International Review of Cytology (eBook)

Cover 1
Xenopus laevis: Practical Uses in Cell and Melecular Biology 4
Copyright Page 5
Contents 6
Contributors 14
Forword 18
Preface 20
PART I. General Information 24
Chapter 1. Raising Xenopus in the Laboratory 26
I. Introduction 26
II. Equipment 28
III. Infectious Diseases 34
IV. Raising Tadpoles from Ovulated Eggs 36
V. Raising and Maintaining Frogs 38
References 40
Chapter 2. Genetics of Xenopus laevis 42
I. Introduction 42
II. Genome Organization 44
III. Estimation of Proportion of Conserved Duplications 47
IV. Gene Mapping 50
V. Conclusion 53
References 54
Chapter 3. Experimentally Induced Homozygosity in Xenopus laevis 58
I. Introduction 58
II. Methods 59
III. Conclusions 66
References 67
Chapter 4. Oogenesis and Oocyte Isolation 68
I. Introduction 68
II. Oogenesis: An Overview 69
III. Isolation of Individual Oocytes from the Ovary 72
IV. Protein Synthesis during Oogenesis: Practical Hints 76
V. Labeling of Oocytes 79
VI. Oocyte Maturation 81
VII. Concluding Comments 82
References 83
Chapter 5 . Early Embryonic Development of Xenopus laevis 86
I. Introduction 87
II. Origins of the Gastrula: Cleavage and Blastula Stages 87
III. Gastrulation and Neurulation 93
IV. Investigating Cell Behaviors 107
V. Experimental Design and the Context-Dependent Function of Cells and Tissues 126
VI. Microsurgical Methods, Tools, and Manipulations 127
VII. Microscopy, Image Processing, and Recording: Epi-illumination and Low-Light Fluorescence Microscopy 133
References 134
PART II. OOCYTES 140
Chapter 6. Vitellogenin Uptake and in Vitro Culture of Oocytes 142
I. Introduction and History 142
II. Methods 144
III. Discussion 155
References 156
Chapter 7. Biochemical Fractionation of Oocytes 158
I. Introduction 158
II. Methods 160
III. Discussion 171
References 172
Chapter 8. Lumpbrush Chromosomes 174
I. Introduction 175
II. Protocol for Lampbrush Chromosomes of Xenopus 176
III. Composition of Isolation and Dispersal Media 182
IV. Alternatives If Germinal Vesicle Contents Fail to Disperse 184
V. Use of Lampbrush Chromosome Preparations 184
VI. Solutions 187
VII. Materials 189
References 190
Chapter 9. Preparation of Synthetic mRNAs and Analyses of Translational Efficiency in Microinjected Xenopus Oocytes 192
I. Introduction 192
II. Methods 196
References 207
Chapter 10. Use of Oligonucleotides for Antisense Experiments in Xenopus laevis Oocytes 210
I. Introduction 210
II. Oligonucleotides Reveal Aspects of snRNA Structure and Function in Oocytes 221
References 233
PART III. EMBRYOS 236
Chapter 11. Fertilization of Cultured Xenopus Oocytes and Use in Studies of Maternally Inherited Molecules 238
I. Introduction 239
II. Protocol for Oocyte Fertilization Using the Host Transfer Technique 241
III. Protocol for Oocyte Fertilization Using in Vitro Fertilization Method 246
IV. Choice of Method 249
V. Applications of the Techniques 249
VI. Summary 254
References 255
Chapter 12. Isolation of Extracellular Matrix Structures from Xenopus laevis Oocytes, Eggs, and Embryos 258
I. Introduction 259
II. Methods 260
III. Discussion 270
References 273
Chapter 13. Analysis of Cellular Signaling Events, the Cytoskeleton, and Spatial Organization of Macromolecules during Early Xenopus Development 276
I. Introduction 276
II. Methodology 279
III. Conclusions 295
References 296
Chapter 14. Generation of Body Plan Phenotypes in Early Embryogenesis 298
I. Introduction 299
II. Axis Perturbations in Early Development 299
III. Scoring of Phenotypes 304
IV. Methods 305
V. Summary 310
References 310
Chapter 15. Fluorescent Dextran Clonal Markers 312
I. Introduction 312
II. Methods 315
III. Practical Considerations 320
References 325
Chapter 16. Nuclear Transplantation in Xenopus 328
I. Introduction and Historical Background 328
II. Results Attainable with Nuclei Transplanted to Eggs 330
III. Methodology 330
References 338
Chapter 17. Mesoderm Induction 340
I. Introduction 340
II. Mesoderm Induction Assay 341
III. Assay End Points: Markers of Mesoderm Induction 345
IV. Mesoderm-Inducing Factors 348
V. Specification of Cell Fate, Organization of Tissues, and Axis Determination 351
VI. Outlook 353
References 354
Chapter 18. Neural Induction 358
I. Neural Induction: Historical Perspective 358
II. Recent Experimental Approaches to Neural Induction 360
III. Signals Initiating Neural Induction 364
IV. Methods 367
V. Summary 371
References 373
Chapter 19. Analysis of Class II Gene Regulation 376
I. Introduction 376
II. Brief Review of Class II Gene Transfer into Frog Embryos 377
III. Gene Selection 378
IV. Initial DNA Injection Experiments 379
V. Preliminary Mapping by Polymerase Chain Reaction Footprinting Analysis 384
VI. Regulatory Element Functional Studies 394
References 395
Chapter 20. Assays for Gene Function in Developing Xenopus Embryos 398
I. Introduction 399
II. Methods 399
III. Results 403
IV. Conclusions 416
References 417
Chapter 21. Histological Preparation of Xenopus laevis Oocytes and Embryos 422
I. Introduction 422
II. Tissue Processing for Light Microscopy 424
III. Light Microscope Level Immunohistochemistry 434
IV. Tissue Preparation for Electron Microscopy 439
V. Immunoelectron Microscopy 442
VI. Photomicrography 447
References 449
Chapter 22. Whole-Mount Staining of Xenopus and Other Vertebrates 454
I. Introduction 455
II. Methods 466
III. Formulations 474
IV. Conclusion 475
References 476
Chapter 23. In Situ Hyridization 480
I. Introduction 481
II . In Situ Hybridization to Sectioned Tissue 482
III. In Situ Hybridization to Whole-Mount Tissue 493
IV. Evaluation of Method 496
References 499
PART IV. MODEL SYSTEMS USING OOCYTES, EGGS, AND EMBRYOS 502
Chapter 24. DNA Recombination and Repair in Oocytes, Eggs, and Extracts 504
I. Introduction 504
II. Methods 506
III. Results and Discussion 513
References 521
Chapter 25. Expression of Ion Channels by Injection of mRNA into Xenopus Oocytes 524
I. Introduction 524
II. Studies Using Oocytes to Express Ion Channels 525
III. Cautionary Notes 527
IV. Preparation of RNA for Injection 530
V. Preparation and Injection of Oocytes 535
VI. Biochemical Analysis of Expression 536
VII. Electrophysiological Analysis 539
VIII. Conclusions 544
References 545
Chapter 26. Tissue Culture of Xenopus Neurons and Muscle Cells as a Model for Studying Synaptic Induction 548
I. Introduction 548
II. Preparation of Neuron and Myotomal Muscle Cultures 549
III. Characteristics of Xenopus Cultures and Development of Neuromuscular Junctions in Vitro 556
IV. Preparation of Myotube Cultures 560
V. Discussion 562
References 563
Chapter 27. The Xenopus Embryo as a Model System for the Study of Cell–Extracellular Matrix Interactions 564
I. Introduction 564
II. Methods 566
III. Concluding Remarks 574
References 576
Chapter 28. Chromatin Assembly 578
I. Introduction 579
II. Preparation of Extracts 580
III. Assays for Chromatin Assembly 585
IV. Unresolved Problems 590
References 594
Chapter 29. DNA Replication in Cell-Free Extracts from Xenopus laevis 598
I. Introduction 598
II. Preparation of Egg and Oocyte Extracts 599
III. Analysis of DNA Replication in Xenopus Cell-Free Systems 607
IV. Selecting an Appropriate System 614
References 617
Chapter 30. Cell Cycle Extracts 620
I. Introduction 620
II. Reagents and Equipment 623
III. Methods 626
IV. Conclusion and Prospects 642
References 643
Chapter 31. Egg Extracts for Nuclear Import and Nuclear Assembly Reactions 646
I. Introduction 647
II. Preparatory Methods 653
III. Nuclear Assembly Reactions and Assays 663
IV. Nuclear Transport Substrates, Reactions, and Assays 664
References 670
Chapter 32. Xenopus Cell Lines 674
I. Introduction 674
II. Uses of Xenopus Cell Lines 675
III. Solutions 676
IV. Preparation of Xenopus Cell Cultures 677
V. Culture Methods 679
VI. Transfection of Xenopus Cell Lines 683
VII. XTC Mesoderm-Inducing Factor 688
VIII. Conclusions 691
References 692
PART V. APPENDIXES 694
Appendix A. Solutions and Protocols 696
Appendix B. Injections of Oocytes and Embryos 702
Appendix C. Mutants of Xenopus laevis 710
Appendix D. Codon Usage for Xenopus laevis 714
Appendix E. Pictorial Collage of Embryonic Stages 718
Appendix F. Xenopus Suppliers in the United States 722
Appendix G. In Situ Hydridization: An Improved Whole Mount Method for Xenopus Embryos 724
Index 736