Drosophila Oogenesis: Methods and Protocols (Methods in Molecular Biology, 2626) 9781071629697, 9781071629703, 1071629697

Table of contents :
Preface
Contents
Contributors
Chapter 1: The Vast Utility of Drosophila Oogenesis
1 Overview
2 Early Ovary Development
3 The Adult Ovary
4 Drosophila GSCs: A Model for Stem Cell Maintenance and Differentiation
5 Cell Cycle Regulation
6 Collective Cell Migration
7 Cytoplasmic Streaming
8 Nurse Cell Dumping
9 Lipid Droplets: Roles in Oogenesis
10 Cell Death
11 Drosophila Oogenesis: A Model for Other Cell Biology Processes
12 Drosophila as a Female Reproductive Model
13 Conclusion
References
Chapter 2: Visualization and Quantification of Drosophila Larval Ovaries
1 Introduction
2 Materials
2.1 Gonad Dissection
2.2 Gonad Staining
2.3 Gonad Mounting
2.4 Gonad Counting
3 Methods
3.1 Dissect Larval Fat Body
3.2 Stain Larval Fat Body
3.3 Mount Gonads
3.4 Method to Count Germline Cells in Larval Gonads
3.4.1 Acquire Images
3.4.2 Analyze Images
4 Notes
Appendix I: Macro Script to Process Entire z-stacks (See Electronic Supplementary Material)
References
Chapter 3: Dissection, Fixation, and Standard Staining of Adult Drosophila Ovaries
1 Introduction
2 Materials
2.1 Fly Husbandry
2.2 Ovary Dissection
2.3 Tissue Fixation and Washing
2.4 DNA and F-Actin Staining
2.5 Immunostaining
2.6 Mounting
3 Methods
3.1 Fly Husbandry
3.2 Ovary Dissection
3.3 Tissue Fixation and Washing
3.4 DNA and F-Actin Staining
3.5 Immunostaining
3.5.1 Day 1: Blocking and Primary Antibody Incubation
3.5.2 Day 2: Washing and Secondary Antibody Incubation
3.6 Mounting
3.6.1 Manual Separation of Ovarioles
3.6.2 Mechanical Disruption of Ovarioles
4 Notes
References
Chapter 4: Utilizing the FLP-Out System for Clonal RNAi Analysis in the Adult Drosophila Ovary
1 Introduction
1.1 Harnessing the Power of Genetic Mosaics in Ovarian Cell Analyses
1.2 Selecting a Genetic Mosaic Generation System
1.3 The FLP-Out Method: Combining UAS/Gal4 and FLP/FRT with RNAi to Knockdown Gene Function in Germ Cells and Somatic Cells in...
2 Materials
2.1 Drosophila Strains and Culture
2.2 Materials for Heat Shock to Induce Clones
2.3 Ovary Dissection and Immunostaining
2.4 Sample Preparation and Confocal Microscopy
3 Methods
3.1 Drosophila Husbandry (See Note 19)
3.2 Heat Shocking Flies to Induce FLP-Out
3.3 Ovary Dissection and Immunostaining
3.4 Sample Preparation and Confocal Imaging
3.5 Data Analysis, Quantification, and Next Steps
4 Notes
References
Chapter 5: Analysis of Physiological Control of Adult Drosophila Oogenesis by Interorgan Communication
1 Introduction
2 Materials
2.1 Drosophila Strains and Culture
2.2 Dissection, Immunostaining, and Slide Preparation
2.3 Image Acquisition and Analysis
3 Methods
3.1 Checking Gal4 Expression Patterns
3.2 Dissection and Immunostaining
3.3 Assaying Interorgan Cross-Talk on Oogenesis
3.4 Data Analysis and Image Acquisition
3.4.1 Number of Laid Eggs
3.4.2 Hatching Percentage Analysis
3.4.3 Block in Ovulation
3.4.4 GSC and Cap Cell Maintenance
3.4.5 GSC and FSC Proliferation
3.4.6 Follicle Cell Proliferation
3.4.7 Early Germline Cyst Survival
3.4.8 Early Germline Cyst Distribution
3.4.9 Survival of Vitellogenic Egg Chambers
4 Notes
References
Chapter 6: Immunohistochemical Analysis of Nuclear Lamina Structures in the Drosophila Ovary Using CRISPR-Tagged Genes
1 Introduction
1.1 Resources for Immunohistochemical NL Analysis in the Ovary
1.2 Targeted Gene Editing Using CRISPR to Expand NL Tools
2 Materials
2.1 Fly Media and Stocks
2.2 Molecular Biology Reagents and Equipment
2.3 Immunohistochemical Analyses Reagents and Equipment
2.4 Web Resources and Tools
3 Methods
3.1 Sequence of the Gene of the Fly Strain Used in Editing
3.2 Generate the gRNA Expression Plasmid
3.3 Preparation of a Donor Plasmid
3.4 Injection and Isolation of CRISPR-Engineered Lines
3.5 Sequence Validation of Tagged Lines
3.6 Confirm Expression of Tagged Fusion Protein by Western Blot Analysis
3.7 Assess the Functionality of the Edited Gene
3.8 Immunohistochemical Analysis of the Tgged Protein
3.9 Confocal Imaging of Antibody-Stained Ovaries
4 Notes
References
Chapter 7: Visualizing Fusome Morphology via Tubulin Immunofluorescence in Drosophila Ovarian Germ Cells
1 Introduction
2 Materials
2.1 Fly Strains and Culture
2.2 Ovary Dissection
2.3 Immunostaining
2.4 Sample Mounting and Microscopy
3 Methods
3.1 Fly Preparation
3.2 Ovary Dissection
3.3 Immunostaining
3.4 Microscopy
4 Notes
References
Chapter 8: Tracking Follicle Cell Development
1 Introduction
2 Materials
2.1 Fly Husbandry and Clone Induction
2.2 Immunostaining
2.3 Fluorescent In Situ Hybridization with Immunostaining
3 Methods
3.1 Generation of GFP-Marked Follicle Cell Clones Using Heat Shock
3.2 Immunostaining of Adult Ovaries
3.3 Fluorescent In Situ Hybridization with Immunostaining
4 Notes
References
Chapter 9: Optimized Fixation and Phalloidin Staining of Basally Localized F-Actin Networks in Collectively Migrating Follicle...
1 Introduction
2 Materials
2.1 Preparing Female Flies for Dissection
2.2 Egg Chamber Dissection
2.3 Fixation, Staining, and Mounting
2.4 Imaging
3 Methods
3.1 Preparing Female Flies for Dissection
3.2 Reagent Preparation
3.3 Egg Chamber Dissection
3.4 Fixation, Staining, and Mounting
3.5 Imaging
4 Notes
References
Chapter 10: Quantitative Image Analysis of Dynamic Cell Behaviors During Border Cell Migration
1 Introduction
2 Materials
2.1 Preparation of Fly Stocks for Time Lapse Imaging
2.2 Ovary Dissection
2.3 Mounting Egg Chambers for Live Imaging
2.4 Live Time-Lapse Imaging and Quantitative Analysis of Live Migrating Border Cells
3 Methods
3.1 Prepare Flies for Dissection
3.2 Dissection of Ovaries for Live Imaging
3.2.1 Pull Out Whole Ovaries
3.2.2 Dissect Ovaries into Ovarioles
3.3 Mounting Egg Chambers for Live Imaging
3.3.1 Mount Egg Chambers for Upright Microscope Live Imaging
3.3.2 Mount Egg Chambers for Inverted Microscope Live Imaging and Immobilize Egg Chambers with a Fibrinogen-Thrombin Clot
3.4 Live Time-Lapse Imaging
3.4.1 Live Imaging Using a Widefield Epifluorescent Microscope
3.4.2 Set Up the Widefield Epifluorescent Time-Lapse Experiment
3.4.3 Live Imaging Using a Confocal Microscope
3.5 Quantitative Analysis of Live Migrating Border Cells
3.5.1 General Image Processing
3.5.2 Measure Cellular Behaviors in Fiji
3.5.3 Measure Protein Localization and Dynamics in Fiji
4 Notes
References
Chapter 11: Live Imaging of Nurse Cell Behavior in Late Stages of Drosophila Oogenesis
1 Introduction
2 Materials
2.1 Materials for Dissection of Individual Follicles
2.2 Imaging
2.3 Image Analysis
3 Methods
3.1 Dissection of Individual Follicles
3.2 Imaging
3.3 Image Analysis
3.3.1 Size Trajectories
3.3.2 Cytoplasmic Flow Behaviors
4 Notes
References
Chapter 12: Visualizing Lipid Droplets in Drosophila Oogenesis
1 Introduction
2 Materials
2.1 Dissecting Ovaries/Follicles
2.2 Detecting LDs in Fixed Samples
2.3 Detecting LDs by Live Imaging
2.4 Enriching LDs by In Vivo Centrifugation for Colocalization Studies
3 Methods
3.1 Dissecting Ovaries or Follicles Out of Adult Females
3.1.1 Preparation
3.1.2 Dissection
3.2 Detecting LDs in Fixed Tissue
3.2.1 Preparation and Dissection
3.2.2 Fixation and Staining with LD Dyes
3.2.3 Mounting
3.2.4 Imaging
3.3 Detecting LDs in Living Follicles
3.3.1 Preparation and Dissection
3.3.2 Staining with BODIPY-FL
3.3.3 Mounting
3.3.4 Imaging
3.4 Detecting Colocalization with LDs Using In Vivo Centrifugation
3.4.1 Preparing a Vessel to Keep Flies/Ovaries Oriented during Centrifugation
3.4.2 In Vivo Centrifugation of Entire Flies
3.4.3 In Vivo Centrifugation of Ovaries
4 Notes
References
Chapter 13: Assessing Ovulation in Drosophila melanogaster
1 Introduction
2 Materials
2.1 Common Equipment and Materials
2.2 Egg-Laying Assay and Counting Mature Follicles
2.3 Egg-Laying Time Assay
2.4 Materials for Ex Vivo Follicle Rupture Assay
3 Methods
3.1 The Egg-Laying Assay
3.1.1 Preparation of Reagents
3.1.2 Egg Laying
3.1.3 Mature Follicle Count
3.2 Egg-Laying Time Assay
3.3 Ex Vivo Follicle Rupture Assay
4 Notes
References
Chapter 14: A Low-Tech Flow Chamber for Live Imaging of Drosophila Egg Chambers During Drug Treatments
1 Introduction
2 Materials
2.1 Preparing Female Flies for Dissection
2.2 Flow Chamber Preparation
2.3 Egg Chamber Dissection
2.4 Egg Chamber Mounting in Flow Chamber
2.5 Exchanging Media and Imaging
3 Methods
3.1 Prepare Female Flies for Dissection
3.2 Prepare Flow Chamber
3.3 Egg Chamber Dissection
3.4 Flow Chamber Construction and Egg Chamber Mounting
3.5 Exchange Fluids in Flow Chamber
4 Notes
References
Chapter 15: Detection and Assessment of Wolbachia pipientis Infection
1 Introduction
2 Materials
2.1 Detection of Wolbachia by PCR or MLST
2.2 Detection of Wolbachia by Western Blotting
2.2.1 Western Sample Preparation
2.2.2 Running the SDS-PAGE Gel for Western Blotting
2.2.3 Membrane Preparation
2.2.4 Assembling and Assessing the Western Blot
2.3 Visualization of Wolbachia Infection in Drosophila Ovaries by Immunohistochemistry
2.3.1 Dissection of Ovaries
2.3.2 Fixation and Application of Antibodies
2.4 Visualization of Wolbachia Infection in Drosophila Ovaries by FISH
2.5 Imaging Wolbachia Within Ovary Tissue
3 Methods
3.1 Detection of Wolbachia by PCR or MLST
3.1.1 DNA Extraction for PCR
3.1.2 PCR
3.1.3 Determination of Wolbachia Strain by MLST
3.2 Detection of Wolbachia by Western Blotting
3.2.1 Western Sample Preparation
3.2.2 Running the SDS-PAGE Gel for Western Blotting
3.2.3 Preparing the Membrane
3.2.4 Assembling and Assessing the Western Blot
3.3 Visualization of Wolbachia Infection in Drosophila Ovaries by Immunohistochemistry
3.3.1 Dissection of Ovaries
3.3.2 Fixation and Application of Antibodies
3.4 Visualization of Wolbachia Infection in Drosophila Ovaries by FISH
3.5 Imaging and Analysis of Wolbachia Within Ovary Tissue
4 Notes
References
Chapter 16: Measuring Transposable Element Activity in Adult Drosophila Ovaries
1 Introduction
2 Materials
2.1 Dissection
2.2 RNA Isolation
2.3 DNase Treatment and Reverse Transcription
2.4 RT-qPCR
3 Methods
3.1 Dissection
3.2 RNA Isolation
3.3 DNase I Treatment
3.4 Two-Step RT-qPCR
3.5 Data Analysis
4 Notes
References
Chapter 17: Preparation of Drosophila Ovarioles for Single-Cell RNA Sequencing
1 Introduction
2 Materials
2.1 Drosophila Culture
2.2 Preparation of Buffers
2.3 Ovary Dissection
2.4 Tissue Digestion and Single-Cell Isolation
2.5 Cell Counting
3 Methods
3.1 Preparation of Flies
3.2 Ovary Dissection and Enrichment of the Early (Non-Vitellogenic) Stages
3.3 Tissue Digestion and Single-Cell Isolation
3.4 Cell Counting
4 Notes
References
Chapter 18: Chromatin Immunoprecipitation Experiments from Drosophila Ovaries
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 Sonication Optimization
2.3 Chromatin Immunoprecipitation
2.4 ChIP-seq Library Preparation
2.5 ChIP-seq Data Processing
3 Methods
3.1 Sample Preparation
3.2 Sonication Optimization
3.3 Chromatin Immunoprecipitation
3.4 ChIP-seq Library Preparation
3.5 ChIP-seq Data Processing
4 Notes
References
Chapter 19: Detection of Actin in Nuclear Protein Fraction Isolated from Adult Drosophila Ovary
1 Introduction
2 Materials
2.1 Dissection
2.2 Isolation of Cytoplasmic and Nuclear Protein Fractions
2.3 Measuring Protein Concentration with Bradford Assay
2.4 Western Blotting
3 Methods
3.1 Isolation of Ovaries
3.2 Isolation of Cytoplasmic and Nuclear Protein Fractions from the Ovaries
3.3 Measuring Protein Concentration with Bradford Assay
3.4 Western Blotting
3.4.1 Preparation of the SDS-Polyacrylamide Gel
3.4.2 Electrophoresis
3.4.3 Wet Transfer to PVDF Membrane
3.4.4 Immunoblotting
4 Notes
References
Chapter 20: STAMP: Spatio-Temporal Association Mapping of Proteins
1 Introduction
2 Materials
2.1 Experimental Instruments
2.2 RNA Isolation and Reverse-Transcription (RT) Kits and Reagents
2.3 Plasmid Preparation and Cloning
2.4 Immunostaining
2.5 Western Blotting
2.6 Mass Spectrometry
2.7 Fly Strains and Fly Culture
3 Methods
3.1 Plasmid Construction and Transgenic Fly Generation
3.1.1 cDNA Generation
3.1.2 Plasmid Preparation and Cloning
3.2 Fly Hybridization and Feeding
3.3 Verifying the Efficiency of Proximity Labeling In Vivo
3.3.1 Verification of Ligase Expression and Labeling Efficiency by Immunofluorescence
3.3.2 Verification of Ligase Expression and Labeling Efficiency by Twice Western Blot
3.4 Mass Spectrometry and Data Analysis
4 Notes
References
Chapter 21: Using Drosophila Oogenesis in the Classroom to Increase Student Participation in Biomedical Research
1 Introduction
1.1 Introduction to Course-Based Undergraduate Research Experiences (CUREs)
1.2 Design and Implementation of an Oogenesis-Based CURE
1.2.1 Experimental Questions and General Approaches
1.2.2 Introduction of the CURE and Preparatory Assignments
1.2.3 CURE Experiments, Data Collection, and Data Analysis
1.2.4 CURE Presentations
1.2.5 Student Learning Outcomes
1.3 Summary of the CURE
2 Materials
2.1 Fly Maintenance and Manipulation
2.2 Ovary Dissection, Staining, and Microscopy
2.3 Hatch Assays
3 Methods
3.1 Implementation of the CURE
3.1.1 Advance Preparation by Instructor
3.1.2 Instructor Guidelines for Implementation of the CURE
3.2 Lab Protocols and Assignments for Students
3.2.1 Preparation of Females for Dissection and Embryo Collection Chambers
3.2.2 Ovary Dissection, Fixation, and Staining
3.2.3 Hatch Assays
4 Notes
References
Chapter 22: Gaining Wings to FLY: Using Drosophila Oogenesis as an Entry Point for Citizen Scientists in Laboratory Research
1 Introduction
1.1 Enter Citizen Science
1.1.1 Contributory and Collaborative Citizen Science for Biomedicine
1.1.2 Co-created Citizen Science
1.2 Enter Oogenesis
1.3 Signaling Pathways in Oogenesis
1.3.1 EGFR Signaling
1.3.2 Planar Cell Polarity (PCP)
1.3.3 Src/Tec/Cytoskeleton Signaling
1.3.4 E-Cadherin
1.3.5 Hedgehog
1.3.6 Discs Large/Lethal (2) Giant Larvae/Scribble
1.3.7 Integrins
1.3.8 Notch
1.3.9 Bcl-2 Family
1.4 New Genes and Phenotypes
1.4.1 Brca2
1.4.2 Cell Cycle Regulators
1.4.3 Hippo/Warts/Yki
1.4.4 Estrogen-Related Receptor (ERR)
1.5 Defining Mechanisms in Molecular Detail
1.6 Moving the Research into the Home Laboratory
1.7 An Untapped Resource
2 Materials
2.1 Pupal Counting
2.2 Ovary Dissection
2.3 Phalloidin and Propidium Iodide Staining (In-Lab)
2.4 Immunostaining (In-Lab)
2.5 Trypan Blue and Methylene Blue Staining (In-Lab and In-Home)
3 Methods
3.1 Pupal Counting (In-Lab and In-Home)
3.1.1 Prepare Fly Food Vials by Adding Test Foods/Beverages
3.1.2 Pupal Counting
3.2 Egg Analysis
3.2.1 Prepare Grape Juice Agar Plates
Microwave Preparation of Grape Juice Agar Plates, Active Time 20 min (In-Lab or In-Home)
Hotplate Version, Active Time 60 min
3.2.2 24-h Egg Lay and Visualization of Egg Structures
3.3 Ovary Dissection and Fixation
3.3.1 Dissection
3.3.2 Fixation (In-Lab)
3.3.3 Propidium Iodide and Phalloidin Staining (In-Lab)
3.3.4 Antibody Staining (In-Lab)
3.3.5 Methylene Blue and Trypan Blue Staining (In-Lab or In-Home)
4 Notes
References
Index

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