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.

1. Collection of Samples
<ol>
	<li>Embryo collection
	<ol>
		<li>In each embryo collection cage, use a minimum of 50 virgin females with 20&#8722;25 males for collections. Incubate these flies in a bottle with cornmeal-agar food (Table of Materials) for 1&#8722;2 days before beginning collections23. 
		NOTE: More flies can be used, but the female-to-male ratio should be kept at 2:1.</li>
		<li>Pre-warm apple juice agar plates (Table 1) at 25 &#176;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.</li>
		<li>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 &#176;C.</li>
		<li>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.</li>
		<li>Allow flies to lay embryos/eggs on the new apple juice agar plate for 1 h at 25 &#176;C. Discard this plate following the 1 h collection and proceed to the next step to collect embryos for injection.</li>
		<li>To collect late stage 17 embryos (20&#8722;21 h AEL), allow flies of desired genotype from steps 1.1.1&#8722;1.1.5 to lay on a new pre-warmed apple juice agar plates with a small smear of yeast paste at 25 &#176;C for 1 h. Age plate for 19 h in a 25 &#176;C incubator, so embryos will be 20&#8722;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.</li>
		<li>Collect embryos from plates into a cell strainer with 70 &#956;m 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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Using a glass pipette, transfer embryos in PBTx to a 2% agarose gel slab (Table 1). Remove excess liquid with filter paper. Align ~6&#8722;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).</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Larval collection
	<ol>
		<li>Set up a cross with 5&#8722;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 &#176;C23.</li>
		<li>After 5&#8722;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.</li>
		<li>Roll larvae on a tissue using a paintbrush to dry them off. Transfer 6&#8722;8 larvae to a slide prepared with double-sided tape using forceps.</li>
	</ol>
	</li>
</ol>
2. Preparation of Needles and Specimen Injection
<ol>
	<li>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.</li>
	<li>Load a needle with 5 &#956;L of 10 kDa dextran conjugated to sulforhodamine 101 acid chloride (Table of Materials) using a 20 &#956;L 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).</li>
	<li>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.</li>
	<li>Set the injection apparatus (Table of Materials) to 50 psi, 5&#8722;10 ms with a range of 10. 
	NOTE: It may be necessary to alter these settings for the particular injection apparatus being used.</li>
	<li>Place the slide on stage and brush edge of the needle against the edge of the double-sided tape at a 45&#176; 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.</li>
	<li>Pump foot pedal until the dye is at the tip of the needle.</li>
	<li>Align the needle so that it is parallel with the embryo or angled slightly downward toward the larva.</li>
	<li>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.</li>
	<li>Note the time of injection for incubation purposes. Incubate embryos for 10 min at room temperature. Incubate larvae for 30 min at room temperature.</li>
	<li>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&#8722;8 specimens can be injected at a time.</li>
</ol>
3. Preparation of Samples for Imaging
<ol>
	<li>Imaging of embryos
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Dissection and imaging of larvae
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Cover the sample with 10 &#956;L of 80% glycerol and place a coverslip on top of the sample for imaging.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 ...

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 cells2,3. 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 system5,6,7,8,9,10,11. The subperineurial glia form septate junctions, which provide the main diffusion barrier between the hemolymph and the nervous system5,6,12. 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 mammals13,14,15,16,17. The perineurial glia divide, grow, and wrap around the subperineurial glia to regulate the diffusion of metabolites and large molecules6,10,18,19. BBB formation is complete by 18.5 h after egg laying (AEL) at 25 &#176;C5,8. Previous studies have identified genes that are critical regulators of BBB formation20,21,22. 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 described5,7. This step-by-step protocol describes methods for assaying BBB integrity during D. melanogaster embryonic and third instar larval stages.

<table>
	<tbody>
		<tr>
			<td>10 kDa sulforhodamine 101 acid chloride (Texas Red) Dextran</td>
			<td>ThermoFisher Scientific</td>
			<td>D1863</td>
			<td>Dextran should be diluted in autoclaved ddH2O to a concentration of 25 mg/mL.</td>
		</tr>
		<tr>
			<td>20 &#956;L Gel-Loading Pipette Tips</td>
			<td>Eppendorf</td>
			<td>22351656</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol (200 proof)</td>
			<td>Pharmco-Aaper</td>
			<td>11000200</td>
			<td></td>
		</tr>
		<tr>
			<td>Active Dry Yeast</td>
			<td>Red Star</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>BP1423</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>BP160-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Air Compressor</td>
			<td>DeWalt</td>
			<td>D55140</td>
			<td></td>
		</tr>
		<tr>
			<td>Apple Juice</td>
			<td>Mott&#39;s Natural Apple Juice</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Household Bleach</td>
			<td></td>
			<td>1-5% Hypochlorite</td>
		</tr>
		<tr>
			<td>Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle Plugs</td>
			<td>Fisher Scientific</td>
			<td>AS-277</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainers</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td>Samples imaged using 20x objective (UPlanSApo 20x/ 0.75)</td>
		</tr>
		<tr>
			<td>Cotton-Tipped Applicator</td>
			<td>Puritan</td>
			<td>19-062614</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-Sided Tape 1/2&#34;</td>
			<td>Scotch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Tweezers; Pattern #5; .05 X .01mm Tip</td>
			<td>Roboz</td>
			<td>RS-5015</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly Food Bottles</td>
			<td>Fisher Scientific</td>
			<td>AS-355</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly Food Vials</td>
			<td>Fisher Scientific</td>
			<td>AS-515</td>
			<td></td>
		</tr>
		<tr>
			<td>Foot Pedal</td>
			<td>Treadlite II</td>
			<td>T-91-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Caster</td>
			<td>Bio-Rad</td>
			<td>1704422</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Tray</td>
			<td>Bio-Rad</td>
			<td>1704436</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pipette</td>
			<td>VWR</td>
			<td>14673-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Granulated sugar</td>
			<td></td>
			<td></td>
			<td>Purchased from grocery store.</td>
		</tr>
		<tr>
			<td>Halocarbon Oil</td>
			<td>Lab Scientific, Inc.</td>
			<td>FLY-7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Light Source</td>
			<td>Schott</td>
			<td>Ace I</td>
			<td></td>
		</tr>
		<tr>
			<td>Manipulator Stand</td>
			<td>World Precision Instruments</td>
			<td>M10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instruments</td>
			<td>KITE-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle Holder</td>
			<td>World Precision Instruments</td>
			<td>MPH310</td>
			<td></td>
		</tr>
		<tr>
			<td>Nightsea Filter Sets</td>
			<td>Electron Microscopy Science</td>
			<td>SFA-LFS-CY</td>
			<td>For visualization of YFP</td>
		</tr>
		<tr>
			<td>Nightsea Full Adapter System w/ Royal Blue Color Light Head</td>
			<td>Electron Microscopy Science</td>
			<td>SFA-RB</td>
			<td>For visualization of GFP</td>
		</tr>
		<tr>
			<td>Paintbrush</td>
			<td>Simply Simmons</td>
			<td>Chisel Blender #6</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetter</td>
			<td>Fisher Scientific</td>
			<td>13-683C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatic Pump</td>
			<td>World Precision Instruments</td>
			<td>PV830</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Fisher Scientific</td>
			<td>BP363-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Embryo Collection Cages</td>
			<td>Genesee Scientific</td>
			<td>59-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic Anhydrous</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel Base Plate</td>
			<td>World Precision Instruments</td>
			<td>5052</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi 2000</td>
			<td>Used for tissue dissection.</td>
		</tr>
		<tr>
			<td>Stereomicroscope with transmitted light source</td>
			<td>Baytronix</td>
			<td></td>
			<td>Used for injection.</td>
		</tr>
		<tr>
			<td>Tegosept (p-hydroxybenzoic acid, methyl ester)</td>
			<td>Genesee Scientific</td>
			<td>20-258</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-500</td>
			<td>Nonionic surfactant</td>
		</tr>
		<tr>
			<td>Vial Plugs</td>
			<td>Fisher Scientific</td>
			<td>AS-273</td>
			<td></td>
		</tr>
	</tbody>
</table>

assay for blood-brain barrier integrity in drosophila melanogaster

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.

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&#8722;21 h old) embryos were injected with 10 kDa dextran conjugated t...

Watch this Scientific Journal Video about Assay for Blood-brain Barrier Integrity in Drosophila melanogaster at JoVE.com

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

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.

Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Proper nervous system development includes the formation of the blood-brain barrier, the diffusion barrier that tightly regulates access to the nervous ...

assay-for-blood-brain-barrier-integrity-in-drosophila-melanogaster

Research

JoVE Journal

Developmental Biology

8.1K Views.  Loyola University Chicago. 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.

Video: Assay for Blood-brain Barrier Integrity in Drosophila melanogaster

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is a model for understanding microbial interactions with animal hosts, facilitated by approaches to rear large sample sizes of Drosophila under microorganism-free (axenic) conditions, or with defined microbial communities (gnotobiotic). This work outlines a method for collection of Drosophila embryos, hypochlorite dechorionation and sterilization, and transfer to sterile diet. Sterilized embryos are transferred to sterile diet in 50 ml centrifuge tubes, and developing larvae and adults remain free of any exogenous microbes until the vials are opened. Alternatively, flies with a defined microbiota can be reared by inoculating sterile diet and embryos with microbial species of interest. We describe the introduction of 4 bacterial species to establish a representative gnotobiotic microbiota in Drosophila. Finally, we describe approaches for confirming bacterial community composition, including testing if axenic Drosophila remain bacteria-free into adulthood.

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

A method for rearing Drosophila melanogaster under axenic and gnotobiotic conditions is presented. Fly embryos are dechorionated in sodium hypochlorite, transferred aseptically to sterile diet, and reared in closed containers. Inoculating diet and embryos with bacteria leads to gnotobiotic associations, and bacterial presence is confirmed by plating whole-body Drosophila homogenates.

The influence of microbes on myriad animal traits and behaviors has been increasingly recognized in recent years. The fruit fly Drosophila melanogaster is ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-&#945;-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Methods to Test Endocrine Disruption in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

Imaging Live Brain Explants from Drosophila melanogaster Third Instar Larvae

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

Author Spotlight: Investigating Asymmetric Cell Division Dynamics: A Protocol for Live-Imaging of Drosophila Larval Brain Explants 

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...