Salamanders: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Salamanders as Key Models for Development and Regeneration Research
	1 Early Studies of Salamanders
	2 Developmental Biology Begins: Entwicklungsmechanik
	3 A Nobel Prize for a Revolutionary Discovery Using Salamander Embryos
	4 Developmental Studies with Ambystoma
	5 Einsteck and Neural Induction: Another Major Contribution from Salamanders
	6 Axolotls Appear and Are Replaced by Xenopus
	7 Regeneration
		7.1 Neurotrophic Control
		7.2 Dedifferentiation
		7.3 Pattern Formation
		7.4 Brain Regeneration
		7.5 Lens Regeneration
	8 Conclusions
	References
Part I: Laboratory Colony Husbandry
	Chapter 2: Establishing a New Research Axolotl Colony
		1 Introduction
		2 Materials
			2.1 Housing
			2.2 Water Supply
			2.3 Diet
			2.4 Water Quality
			2.5 Anesthetic
		3 Methods
			3.1 Housing
			3.2 Water Quality
			3.3 Diet
			3.4 Anesthesia
			3.5 Courtship and Mating
		4 Notes
		References
	Chapter 3: Health Monitoring for Laboratory Salamanders
		1 Introduction
			1.1 Biosecurity and Health Monitoring Program Design
			1.2 Diagnostic Platforms
				1.2.1 Simple Direct Examinations
				1.2.2 Histopathology
				1.2.3 Cytology
				1.2.4 Immunology
				1.2.5 Parasitology
				1.2.6 Microbiology
				1.2.7 Molecular Diagnostics
		2 Materials
			2.1 General Biosecurity
			2.2 Necropsy and Postmortem Sample Collection
			2.3 Antemortem and Environmental Sample Collection
		3 Methods
			3.1 Import Procedures
				3.1.1 Approved Vendors List
				3.1.2 Exclusion Lists
				3.1.3 Quarantine
			3.2 Biosecurity
				3.2.1 Exclusion
				3.2.2 Work and Material Flow
				3.2.3 Disinfection
				3.2.4 Elimination
				3.2.5 Containment
				3.2.6 Eradication
			3.3 Necropsy and Postmortem Sample Collection
				3.3.1 General Considerations
				3.3.2 Skin and Gills
				3.3.3 Fluids
				3.3.4 Internal Organs
			3.4 Routine Colony Health Monitoring
				3.4.1 Selection of Agents to Monitor
				3.4.2 Diagnostic Platforms
				3.4.3 Sample Sizes
				3.4.4 Testing Interval
				3.4.5 Antemortem Sample Collection
				3.4.6 Environmental Sample Collection
		4 Notes
		References
	Chapter 4: Husbandry, Captive Breeding, and Field Survey of Chinese Giant Salamander (Andrias davidianus)
		1 Introduction
		2 Materials
			2.1 Setup and Equipment for Husbandry
			2.2 Maintenance and Food Supply
			2.3 Supplies, Reagents, and Solutions for Disease Treatment and Artificial Insemination
			2.4 Tools and Equipment for Field Survey and Monitoring
		3 Methods
			3.1 Daily Rearing, Feeding, and Cleaning
			3.2 Health Inspection and Disease Treatment
			3.3 Natural-Imitative Breeding and the Following Maintenance
			3.4 Artificial Insemination and the Following Maintenance
			3.5 Field Survey, Capture, and Monitoring
		4 Notes
		References
Part II: Traditional Molecular Techniques
	Chapter 5: Whole-Mount In Situ Hybridization (WISH) for Salamander Embryos and Larvae
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Reagent Preparation
		3 Methods
			3.1 Tissue Harvest and Processing (See Notes 1-3)
			3.2 Primer Design
			3.3 RNA Extraction and cDNA Synthesis
			3.4 Standardizing PCR for Target Amplification
			3.5 PCR Product Purification
			3.6 Gel Excisions and Extraction of PCR Product
			3.7 Ligation of PCR Product into Vector and Bacterial Transformation
			3.8 Colony PCR
			3.9 Plasmid Extraction (Miniprep) and Sequencing
			3.10 Plasmid Extraction (Maxiprep) and Glycerol Stock
			3.11 Plasmid Linearization
			3.12 In Vitro Transcription
			3.13 Purification of In Vitro Transcription Product
			3.14 Whole-Mount In Situ Hybridization (WISH)
		4 Notes
		References
	Chapter 6: Hybridization Chain Reaction Fluorescence In Situ Hybridization (HCR-FISH) in Ambystoma mexicanum Tissue
		1 Introduction
		2 Materials
			2.1 Axolotls
			2.2 Equipment and Consumables
			2.3 Buffers and Solutions
			2.4 Oligo Probe Design and Ordering
			2.5 Ordering Hairpins
		3 Methods
			3.1 Coverslip Functionalization
			3.2 Tissue Cryosectioning and Section Dehydration
			3.3 v3.HCR-FISH on Tissue Sections
			3.4 Probe Wiping and Rehybridization of New Probes
			3.5 Multiround v3.HCR-FISH Image Analysis: Segmentation
			3.6 Multiround v3.HCR-FISH Image Analysis: Dot Counting
			3.7 Whole-Mount v3.HCR-FISH
			3.8 Imaging with Lightsheet Fluorescence Microscopy (LSFM)
		4 Notes
		References
	Chapter 7: Ethyl Cinnamate-Based Tissue Clearing Strategies
		1 Introduction
			1.1 Planning the Tissue Clearing and Imaging Process
		2 Materials
			2.1 Fixation
			2.2 Depigmentation (Optional)
			2.3 Antibody Staining (Optional)
			2.4 Embedding (Optional)
			2.5 Dehydration
			2.6 Refractive Index Matching
		3 Methods
			3.1 Fixation
			3.2 Depigmentation (Optional, See Note 4)
			3.3 Antibody Labeling (Optional, See Note 5)
			3.4 Embedding (Optional, See Note 6)
			3.5 Dehydration
			3.6 Refractive Index Matching
			3.7 Mounting and Imaging
		4 Notes
		References
	Chapter 8: Induction and Characterization of Cellular Senescence in Salamanders
		1 Introduction
		2 Materials
			2.1 Induction of Cellular Senescence in Salamander Cell Culture
			2.2 Evaluating Senescence-Associated Phenotypes in Tissue Culture
			2.3 In Vitro SA-β-Gal Staining
			2.4 In Vivo Senescence Analysis
				2.4.1 Tissue Collection, Processing, and Fixation
				2.4.2 SA-β-Gal Staining
				2.4.3 EdU and Antibody Staining
		3 Methods
			3.1 Induction of Cellular Senescence in Salamander Cell Culture
				3.1.1 Cellular Expansion
				3.1.2 Etoposide-Induced DNA damage (Day 1)
				3.1.3 UV-Induced DNA damage (Day 1)
				3.1.4 p53 Stabilization and Senescence Induction (Days 2-12)
			3.2 Detection of Senescence-Associated Phenotypes In Vitro
				3.2.1 Analysis of Mitochondrial Networks and ROS Production
				3.2.2 Analysis of Lysosomal Networks
				3.2.3 Analysis of DNA Damage by γH2AX Immunostaining
				3.2.4 Proliferation Analysis by EdU Staining
			3.3 SA-β-Gal Staining In Vitro
			3.4 SA-β-Gal Staining, EdU, and Immunostaining: In Vivo
				3.4.1 EdU Treatment, Tissue Collection, Fixation, and Processing
				3.4.2 SA-β-Gal Staining
				3.4.3 EdU and Antibody Staining
		4 Notes
		References
	Chapter 9: Methods for Studying Appendicular Skeletal Biology in Axolotls
		1 Introduction
		2 Materials
			2.1 General Tools and Reagents
			2.2 Alcian Blue/Alizarin Red Staining
			2.3 Sample Preparation for μCT Scan
			2.4 Bone Decalcification
			2.5 In Vivo Skeletal Staining
		3 Methods
			3.1 Alcian Blue/Alizarin Red Staining
			3.2 Sample Preparation for μCT Scan
			3.3 Bone Decalcification
			3.4 In Vivo Skeletal Staining
		4 Notes
		References
	Chapter 10: Fluorescence In Situ Hybridization of DNA Probes on Mitotic Chromosomes of the Mexican Axolotl
		1 Introduction
		2 Materials
			2.1 Spring Water (10% Holtfreter´s Solution)
			2.2 Hypotonic Solution
			2.3 Fixative Solution
			2.4 DNase I Working Solution
			2.5 BSA Working Solution 0.5 mg/ml
			2.6 dNTPs Mixture
			2.7 RNase Solution
			2.8 Pepsin Solution
			2.9 Hybridization Mix
			2.10 Denaturing Solution
			2.11 Washing Solutions
		3 Methods
			3.1 Slide Preparation
			3.2 Probe Labeling
			3.3 Cot-2-3 DNA Isolation
			3.4 Fluorescence In Situ Hybridization
		4 Notes
		References
	Chapter 11: The Use of Small Molecules to Dissect Developmental and Regenerative Processes in Axolotls
		1 Introduction
		2 Materials
		3 Methods
			3.1 Larvae
			3.2 Eggs
		4 Notes
		References
	Chapter 12: COMET Assay for Detection of DNA Damage During Axolotl Tail Regeneration
		1 Introduction
		2 Materials
			2.1 Maintenance and Surgical Procedures
			2.2 Tissue Dissociation
			2.3 Peripheral Blood Collection
			2.4 Pretreatment of Microscope Slides for Electrophoresis and Sample Assembly
			2.5 Lysis and Single-Cell Electrophoresis
		3 Methods
			3.1 Axolotl Tail Amputation to Induce Regeneration
			3.2 Recovery of Regenerating and Non-regenerative Tissue and Dissociation into Single-Cell Suspension
			3.3 Peripheral Blood Cell Isolation
			3.4 Preparation of Individual Cells from Regenerating Tissues and Controls (Peripheral Blood and Non-regenerative Tissue) on M...
				3.4.1 Pretreatment of Microscope Slides for Single-Cell Electrophoresis
				3.4.2 Assembly of Dissociated Cells onto Pretreated Microscope Slides
			3.5 Lysis and Electrophoresis of Individual Axolotl Cells
			3.6 DNA Damage Detection and Analysis
		4 Notes
		References
Part III: Experimental Manipulations and Surgeries
	Chapter 13: In Vivo and Ex Vivo View of Newt Lens Regeneration
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Equipment
			2.3 Optical Coherence Tomography
		3 Methods
			3.1 Housing
			3.2 Lentectomy
			3.3 Explant Tissue Cultures
			3.4 Optical Coherence Tomography
			3.5 Image Data Processing
		4 Notes
		References
	Chapter 14: Bead Implantation and Delivery of Exogenous Growth Factors
		1 Introduction
		2 Materials
			2.1 Obtaining Axolotls and Axolotl Husbandry
			2.2 Limb Amputation
			2.3 Bead Preparation
		3 Methods
			3.1 Preparation of Beads (Movie 1)
			3.2 Protein Absorption into the Beads
			3.3 Delivery of a Bead into a Blastema (Movie 2)
		4 Notes
		References
	Chapter 15: The Accessory Limb Model Regenerative Assay and Its Derivatives
		1 Introduction
			1.1 History of the Study of Nerve-Induced Ectopic Limb Structures
			1.2 The Establishment of the ALM and Its Use in Research
		2 Materials
			2.1 Solution Preparation
			2.2 Surgical Supplies
		3 Methods
			3.1 Standard ALM
			3.2 ALMs in Different Limb Positions
				3.2.1 Identification of Limb Axes
				3.2.2 Location-Specific Nerve Deviations.
		4 Notes
		References
	Chapter 16: Embryonic Tissue and Blastema Transplantations
		1 Introduction
		2 Materials
			2.1 General Tools
			2.2 Reagents
			2.3 Reagent Preparation
		3 Methods
			3.1 Embryo Preparation
			3.2 Limb Blood Vessels and Limb Muscle
			3.3 Limb Connective Tissue and Blood Cells
			3.4 Limb Blastema Transplantations
		4 Notes
		References
	Chapter 17: Retinoic Acid-Induced Limb Duplications
		1 Introduction
		2 Materials
			2.1 Retinoid Solutions
			2.2 Anesthesia Solution
			2.3 Holtfreter´s Solution
			2.4 Victoria Blue Solution
			2.5 Animals
		3 Methods
			3.1 Anesthesia and Amputation of Limbs
			3.2 Administration of Retinoid
			3.3 Fixation and Cartilage Staining of Regenerates
		4 Notes
		References
	Chapter 18: Isolation and Characterization of Peritoneal Macrophages from Salamanders
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Reagents
			2.3 Equipment
		3 Methods
		4 Notes
		References
Part IV: Bioinformatics and Genomics
	Chapter 19: Navigation and Use of Custom Tracks within the Axolotl Genome Browser
		1 Introduction
		2 Methods
			2.1 A Practical Guide to Exploring the Axolotl Genome Using the UCSC Browser
				2.1.1 Retrieving the DNA Sequence
				2.1.2 Exploring the Gene Neighborhood
				2.1.3 Missing Genes and Hi-C Data
			2.2 Looking for Missing Genes
				2.2.1 Track Hubs
		3 Notes
		References
	Chapter 20: Chromosome Conformation Capture for Large Genomes
		1 Introduction
		2 Materials
			2.1 Materials for Hi-C Preparations
			2.2 Equipment for all Hi-C Preparations
			2.3 Additional Materials for Larval Hi-C Preparations
			2.4 Additional Materials for Mitotic Hi-C Preparations
		3 Method
			3.1 Cell Preparation
				3.1.1 Cell Preparation of Cycling Cultured Axolotl Cells
				3.1.2 Protocol for Mitotic Synchronization of Cultured Axolotl Cells
				3.1.3 Cell Preparation (from Larvae)
			3.2 Nuclei Isolation, Fixation, In Situ Chromatin Digestion
			3.3 Biotin Filled-In, Blunt-End Ligation
			3.4 DNA Extraction
			3.5 DNA Shearing
			3.6 Biotin-Streptavidin Immunoprecipitation and Adapter-Ligation
			3.7 Library Amplification and Purification
		4 Notes
		References
Part V: Transgenics and Lineage-Tracing
	Chapter 21: Axolotl Transgenesis via Injection of I-SceI Meganuclease or Tol2 Transposon System
		1 Introduction
		2 Materials
			2.1 Equipment and Materials
			2.2 Reagents
			2.3 Reagent Preparation
			2.4 Injection Needles
			2.5 Injection Plate
		3 Methods
			3.1 Preparation of I-SceI and Tol2-Compatible Transgenic DNA Construct
			3.2 In Vitro Transcription of the Tol2 Transposase mRNA
			3.3 Injection of DNA or RNA-DNA Mix
			3.4 Screening
		4 Notes
		References
	Chapter 22: A Practical Guide for CRISPR-Cas9-Induced Mutations in Axolotls
		1 Introduction
		2 Materials
			2.1 Molecular Biology Materials
			2.2 Bioinformatics
			2.3 Injections
			2.4 Solutions
		3 Methods
			3.1 Bioinformatics and Guide RNA Design (Fig. 1)
			3.2 CRISPR-Cas9 Injections (Fig. 2)
			3.3 Phenotype Crispants
			3.4 Genotype Crispants
		4 Notes
		References
	Chapter 23: Applying a Knock-In Strategy to Create Reporter-Tagged Knockout Alleles in Axolotls
		1 Introduction
		2 Materials
			2.1 Axolotls
			2.2 Targeting Construct and Other Plasmids
			2.3 Primers
			2.4 Preparation of Targeting Construct
			2.5 gRNA Synthesis and Preparation of the Mixture of CAS9 Protein, gRNA, and Targeting Construct
			2.6 Microinjection
			2.7 Genotyping
			2.8 Buffers and Other Reagents
		3 Methods
			3.1 Preparation of Targeting Construct
			3.2 Preparation of gRNA
			3.3 Preparation of the Mixture of CAS9 Protein, gRNA, and Targeting Construct
			3.4 Axolotl Breeding, Egg Collection, and Microinjection
			3.5 Screening of F0 Transgenic Axolotls by Phenotyping
			3.6 Screening of F0 Transgenic Axolotls by Genotyping
			3.7 Germline Transmission and Establishing F1 Transgenic Axolotl Line
			3.8 Mutant Phenotype in F2 Generation Transgenics
		4 Notes
		References
	Chapter 24: Baculovirus Production and Infection in Axolotls
		1 Introduction
		2 Materials
			2.1 Bacmid and pOEM1 Transfer Vector Preparation
			2.2 Recombinant BV Production and Virion Amplification
			2.3 BV Purification and Concentration
			2.4 Titer Determination
			2.5 In Vitro Application of Baculovirus
			2.6 In Vivo Application of Baculovirus
		3 Methods
			3.1 Bacmid and pOEM1 Transfer Vector Preparation (One Day Before Virus Infection)
			3.2 Recombinant BV Production and Virion Amplification
			3.3 BV Purification and Concentration (Day 1-Day 3)
			3.4 Titer Determination
			3.5 In Vitro Application of BV to Axolotl Limb-Derived AL1 Cells (24-Well Format)
			3.6 In Vivo Application of BV
		4 Notes
			4.1 Bacmid and pOEM1 Transfer Vector Preparation
			4.2 Recombinant BV Production and Virion Amplification
			4.3 BV Purification and Concentration
			4.4 Titer Determination
			4.5 In Vitro Application of BV
			4.6 In Vivo Application of BV
		References
	Chapter 25: Cell Dissociation Techniques in Salamanders
		1 Introduction
			1.1 The Importance of Cell Dissociation and Potential Applications
			1.2 Manual Dissociation Approaches
			1.3 Enzymatic Dissociation Approaches
			1.4 Monitoring the Success of Dissociation
		2 Materials
			2.1 General Reagents, Equipment, and Consumables
			2.2 Axolotl Surgery
			2.3 Axolotl Tissue Dissociation Cocktails
			2.4 Antibody Staining and Flow Cytometry
		3 Methods
			3.1 Animal Maintenance and Usage
			3.2 Axolotl Limb Amputation and Generation of Blastema Tissue
			3.3 Blastema/Spleen Isolation and Tissue Dissociation
			3.4 Live-Cell Antibody Staining for Myeloid Cells from the Limb Blastema or Spleen
			3.5 Expected Results and Conclusions
		4 Notes
		References
Part VI: Physiological and Organismal Techniques
	Chapter 26: Axolotl Metabolism: Measuring Metabolic Rate
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Respiratory Chamber
			2.3 Chiller/Heater Tanks
			2.4 Water
			2.5 Hardware and Software for Measuring Respiratory Gases and Related Variables
				2.5.1 Measurement of DO
				2.5.2 Measurement of Atmospheric Oxygen
				2.5.3 Measurement of Atmospheric Carbon Dioxide
				2.5.4 Measurement of Water Conductivity
		3 Methods
		4 Notes
		References
	Chapter 27: Artificial Insemination in Axolotl
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Chemicals and Solutions
		3 Methods
		4 Notes
		References
	Chapter 28: Screening Salamanders for Symbionts
		1 Introduction
		2 Materials
			2.1 Sampling
				2.1.1 Skin Swabs
				2.1.2 Gut/Cloaca
				2.1.3 Egg Capsule Fluid
			2.2 Culturing
				2.2.1 Bacterial Culturing
				2.2.2 Algal Culturing
				2.2.3 Batrachochytrium Culturing
			2.3 DNA Extraction
				2.3.1 Swabs and Tissue Samples
				2.3.2 Egg Capsule Fluid
			2.4 PCR
			2.5 Analytical Software
		3 Methods
			3.1 Sampling
				3.1.1 Collecting Sample Metadata
				3.1.2 Skin Swab
				3.1.3 Cloaca/Gut
				3.1.4 Egg Capsule Fluids
			3.2 Culturing
				3.2.1 Bacterial Culturing
				3.2.2 Algal Culturing
				3.2.3 Batrachochytrium Culturing
			3.3 Sanger Sequencing
				3.3.1 DNA Extraction
				3.3.2 16s PCR
				3.3.3 Sanger Sequencing Analysis
			3.4 Amplicon Sequencing (Metabarcoding)
		4 Notes
		References
	Chapter 29: Assessing Leukocyte Profiles of Salamanders and Other Amphibians: A Herpetologists´ Guide
		1 Introduction
		2 Materials
			2.1 Blood Collection
			2.2 Microscopy
		3 Methods
			3.1 Blood Collection
				3.1.1 Terminal Blood Collection
				3.1.2 Non-lethal Blood Collection
			3.2 Slide Making and Preparation
			3.3 Microscopy
			3.4 Blood Cell Identification Tips
		4 Notes
			4.1 Animal Handling
			4.2 Slide Preparation
			4.3 Microscopy
			4.4 Interpreting Cell Data
		References
Part VII: Epilogue
	Chapter 30: Best Practices to Promote Data Utility and Reuse by the Non-Traditional Model Organism Community
		1 Introduction
		2 Materials
			2.1 Computer and Internet Access
			2.2 Storage
		3 Methods
			3.1 Planning Experiments
			3.2 Conducting Experiments
			3.3 Analyzing Data
			3.4 Archiving and Sharing Data
		4 Notes
		References
	Chapter 31: Now that We Got There, What Next?
		1 Genome Dynamics During Regeneration and Over Evolutionary Timescales
		2 Cellular and Molecular Dynamics of Regeneration
		3 Commonality and Divergence Among Salamanders as Meaningful for Regeneration
		4 Beyond Regeneration
		References
Index