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

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Charles A. Ettensohn, Gregory A. Wray, Gary M. Wessel
Gulf Professional Publishing, 2004 - Science - 883 pages
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This book provides a practical guide to experimental methods for studying the development of invertebrate deuterostomes, such as sea urchins, ascidians, hemichordates, and amphioxus. These model organisms are of contemporary and historical importance to the study of developmental biology, particularly genomic research. 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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Contents

Procurement and Culture
39
Culture of Echinoderm Larvae through Metamorphosis
75
Obtaining and Handling Echinoderm Oocytes
87
Procurement and Culture of Ascidian Embryos
115
Culture of Adult Ascidians and Ascidian Genetics Carolyn Hctulrickson Lionel Christiaen Karine Deschet Di Jiang JcanStephane Joly Laurent Legend...
143
Introduction
144
Culturing Ascidians
145
Induced Developmental Mutants and Natural VariantsMutants
153
Mitochondria
478
Endoplasmic Reticulum
479
Golgi Apparatus
483
Nucleus
485
Microscopy Considerations
486
Future Directions
487
Isolation of Organelles and Components from Sea Urchin Eggs and Embryos Gary M Wessel and Victor D Vacquicr
491
Overview
492

Linkage Analysis and Mapping Genes in Ascidians
160
References
169
Procurement Culture and Basic Methods ChristopherJ Lowe Kuni Tagawa Tom Humphreys Man Kirschner and John Gcrhart
171
Introduction
172
Procurement Spawning and Culture of 5 kowalevskii
173
Procurement Spawning and Culture of Ptychodera flai1a
179
Removal of Vitelline Envelope in 5 kowalevskii
182
WholeMount In Situ Hybridization
183
Preparation of Blocking Reagent for P flava
190
Materials and Reagents
191
References
192
Procurement Culture and Basic Methods Linda Z Holland and JuKa Yu I Overview
195
Introduction
196
Obtaining Gametes of Branchiostoma Jloridae
200
Raising Embryos
202
Manipulating Embryos
206
Amphioxus Resources Available
212
Concluding Remarks
213
Quantitative Microinjection of Oocytes Eggs and Embryos
219
Blastomere Isolation and Transplantation
243
Isolation and Culture of Micromeres and Primary Mesenchyme Cells
273
Rapid Microinjection of Fertilized Eggs
287
Methods for Embryo Dissociation and Analysis of Cell Adhesion
311
Analysis of Sea Urchin Embryo Gene Expression by Immunocytochemistry
333
Light Microscopy of Echinoderm Embryos
371
TEM and SEM Methods
411
Fixation for SEM
426
Quick Freezing and FreezeSubstitution
428
Fixation for Immunohistochemistry and Immunocytochemistry
434
Ultrastructural Immunoperoxidase
436
Preparation of Colloidal Gold Reagents Horisberger 1981
437
References
440
Calcium Imaging Michael Whitaker I Introduction
443
Calcium Signals in Embryos
444
Getting Calcium Sensors into Embryos
450
Measuring Emitted Light
454
Fluorescence Lifetime Imaging
459
Manipulating Intracellular Calcium
460
Conclusions and Perspectives
462
References
463
Labeling of Cell Membranes and Compartments for Live Cell Fluorescence Microscopy Mark Terasaki and Laurinda A Jaffe I Introduction
469
Labeling of Extracellular SpaceEndocytosisExocytosis
471
Labeling of Plasma MembraneExocytosisEndosomes
472
Labeling of the Cytosol
475
Yolk PlateletsReserve Granules
476
Isolation of the Vitelline Layer from Sea Urchin Eggs
496
Isolation of the Cell Surface Complex and the Plasma MembraneVitelline Layer PMVL Complex from Sea Urchin Eggs
498
The Cytolytic Isolation of the Egg Cortex
500
Isolation of Cortical Granules
501
Isolation of Yolk Platelets
503
Isolation of Mitochondria from Eggs and Embryos
504
Isolation of Plasma Membranes and Lipid Rafts from Eggs and Zygotes
505
Isolation of Microsomes Containing the Endoplasmic Rcticulum
507
Removal and Isolation of the Fertilization Envelope
508
Isolation of Cilia from Embryos
511
Isolation of Extracellular Matrices from Embryos and Larvae
512
Isolation of Sea Urchin Larval Skeletons
517
Resources for the Isolation of Additional Organelles
518
References
519
Sea Urchin Spermatozoa Victor D Vacquier and Noritaka Hirohashi
523
Introduction
524
Obtaining Sperm and Removing Coelomocytes
528
Isolating Sperm Heads Flagella and Chromatin
529
The Isolated Sperm Flagellum as a Sealed Compartment
530
Extracting Intact Sperm or Isolated Flagella and Heads with Detergents
531
Wheat Germ Agglutinin WGA Affinity Chromatography of Sperm Membrane Proteins
532
Isolation of Acrosome Reaction Vesicle Membranes ARV
535
Isolating Lipid Rafts from Sperm
536
Scoring the Acrosome Reaction by Phase Contrast Microscopy
537
Sea Urchin Sperm Bindin
539
References
540
Measuring Ion Fluxes in Sperm Alberto Darszon Christopher D Wood Carmen Beltran Daniel Sanchez Esmeralda Rodriguez Julia Gorelik Yuri E Ko...
545
Overview
546
Isolating DNA RNA Polysomes and Protein
579
Detection of mRNA by In Situ Hybridization and RTPCR
601
Using Reporter Genes to Study asRegulatory Elements
622
Identification of SequenceSpecific DNA Binding Proteins
653
Expression of Exogenous mRNAs to Study Gene Function in
677
Disruption of Gene Function Using Antisense Morpholinos
699
Generation and Use of Transgenic Ascidian Embryos
713
Genomic Resources for the Study of Sea Urchin Development
733
SequenceExpression Databases
759
Gene Regulatory Network Analysis in Sea Urchin Embryos
775
Good Experiments
797
Experiments
819
Appendix
825
Index
841
Volumes in Series
877
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