Elsevier S & T

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

Ettensohn, Charles E.

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Table of contents

Cover
Contentsv
Contributorsxvii
Prefacexxiii
Dedicationxxv
Chapter 1. The Invertebrate Deuterostomes: An Introduction to Their Phylogeny, Reproduction, Develop1
I. Introduction1
II. Phylogeny2
III. Reproduction5
IV. Development8
V. Genomics10
References11
Part I: Procurement, Maintenance & Culture of Oocytes, Embryos, Larvae and Adults15
Chapter 2. Care and Maintenance of Adult Echinoderms17
I. Overview18
II. Introduction18
III. Adult Echinoderm Models: Their Reproductive Cycles and Gametogenesis18
IV. Obtaining Adult Echinoderms24
V. Maintenance of Adult Echinoderms in Land-Based Systems26
VI. Care and Handling of Adult Echinoderms28
References33
Chapter 3. Echinoderm Eggs and Embryos: Procurement and Culture39
I. Introduction40
II. Method for Sea Urchins (Class Echinoidea)41
III. Method for Sea Stars (Class Asteroidea)53
IV. Method for Brittle Stars (Class Ophiuroidea, Order Ophiurida)60
V. Method for Sea Cucumbers (Class Holothuroidea)64
VI. Method for Interspecific Crosses68
VII. Summary70
References71
Chapter 4. Culture of Echinoderm Larvae through Metamorphosis75
I. Introduction75
II. Materials for Culturing Algae and Larvae76
III. Establishing Algal Cultures78
IV. Culturing Echinoderm Larvae81
V. Metamorphosis and Beyond84
References85
Chapter 5. Obtaining and Handling Echinoderm Oocytes87
I. Introduction88
II. Experimental Preparation88
III. Methods of Oocyte Collection92
IV. Sea Urchin Oocytes Cultured In Vitro98
V. Labeling of Oocyte Components101
VI. Introduction of Experimental Substances into the Oocytes110
VII. Concluding Remarks/Outlook113
References114
Chapter 6. Procurement and Culture of Ascidian Embryos115
I. Overview116
II. Ascidian Development and Metamorphosis116
III. Larval Tissue Specification126
IV. Experimental Techniques129
V. Protocols132
References139
Chapter 7. Culture of Adult Ascidians and Ascidian Genetics143
I. Overview143
II. Introduction144
III. Culturing Ascidians145
IV. Induced Developmental Mutants and Natural Variants/Mutants153
V. Linkage Analysis and Mapping Genes in Ascidians160
References169
Chapter 8. Hemichordate Embryos: Procurement, Culture, and Basic Methods171
I. Introduction172
II. Procurement, Spawning, and Culture of S. kowalevskii173
III. Procurement, Spawning, and Culture of Ptychodera flava179
IV. Removal of Vitelline Envelope in S. kowalevskii182
V. Whole-Mount In Situ Hybridization183
VI. Preparation of Blocking Reagent for P. flava190
VII. Materials and Reagents191
References192
Chapter 9. Cephalochordate (Amphioxus) Embryos: Procurement, Culture, and Basic Methods195
I. Overview195
II. Introduction196
III. Obtaining Gametes of Branchiostoma floridae200
IV. Raising Embryos202
V. Manipulating Embryos206
VI. Amphioxus Resources Available212
VII. Concluding Remarks213
References213
Part II: Embryological Approaches217
Chapter 10. Quantitative Microinjection of Oocytes, Eggs, and Embryos219
I. Introduction219
II. Methods220
III. Equipment and Supplies (Prices are as of December, 2002)230
References242
Chapter 11. Blastomere Isolation and Transplantation243
I. Introduction244
II. Preparation of Mouth Pipettes and Needles245
III. Removal of Fertilization Envelope of Sea Urchin Eggs248
IV. Isolation and Recombination of Blastomeres in Sea Urchin Embryos249
V. Blastomere Isolation and Transplantation in Starfish Embryos263
VI. Blastomere Isolation and Transplantation in Amphioxus265
VII. Blastomere Isolation and Transplantation in Ascidians266
VIII. Lineage Tracing, Blastomere Isolation, and Transplantation in Hemichordates267
References270
Chapter 12. Isolation and Culture of Micromeres and Primary Mesenchyme Cells273
I. Introduction273
II. Isolation and Culture of Micromeres275
III. Isolation and Culture of Primary Mesenchyme Cells282
References284
Chapter 13. Rapid Microinjection of Fertilized Eggs287
I. Introduction287
II. Equipment289
III. Experimental Protocols292
References310
Chapter 14. Methods for Embryo Dissociation and Analysis of Cell Adhesion311
I. Introduction311
II. Gaining Access to the Egg and Embryo312
III. Cell Adhesion Assays: Nonquantitative and Semiquantitative317
IV. Quantitative Centrifugation Assay320
References328
Part III: Cell Biological Approaches331
Chapter 15. Analysis of Sea Urchin Embryo Gene Expression by Immunocytochemistry333
I. Introduction334
II. Immunocytochemistry on Whole Embryos335
III. Immunostaining Small Numbers of Embryos351
IV. Co-localization359
V. Troubleshooting364
References366
Chapter 16. Light Microscopy of Echinoderm Embryos371
I. Introduction (D. Burgess and L. Strickland)372
II. Formaldehyde Fixation of Cleavage Stage Sea Urchin Embryos372
III. Staining and Imaging Fixed Embryos382
IV. Simultaneous Fixation and Visualization of the Actin and Microtubule Cytoskeletons (G. von Dasso385
V. Observation of Live Embryos (D. Burgess and L. Strickland)393
VI. 4-D Imaging of Fluorescent Markers in Live Starfish Oocytes ( J. Ellenberg and P. Lenart)399
References407
Chapter 17. TEM and SEM Methods411
I. Introduction412
II. Basic Fixation and Preparation412
III. Embedding Media421
IV. Preservation of Extracellular Matrix424
V. Fixation for SEM426
VI. Quick Freezing and Freeze-Substitution428
VII. Fixation for Immunohistochemistry and Immunocytochemistry434
VIII. Immunogold Methods434
IX. Ultrastructural Immunoperoxidase436
X. Preparation of Colloidal Gold Reagents (Horisberger, 1981; Slot and Geuze, 1985) as modified by C437
References440
Chapter 18. Calcium Imaging443
I. Introduction443
II. Calcium Signals in Embryos444
III. Getting Calcium Sensors into Embryos450
IV. Measuring Emitted Light454
V. Fluorescence Lifetime Imaging459
VI. Calibration459
VII. Manipulating Intracellular Calcium460
VIII. Conclusions and Perspectives462
References463
Chapter 19. Labeling of Cell Membranes and Compartments for Live Cell Fluorescence Microscopy469
I. Introduction469
II. Labeling of Extracellular Space/Endocytosis/Exocytosis471
III. Labeling of Plasma Membrane/Exocytosis/Endosomes472
IV. Labeling of the Cytosol475
V. Yolk Platelets/Reserve Granules476
VI. Mitochondria478
VII. Other Organelles478
VIII. Endoplasmic Reticulum479
IX. Golgi Apparatus483
X. Nucleus485
XI. Microscopy Considerations486
XII. Future Directions487
References487
Chapter 20. Isolation of Organelles and Components from Sea Urchin Eggs and Embryos491
I. Overview492
II. Egg Jelly Molecules Affecting Sperm492
III. Isolation of the Vitelline Layer from Sea Urchin Eggs496
IV. Isolation of the Cell Surface Complex and the Plasma Membrane–Vitelline Layer (PMVL) Complex f498
V. The Cytolytic Isolation of the Egg Cortex500
VI. Isolation of Cortical Granules501
VII. Isolation of Yolk Platelets503
VIII. Isolation of Mitochondria from Eggs and Embryos504
IX. Isolation of Plasma Membranes and Lipid Rafts from Eggs and Zygotes505
X. Isolation of Microsomes Containing the Endoplasmic Reticulum507
XI. Nuclear Isolation Procedures507
XII. Removal and Isolation of the Fertilization Envelope508
XIII. Isolation of Cilia from Embryos511
XIV. Isolation of Extracellular Matrices from Embryos and Larvae512
XV. Isolation of Sea Urchin Larval Skeletons517
XVI. Resources for the Isolation of Additional Organelles518
References519
Chapter 21. Sea Urchin Spermatozoa523
I. Introduction524
II. Obtaining Sperm and Removing Coelomocytes528
III. Isolating Sperm Heads, Flagella, and Chromatin529
IV. The Isolated Sperm Flagellum as a Sealed Compartment530
V. Extracting Intact Sperm or Isolated Flagella and Heads with Detergents531
VI. Wheat Germ Agglutinin (WGA) Affinity Chromatography of Sperm Membrane Proteins532
VII. Isolating Sperm Plasma Membranes532
VIII. Isolation of Acrosome Reaction Vesicle Membranes (ARV)535
IX. Isolating Lipid Rafts from Sperm536
X. Scoring the Acrosome Reaction by Phase Contrast Microscopy537
XI. Sea Urchin Sperm Bindin539
References540
Chapter 22. Measuring Ion Fluxes in Sperm545
I. Overview546
II. Introduction546
III. Sperm Physiology is Deeply Influenced by Ion Channels and Transporters547
IV. Strategies to Study Sperm Ion Channels and Transporters549
References573
Part IV: Molecular Biological Approaches577
Chapter 23. Isolating DNA, RNA, Polysomes, and Protein579
I. Overview580
II. Introduction580
III. Purification of Total RNA580
IV. Purification of Cytoplasmic and Polysomal RNA586
V. Purification of Genomic DNA589
VI. Preparation of Protein Samples for One- and Two-Dimensional Gel Electrophoresis592
References598
Chapter 24. Detection of mRNA by In Situ Hybridization and RT-PCR601
I. Introduction601
II. In Situ Hybridization602
III. Quantitative PCR613
References618
Chapter 25. Using Reporter Genes to Study cis-Regulatory Elements621
Overview622
I. Introduction622
II. Using Chloramphenicol Acetyltransferase (CAT) Reporter Gene627
III. Using Green Fluorescent Protein (GFP) Reporter Gene632
IV. Quantitative Imaging of GFP in Living Embryos633
V. Using Luciferase (luc) Reporter Gene640
VI. Using lacZ Reporter Gene643
VII. Summary, Prospects, Concluding Remarks648
References649
Chapter 26. Identification of Sequence-Specific DNA Binding Proteins653
Overview654
I. Introduction654
II. Preparation of Nuclear Extracts655
III. Identification of Transcription Factor Target Sites by Footprint and Electrophoretic Mobility S658
IV. Affinity Purification of Sequence-Specific DNA Binding Proteins662
V. Identification of Affinity-Purified DNA-Binding Proteins in SDS Gels666
VI. Preparation of Affinity-Purified Transcription Factors for Protein Sequencing671
VII. Prospects673
References674
Chapter 27. Expression of Exogenous mRNAs to Study Gene Function in the Sea Urchin Embryo677
Overview677
I. Introduction678
II. Methods for Expressing Synthetic mRNA in the Sea Urchin Embryo688
III. Summary, Prospects, Concluding Remarks695
References696
Chapter 28. Disruption of Gene Function Using Antisense Morpholinos699
I. Introduction699
II. Morpholino-Mediated Loss-of-Function: Advantages and Limitations700
III. Morpholino Methods704
IV. Concluding Remarks709
References710
Chapter 29. Generation and Use of Transgenic Ascidian Embryos713
Overview713
I. Ascidian Embryos as Model Chordate Embryos714
II. Transgenic Ascidian Embryos716
III. Fundamentals of Electroporation719
IV. Transgene Construction720
V. Detailed Protocols for Electroporation of Ascidian Zygotes721
VI. List of Required Chemicals and Supplies725
References726
Part V: Genomics731
Chapter 30. Genomic Resources for the Study of Sea Urchin Development733
Overview734
I. Introduction734
II. Sea Urchin Arrayed Library Resources736
III. Computational Tools746
IV. Sea Urchin Genome Project Web Site750
References755
Chapter 31. Genomic Resources for Ascidians: Sequence/Expression Databases and Genome Projects759
Overview759
I. Introduction760
II. The cDNA Projects: cDNA Sequence and Expression Databases762
III. The Genome Project769
IV. Conclusion771
References772
Chapter 32. Gene Regulatory Network Analysis in Sea Urchin Embryos775
I. Introduction775
II. Perturbation Analysis777
III. Assembling and Testing the GRN Logic Map787
IV. Summary792
References793
Part VI: Echinoderm Eggs and Embryos in the Teaching Lab795
Chapter 33. Sea Urchin Gametes in the Teaching Laboratory: Good Experiments and Good Experiences797
Overview798
I. Introduction798
II. Obtaining Adult Urchins and Gametes for the Classroom799
III. Some Guidelines for Using Sea Urchin Gametes in the Classroom801
IV. Basic Introductory Labs802
V. Inquiry-Based Experiments Using the Basic Fertilization Protocol810
VI. Inquiry-Based Experiments in Later Development: Experiments on Differential Gene Expression819
VII. Experiments Using Morphology as an Endpoint821
VIII. Epilogue822
References822
Appendix825
Index841
Volumes in Series877

Book details

Vendor Elsevier S & T
SKU 9780124802780
ISBN-13 9780080496597
Author Ettensohn, Charles E.
Category Science
Subject Developmental Biology

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This book provides a practical guide to experimental methods for studying the development invertebrate deuterostomes as animal model systems. The chapters provide detailed experimental protocols that cover a broad range of topics in modern experimental methods. Topics covered range from rearing embryos to the care of adult animals, while also presenting the basic experimental methods including light and electron microscopy, used to study gene expression, transgenics, reverse genetics, and genomic approaches.

* Covers a wide range of methods, from classical embryology through modern genomics
* Discusses animals related to vertebrates, providing a valuable evolutionary perspective
* Includes a practical guide to the use of sea urchins in the teaching laboratory

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