The Zebrafish Disease Models and Chemical Screens 3rd Edition by H William Detrich, Monte Westerfield, Leonard I Zon ISBN 0123813204 9780123813206

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ISBN 10: 0123813204 
ISBN 13: 9780123813206
Author: H William Detrich, Monte Westerfield, Leonard I Zon

This volume of Methods in Cell Biology is the 3e, and provides comprehensive compendia of laboratory protocols and reviews covering all the new methods developed since 2004. This new volume on Disease Models and Chemical Screens, covers two rapidly emerging and compelling applications of the zebrafish.

The Zebrafish Disease Models and Chemical Screens 3rd Table of contents:

Disease Models
Zebrafish Models of Germ Cell Tumor
Abstract
Overview
Germline Development
Specification of Primordial Germ Cells
Primordial Germ Cell Migration
Epigenetic Reprogramming of Primordial Germ Cells
Spermatogenesis
Germ Cell Tumors
Human Germ Cell Tumors
Type I Germ Cell Tumors
Type II Germ Cell Tumors
Germ Cell Tumors in Zebrafish
Carcinogenesis and Reverse Genetic Models of GCT
Forward Genetic Screens for Gonadal Phenotypes and GCT
Methods for Studying Zebrafish Germ Cells
Visualization of Primordial Germ Cells
In vitro Culture of the Testis
Profiling Testis DNA Content by FACS
Detection of Cell Proliferation by Antiphosphohistone H3 Immunohistochemistry
Materials
References
Dissecting Mechanisms of Myelinated Axon Formation Using Zebrafish
Abstract
Introduction
Origin of Myelinating Glia: Schwann Cells
Origin of Myelinating Glia: Oligodendrocytes
Cell Biology of Myelination
Node of Ranvier Formation
Zebrafish Models of Disruption to Myelinated Axons in Humans
Methods and Materials
Raising Animals for Analysis of Myelinated Axons
Markers of Myelinating Glia and Myelinated Axons
Brief Practical Notes on Whole-Mount Immunochemistry
Visualization of Myelinated Axons: Transgenesis and Chemical Biology
Visualization of Myelinated Axons: Single Cell Analysis
Visualization of Myelinated Axons: Live Imaging
Visualization of Myelinated Axons: Electron Microscopy
Microwave-Assisted Embryonic and Larval Zebrafish Tissue Processing for Electron Microscopy
Primary Fixation
Rinse
Secondary Fixation
Rinse
En Bloc Stain
Dehydration
Acetone Dehydration: 100% EM Grade Acetone
EMBED: Acetone
EMBED Preinfiltration
Embedding
Staining of Sections Mounted on Grids
Recipes
EMBED solution
Sato’s lead stain
Analysis of Electron Microscopy Images
New Frontiers in Electron Microscopy
Behavioral and Physiological Analyses of Myelinated Axon Function in Zebrafish
Forward Genetic Manipulation
Reverse Genetic Manipulation
Morpholino Antisense Oligonucleotides
TILLING
Zinc Finger Nuclease Engineering
Generation of Genetic Chimeras
Generation of Genetic Chimeras by Cell Transplantation
Timing of Chimera Generation
Cell Mosaicism of Individual Chimeras
Transgenic Generation of Genetic Chimeras
Chemical Biology
Summary
Acknowledgments
References
In vivo Analysis of White Adipose Tissue’in’Zebrafish
Abstract
Introduction
Biomedical Relevance of Adipose Tissue
Cellularity of White Adipose Tissue
Rationale
Materials
Methods
Obtaining Zebrafish Embryos
Rearing Zebrafish to Postembryonic Stages in Preparation for Fluorescent Lipid Staining
Staining Live Zebrafish Larvae/Juveniles with Fluorescent Lipophilic Dyes
In vivo Imaging of Neutral Lipid on a Fluorescence Stereomicroscope
In vivo Imaging of Neutral Lipid on a Confocal Microscope
Recovery of Sample After Imaging of Fluorescent Neutral Lipid
Methods for Analyzing Fixed Zebrafish Adipose Tissues
Summary
Acknowledgments
References
Study of Host–Microbe Interactions in•Zebrafish
Abstract
Introduction
Host–Pathogen Interactions
Host–Microbiota Interactions
Laboratory Protocols
Characterization of Microbial Communities in the Gut
Gut Dissections
Materials
Plating Gut Microbiota
Materials
Bacterial gDNA Extractions from Fish Intestines for 16S rRNA Gene Sequencing
Materials
Fluorescent in situ Hybridization for the Detection of Bacteria
Materials
Rearing Germ-Free or Gnotobiotic Zebrafish to 30 dpf
Derivation of Germ-Free Zebrafish
Materials
Solutions (Make the Same Day as Use)
Bacterial Association of Germ-Free Zebrafish
Infection of Zebrafish with Pathogens
Infections of Embryos and Larvae by Static Immersion with Bacterial Pathogens
Materials
Solutions
Infections of Embryos and Larva by Injection with Bacterial or Viral Pathogens
Materials
Solutions
Infections of Embryos and Larva by Static Immersion with Viral Pathogens
Materials
Methods
Infections of Adults by Intraperitoneal Injection with Viral or Bacterial Pathogens
Materials
Methods
References
Hematopoietic Stem Cell Development: Using the Zebrafish to Identify the Signaling Networks and Phys
Abstract
Developmental Hematopoiesis
Review of the Literature
Patterning of the Ventral Mesoderm
Hemangioblast Formation
Primitive Hematopoiesis
Definitive Hematopoiesis
Use of Zebrafish to Investigate HSC Biology
Zebrafish Mutants
Mesoderm Mutants
Kugelig and spadetail
Hemangioblast Mutants
Scl/tal1
Cloche
Primitive Hematopoietic Mutants
Bloodless
Vlad tepes
Moonshine
Definitive Hematopoietic Mutants
Runx1
Mindbomb
Rumba and samba
Chemical Screens
PGE2 and wnt
Nitric Oxide and Blood Flow
Rescue Screens
Visualization of HSC Emergence
Transgenic Lines, Hematopoietic Cell Culture, FACS Analysis, and Transplantation
Zebrafish Tools and Protocols
Tools – Transgenic Reporter Lines
Protocol – Chemical Screen for HSC Formation
Protocol – Confocal Microscopy for Analysis of HSC Formation and Blood Flow
Protocol – Use of Fluorescence-Activated Cell Sorting to Demonstrate Cellular Processes During
References
Zebrafish as a Model for Hemorrhagic Stroke
Abstract
Introduction and Basic Concepts
Maintaining the Barrier Function of Blood Vessels
Vascular Integrity and Stroke
Zebrafish as a Model for Studying Hemorrhage and Stroke
Studying Vascular Integrity in the Zebrafish
Concluding Remarks
References
A Zebrafish Model for VHL and Hypoxia Signaling
Abstract
VHL Disease
Chuvash Polycythemia
VHL Regulates HIF Signaling
HIF-Independent Functions of pVHL
VHL Animal Models
Zebrafish as a New Model for VHL
Loss of Vhl Leads to a Systemic Hypoxic Response in’Zebrafish
vhl Mutants Develop Chuvash Polycythemia and Blood Cell Maturation Defects
Zebrafish vhl Mutants Develop Angiogenesis Defects
Zebrafish vhl Mutants Develop Pronephros Abnormalities
Translation from Fish to Humans
Dissection of Genotype/Phenotype Correlations
Translation from Fish to Humans – Identification of Biomarkers
Translation from Fish to Humans – Screening for Novel Agents and Combination Therapies
Concluding Remarks
Acknowledgments
References
Basement Membrane Diseases in Zebrafish
Abstract
Introduction
Basement Membrane-Related Zebrafish Pathologies
Notochord
Somites and Muscle
Blood Vessels
Kidney
Central Nervous System
Skin and Fins
Methodology for Zebrafish Studies of Basement Membrane’Composition and Function
Embedding Samples and Sectioning
Durcupan Embedding Protocol
Materials
Protocol
Localization of BM Components
Fixation
Histological Staining
Antibody Staining
Transmission Electron Microscopy
Transplantation Experiments
Forward and Reverse Genetics and Transgenesis
Summary
Acknowledgments
References
Zebrafish Provides a Novel Model for Lymphatic Vascular Research
Abstract
Introduction
Embryonic Lymphangiogenesis in Zebrafish
Comparison Between Teleosts and Other Vertebrates
The Added Value of Zebrafish
The Secondary Vessel System in Teleosts
Concluding Remarks
Methods
Lymphangiography
Acknowledgments
References
Not All Bones are Created Equal – Using Zebrafish and Other Teleost Species in Osteogenesis Resear
Abstract
Case Studies – Using Zebrafish for Addressing Biomedical Questions
The Evolution of Skeletal Tissues
Cartilage and Bone in Teleost Fish
Intermediate Skeletal Tissues
Osteocyte-Containing Bone and Acellular Bone
Development of Teleost Vertebral Bodies, A Derived Process
Remodeling of the Teleost Skeleton
Conclusions
Acknowledgments
References
Zebrafish Assays of Ciliopathies
Abstract
Introduction
Methods
Visualization of Cilia in Zebrafish Embryos
Whole-mount Fluorescence Imaging of Cilia
Visualization of Kupffer’s Vesicle
Phenotypic Assays of Ciliary Dysfunction
Live Embryo Phenotypes
Measurement of Convergent Extension Defects by ISH
Epiboly Cell Tracking
Assaying Human Gene Variation in Zebrafish
Zebrafish Ciliary Mutant Lines
General Considerations/Future Development
Acknowledgment
References
Infectious Disease Modeling and Innate Immune Function in Zebrafish Embryos
Abstract
Introduction
Observation and Isolation of Innate Immune Cells
Innate Immune Cell Development in Zebrafish
Detection of Immune Cells Using Cell-Specific Markers
Colocalization Studies of Immune Cell Markers by in situ Hybridization
Two-Step Double FISH Protocol
One-Step Double FISH Protocol
Identification of Cell Types Using Immunodetection and Histochemical Staining
Combined l-Plastin Immunofluorescence and Chromogenic Mpx Activity Detection
Transgenic Reporter Lines for Live Imaging of Immune Cell Behavior
Isolation of Immune Cells from Reporter Lines
Embryo Dissociation for FACS
FACS
RNA Extraction from FACS-Sorted Cells
Bacterial Infection Methods
Routes of Infection
Yolk Injection
Tail Muscle Injection
Hindbrain Ventricle Injection
Otic Vesicle Injection
Systemic Infection via the Blood Island
Infection via Duct of Cuvier
Quantification of Bacterial Burden
CFU Determination
Pixel Count Analysis Using Fluorescent Images
High-Throughput Quantification Using the COPAS System
Model Systems for Infectious Diseases
S. typhimurium Infections
M. marinum Infections
Analysis of the Innate Immune Response
Bioassays for the Innate Immune Response
Transcriptomic Analysis
Microarray-Based Transcriptome Analysis
Next-Generation Sequencing
RNA Isolation Protocol
RNA Isolation Protocol for Pools of 15–20 Embryos
Morpholino Knockdown of Innate Immunity Mediators
Conclusions
Acknowledgments
References
Zebrafish as a Model for the Study of Human Cancer
Abstract
Introduction: Zebrafish as a Cancer Model
The Genetically Tractable Zebrafish
Transgenesis
Mutagenesis
Transgenic Models of Oncogenesis
Zebrafish Models of Leukemia
Pancreatic Cancer Models
Melanoma Models
Embryonic Rhabdomyosarcoma Model
Modeling the Loss of Tumor Suppression
tp53
rp
apc
pten
mybl2
hlk, srk, and otg mutants
Modeling Tumor Cell Intravasation and Metastasis
Tumor-Initiating Cells
In vivo Small-Molecule Screens and Drug Discovery
Conclusions
References
Generating and Analyzing Fish Models of Melanoma
Abstract
Introduction
Melanoma
Melanoma Genetics in Fish
Xiphophorus
Zebrafish
Medaka
Rationale
Transgenic Melanoma Lines in Zebrafish and Medaka
Medaka Microinjection Protocol
Crossing Procedures for Obtaining Xiphophorus Tumors
Spontaneous Melanoma and Pigment Lesions
UV-induced Melanoma Crosses
Ultra-violet Irradiation Treatments
Protocol for UVB Treatment of Laboratory Fish
Basic Melanoma Pathology
Morphological Assessment of Tumors
Histology
From the Tank to the Slide: Tissue Preparation and Sectioning
Hematoxylin and Eosin (H&E) Staining
Molecular Analysis
Immunohistochemistry Protocol
Pigmentation
Melanocytic Markers
Histogenetic Markers for Other Cell Types
Markers of Proliferation and Cell Cycle Activity
RNA and Protein Isolation From Adult Fish Tumors
RNA Isolation From Melanoma
Preparation of Protein Lysate From Fish Tissue
Conclusions
Acknowledgments
References
Screening Pancreatic Oncogenes in Zebrafish Using the Gal4/UAS System
Abstract
Introduction
Rationale
Transgenic Zebrafish with Gal4/UAS-Mediated eGFP-KRASG12V Expression in the Exocrine Pancreas
Generation of UAS Regulated eGFP-KRASG12V Transgene
Gal4/UAS-Mediated Expression of eGFP-KRASG12V Transgene in the Exocrine Pancreas
Identification and Characterization of Pancreatic Tumors
Identifying Pancreatic Tumors Expressing eGFP-KRASG12V in Living Fish
Dissection and Fixation of Transgenic Fish Bearing KRAS-Initiated Pancreatic Tumors
Dissection of Visceral Organs
Fixation
Preparing Paraffin-Embedded Sections of Tumors
Decalcification (optional)
Preliminary Embedding in Agar (Optional)
Paraffin Embedding and Sectioning
Preparing Frozen Sections of Dissected Tumors
Sucrose Treatment
O.C.T Compound Treatment
Sectioning
Rapid Evaluation of eGFP-KRASG12V Expression
Hematoxylin and Eosin (H&E) Staining
Deparaffinization/Rehydration
H&E staining
Immunohistochemical (IHC) Staining for Paraffin-Embedded Sections
IHC Staining Using ABC Avidin/Biotin Method
Immunofluorescent (IF) Staining
Blocking
Incubation in Primary Antibody
Detection
Conclusions
Acknowledgments
References
Zebrafish Models of Rhabdomyosarcoma
Abstract
Introduction
Rationale
Material and Methods
Generation of Zebrafish Rhabdomyosarcoma Mosaic Transgenic Approach
Generation of Stable Transgenic Lines
Heat Shock-Inducible Cre-Lox Approaches to Induce Rhabdomyosarcoma in Zebrafish
Identification of Genetic Modifiers of Cancer Initiation and Maintenance
Co-Injection Strategies to Label ERMS Cell Subpopulations
Identifying the Serially Transplantable ERMS-Propagating Cell Population
Short-Term Irradiation Assay
Transplanting into Clonally Syngeneic Fish
Bioinformatics Approaches to Identify Conserved Pathways in Zebrafish and Human ERMS
Microarray Analysis and Gene Set Enrichment Analysis (GSEA)
Array CGH Approach
Discussion
Acknowledgments
References
Transplantation in Zebrafish
Abstract
Introduction
Rationale
Mosaic Analysis and Transplantation in Developmental Biology
Transplantation in Stem Cell Biology
Transplantation in Cancer Biology
Assessment of Engrafted Populations in the casper Strain
Methods
Blastula/Gastrula Transplantation in Zebrafish Embryos for Mosaic Analysis
Blastula Transplantation
Gastrula Transplantation
Resources and Protocols
Adult Stem Cell Transplantation in Zebrafish
Transplanting HSCs into Zebrafish Embryos
Transplanting HSCs into Adult Zebrafish
Resources and Protocols
Tumor Transplantation in Zebrafish
Resources and Protocols
Discussion
Zebrafish Immunology and Clonal Zebrafish
Identifying Bona Fide Stem Cells
Genetic and Chemical Screening with Transplantation Model
Acknowledgment
References
Disease Modeling by Gene Targeting Using MicroRNAs
Abstract
Introduction
Mechanisms of MicroRNA Silencing
Dicer and MicroRNAs in Zebrafish Development
Development of Vector-Based RNA Interference
RNA Interference Work in Zebrafish
Use of siRNAs in the Zebrafish
Materials and Methods
MicroRNA Cassette Design
Annealing DNA Oligonucleotides
Generation of MicroRNA Containing Vectors
Synthesis of Runoff Transcripts From the MicroRNA Containing Vector and the Reporter Vector
Microinjection of Runoff Transcripts
Fluorescence Imaging of Injected Embryos
Results
Future Directions
References
Fluorescent Imaging of Cancer in Zebrafish
Abstract
Introduction
Fluorescent Proteins and Transgenic Models of Cancer
Types of Fluorescent Proteins and Practical Considerations
Creation of Stable Transgenic Cancer Models
Mosaic Transgenic Approaches
Selecting Strains of Zebrafish for Imaging Cancer
Macroscopic Observation of Tumor Growth
Stereomicroscopy to Image Tumor Growth
LED Fluorescence Macroscope Imaging
Microscopic Observation in Tumorigenesis
Confocal Microscopy
Spinning-Disc Confocal Microscopy
Two-Photon Microscopy
Confirming Transformation of Fluorescent-Labeled Tumor Cells by Cell Transplantation into Irradiated
Method
Identifying Tumor-Propagating Cell Subpopulations by Fluorescent Protein Expression and Cell Transpl
Use of Syngeneic Zebrafish for Cell Transplantation of Fluorescent-Labeled Tumors and Drug Discovery
Cell Transplantation into Syngeneic Zebrafish to Accurately Assess Self-Renewal in Fluorescent-Label
Use of Syngeneic Zebrafish for Cell Transplantation: Single Cell Transplants
Xenograft Transplantation of Fluorescently Labeled Cells into Zebrafish
Conclusions
Acknowledgments
References
The Role of Fanconi Anemia/BRCA Genes in Zebrafish Sex Determination
Abstract
Introduction
Fanconi Anemia: A Disease of DNA Repair
Fanconi Anemia Genes
The FA/BRCA Network
FANC Genes do More than Repair DNA
Results and Discussion
The FA/BRCA Gene Network is Conserved between Humans and Zebrafish
Zebrafish Fanconi Mutants and Sex-Determination
Mechanisms of Sex Determination
Master Sex-Determining Genes
Gonad Development in Fish
Sex Determination and Gonad Development in Zebrafish
Gonad Differentiation Determines Sex in Zebrafish: Animals Lacking Germ Cells Develop Testes and Bec
Mutations in FA Genes Induce Female-to-Male Sex Reversal in Zebrafish
Zebrafish FA Mutants Show that the Mere Presence of Germ Cells is not Sufficient to Feminize the Gon
Oocytes Passing Through Meiosis are Essential to Support the Differentiation of Ovaries in Zebrafish
The Role of Meiotic Oocytes in Preventing Female Somatic Cells (Pregranulosa) from Transdifferentiat
Increased Tp53-Dependent Germ Cell Apoptosis Causes Sex Reversal in fancl Mutants by Compromising Oo
A Threshold Number of Developing Oocytes could be a Determinant of Ovarian Fate
A Model for Zebrafish Sex Determination: Tp53-Mediated Germ Cell Apoptosis can Regulate Oocyte Survi
Zebrafish as a Model for Understanding the Molecular Basis of FA Disease and Gonadal Cancers
Alteration of the FA/BRCA Network Abnormally Activates Tp53-Dependent Apoptosis in Zebrafish as it d
Postembryonic Roles of fancd1(brca2) in Zebrafish Gametogenesis and Oocyte Nuclear Architecture Prov
Zebrafish FA Mutants as Useful Tools for Drug Screening
Summary
Acknowledgments
References
Chemical Screens
Chemical Screening in Zebrafish for Novel Biological and Therapeutic Discovery
Abstract
Introduction
Rationale
Materials and Methods
Zebrafish Screen Scoring Phenotypes
Developmental Morphology Scoring – Specific
Developmental Morphology Scoring – Nonspecific
Behavioral Scoring
Cell State Scoring
Choice of Small Molecule Library
Commercial Libraries
Bioactive Libraries
In-House Synthetic Libraries
Diversity-Oriented Synthesis (DOS) Libraries
Natural Product Libraries
Chemical Screening Platform
Target Identification (Target ID)
Candidate-Based Identification
Annotated Bioactive Libraries
Chemoinformatics
Genetics
General Target ID Methods Applied to Zebrafish Screens
Protein Pull-Down
Cell-Based Assays
In vitro Biochemical Assays
Computer Docking Simulations
Discussion/Caveats
Biological Relevance of Zebrafish Screening
Screening Technology Caveats
The Feasibility of Juvenile/Adult Chemical Screens
Hit Detection Caveats
Summary
Acknowledgments
References
Using the Zebrafish Photomotor Response for Psychotropic Drug Screening
Abstract
Introduction
The PMR Behavior
Methods
Aquaculture and Multiwell Plates
Chemical Treatment
Dark Adaptation
Stimulus Presentation
Digital Video Capture
Measuring Motor Activity
Behavioral Barcoding, Hit Identification and Phenoclustering
Discussion
Acknowledgments
References
Designing Zebrafish Chemical Screens
Abstract
Rationale for Conducting Small Molecule Screens With Zebrafish
Selection of Small Molecule Libraries
Assay Design: Major Screen Types
Identifying Novel Probes for Defined Biological Pathways
Discovering Unexpected Connections Between Known Biological Pathways
Identifying Novel Modifiers of Disease-Related Phenotypes
Annotating Chemical Libraries
Screening Methods
Handling Embryos
Compound Handling
Phenotype Detection
Mechanism of Action Studies
Beginning With the End in Mind
Computational Approaches to Mechanism
Experimental Approaches to Mechanism

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Tags: H William Detrich, Monte Westerfield, Leonard I Zon, Zebrafish Disease, Chemical Screens