Name: methods to test endocrine disruption in drosophila melanogaster
Uploaded: 2019-07-03T14:00:00
Description: Watch this Scientific Journal Video about Methods to Test Endocrine Disruption in Drosophila melanogaster at JoVE.com

The authors thank Orsolina Petillo for technical support. The authors thank Dr. Mariarosaria Aletta (CNR) for bibliographic support. The authors thank Dr. Gustavo Damiano Mita for introducing them to the EDC world. The authors thank Leica Microsystems and Pasquale Romano for their assistance. This research was supported by Project PON03PE_00110_1. &#8220;Sviluppo di nanotecnologie Orientate alla Rigenerazione e Ricostruzione Tissutale, Implantologia e Sensoristica in Odontoiatria/oculistica&#8221; acronimo &#8220;SORRISO&#8221;; Committente: PO FESR 2014-2020 CAMPANIA; Project PO FESR Campania 2007-2013 &#8220;NANOTECNOLOGIE PER IL RILASCIO CONTROLLATO DI MOLECOLE BIO-ATTIVE NANOTECNOLOGIE&#8221;.

1. Food Preparation
<ol>
	<li>For stock maintenance and for larval growth, use a cornmeal medium containing 3 % powdered yeast, 10 % sucrose, 9 % precooked cornmeal, 0.4 % agar, thereafter called Cornmeal medium (CM).
	<ol>
		<li>Put 30 g of yeast into 100 mL of tap water, bring it to a boil and let it boil for 15 min.</li>
		<li>Separately, mix well 90 g of precooked cornmeal, 100 g of sugar, and 4 g of agar into 900 mL of tap water.</li>
		<li>Bring the solution to a boil, lower the heat and cook for...

The fruit fly D. melanogaster has been extensively employed as an in vivo model system to investigate the potential effects of environmental EDCs such as DBP28, BPA, 4-NP, 4-tert-OP29, MP30, EP31,32, DEHP33, and EE234. Several reasons have led its use as a model in this field of research. Apart from its undisputed advantages as a model ...

Human activities have been releasing into the environment a massive amount of chemicals, which represent a serious problem for organisms and for natural ecosystems1. Of these pollutants, it is estimated that about 1,000 different chemicals may alter the normal balance of endocrine systems; according to this property, they are classified as endocrine disrupting chemicals (EDCs). Specifically, based on a recent definition by the Endocrine Society, the EDCs are &#8220;an exogenous chemical, or mixture of chemicals, that can interfere with any aspect of hormone action&#8221;2. Over the last three decades, there has been growing scientific evidence that EDCs can affect the reproduction and development of animals and plants3,4,5,6,7,8. Further, EDC exposure has been related to the increasing prevalence of some human diseases, including cancer, obesity, diabetes, thyroid diseases, and behavioral disorders9,10,11.
General mechanisms of EDC
Due to their molecular properties, EDCs behave like hormones or hormone precursors3,4,5,6,7,8,9,10,11,12. In this sense, they can bind to a hormone&#8217;s receptor and disrupt endocrine systems either by mimicking hormone activity or by blocking endogenous hormones binding. In the first case, after binding to the receptor, they can activate it as its natural hormone would do. In the other case, binding of the EDC to the receptor prevents the binding of its natural hormone, so the receptor is blocked and can no longer be activated, even in the presence of its natural hormone3. As a consequence, EDCs can affect several processes, such as the synthesis, secretion, transport, metabolism, or peripheral action of endogenous hormones that are responsible for the maintenance of homeostasis, reproduction, development, and/or behavior of the organism. Receptor binding is not the only way of action described so far for the EDCs. It is now clear that they can also act by recruiting coactivators or corepressors in enzymatic pathways or by modifying epigenetic markers deregulating gene expression10,11,12,13,14, with consequences not only for the current generation but also for the health of generations to come8.
Drosophila hormones
The potential effects of selected EDCs have been studied widely, both in wildlife species and in several model systems in which endocrine mechanisms are reasonably well known. For invertebrates, endocrine systems that influence growth, development, and reproduction have been extensively characterized in insects for several reasons, involving their extensive use in the field of biological research, their economic importance, and finally the development of insecticides able to interfere specifically with the hormone system of pest insects.
In particular, among insects, the fruit fly D. melanogaster has proven to be a very powerful model system to evaluate the potential endocrine effects of EDCs. In D. melanogaster, as well as in vertebrates, hormones play an important role throughout the entire life cycle. In this organism, there are three main hormonal systems, which involve the steroid hormone 20-hydroxyecdysone (20E)15,16, the sesquiterpenoid juvenile hormone (JH)17, and the neuropeptides and peptide/protein hormones18. This third group consists of several peptides discovered more recently but clearly involved in a huge variety of physiological and behavioral processes, such as longevity, homeostasis, metabolism, reproduction, memory, and locomotor control. 20E is homologous to cholesterol-derived steroid hormones such as estradiol, while JH shares some similarities with retinoic acid; both of them are the better-known hormones in Drosophila19,20. Their balance is vital in coordinating molting and metamorphosis, as well as in controlling several postdevelopmental processes, such as reproduction, lifespan, and behavior21, thus offering different possibilities for testing endocrine disruption in Drosophila. Further, ecdysteroid hormones and JHs are the main targets of the so-called third-generation insecticides, developed to interfere with developmental and reproductive endocrine-mediated processes in insects. The agonist or antagonist mode of action of these chemicals is well known, and thus they can serve as reference standards for evaluating the effects of potential EDCs on the growth, reproduction, and development of insects22. For example, methoprene, which has been widely used in controlling mosquitoes and other aquatic insects23,24, works as a JH agonist and represses 20E-induced gene transcription and metamorphosis.
In addition to hormones, the nuclear receptor (NR) superfamily in Drosophila is also well known; it consists of 18 evolutionarily conserved transcription factors involved in controlling hormone-dependent developmental pathways, as well as reproduction and physiology25. These hormone NRs belong to all six NR superfamily subtypes, including those involved in neurotransmission26, two for retinoic acid NRs, and those for steroid NRs that, in vertebrates, represent one of the primary targets of EDCs27.
Drosophila as a model system for studying EDCs
Currently, on the basis of molecular properties, several environmental agencies around the world are attributing the potential to interfere with the endocrine systems to different man-made chemicals. Given that the EDCs are a global and ubiquitous problem for the environment and for organisms, the overall goal of the research in this field is to reduce their disease burden, as well as to protect living organisms from their adverse effects. In order to deepen the understanding about the potential endocrine effects of a chemical, it is necessary to test it in vivo. To this end, D. melanogaster represents a valid model system. To date, the fruit fly has been extensively used as in vivo model to evaluate the effects of several environmental EDCs; it has been reported that exposure to several EDCs, such as dibutyl phthalate (DBP)28, bisphenol A (BPA), 4-nonylphenol (4-NP), 4-tert-octylphenol (4-tert-OP)29, methylparaben (MP)30, ethylparaben (EP)31,32, bis-(2-ethylhexyl) phthalate (DEHP)33, and 17-&#945;-ethinylestradiol (EE2)34, influences metabolism and endocrine functions as in vertebrate models. Several reasons have led to its use as a model in this field of research. Beyond an excellent knowledge of its endocrine systems, further advantages include its short lifecycle, low cost, easily manipulable genome, a long history of research, and several technical possibilities (see the FlyBase website, http://flybase.org/). D. melanogaster also provides a powerful model for easily studying transgenerational effects and population responses to environmental factors8 and avoids ethical issues relevant for in vivo studies in higher animals. In addition, the fruit fly shares a high degree of gene conservation with humans which might make it possible for Drosophila EDC assays to help in predicting or suggesting potential effects of these chemicals for human health. Besides expanding the understanding about human health effects, Drosophila can help to assess risks of EDC exposure to the environment, such as biodiversity loss and environmental degradation. Finally, the fruit fly offers the additional advantage of being used in laboratories, where the factors potentially affecting its development, reproduction, and lifespan can be kept under control in order to attribute any variation to the substance to be tested.
With this in mind, we have optimized simple and robust fitness assays for determining EDC effects on some Drosophila hormonal traits, such as fecundity/fertility, developmental timing, and adult lifespan. These assays have been widely used for some EDCs23,24,25,26,27. In particular, we have used the following protocols to evaluate the effects of the exposure to the synthetic estrogen EE234 and to BPA and to bisphenol AF (BPA F) (unpublished data). These protocols may be easily modified to investigate the effects of a given EDC at a time, as well as the combined effects of multiple EDCs in D. melanogaster.

<table>
	<tbody>
		<tr>
			<td>17&#945;-Ethinylestradiol</td>
			<td>Sigma</td>
			<td>E4876-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar for Drosophila medium</td>
			<td>BIOSIGMA</td>
			<td>789148</td>
			<td></td>
		</tr>
		<tr>
			<td>Bisphenol A</td>
			<td>Sigma</td>
			<td>239658-50G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bisphenol AF</td>
			<td>Sigma</td>
			<td>90477-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Cornmeal</td>
			<td>CA&#39; BIANCA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila Vials</td>
			<td>BIOSIGMA</td>
			<td>789008</td>
			<td>25x95 mm</td>
		</tr>
		<tr>
			<td>Drosophila Vials</td>
			<td>BIOSIGMA</td>
			<td>789009</td>
			<td>29x95 mm</td>
		</tr>
		<tr>
			<td>Drosophila Vials</td>
			<td>Kaltek</td>
			<td>187</td>
			<td>22X63</td>
		</tr>
		<tr>
			<td>Embryo collection cage</td>
			<td>Crafts</td>
			<td></td>
			<td>Plexiglass cylinder (12,5 x7 cm) with an open end and the other end closed by a rectangular base in which a slot allows the insertion of special trays for laying</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>FLUKA</td>
			<td>2860</td>
			<td></td>
		</tr>
		<tr>
			<td>Etherizer</td>
			<td>Crafts</td>
			<td></td>
			<td>cylindrical glass container with a cotton plug</td>
		</tr>
		<tr>
			<td>Glass Bottle</td>
			<td></td>
			<td></td>
			<td>250mL Bottles</td>
		</tr>
		<tr>
			<td>Glass Vials</td>
			<td>Microtech</td>
			<td>ST 10024</td>
			<td>FLAT BOTTOM TUBE 100X24</td>
		</tr>
		<tr>
			<td>Hand blender Pimmy</td>
			<td>Ariete</td>
			<td></td>
			<td>food processor</td>
		</tr>
		<tr>
			<td>Instant Success yeast</td>
			<td>ESKA</td>
			<td></td>
			<td>Powdered yeast</td>
		</tr>
		<tr>
			<td>Laying tray</td>
			<td>Crafts</td>
			<td></td>
			<td>plexiglass trays (11 x 2,6 cm) in wich to pour medium for laying</td>
		</tr>
		<tr>
			<td>Methyl4-hydroxybenzoate</td>
			<td>SIGMA</td>
			<td>H5501</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Falcon</td>
			<td>351016</td>
			<td>60x5</td>
		</tr>
		<tr>
			<td>Red dye no. 40</td>
			<td>SIGMA</td>
			<td>16035</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope with LED lights</td>
			<td>Leica</td>
			<td></td>
			<td>S4E</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>HIMEDIA</td>
			<td>MB025</td>
			<td></td>
		</tr>
		<tr>
			<td>Tomato sauce</td>
			<td>Cirio</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In this section, key steps of the above protocols are reported in the form of simplified schemes. Given that flies tend to avoid unpalatable compounds, the first thing to do is to assay the taste of the selected EDC. This can be done by mixing a food coloring (for example, red food dye no. 40)35 with the food supplemented with the selected EDC at various doses or with the solvent alone. Flies fed on these media are examined under a stereomicroscope and the food intake is estimated by their abdomin...

Watch this Scientific Journal Video about Methods to Test Endocrine Disruption in Drosophila melanogaster at JoVE.com

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.

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 ...

Drosophila melanogaster is a powerful model to evaluate in vivo the potential toxic and endocrine effects of chemicals, such as endocrine disruptors, that represent a serious problem for human health and natural environments. Addressing the effect of EDCs on the hormonal life traits of the fly, such as fecundity, fertility, developmental time, and the lifespan. It's an easy, cheap, and fast way to investigate endocrine disruption in vivo.The fertility, development, and lifespan in Drosophila can be affected by several factor that have to be carefully controlled to ensure the successful outcome of this protocol. Therefore, maximum attention in rearing and handling flies is required. To assay the taste of the selected EDC, after anesthetization, transfer 15 young flies from the fly bed to four parallel vials containing cornmeal medium mixed with a food coloring.The control vial contains the solvent alone, and the three other vials are supplemented with different doses of the selected EDCs. Keep the vials in a humidified incubator for one day with a natural 12-hour light. After anesthetizing the flies again, transfer the immobilized flies to a white paper, and place it under a stereo microscope to observe.Compare the abdominal coloring of each treatment group with respect to the control group. To assay the reproductive performance of the flies, prepare three vials of flies for each EDC as parental vials, with eight females and four males in 10 milliliters of cornmeal medium supplemented with EE2. For the control, prepare six vials of flies, each with eight females and four males in 10 milliliters of cornmeal medium food supplemented with EE2 solvent.Rear flies in an incubator at 25 degrees Celsius. During their development, avoid overcrowding the larvae. After four days, invert each vial over an etherizer to remove the parents and return the vials to the incubator for five more days.In the late afternoon of day nine, invert each vial over an etherizer to remove all newly-emerged flies from the vials and put the vials in another incubator at 18 degrees Celsius overnight. On the morning of day 10, on a Drosophila carbon dioxide work station, carefully invert the culture vial to prevent anesthetized flies from falling onto medium and insert a rubber tube with a needle into the vial between the cotton stopper and the vial wall. Open the valve to deliver carbon dioxide.Within a matter of seconds, transfer the flies to a fly bed under a microscope. Collect virgin females and young males separately into two groups on the fly bed. Randomly subdivide each group of flies in small subgroups, with 10 females or 20 males per vial filled with fresh corresponding cornmeal medium.Continue incubation and collect 30 virgin females and 30 males for each EDC, and 90 virgin females and 90 males for the control. House these groups of flies at 25 degrees Celsius until they are aged four days post-eclosion. Then transfer them into new vials containing fresh corresponding medium every two days.Check there are no larvae in the vials of females. Then label vials with a different series of sequential numbers for the following treatment. After anesthetization, transfer one EE2 solvent-treated female into a small vial containing fresh cornmeal tomato medium without EE2 and add one EE2 solvent-treated male for the control across.Pair an EE2-treated male with EE2 solvent-treated female or EE2-treated female with EE2 solvent-treated male for each treatment. House 20 single crosses at 25 degrees Celsius. Transfer each mating pair to fresh cornmeal tomato medium vials without EDC every day for the subsequent 10 days.Visually inspect each vial every day for the eggs and report their number on the fertility spreadsheet. In each vial, check closely for newly-emerged flies and record the daily number of adult progenies over the 10-day period. After 10 days from the initial mating, remove the parents from the last series of vials.In eclosion assay protocols, first, for each treatment group, set up 10 vials of young, healthy flies less than two days old, each with six females and three males in 10 milliliters of cornmeal food without EDC. Rear the flies on food for 24 hours and allow them to mate. Then prepare another 10 parallel vials per treatment group with 10 milliliters each of fresh cornmeal food supplemented with different EDC concentrations or the EE2 solvent alone for the control.Transfer the mated flies to these new vials. Make a developmental spreadsheet to record the different series. Allow the flies to lay eggs for 16 hours.Then, remove the parents from the vials. Incubate the vials at 25 degrees Celsius for three to four days, or until wandering larvae are visible. Every day, count the number of new pupae in each vial by writing a number in sequence by each pupa on the outside of the vial to avoid counting the same pupa twice.Report the number of newly-emerged pupae for three to four days, or until no more pupae form. Starting from day nine, count the number of emerging adults daily, until no more adults emerge. Report it on the developmental spreadsheet and perform the rest of the eclosion assay according to the manuscript.In the lifespan protocol, first set up 20 vials with eight females and four males, each filled with 10 milliliters of cornmeal food and house them at 25 degrees Celsius. After four days, discard the flies and place the vials back into the incubator. In the late afternoon of day nine, remove all newly-emerged flies from the vials and return the vials to the incubator.After 16 to 24 hours, divide 250 one-day-old adult flies of both sexes into four groups under light carbon dioxide anesthetization and transfer them to 250-milliliter bottles containing adult medium food, three supplemented with different EDC concentrations and one with the EE2 solvent alone. Maintain the flies at 25 degrees Celsius for two to three days to allow them to mate. Then, sort each cohort of flies by sex into two groups.Within each treatment condition, for both sexes, randomly subdivide each group into five parallel vials at a density of 20 individuals per vial. Prepare a lifespan spreadsheet in which the number of dead flies is subtracted from the number of surviving flies from the previous transfer, so that the number of survivors at each transfer is automatically obtained. After three days, transfer the flies without anesthesia to new vials containing the corresponding food at the same time and check for death.Record the age of the flies and the number of dead flies. Repeat the transfer every three days until all of the flies die. In this experiment, lifespan curves were plotted as the cumulative survivorship versus the elapsed days for each EDC concentration and the control.For the control, after a long initial period in which the survivorship curve remained relatively high, it declined exponentially after about 60 days. Following EDC exposure, the survivorship curve of the treated flies was affected significantly and showed dramatic decline after 36 days. It's advised about that the parallel vials under analysis should be very similar, otherwise it will be difficult to understand which to discard.Therefore, we suggest taking great care, adopting a good experimental practice, using a large sample size. Following this protocols, it is also possible to evaluate the transgenerational impact of a selected endocrine disruptor as well as to test the potential additive effects of a combination of different endocrine disruptors.

methods-to-test-endocrine-disruption-in-drosophila-melanogaster

Research

JoVE Journal

Developmental Biology

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 ...

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 ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

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 ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

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.

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.

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

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 &lt;em&gt;Drosophila melanogaster&lt;/em&gt; 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 ...

﻿Staging and Collecting Drosophila melanogaster Third Instar Larvae

Staging and Collecting Drosophila melanogaster Third Instar Larvae  

Begin by crossing one to five-day-old female virgin flies with one to seven-day-old adult male flies to produce progeny with the desired genotype. To do so, place the flies in a fly cage with a meal cap. Use 10 to 15 female virgins with five to 10 males to get optimal yield.Incubate the fly cage containing flies at 25 degrees Celsius. Change the meal cap daily to prevent overcrowding of larvae which reduces the quality of the dissected brains. If the meal cap has more than 30 larvae, split it into half and replace one portion with a fresh meal cap cut in half.Incubate the meal caps with larvae at 25 degrees Celsius until the larvae reached the desired age.