Craniofacial Development: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Using Caenorhabditis elegans as a Model for Mechanistic Insights of Craniofacial Development
	1 Introduction
		1.1 Background
		1.2 Egg-Laying Assays
		1.3 Constipation Assays
		1.4 C. elegans Mutants
	2 Materials
		2.1 C. elegans Growth Plates
			2.1.1 Nematode Growth Medium (NGM)  Agar
			2.1.2 Modified Youngren´s, Only Bacto-peptone (MYOB) Plates
		2.2 C. elegans  Food
			2.2.1 OP50 E. coli Strain
		2.3 C. elegans Manipulation (Fig. 2)
		2.4 Agarose Pad Preparation for Imaging in a Compound Microscope
	3 Methods
		3.1 C. elegans Husbandry
		3.2 Brood Size Assay (Fig. 6)
		3.3 Embryo Retention (Ret) Assay
		3.4 Constipation Assay (Fig. 6)
		3.5 Examining Phenotypes in Heterozygotes
	4 Notes
	References
Chapter 2: Wholemount In-Situ Hybridization (WISH) in Zebrafish Embryos to Analyze Craniofacial Development
	1 Introduction
	2 Materials
		2.1 Preparation of Embryos
		2.2 RNA Probe Transcription
		2.3 RNA Probe Hybridization
	3 Methods
		3.1 Preparation of Embryos
		3.2 RNA Probe Synthesis
		3.3 Probe Hybridization
			3.3.1 Embryo Rehydration
			3.3.2 Proteinase Digestion and Postfixation
			3.3.3 Prehybridization
			3.3.4 Hybridization
		3.4 Probe Removal and Antibody Detection
			3.4.1 Probe Removal and Posthybridization Washes
			3.4.2 Detection
		3.5 DIG Color Development and Imaging
		3.6 Fluorescein Color Development and Imaging
		3.7 Imaging
	4 Notes
	References
Chapter 3: Elucidation of Gene Expression Patterns in the Craniofacial Tissues of Mouse Embryos by Wholemount In Situ Hybridiz...
	1 Introduction
	2 Materials
		2.1 PB1 Media
		2.2 Dissection
		2.3 Embryo Dehydration and Permeabilization
		2.4 Embryo Rehydration and Refixation
		2.5 Prehybridization
		2.6 Post Antibody  Wash
		2.7 Histochemistry
	3 Methods
		3.1 Dissection
		3.2 Embryo Dehydration and Permeabilization
		3.3 Embryo Rehydration and Refixation
		3.4 Prehybridization
		3.5 Post Antibody  Wash
		3.6 Histochemistry
	4 Notes
	References
Chapter 4: Visualization of the Cartilage and Bone Elements in the Craniofacial Structures by Alcian Blue and Alizarin Red Sta...
	1 Introduction
	2 Materials
		2.1 PB1 Media
		2.2 Embryo Dissection
		2.3 Skin Removal and Evisceration
		2.4 Dehydration and Staining
	3 Methods
		3.1 Dissection
		3.2 Skin Removal and Evisceration
		3.3 Dehydration and Staining
	4 Notes
	References
Chapter 5: Methodology for Free-Floating Organ Culture of Mid-gestation Maxillary Primordial Tissue
	1 Introduction
	2 Materials
		2.1 Recommended Equipment
		2.2 Solutions and Chemical Components
	3 Methods
		3.1 Experimental Animal Generation
		3.2 Preparation for Dissection
		3.3 Tissue Dissection
		3.4 Tissue Culture
		3.5 Postculture Processing for Histological Analysis
	4 Notes
	References
Chapter 6: Methods of Palate Culture in Later Palatogenesis: Elevation, Horizontal Outgrowth, and Fusion
	1 Introduction
	2 Materials
		2.1 Culture Media and Supplementation
		2.2 Dissection Materials
		2.3 Gas and Apparatus/Other (Fig. 1)
		2.4 Culture Media and Supplementation
		2.5 Dissection Materials
		2.6 Culture Apparatus/Other (Fig. 5)
	3 Methods
		3.1 Preparing Culture Media/Gas Mixture
		3.2 Embryo Collection and Dissection
		3.3 Culture Maintenance and Explant Documentation
		3.4 Preparing Culture Media and Equipment Setup
		3.5 Embryo Collection and Dissection
		3.6 Culture Maintenance and Explant Documentation
		3.7 Using Indian Ink as an Exogenous Contrasting Agent for Whole-Mount Photomicrography
	4 Notes
	References
Chapter 7: Single Cell RNA-Seq: Cell Isolation and Data Analysis
	1 Introduction
	2 Materials
		2.1 Solutions and Reagents
		2.2 Equipment and Consumables
	3 Methods
		3.1 Isolating Cells from Tissues
		3.2 Preparing Single-Cell Suspension for Sequencing
		3.3 Analyzing and Exploring scRNA-Seq Data
	4 Notes
	References
Chapter 8: Generating Zebrafish RNA-Less Mutant Alleles by Deleting Gene Promoters with CRISPR/Cas9
	1 Introduction
	2 Materials
	3 Methods
		3.1 Establish the Promoter(s) Sequence of the Gene of Interest
		3.2 Design gRNAs (See Note 9)
		3.3 Prepare sgRNAs and RNP Complexes for Injection into Embryos
		3.4 Inject Embryos with  gRNA
		3.5 Genotype F0 Embryos
		3.6 Generate and Raise F1  Fish
		3.7 Generate F2 Embryos and Assess if Those with Abnormal Phenotypes Harbour the Targeted Promoter Mutation
		3.8 Determine mRNA Levels of the Targeted  Gene
	4 Notes
	References
Chapter 9: Craniofacial Phenomics: Three-Dimensional Assessment of the Size and Shape of Cranial and Dentofacial Structures
	1 Introduction
	2 Materials
		2.1 Laboratory Supplies
		2.2 Examples of Micro-CT Scanners and Reconstruction Software (See Note 1)
		2.3 Examples of 3D Image Processing Software
		2.4 Examples of Geometric Morphometric Analysis Software
	3 Methods
		3.1 Sample Preparation
		3.2 Micro-CT Imaging Parameters
		3.3 Image Reconstruction
		3.4 Traditional Morphometric Analysis
			3.4.1 Cranium
			3.4.2 Midface
			3.4.3 Maxilla
			3.4.4 Mandible
			3.4.5 Dentition
			3.4.6 Surface and Volume Analyses
			3.4.7 Measurement Error
			3.4.8 Suture Patency
		3.5 Geometric Morphometric Analysis
			3.5.1 Landmark Digitization
			3.5.2 Measurement Error
			3.5.3 Ordination
		3.6 Statistical Tests
	4 Notes
	References
Chapter 10: Micro-CT-Based Bone Microarchitecture Analysis of the Murine Skull
	1 Introduction
	2 Materials
		2.1 Laboratory Supplies
		2.2 Examples of Micro-CT Scanners and Reconstruction Software
		2.3 Image Processing Software
	3 Methods
		3.1 Sample Preparation
		3.2 Micro-CT Imaging
		3.3 Image Realignment
		3.4 Bone Microarchitecture Analysis
		3.5 Statistical Analysis
	4 Notes
	References
Chapter 11: Characterization of Mammalian In Vivo Enhancers Using Mouse Transgenesis and CRISPR Genome Editing
	1 Introduction
	2 Materials
		2.1 Vector Cloning and Preparation for In Vivo Transgenic Reporter Analysis
		2.2 Preparation of CRISPR Injection Mixes for Site-Directed Transgenesis
		2.3 Preparation of CRISPR Injection Mixes for Genomic Deletions
		2.4 Colony Set-up for Transgenic Mouse Production, Superovulation, and Egg Collection
		2.5 Pronuclear Injection and Embryo Transfer
		2.6 LacZ Transgenic Embryo Collection and X-gal Staining
		2.7 Genotyping
	3 Methods
		3.1 Vector Cloning and Preparation for In Vivo Transgenic Reporter Analysis
		3.2 Preparation of CRISPR Injection Mixes for Site-Directed Transgenesis
		3.3 Preparation of CRISPR Injection Mixes for Genomic Deletions
		3.4 Colony Set-up for Transgenic Mouse Production, Superovulation, and Egg Collection
		3.5 Pronuclear Injection and Embryo Transfer
		3.6 LacZ Transgenic Embryo Collection and X-Gal Staining
		3.7 Genotyping
	4 Notes
	References
Chapter 12: Histological Techniques for Sectioning Bones of the Vertebrate Craniofacial Skeleton
	1 Introduction
	2 Materials
		2.1 Tissue Preparation
		2.2 Tissue Processing
		2.3 Sectioning
		2.4 Staining
	3 Methods
		3.1 Tissue Preparation
		3.2 Tissue Processing
		3.3 Sectioning
		3.4 Staining
			3.4.1 Deparaffinization and Rehydration
			3.4.2 Hematoxylin and Eosin
			3.4.3 Van Gieson´s Stain
			3.4.4 Picrosirius Red
			3.4.5 Orcein
			3.4.6 Safranin O
			3.4.7 Tartrate-Resistant Acid Phosphatase (TRAP)
			3.4.8 Dehydration and Mounting
	4 Notes
	References
Chapter 13: Engineering Epithelial-Mesenchymal Microtissues to Study Cell-Cell Interactions in Development
	1 Introduction
	2 Materials
		2.1 Device Design and Construction
		2.2 Device Sterilization and Preculture Treatment
		2.3 Culturing Cell Lines for Devices
		2.4 Making 3D HA Cell Suspensions and Device Loading of 09-1 or 3T3 Cells
		2.5 Experimental Procedure for Gene Expression
		2.6 Evaluation of Gli-Driven Luciferase
		2.7 H&E Staining
	3 Methods
		3.1 Device Design and Construction
		3.2 Device Sterilization and Preparation for Culture
		3.3 Culture and Preparation of 09-1, GMSM-K, and 3T3 Shh-Light2 Cells for Seeding in Devices
		3.4 Making 3D Hyaluronic Acid Cell Suspensions and Device Loading of 09-1 or 3T3 Cells
		3.5 Experimental Procedure for Gene Expression
		3.6 Evaluation of Gli-Driven Luciferase
		3.7 H&E Staining
	4 Notes
	References
Chapter 14: Ex Vivo Culture of Human Cranial Suture Cells
	1 Introduction
	2 Materials
	3 Methods
		3.1 Culture of Human Cranial Suture Cells
		3.2 Trypsinizing Cells for Passaging and Cryopreserving
		3.3 Passaging Cells
		3.4 Cryopreserving Cells
	4 Notes
	References
Chapter 15: Scaffolds for Use in Craniofacial Bone Regeneration
	1 Introduction
	2 Materials
		2.1 Hydrogels
		2.2 Cryogels
		2.3 Electrospinning
	3 Methods
		3.1 Hydrogel Fabrication Process
		3.2 Cryogel Fabrication Process
		3.3 Electrospinning Process
	4 Notes
	References
Chapter 16: Mandible Explant Assay for the Analysis of Meckel´s Cartilage Development
	1 Introduction
	2 Materials
		2.1 Mandible Explant and Tissue Culture
		2.2 Agarose Bead Implantation
		2.3 Alcian Blue Staining of Meckel´s Cartilage
		2.4 Immunohistochemistry
	3 Methods
		3.1 Preparation of Tissue Culture Dishes
		3.2 Dissection of Mouse Embryos
		3.3 Mandible Dissection and Explant
		3.4 Agarose Bead Implantation
		3.5 Alcian Blue Staining of Meckel´s Cartilage
		3.6 Immunohistochemistry
	4 Notes
	References
Chapter 17: High-Resolution Histology for Craniofacial Studies on Zebrafish and Other Teleost Models
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Anesthesia
		2.3 Fixation, Rinsing, and Postfixation
		2.4 Epon Embedding (; See Note 3)
		2.5 Staining
	3 Methods
		3.1 Anesthesia, Fixation, and Decalcification
		3.2 Dehydration and Embedding
		3.3 Sectioning and Staining
	4 Notes
	References
Chapter 18: Live Imaging of the Dynamics of Mammalian Neural Crest Cell Migration
	1 Introduction
	2 Materials
		2.1 Isolation of Post-implantation Stage Mouse Embryos
		2.2 Neural Plate Dissection and 2D Culture
		2.3 Neural Plate Dissection and 3D Culture
		2.4 Whole Embryo Culture for Live Imaging
		2.5 Imaging and Analysis
	3 Methods
		3.1 Isolation of Post-implantation Stage Mouse Embryos
		3.2 Neural Plate Dissection and 2D Culture
		3.3 Neural Plate Dissection and 3D Culture
		3.4 Mounting Embryos for Static Culture and Live Imaging
		3.5 Imaging and Analysis for Whole Embryo Culture
	4 Notes
	References
Chapter 19: Salivary Gland Development in Culture
	1 Introduction
	2 Materials
		2.1 Organ Dissection and Culture
			2.1.1 Organ Dissection Instruments
			2.1.2 Culture Medium and Liquids
			2.1.3 Plastic Material
			2.1.4 Metal Grids
			2.1.5 Transparent Polyethylene Terephthalate (PET) Permeable Membrane
			2.1.6 Equipment
		2.2 Reagents for Specific Uses in Culture
		2.3 Solutions for Wholemount Immunofluorescence & BrdU Detection
	3 Methods
		3.1 Whole and Slice Salivary Gland Culture
			3.1.1 Setting up the Culture
			3.1.2 Mandibular Dissection
		3.2 Whole Organ Culture of Embryonic Salivary Gland
		3.3 Slice Organ Culture of Embryonic Salivary Gland
			3.3.1 Setting up the Explant Culture
		3.4 Salivary Gland Culture in Snake Embryos
		3.5 Specific Methods for Cultured Samples
			3.5.1 LysoTracker
			3.5.2 Wholemount Immunofluorescence
			3.5.3 Dispase
			3.5.4 BrdU
		3.6 Morphovideos and Morphometric Analysis
			3.6.1 Morphovideos
			3.6.2 Spooner Ratios
	4 Notes
	References
Chapter 20: Antenatal Ultrasound Imaging for Analysis of Human Craniosynostosis
	1 Introduction: The Normal Fetal Cranium
	2 Materials: Abnormal Fetal Head Shape
	3 Methods: Diagnosis of Craniosynostosis In Utero
	4 Notes
	References
Index