Emerging Model Organisms

Preface to the Series
Preface
Contents
Contributors
Part I: African Killifish (Nothobranchius furzeri)
Chapter 1: Establishing a Fishroom for African Turquoise Killifish Nothobranchius furzeri
1 Introduction
2 Materials
2.1 Fishroom Operation
2.1.1 Fishroom Planning and Building
2.1.2 Fishroom Running
2.2 Husbandry System
3 Method and Notes
3.1 Factors to Consider Before Setting Up a Killifish Fishroom
3.1.1 Location of the Fishroom
3.1.2 Construction of the Fishroom
3.1.3 Additional Features to Consider for the Fishroom
3.2 Deciding on the Fish Husbandry System and Fishroom Supporting Components
3.2.1 Components of a System
3.2.2 Layout of a Killifish Fishroom
3.3 Fishroom Conditions
3.3.1 Fishroom Operations
3.3.2 Husbandry System
References
Chapter 2: Gene Editing and Transgenic Reporter Assays in African Killifish
1 Introduction
2 Materials
2.1 Reagents
2.2 Preparation of Solutions
2.3 Preparation of Microinjection Needles and Plate
2.4 Equipment and Tools
3 Method
3.1 CRISPR/Cas9-Mediated Gene Knockout
3.2 Set Up Breeding Tanks
3.3 Preparation of Microinjection Mix
3.4 Collection of One-Cell-Stage African Killifish Embryos
3.5 Microinjection
3.6 African Killifish Hatching, Maintenance, and Genotyping
3.7 Transgenic Reporter Assays
References
Chapter 3: Spinal Cord Injury in African Killifish
1 Introduction
2 Materials
2.1 Reagents
2.2 Solution Preparation
2.3 Instruments
2.4 Animals
3 Methods
3.1 Spinal Cord Injury in Adult Killifish
3.1.1 Preparation of Tools
3.1.2 Anesthetization of Adult Killifish
3.1.3 SCI Surgery of Adult Killifish
3.1.4 Maintenance of Fish After SCI
3.1.5 Monitoring Spinal Cord Injury Through Immunofluorescence Staining
3.2 Spinal Cord Injury in Larval Killifish
3.2.1 Preparation of Tools
3.2.2 Spinal Cord Transection
3.2.3 Monitoring Spinal Cord Regeneration of Larval Killifish
4 Notes
References
Chapter 4: Whole-Brain Clearing and Immunolabelling in the African Killifish Nothobranchius furzeri
1 Introduction
2 Materials
2.1 Equipment
2.2 Fixation, Washes, and Quenching
2.3 Clearing and Mounting
2.4 Immunolabelling
3 Methods
3.1 Sample Collection and Fixation
3.2 Quenching
3.3 SHIELD Hydrogel Matrix Exchange
3.4 Active Tissue Delipidation with Stochastic Electrotransport
3.5 Whole Killifish Brain GFAP Immunostaining
3.6 Refractive Index Matching
3.7 Imaging
4 Notes
5 Conclusion
References
Chapter 5: Studying Mating Behaviors in Nothobranchius furzeri
1 Introduction
2 Materials
2.1 Behavioral Tank (See Subheading 3.4)
2.2 Video Cameras
2.3 Camera Accessories
2.4 Behavioral Room Supplies
2.5 Animated Fish Displays
3 Methods
3.1 Acquiring Fish
3.2 Housing and Husbandry
3.3 Use of Live Individuals or Animations
3.4 Experimental Tank Setup
3.4.1 Dimensions
3.4.2 Zone Partitions
3.4.3 Background
3.4.4 Recording Angle
3.5 Behavioral Experiment
3.6 Behavioral Analysis
4 Typical Results
5 Limitations
6 Final Remarks and Considerations
References
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Chapter 6: The Chromatin Immunoprecipitation (ChIP) Protocol for African Turquoise Killifish
1 Introduction
2 Materials
2.1 Equipment and Supplies
2.2 Reagents and Buffers
3 Methods
3.1 Collection and Fixation of Fin Tissues
3.2 Isolation of Cell Nuclei
3.3 Sonication and Immunoprecipitation
3.4 Extraction of DNA
3.5 Library Construction
3.6 Next-Generation Sequencing and Analysis
4 Notes
References
Chapter 7: EdU Labeling of Proliferating Cells in the African Killifish Nothobranchius furzeri
1 Introduction
2 Materials
2.1 Husbandry
2.2 IP Injection
2.3 Click Chemistry Development
2.4 Fixation, Washes, Bleaching, Clearing, and Mounting (Whole-Mount)
2.5 Sample Processing for Cryo-Sectioning
3 Methods
3.1 IP Injection
3.2 Sample Collection, Fixation, and Bleaching (Whole-Mount)
3.3 Click Chemistry Development (Whole-Mount)
3.4 iDISCO Clearing and Mounting (Whole-Mount)
3.5 Imaging (Whole-Mount)
3.6 Sample Collection, Fixation, and Decalcification (Cryo-Sectioning)
3.7 Embedding and Sectioning (Cryo-Sectioning)
3.8 Staining (Cryo-Sectioning)
3.9 Imaging (Cryo-Sectioning)
4 Notes
5 Conclusion
References
Chapter 8: Immunofluorescence Staining on Cryosections with High Autofluorescence in African Killifish
1 Introduction
1.1 Sample Preparation
1.1.1 Fixative Selection
1.1.2 Tissue Embedding and Sectioning
1.2 Autofluorescence Reduction
1.3 Selection of Mounting Medium
2 Materials
2.1 Reagents
2.2 Solution Preparation
2.3 Equipment
3 Methods
3.1 Sample Collection and Fixation
3.2 Embedding
3.3 Cryosectioning
3.4 Antibody Staining
4 Notes
References
Chapter 9: Rapid Visualization of Gene Expression Using Chromogenic RNA In Situ Hybridization in African Killifish
1 Introduction
2 Materials
2.1 Reagents
2.2 Solution Preparation
2.3 Equipment
3 Methods
3.1 RNA Probe Design and Preparation
3.2 Sample Collection and Fixation
3.3 Sample Rehydration and Bleaching
3.4 In Situ Hybridization
3.5 Wash and Anti-DIG Antibody Incubation
3.6 NBT/BCIP-Based Signal Development
4 Notes
References
Chapter 10: Profiling Gene Expression in African Turquoise Killifish Nothobranchius furzeri Embryos with RNA Fluorescence In S...
1 Introduction
2 Materials
2.1 Samples
2.2 Buffer and Solution Composition
2.2.1 Self-Prepared Buffers and Solutions
2.2.2 Commercially Available Buffers From Molecular Instruments, Inc.
2.3 Microscopy
3 Methods
3.1 Experimental Design
3.2 Sample Preparation
3.3 Probe Hybridization and Wash
3.4 Amplifier Hybridization and Wash
3.5 Microscopy
4 Notes
References
Chapter 11: Cell Dissociation of Tissues for Single-Cell Analysis in African Killifish
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 Tail Amputation and Tissue Collection
3.2 Mechanical Dissociation and Enzymatic Digestion
3.3 Single-Cell Sorting by FACS
4 Discussion
References
Part II: Mexican Cavefish (Astyanax mexicanus)
Chapter 12: Cryopreservation of the Mexican Tetra Astyanax mexicanus
1 Introduction
1.1 History
1.2 Benefits
1.3 Program Components
2 Materials
2.1 Animal Preparation
2.2 Sperm Collection Preparation
2.3 Cryopreservation Preparation and Setup
2.4 CASA Preparation
3 Methods
3.1 Milt Collection Procedure
3.2 Freezing Procedure
3.3 Test Thaw Procedure
4 CASA Analysis
4.1 Thaw and Fertilization Procedure
5 Conclusion
References
Chapter 13: Whole-Mount Multicolor Fluorescent Labeling by In Situ Hybridization in Astyanax mexicanus Embryos and Larvae
1 Introduction
2 Materials
2.1 Equipment
2.2 Equipment Setup
2.3 Reagents and Solutions Setup
3 Procedure Step by Step (Fig. 1c)
4 Troubleshooting (TS)
5 Critical Steps (!x)
6 Expected Results
7 Conclusion
References
Chapter 14: Application of CRISPR-Cas9 for Functional Analysis in A. mexicanus
1 Introduction
2 Materials
2.1 A. mexicanus Animals
2.2 Generation of Reagents for CRISPR-Cas9 Mutagenesis
2.3 Microinjections
2.4 Genotyping
2.5 Phenotyping
3 Methods
3.1 Designing gRNAs
3.2 Synthesizing gRNAs/Cas9
3.2.1 Cas9 mRNA Synthesis
3.3 Cas9 Protein Preparation
3.4 Collecting/Injecting Embryos
3.5 Genotyping/Phenotyping Injected Individuals (CRISPants)
3.6 Identifying Founder Fish
3.7 Establishing a Line
4 Conclusion
References
Chapter 15: Immunohistochemistry and In Vivo Neural Imaging in A. mexicanus
1 Introduction
2 Immunohistochemistry
2.1 Stock Solutions
2.2 Procedure
2.2.1 Immunohistochemistry
3 Sample Prep and Imaging
3.1 Stock Solutions
3.1.1 Mounting
3.1.2 Imaging
3.1.3 Post-Processing
4 Live Imaging
4.1 Stock Solutions
4.2 Procedure
4.2.1 Husbandry and Subject Preparation
4.2.2 Imaging
4.2.3 Image Analysis
5 Conclusion
References
Chapter 16: Sleep Behavior Analysis in Astyanax mexicanus
1 Introduction
2 Materials
3 Methods
3.1 Sleep Analysis in Larvae and Juveniles (4-30 dpf)
3.2 Sleep Analysis in Adults
4 Conclusion
References
Chapter 17: A New Method to Analyze Nonvisual-Based Social-Like Interactions in Asocial Cave Fish
1 Introduction: Sociality in Fish
1.1 Nearby Interaction in Astyanax mexicanus Under Dark Conditions
2 Materials
2.1 Custom-Made Recording System
2.2 Animal Rearing Conditions
2.3 Animal Preparation for Behavior Recording
2.4 Analysis Tool
3 Methods
3.1 Recording Social Behavior
3.2 Image Preprocessing for ID Tracking
3.3 Fish ID Tracking Using idTracker Software
3.4 Post-Tracking ID Correction
3.5 Social Analysis
4 Conclusion
References
Chapter 18: A Generalist´s Guide to Quantifying Swimming Behavior Within Traditional and Nontraditional Teleost Models
1 Introduction
2 Materials
3 Methods
3.1 Mean Basal Swimming Velocity and Total Swimming Distance
3.2 Training MS COCO Model for Fish
3.3 Fish Detection Validation
3.4 Tracking Validation with Astyanax mexicanus
3.5 Individual Fish Burst Velocity
4 Conclusion
References
Chapter 19: Recording Acoustic Behavior in Astyanax mexicanus Fish: Acquisition, Decryption, and Interpretation
1 Introduction
2 Material
3 Methods
3.1 Acoustic Signal and Video Acquisition in Astyanax mexicanus
3.1.1 Isolated Environment and Tank Setting
3.2 Acoustic Recording
3.3 Video Recording
3.4 Playback to Record Behavioral Response
3.5 Signal Decryption: Extracting Relevant Sound Parameters
4 Acoustic Behavioral Analysis
5 Notes
References
Part III: Mini-Brain
Chapter 20: Generation of Mini-Brains From hiPSCs
1 Prepare hiPSCs
1.1 Materials
1.1.1 hiPSC Lines
1.1.2 Reagents and Equipment
1.2 Passage hiPSCs as Clumps (1-Well of 6-Well Plate)
1.3 Passage hiPSCs as Single Cells (1-Well of 6-Well Plate)
1.4 Stem Cell Characterization
2 Mini-Brain Differentiation
2.1 Reagents and Equipment
2.2 Prepare Mini-Brain Differentiation Reagent
2.3 Day 0 EB Seeding
2.4 Day 2-4 Media Feeding
2.5 Day 5 Induction
2.6 Day 7 Expansion
2.7 Day 10 ~Day 40 Maturation
2.8 Beyond Day 40 Maturation
3 Trouble Shooting of Mini-Brain Differentiation
4 Mini-Brain Sample Preparation for Single-Cell RNA Sequencing (SC-RNA-seq) Analysis
4.1 Materials
4.2 Preparation
4.3 Method
References
Chapter 21: Generating Human Pluripotent Stem Cell-Derived Neural AssemBloids to Model Interneuron Migration and Immune Cell I...
1 Introduction
2 Dorsal-Ventral Forebrain AssemBloids to Model Interneuron Migration
2.1 Materials
2.2 Equipment and Supplies
2.3 Preparation of Complete Media
2.4 Generating Dorsal Forebrain and Ventral Forebrain BROs
2.5 Generating Dorsal-Ventral Forebrain AssemBloids
2.6 Live Cell Imaging Interneuron Migration in Dorsal-Ventral Forebrain AssemBloids
3 BRO-Microglia AssemBloids to Model Brain-Immune Cell Interactions
3.1 Materials
3.2 Equipment and Supplies
3.3 Preparation of hPSC-Derived Microglia
3.4 Forming BRO-Microglia AssemBloids and Inflicting Injury
4 Notes
References
Chapter 22: Multiplexed Quantitative Proteomics Analysis of Developing Human Brain Organoids
1 Introduction
2 Materials
2.1 Samples
2.2 Sample Processing
2.3 Liquid Chromatography-Mass Spectrometry (LCMS)
2.4 Data Processing
3 Methods
3.1 Human Brain Organoids
3.1.1 Pluripotent Stem Cell Culture
3.1.2 Organoids Differentiation
3.2 Multiplexed Quantitative Proteomics Analysis
3.2.1 Extraction of Soluble Proteins From Cell Pellets and Organoids
3.2.2 Quantitation of Soluble Proteins Extracted From Cell Pellets and Organoids
3.2.3 TCA Precipitation
3.2.4 Protein Digestion
3.2.5 Solid-Phase Extraction
3.2.6 Peptide Quantitation
3.2.7 Tandem Mass Tag (TMT) Labeling
3.2.8 Preparing Custom-Made Analytical Reverse-Phase Columns
3.2.9 Checking TMT Labeling Efficiency
3.2.10 Multiplexed LC/SPS-MS3 Data Acquisition
3.2.11 MS Data Processing
4 Conclusion
5 Notes
References
Index