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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.
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.
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....
1. Collection of Samples
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.......
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 .......
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.
....Name | Company | Catalog Number | Comments |
10 kDa sulforhodamine 101 acid chloride (Texas Red) Dextran | ThermoFisher Scientific | D1863 | Dextran should be diluted in autoclaved ddH2O to a concentration of 25 mg/mL. |
20 μL Gel-Loading Pipette Tips | Eppendorf | 22351656 | |
100% Ethanol (200 proof) | Pharmco-Aaper | 11000200 | |
Active Dry Yeast | Red Star | ||
Agar | Fisher Scientific | BP1423 | |
Agarose | Fisher Scientific | BP160-500 | |
Air Compressor | DeWalt | D55140 | |
Apple Juice | Mott's Natural Apple Juice | ||
Bleach | Household Bleach | 1-5% Hypochlorite | |
Borosilicate Glass Capillaries | World Precision Instruments | 1B100F-4 | |
Bottle Plugs | Fisher Scientific | AS-277 | |
Cell Strainers | BD Falcon | 352350 | |
Confocal Microscope | Olympus | FV1000 | Samples imaged using 20x objective (UPlanSApo 20x/ 0.75) |
Cotton-Tipped Applicator | Puritan | 19-062614 | |
Double-Sided Tape 1/2" | Scotch | ||
Dumont Tweezers; Pattern #5; .05 X .01mm Tip | Roboz | RS-5015 | |
Fly Food Bottles | Fisher Scientific | AS-355 | |
Fly Food Vials | Fisher Scientific | AS-515 | |
Foot Pedal | Treadlite II | T-91-S | |
Gel Caster | Bio-Rad | 1704422 | |
Gel Tray | Bio-Rad | 1704436 | |
Glass Pipette | VWR | 14673-010 | |
Glycerol | Fisher Scientific | BP229-1 | |
Granulated sugar | Purchased from grocery store. | ||
Halocarbon Oil | Lab Scientific, Inc. | FLY-7000 | |
Light Source | Schott | Ace I | |
Manipulator Stand | World Precision Instruments | M10 | |
Micromanipulator | World Precision Instruments | KITE-R | |
Micropipette Puller | Sutter Instrument Co. | P-97 | |
Needle Holder | World Precision Instruments | MPH310 | |
Nightsea Filter Sets | Electron Microscopy Science | SFA-LFS-CY | For visualization of YFP |
Nightsea Full Adapter System w/ Royal Blue Color Light Head | Electron Microscopy Science | SFA-RB | For visualization of GFP |
Paintbrush | Simply Simmons | Chisel Blender #6 | |
Pipetter | Fisher Scientific | 13-683C | |
Pneumatic Pump | World Precision Instruments | PV830 | This 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 Chloride | Fisher Scientific | BP366-500 | |
Potassium Phosphate Dibasic | Fisher Scientific | BP363-500 | |
Small Embryo Collection Cages | Genesee Scientific | 59-100 | |
Sodium Chloride | Fisher Scientific | BP358-212 | |
Sodium Phosphate Dibasic Anhydrous | Fisher Scientific | BP332-500 | |
Steel Base Plate | World Precision Instruments | 5052 | |
Stereomicroscope | Carl Zeiss | Stemi 2000 | Used for tissue dissection. |
Stereomicroscope with transmitted light source | Baytronix | Used for injection. | |
Tegosept (p-hydroxybenzoic acid, methyl ester) | Genesee Scientific | 20-258 | |
Triton X-100 | Fisher Scientific | BP151-500 | Nonionic surfactant |
Vial Plugs | Fisher Scientific | AS-273 |
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