Medaka

About the Editors Kenji Murata, Center for Health and the Environment, University of California Davis, USA. Masato Kinoshita, Department of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Japan. Kiyoshi Naruse, Laboratory of Bioresources/IBBP Center, National Institute for Basic Biology, Japan. Minoru Tanaka, Division of Biological Science, Nagoya University, Japan. Yasuhiro Kamei, Spectrography and Bioimagin Facility, NIBB Core Facilities, National, Institute for Basic Biology, Japan.

• List of Contributors xvPreface xxi1 Medaka Management 11.1 Introduction 11.2 Medaka Management for Scientific Research 11.2.1 Outline of medaka life‐cycle in the wild 21.2.2 Preparation of normal rearing conditions of medaka in the laboratory and procedures for breeding 21.2.2.1 Breeding system set‐up 21.2.2.2 Obtaining medaka 31.2.2.3 Collecting eggs in a laboratory setting 31.2.2.4 Daily care and maintenance of eggs 41.2.2.5 Rearing medaka from the larval stage to adulthood 41.2.2.6 Anesthesia and euthanasia 41.3 Standardized Culture and Growth Curve 71.3.1 Characteristics and selection of strains 71.3.2 Management of medaka eggs and fish 81.3.2.1 Mating 81.3.2.2 Management of embryos 81.3.2.3 Management of embryos before hatching 131.3.2.4 Rearing from the larval stage to adulthood (to induce earlier maturation) 141.3.3 Maintenance of breeding tanks during breeding 231.3.3.1 Judgment of water quality 231.3.3.2 Maintenance of breeding water 241.3.4 Anesthesia 251.3.4.1 Behavior under each anesthesia stage 261.3.4.2 Difference in sensitivity to anesthesia among strains 261.3.4.3 Growth stage specificity in sensitivity to MS‐222 271.3.4.4 Eugenol is recommended as an anesthetic reagent 281.3.4.5 Euthanasia 281.3.4.6 Important reminders for euthanasia 292 Medaka and Oryzias Species as Model Organisms and the Current Status of Medaka Biological Resources 312.1 Introduction 312.2 Common and Unique Futures of Medaka and Related Species as Model Organisms 312.3 Phylogenetic Relationships of Medaka and Related Species 352.3.1 The javanicus species group 352.3.2 The latipes species group 402.3.3 The celebensis species group 422.4 BAC Resources of Species Related to Medaka 432.5 National Bio‐Resource Project Medaka (NBRP Medaka) 432.5.1 Support for visiting researchers 453 Looking at Adult Medaka 493.1 General Morphology 493.1.1 Secondary sexual characters 493.1.1.1 Dorsal fin 493.1.1.2 Anal fin 493.1.1.3 Papillar processes 503.1.1.4 Urogenital papillae 503.1.2 Body color 513.1.2.1 Pigment cells (chromatophores) 513.1.2.2 Structures of the chromatophores 513.1.2.3 Chromatophores in medaka 513.1.2.4 Chromatophore distribution in medaka 553.1.2.5 See‐through medaka 563.2 Anatomy and Histology 563.2.1 Observations of internal organs 563.2.1.1 Observations of internal organs in the live see‐through medaka 563.2.1.2 Dissection of adult medaka 583.2.2 Horizontal and sagittal sections of juvenile medaka 583.2.3 Nervous system 583.2.3.1 Adult central nervous system 583.2.3.2 Adult peripheral nervous system 673.2.4 Endocrine system 743.2.4.1 Hypothalamo‐pituitary system 763.2.4.2 Pineal organ (epiphysis) 783.2.4.3 Thyroid gland 793.2.4.4 Heart 813.2.4.5 Interrenal gland and chromaffin cells 813.2.4.6 Gonads 813.2.4.7 Endocrine pancreas (islets of Langerhans) 813.2.4.8 Gastrointestinal tract 813.2.4.9 Ultimobranchial gland 823.2.4.10 Corpuscle of Stannius 823.2.4.11 Urophysis 833.2.4.12 Thymus 833.2.5 Gonads 833.2.5.1 Ovary 833.2.5.2 Testis 853.2.6 Kidney 853.2.6.1 Pronephros 863.2.6.2 Mesonephros 863.2.6.3 Histology of the kidney 86Column 3.1 How to make sections of a mature ovary for histological analysis 884 Looking at Medaka Embryos 974.1 Development of Various Tissues and Organs 974.1.1 Developmental stages 974.1.1.1 Stage 0: unfertilized egg (Figure 4-1) 974.1.1.2 Stage 1: activated egg stage (3 minutes) (Figure 4-1) 994.1.1.3 Stage 2: blastodisc stage (Figure 4-1) 994.1.1.4 Stage 3: two‐cell stage (1 hour 5 minutes) (Figure 4-1) 994.1.1.5 Stage 4: four‐cell stage (1 hour 45 minutes) (Figure 4-1) 1004.1.1.6 Stage 5: eight‐cell stage (2 hours 20 minutes) (Figure 4-1) 1004.1.1.7 Stage 6: 16‐cell stage (2 hours 55 minutes) (Figure 4-2) 1004.1.1.8 Stage 7: 32‐cell stage (3 hours 30 minutes) (Figure 4-2) 1004.1.1.9 Stage 8: early morula stage (4 hours 5 minutes) (Figure 4-2) 1004.1.1.10 Stage 9: late morula stage (5 hours 15 minutes) (Figure 4-2) 1004.1.1.11 Stage 10: early blastula stage (6 hours 30 minutes) (Figure 4-2) 1004.1.1.12 Stage 11: late blastula stage (8 hours 15 minutes) (Figure 4-2) 1024.1.1.13 Stage 12: pre‐early gastrula stage (10 hours 20 minutes) (Figure 4-3) 1024.1.1.14 Stage 13: early gastrula stage (13 hours) (Figure 4-3) 1024.1.1.15 Stage 14: pre‐mid‐gastrula stage (15 hours) (Figure 4-3) 1024.1.1.16 Stage 15: mid‐gastrula stage (17 hours 30 minutes) (Figure 4-3) 1024.1.1.17 Stage 16: late gastrula stage (21 hours) (Figure 4-3) 1024.1.1.18 Stage 17: early neurula stage (1 day 1 hour) (Figure 4-3) 1034.1.1.19 Stage 18: late neurula stage (1 day 2 hours) (Figure 4-4) 1044.1.1.20 Stage 19: two‐somite stage (1 day 3 hours 30 minutes) (Figure 4-4) 1044.1.1.21 Stage 20: four‐somite stage (1 day 7 hours 30 minutes) (Figure 4-4) 1044.1.1.22 Stage 21: six‐somite stage (1 day 10 hours) (Figure 4-4) 1044.1.1.23 Stage 22: nine‐somite stage (1 day 14 hours) (Figure 4-4) 1044.1.1.24 Stage 23: 12‐somite stage (1 day 17 hours) (Figure 4-4) 1044.1.1.25 Stage 24: 16‐somite stage (1 day 20 hours) (Figure 4-5) 1064.1.1.26 Stage 25: 18–19‐somite stage (2 days 2 hours) (Figure 4-5) 1074.1.1.27 Stage 26: 22‐somite stage (2 days 6 hours) (Figure 4-5) 1074.1.1.28 Stage 27: 24‐somite stage (2 days 10 hours) (Figure 4-5) 1074.1.1.29 Stage 28: 30‐somite stage (2 days 16 hours) (Figure 4-5) 1074.1.1.30 Stage 29: 34‐somite stage (3 days 2 hours) (Figure 4-5) 1084.1.1.31 Stage 30: 35‐somite stage (3 days 10 hours) (Figure 4-6) 1084.1.1.32 Stage 31: gill blood vessel formation stage (3 days 23 hours) (Figure 4-6) 1084.1.1.33 Stage 32: somite completion stage (4 days 5 hours) (Figure 4-6) 1084.1.1.34 Stage 33: stage at which notochord vacuolization is completed (4 days 10 hours) (Figure 4-6) 1084.1.1.35 Stage 34: pectoral fin blood circulation stage (5 days 1 hour) (Figure 4-6) 1104.1.1.36 Stage 35: stage at which visceral blood vessels form (5 days 12 hours) (Figure 4-6) 1104.1.1.37 Stage 36: heart development stage (6 days) (Figure 4-7) 1104.1.1.38 Stage 37: pericardial cavity formation stage (7 days) (Figure 4-7) 1104.1.1.39 Stage 38: spleen development stage (8 days) (Figure 4-7) 1104.1.1.40 Stage 39: hatching stage (9 days) (Figure 4-7) 1104.1.1.41 Stage 40: first larval stage (Figure 4-8) 1124.1.1.42 Stage 41: second larval stage (Figure 4-8) 1134.1.1.43 Stage 42: third larval stage (Figure 4-8) 1134.1.1.44 Stage 43: first juvenile stage (Figure 4-8) 1134.1.1.45 Stage 44: second juvenile stage (Figure 4-8) 1134.1.1.46 Stage 45 (Figure 4-8) 1134.1.2 Brain 1134.1.2.1 Gastrula step (stages 13–17) 1144.1.2.2 Neurula step (stages 17–18) 1144.1.2.3 Neural rod step (stages 19–22) 1164.1.2.4 Neural tube step (stages 23–27) 1164.1.2.5 Late embryonic brain step (stages 28–34) 1174.1.2.6 Larval brain step (stages 35–42) 1194.1.3 Hatching gland 1194.1.3.1 Origin of fish hatching gland cells 1194.1.3.2 Secretion of hatching enzymes from hatching gland cells 1224.1.4 Eye development 1244.1.4.1 Specification of the anterior neural plate 1244.1.4.2 Eye field determination and establishment of retinal identity 1254.1.4.3 Splitting of the retinal anlage into two retinal primordia 1254.1.4.4 Morphogenesis I: evagination of the optic vesicle 1254.1.4.5 Morphogenesis II: formation of the optic cup 1274.1.4.6 Retinal differentiation I: central retina 1274.1.4.7 Retinal differentiation II: Ciliary Marginal Zone 1274.1.4.8 Retinotectal projection 1284.1.5 Branchial arch and jaws 1284.1.5.1 Skeletal development 1284.1.5.2 Muscle development 1304.1.6 Vasculature 1314.1.6.1 Vascular anatomy of the developing medaka 1314.1.6.2 Origin of the medaka endothelial lineage 1424.1.6.3 Abbreviations 1424.1.6.4 Acknowledgment 1434.1.7 Blood cells (hematopoiesis) 1434.1.7.1 Overview 1434.1.7.2 Observation of embryonic and adult blood cells 1444.1.8 Heart 1464.1.8.1 Overview 1464.1.8.2 Heart architecture 1464.1.8.3 Heart morphogenesis 1474.1.8.4 Observation of the developing heart 1564.1.9 Kidney 1594.1.9.1 Overview 1594.1.9.2 Nephrogenesis 1594.1.9.3 Pronephros 1604.1.9.4 Mesonephros 1604.1.10 Thymus 1604.1.10.1 Overview 1604.1.10.2 Early development of the thymus 1604.1.10.3 Cortex and medulla 1614.1.10.4 Involution of the thymus 1624.1.11 Gut and liver 1624.1.12 Bones 1644.1.12.1 Vertebral column 164Column 4.1 Key words in bone formation 1724.1.13 Fins 1734.1.13.1 Overview 1734.1.13.2 Fin anatomy 1734.1.13.3 Embryonic fin development (from fertilization to stage 39 [hatching stage]) 1744.1.13.4 Fin development after hatching (after stage 39) 1754.1.13.5 Gene expression during fin development 1754.1.14 Gonads 1764.1.14.1 Overview 1764.1.14.2 PGC specification 1774.1.14.3 Formation of gonadal primordium (Figure 4-60b) 1774.1.14.4 Sexual dimorphism in germ cell proliferation (Figure 4-61) 1794.1.14.5 Posthatching period in XX gonads 1804.1.14.6 Posthatching period in XY gonads 1804.2 Medaka EGG Envelope and Hatching Enzyme 1814.2.1 Overview 1814.2.2 Preparation of a hatching enzyme solution from hatching liquid 1824.2.2.1 Procedure 1824.2.3 Simple method for preparing hatching enzyme solution 1834.2.3.1 Procedure 1834.2.4 Solubilization of the egg envelope using hatching enzyme 183Column 4.2 Easy method for preparation of a small amount of hatching enzyme solution (see website for figure) 1844.3 Observation of Embryos (Embedding Embryos) 1854.3.1 Anesthesia of embryos using MS‐222 1854.3.1.1 Equipment and reagents 1854.3.2 Observation of embryos (mounting) 1854.3.2.1 Living embryos 1854.3.2.2 Processed embryos 1884.4 Whole‐Mount In Situ Hybridization (see section 4.1.8.4 for a similar protocol) 1894.4.1 Fixation and storage 1894.4.1.1 Procedure 1 1894.4.2 Rehydration, proteinase K treatment, and postfixation at RT 1904.4.2.1 Procedure 2 1904.4.3 Hybridization and washing 1904.4.3.1 Procedure 3 1904.4.4 Immunoreaction and washing antibodies 1914.4.4.1 Procedure 4 1914.4.5 Staining 1914.4.5.1 Procedure 5 1914.5 Embedding in a Plastic Resin (Technovit 7100) 1924.5.1 Equipment and reagents 1924.5.2 Agarose mounting (Figure 4-68) 1924.5.2.1 Procedure 1 1924.5.3 Dehydration and infiltration (Figure 4-68) 1924.5.3.1 Procedure 2 1924.5.4 Polymerization (Figure 4-68) 1934.5.4.1 Procedure 3 193Column 4.3 Pigment Cells (Figure 4-69) 194Column 4.4 Kupffer’s Vesicle 1955 Reproductive Behavior of Wild Japanese Medaka 2055.1 Wild Japanese Medaka 2055.2 Reproductive Behavior of Wild Medaka 2065.2.1 Aggressive behavior 2075.2.2 Spawning behavior 2075.2.3 Egg deposition behavior 2105.2.4 Egg discarding behavior 2105.2.5 School and aggregation 2115.3 Conclusion 2116 Cryopreservation and Transplantation of Medaka Germ Cells 2156.1 Introduction 2156.2 Cryopreservation of Medaka Testes 2156.2.1 Solutions 2166.2.2 Materials 2176.2.3 Procedures 2176.3 Transplantation of Thawed Testicular Cells into Recipient Larvae 2186.3.1 Solutions 2186.3.2 Materials 2196.3.3 Procedures 219Column 6.1 Production of triploid medaka 2227 Genome Editing 2257.1 Introduction 2257.2 Outline of Targeted Genome Editing Using Nucleases 2257.3 Preparation of CRISPR/Cas9 Genome Editing Tools 2267.3.1 Materials 2277.3.2 Production of custom‐designed sgRNA 2287.3.2.1 Preparation of the bsai‐digested sgRNA backbone 2287.3.2.2 Design and production of customized sgRNA 2287.3.3 Production of capped RNA encoding a Cas9 nuclease 2327.4 Preparation of Custom‐Designed TALENs 2347.4.1 Materials 2347.4.2 Preparation of the TALEN assembly system 2367.4.2.1 Preparation of TAL modules (HD1‐6, NG1‐6, NI1‐6, and NN1‐6) 2367.4.2.2 Preparation of array backbone plasmids (pFUS vectors) 2367.4.2.3 Preparation of last repeat modules (LR‐HD, NG, NI, and NN) 2377.4.2.4 Preparation of TALEN backbone plasmids (pCS2TAL3DD and RR vectors) 2387.4.3 Design and construction of custom‐designed TALENs 2397.4.3.1 Design of TALEN using TALE‐NT 2397.4.3.2 First assembly: construction of 6‐modules array vectors 2407.4.3.3 Second assembly: construction of TALEN expression vectors 2427.5 Heteroduplex Mobility Assay – A Simple Method to Detect Targeted Genome Modification 2447.5.1 Materials 2467.5.2 Procedure 2467.5.2.1 Identification of the wild type, heterozygotes, and homozygotes 2467.5.2.2 Evaluation of the efficiency of targeted genome modifications 2467.6 How to Establish Gene Knock‐out Strains 2477.6.1 Design and synthesis of genome‐editing tools 2477.6.2 Evaluation of genome‐editing activity with fertilized medaka eggs 2477.6.3 Microinjection of the selected genome‐editing tool(s) 2487.6.4 Selection of founder fish by genotyping F1 embryos 2497.6.5 Selection of F1 fish carrying the same mutation and the establishment of mutant strain 2497.6.6 Selection of homozygous mutant fish in the F2 family 2517.7 How to Establish Gene Knock‐in Strains 2527.7.1 Design and synthesis of CRISPR/Cas9 components 2537.7.2 Evaluation of genome‐editing activity with fertilized medaka eggs 2537.7.3 Construction of donor plasmid with homology arms (Ca. 0.5 kbp) and bait sequences 2537.7.4 Microinjection for establishing knock‐in strains 2547.7.5 Selecting G0 founders harboring the insert gene in the genomic target site 254Column 7.1 Utilization of crRNA, tracrRNA, Cas9 Protein 2557.A Simple Genomic DNA Preparation by an Alkaline Lysis Method 2567.A.1 Materials 2567.A.2 Procedure 2568 Photo‐Inducible Gene Expression in Medaka 2618.1 Outline of IR‐LEGO 2618.2 Practical Strategies of IR‐LEGO in Medaka Study 2628.2.1 Selection of heat shock promoters and application studies 2628.3 Laser Irradiation Conditions and Sample Preparation 2658.4 Caution in Maintaining Strains 2678.5 Other Uses of IR‐LEGO 2678.6 Summary and Future Prospects 2689 Screening and Testing Methods of Endocrine‐Disrupting Chemicals Using Medaka 2719.1 Applied Toxicity Tests for Endocrine Disruptors 2719.2 Detection of Androgenic and Antiandrogenic Chemicals Using Medaka 2759.2.1 The formation of papillary processes on anal fin rays as an indicative phenotype for exposure of androgenic and/or antiandrogenic chemicals 2759.2.2 Candidate biomarkers for assessing the action of androgenic and antiandrogenic chemicals 2769.2.3 Visualization of androgenic and antiandrogenic activity as green fluorescence with spiggin‐GFP medaka 27610 Application of the Seawater Medaka Oryzias melastigma (McClelland) for Marine Ecotoxicology 28110.1 Background and Development of Oryzias melastigma for Marine Ecotoxicology 28110.2 Marine Medaka Developmental Staging 28310.3 Standard Breeding and Rearing Conditions 28410.3.1 Seawater 28510.3.2 Temperature 28510.3.3 Photoperiod 28510.3.4 Feeding 28610.3.5 Embryo collection and rearing 28610.3.6 Hatching and larvae collection 28710.3.7 Larvae rearing 28810.3.8 Larvae feeding 28810.4 Raising Marine Medaka for Experimental Use 28910.4.1 Experiments using adult fish 28910.4.2 Experiments using larvae 28910.5 Troubleshooting 28910.5.1 Mass mortality 28910.5.2 Low egg production 28910.5.3 Extensive algal growth 29010.6 How to Obtain Marine Medaka O. melastigma 29010.7 Experimental Protocols Using Marine Medaka 29010.8 Immunotoxicity Assessment: Bacteria Challenge Assays 29010.8.1 SOP for adult bacterial challenge assay 29110.8.2 SOP for larval bacterial challenge assay 29210.8.3 Age selection for larval bacterial challenge 29310.9 Fish Dissection and the Whole Adult Histoarray 29410.9.1 SOP for fish dissection 29510.9.2 SOP for adult medaka histoarray 29510.10 Embryo Chip 29710.10.1 SOP for embryo and larvae histoarray 29710.A Materials for SOP for Adult Medaka Histoarray (see section 10.9.2) 29911 Telomerase and Telomere Biology in Medaka 30311.1 Introduction 30311.2 SOP for Quantification of Telomerase Activity Using the Real‐Time Quantitative Telomeric Repeat Amplification Protocol (RTQ‐TRAP) 30811.2.1 Procedures for sample extraction 30811.2.2 Procedures for determination of protein concentration 30811.2.3 Procedures for RTQ‐TRAP linearity test 30811.2.4 Calculation of telomerase activity 30911.3 SOP for Quantification of Telomere Length Using Southern Blotting Analysis 30911.3.1 Procedures for genomic DNA extraction and digestion with restriction enzymes 30911.3.2 Procedures for probe preparation 31111.3.3 Procedures for electrophoresis and southern blotting 31111.3.4 Procedures for hybridization and detection 31211.3.5 Procedures for computerized telomere analysis 31211.4 SOP for Quantification of Telomere Length Using Fluorescence In Situ Hybridization 31311.4.1 Procedures for fluorescence in situ hybridization 31311.4.2 Procedures for confocal microscopy detection 31311.4.3 Procedures for ImageJ analysis 31412 Assessments of Medaka Skeletal Toxicity 31712.1 Introduction 31712.2 Methods 31812.2.1 Embryonic exposures: dioxin 31912.2.2 Embryonic exposure: dithiocarbamates 31912.2.3 Whole‐mount alcian blue staining of hatchlings/larvaea 32012.2.4 Whole‐mount Alizarin red S staining of hatchlings/larvaec 32012.2.5 In vivo Alizarin complexone fluorescent staining for mineralized bone matrix 32112.2.6 In vivo calcein fluorescent staining for mineralized bone matrix 32112.2.7 Confocal imaging of embryo/hatchling medaka 32112.2.8 Morphological assessments 32312.3 Results and Discussion 32412.3.1 Dithiocarbamates 32412.3.2 Dioxin 325Appendix A Solutions 329Attributions 331Index 335