Developmental Biology of the Sea Urchin and Other Marine Invertebrates: Methods and Protocol

Preface
Contents
Contributors
Chapter 1: Laboratory Culture and Mutagenesis of Amphioxus (Branchiostoma floridae)
	1 Introduction
	2 Materials
		2.1 Materials for Aquaculture of Amphioxus
		2.2 General Materials for Transgenesis
			2.2.1 Materials for TALEN Construct Assembly
			2.2.2 Materials for TALEN mRNA Synthesis and Egg Injection
			2.2.3 Materials for Tol2 Mutagenesis
	3 Methods
		3.1 Method for Amphioxus Aquaculture (Figs. 2, 3, and 4)
			3.1.1 Obtaining Adult Amphioxus and Establishing a Culture
			3.1.2 Salinity
			3.1.3 Parasites and Diseases
			3.1.4 Laboratory Breeding Culture (see Note 1)
			3.1.5 Feeding Adults and Algal Culture (Fig. 2)
			3.1.6 Breeding (Fig. 3)
			3.1.7 Raising Larvae of B. floridae (Fig. 4)
		3.2 Generation of Amphioxus Mutants with TALEN Method (Figs. 5, 6, and 7)
			3.2.1 TALEN Target Site Design
			3.2.2 TALEN Construct Assembly (Figs. 5 and 6)
			3.2.3 Before Golden Gate Assembly
			3.2.4 Golden Gate Assembly: Day 1
			3.2.5 Golden Gate Assembly: Day 2 (Fig. 6)
			3.2.6 Golden Gate Assembly: Day 3
			3.2.7 Golden Gate Assembly: Day 4
			3.2.8 Golden Gate Assembly: Day 5
			3.2.9 TALEN mRNA Synthesis, Microinjection and Analysis of Somatic Mutations (Figs. 6 and 7)
			3.2.10 Detection of Mutations in Founder and F1 Amphioxus (Fig. 7)
			3.2.11 Obtaining and Analyzing Homozygous Mutant Embryos (Fig. 7)
		3.3 Generation of Amphioxus Transgenic Line with Tol2 Transposon System
			3.3.1 Constructs
			3.3.2 Preparation of the Injection Reagent and Embryonic Injection
			3.3.3 Detection of Germline Transmission in F0 Founders
			3.3.4 Identification of F1 Transgenic Amphioxus
	4 Notes
	References
Chapter 2: Handling and Manipulation of Gametes and Embryos of the Annelidan Worm Pseudopotamilla occelata
	1 Introduction
	2 Materials
		2.1 Culture of Adults and Collection of Gametes
		2.2 Induction of Oocyte Maturation and Fertilization
		2.3 Microinjection
		2.4 Ca2+ Imaging at Fertilization
	3 Methods
		3.1 Culture of Adults and Collection of Gametes
		3.2 Induction of Oocyte Maturation and Fertilization
		3.3 Microinjection
		3.4 Ca2+ Measurements at Fertilization
	4 Notes
	References
Chapter 3: The Starfish Asterina pectinifera: Collection and Maintenance of Adults and Rearing and Metamorphosis of Larvae
	1 Introduction
	2 Materials
		2.1 Collection and Selection of Starfish
		2.2 Laboratory Aquaria
		2.3 Seawater for Aquaria
		2.4 Oocyte Maturation, Insemination, and Embryonic Development
		2.5 Collection of Normal Embryos
		2.6 Diatom Culture
		2.7 Induction of Metamorphosis
	3 Methods
		3.1 Collection of Adult Starfish
		3.2 Getting Collection Permits from Local Agencies
		3.3 SCUBA Diving for Starfish
		3.4 Selection of Starfish
		3.5 Packing and Transport
		3.6 Maintenance of Adult Starfish in Laboratory Aquaria
		3.7 Managing Laboratory Populations of Starfish
		3.8 Quality Monitoring of Aquarium Seawater
		3.9 Seawater Temperature
		3.10 Feeding Adults
		3.11 Preparation of ASW for Embryonic and Larval Cultures
		3.12 Oocyte Maturation, Fertilization, and Culture of Embryos
		3.13 Collection of Normally Developing Embryos
		3.14 Culture of Diatoms (Chaetoceros Sp.) as Food for Larvae
		3.15 Rearing Larvae
		3.16 Induction of Metamorphosis by Pebbles Associated with Adults
	4 Notes
	References
Chapter 4: Experimental Tools to Study Regeneration in the Sea Anemone Nematostella vectensis
	1 Introduction
	2 Materials
		2.1 Animal Care
		2.2 Induction of Regeneration
		2.3 Fixation, Permeabilization, Coating, and Washing Buffer for Immunostaining and EdU/EU Staining
		2.4 Membrane and Nuclei Staining
		2.5 EdU (5-Ethynyl-2′-Deoxyuridine) and EU (5-Ethynyl-Uridine) Staining
		2.6 TUNEL Staining
		2.7 Immunostaining
	3 Methods
		3.1 Animal Care
		3.2 Induction of Regeneration
		3.3 In Vivo Analysis of Wound Healing and Pharynx Reformation
		3.4 Tissue Morphology, Cellular Proliferation, Neotranscription, and Apoptosis Staining
			3.4.1 Tissue Morphology
			3.4.2 Cellular Proliferation (EdU) and Neotranscription (EU)
			3.4.3 Apoptosis
			3.4.4 Immunostaining
	4 Notes
	References
Chapter 5: Staining and Tracking Methods for Studying Sponge Cell Dynamics
	1 Introduction
	2 Materials
		2.1 Biological Material
			2.1.1 Amphimedon queenslandica Biological Samples
			2.1.2 Lycopodina hypogea Biological Samples
			2.1.3 Oscarella lobularis Biological Samples
		2.2 Antibody-Based Staining on Whole Mount Biological Samples (See Note 2)
			2.2.1 A. queenslandica
			2.2.2 L. hypogea
			2.2.3 O. lobularis
		2.3 EDU Incorporation Assays
		2.4 Live Cell Staining and Imaging Assays in O. lobularis and A. queenslandica
	3 Methods
		3.1 Obtaining Amphimedon queenslandica Juveniles
		3.2 Stalling and Nutrition of Lycopodina hypogea Adults
		3.3 Obtaining Oscarella lobularis Buds
		3.4 Detection of Phosphohistone 3 (PH3) by Immunofluorescence (See Note 6)
			3.4.1 A. queenslandica
			3.4.2 L. Hypogea
			3.4.3 O. lobularis
		3.5 EdU Incorporation Assays
			3.5.1 A. queenslandica
			3.5.2 L. hypogea
			3.5.3 O. lobularis
		3.6 TUNEL Assays (See Note 7)
			3.6.1 O. lobularis and L. hypogea
		3.7 MAPLC3 Staining in L. hypogea (See Note 8)
		3.8 Staining Choanocytes with the Lipidic Marker CM-DiI Dye
			3.8.1 A. queenslandica
			3.8.2 O. lobularis
		3.9 Staining O. lobularis Choanocytes with Fluorescent Microspheres (See Note 9)
		3.10 Staining O. lobularis Choanocytes with Fluorescent Lectins (See Note 9)
		3.11 Live Imaging in White Light
			3.11.1 A. queenslandica
	4 Notes
	References
Chapter 6: Microscopy Studies of Placozoans
	1 Introduction
	2 Materials
		2.1 Culturing and Maintenance
		2.2 High Pressure Freezing for Thin Section EM
		2.3 Freeze Substitution for Thin Section EM
		2.4 Chemical Fixation for SEM
		2.5 Freezing, Freeze Substitution, and Immunolabeling for Light Microscopy
	3 Methods
		3.1 Collection and Maintenance
		3.2 Algal Cultures
		3.3 Trichoplax Cultures
		3.4 High Pressure Freezing for Thin Section EM
		3.5 Freeze Substitution for Thin Section EM
		3.6 Freezing, Freeze-Substitution and Immunolabeling for Light Microscopy
		3.7 Fixation for SEM
		3.8 Freeze Fracturing Tissues for SEM
		3.9 Critical Point Drying and SEM
	4 Notes
	References
Chapter 7: Identification of SH2 Domain-Mediated Protein Interactions that Operate at Fertilization in the Sea Star Patiria mi...
	1 Introduction
	2 Materials
		2.1 Starfish Egg and Sperm Preparation
		2.2 Mutagenesis
		2.3 GST Fusion Protein Expression
		2.4 Affinity Interactions, Gel Electrophoresis, and Western Blotting
	3 Methods
		3.1 Obtaining Oocytes
		3.2 Obtaining Sperm
		3.3 Oocyte Maturation and Fertilization
		3.4 Mutagenesis of GST-Wild-Type SH2 Domain Fusion Protein Constructs
		3.5 Fusion Protein Expression and Purification
		3.6 Affinity Interactions
		3.7 SDS-PAGE and Silver Staining
		3.8 Western Blotting
	4 Notes
	References
Chapter 8: Marine Nemertean Worms for Immunoblotting Studies of Oocyte Aging
	1 Introduction
	2 Materials
		2.1 General Solutions
		2.2 SDS-Polyacrylamide Gel Components
		2.3 Transfer Components
		2.4 Supplies to Take to the Darkroom When Developing Film
	3 Methods
		3.1 Maintaining Adult Specimens
		3.2 Obtaining Gametes and Fertilized Embryos
		3.3 General Cell Biological Applications
		3.4 Incubating Oocytes During Aging Experiments
		3.5 Sample Processing for Immunoblotting Analyses
		3.6 Gels
		3.7 Electrophoresis
		3.8 Transfer
		3.9 Antibody Probing
		3.10 Film Developing
		3.11 Reprobing Stripped Membranes
		3.12 Quantification of Blot Intensities
	4 Notes
	References
Chapter 9: Recovery of Sea Star Egg Cell Surface Proteins Released at Fertilization
	1 Introduction
	2 Materials
		2.1 Obtaining and Culturing Oocytes/Testes
		2.2 Biotinylation
		2.3 Streptavidin-Biotin Affinity Interaction
		2.4 SDS-PAGE Gel and Western Blotting
		2.5 Miscellaneous
	3 Methods
		3.1 Oocyte/Sperm Removal and Preparation
		3.2 Biotinylation of Eggs
		3.3 Fertilization
		3.4 Affinity Interaction Using Streptavidin Mag Sepharose Beads (See Note 11)
		3.5 SDS-PAGE Gel and Western Blotting
	4 Notes
	References
Chapter 10: Quantifying Cell Proliferation During Regeneration of Aquatic Worms
	1 Introduction
	2 Materials
		2.1 Buffers and Relaxation Solutions
		2.2 Immunohistochemistry and Nuclear Counterstains
		2.3 Thymidine Analogues
		2.4 Peroxidase Development
		2.5 Click Chemistry
	3 Methods
		3.1 Worm Relaxation and Fixation
		3.2 PH3/PCNA Detection
		3.3 BrdU Proliferation Assay
		3.4 EdU Proliferation Assay
	4 Notes
	References
Chapter 11: In Situ Hybridization Techniques in the Homoscleromorph Sponge Oscarella lobularis
	1 Introduction
	2 Materials
		2.1 Biological Material
		2.2 Probe Synthesis
		2.3 Sample Fixation
		2.4 Paraffin Fixation and Cuts
		2.5 In Situ Hybridization
		2.6 Embedding and Sectioning Tissues
	3 Methods (see Notes 2 and 3)
		3.1 Probe Synthesis
		3.2 Fixation (See Note 4)
			3.2.1 To Fix Adults
			3.2.2 To Fix Buds and Larvae
		3.3 Paraffin Embedding and Sectioning (for SISH) (See Note 5)
		3.4 In Situ Hybridization: Section ISH (SISH)
		3.5 In Situ Hybridization: Whole mount ISH (WISH) on buds and larvae (See Note 8)
		3.6 In Situ Hybridization: Whole Mount ISH on Adult Tissue (See Note 8)
		3.7 In Situ Hybridization: Whole Mount ISH Using InsituPro VSi (INTAVIS)
		3.8 Embedding and Sectioning for Light Microscopy after WMISH (See Note 9)
			3.8.1 Epoxy Embedding
			3.8.2 L.R. White
	4 Notes
	References
Chapter 12: Methodology for Whole Mount and Fluorescent RNA In Situ Hybridization in Echinoderms: Single, Double, and Beyond
	1 Introduction
	2 Materials
	3 Methods
		3.1 Fixation Protocol
			3.1.1 Harvest of Embryos and Larvae
			3.1.2 Fixation
		3.2 Riboprobe Synthesis
		3.3 Sea Urchin WMISH Protocols
		3.4 Sea Urchin FISH Protocol
		3.5 Second Probe Detection for Double-FISH
		3.6 P. miniata WMISH and FISH Protocols
		3.7 Positive and Negative Controls
		3.8 Combination of Immunolabeling and In Situ Hybridization
		3.9 Conclusions
	4 Notes
	References
Chapter 13: Gene Editing in the Ascidian Phallusia mammillata and Tail Nerve Cord Formation
	1 Introduction
	2 Materials
		2.1 Microinjection of mRNA or CRISPR-Cas9 Protein Mixtures
		2.2 CRISPR-Cas9 Protein or mRNA Microinjection
	3 Methods
		3.1 Microinjection
		3.2 Preparing and Injecting CRISPR-Cas9
		3.3 Imaging the Nerve Cord
		3.4 Analyzing the Nerve Cord Imaging Data Using Freeware: Fiji, Ilastik, and ICY
	4 Notes
	References
Chapter 14: Transcriptomic Analysis in the Sea Anemone Nematostella vectensis
	1 Introduction
	2 Materials
		2.1 RNA Isolation
		2.2 Quantification of Transcriptomic Data
	3 Methods
		3.1 RNA Isolation and Quality Control
		3.2 Quantification of Transcriptomic Data
	4 Notes
	References
Chapter 15: RNA Interference on Regenerating Holothurian Gut Tissues
	1 Introduction
	2 Materials
		2.1 Tissue Selection
		2.2 Gut Rudiments Culture
		2.3 Gut Rudiment Electroporation
		2.4 Determining Electroporation Efficiency Using Histological Approaches
		2.5 DsiRNAs
		2.6 Determination of RNA Abundance Using  qPCR
	3 Methods
		3.1 Sea Cucumber Collection and Evisceration
		3.2 Sea Cucumber Disinfection and Explant Preparation
		3.3 Optimization of the Electroporation Protocol
		3.4 Histological Studies for Electroporation Efficiency
		3.5 siRNA Design
		3.6 Electroporation of Gut Rudiments with DsiRNAs
		3.7 RNA Extraction
		3.8 cDNA Synthesis
		3.9 qPCR to Determine the Efficiency of the Interference
	4 Notes
	References
Chapter 16: ATAC-Seq for Assaying Chromatin Accessibility Protocol Using Echinoderm Embryos
	1 Introduction
	2 Materials
		2.1 Animal Origin
		2.2 Equipment
			2.2.1 In Vitro Fertilization and Embryo Collection
			2.2.2 ATAC-Seq Library Preparation
		2.3 Reagents
		2.4 Downloading Genome and Annotation
	3 Methods
		3.1 In Vitro Fertilization
		3.2 Collection of Embryos
		3.3 Cell Lysis
		3.4 Tagmentation
		3.5 PCR Amplification
		3.6 Concentration Check and Electrophoresis Gel
		3.7 Data Analysis
		3.8 Anticipated Results
	4 Notes
	References
Chapter 17: Usage of the Sea Urchin Hemicentrotus pulcherrimus Database, HpBase
	1 Introduction
	2 Materials
	3 Methods
		3.1 Gene Search Page
		3.2 Homology Search Page
		3.3 Genome Viewer Page (Genome Explorer)
			3.3.1 How to Display a Specific Genome Position
			3.3.2 How to Search for a Gene
			3.3.3 How to Retrieve a Sequence
			3.3.4 How to Change Color and Thickness of Boxplot
			3.3.5 How to Obtain the Image of Genome Map
		3.4 Data Download Page
		3.5 Protocol Page
			3.5.1 Protocol for Immunohistochemistry
			3.5.2 Protocol for In Situ Hybridization
			3.5.3 Reagents for Microinjection
			3.5.4 Protocol for qPCR
			3.5.5 Protocol for Luciferase Assay (TOPFlash Assay)
	4 Notes
	References
Chapter 18: Functional Studies of Trichoplax adhaerens Voltage-Gated Calcium Channel Activity
	1 Introduction
	2 Materials
		2.1 Extraction of RNA from Trichoplax adhaerens
		2.2 Cloning of the Trichoplax T-Type Voltage-Gated Calcium Channel (TCaV3) cDNA into the pIRES2-EGFP Vector
		2.3 Transfection of TCaV3-pIRES-EGFP Vectors into HEK-293T Cells
		2.4 Whole-Cell Patch Clamp Recording of TCaV3-pIRES2-EGFP Transfected HEK-293T Cells
	3 Methods
		3.1 Extraction of RNA from Trichoplax adhaerens
		3.2 Cloning of the Trichoplax T-Type Voltage-Gated Calcium Channel (TCaV3) cDNA into the pIRES2-EGFP Vector
		3.3 Transfection of the pTCaV3-IRES2-EGFP Vector into HEK-293 T Cells
		3.4 Whole-Cell Patch Voltage Clamp Recording of pTCaV3-IRES2-EGFP Transfected HEK-293T Cells
	4 Notes
	References
Chapter 19: A Bioinformatics Tutorial for Comparative Development Genomics in Diverse Meiofauna
	1 Introduction
	2 Materials
		2.1 Computers
		2.2 Required Software
		2.3 Database
	3 Methods
		3.1 Genome Assembly and Assessment Tutorial Overview
		3.2 Examining the Raw Data
		3.3 Adapter and Quality Trimming
		3.4 Genome Assembly
		3.5 Genome Assessment
			3.5.1 Part 2: Comparative Genomics
	4 Notes
	References
Index