Whole-Body Regeneration: Methods and Protocols (Methods in Molecular Biology, 2450)

Preface
Contents
Contributors
Part I: Historical Overviews
	Chapter 1: The Hazards of Regeneration: From Morgan´s Legacy to Evo-Devo
		1 Introduction
		2 The Legacy of Morgan´s Regeneration
			2.1 Partial Versus Whole-Body Regeneration
			2.2 Regeneration: Function Versus Process
			2.3 Help from Evo-Devo Theoretical Tools
		3 The Difficult Task of Reconstructing WBR Evolution
			3.1 Far from Basal: Diversity of Regeneration in Sponges
			3.2 Acoels and Planarians: Lessons from Faraway Cousins
			3.3 Plastic Families: Convergent Acquisition of WBR in Tunicates
			3.4 A Roadmap to Investigate WBR Evolution
		4 What Is the Significance of WBR? An Integrative and Practical Approach
			4.1 The Puzzle of ``Restriction and Absence´´ of WBR: Eco-Evo-Evo Perspectives
			4.2 Questions and Approaches to Investigate WBR Evolution
		5 Conclusions
		References
	Chapter 2: Studying Regeneration in Ascidians: An Historical Overview
		1 Introduction
		2 Regeneration in Solitary Ascidians
		3 Regeneration in Colonial Ascidians
			3.1 WBR
			3.2 Partial Body Regeneration
			3.3 Tunic and Colonial Circulatory System Regeneration
		4 Methodological Approaches to Induce Regeneration: An Historical Overview
			4.1 Solitary Ascidians
			4.2 Colonial Ascidians
		5 Concluding Remarks
		References
Part II: Zoological Approaches
	Chapter 3: Studying Protista WBR and Repair Using Physarum polycephalum
		1 Introduction
		2 Materials
			2.1 Physarum Culture Environments
			2.2 Physarum Imaging and Analysis
		3 Methods
			3.1 Physarum Culture
			3.2 Models of Vein Repair After Injury & Amputation
			3.3 Generation in Complex Environments: Maze & Salt Bridge Experiments
			3.4 Structural & Functional Imaging
			3.5 Quantification of Growth & Repair
			3.6 Sampling Physarum
		4 Notes
		References
	Chapter 4: Studying Porifera WBR Using the Calcerous Sponges Leucosolenia
		1 Introduction
		2 Materials
		3 Methods
			3.1 Collection, Manipulation, and Laboratory Maintenance of Sponges
			3.2 Surgical Operations and Subsequent Cultivation
				3.2.1 Excision of a Small Part
				3.2.2 Amputation of Oscular Tubes
				3.2.3 Whole-Body Regeneration
				3.2.4 Cell Reaggregation
			3.3 Time-Lapse Recordings
			3.4 Methods of Tissue Fixation and Processing for Histological Investigation
			3.5 Methods of Tissue Fixation and Processing for Transmission Electron Microscopy (TEM)
			3.6 Methods of Tissue Fixation and Processing for Scanning Electron Microscopy (SEM)
			3.7 Cell Proliferation and Immunohistochemical Studies
			3.8 Cell Proliferation Blocking
			3.9 Apoptosis Studies
			3.10 Skeleton Synthesis Studies
		4 Notes
		References
	Chapter 5: Studying Ctenophora WBR Using Mnemiopsis leidyi
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Reagents
		3 Methods
			3.1 Sources and Collection
			3.2 Laboratory Setup for Mnemiopsis leidyi Culture
			3.3 Culturing Live Feed for Mnemiopsis leidyi Husbandry
			3.4 Spawning and Husbandry of Mnemiopsis leidyi
			3.5 Animal Surgeries to Study Wound Healing and Whole-Body Regeneration
			3.6 Cell Proliferation Inhibitor Treatment with Hydroxyurea (HU)
			3.7 Fixation of Mnemiopsis leidyi Cydippids
			3.8 Live Imaging During Wound Healing and Regeneration
		4 Notes
		References
	Chapter 6: Studying Placozoa WBR in the Simplest Metazoan Animal, Trichoplax adhaerens
		1 Introduction
		2 Materials
		3 Methods
			3.1 Animal Cultivation
			3.2 DiI Staining
			3.3 Methylene Blue Staining
			3.4 Mechanical Manipulation
			3.5 Animal Fixation
			3.6 Animal Body Dissociation and Reaggregation
		4 Notes
		References
	Chapter 7: Collecting and Culturing Kamptozoans for Regenerative Studies
		1 Introduction
		2 Material
			2.1 Barentsia benedeni Collection and Culture
			2.2 Live Cryptophyta Algae Stock
			2.3 Fixation for F-Actin Staining
			2.4 RNA Extraction and Target Gene Amplification
		3 Methods
			3.1 Collection and Identification
			3.2 Food Algae Culture
			3.3 Barentsia benedeni Culture
			3.4 Tissue Isolation and Regeneration
			3.5 Fixation and F-Actin Staining
			3.6 Total RNA Extraction and Target Gene Amplification
		4 Notes
		References
	Chapter 8: Collecting and Culturing Bryozoans for Regenerative Studies
		1 Introduction
		2 Materials
			2.1 Colony Collection
			2.2 Identification of Bryozoans
			2.3 Culture and Feeding
			2.4 Marking
			2.5 Anesthetizing and Fixing for Histology
		3 Methods
			3.1 Onshore Collection
			3.2 Offshore Collection
			3.3 Postcollection Sorting
			3.4 Identification
			3.5 Culture and Feeding
			3.6 Spawning
			3.7 Marking Colonies
			3.8 Anesthetizing and Fixing for Histology
		4 Notes
		References
	Chapter 9: Studying Annelida Regeneration in a Novel Model Organism: The Freshwater Aeolosoma viride
		1 Introduction
		2 Materials
			2.1 Regeneration in Aeolosoma viride
			2.2 Cell Proliferation Assay
			2.3 Whole-Mount In Situ Hybridization (WISH)
			2.4 RNA Interference (RNAi)
		3 Methods
			3.1 Animal Husbandry
			3.2 Regeneration in Aeolosoma viride
			3.3 Animal Fixation
			3.4 Cell Proliferation Assay
			3.5 Whole-Mount In Situ Hybridization (ISH)
			3.6 RNA Interference (RNAi) by Feeding Method
			3.7 RNAi by Microinjection
		4 Notes
		References
	Chapter 10: Studying Annelida Body Regeneration Under Environmental Stress in Diopatra neapolitana
		1 Introduction
		2 Materials
		3 Methods
			3.1 Collection of Organisms and Acclimation
			3.2 Regeneration Assay
		4 Notes
		References
	Chapter 11: Studying Annelida Regeneration Using Platynereis dumerilii
		1 Introduction
		2 Materials
			2.1 Platynereis Worms Culture and Biological Material Production
			2.2 Whole Mount In Situ Hybridization and EdU Labelling
		3 Methods
			3.1 Platynereis Worms Culture
			3.2 Production and Fixation of Samples at Specific Stages of Regeneration
			3.3 Whole Mount In Situ Hybridization
			3.4 EdU Labelling for Investigating Cell Proliferation During Platynereis Regeneration
			3.5 Pharmacological Treatments for Functional Studies During Platynereis Regeneration
		4 Notes
		References
	Chapter 12: Collecting and Culturing Lineus sanguineus to Study Nemertea WBR
		1 Introduction
		2 Materials
			2.1 Field Collection of Specimens
			2.2 Specimen Rearing
		3 Methods
			3.1 Field Collection of Specimens
			3.2 Hypoxia-Induced Migration
			3.3 Specimen Rearing
			3.4 Feeding
			3.5 Propagation
		4 Notes
		References
	Chapter 13: Studying Xenacoelomorpha WBR Using Isodiametra pulchra
		1 Introduction
		2 Materials
			2.1 Nucleic Acid Extractions
			2.2 Antibody and Cytochemical Stainings
			2.3 In Situ Hybridization and RNA Interference
		3 Methods
			3.1 Anesthesia (Relaxation), Amputation, and Fixation
			3.2 In Situ Hybridization
			3.3 RNA Interference (RNAi)
			3.4 Antibody Stainings
			3.5 Cytochemical Stainings
			3.6 RNA Extraction
			3.7 DNA Extraction
		4 Notes
		References
	Chapter 14: Studying Echinodermata Arm Explant Regeneration Using Echinaster sepositus
		1 Introduction
		2 Materials
			2.1 Starfish Collection and Husbandry
			2.2 Fixation and Epoxy Resin Embedding
			2.3 Semithin and Ultrathin Sectioning and Staining
			2.4 RNA Extraction
		3 Methods
			3.1 Starfish Collection and Husbandry
			3.2 Arm Explants
			3.3 Fixation for Transmission Electron Microscopy (TEM)
			3.4 Semithin Sectioning and Staining
			3.5 Ultrathin Sectioning
			3.6 Ultrathin Section Staining and TEM Grid Carbon-Coating
			3.7 RNA Extraction
		4 Notes
		References
	Chapter 15: Studying Hemichordata WBR Using Ptychodera flava
		1 Introduction
		2 Materials
			2.1 Animal Husbandry
			2.2 Preparation of RNA Probes
			2.3 Preparation of Preabsorbed Antibodies
			2.4 Whole Mount In Situ Hybridization
		3 Methods
			3.1 Animal Collection
			3.2 Preparation of Regenerating Tissues
			3.3 Preparation of RNA Probes
			3.4 Preparation of Preabsorbed Antibodies
			3.5 Whole Mount In Situ Hybridization
		4 Notes
		References
	Chapter 16: Studying Tunicata WBR Using Botrylloides anceps
		1 Introduction
		2 Materials
			2.1 Animals Collection, Handling, Feeding, and DNA Barcoding
			2.2 Histological Cryosectioning and Staining
		3 Methods
			3.1 Sampling and Adaptation
			3.2 DNA Barcoding
			3.3 Colonies Maintenance
			3.4 Whole-Body Regeneration
			3.5 Histological Sectioning
			3.6 Hematoxylin-Eosin Stain
		4 Notes
		References
Part III: Cellular Approaches
	Chapter 17: In Situ Hybridization to Identify Stem Cells in the Freshwater Sponge Ephydatia fluviatilis
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Sponge Samples
			3.2 RNA Probe Synthesis
			3.3 Whole Mount In Situ Hybridization (BCIP-NBT Detection)
			3.4 Dual-Color Fluorescent Whole Mount In Situ Hybridization
		4 Notes
		References
	Chapter 18: Isolation and Maintenance of In Vitro Cell Cultures from the Ctenophore Mnemiopsis leidyi
		1 Introduction
		2 Materials
		3 Methods
			3.1 Ctenophore Mesogleal Serum (CMS)
			3.2 Animal Preparation
			3.3 Tissue Explant Preparation for Small-Scale Cultures
			3.4 Mechanical Cell Dissociation for Small-to-Medium Scale Cultures
			3.5 Mechanical Cell Dissociation for Large-Scale Cultures
			3.6 Preparing Enzymatically Dissociated Cells
			3.7 Primary Cell Culture Maintenance
		4 Notes
		References
	Chapter 19: Analysis of Spatial Gene Expression at the Cellular Level in Stony Corals
		1 Introduction
		2 Materials
			2.1 Removal of Paraffin
			2.2 Hybridization of RNA Probe
			2.3 Visualization of RNA Probe
		3 Methods
			3.1 Removal of Paraffin
			3.2 Slide Pretreatment and Prehybe Preparation
			3.3 RNA Probe Hybridization
			3.4 RNA Probe Visualization
		4 Notes
		References
	Chapter 20: Studying Stem Cell Biology in Intact and Whole-Body Regenerating Hydra by Flow Cytometry
		1 Introduction
			1.1 Hydra and the Unusual Properties of Its Stem Cells
			1.2 Stem Cell Sorting and Methods to Monitor the Cell Cycle Activity in Hydra
		2 Materials
			2.1 Dissociation of Hydra Live Cells
			2.2 PI Nuclei Staining for Cell Cycle Analysis
			2.3 Click-iT EdU-Detection of Proliferating Hydra Cells
			2.4 Equipment
		3 Methods
			3.1 Live Hydra Cell Suspension
			3.2 Flow Cytometry Sorting of Hydra GFP-Expressing Stem Cell (FACS Protocol)
			3.3 Cell Cycle Measurement: PI Staining of Trypsin-Dissociated Cells
			3.4 Cell Cycle Measurement: Click-iT EdU Labeling of Hydra Cells Replicating Their Genomic DNA (S-Phase)
		4 Notes
		References
	Chapter 21: Noninvasive Intravascular Microtransfusion in Colonial Tunicates
		1 Introduction
		2 Materials
		3 Methods
			3.1 Intravascular Collection of Hemolymph
			3.2 Characterization of Hemolymph Collection
			3.3 Flow Cytometry Analysis of Hemolymph
			3.4 Injection of Compounds and Cells
		4 Notes
		References
Part IV: Genetic Approaches
	Chapter 22: Gene Manipulation in Hydractinia
		1 Introduction
		2 Materials
			2.1 Animal Maintenance, Spawning, and Metamorphosis
			2.2 Embryo Microinjection
			2.3 shRNA
			2.4 CRISPR-Cas9 Editing and Genotyping
		3 Methods
			3.1 Animal Maintenance, Spawning and Metamorphosis
			3.2 Capillaries´ Preparation and Injection
			3.3 Strategy for Reporter Line Cloning
			3.4 Strategy for Ectopic Expression Construct Cloning
			3.5 Designing mRNA Constructs
			3.6 Synthesizing the mRNA Template Construct
				3.6.1 Generating Plasmid for Template Construct
				3.6.2 Synthesizing Gibson Assembled dsDNA Fragment for Template Construct
			3.7 T7 In Vitro Transcription mRNA Synthesis, Microinjection, and Evaluation
			3.8 Short-Hairpin RNA Interference
			3.9 CRISPR/Cas9 Design, Microinjection, and Genotyping
		4 Notes
		References
	Chapter 23: Manipulation of Gene Activity in the Regenerative Model Sea Anemone, Nematostella vectensis
		1 Introduction
		2 Materials
			2.1 shRNA Production
			2.2 CRISPR/Cas9 Targeted Mutagenesis
			2.3 Microinjection
			2.4 Electroporation of shRNA
		3 Methods
			3.1 shRNA Production
			3.2 shRNA Microinjection
			3.3 shRNA Electroporation
			3.4 Screening and Care of shRNA-Treated Animals
			3.5 sgRNA Production for CRISPR/Cas9-Targeted Mutagenesis
			3.6 sgRNA/Cas9 Delivery by Microinjection
			3.7 Genotypic Screening and Care of CRISPR/Cas9 Injected Animals
		4 Notes
		References
	Chapter 24: Monitoring Telomere Maintenance During Regeneration of Annelids
		1 Introduction
		2 Materials
			2.1 Telomeric Repeat Amplification Protocol (TRAP) Assay
			2.2 Terminal Restriction Fragment (TRF) Assay
			2.3 Telomere Fluorescence In Situ Hybridization (Telomere FISH)
		3 Methods
			3.1 Protein Extraction from Regenerating Tissues of Aeolosoma Viride
			3.2 Telomeric Repeat Amplification Protocol (TRAP) Assay
			3.3 Genomic DNA Extraction from Regenerating Tissues
			3.4 Terminal Restriction Fragment (TRF) Assay
			3.5 Telomere Fluorescence in Situ Hybridization (Telomere FISH)
		4 Notes
		References
	Chapter 25: Analysis of DNA Double-Stranded Breaks Using the Comet Assay in Planarians
		1 Introduction
		2 Materials
			2.1 Handling Equipment
			2.2 Comet Assay Solutions
		3 Methods
			3.1 Agarose Slide Preparation
			3.2 Planarian Dissociation and Cell Recovery
			3.3 Slide Preparation
			3.4 Cell Lysis
			3.5 Comet Slide Electrophoresis
			3.6 Comet Slide Neutralization and Fixation
			3.7 Comet Slide Staining and Visualization
			3.8 Comet Slide Storage
		4 Notes
		References
	Chapter 26: Random Integration Transgenesis in a Free-Living Regenerative Flatworm Macrostomum lignano
		1 Introduction
		2 Materials
		3 Methods
			3.1 Setting Up Egg Producing Worm Cultures
			3.2 Preparing Single Wild-Type Worms for Crosses
			3.3 Preparation of Plastic Pickers for Egg Collection
			3.4 Preparation of the Holders
			3.5 Preparation of the Microinjection Needles
			3.6 Design of Transgenic Constructs
			3.7 Microinjection Mix: DNA for Random Integration
			3.8 Microinjection Procedure
			3.9 Irradiation of Injected Eggs
			3.10 Transgenic Eggs Maintenance and Selection of Homozygous Lines
		4 Notes
		References
	Chapter 27: RNAi Screening to Assess Tissue Regeneration in Planarians
		1 Introduction
		2 Materials
			2.1 Template Preparation
			2.2 dsRNA Synthesis
			2.3 RNAi Treatment and Amputation
		3 Methods
			3.1 Template Preparation by  PCR
			3.2 In Vitro dsRNA Synthesis
			3.3 dsRNA Feeding for RNA Interference (RNAi)
			3.4 Strategies for Assessing Regeneration After  RNAi
		4 Notes
		References
	Chapter 28: Monitoring Chromatin Regulation in Planarians Using Chromatin Immunoprecipitation Followed by Sequencing (ChIP-seq)
		1 Introduction
		2 Materials
			2.1 Cell Dissociation and Isolation of Stem Cells
			2.2 Chromatin Extraction and Sonication
			2.3 Immunoprecipitation and Reverse Crosslinking
			2.4 Preparation of ChIP Libraries for Sequencing
		3 Methods
			3.1 Dissociation and Isolation of Stem Cells
			3.2 Chromatin Extraction and Sonication
			3.3 Chromatin Immunoprecipitation
			3.4 Preparation of ChIP Libraries for Sequencing
			3.5 PCR Enrichment and Purification of DNA
		4 Notes
		References
	Chapter 29: Assessing Chromatin Accessibility During WBR in Acoels
		1 Introduction
		2 Materials
		3 Methods
			3.1 Library Preparation
			3.2 PCR Amplification of Library and Sequencing
			3.3 Data Analysis
		4 Notes
		References
Part V: OMICS Approaches
	Chapter 30: Single-Cell Transcriptomic Analysis in the Regenerating Cnidarian Nematostella vectensis
		1 Introduction
		2 Materials
		3 Methods
			3.1 Cell Dissociation of Regenerated and Control Samples
			3.2 Live Single-Cell Sorting Using Flow Cytometry for Single-Cell RNA-Sequencing
			3.3 Single-Cell RNA-Seq Library Generation, QC, and Initial Characterization
		4 Notes
		References
	Chapter 31: Characterization of Soluble Cell-Free Coelomic Fluid Proteome from the Starfish Marthasterias glacialis
		1 Introduction
		2 Materials
			2.1 Sample Preparation
			2.2 Denaturing Polyacrylamide Gel Electrophoresis (SDS-PAGE)
			2.3 Protein In-Gel Trypsination
			2.4 Untargeted Proteomics and Data Analysis
		3 Methods
			3.1 Cell-Free Coelomic Fluid Collection and Protein Precipitation
			3.2 Peptide-N-Glycosidase F Enzymatic Treatment and SDS-PAGE Sample Preparation
			3.3 Denaturing Polyacrylamide Gel Electrophoresis (SDS-PAGE): Gel Casting and Sample Running
			3.4 Coomassie Blue Gel Staining
			3.5 Protein In-Gel Trypsination
			3.6 Liquid Chromatography-Mass Spectrometry Untargeted Proteomics Analysis
		4 Notes
		References
	Chapter 32: Using RNA-Seq for Transcriptome Profiling of Botrylloides sp. Regeneration
		1 Introduction
		2 Materials
		3 Methods
			3.1 Quality Control and Trimming
			3.2 Mapping
			3.3 Normalizing Count Data with DESeq2
			3.4 Differential Gene Expression Analysis Using DESeq2
			3.5 Gene Ontology Enrichment Analysis
		4 Notes
		References
Part VI: Integrative Approaches
	Chapter 33: Studying Mechanical Oscillations During Whole-Body Regeneration in Hydra
		1 Introduction
		2 Materials
			2.1 Culture Media and Animal Handling
			2.2 Cutting and Dissociating Animals
		3 Methods
			3.1 Spheroid Preparation from Cut Tissue Pieces
			3.2 Spheroid Preparation from Dissociated Body Tissue
			3.3 Imaging
			3.4 Image Segmentation and Quantification of Oscillation Parameters
		4 Notes
		References
	Chapter 34: Combining RNAi-Mediated β-Catenin Inhibition and Reaggregation to Study Hydra Whole-Body Regeneration
		1 Introduction
		2 Materials
			2.1 Electroporation and Reaggregation
			2.2 RNA Extraction, Small Interfering RNAs, and Real-Time PCR
			2.3 Kits and Equipment
		3 Methods
			3.1 Hydra Electroporation
			3.2 Hydra Dissociation and Reaggregation
			3.3 RNA Extraction and Real-Time PCR (qPCR)
		4 Notes
		References
	Chapter 35: Creating a User-Friendly and Open-Access Gene Expression Database for Comparing Embryonic Development and Regenera...
		1 Introduction
		2 Materials
			2.1 Data Resource Requirements
			2.2 Technical Requirements
		3 Methods
			3.1 Defining the Scope and Application of the Tool
			3.2 Setup the Relational Database
				3.2.1 Database Design
				3.2.2 Build the Database Structure
				3.2.3 Populate the Database
			3.3 Build the Website
				3.3.1 Design and Create the Main Webpages
				3.3.2 Implementing Data Visualization and Mining Tools
			3.4 Data and Services Availability
				3.4.1 Accessibility
				3.4.2 Maintenance
				3.4.3 Documentation
				3.4.4 Evaluating the Impact of the Database
		4 Notes
		References
	Chapter 36: Formalizing Phenotypes of Regeneration
		1 Introduction
		2 Materials
		3 Methods
			3.1 Formalizing Planarian Regeneration Phenotypes with Planform
			3.2 Formalizing Limb Regeneration Phenotypes with Limbform
			3.3 Formalizing Planarian Gene Expression Patterns with PlanGexQ
		4 Notes
		References
Index