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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Blood-brain barrier integrity is critical for nervous system function. In Drosophila melanogaster, the blood-brain barrier is formed by glial cells during late embryogenesis. This protocol describes methods to assay for blood-brain barrier formation and maintenance in D. melanogaster embryos and third instar larvae.

Abstract

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous system and protects neural tissue from toxins and pathogens. Defects in the formation of this barrier have been correlated with neuropathies, and the breakdown of this barrier has been observed in many neurodegenerative diseases. Therefore, it is critical to identify the genes that regulate the formation and maintenance of the blood-brain barrier to identify potential therapeutic targets. In order to understand the exact roles these genes play in neural development, it is necessary to assay the effects of altered gene expression on the integrity of the blood-brain barrier. Many of the molecules that function in the establishment of the blood-brain barrier have been found to be conserved across eukaryotic species, including the fruit fly, Drosophila melanogaster. Fruit flies have proven to be an excellent model system for examining the molecular mechanisms regulating nervous system development and function. This protocol describes a step-by-step procedure to assay for blood-brain barrier integrity during the embryonic and larval stages of D. melanogaster development.

Introduction

During development, cell-cell communication and interactions are critical for the establishment of tissue and organ structure and function. In some cases, these cell-cell interactions seal off organs from the surrounding environment to ensure proper organ function. This is the case for the nervous system, which is insulated by the blood-brain barrier (BBB). Dysfunction of the BBB in humans has been linked to neurological disorders including epilepsy, and breakdown of the barrier has been observed in neurodegenerative diseases including multiple sclerosis and amyotrophic lateral sclerosis1. In mammals, the BBB is formed by tight junctions betwee....

Protocol

1. Collection of Samples

  1. Embryo collection
    1. In each embryo collection cage, use a minimum of 50 virgin females with 20−25 males for collections. Incubate these flies in a bottle with cornmeal-agar food (Table of Materials) for 1−2 days before beginning collections23.
      NOTE: More flies can be used, but the female-to-male ratio should be kept at 2:1.
    2. Pre-warm apple juice agar plates (Table.......

Representative Results

The methods described here allow for the visualization of the integrity of the BBB throughout the CNS in D. melanogaster embryos and larvae (Figure 1). Upon completion of BBB formation in late embryogenesis, the BBB functions to exclude large molecules from the brain and VNC5. This protocol takes advantage of this function to assay BBB formation. When wild-type (Oregon R) late stage 17 (20−21 h old) embryos were injected with 10 kDa dextran conjugated t.......

Discussion

This protocol provides a comprehensive description of the steps needed to assay for BBB integrity during the late embryonic and third instar larval stages of D. melanogaster development. Similar approaches have been described elsewhere to assay the integrity of the BBB during development, as well as in adult stages5,7,29,30. However, descriptions of procedures in materials and methods .......

Acknowledgements

The authors thank Dr. F. Bryan Pickett and Dr. Rodney Dale for use of equipment for injection. This work was funded by research funding from Loyola University Chicago to M.D., D.T., and J.J.

....

Materials

NameCompanyCatalog NumberComments
10 kDa sulforhodamine 101 acid chloride (Texas Red) DextranThermoFisher ScientificD1863Dextran should be diluted in autoclaved ddH2O to a concentration of 25 mg/mL.
20 μL Gel-Loading Pipette TipsEppendorf22351656
100% Ethanol (200 proof)Pharmco-Aaper11000200
Active Dry YeastRed Star
AgarFisher ScientificBP1423
AgaroseFisher ScientificBP160-500
Air CompressorDeWaltD55140
Apple JuiceMott's Natural Apple Juice
BleachHousehold Bleach1-5% Hypochlorite
Borosilicate Glass CapillariesWorld Precision Instruments1B100F-4
Bottle PlugsFisher ScientificAS-277
Cell StrainersBD Falcon352350
Confocal MicroscopeOlympusFV1000Samples imaged using 20x objective (UPlanSApo 20x/ 0.75)
Cotton-Tipped ApplicatorPuritan19-062614
Double-Sided Tape 1/2"Scotch
Dumont Tweezers; Pattern #5; .05 X .01mm TipRobozRS-5015
Fly Food BottlesFisher ScientificAS-355
Fly Food VialsFisher ScientificAS-515
Foot PedalTreadlite IIT-91-S
Gel CasterBio-Rad1704422
Gel TrayBio-Rad1704436
Glass PipetteVWR14673-010
GlycerolFisher ScientificBP229-1
Granulated sugarPurchased from grocery store.
Halocarbon OilLab Scientific, Inc.FLY-7000
Light SourceSchottAce I
Manipulator StandWorld Precision InstrumentsM10
MicromanipulatorWorld Precision InstrumentsKITE-R
Micropipette PullerSutter Instrument Co.P-97
Needle HolderWorld Precision InstrumentsMPH310
Nightsea Filter SetsElectron Microscopy ScienceSFA-LFS-CYFor visualization of YFP
Nightsea Full Adapter System w/ Royal Blue Color Light HeadElectron Microscopy ScienceSFA-RBFor visualization of GFP
PaintbrushSimply SimmonsChisel Blender #6
PipetterFisher Scientific13-683C
Pneumatic PumpWorld Precision InstrumentsPV830This is also referred to as a microinjector or pressure regulator. Since the model used in our study is no longer available this is one alternative.
Potassium ChlorideFisher ScientificBP366-500
Potassium Phosphate DibasicFisher ScientificBP363-500
Small Embryo Collection CagesGenesee Scientific59-100
Sodium ChlorideFisher ScientificBP358-212
Sodium Phosphate Dibasic AnhydrousFisher ScientificBP332-500
Steel Base PlateWorld Precision Instruments5052
StereomicroscopeCarl ZeissStemi 2000Used for tissue dissection.
Stereomicroscope with transmitted light sourceBaytronixUsed for injection.
Tegosept (p-hydroxybenzoic acid, methyl ester)Genesee Scientific20-258
Triton X-100Fisher ScientificBP151-500Nonionic surfactant
Vial PlugsFisher ScientificAS-273

References

  1. Obermeier, B., Daneman, R., Ransohoff, R. M. Development, maintenance and disruption of the blood-brain barrier. Nature Medicine. 19 (12), 1584-1596 (2013).
  2. Brightman, M. W., Reese, T. S. Junctions between inti....

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