Explains the advantages of using medaka in experimental designs, to facilitate research, and to stimulate progress by adopting medaka as a model animal
The second volume of Medaka: Biology, Management, and Experimental Protocols, together with the first volume, helps to familiarize scientists with the advantages of using medaka in experimental designs, to facilitate research using medaka, and to stimulate progress by adopting medaka as a model animal. The second edition expands on the first by providing additional information and current protocols that have been recently developed, or modified, to successfully raise medaka fish under stable culture conditions in the laboratory.
This volume explores new technologies developed after 2009, using the fish as a molecular tool in the fields of life science, evolution, ecology, and toxicology. The authors - noted experts in the field - provide the latest information that spans the varied research disciplines and addresses the value to science of medaka's adoption as a model animal. This important book:
- Explores the advantages of using medaka in experimental designs, to facilitate research
- Details the most recent protocols to successfully raise medaka fish under stable conditions in the laboratory
- Explores the most recent developments in the field
- Provides step-by step specifics for each protocol, allowing researchers to adapt them for use in their own work
Written for students and researchers in fish biology and aquaculture, Medaka: Biology, Management, and Experimental Protocols, Volume 2 introduces the cutting edge research in basic and applied biology using medaka as a model animal as well as descriptions of experimental methods and protocols.
Table of Contents
List of Contributors xv
Preface xxi
1 Medaka Management 1
1.1 Introduction 1
1.2 Medaka Management for Scientific Research 1
1.2.1 Outline of medaka life‐cycle in the wild 2
1.2.2 Preparation of normal rearing conditions of medaka in the laboratory and procedures for breeding 2
1.2.2.1 Breeding system set‐up 2
1.2.2.2 Obtaining medaka 3
1.2.2.3 Collecting eggs in a laboratory setting 3
1.2.2.4 Daily care and maintenance of eggs 4
1.2.2.5 Rearing medaka from the larval stage to adulthood 4
1.2.2.6 Anesthesia and euthanasia 4
1.3 Standardized Culture and Growth Curve 7
1.3.1 Characteristics and selection of strains 7
1.3.2 Management of medaka eggs and fish 8
1.3.2.1 Mating 8
1.3.2.2 Management of embryos 8
1.3.2.3 Management of embryos before hatching 13
1.3.2.4 Rearing from the larval stage to adulthood (to induce earlier maturation) 14
1.3.3 Maintenance of breeding tanks during breeding 23
1.3.3.1 Judgment of water quality 23
1.3.3.2 Maintenance of breeding water 24
1.3.4 Anesthesia 25
1.3.4.1 Behavior under each anesthesia stage 26
1.3.4.2 Difference in sensitivity to anesthesia among strains 26
1.3.4.3 Growth stage specificity in sensitivity to MS‐222 27
1.3.4.4 Eugenol is recommended as an anesthetic reagent 28
1.3.4.5 Euthanasia 28
1.3.4.6 Important reminders for euthanasia 29
2 Medaka and Oryzias Species as Model Organisms and the Current Status of Medaka Biological Resources 31
2.1 Introduction 31
2.2 Common and Unique Futures of Medaka and Related Species as Model Organisms 31
2.3 Phylogenetic Relationships of Medaka and Related Species 35
2.3.1 The javanicus species group 35
2.3.2 The latipes species group 40
2.3.3 The celebensis species group 42
2.4 BAC Resources of Species Related to Medaka 43
2.5 National Bio‐Resource Project Medaka (NBRP Medaka) 43
2.5.1 Support for visiting researchers 45
3 Looking at Adult Medaka 49
3.1 General Morphology 49
3.1.1 Secondary sexual characters 49
3.1.1.1 Dorsal fin 49
3.1.1.2 Anal fin 49
3.1.1.3 Papillar processes 50
3.1.1.4 Urogenital papillae 50
3.1.2 Body color 51
3.1.2.1 Pigment cells (chromatophores) 51
3.1.2.2 Structures of the chromatophores 51
3.1.2.3 Chromatophores in medaka 51
3.1.2.4 Chromatophore distribution in medaka 55
3.1.2.5 See‐through medaka 56
3.2 Anatomy and Histology 56
3.2.1 Observations of internal organs 56
3.2.1.1 Observations of internal organs in the live see‐through medaka 56
3.2.1.2 Dissection of adult medaka 58
3.2.2 Horizontal and sagittal sections of juvenile medaka 58
3.2.3 Nervous system 58
3.2.3.1 Adult central nervous system 58
3.2.3.2 Adult peripheral nervous system 67
3.2.4 Endocrine system 74
3.2.4.1 Hypothalamo‐pituitary system 76
3.2.4.2 Pineal organ (epiphysis) 78
3.2.4.3 Thyroid gland 79
3.2.4.4 Heart 81
3.2.4.5 Interrenal gland and chromaffin cells 81
3.2.4.6 Gonads 81
3.2.4.7 Endocrine pancreas (islets of Langerhans) 81
3.2.4.8 Gastrointestinal tract 81
3.2.4.9 Ultimobranchial gland 82
3.2.4.10 Corpuscle of Stannius 82
3.2.4.11 Urophysis 83
3.2.4.12 Thymus 83
3.2.5 Gonads 83
3.2.5.1 Ovary 83
3.2.5.2 Testis 85
3.2.6 Kidney 85
3.2.6.1 Pronephros 86
3.2.6.2 Mesonephros 86
3.2.6.3 Histology of the kidney 86
Column 3.1 How to make sections of a mature ovary for histological analysis 88
4 Looking at Medaka Embryos 97
4.1 Development of Various Tissues and Organs 97
4.1.1 Developmental stages 97
4.1.1.1 Stage 0: unfertilized egg (Figure 4-1) 97
4.1.1.2 Stage 1: activated egg stage (3 minutes) (Figure 4-1) 99
4.1.1.3 Stage 2: blastodisc stage (Figure 4-1) 99
4.1.1.4 Stage 3: two‐cell stage (1 hour 5 minutes) (Figure 4-1) 99
4.1.1.5 Stage 4: four‐cell stage (1 hour 45 minutes) (Figure 4-1) 100
4.1.1.6 Stage 5: eight‐cell stage (2 hours 20 minutes) (Figure 4-1) 100
4.1.1.7 Stage 6: 16‐cell stage (2 hours 55 minutes) (Figure 4-2) 100
4.1.1.8 Stage 7: 32‐cell stage (3 hours 30 minutes) (Figure 4-2) 100
4.1.1.9 Stage 8: early morula stage (4 hours 5 minutes) (Figure 4-2) 100
4.1.1.10 Stage 9: late morula stage (5 hours 15 minutes) (Figure 4-2) 100
4.1.1.11 Stage 10: early blastula stage (6 hours 30 minutes) (Figure 4-2) 100
4.1.1.12 Stage 11: late blastula stage (8 hours 15 minutes) (Figure 4-2) 102
4.1.1.13 Stage 12: pre‐early gastrula stage (10 hours 20 minutes) (Figure 4-3) 102
4.1.1.14 Stage 13: early gastrula stage (13 hours) (Figure 4-3) 102
4.1.1.15 Stage 14: pre‐mid‐gastrula stage (15 hours) (Figure 4-3) 102
4.1.1.16 Stage 15: mid‐gastrula stage (17 hours 30 minutes) (Figure 4-3) 102
4.1.1.17 Stage 16: late gastrula stage (21 hours) (Figure 4-3) 102
4.1.1.18 Stage 17: early neurula stage (1 day 1 hour) (Figure 4-3) 103
4.1.1.19 Stage 18: late neurula stage (1 day 2 hours) (Figure 4-4) 104
4.1.1.20 Stage 19: two‐somite stage (1 day 3 hours 30 minutes) (Figure 4-4) 104
4.1.1.21 Stage 20: four‐somite stage (1 day 7 hours 30 minutes) (Figure 4-4) 104
4.1.1.22 Stage 21: six‐somite stage (1 day 10 hours) (Figure 4-4) 104
4.1.1.23 Stage 22: nine‐somite stage (1 day 14 hours) (Figure 4-4) 104
4.1.1.24 Stage 23: 12‐somite stage (1 day 17 hours) (Figure 4-4) 104
4.1.1.25 Stage 24: 16‐somite stage (1 day 20 hours) (Figure 4-5) 106
4.1.1.26 Stage 25: 18-19‐somite stage (2 days 2 hours) (Figure 4-5) 107
4.1.1.27 Stage 26: 22‐somite stage (2 days 6 hours) (Figure 4-5) 107
4.1.1.28 Stage 27: 24‐somite stage (2 days 10 hours) (Figure 4-5) 107
4.1.1.29 Stage 28: 30‐somite stage (2 days 16 hours) (Figure 4-5) 107
4.1.1.30 Stage 29: 34‐somite stage (3 days 2 hours) (Figure 4-5) 108
4.1.1.31 Stage 30: 35‐somite stage (3 days 10 hours) (Figure 4-6) 108
4.1.1.32 Stage 31: gill blood vessel formation stage (3 days 23 hours) (Figure 4-6) 108
4.1.1.33 Stage 32: somite completion stage (4 days 5 hours) (Figure 4-6) 108
4.1.1.34 Stage 33: stage at which notochord vacuolization is completed (4 days 10 hours) (Figure 4-6) 108
4.1.1.35 Stage 34: pectoral fin blood circulation stage (5 days 1 hour) (Figure 4-6) 110
4.1.1.36 Stage 35: stage at which visceral blood vessels form (5 days 12 hours) (Figure 4-6) 110
4.1.1.37 Stage 36: heart development stage (6 days) (Figure 4-7) 110
4.1.1.38 Stage 37: pericardial cavity formation stage (7 days) (Figure 4-7) 110
4.1.1.39 Stage 38: spleen development stage (8 days) (Figure 4-7) 110
4.1.1.40 Stage 39: hatching stage (9 days) (Figure 4-7) 110
4.1.1.41 Stage 40: first larval stage (Figure 4-8) 112
4.1.1.42 Stage 41: second larval stage (Figure 4-8) 113
4.1.1.43 Stage 42: third larval stage (Figure 4-8) 113
4.1.1.44 Stage 43: first juvenile stage (Figure 4-8) 113
4.1.1.45 Stage 44: second juvenile stage (Figure 4-8) 113
4.1.1.46 Stage 45 (Figure 4-8) 113
4.1.2 Brain 113
4.1.2.1 Gastrula step (stages 13-17) 114
4.1.2.2 Neurula step (stages 17-18) 114
4.1.2.3 Neural rod step (stages 19-22) 116
4.1.2.4 Neural tube step (stages 23-27) 116
4.1.2.5 Late embryonic brain step (stages 28-34) 117
4.1.2.6 Larval brain step (stages 35-42) 119
4.1.3 Hatching gland 119
4.1.3.1 Origin of fish hatching gland cells 119
4.1.3.2 Secretion of hatching enzymes from hatching gland cells 122
4.1.4 Eye development 124
4.1.4.1 Specification of the anterior neural plate 124
4.1.4.2 Eye field determination and establishment of retinal identity 125
4.1.4.3 Splitting of the retinal anlage into two retinal primordia 125
4.1.4.4 Morphogenesis I: evagination of the optic vesicle 125
4.1.4.5 Morphogenesis II: formation of the optic cup 127
4.1.4.6 Retinal differentiation I: central retina 127
4.1.4.7 Retinal differentiation II: Ciliary Marginal Zone 127
4.1.4.8 Retinotectal projection 128
4.1.5 Branchial arch and jaws 128
4.1.5.1 Skeletal development 128
4.1.5.2 Muscle development 130
4.1.6 Vasculature 131
4.1.6.1 Vascular anatomy of the developing medaka 131
4.1.6.2 Origin of the medaka endothelial lineage 142
4.1.6.3 Abbreviations 142
4.1.6.4 Acknowledgment 143
4.1.7 Blood cells (hematopoiesis) 143
4.1.7.1 Overview 143
4.1.7.2 Observation of embryonic and adult blood cells 144
4.1.8 Heart 146
4.1.8.1 Overview 146
4.1.8.2 Heart architecture 146
4.1.8.3 Heart morphogenesis 147
4.1.8.4 Observation of the developing heart 156
4.1.9 Kidney 159
4.1.9.1 Overview 159
4.1.9.2 Nephrogenesis 159
4.1.9.3 Pronephros 160
4.1.9.4 Mesonephros 160
4.1.10 Thymus 160
4.1.10.1 Overview 160
4.1.10.2 Early development of the thymus 160
4.1.10.3 Cortex and medulla 161
4.1.10.4 Involution of the thymus 162
4.1.11 Gut and liver 162
4.1.12 Bones 164
4.1.12.1 Vertebral column 164
Column 4.1 Key words in bone formation 172
4.1.13 Fins 173
4.1.13.1 Overview 173
4.1.13.2 Fin anatomy 173
4.1.13.3 Embryonic fin development (from fertilization to stage 39 [hatching stage]) 174
4.1.13.4 Fin development after hatching (after stage 39) 175
4.1.13.5 Gene expression during fin development 175
4.1.14 Gonads 176
4.1.14.1 Overview 176
4.1.14.2 PGC specification 177
4.1.14.3 Formation of gonadal primordium (Figure 4-60b) 177
4.1.14.4 Sexual dimorphism in germ cell proliferation (Figure 4-61) 179
4.1.14.5 Posthatching period in XX gonads 180
4.1.14.6 Posthatching period in XY gonads 180
4.2 Medaka EGG Envelope and Hatching Enzyme 181
4.2.1 Overview 181
4.2.2 Preparation of a hatching enzyme solution from hatching liquid 182
4.2.2.1 Procedure 182
4.2.3 Simple method for preparing hatching enzyme solution 183
4.2.3.1 Procedure 183
4.2.4 Solubilization of the egg envelope using hatching enzyme 183
Column 4.2 Easy method for preparation of a small amount of hatching enzyme solution (see website for figure) 184
4.3 Observation of Embryos (Embedding Embryos) 185
4.3.1 Anesthesia of embryos using MS‐222 185
4.3.1.1 Equipment and reagents 185
4.3.2 Observation of embryos (mounting) 185
4.3.2.1 Living embryos 185
4.3.2.2 Processed embryos 188
4.4 Whole‐Mount In Situ Hybridization (see section 4.1.8.4 for a similar protocol) 189
4.4.1 Fixation and storage 189
4.4.1.1 Procedure 1 189
4.4.2 Rehydration, proteinase K treatment, and postfixation at RT 190
4.4.2.1 Procedure 2 190
4.4.3 Hybridization and washing 190
4.4.3.1 Procedure 3 190
4.4.4 Immunoreaction and washing antibodies 191
4.4.4.1 Procedure 4 191
4.4.5 Staining 191
4.4.5.1 Procedure 5 191
4.5 Embedding in a Plastic Resin (Technovit 7100) 192
4.5.1 Equipment and reagents 192
4.5.2 Agarose mounting (Figure 4-68) 192
4.5.2.1 Procedure 1 192
4.5.3 Dehydration and infiltration (Figure 4-68) 192
4.5.3.1 Procedure 2 192
4.5.4 Polymerization (Figure 4-68) 193
4.5.4.1 Procedure 3 193
Column 4.3 Pigment Cells (Figure 4-69) 194
Column 4.4 Kupffer’s Vesicle 195
5 Reproductive Behavior of Wild Japanese Medaka 205
5.1 Wild Japanese Medaka 205
5.2 Reproductive Behavior of Wild Medaka 206
5.2.1 Aggressive behavior 207
5.2.2 Spawning behavior 207
5.2.3 Egg deposition behavior 210
5.2.4 Egg discarding behavior 210
5.2.5 School and aggregation 211
5.3 Conclusion 211
6 Cryopreservation and Transplantation of Medaka Germ Cells 215
6.1 Introduction 215
6.2 Cryopreservation of Medaka Testes 215
6.2.1 Solutions 216
6.2.2 Materials 217
6.2.3 Procedures 217
6.3 Transplantation of Thawed Testicular Cells into Recipient Larvae 218
6.3.1 Solutions 218
6.3.2 Materials 219
6.3.3 Procedures 219
Column 6.1 Production of triploid medaka 222
7 Genome Editing 225
7.1 Introduction 225
7.2 Outline of Targeted Genome Editing Using Nucleases 225
7.3 Preparation of CRISPR/Cas9 Genome Editing Tools 226
7.3.1 Materials 227
7.3.2 Production of custom‐designed sgRNA 228
7.3.2.1 Preparation of the bsai‐digested sgRNA backbone 228
7.3.2.2 Design and production of customized sgRNA 228
7.3.3 Production of capped RNA encoding a Cas9 nuclease 232
7.4 Preparation of Custom‐Designed TALENs 234
7.4.1 Materials 234
7.4.2 Preparation of the TALEN assembly system 236
7.4.2.1 Preparation of TAL modules (HD1‐6, NG1‐6, NI1‐6, and NN1‐6) 236
7.4.2.2 Preparation of array backbone plasmids (pFUS vectors) 236
7.4.2.3 Preparation of last repeat modules (LR‐HD, NG, NI, and NN) 237
7.4.2.4 Preparation of TALEN backbone plasmids (pCS2TAL3DD and RR vectors) 238
7.4.3 Design and construction of custom‐designed TALENs 239
7.4.3.1 Design of TALEN using TALE‐NT 239
7.4.3.2 First assembly: construction of 6‐modules array vectors 240
7.4.3.3 Second assembly: construction of TALEN expression vectors 242
7.5 Heteroduplex Mobility Assay - A Simple Method to Detect Targeted Genome Modification 244
7.5.1 Materials 246
7.5.2 Procedure 246
7.5.2.1 Identification of the wild type, heterozygotes, and homozygotes 246
7.5.2.2 Evaluation of the efficiency of targeted genome modifications 246
7.6 How to Establish Gene Knock‐out Strains 247
7.6.1 Design and synthesis of genome‐editing tools 247
7.6.2 Evaluation of genome‐editing activity with fertilized medaka eggs 247
7.6.3 Microinjection of the selected genome‐editing tool(s) 248
7.6.4 Selection of founder fish by genotyping F1 embryos 249
7.6.5 Selection of F1 fish carrying the same mutation and the establishment of mutant strain 249
7.6.6 Selection of homozygous mutant fish in the F2 family 251
7.7 How to Establish Gene Knock‐in Strains 252
7.7.1 Design and synthesis of CRISPR/Cas9 components 253
7.7.2 Evaluation of genome‐editing activity with fertilized medaka eggs 253
7.7.3 Construction of donor plasmid with homology arms (Ca 0.5 kbp) and bait sequences 253
7.7.4 Microinjection for establishing knock‐in strains 254
7.7.5 Selecting G0 founders harboring the insert gene in the genomic target site 254
Column 7.1 Utilization of crRNA, tracrRNA, Cas9 Protein 255
7.A Simple Genomic DNA Preparation by an Alkaline Lysis Method 256
7.A.1 Materials 256
7.A.2 Procedure 256
8 Photo‐Inducible Gene Expression in Medaka 261
8.1 Outline of IR‐LEGO 261
8.2 Practical Strategies of IR‐LEGO in Medaka Study 262
8.2.1 Selection of heat shock promoters and application studies 262
8.3 Laser Irradiation Conditions and Sample Preparation 265
8.4 Caution in Maintaining Strains 267
8.5 Other Uses of IR‐LEGO 267
8.6 Summary and Future Prospects 268
9 Screening and Testing Methods of Endocrine‐Disrupting Chemicals Using Medaka 271
9.1 Applied Toxicity Tests for Endocrine Disruptors 271
9.2 Detection of Androgenic and Antiandrogenic Chemicals Using Medaka 275
9.2.1 The formation of papillary processes on anal fin rays as an indicative phenotype for exposure of androgenic and/or antiandrogenic chemicals 275
9.2.2 Candidate biomarkers for assessing the action of androgenic and antiandrogenic chemicals 276
9.2.3 Visualization of androgenic and antiandrogenic activity as green fluorescence with spiggin‐GFP medaka 276
10 Application of the Seawater Medaka Oryzias melastigma (McClelland) for Marine Ecotoxicology 281
10.1 Background and Development of Oryzias melastigma for Marine Ecotoxicology 281
10.2 Marine Medaka Developmental Staging 283
10.3 Standard Breeding and Rearing Conditions 284
10.3.1 Seawater 285
10.3.2 Temperature 285
10.3.3 Photoperiod 285
10.3.4 Feeding 286
10.3.5 Embryo collection and rearing 286
10.3.6 Hatching and larvae collection 287
10.3.7 Larvae rearing 288
10.3.8 Larvae feeding 288
10.4 Raising Marine Medaka for Experimental Use 289
10.4.1 Experiments using adult fish 289
10.4.2 Experiments using larvae 289
10.5 Troubleshooting 289
10.5.1 Mass mortality 289
10.5.2 Low egg production 289
10.5.3 Extensive algal growth 290
10.6 How to Obtain Marine Medaka O. melastigma 290
10.7 Experimental Protocols Using Marine Medaka 290
10.8 Immunotoxicity Assessment: Bacteria Challenge Assays 290
10.8.1 SOP for adult bacterial challenge assay 291
10.8.2 SOP for larval bacterial challenge assay 292
10.8.3 Age selection for larval bacterial challenge 293
10.9 Fish Dissection and the Whole Adult Histoarray 294
10.9.1 SOP for fish dissection 295
10.9.2 SOP for adult medaka histoarray 295
10.10 Embryo Chip 297
10.10.1 SOP for embryo and larvae histoarray 297
10.A Materials for SOP for Adult Medaka Histoarray (see section 10.9.2) 299
11 Telomerase and Telomere Biology in Medaka 303
11.1 Introduction 303
11.2 SOP for Quantification of Telomerase Activity Using the Real‐Time Quantitative Telomeric Repeat Amplification Protocol (RTQ‐TRAP) 308
11.2.1 Procedures for sample extraction 308
11.2.2 Procedures for determination of protein concentration 308
11.2.3 Procedures for RTQ‐TRAP linearity test 308
11.2.4 Calculation of telomerase activity 309
11.3 SOP for Quantification of Telomere Length Using Southern Blotting Analysis 309
11.3.1 Procedures for genomic DNA extraction and digestion with restriction enzymes 309
11.3.2 Procedures for probe preparation 311
11.3.3 Procedures for electrophoresis and southern blotting 311
11.3.4 Procedures for hybridization and detection 312
11.3.5 Procedures for computerized telomere analysis 312
11.4 SOP for Quantification of Telomere Length Using Fluorescence In Situ Hybridization 313
11.4.1 Procedures for fluorescence in situ hybridization 313
11.4.2 Procedures for confocal microscopy detection 313
11.4.3 Procedures for ImageJ analysis 314
12 Assessments of Medaka Skeletal Toxicity 317
12.1 Introduction 317
12.2 Methods 318
12.2.1 Embryonic exposures: dioxin 319
12.2.2 Embryonic exposure: dithiocarbamates 319
12.2.3 Whole‐mount alcian blue staining of hatchlings/larvaea 320
12.2.4 Whole‐mount Alizarin red S staining of hatchlings/larvaec 320
12.2.5 In vivo Alizarin complexone fluorescent staining for mineralized bone matrix 321
12.2.6 In vivo calcein fluorescent staining for mineralized bone matrix 321
12.2.7 Confocal imaging of embryo/hatchling medaka 321
12.2.8 Morphological assessments 323
12.3 Results and Discussion 324
12.3.1 Dithiocarbamates 324
12.3.2 Dioxin 325
Appendix A Solutions 329
Attributions 331
Index 335
