Methods in Molecular Biology
Part I: Imaging Morphogenesis
1 Probing Regional Mechanical Properties of Embryonic Tissue Using Microindentation and Optical Coherence Tomography
2.1 Chicken Embryo Preparation, Labeling, and Culture
2.2 Optical Coherence Tomography (OCT) and Microindentation
3.1 Chicken Embryo Preparation, Labeling, and Culture
3.2 Optical Coherence Tomography and Microindentation
3.3 Stiffness and Displacement Analysis
3.4 Deformation Analysis (Strains)
2 Hemodynamic Flow Visualization of Early Embryonic Great Vessels Using PIV
2.1 Transgenic Embryo Sources and Staging
2.2 Solutions and Seeding Particles
2.3 Zebrafish Confocal Microscopy and Microfluidic Platform
2.4 Chick Embryo Microscopy and Instrumentation
2.5 Chick Embryo Optical Coherence Tomography
3.2 Microfluidic Platform Fabrication
3.3 Volumetric Confocal Microscopy Imaging of Zebrafish
3.4 Microinjection of Seeding Particles into the Chick Embryo
3.5 Optical Coherence Tomography Acquisition for PIV
3.6 Time-Lapse Flow Velocity Measurement
3.7 Wall Shear Stress (WSS) Calculation
3 Using Correlative Light and Electron Microscopy to Study Zebrafish Vascular Morphogenesis
2.1 Zebrafish (ZF) Embryo Anesthesia, Live Imaging, and Image Reconstruction (Fig.2)
2.2 Fixation, Post-fixation, En Bloc Staining (Figs.3 and 4)
2.3 Resin Embedding, Laser Etching, and Serial Sectioning (Fig.5)
2.4 Contrasting Agents (See Note 7)
2.6 Software for Image Processing
3.1 Zebrafish Embryo: Need for Fluorescent Lines Compatible with Live Imaging
3.2 Embryo Anesthesia and Montage (Fig.2)
3.3 Live Imaging and Image Processing: Capturing the Event of Interest Together with Anatomical Landmarks
3.4 Sample Preparation for Electron Microscopy
3.4.1 Design of a Multi-Flow-Through Chamber Stack (Fig.3)
3.4.2 Fixation/Dehydration/Infiltration
3.4.3 Flat Embedding (Fig.4) (See Also Refs. 6, 7)
3.4.4 Laser Etching (Fig.5) (See Also Refs. 6, 7)
3.5 Remounting of the Bloc for Parallel Serial Sectioning (Fig.5)
3.6 Serial Thick Sectioning (Fig.5)
3.7 EM Acquisition, Electron Tomography, and Image Processing
4 Micro/Nano-Computed Tomography Technology for Quantitative Dynamic, Multi-scale Imaging of Morphogenesis
2.1 General Soft Tissue Contrast Agents
2.1.2 Phosphotungstic Acid (PTA)
2.1.4 Gallocyanin-Chromalum
2.2 Antigen-Specific Contrast
2.2.1 Enzyme Metallography Immunostaining (Adapted from [8])
2.5 Image Filtering, Rendering, and Analysis
3.1 General Soft Tissue Contrast Agents
3.1.2 Phosphotungstic Acid
3.1.3 Osmium Tetroxide (see Note 2)
3.1.4 Gallocyanin-Chromalum
3.2 Antigen-Specific Contrast
3.2.1 Enzyme Metallography Immunostaining (Adapted from [8])
3.3 Microinjection (see Note 5)
3.5 Image Filtering, Rendering, and Analysis
5 Imaging the Dorsal-Ventral Axis of Live and Fixed Drosophila melanogaster Embryos
2 Manual Cross Sectioning
2.1 Mounting Living Embryos
3.1 Manual Cross Sectioning
3.2 Mounting Live Embryos
6 Light Sheet-Based Imaging and Analysis of Early Embryogenesis in the Fruit Fly
2.1 Materials for Fly Embryo Collection and Embedding
3.1 Light Sheet Microscopy for Simultaneous Multiview Imaging
3.2 Methods for Fly Embryo Imaging
3.2.1 Embryo Collection and Embedding
3.3.1 Image Pre-processing
3.3.2 Nuclei Segmentation
3.3.3 Membrane Segmentation
3.4 Computational Modeling of the Mechanics of Embryogenesis in the Fruit Fly
7 Quantitative Image Analysis of Cell Behavior and Molecular Dynamics During Tissue Morphogenesis
3.1 Cell Segmentation and Tracking
3.2 Morphological and Molecular Analyses
3.3 Quantification of Planar Cell Polarity
3.4 Representative Results
8 A Multiplex Fluorescent In Situ Hybridization Protocol for Clonal Analysis of Drosophila Oogenesis
2.1 Labeled RNA Probe Preparation
2.2 Ovary Sample Preparation
2.3 Hybridization of RNA Probes
3.1 Labeled RNA Probe Preparation
3.2 Ovary Sample Preparation
3.3 Hybridization of RNA Probes
9 Active Cell and ECM Movements During Development
3 In Vivo Labeling of ECM Antigens
5 Epifluorescence Time-Lapse Image Acquisition
7 Particle Image Velocimetry
10 Subtracting Tissue Movements from Cell Locomotion
9 Active Cell Motion Versus Tissue Motion
11 Tissue Flow as Low-Pass Filtered ECM Movement
Part II: Culture Models of Morphogenesis
10 3D Culture Assays of Murine Mammary Branching Morphogenesis and Epithelial Invasion
2.3 Instructions for Preparing Solutions
2.4 Tools and Instruments
3.1 Collecting Mouse Mammary Glands
3.2 Isolating Mammary Epithelial Organoids
3.3 Organoid Density Determination
3.4 Plating Mammary Organoids in Matrigel
3.5 Preparing Collagen I Solution
3.6 Plating Mammary Organoids in Collagen I
3.7 Plating Mammary Organoids in a Mixture of Matrigel and Collagen I
3.8 Immunofluorescence Staining of 3D Culture Samples
3.8.1 Staining Whole Gels
3.8.2 Staining Sections on Slides
3.9 Immunofluorescence Staining of Mammary Gland Tissue Sections
3.10.1 Cyst Formation Assay
3.10.2 Branching Morphogenesis Assay in Matrigel
3.10.3 Branching Morphogenesis Assay in a Mix of Matrigel and Collagen I
11 Culture of Mouse Embryonic Foregut Explants
2.1 Isolation of Foregut from Embryonic Mice
2.2 Culture of Foregut Explants
3.1 Isolation of Foregut Explants
3.2 Culture of Foregut Explants
12 Investigating Human Vascular Tube Morphogenesis and Maturation Using Endothelial Cell-Pericyte Co-cultures and a Doxycycline-Inducible Genetic System in 3D
Extracellular Matrices
2.1 Endothelial Cells and Pericytes
2.2 Rat Tail Collagen Type I
2.3 Reduced Serum Supplement II (RSII)
2.4 Recombinant Growth Factors, Cytokines, and Other Media Additives
2.6 Lentivirus Production
3.1 EC Tube Morphogenesis Assay in 3D Collagen Matrices Using HUAECs
3.2 EC-Pericyte Tube Co-assembly Assay in 3D Collagen Matrices
3.3 Pericyte Invasion into 3D Collagen Matrices in Response to PDGF-BB
3.4 Priming of ECs with VEGF and FGF-2 to Activate EC Morphogenic Responses
3.5 Analysis of Vascular Basement Membrane Deposition Resulting from EC-Pericyte Interactions During Tube Co-assembly in 3D Collagen
Matrices
3.6 Lentiviral Infection of ECs or Pericytes with Doxycycline-Inducible Gene Vectors Facilitates Analysis of Genes of Interest During Specific
Stages of Vascular
Morphogenesis and
Maturation
13 Three-Dimensional Traction Force Microscopy of Engineered Epithelial Tissues
1.1 History and Importance of Traction Force Microscopy (TFM)
2.2 Micropatterning Materials
2.3 Confocal Fluorescence Microscopy
2.4 Tracking Bead Displacements
2.5 Calculating Average Displacement Fields
2.6 Reconstructing Tissue Geometry
2.7 Computing Traction Forces
3.1 Preparation of PDMS Stamps for 3D Micropatterning
3.2 Micropatterning of 3D Epithelial Tissues
3.3 Confocal Time-Lapse Imaging of Fluorescent Bead Displacements and Tissue Morphogenesis
3.4 Tracking Bead Displacements
3.5 Computing Average Displacement Field (Using Data from Multiple Tissues)
3.6 Reconstructing and Exporting Tissue Geometry
3.7 Calculating Traction Forces
Part III: Manipulating Cells and Tissues In Vivo
14 Probing Cell Mechanics with Subcellular Laser Dissection of Actomyosin Networks in the Early Developing Drosophila Embryo
2.2 Laser, Optical, and Mechanical Components
2.3 Software and Controllers
2.4 Sample Preparation Equipment
2.4.1 Laser Alignment Sample
3.2 Setting Up the Laser Dissection Optical Path
3.3 Alignment Sample Preparation
3.4 Focal Ablation Spot Alignment
3.5 Computer Communication with the External Mechanical Devices
3.6 Biological Sample Preparation
3.7 Laser Dissection Experiments of Actomyosin Bundles
3.8 Probing Cell Membrane Integrity After Laser Ablation
3.9 Image Analysis for Force Measurement
15 UV Laser Ablation to Measure Cell and Tissue-Generated Forces in the Zebrafish Embryo In Vivo and Ex Vivo
2.3 Dechorionating and Mounting Embryos
2.4 Spinning Disk with UV Laser Cutter Setup
2.5 Preparation and Culturing of Induced Mesoderm Cells
3.1 UV Laser Ablation of the Actomyosin Cortex In Vivo
3.1.2 Dechorionating Embryos
3.1.3 Handling and Mounting Embryos in Agarose Solution
3.1.4 UV Laser Ablation and High-Speed Imaging of the Actomyosin Cortex of the YSL and EVL During Epiboly
UV Laser Ablation of the Actomyosin Cortex Within the YSL
UV Laser Ablation of the Actomyosin Cortex Within the EVL
3.2 UV Laser Ablation of Cell-Cell Interfaces of Mesoderm Cells Ex Vivo
3.2.1 Obtaining Mesoderm Progenitor Cells from Zebrafish Embryos
3.2.2 Culturing of Mesoderm Cells In Vitro
3.2.3 UV Laser Ablation of Cell-Cell Interfaces Between Migrating Mesoderm Cells In Vitro
3.3.1 Analysis of Laser Cuts for Measurement of Cortical Tension in the YSL and EVL
3.3.2 Analysis of Tension at the Cell-Cell Interface
16 Measurement of Intercellular Cohesion by Tissue Surface Tensiometry
2.1 The Tissue Surface Tensiometer
2.2 Other Equipment/Supplies
3.1 Generation of Spherical Aggregates
3.2 Setting Up of the Compression Cell
3.3 Loading the Aggregates
3.4 Attaching the UCP to the Balance Arm
3.5 Establishing a Pre-compression UCP Weight Baseline
3.6 Aligning the LCP, Aggregate, and UCP
3.7 Aggregate Compression
3.8 Calculation of Aggregate Surface Tension
3.9 Confirmation of Aggregate Liquidity
17 Quail-Chick Chimeras and Eye Development
2.2 Preparation of Donor Grafts (Quail) and Host Embryos (Chick)
2.3 Histological Analysis of the Chimeric Embryos
3.2 Preparation of Eggs for Dissection and Windowing
3.3 Prepare the Donor (Quail) Graft Tissue
3.4 Prepare the Host (Chick) Embryo to Receive the Graft
3.7 Analysis of Chimeric Embryos
3.8 Analysis of Quail-Chick Chimeras for Neural Crest and Ectoderm Contribution to the Eye
Part IV: Emerging Models of Tissue Morphogenesis
18 Studying Epithelial Morphogenesis in Dictyostelium
2.1 Cell Growth and Transformation with Fluorescent Protein Fusions
2.2 Development of D. discoideum Under Controlled Conditions
2.4 Whole-Mount Immunofluorescence of Culminants
3.1 Obtaining Homogenous Expression of Fluorescent Fusion Proteins
3.2 Development of D. discoideum Under Controlled Conditions
3.3 RNA Interference During Multicellular Development
3.4 Whole-Mount Immunofluorescence of Culminants
19 Primary Cell Cultures of Regenerating Holothurian Tissues
2.1 Sea Cucumber Collection and Evisceration
2.2 Sea Cucumber Disinfection and Gut Dissection
2.3 Gut Rudiment Dissociation and Cell Culture
2.4 Cell Identification by Indirect Immunohistochemistry
2.5 Cell Identification by Scanning Electron Microscopy
3.1 Sea Cucumber Collection and Evisceration
3.2 Sea Cucumber Disinfection and Gut Dissection
3.3 Gut Rudiment Dissociation and Cell Culture
3.4 Cell Identification by Indirect Immunohistochemistry
3.5 Cell Identification by Scanning Electron Microscopy (See Note 7)
Part V: Computational Models of Tissue Morphogenesis
20 Large-Scale Parameter Studies of Cell-Based Models of Tissue Morphogenesis Using CompuCell3D or VirtualLeaf
3.1 Organize Project Directory
3.2 Run the CompuCell3D Model from the Command Line
3.3 Analyzing a Single CompuCell3D Simulation
3.4 Setting Up and Running a Parameter Sweep with CompuCell3D
3.5 Analyzing a CompuCell3D Parameter Sweep
3.6 Adapting the Protocol to Alternative Simulation Packages
21 Simulating Tissue Morphogenesis and Signaling
2 Signaling Models on Moving Domains
2.1 The Lagrangian Framework
2.2 Arbitrary Lagrangian-Eulerian (ALE) Method
3.1.1 Model-Based Displacement Field
3.1.2 Image-Based Displacement Field
3D Image and Meshes of Tissue
Calculating the Displacement Field
Simulation of Signaling Dynamics Using FEM
3.3 Cell-Based Tissue Models
3.3.1 Viscoelastic Cell Model
3.3.2 Cellular Potts Model
22 Elasticity-Based Targeted Growth Models of Morphogenesis
2.1 Elasticity of Non-growing Bodies
2.2 Elasticity of Growing Bodies
3.1 Applying Growth Theory to Basic Tissue Morphogenesis
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