Development of Sea Urchins, Ascidians, and Other Invertebrate Deuterostomes: Experimental Approaches (eBook)

Cover 1
Contents 6
Contributors 18
Preface 24
Dedication 26
Chapter 1. The Invertebrate Deuterostomes: An Introduction to Their Phylogeny, Reproduction, Development, and Genomics 27
I. Introduction 27
II. Phylogeny 28
III. Reproduction 31
IV. Development 34
V. Genomics 36
References 37
Part I: Procurement, Maintenance & Culture of Oocytes, Embryos, Larvae and Adults
Chapter 2. Care and Maintenance of Adult Echinoderms 43
I. Overview 44
II. Introduction 44
III. Adult Echinoderm Models: Their Reproductive Cycles and Gametogenesis 44
IV. Obtaining Adult Echinoderms 50
V. Maintenance of Adult Echinoderms in Land-Based Systems 52
VI. Care and Handling of Adult Echinoderms 54
References 59
Chapter 3. Echinoderm Eggs and Embryos: Procurement and Culture 65
I. Introduction 66
II. Method for Sea Urchins (Class Echinoidea) 67
III. Method for Sea Stars (Class Asteroidea) 79
IV. Method for Brittle Stars (Class Ophiuroidea, Order Ophiurida) 86
V. Method for Sea Cucumbers (Class Holothuroidea) 90
VI. Method for Interspecific Crosses 94
VII. Summary 96
References 97
Chapter 4. Culture of Echinoderm Larvae through Metamorphosis 101
I. Introduction 101
II. Materials for Culturing Algae and Larvae 102
III. Establishing Algal Cultures 104
IV. Culturing Echinoderm Larvae 107
V. Metamorphosis and Beyond 110
References 111
Chapter 5. Obtaining and Handling Echinoderm Oocytes 113
I. Introduction 114
II. Experimental Preparation 114
III. Methods of Oocyte Collection 118
IV. Sea Urchin Oocytes Cultured In Vitro 124
V. Labeling of Oocyte Components 127
VI. Introduction of Experimental Substances into the Oocytes 136
VII. Concluding Remarks/Outlook 139
References 140
Chapter 6. Procurement and Culture of Ascidian Embryos 141
I. Overview 142
II. Ascidian Development and Metamorphosis 142
III. Larval Tissue Specification 152
IV. Experimental Techniques 155
V. Protocols 158
References 165
Chapter 7. Culture of Adult Ascidians and Ascidian Genetics 169
I. Overview 169
II. Introduction 170
III. Culturing Ascidians 171
IV. Induced Developmental Mutants and Natural Variants/Mutants 179
V. Linkage Analysis and Mapping Genes in Ascidians 186
References 195
Chapter 8. Hemichordate Embryos: Procurement, Culture, and Basic Methods 197
I. Introduction 198
II. Procurement, Spawning, and Culture of S. kowalevskii 199
III. Procurement, Spawning, and Culture of Ptychodera flava 205
IV. Removal of Vitelline Envelope in S. kowalevskii 208
V. Whole-Mount In Situ Hybridization 209
VI. Preparation of Blocking Reagent for P. flava 216
VII. Materials and Reagents 217
References 218
Chapter 9. Cephalochordate (Amphioxus) Embryos: Procurement, Culture, and Basic Methods 221
I. Overview 221
II. Introduction 222
III. Obtaining Gametes of Branchiostoma floridae 226
IV. Raising Embryos 228
V. Manipulating Embryos 232
VI. Amphioxus Resources Available 238
VII. Concluding Remarks 239
References 239
Part II: Embryological Approaches 243
Chapter 10. Quantitative Microinjection of Oocytes, Eggs, and Embryos 245
I. Introduction 245
II. Methods 246
III. Equipment and Supplies (Prices are as of December, 2002) 256
References 268
Chapter 11. Blastomere Isolation and Transplantation 269
I. Introduction 270
II. Preparation of Mouth Pipettes and Needles 271
III. Removal of Fertilization Envelope of Sea Urchin Eggs 274
IV. Isolation and Recombination of Blastomeres in Sea Urchin Embryos 275
V. Blastomere Isolation and Transplantation in Starfish Embryos 289
VI. Blastomere Isolation and Transplantation in Amphioxus 291
VII. Blastomere Isolation and Transplantation in Ascidians 292
VIII. Lineage Tracing, Blastomere Isolation, and Transplantation in Hemichordates 293
References 296
Chapter 12. Isolation and Culture of Micromeres and Primary Mesenchyme Cells 299
I. Introduction 299
II. Isolation and Culture of Micromeres 301
III. Isolation and Culture of Primary Mesenchyme Cells 308
References 310
Chapter 13. Rapid Microinjection of Fertilized Eggs 313
I. Introduction 313
II. Equipment 315
III. Experimental Protocols 318
References 336
Chapter 14. Methods for Embryo Dissociation and Analysis of Cell Adhesion 337
I. Introduction 337
II. Gaining Access to the Egg and Embryo 338
III. Cell Adhesion Assays: Nonquantitative and Semiquantitative 343
IV. Quantitative Centrifugation Assay 346
References 354
Part III: Cell Biological Approaches 357
Chapter 15. Analysis of Sea Urchin Embryo Gene Expression by Immunocytochemistry 359
I. Introduction 360
II. Immunocytochemistry on Whole Embryos 361
III. Immunostaining Small Numbers of Embryos 377
IV. Co-localization 385
V. Troubleshooting 390
References 392
Chapter 16. Light Microscopy of Echinoderm Embryos 397
I. Introduction (D. Burgess and L. Strickland) 398
II. Formaldehyde Fixation of Cleavage Stage Sea Urchin Embryos 398
III. Staining and Imaging Fixed Embryos 408
IV. Simultaneous Fixation and Visualization of the Actin and Microtubule Cytoskeletons (G. von Dassow and V. Foe) 411
V. Observation of Live Embryos (D. Burgess and L. Strickland) 419
VI. 4-D Imaging of Fluorescent Markers in Live Starfish Oocytes ( J. Ellenberg and P. Lenart) 425
References 433
Chapter 17. TEM and SEM Methods 437
I. Introduction 438
II. Basic Fixation and Preparation 438
III. Embedding Media 447
IV. Preservation of Extracellular Matrix 450
V. Fixation for SEM 452
VI. Quick Freezing and Freeze-Substitution 454
VII. Fixation for Immunohistochemistry and Immunocytochemistry 460
VIII. Immunogold Methods 460
IX. Ultrastructural Immunoperoxidase 462
X. Preparation of Colloidal Gold Reagents (Horisberger, 1981 Slot and Geuze, 1985) as modified by Campbell (1990) and Reimer (1994)
References 466
Chapter 18. Calcium Imaging 469
I. Introduction 469
II. Calcium Signals in Embryos 470
III. Getting Calcium Sensors into Embryos 476
IV. Measuring Emitted Light 480
V. Fluorescence Lifetime Imaging 485
VI. Calibration 485
VII. Manipulating Intracellular Calcium 486
VIII. Conclusions and Perspectives 488
References 489
Chapter 19. Labeling of Cell Membranes and Compartments for Live Cell Fluorescence Microscopy 495
I. Introduction 495
II. Labeling of Extracellular Space/Endocytosis/Exocytosis 497
III. Labeling of Plasma Membrane/Exocytosis/Endosomes 498
IV. Labeling of the Cytosol 501
V. Yolk Platelets/Reserve Granules 502
VI. Mitochondria 504
VII. Other Organelles 504
VIII. Endoplasmic Reticulum 505
IX. Golgi Apparatus 509
X. Nucleus 511
XI. Microscopy Considerations 512
XII. Future Directions 513
References 513
Chapter 20. Isolation of Organelles and Components from Sea Urchin Eggs and Embryos 517
I. Overview 518
II. Egg Jelly Molecules Affecting Sperm 518
III. Isolation of the Vitelline Layer from Sea Urchin Eggs 522
IV. Isolation of the Cell Surface Complex and the Plasma Membrane–Vitelline Layer (PMVL) Complex from Sea Urchin Eggs 524
V. The Cytolytic Isolation of the Egg Cortex 526
VI. Isolation of Cortical Granules 527
VII. Isolation of Yolk Platelets 529
VIII. Isolation of Mitochondria from Eggs and Embryos 530
IX. Isolation of Plasma Membranes and Lipid Rafts from Eggs and Zygotes 531
X. Isolation of Microsomes Containing the Endoplasmic Reticulum 533
XI. Nuclear Isolation Procedures 533
XII. Removal and Isolation of the Fertilization Envelope 534
XIII. Isolation of Cilia from Embryos 537
XIV. Isolation of Extracellular Matrices from Embryos and Larvae 538
XV. Isolation of Sea Urchin Larval Skeletons 543
XVI. Resources for the Isolation of Additional Organelles 544
References 545
Chapter 21. Sea Urchin Spermatozoa 549
I. Introduction 550
II. Obtaining Sperm and Removing Coelomocytes 554
III. Isolating Sperm Heads, Flagella, and Chromatin 555
IV. The Isolated Sperm Flagellum as a Sealed Compartment 556
V. Extracting Intact Sperm or Isolated Flagella and Heads with Detergents 557
VI. Wheat Germ Agglutinin (WGA) Affinity Chromatography of Sperm Membrane Proteins 558
VII. Isolating Sperm Plasma Membranes 558
VIII. Isolation of Acrosome Reaction Vesicle Membranes (ARV) 561
IX. Isolating Lipid Rafts from Sperm 562
X. Scoring the Acrosome Reaction by Phase Contrast Microscopy 563
XI. Sea Urchin Sperm Bindin 565
References 566
Chapter 22. Measuring Ion Fluxes in Sperm 571
I. Overview 572
II. Introduction 572
III. Sperm Physiology is Deeply Influenced by Ion Channels and Transporters 573
IV. Strategies to Study Sperm Ion Channels and Transporters 575
References 599
Part IV: Molecular Biological Approaches 603
Chapter 23. Isolating DNA, RNA, Polysomes, and Protein 605
I. Overview 606
II. Introduction 606
III. Purification of Total RNA 606
IV. Purification of Cytoplasmic and Polysomal RNA 612
V. Purification of Genomic DNA 615
VI. Preparation of Protein Samples for One- and Two-Dimensional Gel Electrophoresis 618
References 624
Chapter 24. Detection of mRNA by In Situ Hybridization and RT-PCR 627
I. Introduction 627
II. In Situ Hybridization 628
III. Quantitative PCR 639
References 644
Chapter 25. Using Reporter Genes to Study cis-Regulatory Elements 647
Overview 648
I. Introduction 648
II. Using Chloramphenicol Acetyltransferase (CAT) Reporter Gene 653
III. Using Green Fluorescent Protein (GFP) Reporter Gene 658
IV. Quantitative Imaging of GFP in Living Embryos 659
V. Using Luciferase (luc) Reporter Gene 666
VI. Using lacZ Reporter Gene 669
VII. Summary, Prospects, Concluding Remarks 674
References 675
Chapter 26. Identification of Sequence-Specific DNA Binding Proteins 679
Overview 680
I. Introduction 680
II. Preparation of Nuclear Extracts 681
III. Identification of Transcription Factor Target Sites by Footprint and Electrophoretic Mobility Shift Analysis (EMSA) 684
IV. Affinity Purification of Sequence-Specific DNA Binding Proteins 688
V. Identification of Affinity-Purified DNA-Binding Proteins in SDS Gels 692
VI. Preparation of Affinity-Purified Transcription Factors for Protein Sequencing 697
VII. Prospects 699
References 700
Chapter 27. Expression of Exogenous mRNAs to Study Gene Function in the Sea Urchin Embryo 703
Overview 703
I. Introduction 704
II. Methods for Expressing Synthetic mRNA in the Sea Urchin Embryo 714
III. Summary, Prospects, Concluding Remarks 721
References 722
Chapter 28. Disruption of Gene Function Using Antisense Morpholinos 725
I. Introduction 725
II. Morpholino-Mediated Loss-of-Function: Advantages and Limitations 726
III. Morpholino Methods 730
IV. Concluding Remarks 735
References 736
Chapter 29. Generation and Use of Transgenic Ascidian Embryos 739
Overview 739
I. Ascidian Embryos as Model Chordate Embryos 740
II. Transgenic Ascidian Embryos 742
III. Fundamentals of Electroporation 745
IV. Transgene Construction 746
V. Detailed Protocols for Electroporation of Ascidian Zygotes 747
VI. List of Required Chemicals and Supplies 751
References 752
Part V: Genomics 757
Chapter 30. Genomic Resources for the Study of Sea Urchin Development 759
Overview 760
I. Introduction 760
II. Sea Urchin Arrayed Library Resources 762
III. Computational Tools 772
IV. Sea Urchin Genome Project Web Site 776
References 781
Chapter 31. Genomic Resources for Ascidians: Sequence/Expression Databases and Genome Projects 785
Overview 785
I. Introduction 786
II. The cDNA Projects: cDNA Sequence and Expression Databases 788
III. The Genome Project 795
IV. Conclusion 797
References 798
Chapter 32. Gene Regulatory Network Analysis in Sea Urchin Embryos 801
I. Introduction 801
II. Perturbation Analysis 803
III. Assembling and Testing the GRN Logic Map 813
IV. Summary 818
References 819
Part VI: Echinoderm Eggs and Embryos in the Teaching Lab 821
Chapter 33. Sea Urchin Gametes in the Teaching Laboratory: Good Experiments and Good Experiences 823
Overview 824
I. Introduction 824
II. Obtaining Adult Urchins and Gametes for the Classroom 825
III. Some Guidelines for Using Sea Urchin Gametes in the Classroom 827
IV. Basic Introductory Labs 828
V. Inquiry-Based Experiments Using the Basic Fertilization Protocol 836
VI. Inquiry-Based Experiments in Later Development: Experiments on Differential Gene Expression 845
VII. Experiments Using Morphology as an Endpoint 847
VIII. Epilogue 848
References 848
Appendix 851
Index 867
Volumes in Series 903