This revised edition will continue to serve as the most complete and up-to-date guide to the use of the avian embryo in studies of vertebrate development. It will include new approaches to analysis of the chick genome, gene knock-out studies using RNA interference, morpholinos, and other cutting edge techniques. As with the original edition, emphasis has been placed on providing practical guidance, highlighting potentials and pitfalls of all key cell biological and embryological techniques.

* Fully revised second edition, organized into basic and advanced methods.
* New chapters with modern techniques only in use in the past decade, with an emphasis placed on providing practical guidance highlighting potentials and pitfalls.
* International team of contributors with broad expertise in using the avian embryo to study vertebrate development.
* Includes new approaches including analysis of the chick genome, gene knock-out studies using RNAi, morpholinos, and other cutting edge techniques.

Contributors         xi   
Preface         xv   

Part I Embryological Microsurgery and Tissue Culture Methods
Operations on Primitive Streak Stage Avian Embryos
    Andrea Streit
    Claudio D. Stern
    Introduction
    Staging of Embryos
    Anatomy of the Primitive Streak Stage Embryo
    New Culture
    Hensen's Node Grafts and Other Induction Assays
    Other Types of Graft
    Fixing Operated Embryos for Analysis
    References
    Quail-Chick Transplantations
   
    Nicole Le Douarin
    Francoise Dieterlen-Lievre
    Sophie Creuzet
    Marie-Aimee Teillet
    Introduction
        20    (3)
    Differential Diagnosis of Quail and Chick Cells
        23    (4)
    Material and Equipment
        27    (1)
    Egg Holders
        28    (3)
    Preparation and Sealing of Eggs
        31    (1)
    Neural Tissue Transplantations
        31    (8)
    Early Transplantations in Blastodiscs
        39    (1)
    Transplantation of Epiblast or Primitive Streak Fragments
        40    (1)
    Hemopoietic Organ Rudiment Transplantations
        40    (7)
    Genetic Manipulation by Electroporation in the Quial-Chick System
        47    (2)
    Results, Discussion, and Perspectives
        49    (11)
    References
        52    (8)
    Other Chimeras: Quail-Duck and Mouse-Chick
   
    Peter Y. Lwigale
    Richard A. Schneider
    Introduction
        60    (2)
    Preparation of Quail, Duck, Chick, and Mouse Embryos
        62    (3)
    Generation and Analysis of Quail-Duck Chimeras
        65    (2)
    Generation and Analysis of Mouse-Chick Chimeras
        67    (4)
    Conclusion
        71    (5)
    References
        71    (5)
    Manipulations of Neural Crest Cells or Their Migratory Pathways
   
    Marianne Bronner-Fraser
    Martin Garcia-Castro
    Introduction
        76    (1)
    Preparation of Avian Neural Crest Cultures
        76    (8)
    Induction and Specification Assays for Neural Crest Cells
        84    (4)
    Microinjection of Cells and Antibodies into Embryos
        88    (2)
    Labeling of Neural Crest Cells In Vivo with Vital Dyes
        90    (2)
    Grafting Techniques
        92    (2)
    Conclusions
        94    (4)
    References
        95    (3)
    Embryo Slices and Strips: Guidance and Adhesion Assays in the Avian Embryo
   
    Catherine E. Krull
    Kathryn Tosney
    Introduction
        98    (1)
    Preparing and Culturing Embryo Slices
        98    (6)
    Preparing and Culturing Somite Strips
        104    (4)
    Perspectives
        108    (8)
    References
        112    (4)
    Neural Crest, Sensory Neuron, and Muscle Cultures
   
    Vivian M. Lee
    Peter Y. Lwigale
    Introduction
        116    (1)
    Materials
        117    (2)
    Methods
        119    (1)
    Neural Crest Culture
        120    (2)
    Sensory Neuron Culture
        122    (4)
    Pectoral Muscle Culture
        126    (1)
    Results and Discussions
        126    (10)
    References
        130    (6)
    Methods in Avian Embryology Experimental and Molecular Manipulation of the Embryonic Chick Limb
   
    Lee Niswander
    Introduction
        136    (1)
    Visualization of the Limb Bud and Analysis of the Experimental Outcome
        137    (2)
    Proximal-Distal Limb Development
        139    (8)
    Anterior-Posterior Patterning
        147    (3)
    Dorsal-Ventral (D-V) Patterning
        150    (3)
    References
        150    (3)
    Cell Division, Differentiation, and Death in Avian Embryos
   
    Sara Ahlgren
    Introduction
        153    (1)
    Studies in Cell Death
        154    (1)
    Studies in Cell Proliferation and Differentiation
        155    (1)
    Selected Protocols
        156    (14)
    References
        164    (6)
    Part II Labeling and Transgenesis Approaches
   
    In Situ Hybridization Analysis of Chick Embryos in Whole-Mount and Tissue Sections
   
    Herve Acloque
    David G. Wilkinson
    M. Angela Nieto
    Introduction
        170    (1)
    Solutions
        171    (1)
    Single and Multiple Detection of RNA in Floating Sections or Whole-Mount Embryos
        172    (7)
    Photography and Sectioning
        179    (1)
    Whole-Mount Fluorescent In Situ Hybridization
        179    (3)
    Photography and Sectioning
        182    (1)
    In Situ Hybridization to Tissue Sections
        182    (6)
    References
        185    (3)
    Vital Labeling of Embryonic Cells Using Fluorescent Dyes and Proteins
   
    Sujata Bhattacharyya
    Paul M. Kulesa
    Scott E. Fraser
    Introduction
        188    (1)
    Iontophoretic Microinjection of Lineage Tracers
        189    (9)
    Iontophoretic Application of Dil
        198    (2)
    Relative Advantages of Dextran and Dil
        200    (1)
    Photoactivation of Fluorescent Proteins in Single Cells
        200    (8)
    Conclusions and Emerging Technologies
        208    (4)
    References
        208    (4)
    Time-Lapse Imaging of the Early Avian Embryo
   
    Max Ezin
    Scott Fraser
    Introduction
        212    (2)
    The Technology of Time-Lapse Imaging
        214    (4)
    Considerations Before Time-Lapse Imaging
        218    (4)
    Methods
        222    (17)
    References
        233    (6)
    Gain-and Loss-of-Function Approaches in the Chick Embryo
   
    Tatjana Sauka-Spengler
    Meyer Barembaum
    Gain-of-Function by Electroporation
        239    (4)
    Loss-of-Function by Electroporation
        243    (8)
    Retrovirus-Mediated Protein Expression
        251    (7)
    References
        254    (4)
    Manipulation and Electroporation of the Avian Segmental Plate and Somites In Vitro
   
    Tadahiro Iimura
    Olivier Pourquie
    Introduction
        258    (1)
    Rationale
        259    (1)
    Methods
        260    (6)
    Materials
        266    (1)
    Discussion
        267    (2)
    Summary
        269    (3)
    References
        269    (3)
    Transposon-Mediated Stable Integration and Tetracycline-Inducible Expression of Electroporated Transgenes in Chicken Embryos
   
    Yoshiko Takahashi
    Tadayoshi Watanabe
    Shinichi Nakagawa
    Koichi Kawakami
    Yuki Sato
    Introduction
        272    (1)
    Tet-Inducible Expression of Electroporated Transgenes
        272    (4)
    Stable Integration of Electroporated Transgenes
        276    (2)
    Stage-Specific Manipulation of Stably Integrated Transgenes
        278    (1)
    Summary
        278    (4)
    References
        280    (2)
    Generating Transgenic Quail using Lentiviruses
   
    Greg Poynter
    Rusty Lansford
    Introduction
        282    (2)
    Experimental Procedures
        284    (8)
    Concluding Remarks
        292    (6)
    References
        292    (6)
    Part III Functional Genomics
   
    Gene Discovery: Macroarrays and Microarrays
   
    Laura S. Gammill
    Vivian M. Lee
    Introduction
        298    (1)
    Chicken as a Model System: Integrating Embryology and Genomics
        298    (1)
    The Array: Choosing a Platform
        299    (3)
    Strategy: Devising the Best Screen Possible
        302    (1)
    Analysis: Sorting through Several Thousand Data Points
        303    (1)
    Protocol: Macroarray Screening
        304    (10)
    References
        311    (3)
    Dissection of Chick Genomic Regulatory Regions
   
    Hisato Kondoh
    Mesanori Uchikawa
    Introduciton
        314    (1)
    Rationale
        315    (4)
    Methods
        319    (10)
    Discussion
        329    (9)
    References
        335    (3)
    Computational Approaches to Finding and Analyzing cis-Regulatory Elements
   
    C. Titus Brown
    Structure and Funciton of cis-Regulatory Elements
        338    (1)
    Effective cis-Regulatory Sequence Analysis
        339    (3)
    Approaches and Tools for Finding Conserved Sequence Elements
        342    (10)
    Identifying Transcription Factor Binding Sites Computationally
        352    (16)
    References
        363    (5)
    Investigating Regulatory Factors and Their DNA Binding Affinities Through Real Time Quantitative PCR (RT-QPCR) and Chromatin Immunoprecipitation (ChIP) Assays
   
    Lisa A. Taneyhill
    Meghan S. Adams
    Introduction
        368    (5)
    Materials for RT-OPCR
        373    (3)
    General Principles and Definitions
        376    (1)
    Types of Assays
        376    (2)
    Methods of Analysis
        378    (2)
    Assay Setup
        380    (1)
    ChIP and RT-QPCR
        381    (6)
    Conclusions and Perspectives
        387    (4)
    References
        389    (2)
Index         391    (10)
Volumes in Series

Marianne Bronner-Fraser, Developmental Biology Center, University of California, Irvine, USA