Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

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 between endothelial cells^2,3. Other animals, including the fruit fly, Drosophila melanogaster, have a BBB composed of glial cells. These glial cells form a selectively permeable barrier to control movement of nutrients, waste products, toxins, and large molecules into and out of the nervous system⁴. This allows for the maintenance of the electrochemical gradient necessary to fire action potentials, allowing for mobility and coordination⁴. In D. melanogaster, the glia protect the nervous system from the potassium-rich, blood-like hemolymph⁵.

In the central nervous system (CNS) and peripheral nervous system (PNS) of D. melanogaster, two outer glial layers, the subperineurial glia and the perineurial glia, as well as an outer network of extracellular matrix, the neural lamella, form the hemolymph-brain and hemolymph-nerve barrier⁶, referred to as the BBB throughout this article. During development subperineurial glia become polyploid and enlarge to surround the nervous system^{5,6,7,8,9,10,11}. The subperineurial glia form septate junctions, which provide the main diffusion barrier between the hemolymph and the nervous system^5,6,12. These junctions are molecularly similar to the septate-like junctions found at the paranodes of myelinating glia in vertebrates, and they perform the same function as tight junctions in the BBB of mammals^{13,14,15,16,17}. The perineurial glia divide, grow, and wrap around the subperineurial glia to regulate the diffusion of metabolites and large molecules^6,10,18,19. BBB formation is complete by 18.5 h after egg laying (AEL) at 25 °C^5,8. Previous studies have identified genes that are critical regulators of BBB formation^20,21,22. To better understand the exact roles of these genes, it is important to examine the effect of mutation of these potential regulators on BBB integrity. While previous studies have outlined approaches for assaying BBB integrity in embryos and larvae, a comprehensive protocol for this assay has yet to be described^5,7. This step-by-step protocol describes methods for assaying BBB integrity during D. melanogaster embryonic and third instar larval stages.

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 1) at 25 °C overnight.
      NOTE: This is required for proper staging of embryos. If plates are drying out quickly, add a bowl with water to the incubator to increase the humidity of the chamber.
   3. Anesthetize flies from step 1.1.1 with CO₂ and transfer flies to a collection cage. Place a pre-warmed apple juice agar plate with a small smear of yeast paste on the open end and secure to the cage with the red sleeve (Table of Materials). In order to clear older embryos, allow flies to lay embryos/eggs on an apple juice agar plate for 1 h at 25 °C.
   4. Remove the collection cage from the incubator. Invert the cage mesh side down and tap flies down to the bottom of the cage. Replace the apple juice agar with a new pre-warmed apple juice agar plate with a small smear of yeast paste. Discard the first plate.
   5. Allow flies to lay embryos/eggs on the new apple juice agar plate for 1 h at 25 °C. Discard this plate following the 1 h collection and proceed to the next step to collect embryos for injection.
   6. To collect late stage 17 embryos (20−21 h AEL), allow flies of desired genotype from steps 1.1.1−1.1.5 to lay on a new pre-warmed apple juice agar plates with a small smear of yeast paste at 25 °C for 1 h. Age plate for 19 h in a 25 °C incubator, so embryos will be 20−21 h of age at the time of imaging.
      NOTE: This step can be repeated as desired for multiple rounds of sample collection, injection, and imaging.
   7. Collect embryos from plates into a cell strainer with 70 μm nylon mesh by adding phosphate-buffered saline (PBS) with 0.1% nonionic surfactant (PBTx; Table 1) to cover the surface of the plate and loosen embryos from the surface using a paintbrush.
   8. Dechorionate embryos collected in the cell strainer in a 50% bleach solution (Table 1) in a 100 mm Petri dish for 5 min with occasional agitation at room temperature. Rinse embryos 3x by swirling the cell strainer in PBTx in a Petri dish, using a fresh dish of PBTx each time.
   9. If all embryos are of the correct genotype, proceed directly to step 1.1.10. If generation of embryos of the correct genotype requires a cross with heterozygous flies, select embryos of the correct genotype using the presence or absence of fluorescently marked balancer chromosomes. Use a stereomicroscope with fluorescent capabilities for genotype selection.
      NOTE: Balancer chromosomes marked with Deformed-yellow fluorescent protein; Kruppel-Gal4, UAS-green fluorescent protein (GFP); and twist-Gal4, UAS-GFP work well for genotype selection in late embryogenesis (Table 2)^24,25,26.
   10. Using a glass pipette, transfer embryos in PBTx to a 2% agarose gel slab (Table 1). Remove excess liquid with filter paper. Align ~6−8 embryos on the 2% agarose gel slab with posterior to the right and dorsal side facing up (Figure 1B).
       NOTE: The micropyle, the small hole through which spermatozoa enter the egg, is located at the anterior end of the embryo. The posterior end is more rounded. The trachea appears white and is located dorsally in the embryo, allowing for the distinction of the dorsal and ventral sides of the embryo (Figure 1B).
   11. Prepare a slide with one piece of double-sided tape. Firmly press the slide on top of the embryos to transfer them to the double-sided tape.
   12. Desiccate embryos by incubation at room temperature for ~25 min (no desiccant is used). Following desiccation, cover embryos with halocarbon oil.
       NOTE: Desiccation periods may vary depending on the temperature, humidity, and ventilation in the room. The incubation period should be used to set up the apparatus for injection and the confocal microscope for imaging, as described in sections 2 and 3 of this protocol.
2. Larval collection
   1. Set up a cross with 5−10 virgin female flies of the desired genotype and half as many males of the desired genotype in a vial with cornmeal-agar food and incubate at 25 °C²³.
   2. After 5−7 days, depending on the genotype, collect wandering third instar larvae from the vial gently with forceps. Rinse larvae in 1x PBS to remove food stuck to the larvae. Transfer larvae to an apple juice agar plate for genotyping as described in step 1.1.9 if necessary.
   3. Roll larvae on a tissue using a paintbrush to dry them off. Transfer 6−8 larvae to a slide prepared with double-sided tape using forceps.

2. Preparation of Needles and Specimen Injection

1. Pull needles on a micropipette puller prior to the initiation of this protocol. Secure capillary tubes into the needle puller and pull according to the standard needle shape and parameters for D. melanogaster injections (Table 3)²⁷. Store needles in a Petri dish by anchoring in clay until use for injection.
2. Load a needle with 5 μL of 10 kDa dextran conjugated to sulforhodamine 101 acid chloride (Table of Materials) using a 20 μL gel-loading pipette tip during the 25-min desiccation period for embryos (step 1.1.12), or immediately following the transfer of larvae to the slide (step 1.2.3).
3. Load the needle into a needle holder and position in a micromanipulator secured to a steel base (Table of Materials).
   NOTE: The needle should be nearly parallel to the microscope stage for embryo injection and angled slightly downward for larval injections.
4. Set the injection apparatus (Table of Materials) to 50 psi, 5−10 ms with a range of 10.
   NOTE: It may be necessary to alter these settings for the particular injection apparatus being used.
5. Place the slide on stage and brush edge of the needle against the edge of the double-sided tape at a 45° angle to create an angled, broken tip.
   NOTE: For embryos it is only necessary to break the tip enough to allow for flow of the 10 kDa dextran. A perfect needle has a slightly angled tip and only a small drop of dye will come out with each injection. For larvae, it is necessary to break the tip more, but with an angled tip to penetrate the larval body wall. A larger drop of dye will come out.
6. Pump foot pedal until the dye is at the tip of the needle.
7. Align the needle so that it is parallel with the embryo or angled slightly downward toward the larva.
8. Move the needle to puncture the posterior end of the specimen and inject the specimen by pumping the foot pedal. Inject ~2 nL of dye into embryos, and ~220 nL of dye into larvae.
   NOTE: The embryo or larva should flood with dye if the injection is successful.
9. Note the time of injection for incubation purposes. Incubate embryos for 10 min at room temperature. Incubate larvae for 30 min at room temperature.
10. Continue down the slide to inject additional specimens, noting the time of injection for each specimen.
    NOTE: Depending on the speed with which subsequent dissection and imaging steps can be performed, 4−8 specimens can be injected at a time.

3. Preparation of Samples for Imaging

1. Imaging of embryos
   1. Following injection, prepare embryos for imaging. Apply petroleum jelly with a cotton-tipped applicator on the right and left sides of the samples on the slide as a spacer to prevent damage to the embryos upon placement of the coverslip.
   2. Image samples using a confocal microscope throughout the depth of the embryo with a 20x objective. Calculate the percentage of total samples with dye observed in the ventral nerve cord (VNC) using the following equation: % of samples with compromised BBB = Number of samples with dye accumulation in the VNC/total number of samples assayed.
2. Dissection and imaging of larvae
   1. Prepare slides for larval samples ahead of time. Mount two coverslips spaced approximately 0.5 cm apart to the slide with nail polish.
      NOTE: The coverslips function as spacers for the brain, so it is not damaged during the mounting process.
   2. Following the 30 min incubation, dissect the larvae in 1x PBS directly on the slide that will be used in imaging. First, use one pair of forceps to grab the larva halfway down the larval body, and use a second pair of forceps to separate the anterior and posterior halves of the larva.
   3. Next, use one pair of forceps to grip the anterior region at the mouth hooks, and use a second pair of forceps to invert the body wall over the tip of the forceps gripping the mouth hooks. The brain and VNC will be exposed.
   4. Separate the brain and VNC from the body wall by severing the nerves, and remove the body wall from the slide (Figure 1C,D). Remove imaginal discs if desired.
   5. Cover the sample with 10 μL of 80% glycerol and place a coverslip on top of the sample for imaging.
   6. Image through the depth of the nervous system tissue using a 20x objective. Calculate the percentage of total samples with dye observed in the VNC.

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 to sulf...

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 stages^5,7,29,30. However, descriptions of procedures in materials and methods ...

Materials

List of materials used in this article

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 .01 mm 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