Germline Development in the Zebrafish: Methods and Protocols (Methods in Molecular Biology, 2218)

✍ Scribed by Roland Dosch (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 380
• Category: Library

✦ Synopsis

This volume details a wide range of methods, ranging from beginner through advanced, used to

further study zebrafish and fish germline.. Chapter guide readers through cultivating and manipulating germ cells, imaging of germline processes and the molecular analysis of their, protein, and RNA. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, application details for both the expert and non-expert reader, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and cutting-edge, Germline Development in the Zebrafish: Methods and Protocols aims to ensure successful results in the further study of this vital field.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Separation of Oocyte and Follicle Layer for Gene Expression Analysis in Zebrafish
1 Introduction
2 Materials
2.1 Zebrafish
2.2 Isolation and Separation of Follicles
2.3 RNA Extraction and RT-PCR
3 Methods
3.1 Isolation of FG Follicles from Zebrafish
3.2 Pretreatment of FG Follicles
3.3 Mechanic Separation of Oocyte and Follicle Layer
3.4 Purity Check by Molecular Markers gdf9 and lhcgr
4 Notes
References
Chapter 2: The Xenopus Oocyte as an Expression System for Functional Analyses of Fish Aquaporins
1 Introduction
2 Materials
2.1 DNA and cRNA Preparation
2.2 Xenopus laevis Oocyte Extraction and Microinjection
2.3 Determination of Oocyte Water and Solute Permeability
2.4 Extraction of Plasma and Total Membrane Proteins from Oocytes
2.5 Co-immunoprecipitation (Co-IP)
2.6 Immunofluorescence Microscopy
3 Methods
3.1 cRNA Synthesis
3.2 Ovarian Follicle Isolation and Microinjection
3.3 Measurement of Oocyte Water and Solute Permeability
3.3.1 Volumetric Assays and Calculation of Oocyte Permeability Coefficients
3.3.2 Solute Uptake Assays Using Radiolabeled Compounds
3.4 Assessment of Protein Levels of Expressed Aquaporins in Xenopus Oocytes
3.4.1 Total Membrane Protein Extraction
3.4.2 Plasma Membrane Protein Extraction
3.4.3 Co-IP Assay
3.5 Immunofluorescence Microscopy
4 Notes
References
Chapter 3: Computer-Assisted Sperm Analysis to Test Environmental Toxicants
1 Introduction
2 Materials
3 Methods
3.1 Stripping
3.2 Assessment of Fresh Sperm Motility
3.3 Sperm Dilution and Exposure
3.4 Assessment of Sperm Motility
3.5 Statistical Analysis
4 Notes
References
Chapter 4: Cryopreservation and Transplantation of Spermatogonial Stem Cells
1 Introduction
2 Materials
2.1 Equipment
2.1.1 Dissection
2.1.2 Cryopreservation
2.1.3 Microinjection and Transplantation
2.2 Chemicals and Buffers
2.3 Zebrafish
3 Methods
3.1 Anesthesia, Dissection, and Testis Collection
3.2 Freezing of Testes
3.3 Tissue Dissociation
3.4 Preparation of Needles
3.4.1 Pulling of Glass Needles
3.4.2 Grinding the Tip of the Needle
3.5 Microinjection of Embryos with MO1-dnd
3.6 Labeling Cells with PKH-26 Linker Dye
3.7 Spermatogonia Transplantation
4 Notes
References
Chapter 5: Masculinization of Zebrafish Through Partial Depletion of Primordial Germ Cells by Injecting Diluted Morpholino Oli...
1 Introduction
2 Materials
2.1 Preparation of the Microinjection Needle
2.2 Microinjection of Embryos with Diluted dnd-MO
2.3 Dechorionation
2.4 Sorting the Embryos According to Their PGC Number
2.5 Dissection of Larvae
3 Methods
3.1 Preparation for Microinjection
3.2 Microinjection of Embryos with Diluted dnd-MO
3.3 Dechorionation of Microinjected Embryos
3.4 Sorting the Embryos According to Their PGC Number
3.5 Dissection of Larvae
3.6 Gene Expression Profiling
4 Notes
References
Chapter 6: Chemical Genetics: Manipulating the Germline with Small Molecules
1 Introduction
2 Materials
2.1 Zebrafish Lines
2.2 In Vitro Transcription of the Reporter mRNA
2.3 Microinjection
2.4 Small-Molecule Preparation
2.5 Assessment of PGC Development
2.6 Common Materials
3 Methods
3.1 Preparation of Zebrafish Embryos
3.2 In Vitro Transcription of the Reporter mRNA
3.3 Microinjection
3.4 Treatment of Embryos with Small-Molecule Libraries
3.5 Qualitative Assessment of PGCs by Imaging GFP Fluorescence
3.6 Quantitative Assessment of PGC by Counting the GFP Fluorescence
3.7 Quantitative Assessment of PGC by Measuring Luminescence
4 Notes
References
Chapter 7: In Vitro Induction of Teleost PGCs
1 Introduction
2 Materials
2.1 Zebrafish Embryo Maintenance
2.2 Embryonic Cell Recovery
2.3 Embryonic Cell Culture
2.4 Embryonic Cell Differentiation to Primordial Germ Cells (PGCs)
2.5 Analysis of Migration Capacity of In Vitro Generated Primordial Germ Cells
3 Methods
3.1 Zebrafish Embryo Maintenance
3.2 Embryonic Cell Recovery
3.3 Embryonic Cell Differentiation to Primordial Germ Cell (PGC)
3.4 Analysis of Migration Capacity of In Vitro Generated Primordial Germ Cell
3.4.1 Recipient Sterilization
3.4.2 In Vitro Generated PGC Transplant
4 Notes
References
Chapter 8: Applying Rho Pathway Inhibitors to Investigate Germ Plasm Localization
1 Introduction
2 Materials
2.1 Injections
2.2 Immunofluorescence Staining
2.2.1 Nuclei Staining
2.3 In Situ Hybridization
3 Methods
3.1 Inhibitor Injections
3.1.1 Immunofluorescence
3.1.2 Tubulin Immunofluorescence
3.1.3 Active RhoA Immunofluorescence
3.1.4 Nuclei Staining
3.2 In Situ Hybridization
3.3 Fluorescent In Situ and Immunofluorescence
4 Notes
References
Chapter 9: Cryopreservation of Pooled Sperm Samples
1 Introduction
2 Materials
2.1 Sperm Collection
2.2 Cryopreservation
2.3 Thawing and IVF
3 Methods
3.1 Collecting and Pooling of Sperm
3.2 Estimation of Sperm Cell Densities and Sample Dilution
3.3 Pooled Sperm Freezing
3.4 Sperm Motility Assessment
3.4.1 Pre-freeze Motility
3.4.2 Post-thaw Sperm Motility
3.5 Obtaining Eggs, Thawing Sperm, and In Vitro Fertilization (IVF) Procedure
3.5.1 Egg Collection
3.5.2 Sperm Sample Thawing
3.5.3 In Vitro Fertilization
4 Notes
References
Chapter 10: Quantifying Tissue Tension in the Granulosa Layer After Laser Surgery
1 Introduction
2 Materials
2.1 Transgenic Fish Lines
2.2 Media
2.3 Ovarian Follicle Isolation
2.4 Laser-Cutter Microscope Setup
2.5 Sample Mounting
2.6 Data Analysis
3 Methods
3.1 Ovary Dissection
3.2 Follicle Isolation
3.3 Prepare the Laser-Cutter Microscope
3.4 Mounting Follicles in Agarose
3.5 UV Laser Ablation of Granulosa Cellular Junctions
3.6 Data Analysis
4 Notes
References
Chapter 11: In Vivo and Ex Vivo CT Imaging of Zebrafish Gonads
1 Introduction
2 Materials
2.1 Devices
2.2 Consumables
2.3 Chemicals
3 Methods
3.1 Anesthesia for In Vivo CT Scans of Zebrafish
3.2 In Vivo CT Scanning
3.3 X-Ray Contrast Agents for In Vivo Imaging of Gonads
3.4 Ex Vivo Staining of the Adult Zebrafish for High-Resolution MicroCT Acquisition
3.5 High-Resolution Ex Vivo Synchrotron CT Imaging of Adult Zebrafish
4 Notes
References
Chapter 12: Live and Time-Lapse Imaging of Early Oogenesis and Meiotic Chromosomal Dynamics in Cultured Juvenile Zebrafish Ova...
1 Introduction
2 Materials
2.1 Ovary Dissection and Culture
2.2 Mounting
2.3 Microscopy
3 Methods
3.1 Ovarian Isolation, Staining, and Culturing
3.2 Mounting Ovaries for Live Imaging
3.3 Microscopy and Live Time-Lapse Image Acquisition
4 Notes
References
Chapter 13: Detection of the Polar Body After Fertilization
1 Introduction
2 Materials
2.1 Zebrafish and Fish Facility
2.2 Zebrafish Embryo Collection
2.3 Dechorionated Embryo Fixation
2.4 Phalloidin and DAPI Staining
2.5 Stained Embryo Imaging
3 Methods
3.1 Zebrafish Natural Spawning
3.2 Synchronous Fertilized Embryo Collection
3.3 Dechorionated Embryo Fixation
3.4 Phalloidin and DAPI Staining
3.5 Stained Embryo Imaging
4 Notes
References
Chapter 14: Labeling the Micropylar Cell in Zebrafish Whole-Mount and Cryo-sectioned Follicles
1 Introduction
2 Materials
2.1 Ovary Dissection and Fixation
2.2 Cryosections
2.3 Immunostaining
2.4 Whole-Mount In Situ Hybridization (WISH)
2.5 Mounting and Imaging
3 Methods
3.1 Zebrafish Husbandry
3.2 Euthanizing a Zebrafish Female and Dissecting the Ovary
3.3 Preparing Ovarian Tissue for Cryosections
3.4 Cryo-sectioning
3.5 Immunostaining on Cryosections
3.6 Imaging of Immunostained Cryosections
3.7 Preparation of Follicles for Whole-Mount Immuno-Fluorescence and In Situ Hybridization (WISH)
3.8 Whole-Mount Immuno-Fluorescence
3.9 Whole-Mount In Situ Hybridization (WISH)
3.10 Mounting and Imaging the Stained Whole-Mount Follicles
4 Notes
References
Chapter 15: Visualization of Transcriptional Activity in Early Zebrafish Primordial Germ Cells
1 Introduction
2 Materials
2.1 Adult Zebrafish Cross and Embryo Collection
2.2 Microinjection of Zebrafish Embryos
2.3 Embryo Preparation for Live Imaging
2.4 Embryo Preparation for Fixed-Embryo Imaging
3 Methods
3.1 Embryo Collection and Dechorionation
3.2 Microinjection of Morpholinos in Zebrafish Embryos
3.3 Sample Preparation and Imaging Acquisition
3.3.1 Embryo Fixation and Imaging
3.3.2 Live Embryo Imaging via Light Sheet Z1 Microscope
3.4 Image Acquisition
4 Notes
References
Chapter 16: Experimental Methodologies to Visualize Early Cell Biology in Zebrafish Embryos
1 Introduction
2 Materials
2.1 Obtaining Synchronously Fertilized Zebrafish Embryos
2.1.1 Manual Extrusion of Mature Eggs from Female Zebrafish
2.1.2 Preparation of Sperm Solution from Male Zebrafish
2.1.3 In Vitro Fertilization of Zebrafish Eggs
2.2 Immunofluorescence Labeling of Zebrafish Embryos
2.2.1 Dechorionation of Live Embryos
2.2.2 Fixation and Storage of Fixed Embryos
2.2.3 Preparation of Embryos for Immunofluorescence Labeling
2.2.4 Incubation of Embryos with Primary Antibodies
2.2.5 Incubation of Embryos with Secondary Antibodies and Nuclei Labeling
2.2.6 Mounting Immunolabeled Zebrafish Embryos for Imaging
3 Methods
3.1 Obtaining Synchronously Fertilized Zebrafish Embryos
3.1.1 Manual Extrusion of Mature Eggs from Female Zebrafish
3.1.2 Preparation of Sperm Solution from Male Zebrafish
3.1.3 In vitro Fertilization of Zebrafish Eggs
3.2 Immunofluorescence Labeling of Zebrafish Embryos
3.2.1 Dechorionation of Live Embryos
3.2.2 Fixation and Storage of Fixed Embryos
3.2.3 Preparation of Embryos for Immunofluorescence Labeling
3.2.4 Incubation of Embryos with Primary Antibodies
3.2.5 Incubation of Embryos with Secondary Antibodies and Nuclei Labeling
3.2.6 Mounting Immunolabeled Zebrafish Embryos for Imaging
4 Notes
References
Chapter 17: Observation of Medaka Larval Gonads by Immunohistochemistry and Confocal Laser Microscopy
1 Introduction
2 Materials
2.1 Dissection and Fixation
2.2 Immunohistochemistry
2.3 Preparation of Agarose Pads
2.4 Mounting on the Agar Pad and Observation
3 Methods
3.1 Dissection and Fixation
3.2 Immunohistochemistry
3.3 Preparation of Agarose Pads
3.4 Mounting and Observation
4 Notes
References
Chapter 18: Methods for Visualization of RNA and Cytoskeletal Elements in the Early Zebrafish Embryo
1 Introduction
2 Materials
2.1 Fixation of Zebrafish Embryos
2.2 De Novo RNA Probe Design and Synthesis
2.3 Two-Color Fluorescence In Situ Hybridization with Microtubule Immunofluorescence
2.4 Phalloidin Labeling for Preservation and Visualization of F-Actin Structures in the Early Zebrafish Embryo (with Optional ...
3 Methods
3.1 Fixation of Zebrafish Embryos for Cytoskeleton Visualization
3.2 De Novo RNA Probe Design and Synthesis
3.3 Two-Color Fluorescence In Situ Hybridization with Microtubule Immunofluorescence
3.4 Rapid Phalloidin Labeling for Preservation and Visualization of F-Actin Structures in the Early Zebrafish Embryo (with Opt...
4 Notes
References
Chapter 19: Glyoxal Fixation as an Alternative for Zebrafish Embryo Immunostaining
1 Introduction
2 Materials
2.1 Zebrafish Embryo Collection and Handling
2.2 Embryo Fixation
2.3 Blocking and Washings
2.4 Staining
2.5 Imaging
3 Methods
3.1 Zebrafish Embryo Collection and Handling
3.2 Embryo Fixation
3.2.1 Glyoxal-Based Embryo Fixation
3.2.2 PFA-Based Embryo Fixation
3.3 Immunostaining
3.4 Imaging
4 Notes
References
Chapter 20: Histological Analysis of Gonads in Zebrafish
1 Introduction
2 Materials
2.1 Dissection and Fixation
3 Methods
3.1 Fixation
3.2 Decalcification (Not Necessary for Bouin´s Fixed Samples or Isolated Gonads)
3.3 Infiltration and Embedding
3.4 Sectioning
3.5 Histological Staining
3.5.1 Hematoxylin and Eosin (see Note 16)
3.5.2 Periodic Acid Schiff´s With Hematoxylin (see Note 16)
4 Notes
References
Chapter 21: Manipulating and Visualizing the Germline with Transgenic Lines
1 Introduction
2 Materials
3 Methods
3.1 Generation of Germ-Line Labeling Transgenic Zebrafish
3.1.1 Harvest Fertilized Eggs for Microinjection
3.1.2 Injection of Transgenic Constructs (Fig. 1)
3.1.3 Raising Transgenic Founders
3.1.4 Maintenance of Tg(piwil1:egfp-UTRnanos3) Transgenic Line
3.2 Observation on Live Embryo
3.3 Mounting Embryos for PGCs Imaging (Fig. 2)
3.4 Imaging Fixed PGCs
3.5 Imaging Stained Larval Gonads
3.6 Confocal Imaging of Gonads and Counting PGCs
4 Notes
References
Chapter 22: Liquid Chromatography and Tandem Mass Spectrometry in Label-Free Protein Quantification of Zebrafish (Danio rerio)...
1 Introduction
2 Materials
2.1 Sample Collection and Embryo Culture for Egg Quality Assessments
2.2 Tissue Homogenization, Total Protein Extraction and Measurement
2.3 Determination of Protein Concentration (Bradford Assay)
2.4 SDS-PAGE
2.5 In Gel Tryptic Digestion
2.6 Peptide Extraction
2.7 LC-MS/MS Instrumentation and Software
3 Methods
3.1 Sample Collection and Egg Quality Assessments
3.2 Tissue Homogenization and Total Protein Extraction
3.3 Bradford Assay
3.4 SDS-PAGE
3.5 In Gel Tryptic Digestion
3.5.1 Destaining of Gel Pieces
3.5.2 Alkylation and Reduction
3.5.3 Tryptic Digestion
3.5.4 Extraction of Digested Peptides
3.6 LC-MS/MS, Mascot Search and Data Retrieval and Analysis
3.6.1 LC-MS/MS
3.6.2 Mascot Search and Data Retrieval
3.6.3 Data Analysis
4 Notes
References
Chapter 23: In Vivo Protein Lifetime Measurements Across Multiple Organs in the Zebrafish
1 Introduction
2 Materials
2.1 Feeding Zebrafish
2.2 Tissue Preparation and Protein Extraction
2.3 Protein Digestion and Desalting
2.4 Basic Reversed-Phase Chromatography
2.5 Mass Spectrometry
2.6 Online Tools and Tutorials
3 Methods
3.1 Feeding and Dissecting Zebrafish
3.2 Tissue Preparation and Protein Extraction
3.3 Protein Digestion and Desalting
3.4 Fractionation of Unlabeled Lysate Peptides for Setting the Library
3.5 DDA Runs of Unlabeled to Set Up the Library
3.6 BoxCar Mass Spectrometry Runs for the Labeled Lysate Digest
3.7 Data Analysis and Protein Turnover Interpretation
4 Notes
References
Chapter 24: In Vivo Imaging of Protein Interactions in the Germplasm with Bimolecular Fluorescent Complementation
1 Introduction
2 Materials
2.1 Common Materials
2.2 Gateway Cloning BP Cloning Reaction
2.3 Gateway Cloning LR Cloning Reaction
2.4 Restriction Enzyme Digestion
2.5 In Vitro Transcription
2.6 Microinjection
3 Method
3.1 Construct Gateway DONR Vectors
3.2 Transformation of Gateway Cloning BP Reaction
3.3 Plating and Making Mini-Cultures for Sequencing
3.4 Plasmid Isolation
3.5 Construct Gateway BiFC Destination Vectors
3.6 Transformation Gateway Cloning LR Reaction
3.7 Plating and Making Mini-Cultures for Sequencing
3.8 Plasmid Isolation
3.9 Plasmid Linearization
3.10 In Vitro Transcription of BiFC Destination Vectors
3.11 Microinjection
3.12 Visualization in Live Embryos
4 Notes
References
Chapter 25: Zygotic Genome Activation: Critical Prelude to the Most Important Time of Your Life
1 Introduction
2 Early Descriptive Period
3 Molecular Studies of ZGA
4 Conclusion
References
Chapter 26: Identification of RNA-Binding Protein Landscapes Across Zebrafish Embryonic Transcriptome via iCLIP Approach
1 Introduction
2 Materials
2.1 Staging and Collection of Zebrafish Embryos
2.2 Preparation of the Embryonic Lysate
2.3 DNase and RNase Treatment
2.4 Immunoprecipitation
2.5 3′-End RNA Dephosphorylation
2.6 3′-End Adapter Ligation
2.7 5′-End Radioactive Labeling of RNA
2.8 SDS-PAGE
2.9 Reverse Transcription
2.10 cDNA Size Selection
2.11 cDNA Circularization and Linearization
2.12 PCR Amplification
3 Methods
3.1 Staging and Collection of Zebrafish Embryos
3.2 Preparation of the Embryonic Lysate
3.3 DNase and RNase Treatment
3.4 Immunoprecipitation
3.5 3′-End RNA Dephosphorylation
3.6 3′-End Adapter Ligation
3.7 5′-End Radioactive Labeling of RNA
3.8 SDS-PAGE, Nitrocellulose Transfer, and RNA Isolation
3.9 Reverse Transcription
3.10 cDNA Size Selection
3.11 cDNA Circularization and Linearization
3.12 PCR Amplification
3.13 Bioinformatic Analysis of iCLIP Data
4 Notes
References
Chapter 27: Tethered Function Assay to Study RNA-Regulatory Proteins in Zebrafish Embryos
1 Introduction
2 Materials
2.1 Plasmid Construction and mRNA Preparation
2.2 mRNA Injection
2.3 Luciferase Assay
3 Methods
3.1 Plasmid Construction and mRNA Preparation
3.2 mRNA Injection
3.3 Luciferase Assay
4 Notes
References
Chapter 28: Massively Parallel Analysis of Regulatory RNA Sequences
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
2.3 Software
3 Methods
3.1 Design and Synthesis of the Oligonucleotide Library
3.2 Reporter Library Cloning and Transcription
3.3 Control RNA Cloning and Transcription
3.4 Fish Microinjection and Sample Collection
3.5 RNA-Seq Library Construction and Sequencing
3.6 RNA-Seq Data Processing
3.7 Extracting Sequence Elements That Regulate RNA Stability
4 Notes
References
Chapter 29: Exploring Translational Control of Maternal mRNAs in Zebrafish
1 Introduction
2 Materials
2.1 Zebrafish Maintenance and Embryonic Staging
2.2 Cell Lysate Isolation and Polysome Fractionation
2.3 RNA Isolation and Quality Assessment
2.4 Ribo-Depleted Sequencing Library Preparation
2.5 Poly(A) Tail Length Assay
3 Methods
3.1 Isolation of Cell Lysates from Zebrafish Embryos
3.2 Preparation of Sucrose Gradients
3.3 Total RNA Isolation from Polysome Fractions
3.4 Preparation of RNA-Seq Library and Sequencing
3.5 Assessment of Translation Rate and Translational Regulation
3.6 Poly(A) Tail Length Assay
3.6.1 Isolate Total RNA from Embryos
3.6.2 Perform Poly(A) Tail Length Assay
4 Notes
References
Index