 5.1 Gy irradiation for 30 min. TUNEL-positive cells were found in 23.9% of the wild-type embryos and 63.6% of the ric1 homozygous embryos
Fig. 2. Effects of the maternal genotype ric1 on hatchability after g-irradiation. Hatchabilities were examined in embryos obtained from four types of crosses: wt C £ wt F; wt C £ ric1 F; ric1 C £ wt F and ric1 C £ ric1 F. We irradiated stage 8 embryos with 2 Gy and stage 25 embryos with 10.2 Gy of g-rays. Vertical bars indicate upper and lower 95% confidence limits.
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Table 3 Malformation of embryos irradiated at early embryonic stage and organogenesis stage Irradiated stage (dose)
Morula (1.0 Gy) Pre-early gastrula (5.1 Gy) Organosenesis stage (10.2 Gy)
Strain
CAB ric1 CAB ric1 CAB ric1
Malformation rate (%)
0 54.3 4.7 71.5 0.6 70.9
Number of examined embryos
396 320 380 288 348 247
Malformation c.t.
b.t.
s.e.
p.l.
b.e.
c.e.
r.e.
s.h.
0 10 1 32 0 104
0 84 3 96 2 59
0 51 1 20 0 12
0 12 0 17 0 18
0 23 1 26 0 20
0 0 0 0 0 2
0 8 0 5 0 1
0 36 12 26 0 0
c.t., curly tail; b.t., bent tail; s.e., spherical eyes; p.e., protruding lens; b.e., enlarged ear vesicle; c.e., cross-eyed; r.e., red eyes; s.h., small head.
(Fig. 3). Thus, the ric1 mutant shows high incidence of apoptosis after g-irradiation. 2.6. Rejoining of DNA double-strand breaks at the pre-early gastrula stage To compare the extent of rejoining of DNA DSBs in early embryonic cells of wild type and the ric1 homozygous embryos exposed to g-irradiation, we performed the neutral single cell microgel electrophoresis assay (neutral comet assay). The relative incidence of DNA DSBs in each cell can be quantified sensitively using this method. The radiation dose used in this study was 15.2 Gy because of the difficulty in detecting DSBs by neutral comet assay at lower doses of irradiation quantitatively. Tail moment (TM) was used as an index to evaluate the relative number of DNA strand breaks. The typical results of neutral comet assay at 0, 30 min and 1 h after irradiation are shown in Fig. 4A. The comet TMs of extracted DNA fragments immediately after 15.2 Gy g-irradiation in both the wildtype and the ric1 embryos were 58.5 ^ 7.2 and 61.3 ^ 5.4,
respectively (Fig. 4B). The TM for the wild-type embryo decreased to the control level 30 min after irradiation (26.7 ^ 3.6). This suggests that almost all DSBs induced in early embryonic cells of wild-type embryos were rejoined within 30 min after irradiation. However, the TM for the ric1 homozygous embryos was not decreased to control levels even 1 h after irradiation (48.4 ^ 4.2). This suggests that the early embryonic cells have high capacity of DSBs repair in which ric1 products are involved.
3. Discussion DNA repair is involved in processes that minimize cell killing, mutations, replication errors, persistence of DNA damage and genomic instability. Abnormalities in these processes found in DNA repair deficient mutants of mammals have been implicated in cancer and aging. The ric1 is the first radiation-sensitive fish strain with the defect in DNA double strand break repair. The fish combine the benefits of both a distinct and easily
Fig. 3. Gamma-irradiation induced apoptotic cells as detected by TUNEL assays. (A) Typical flattened whole mount specimen (ric1 strain; 30 min after exposure to 5.1 Gy of g-rays. TUNEL positive cells were found (arrow). (B) Frequency of embryos with TUNEL positive cells after g-irradiation. Embryos were irradiated at pre-early gastrula stage (5.1 Gy). The TUNEL assay was performed 30 min later. The presence of more than five TUNEL-positive cells was used to define an embryo as having apoptosis. Vertical bars indicate upper and lower 95% confidence limits.
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Fig. 4. DNA double-strand break (DSB) repair at the pre-early gastrula stage after g-irradiation, showing typical comet tails produced by the neutral comet assay. (a) Non-irradiated control comet (wild-type CAB strain); (b) Immediately after exposure to 15.2 Gy of g-rays (CAB strain); (c) One hour after exposure to 15.2 Gy of g-rays (CAB strain); (d) Immediately after exposure to 15.2 Gy of g-rays (ric1 mutant strain); (e) One hour after exposure to 15.2 Gy of g-rays (ric1). (B) Tail moments in early embryonic cells exposed to 15.2 Gy of g-rays as a function of post-exposure incubation time. Each point represents the mean tail moment (mean ^ SEM).
identifiable phenotype for a mutation, with a greater sensitivity to induction of mutations (at least to radiation), in a fish species with a proven record as a valuable model for germ-line mutagenesis, radiation biology, teratogenesis, carcinogenesis, aging and environmental monitoring. We examined hatchability and malformation rates of the ric1 homozygous embryos irradiated at the early embryonic stage (morula and pre-early gastrula stage) and the organogenesis stage. A dose that had no effect on the hatchability of wild-type embryos was selected for each stage. The mutant embryos showed lower hatchability and higher malformation rates upon exposure to g-irradiation, not only at the organogenesis stage used for screening but also at the morula and pre-early gastrula stages. Few embryos irradiated at the early embryonic stage showed a high incidence of radiation induced curly tailed malformation comparable to the embryos irradiated at the organogenesis stage, but many embryos did show a small head malformation. These results suggest that the ric1 homozygous embryo is radiation-sensitive even at the early embryonic stage and at organogenesis stage. Highly proliferating cells in the tail at organogenesis stage are sensitive to radiation and loss of these cells could be a major cause for high incidence of malformation in ric1 mutants. We further investigated the maternal effects of the ric1 gene by examining hatchabilities of the embryos obtained by four types of crosses. It was suggested that the ric1 gene encodes for maternal factors that determine survival rate after irradiation not only before MBT but also at the organogenesis stage.
We performed the neutral comet assay at the pre-early gastrula stage to examine the rejoining of DSBs induced by g-irradiation and found the high capacity of rapid DSB repair in early embryos of the Medaka. On the other hand, the ric1 mutant strain is defective in DSB repair. Shoji et al. (1998) reported that intrauterine deaths in scid mouse embryos, which are deficient in rejoining radiation-induced DSBs, increased with radiation dose and that the mortality was substantially higher than in the wild-type strain. It has been suggested that radiationinduced apoptosis is a p53-dependent event in the late period of mouse organogenesis but that p53-dependent apoptosis prevents malformations in the preimplantation stages as well as in early organogenesis (Norimura et al., 1996; Wang, 2001). Moreover, it has been found that the mouse embryo becomes hypersensitive to DNA damage caused by X-rays and undergoes p53-dependent apoptosis without cell cycle arrest during gastrulation (Heyer et al., 2000). In the present study in the Medaka, apoptotic cells were detected at the pre-early gastrula stage and it is possible that the ric1 gene may also play a role in DNA surveillance mechanisms to ensure genomic integrity, both via the p53-mediated apoptotic pathway and DSBs repair. If this is a case, cloning the ric1 mutation will open an avenue for understanding the molecular mechanisms underlying the repair of DNA damage caused by g-irradiation. We are now establishing cultured cell lines from ric1 embryos that show higher radiosensitivity to make it possible to identify the mutated gene by candidate positional cloning method using the high density linkage map that we established (Naruse et al., 2004).
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4. Experimental procedures 4.1. Medaka strain The Medaka strain CAB originated from the Carolina Biological Supply Company (NC) and has been inbred for more than 30 generations (Loosli et al., 2000). Fish were kept in the system with recirculating water under a 14 h light and 10 h dark cycle at 27 8C. Fish were fed powdered fish food (Tetra-min, Tetra Werke Co., Mells, Germany) and brine shrimp (Artemia franciscana) three times a day. Embryos from natural matings in a plastic aquarium (water temperature was kept at 27 ^ 2 8C) were collected and incubated at 27 8C. Embryos were staged according to Iwamatsu (1994). The main features of each stage of embryo development were as follows. Stage 8, early morula: blastomeres (64 –128) are arranged in three layers. Stage 12, pre-early gastrula: the blastomeres have flattened onto the yolk sphere, and the cell layers are slightly thicker on one side. Stage 25: 18 –19 somites are present (onset of blood circulation). 4.2. Irradiation and incubation of embryos In radiation biology, ‘dose’ has a more specific meaning—it is the energy ionizing radiation absorbed per unit mass of any material. Special units exist for dose, including the ‘rad’, which is defined as 100 erg/g, and the ‘gray’(Gy), which defined as absorption of 1 J/kg. The absorbed-dose rate was monitored by both a radiophotoluminescent glass dosimeter (SC-1 with a Reader FGD-201, Toshiba Glass Co.) and a Fricke dosimeter. Embryos in water at room temperature were exposed to g-rays from a 137Cs source with dose rates of 3.8 Gy/min (used for first screening at Radiation Biology Center, Kyoto University, Japan) 0.95 Gy/min (used for second screening Gammacell 6000 Elan, MDS Nordion, Ottawa, Canada at Research Center for Nuclear Science and Engineering, University of Tokyo, Japan), or 10.2 Gy/min (used for other experiments, Gammacell 3000 Elan, MDS Nordion, Ottawa, Canada). Clusters of fertilized eggs were collected every morning. Embryos were subjected to g-irradiation in a plastic tube. After irradiation, embryos were placed into 96-well plastic microtiter plates (one embryo per well) and incubated at 27 8C. It is difficult for us to set the same dose of g-irradiation because we used different machines with different dose rate. Results of the first screening (9.1 Gy) were confirmed in the second and third screenings (10.2 Gy). 4.3. TUNEL assay A commercial kit (Apoptosis In Situ Detection Kit, Wako Pure Chemical Co., Osaka, Japan) was used for the assay (Yager et al., 1998). Embryos were fixed in Bouin’s solution. After treatment with proteinase K, end labeling
901
was performed using TdT. Strongly colored cells were counted as TUNEL-positive cells, and embryos with more than five TUNEL-positive cells were counted as TUNELpositive embryos. 4.4. Neutral comet assay Immediately after removing the chorion of the embryo at 4 8C, a cell suspension was prepared from the blastoderm in phosphate buffered saline (PBS, pH 7.4), and 100 ml of the cell suspension was added to 100 ml of 2% (w/v) lowmelting-point agarose (Nacalai Tesque, Kyoto, Japan). Then, 150 ml of this suspension was applied to the surface of a microscope slide (Matsunami Glass Inc., Osaka, Japan) to form a microgel and was allowed to set at 4 8C for 5 min. Slides were submersed in cell lysis buffer (2% SDS, 0.03 M EDTA, pH 8.0) for 30 min at 4 8C in the dark. Following cell lysis, all slides were washed through three changes of deionized water at 10 min intervals to remove salt and detergent from the microgels. Slides were placed in a horizontal electrophoresis unit and allowed to equilibrate for 5 min with TAE buffer before electrophoresis (50 V) for 7 min. After electrophoresis, slides were rinsed three times with deionized water and stored protected from light until analysis. Microgels were stained with SYBR Green (1:10,000 dilution; BioWhittaker Molecular Applications, Rockland, ME, USA) for 5 min. Slides were rinsed briefly with distilled water and covered with coverslips before image analysis. More than 70 cells were randomly analyzed per slide and scored for comet tail parameters as previously defined (Bocker et al., 1997). TM was used in this study to evaluate the relative DNA damage. TM ¼ (the percentage of tail DNA) £ (the tail length), where comet tail length is the maximum distance the damaged DNA migrates from the center of the cell nucleus, and the percentage of tail DNA is the total percentage of nuclear DNA that migrates from the nucleus into the comet tail. However, the DNA patterns per se, formed from the arrays of DNA fragments that migrate away from the nucleus, were not analyzed (Tice et al., 2000; Czene et al., 2002).
Acknowledgements We thank Yasuko Okamoto, Yasuko Kota, Toshiyuki Yamanaka, Ayako Yamaguchi, Emiko Nishimura (ERATO) and Tomoko Ishikawa, Jun Hirayama, Yuri Kobayashi, Junya Tomida, Haruka Tomida, Kazunori Jikihara (Kyoto University) for taking care of the fish facility and Hoshio Eguchi at the Research Center for Nuclear Science and Engineering, The University of Tokyo, for the use of a g-ray irradiator. We also thank Dr Richard Winn (Aquatic Biotechnology and Environmental Lab, University of Georgia) for many valuable comments and editing on an earlier version of the manuscript. This work was supported by the ERATO grant from Japan Science and
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Technology Agency, Japan to H.K. and by Grant-in-Aid for Scientific Research Priority Area #813, from the Ministry of Education, Science, Sports, Culture and Technology (MEXT), Japan to H.M. and A.S.
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Ishikawa, Y., Hyodo-Taguchi, Y., 1997. Heritable malformations in the progeny of the male medaka (Oryzias latipes) irradiated with X-rays. Mutat. Res. 389, 149 –155. Iwamatsu, T., 1994. Stages of normal development in the medaka Oryzias latipes. Zool. Sci. 11, 825– 839. Loosli, F., Koster, R.W., Carl, M., Kuhnlein, R., Henrich, T., Mucke, M., et al., 2000. A genetic screen for mutations affecting embryonic development in medaka fish (Oryzias latipes). Mech. Dev. 97, 133 –139. Naruse, K., Tanaka, K., Mita, K., Shima, A., Postlethwait, J., Mitani, H., 2004. A medaka gene map: the trace of ancestral vertebrate protochromosomes revealed by comparative gene mapping. Genome Res. in press. Norimura, T., Nomoto, S., Katsuki, M., Gondo, Y., Kondo, S., 1996. p53 dependent apoptosis suppresses radiation induced teratogenesis. Nat. Med. 2, 577–580. Russell, L.B., Russell, W.L., 1957. An analysis of the changing radiation response of the developing mouse embryo. J. Cell Physiol. 43, 1030–1049. Shima, A., Shimada, A., 1991. Development of a possible nonmammalian test system for radiation-induced germ-cell mutagenesis using a fish, the Japanese medaka (Oryzias latipes). Proc. Natl Acad. Sci. USA 88, 2545–2549. Shoji, S., Watanabe, H., Katoh, O., Masaoka, Y., Matsuura, S., Tauchi, H., et al., 1998. Developmental malformation and intrauterine deaths in gray-irradiated scid mouse embryos. Int. J. Radiat. Biol. 73, 705–709. Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., et al., 2000. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen 35, 206–221. Wang, B., 2001. Involvement of p53-dependent apoptosis in radiation teratogenesis and in the radioadaptive response in the late organogenesis of mice. J. Radiat. Res. 42, 1–10. Yager, T.D., Ikegami, R., Rivera-Bennetts, A.K., Zhao, C., Brooker, D., 1998. High-resolution imaging at the cellular and subcellular levels in flattened whole mounts of early zebrafish embryos. Biochem. Cell Biol. 75, 535–550.
Research paper
Identification of radiation-sensitive mutants in the Medaka, Oryzias latipes Kouichi Aizawaa, Hiroshi Mitania,*, Nozomi Kogurea, Atsuko Shimadab, Yukihiro Hirosec, Takao Sasadod, Chikako Morinagad, Akihito Yasuokae, Hiroki Yodaf, Tomomi Watanabeg, Norimasa Iwanamih,i, Sanae Kunimatsuh, Masakazu Osakadaj, Hiroshi Suwad, Katsutoshi Niwad, Tomonori Deguchif, Thorsten Hennrichd, Takeshi Todoj, Akihiro Shimaa, Hisato Kondohd,f, Makoto Furutani-Seikid a
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa-no-ha 5-1-5, Kashiwa, Chiba 277-8572, Japan b Department of Biological Sciences, The University of Tokyo, Tokyo, Japan c Graduate School of Biostudies, Kyoto University, Kyoto, Japan d Kondoh Differentiation Signaling Project, JST, Sakyo-ku Kyoto, Japan e Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan f Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan g Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan h Division of Experimental Immunology, Institute for Genome Research, The University of Tokushima, Tokushima, Japan i Research Center for Allergy and Immunology, RIKEN, Saitama, Japan j Radiation Biology Center, Kyoto University, Kyoto, Japan Received 26 December 2003; received in revised form 5 March 2004; accepted 3 April 2004
Abstract We screened populations of N-ethyl-N-nitrosourea (ENU)-mutagenized Medaka, (Oryzias latipes) for radiation-sensitive mutants to investigate the mechanism of genome stability induced by ionizing radiation in developing embryos. F3 embryos derived from male founders that were homozygous for induced the mutations were irradiated with g-rays at the organogenesis stage (48 hpf) at a dose that did not cause malformation in wild-type embryos. We screened 2130 F2 pairs and identified three types of mutants with high incidence of radiationinduced curly tailed (ric) malformations using a low dose of irradiation. The homozygous strain from one of these mutants, ric1, which is highly fertile and easy to breed, was established and characterized related to g-irradiation response. The ric1 strain also showed higher incidence of malformation and lower hatchability compared to the wild-type CAB strain after g-irradiation at the morula and pre-early gastrula stages. We found that the decrease in hatching success after g-irradiation, depends on the maternal genotype at the ric1 locus. Terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end-labeling assays showed a high frequency of apoptosis in the ric1 embryos immediately after g-irradiation at the pre-early gastrula stage but apoptotic cells were not observed before midblastula transition (MBT). The neutral comet assay revealed that the ric1 mutant has a defect in the rapid repair of DNA double-strand breaks induced by g-rays. These results suggest that RIC1 is involved in the DNA double strand break repair in embryos from morula to organogenesis stages, and unrepaired DNA double strand breaks in ric1 trigger apoptosis after MBT. These results support the use of the ric1 strain for investigating various biological consequences of DNA double strand breaks in vivo and for sensitive monitoring of genotoxicity related to low dose radiation. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Medaka; Oryzias latipes; Development; Mutagenesis; Radiation; ric; Apoptosis; Repair; Malformation; Double strand break
1. Introduction * Corresponding author. Tel./fax: þ 81-471-36-3670. E-mail address: [email protected] (H. Mitani). 0925-4773/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mod.2004.04.002
Exposure to ionizing radiation at embryonic stages may predispose organisms to a high risk of developing diseases
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(Carlson and Desnick, 1979), and harmful mutations in germ cells can be transmitted to descendants (Bakker et al., 1987). Russell and Russell (1957) reported that lethality and malformations in mouse embryos induced by ionizing irradiation was highly dependent on the developmental stage. The preimplantation stage was highly sensitive to lethal irradiation effects, whereas very little increase in malformation frequency was due to irradiation during the preimplantation period. Most experimental data on the effects of radiation on developing embryos have been obtained from studies on the mouse or rat in utero because these species have high rates of reproduction. However, our knowledge of the DNA repair and cell cycle regulation in utero is limited. For example, it is not known which DNA repair pathway provides the greatest protection against malformation or lethality, or how the apoptosis pathway is activated in embryos before and after MBT. The small fish species, Medaka (Oryzias latipes), is a useful experimental animal because transparent embryos (. 20 embryos/female) that develop outside of the mother’s body are obtained every day thereby allowing the cellular effects of radiation—such as cell death—to be easily be observed during all stages of embryogenesis. The availability of inbred strains also enables qualitative and quantitative analysis of the effects of radiation using uniform genetic backgrounds. There are many reports on hatchability and developmental abnormalities of Medaka embryos irradiated at early developmental stages and in F1 embryos obtained by mating irradiated males with non-irradiated females (Hyodo-Taguchi et al., 1973; Ishikawa and Hyodo-Taguchi, 1997). Shima and Shimada (1991) reported extensive data for the Medaka specific-locus test system, which can detect the germ line mutagenesis after exposure to environmental mutagens, including ionizing radiation. Mutant Medaka strains that are sensitive to ionizing radiation will thus enable us to examine the molecular functions of genes responsible for genomic stability in early embryos. Prospective genetic screening is an ideal approach to search for functions of genes responsible for genome stability in vivo. N-ethyl-N-nitrosourea (ENU), which predominantly induces point mutations (Harbach et al., 1992), has been used for producing and isolating mutations that produce developmental anomalies during embryonic development in the mouse, zebrafish and Medaka (Hrabe de Angelis et al., 2000; Haffter et al., 1996; Loosli et al., 2000). Here, we report the isolation of three mutations associated with a high incidence of radiation-induced malformations of the tail. One of the three mutants, ric1, exhibited a defect in rejoining of DNA double-strand breaks (DSBs) at the pre-early gastrula stage. We also found that the maternal genotype at the ric1 locus determines hatchability after g-irradiation, even at the organogenesis stage, which is a developmental stage at high risk of radiation-induced malformation in vertebrates.
2. Results 2.1. Design of screening by g-ray irradiation The dose of g-ray irradiation used for screening was determined by examining lethality rates in wild-type CAB strain embryos irradiated at the organogenesis stage (stage 25). We selected this stage because irradiation at the corresponding stage in mouse induces high incidences of both abnormalities and neonatal death (Russell and Russell, 1957). After irradiating with 25 Gy (see Section 4) at the organogenesis stage, lethality in wild-type CAB strain embryos was about 80% (data not shown). After irradiation with lower doses (10.2 Gy), lethality rates were less than 10%. 2.2. Isolation of three mutants associated with a high incidence of radiation-induced curly tail malformation The first screening for radiation-sensitive mutants was carried out by irradiating F3 embryos of the mutagenized male founders with g-rays (9.1 Gy) at Radiation Biology Center, Kyoto University, at 48 h post-fertilization (hpf), which corresponds to the organogenesis stage of mouse. Morphological abnormalities were examined at 5 or 6 days post-fertilization (dpf). After screening 2130 F2 pairs, we identified 352 F2 pairs whose F3 embryos showed severe malformations, mainly related to tail or eye development. The typical malformation phenotypes (Fig. 1) of these candidate mutants were roughly similar to those frequently observed in the wild-type CAB strain after severe irradiation of g-rays (data not shown). We then examined whether the F3 embryos display malformations without g-irradiation. For the 122 F2 pairs whose non-irradiated F3 embryos developed normally, we investigated whether there were significant differences in lethality between F3 embryos irradiated or not irradiated with 8.4 Gy to select pairs showing higher sensitivity to g-irradiation. Three pairs were selected, whose progenies showed high and stable sensitivity in terms of induced severe malformations such as curly tail malformations and were also easy to maintain as homozygotes for each of the mutations. Complementation analyses from crosses of different homozygous strains demonstrated that these three strains have mutation at three different genetic loci (Table 1). Therefore, these strains were named ric (radiation-induced curly tailed) 1, ric2 and ric3, respectively. 2.3. Homozygous ric1 embryos show radiation sensitivity at various developmental stages A strain homozygous for ric1, which is highly fertile and easy to breed, was established and characterized related to response to g-irradiation at various developmental stages. We examined hatchabilities and malformation rates of the embryos irradiated at the morula (stage 8), pre-early gastrula (stage 12) and organogenesis
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Fig. 1. Typical morphogenetic abnormalities of the ric1 homozygotes after. g-irradiation (a) A wild-type (CAB strain) embryo at 2 days post-fertilization (dpf); (b) A wild-type embryo at 5 dpf; (c) A wild-type embryo at 5 dpf irradiated with 9.1 Gy of g-rays at 48 h after fertilization; (d) A ric1 homozygote embryo at 2 dpf; (e) ric1 homozygote embryos at 5 dpf; (f)–(k) Examples of ric1 embryos at 5 dpf irradiated with 9.1 Gy of g-rays at 48 h after fertilization showing (f) curly tail; (g) bent tail; (h) spherical eyes; (i) protruding lens; (j) enlarged ear vesicle; (k) cross-eyes and (l) red eyes. More than 50% of malformed embryos had curly tails.
stages (stage 25) (Table 2). After irradiation with 1.0 Gy at the radiation sensitive morula stage, although no significant increase of embryonic abnormality was observed for CAB embryos (hatchability, 90.4%, malformation rate, 0%), the ric1 embryos exhibited reduced hatchability (23.8%) and elevated malformation rates (54.3%). The embryos homozygous for ric1 irradiated with 5.1 Gy at the pre-early gastrula stage, showed reduced hatchability (36.1 vs. 94.2%) and an elevated malformation rate (71.5 vs. 4.7%) compared with wildtype embryos. Similarly, after irradiation with 10.2 Gy at the organogenesis stage, hatchability was decreased (4.1%) and the malformation rate increased (70.9%) in the ric1 homozygous embryos (Fig. 2). Malformations of the tail (curly and bent tails) were typically observed, with curly tails being the most frequent ric1 malformation (Table 3). These results suggest that the ric1 homozygous embryo is sensitive to radiation beginning at the molura and extending through the organogenesis stage. We also investigated the radiation sensitivity of embryos homozygous for ric2 and ric3 mutations. After irradiating ric2 homozygous embryos with 10.2 Gy at the organogenesis stage, we observed a reduction in hatchability (4.9%) and an increase in malformation rate (53.5%). Similar results were found for the embryos homozygous for ric3 (hatchability 6.3%; malformation rate 72.9%, Table 2).
Table 1 Hatchability and malformation rate of embryos irradiated at the organogenesis stage Genotype
Dose Number of Malformation Hatchability (Gy) examined rate (%) (%) embryos
(þ /þ ) £ (þ /þ )
0 10.2 0 10.2 0 10.2 0 10.2 0 10.2 0 10.2 0 10.2 0 10.2 0 10.2 0 10.2
(ric1/þ) £ (ric1/þ) (ric1/ric1) £ (ric1/ric1) (ric2/þ) £ (ric2/þ) (ric2/ric2) £ (ric2/ric2) (ric3/þ) £ (ric3/þ) (ric3/ric3) £ (ric3/ric3) (ric1/ric1) £ (ric2/ric2) (ric2/ric2) £ (ric3/ric3) (ric3/ric3) £ (ric1/ric1)
86 348 76 106 72 247 53 54 153 144 91 89 47 48 48 176 53 139 61 170
0 0.6 0 20.8 2.8 70.9 0 11.2 4.6 53.5 0 16.9 4.3 72.9 6.3 21.5 5.7 30.9 6.6 12.9
97.7 90.2 97.4 67.9 94.5 4.1 98.1 70.4 97.4 4.9 98.9 76.4 93.6 6.3 95.8 51.1 94.3 64.0 95.1 50.6
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Table 2 Hatchability and malformation rate of embryos irradiated at early embryonic stage and organogenesis stage
2.4. The maternal genotype in ric1 affects on hatchability after g-irradiation
Irradiated stage
Strain Dose Number of Malformation Hatchability (Gy) examined rate (%) (%) embryos
Morula stage (stage 8)
CAB (WT)
We next examined the hatchability of embryos obtained by the four types of crosses: C (þ /þ ) £ F (þ /þ ), C (þ /þ ) £ F (ric1/ric1), C (ric1/ric1) £ F (þ /þ ) and C (ric1/ric1) £ F (ric1/ric1). To examine whether the maternal genotype in ric1 could modify the radiosensitivity of embryos before MBT. After 2 Gy g-irradiation at stage 8, the hatchabilities of wild-type and the ric1 homozygous embryos were 66.7 and 0%, respectively. The hatchability of the embryos produced from wild-type eggs fertilized with sperm from the males homozygous for ric1 was 65.7%, which is similar to that of wild-type embryos, whereas the hatchability of the embryos obtained from the reverse cross was significantly lower (31.5%). We found that maternal genotype in ric1 locus could modify the hatchability after 10.2 Gy g-irradiation at stage 25, the organogenesis stage.
ric1
Pre-early gastrula CAB stage (stage 12) (WT) ric1
Organogenesis stageb (stage 25)
CAB (WT)
ric1 ric2 ric3 a b
0
104
0
94.2
1.0 0 1.0
396 84 320
0 3.6 54.3a
90.4 84.5 23.8
84
0
98.8
380 74 288
4.7 8.1 71.5a
94.2 81.1 36.1
0
86
0
97.7
10.2 0 10.2 0 10.2 0 10.2
348 72 247 153 144 47 48
0.6 2.8 70.9 4.6 53.5 4.3 72.9
90.2 94.5 4.1 97.4 4.9 93.6 6.3
0 5.1 0 5.1
Low malformation rate of curly tail. These results are entered in Table 1.
2.5. g-Irradiation induces apoptosis as an immediate response in ric1 homozygous embryos To examine the immediate responses of the ric1 homozygous embryos to g-irradiation, we carried out the terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick end-labeling (TUNEL) assay on whole mount embryos. Radiation-induced apoptotic cells are shown as TUNEL positive cells. No TUNEL-positive cells were detected in embryos irradiated at the morula stage. We therefore selected the pre-early gastrula stage for the TUNEL assay, and used 5.1 Gy irradiation for 30 min. TUNEL-positive cells were found in 23.9% of the wild-type embryos and 63.6% of the ric1 homozygous embryos
Fig. 2. Effects of the maternal genotype ric1 on hatchability after g-irradiation. Hatchabilities were examined in embryos obtained from four types of crosses: wt C £ wt F; wt C £ ric1 F; ric1 C £ wt F and ric1 C £ ric1 F. We irradiated stage 8 embryos with 2 Gy and stage 25 embryos with 10.2 Gy of g-rays. Vertical bars indicate upper and lower 95% confidence limits.
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Table 3 Malformation of embryos irradiated at early embryonic stage and organogenesis stage Irradiated stage (dose)
Morula (1.0 Gy) Pre-early gastrula (5.1 Gy) Organosenesis stage (10.2 Gy)
Strain
CAB ric1 CAB ric1 CAB ric1
Malformation rate (%)
0 54.3 4.7 71.5 0.6 70.9
Number of examined embryos
396 320 380 288 348 247
Malformation c.t.
b.t.
s.e.
p.l.
b.e.
c.e.
r.e.
s.h.
0 10 1 32 0 104
0 84 3 96 2 59
0 51 1 20 0 12
0 12 0 17 0 18
0 23 1 26 0 20
0 0 0 0 0 2
0 8 0 5 0 1
0 36 12 26 0 0
c.t., curly tail; b.t., bent tail; s.e., spherical eyes; p.e., protruding lens; b.e., enlarged ear vesicle; c.e., cross-eyed; r.e., red eyes; s.h., small head.
(Fig. 3). Thus, the ric1 mutant shows high incidence of apoptosis after g-irradiation. 2.6. Rejoining of DNA double-strand breaks at the pre-early gastrula stage To compare the extent of rejoining of DNA DSBs in early embryonic cells of wild type and the ric1 homozygous embryos exposed to g-irradiation, we performed the neutral single cell microgel electrophoresis assay (neutral comet assay). The relative incidence of DNA DSBs in each cell can be quantified sensitively using this method. The radiation dose used in this study was 15.2 Gy because of the difficulty in detecting DSBs by neutral comet assay at lower doses of irradiation quantitatively. Tail moment (TM) was used as an index to evaluate the relative number of DNA strand breaks. The typical results of neutral comet assay at 0, 30 min and 1 h after irradiation are shown in Fig. 4A. The comet TMs of extracted DNA fragments immediately after 15.2 Gy g-irradiation in both the wildtype and the ric1 embryos were 58.5 ^ 7.2 and 61.3 ^ 5.4,
respectively (Fig. 4B). The TM for the wild-type embryo decreased to the control level 30 min after irradiation (26.7 ^ 3.6). This suggests that almost all DSBs induced in early embryonic cells of wild-type embryos were rejoined within 30 min after irradiation. However, the TM for the ric1 homozygous embryos was not decreased to control levels even 1 h after irradiation (48.4 ^ 4.2). This suggests that the early embryonic cells have high capacity of DSBs repair in which ric1 products are involved.
3. Discussion DNA repair is involved in processes that minimize cell killing, mutations, replication errors, persistence of DNA damage and genomic instability. Abnormalities in these processes found in DNA repair deficient mutants of mammals have been implicated in cancer and aging. The ric1 is the first radiation-sensitive fish strain with the defect in DNA double strand break repair. The fish combine the benefits of both a distinct and easily
Fig. 3. Gamma-irradiation induced apoptotic cells as detected by TUNEL assays. (A) Typical flattened whole mount specimen (ric1 strain; 30 min after exposure to 5.1 Gy of g-rays. TUNEL positive cells were found (arrow). (B) Frequency of embryos with TUNEL positive cells after g-irradiation. Embryos were irradiated at pre-early gastrula stage (5.1 Gy). The TUNEL assay was performed 30 min later. The presence of more than five TUNEL-positive cells was used to define an embryo as having apoptosis. Vertical bars indicate upper and lower 95% confidence limits.
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Fig. 4. DNA double-strand break (DSB) repair at the pre-early gastrula stage after g-irradiation, showing typical comet tails produced by the neutral comet assay. (a) Non-irradiated control comet (wild-type CAB strain); (b) Immediately after exposure to 15.2 Gy of g-rays (CAB strain); (c) One hour after exposure to 15.2 Gy of g-rays (CAB strain); (d) Immediately after exposure to 15.2 Gy of g-rays (ric1 mutant strain); (e) One hour after exposure to 15.2 Gy of g-rays (ric1). (B) Tail moments in early embryonic cells exposed to 15.2 Gy of g-rays as a function of post-exposure incubation time. Each point represents the mean tail moment (mean ^ SEM).
identifiable phenotype for a mutation, with a greater sensitivity to induction of mutations (at least to radiation), in a fish species with a proven record as a valuable model for germ-line mutagenesis, radiation biology, teratogenesis, carcinogenesis, aging and environmental monitoring. We examined hatchability and malformation rates of the ric1 homozygous embryos irradiated at the early embryonic stage (morula and pre-early gastrula stage) and the organogenesis stage. A dose that had no effect on the hatchability of wild-type embryos was selected for each stage. The mutant embryos showed lower hatchability and higher malformation rates upon exposure to g-irradiation, not only at the organogenesis stage used for screening but also at the morula and pre-early gastrula stages. Few embryos irradiated at the early embryonic stage showed a high incidence of radiation induced curly tailed malformation comparable to the embryos irradiated at the organogenesis stage, but many embryos did show a small head malformation. These results suggest that the ric1 homozygous embryo is radiation-sensitive even at the early embryonic stage and at organogenesis stage. Highly proliferating cells in the tail at organogenesis stage are sensitive to radiation and loss of these cells could be a major cause for high incidence of malformation in ric1 mutants. We further investigated the maternal effects of the ric1 gene by examining hatchabilities of the embryos obtained by four types of crosses. It was suggested that the ric1 gene encodes for maternal factors that determine survival rate after irradiation not only before MBT but also at the organogenesis stage.
We performed the neutral comet assay at the pre-early gastrula stage to examine the rejoining of DSBs induced by g-irradiation and found the high capacity of rapid DSB repair in early embryos of the Medaka. On the other hand, the ric1 mutant strain is defective in DSB repair. Shoji et al. (1998) reported that intrauterine deaths in scid mouse embryos, which are deficient in rejoining radiation-induced DSBs, increased with radiation dose and that the mortality was substantially higher than in the wild-type strain. It has been suggested that radiationinduced apoptosis is a p53-dependent event in the late period of mouse organogenesis but that p53-dependent apoptosis prevents malformations in the preimplantation stages as well as in early organogenesis (Norimura et al., 1996; Wang, 2001). Moreover, it has been found that the mouse embryo becomes hypersensitive to DNA damage caused by X-rays and undergoes p53-dependent apoptosis without cell cycle arrest during gastrulation (Heyer et al., 2000). In the present study in the Medaka, apoptotic cells were detected at the pre-early gastrula stage and it is possible that the ric1 gene may also play a role in DNA surveillance mechanisms to ensure genomic integrity, both via the p53-mediated apoptotic pathway and DSBs repair. If this is a case, cloning the ric1 mutation will open an avenue for understanding the molecular mechanisms underlying the repair of DNA damage caused by g-irradiation. We are now establishing cultured cell lines from ric1 embryos that show higher radiosensitivity to make it possible to identify the mutated gene by candidate positional cloning method using the high density linkage map that we established (Naruse et al., 2004).
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4. Experimental procedures 4.1. Medaka strain The Medaka strain CAB originated from the Carolina Biological Supply Company (NC) and has been inbred for more than 30 generations (Loosli et al., 2000). Fish were kept in the system with recirculating water under a 14 h light and 10 h dark cycle at 27 8C. Fish were fed powdered fish food (Tetra-min, Tetra Werke Co., Mells, Germany) and brine shrimp (Artemia franciscana) three times a day. Embryos from natural matings in a plastic aquarium (water temperature was kept at 27 ^ 2 8C) were collected and incubated at 27 8C. Embryos were staged according to Iwamatsu (1994). The main features of each stage of embryo development were as follows. Stage 8, early morula: blastomeres (64 –128) are arranged in three layers. Stage 12, pre-early gastrula: the blastomeres have flattened onto the yolk sphere, and the cell layers are slightly thicker on one side. Stage 25: 18 –19 somites are present (onset of blood circulation). 4.2. Irradiation and incubation of embryos In radiation biology, ‘dose’ has a more specific meaning—it is the energy ionizing radiation absorbed per unit mass of any material. Special units exist for dose, including the ‘rad’, which is defined as 100 erg/g, and the ‘gray’(Gy), which defined as absorption of 1 J/kg. The absorbed-dose rate was monitored by both a radiophotoluminescent glass dosimeter (SC-1 with a Reader FGD-201, Toshiba Glass Co.) and a Fricke dosimeter. Embryos in water at room temperature were exposed to g-rays from a 137Cs source with dose rates of 3.8 Gy/min (used for first screening at Radiation Biology Center, Kyoto University, Japan) 0.95 Gy/min (used for second screening Gammacell 6000 Elan, MDS Nordion, Ottawa, Canada at Research Center for Nuclear Science and Engineering, University of Tokyo, Japan), or 10.2 Gy/min (used for other experiments, Gammacell 3000 Elan, MDS Nordion, Ottawa, Canada). Clusters of fertilized eggs were collected every morning. Embryos were subjected to g-irradiation in a plastic tube. After irradiation, embryos were placed into 96-well plastic microtiter plates (one embryo per well) and incubated at 27 8C. It is difficult for us to set the same dose of g-irradiation because we used different machines with different dose rate. Results of the first screening (9.1 Gy) were confirmed in the second and third screenings (10.2 Gy). 4.3. TUNEL assay A commercial kit (Apoptosis In Situ Detection Kit, Wako Pure Chemical Co., Osaka, Japan) was used for the assay (Yager et al., 1998). Embryos were fixed in Bouin’s solution. After treatment with proteinase K, end labeling
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was performed using TdT. Strongly colored cells were counted as TUNEL-positive cells, and embryos with more than five TUNEL-positive cells were counted as TUNELpositive embryos. 4.4. Neutral comet assay Immediately after removing the chorion of the embryo at 4 8C, a cell suspension was prepared from the blastoderm in phosphate buffered saline (PBS, pH 7.4), and 100 ml of the cell suspension was added to 100 ml of 2% (w/v) lowmelting-point agarose (Nacalai Tesque, Kyoto, Japan). Then, 150 ml of this suspension was applied to the surface of a microscope slide (Matsunami Glass Inc., Osaka, Japan) to form a microgel and was allowed to set at 4 8C for 5 min. Slides were submersed in cell lysis buffer (2% SDS, 0.03 M EDTA, pH 8.0) for 30 min at 4 8C in the dark. Following cell lysis, all slides were washed through three changes of deionized water at 10 min intervals to remove salt and detergent from the microgels. Slides were placed in a horizontal electrophoresis unit and allowed to equilibrate for 5 min with TAE buffer before electrophoresis (50 V) for 7 min. After electrophoresis, slides were rinsed three times with deionized water and stored protected from light until analysis. Microgels were stained with SYBR Green (1:10,000 dilution; BioWhittaker Molecular Applications, Rockland, ME, USA) for 5 min. Slides were rinsed briefly with distilled water and covered with coverslips before image analysis. More than 70 cells were randomly analyzed per slide and scored for comet tail parameters as previously defined (Bocker et al., 1997). TM was used in this study to evaluate the relative DNA damage. TM ¼ (the percentage of tail DNA) £ (the tail length), where comet tail length is the maximum distance the damaged DNA migrates from the center of the cell nucleus, and the percentage of tail DNA is the total percentage of nuclear DNA that migrates from the nucleus into the comet tail. However, the DNA patterns per se, formed from the arrays of DNA fragments that migrate away from the nucleus, were not analyzed (Tice et al., 2000; Czene et al., 2002).
Acknowledgements We thank Yasuko Okamoto, Yasuko Kota, Toshiyuki Yamanaka, Ayako Yamaguchi, Emiko Nishimura (ERATO) and Tomoko Ishikawa, Jun Hirayama, Yuri Kobayashi, Junya Tomida, Haruka Tomida, Kazunori Jikihara (Kyoto University) for taking care of the fish facility and Hoshio Eguchi at the Research Center for Nuclear Science and Engineering, The University of Tokyo, for the use of a g-ray irradiator. We also thank Dr Richard Winn (Aquatic Biotechnology and Environmental Lab, University of Georgia) for many valuable comments and editing on an earlier version of the manuscript. This work was supported by the ERATO grant from Japan Science and
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Technology Agency, Japan to H.K. and by Grant-in-Aid for Scientific Research Priority Area #813, from the Ministry of Education, Science, Sports, Culture and Technology (MEXT), Japan to H.M. and A.S.
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