Front Cover 
1 
Essential Zebrafish Methods: Cell and Developmental Biology 4
Copyright Page 5
Dedication Page 6
Contents 8
Contributors 14
Preface 
18 
Chapter 1: Overview of the Zebrafish System 20
I. Introduction 20
II. History of the Zebrafish System and Its Advantages and Disadvantages 21
III. Cell and Developmental Biology, Organogenesis, and Human Disease 23
IV. Genetics and Genomics 24
V. Future Prospects 25
VI. Conclusions 25
VII. Epilogue: Volumes 76 and 77 and Technological Advances to Come 26
References 26
Chapter 2: Cell Cycles and Development in the Embryonic Zebrafish 30
I. Introduction 30
II. Terminology and the Staging Series 31
III. The Zygote Period 31
IV. The Cleavage Period 33
V. The Blastula Period 35
VI. The Gastrula Period 39
VII. The Segmentation Period 44
References 45
Chapter 3: Primary Fibroblast Cell Culture 48
I. Introduction 48
II. Material and Methods 49
A. Basic Tissue Culture Techniques 
49 
B. Zebrafish Strains 50
C. Caudal Fin Amputation 50
D. Cell Culture 50
E. Cryopreservation 50
F. Cell Lines 51
III. Results and Discussion 51
Acknowledgments 52
References 52
Chapter 4: Production of Haploid and Diploid Androgenetic Zebrafish (Including Methodology for Delayed In Vitro Fertilization) 
54 
I. Introduction 55
II. Equipment and Materials 57
A. Collection of Salmonid Ovarian Fluid 57
B. Irradiation Source 58
C. Water Baths 59
D. Solutions 59
III. Methods 59
A. Collection of Zebrafish Eggs for Delayed In Vitro Fertilization 59
B. Collection of Sperm for Delayed In Vitro Fertilization 60
C. Irradiation of Eggs 61
D. In Vitro Fertilization 61
E. Production of Diploid Androgenotes by Heat Shock 62
IV. Results and Discussion 63
A. Properties of Androgenetic Embryos 63
B. Are Maternal Genes Transmitted to Androgenotes? 64
C. Alternative Sources of Irradiation 66
V. Conclusions and Perspectives 67
Acknowledgments 67
References 68
Chapter 5: Analysis of Protein and Gene Expression 70
I. In Situ Hybridization to RNA and Immunolocalization of Proteins 71
II. Probe Synthesis 72
A. RNase-Free Conditions 72
B. Antisense RNA Probe S 72
1. Protocol 1: Linearization of Plasmid and Probe Synthesis 73
III. Fixation 74
A. Protocol 2: Fixation of Zebrafish Embryos for In Situ Hybridization 75
IV. Hybridization to Whole-Mount Embryos 75
A. Pretreatments and Hybridization of Zebrafish Embryos 75
1. Protocol 3: Pretreatments and Hybridization of Zebrafish Embryos 76
B. Posthybridization Washes of Zebrafish Embryos 77
1. Protocol 4: Posthybridization Washes for Zebrafish Embryos 77
V. Immunolocalization of Probes 78
A. Protocol 5: Staining Zebrafish Whole-Mounts with NBT/BCIP 79
B. Protocol 6: Staining Zebrafish Whole-Mounts with Diaminobenzidine 79
VI. Two-Color In Situ Hybridizations 80
A. Incubation with Antibodies Conjugated to Horseradish Peroxidase and Alkaline Phosphatase 
80 
1. Protocol 7: Two-Color Whole-Mount Staining with DAB and BCIP/NBT 81
B. Sequential Incubation in Antibodies Conjugated with Alkaline Phosphatase 82
1. Protocol 8: Sequential Alkaline Phosphatase Staining with Chromogenic Substrates 83
VII. Double-Fluorescent In Situ Hybridization 84
A. Protocol 9: Sequential Alkaline Phosphatase Staining with Fluorescent Substrates 84
VIII. Simultaneous Localization of Transcription and Translation Gene Products 85
A. Protocol 10: Immunolocalization with a Horseradish Peroxidase-Conjugated Secondary Antibody 
86 
B. Protocol 11: Immunolocalization by the Peroxidase Antiperoxidase (PAP) Method 87
C. Protocol 12: Vector Avidin Biotinylated Enzyme Complex (ABC) Antibody Staining of Zebrafish Embryos 
88 
IX. Embedding and Sectioning Whole-Mount Embryos 89
A. Protocol 13: Paraffin-Embedding of Whole Embryos after In Situ Hybridization 89
X. Solutions and Reagents 89
References 91
Chapter 6: Analysis of Zebrafish Development Using Explant Culture Assays 92
I. Introduction 93
A. Basic Questions: Lineage Commitment and Inductive Interactions 93
B. Application of Explant Assays to Zebrafish 94
II. Zebrafish Explants: General Considerations 96
A. Tissue Choice 96
B. Reproducibility in Isolating Tissue 96
C. The Size of the Explant 97
D. The Method of Assay 97
III. Materials Required 97
A. Equipment 97
1. Warm Room 97
2. Microscope 97
3. Microinjectors and Micromanipulators 98
4. Micropipette Puller 98
B. Solutions and Culture Media 98
1. Water 98
2. Culture Media 98
a. 10times MBS Stock Solution 
99 
b. 1times MBS 
99 
c. 1 M CaCl2 99
d. 3% Methyl Cellulose 99
C. Dissection and Culture Dishes 100
1. Dissection Dishes 100
2. Culture Dishes 100
D. Dissection Tools 100
1. Eyebrow Knives 100
a. Preparation 102
2. Glass Knives 102
a. Preparation 102
3. Hairloops 103
a. Preparation 103
4. Forceps 103
E. Embryos 103
1. Age 103
2. Feeding and Density 104
3. Water Quality 104
4. Mating 104
5. Embryo Preparation 104
IV. Guide to Explant Isolation and Culture 105
A. Common Procedures 105
1. Dechorionation 105
a. Protocol 9.1 105
2. Temperature Control 105
3. Evaluating Explant Health 106
4. Suggestions for Improving Survival of Fragile Explants 106
5. Assaying Explant Fate After Culture 107
a. RT-PCR Assay 107
b. In situ Hybridization 108
B. Guide to Specific Explants 109
1. The Late Blastula (Sphere Stage) Animal Cap 109
a. Protocol 9.2 109
b. Helpful Hints 111
2. The Early Gastrula (Shield Stage) Animal Cap 111
a. Protocol 9.3 111
b. Helpful Hints 112
3. The Early Gastrula Shield 112
a. Protocol 9.4 112
b. Helpful Hints 113
V. Using Explants to Assay Induction 113
A. Common Procedures and Considerations 113
1. Lineage Labeling 113
2. Detection of Lineage Label During In Situ Hybridization 114
a. Protocol 9.5 114
B. Guide to the Animal Cap/Shield Induction Assay 114
1. Protocol 9.6 115
2. Helpful Hints 115
C. Assaying Purified Molecules as Inducers 115
VI. Illustrations of Specification and Induction Assays 116
A. Specification Analysis 116
1. Aims 116
2. Procedure 116
3. Outcome 116
B. Induction Analysis 117
1. Aims 117
2. Procedure 118
3. Outcome 118
VII. Future Directions 119
Acknowledgments 119
References 119
Chapter 7: Confocal Microscopic Analysis of Morphogenetic Movements 122
I. Introduction 123
II. Confocal Imaging of Embryos 124
III. General Principles of Vital Staining 124
A. Vital Stains and Vital Labels for Zebrafish Embryos 125
1. BODIPY 505/515 125
2. BODIPY-Ceramide 128
3. SYTO-11 128
4. BODIPY-HPC 131
5. Vital Staining and Confocal Imaging Procedures 132
B. Materials 
132 
C. Vital-Staining Procedure 133
D. Additional Vital Stains 134
IV. Mounting Embryos for Imaging 135
A. Imaging Chambers 135
B. Spatial Orientation of Embryos 136
C. pH Stabilization 137
V. Imaging Procedures 137
A. Selection of Optics 137
B. Acquiring Confocal Images in Time-Lapse Form 138
C. Storage and Analysis of Time-Lapse Recordings 139
VI. Multilevel Time-Lapse Confocal Analysis 139
A. Embryo Labeling 139
B. Multilevel Time-Lapse Data Sets 140
1. Simultaneous Two-Channel Imaging 142
2. Saving the Data Set 142
3. Image Processing 142
4. Creating Color-Merged Stacks of the Data Set 142
C. Analysis of Cell Movements During Morphogenesis 143
1. 3D (Stereo Pair) Movie Generation 143
2. 4D-Turnaround and 4D-Viewer 144
D. Analysis of Cellular Trajectories 144
VII. Distribution of Visual Information 146
VIII. Confocal Imaging of Embryos Expressing Green Fluorescent Protein (GFP) 148
IX. Summary 149
Acknowledgments 150
References 150
Chapter 8: Cytoskeletal Dynamics of the Zebrafish Embryo 152
I. Introduction 153
II. Cytoskeleton of the Unfertilized Egg 154
III. Organization and Function of the Cytoskeleton in the Zygote 155
A. Cortical Granule Exocytosis 157
B. Ooplasmic Segregation 158
IV. Cleavage and Blastula Period 159
A. The Role of a Vegetal Array of Parallel Microtubules in the Directed Transport of Dorsal Determinants at the Zygote and Cleavage Stages 
159 
V. Yolk Cell Microtubules during Epiboly 161
VI. Tubulin Dynamics in Neuronal Axons of Living Zebrafish Embryos 165
VII. Intermediate Filaments in Zebrafish 165
VIII. Methods 166
A. Collecting Oocytes, Eggs, and Embryos 166
1. Protocol 12.1 166
2. Protocol 12.2 166
3. Protocol 12.3 168
B. Preparation of Egg Extracts (Becker and Hart, 1996) 168
1. Protocol 12.4 168
2. Protocol 12.5 168
3. Protocol 12.6 169
4. Protocol 12.7 169
5. Protocol 12.8 169
6. Protocol 12.9 170
C. Whole Mount Cytoskeleton Staining 170
1. Protocol 12.10 170
2. Protocol 12.11 171
3. Protocol 12.12 171
Acknowledgments 173
References 173
Chapter 9: Analyzing Axon Guidance in the Zebrafish Retinotectal System 178
I. Introduction 178
II. Retinotectal Mutants 179
III. Labeling the Retinotectal System 182
A. Overview 182
B. Antibody Labeling 184
C. Lipophilic Dye Labeling 184
D. Using GFP or Other Fluorescent Proteins for Labeling 185
1. Method 1: Whole Eye Fills in Fixed Embryos 187
a. Summary 187
b. Solutions Needed 187
c. Protocol 187
2. Method 2: Live Axon Labeling for Time-Lapse 190
a. Summary 190
b. Solutions and Materials Needed 190
c. Protocol 190
3. Method 3: Single Axon Labeling Using Xfp Constructs 191
a. Summary 191
b. Solutions and Materials Needed 192
c. Protocol 192
IV. Perturbing the Retinotectal System 193
A. Overview 193
1. Method 4: Eye Transplants 195
a. Summary 195
b. Solutions and Materials 195
c. Protocol 196
V. Future Directions 197
Acknowledgments 198
References 198
Chapter 10: Analysis of Cell Proliferation, Senescence, and Cell Death in Zebrafish Embryos 
202 
I. Introduction: The Cell Cycle in Zebrafish 
202 
A. Forward-Genetic Screens 
204 
II. Zebrafish Embryo Cell-Cycle Protocols1 
205 
A. Analysis of Cell Proliferation and Mitosis 
205 
1. DNA Content Analysis 205
a. Protocol 205
2. Whole-Mount Immunohistochemistry with Mitotic Marker Phosphohistone H3 206
a. Protocol 208
3. Mitotic Spindle/Centrosome Detection 208
a. Protocol 208
4. BrdU Incorporation 209
a. Protocol 209
B. Analysis of DNA Damage, Senescence, and Apoptosis 210
1. COMET Assay 210
a. Protocol 211
2. Detection of Senescence-Associated Beta Galactosidase 212
a. Protocol 212
3. Apoptosis Detection by TUNEL Staining 213
a. Protocol 213
4. Apoptosis Detection by Acridine Orange 214
a. Protocol 214
C. In Situ Hybridization 214
III. Screening for Chemical Suppressors of Zebrafish Cell-Cycle Mutants 
215 
A. Protocol 
216 
IV. Conclusions 
218 
V. Reagents and Supplies 
218 
Acknowledgments 219
References 219
Chapter 11: Cellular Dissection of Zebrafish Hematopoiesis 
224 
I. Introduction 
224 
II. Zebrafish Hematopoiesis 
225 
A. Primitive Hematopoiesis 
225 
B. Definitive Hematopoiesis 
227 
C. Adult Hematopoiesis 
229 
III. Hematopoietic Cell Transplantation 
234 
A. Embryonic Donor Cells 
234 
1. Protocol for Isolating Hematopoietic Cells from Embryos 
236 
2. Transplanting Purified Cells into Embryonic Recipients 
237 
3. Transplanting Cells into Blastula Recipients 
238 
4. Transplanting Cells into 48hpf Embryos 
238 
B. Adult Donor Cells 
239 
1. Protocols for Isolating Hematopoietic Cells from Adult Zebrafish 
239 
2. Transplanting Whole Kidney Marrow 
240 
3. Transplanting Cells into Irradiated Adult Recipients 
241 
4. Irradiation 
241 
5. Transplantation 
241 
IV. Enrichment of HSCs 
242 
V. In Vitro Culture of Hematopoietic Progenitors 
245 
A. Zebrafish Kidney Stroma (ZKS) 
245 
B. Protocol for Generation, Culture, and Maintenance of ZKS Cells 
246 
C. Protocols for In Vitro Proliferation and Differentiation Assays 
246 
VI. Conclusions 
247 
References 248
Chapter 12: Culture of Embryonic Stem and Primordial Germ Cell Lines from Zebrafish 
252 
I. Introduction 
253 
II. Methods 
254 
A. General Characteristics of Zebrafish ES Cell Cultures 
254 
B. Derivation of ES Cell Cultures from Blastula-Stage Embryos 
257 
C. Derivation of ES Cell Cultures from Zebrafish Gastrula-Stage Embryos 
258 
D. Electroporation of Plasmid DNA into the ES Cell Cultures 
259 
III. Materials 
260 
A. Reagents 
260 
B. Feeder Cell Lines 
261 
1. Growth-Arrested Feeder Cells 261
2. Drug Resistant Feeder Cells 261
3. RTS34ST Cell-Conditioned Medium 262
Acknowledgments 262
References 262
Chapter 13: Neurogenesis 
264 
I. Introduction 
264 
II. The Primary Neuronal Scaffold 
265 
III. Early Development of the Zebrafish Neural Plate 
268 
A. Morphogenesis 
268 
B. Neural Induction 
270 
C. Delimitation of Proneural Fields by Prepattern Factors 
271 
D. Neural Tube Organizers as Neurogenesis Signals? 
274 
IV. Lateral Inhibition and the Neurogenesis Cascade 
275 
A. Primary Neurogenesis 
275 
B. Molecular Control of Secondary Neurogenesis and Gliogenesis 
277 
C. Adult Neurogenesis 
278 
V. Establishment of Neuronal Identity 
280 
VI. Lab Methods to Study Adult Neurogenesis 
281 
A. BrdU Injections 
281 
B. Staining Methods 281
1. Immunohistochemistry on Vibratome Sections 
281 
a. Pretreatments, antigen retrieval 
282 
2. In Situ Hybridization Starting on Whole-Mount Adult Brains 
282 
a. In Situ Hybridization on Gelatine Albumine Sections 
282 
b. Immediately Before Embedding the Brain 
283 
C. DNA Electroporation of the Adult Brain 
283 
D. Slice Culture 
284 
VII. Useful Tools for the Study of Zebrafish Neurogenesis 
284 
VIII. Conclusion 
284 
Acknowledgments 295
References 295
Chapter 14: Time-Lapse Microscopy of Brain Development 
312 
I. Introduction: Why and When to Use Intravital Imaging 
313 
II. Techniques for Vital Staining of the Nervous System 
314 
A. Overview of Techniques 
314 
1. Ubiquitous Labeling 314
2. Labeling Cell Clusters Randomly 315
3. Targeted Labeling of Specific Cell Clusters 315
4. Targeted Single Cell Labeling 316
5. Targeted Labeling of Entire Cell Populations 316
B. Details of Techniques 317
1. Soaking Embryos in Dyes for Ubiquitous Labeling 317
a. Protocol 317
2. Pressure Injection of Dyes for Labeling Groups of Cells 319
a. Protocol 319
b. Protocol 320
3. Iontophoretic Labeling of Single Cells 320
4. Quantum Dots 321
5. Genetic Labeling 321
III. Preparation of the Zebrafish Specimen 324
A. Imaging Chambers 324
B. Stabilizing the Embryo 325
1. Methylcelluose-Embedding 325
a. Protocol 326
2. Agarose-Embedding 326
a. Protocol 326
3. Plasma Clot-Embedding 327
a. Protocol 327
IV. The Microscopic System 327
A. Heating Chamber 327
B. Heater 328
V. Data Recording 329
A. Preparations 329
B. Mounting 329
C. Choice of Specimen 329
D. Saturation 329
E. Pinhole 330
F. Time Interval 330
G. Localizing Cells 330
H. Refocusing 331
VI. Data Analysis 331
A. NIH-Image 331
B. Projections 332
C. LSM Software 333
D. Three-Dimensional Renderings Over Time 334
E. Cell-Tracking 334
VII. Pitfalls to Avoid 335
A. Technical Pitfalls 335
1. DNA-Purification 335
2. Fixation of Mounted Embryo 335
3. Colocalization of Fluorophores 336
4. Storage During Data Recording 336
5. Image Resolution 336
B. Analytical Pitfalls 336
1. Annotation of Recorded Data 336
2. Reference Points 337
3. Identifying Individual Cells 337
VIII. Summary 337
Acknowledgments 337
References 338
Further Reading 342
Chapter 15: Development of the Peripheral Sympathetic Nervous System in Zebrafish 
344 
I. Introduction 
344 
II. The Peripheral Autonomic Nervous System 
345 
A. Overview 
345 
B. Molecular Pathways Underlying PSNS Development 
346 
III. The Zebrafish as a Model System for Studying PSNS Development 
349 
A. Overview 
349 
B. Development of the PSNS in Zebrafish 
350 
1. Neural Crest Development and Migration 
350 
2. Gene Expression in Migrating SA Progenitors 
351 
3. Neuronal Differentiation and Coalescence into Sympathetic Ganglia 
352 
4. Differentiation of Noradrenergic Neurons 
355 
5. Modeling of Sympathetic Ganglia 
356 
C. Mutations Affecting PSNS Development 
358 
1. Introduction 
358 
2. Mutations Affecting Early PSNS Development 
358 
3. Mutations Affecting Later Stages of PSNS Development 
360 
4. Mutations Affecting Sympathetic Ganglia Modeling 
361 
IV. Zebrafish as a Novel Model for Studying Neuroblastoma 
361 
V. Conclusion and Future Directions 
362 
Acknowledgments 363
References 363
Chapter 16: Approaches to Study the Zebrafish Retina 
368 
I. Introduction 
368 
II. Development of the Zebrafish Retina 
370 
A. Early Morphogenetic Events 
370 
B. Neurogenesis 
373 
C. Development of Retinotectal Projections 
376 
D. Nonneuronal Tissues 
377 
III. Analysis of Wild-Type and Mutant Visual System 
378 
A. Histological Analysis 
378 
B. The Use of Molecular Markers 
381 
1. Antibodies 381
2. mRNA Probes 387
3. Lipophilic Tracers 387
4. Fluorescent Proteins 389
C. Analysis of Cell Movements and Lineage Relationships 
391 
D. Analysis of Cell and Tissue Interactions 
392 
E. Analysis of Cell Proliferation 
394 
F. Behavioral Studies 
395 
G. Electrophysiological Analysis of Retinal Function 
395 
H. Biochemical Approaches 
396 
I. Chemical Screens 
397 
IV. Analysis of Gene Function in the Zebrafish Retina 
397 
A. Reverse Genetic Approaches 
397 
1. Loss-of-function Analysis 397
2. Approaches to Gene Overexpression 399
B. Forward Genetics 
401 
1. Mutagenesis Approaches 401
2. Breeding Schemes 402
3. Phenotype Detection Methods 403
4. Positional and Candidate Cloning 406
5. Mutant Strains Available 406
V. Summary 
407 
Acknowledgments 407
References 407
Chapter 17: Instrumentation for Measuring Oculomotor Performance and Plasticity in Larval Organisms 
420 
I. Introduction 
421 
II. Methods 
424 
A. Vestibular Turntable and Drum (Supplemental Movie 1) 
424 
B. Specimen Holder and Mounting (Supplemental Movie 2) 
426 
C. Measuring Eye Movements (Supplemental Movie 3) 
426 
D. Data Acquisition and Analysis 
429 
E. Experimental Animals 
430 
F. Updates on the methodology 
430 
III. Results and Discussion 
431 
A. Table and Drum (Supplemental Movie 1) 
431 
B. Animal Immobilization (Supplemental Movie 2) 
431 
C. Eye Position Measurements (Supplemental Movies 2-4) 
433 
D. Behavioral Recordings (Supplemental Movies 6-10) 
437 
1. Optokinetic Measurements 
437 
2. Negative Effect of Methylcellulose on Optokinetic Performance 
440 
3. Summary of Optokinetic and Vestibular Performance in Zebrafish 
440 
IV. Conclusion 
444 
Acknowledgments 444
References 445
Supplemental Movie Descriptions 447
Chapter 18: Development of Cartilage and Bone 
448 
I. Introduction 
448 
A. The Zebrafish Model 
448 
B. Zebrafish Skeletal Anatomy 
449 
C. Mutations that Disrupt the Skeleton 
452 
II. Cartilage Visualization Techniques 
457 
A. Alcian Blue Staining 
457 
1. Protocol 1: Alcian Blue Staining of Cartilage 458
B. Microdissection of Larval Craniofacial Cartilage 
458 
1. Protocol 2: Microdissection and Mounting of Stained Cartilage 459
a. Suggested Preparations 459
C. BrdU Labeling 
461 
1. Protocol 3: BrdU Labeling of Dividing Cartilage Cells Counter Stained with Alcian Blue 461
III. Bone Visualization Techniques 
463 
A. Alizarin Red Staining 
463 
1. Protocol 4a: Alizarin Red Staining of Bone 463
2. Protocol 4b: Acid-Free Double Cartilage and Bone Stain 464
3. Protocol 4c: Live Alizarin Red Staining 464
B. Adult/Larval Calcein Staining 
464 
1. Protocol 5: Calcein Staining 464
C. Radiographic Visualization of Adult Zebrafish Skeleton 
465 
1. Protocol 6: Radiography of Adult Zebrafish 465
D. Osteoblast and Osteoclast Histology 
465 
1. Protocol 7: TRAP Staining to Visualize Osteoclasts 466
IV. Molecular Markers and Transgenic Lines 
467 
A. Molecular Markers of Progenitor Populations 
467 
B. Molecular Markers of Cartilage and Bone Lineages 
467 
C. Transgenics 
468 
V. Strategy and Potential of Future Screens for Skeletal Mutants 
469 
Acknowledgments 470
References 470
Chapter 19: Morphogenesis of the Jaw: Development Beyond the Embryo 
476 
I. Larval Zebrafish Craniofacial Cartilage Development 
476 
II. Analysis of Craniofacial Skeletal and Replacement Tooth Development 
478 
A. Zebrafish Pharyngeal Tooth Development 
480 
B. Bone Development 
481 
C. Paradigm Shift: Analyses of Postlarval Tooth and Bone Development 
483 
D. Use of Geometric Morphometric Tools for Quantification of Skeletal Defects 
485 
III. Conclusion 
490 
IV. Updates and Recent Advances 
491 
References 493
Chapter 20: Cardiac Development 
498 
Update 498
I. Introduction 
499 
II. Stages of Heart Tube Morphogenesis 
499 
A. Formation of the Heart Fields 
500 
B. Migration to the Midline 
502 
C. Heart Tube Elongation 
505 
D. Heart Looping 
506 
E. Valve Formation 
507 
F. Myocardial Remodeling 
510 
III. Gene Expression 
510 
A. Lateral Plate Mesoderm Gene Expression 
510 
B. Myocardial Gene Expression 
512 
C. Endocardial Gene Expression 
513 
IV. Conclusion and Future Directions 
514 
References 515
Chapter 21: Zebrafish Kidney Development 
520 
I. Introduction 
520 
II. Pronephric Structure and Function 
521 
III. Pronephric Development 
523 
A. Patterning the Intermediate Mesoderm 
524 
1. Origins and Patterning of Nephrogenic Mesoderm 524
2. Pronephric Nephron Cell Lineage 525
3. Gene Expression in the Intermediate Mesoderm 526
4. Adjacent Tissues and Kidney Cell Specification 528
B. Development of the Pronephric Nephrons 
530 
1. Epithelial Tubule Formation 530
2. Nephron Cell DiVerentiation and Segmentation 532
3. Nephron Segment Boundary Patterning 532
4. Pronephric Tubule Isolation 536
C. Pronephric Vascularization 
537 
1. The Role of Podocytes 537
2. The Role of Endothelial Cells 538
3. The Role of Vascular Flow 539
4. A Simple Assay for Glomerular Filtration 539
IV. The Zebrafish Pronephros as a Model of Human Disease 
541 
A. Disease of the Glomerular Filtration Apparatus 
541 
B. Disease of the Tubules 
541 
V. Conclusions 
544 
Acknowledgments 545
References 545
Index 554
Color Plates 
566