We thank Reiko Kise and Tomotsune Ameku for their technical support for this work. We are also grateful to Kei Ito, Olga Alekseyenko, Akiko Koto, Masayuki Miura, the Bloomington Drosophila Stock Center, KYOTO Stock Center (DGRC), and the Developmental Studies Hybridoma Bank for stocks and reagents. This work was supported by grants to Y.S.N. from JSPS KAKENHI Grant Number 16K20945, The Naito Foundation, and Inoue Science Research Award; and by a grant to R.N. from MEXT KAKENHI Grant Number 16H04792.

NOTE: The overall scheme of protocols is shown in Figure 1.
1. The Dissection of the Larval Ring Gland (RG)
NOTE: In D. melanogaster, which belongs to cyclorrhaphous Diptera, the PG is within a composite endocrine organ called the ring gland (RG, Figure 2D).&#160;Since it is unfeasible that the PG is surgically separated from other types of cells (discussed later), a practical target is to i...

<ol>
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We studied ecdysteroid biosynthesis and its regulatory mechanism in D. melanogaster, and devised a protocol for dissection and immunostaining. The timing of ecdysteroid biosynthesis is affected by environmental cues through neuronal inputs33, so it is essential to maintain the innervation of the ecdysteroidogenic organs along with the brain, VNC, and other tissues during dissection.
As described above, the&#160;D. melanogaster PG forms a complex endocr...

In multicellular organisms, a group of cells is endowed with a specialized function in their biogenic activity that is essential for the whole body. To fulfill their missions, each tissue or organ expresses a series of genes related to their functions and communicates with other tissues to orchestrate their activities in the context of development. To characterize such specialized cellular functions and inter-organ interactions, we need to specify a group of cells along with other types of cells being kept intact in the multicellular architecture.
One example of such specialized organs is a steroidogenic organ, where many biosynthetic enzymes mediate the conversion steps from cholesterol to active steroid hormones1. Most of these enzyme genes are specifically expressed in steroidogenic organs, and the biosynthesis pathway is tightly regulated by many external stimuli via humoral inputs and neuronal inputs. Once synthesized, steroid hormones are secreted into the hemolymph and are targeted to many tissues and organs for regulating the expression of a variety of genes2. Therefore, the action of a steroid hormone induces a systemic response to maintain homeostasis, growth, and reproduction.
To investigate the functions of steroid hormone biosynthesis and the pleiotropic actions of steroid hormones, Drosophila melanogaster can be utilized as a suitable model system. During the larval stages, the insect steroid hormone, ecdysteroid, is biosynthesized in a specialized endocrine organ called the prothoracic gland (PG)3. In the PG, several ecdysteroidogenic enzymes specifically catalyze the multiple conversion steps from cholesterol to ecdysone, which controls molting and metamorphosis at the appropriate developmental stages4. Therefore, a dynamic change in&#160;ecdysteroid titer is regulated by many signaling pathways in response to environmental cues. On the other hand, in the adult stage, ecdysteroid plays essential roles in physiology, including reproduction, sleep, memory, and lifespan5,6,7,8. It is known that ecdysteroid is actively biosynthesized in the ovary, regulating the progression of oogenesis6,7,8,9,10,11. Recently we have reported that the number of germline stem cells (GSCs) is affected by ecdysteroid and sex peptide signaling in response to mating stimuli12.
Powerful tools of D. melanogaster genetics and cell biology, including well-annotated genome information, binary gene expression systems, and transgenic RNAi techniques, have enabled us to identify genes essential to ecdysteroid biosynthesis in the PG and the ovary13,14,15. Once the ecdysteroidogenic genes are identified, the transcriptional regulation of these genes and the dynamic localizations of gene products can be examined in the biosynthesis pathway16. For this purpose, quantitative-reverse transcription-PCR, RNA in situ hybridization, and immunohistological analysis are conducted. The application of these techniques includes a challenging task; the&#160;elaborate dissection of the PG or the ovary. In particular, the PG of the fruit fly is relatively smaller than that of other insects (e.g. the silkworm and the blow fly), so one needs to practice the vital skill of fruit fly dissection for sampling. Furthermore, both ecdysteroidogenic organs receive innervations from the central nervous system (CNS)17,18,19,20. Thus, for accurate anatomical analyses, the ecdysteroidogenic organs should be kept intact along with the CNS and other organs, not to disrupt their neuronal connections.
Here we provide protocols for the dissection and visualization of steroidogenic organs in D. melanogaster. Learning the dissection technique is the key starting point for these&#160;experiments. In addition, one can successfully label the steroidogenic organs as well as their interactive organs with several antibodies and GAL4 driver lines. Taking advantage of these techniques, materials, and genetics, one can study the comprehensive mechanisms of steroid hormone biosynthesis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>egg collection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tissue culture dish (55 mm)</td>
			<td>AS ONE</td>
			<td>1-8549-02</td>
			<td>&#160;for grape-juice agar plates</td>
		</tr>
		<tr>
			<td>collection cup</td>
			<td>HIKARI KAGAKU</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>yeast paste</td>
			<td>Oriental dry yeast, Tokyo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100% grape juice</td>
			<td>Welch Food Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>rearing larvae</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>small vials (12ml, 40&#215;23.5 mm, PS)</td>
			<td>SARSTEDT</td>
			<td>58.487</td>
			<td></td>
		</tr>
		<tr>
			<td>disposable loop</td>
			<td>AS ONE</td>
			<td>6-488-01</td>
			<td></td>
		</tr>
		<tr>
			<td>standard fly food</td>
			<td></td>
			<td></td>
			<td>&#160;the recepi us on the website of Blooington stock center.</td>
		</tr>
		<tr>
			<td>dissection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dissecting microscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi 2000-C</td>
			<td></td>
		</tr>
		<tr>
			<td>dissecting microscope</td>
			<td>Leica</td>
			<td>S8 AP0</td>
			<td></td>
		</tr>
		<tr>
			<td>tissue culture dish (35 x 10 mm, non-treated)</td>
			<td>IWAKI</td>
			<td>1000-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard</td>
			<td>TORAY</td>
			<td></td>
			<td>coarting silicon inside dishes</td>
		</tr>
		<tr>
			<td>Terumo needle (27G, 0.40 x 19 mm)&#160;</td>
			<td>TERUMO</td>
			<td>NN-2719S</td>
			<td>A &#34;knife&#34; to cut the tissue</td>
		</tr>
		<tr>
			<td>Terumo syringe,&#12288;1ml</td>
			<td>TERUMO</td>
			<td>SS-01T</td>
			<td></td>
		</tr>
		<tr>
			<td>forceps, Inox, #5</td>
			<td>Dumont, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>insect pin (0.18 mm in diameter)</td>
			<td>Shiga Brand</td>
			<td></td>
			<td>for fillet dissection</td>
		</tr>
		<tr>
			<td>micro scissors</td>
			<td>NATSUME SEISAKUSHO CO LTD.&#160;</td>
			<td>MB-50-10</td>
			<td></td>
		</tr>
		<tr>
			<td>fixation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ultrapure water</td>
			<td>Merck Millipore</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Nacalai tesque</td>
			<td>16222-65</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Nacalai tesque</td>
			<td>02890-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Nacalai tesque</td>
			<td>35501-15</td>
			<td></td>
		</tr>
		<tr>
			<td>microtubes (1.5 ml)</td>
			<td>INA OPTIKA</td>
			<td>CF-0150</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>As one swist mixer TM-300 (rocker)</td>
			<td>As one</td>
			<td>TM-300</td>
			<td>rocker</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>SIGMA</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>primary antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Sro (guinea pig), 1:1000</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>anti-GFP (rabbit), 1:1000</td>
			<td>Molecular Probes</td>
			<td>A6455</td>
			<td>Shimada-Niwa ans Niwa, 2014</td>
		</tr>
		<tr>
			<td>anti-GFP (mouse mAb, GF200), 1:100</td>
			<td>Nakarai tesque</td>
			<td>04363-66</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-5HT (rabbit), 1:500</td>
			<td>SIGMA</td>
			<td>S5545</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Hts 1B1 (mouse)</td>
			<td>Developmental Studies Hybridoma Bank (DSHB)</td>
			<td>1B1</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-DE-cadherin (rat), 1:20</td>
			<td>DSHB</td>
			<td>DCAD2</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-nc82 (mouse), 1:50</td>
			<td>DSHB</td>
			<td>nc82</td>
			<td></td>
		</tr>
		<tr>
			<td>secondary antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td>
			<td>Life Technologies</td>
			<td>A-11001</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 546 conjugate</td>
			<td>Life Technologies</td>
			<td>A-11081</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Guinea Pig IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate</td>
			<td>Life Technologies</td>
			<td>A-21435</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 dye-conjugated phalloidin</td>
			<td>Life Technologies</td>
			<td>A-22283</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro slide glass</td>
			<td>Matsunami Glass Ind.,Ltd.</td>
			<td>SS7213</td>
			<td></td>
		</tr>
		<tr>
			<td>Square microscope cover glass</td>
			<td>Matsunami Glass Ind.,Ltd.</td>
			<td>C218181</td>
			<td></td>
		</tr>
		<tr>
			<td>FluorSave reagent (Mounting reagent)</td>
			<td>Calbiochem</td>
			<td>345789</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette 1 ml (Disposable dropper)</td>
			<td>WATSON</td>
			<td>5660-222-1S</td>
			<td></td>
		</tr>
		<tr>
			<td>imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 laser scanning microscope system</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>inverted Axio Observer. Z1 SP left</td>
		</tr>
		<tr>
			<td>image processing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 ZEN</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>It is a special user interface based on the 64 bit Microsoft Windows7 operating system</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fly stocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>w; GMR45C06-GAL4</td>
			<td></td>
			<td></td>
			<td>&#160;from Bloomington Drosophila Stock Center. (#46260)</td>
		</tr>
		<tr>
			<td>UAS&#8211;GFP; UAS&#8211;mCD8::GFP</td>
			<td></td>
			<td></td>
			<td>gifts from K. Ito, The University of Tokyo.</td>
		</tr>
		<tr>
			<td>w[1118]</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>w; phantom-GAL4#22/UAS-turboRFP</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>w; UAS-mCD8::GFP; TRH-GAL4</td>
			<td></td>
			<td></td>
			<td>see in Ref29, Alekseyenko, O. V, Lee, C. &#38; Kravitz, E. A.(2010)</td>
		</tr>
		<tr>
			<td>w; UAS-mCD8::GFP</td>
			<td></td>
			<td></td>
			<td>&#160;from Bloomington Drosophila Stock Center. (#32188)</td>
		</tr>
		<tr>
			<td>yw;; nSyb-GAL4</td>
			<td></td>
			<td></td>
			<td>&#160;from Bloomington Drosophila Stock Center. (#51941)</td>
		</tr>
	</tbody>
</table>

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.

We used the above protocols to visualize steroidogenic organs and their interactive organs in D. melanogaster larvae and adult females. The overall scheme of protocols is shown in Figure 1.
The RG, including the PG (Figure 2D), is smaller and more transparent than the brain and is located at the anterior-dorsal side of the brain (Figure 2A-C and 3A-E). To label the PG cells, several groups have generated various types of antibodies against ecdysteroidogenic e...

Watch this Scientific Journal Video about Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster at JoVE.com

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.

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 ...

The overall goal of this procedure is to visualize the steroidogenic organs and their interactive organs in larvae or adult females of the fruit fly, Drosophila melanogaster. This method can help understand insect steroid hormone biosynthesis, and the neuronal regulatory mechanism underlying mating and metamorphosis. The main advantage of this technique is that the innervation of steroidogenic organs and the relative portion of their interactive organs remains intact.Generally, individuals new to this method will struggle because the steroidogenic organs in fly larvae are quite tiny and transparent. My lab and I are pleased to share our dissection protocols. So, let's get started.After preparing the larvae, start the course dissection in a three centimeter dish filled with PBS, under a dissecting microscope. First, grip the mouth hook with forceps. Then, using micro scissors, sever the body at about a third of the length from the anterior tip.Next, hold the anterior length with one pair of forceps and use a second pair to gently push the tip of the head into the body, to ultimately turn the larval body inside out. Prepare up to 20 larvae in this manner within 10 minutes, and then transfer them into fixative solution. After 30 minutes, wash the preparations, and proceed with immunostaining.To begin, leave the larvae immersed in water for about one hour, to asphyxiate and thus immobilize the larvae. Then, place a larva dorsal side up in a drop of PBS on a silicone-coated dish. The dorsal side has the two tracheal tubes.Under a dissecting microscope, use forceps to insert an insect pin into the anterior tip. Then, stretch the body to the posterior side and put a second pin into the posterior tip. Next, make a small incision near the tail using micro scissors.From the incision, carefully cut the dorsal cuticle longitudinally along the dorsal midline, toward the head, while not damaging any of the tissues under the cuticle. After making the incision, stretch the body walls apart, and secure them with insect pins at the corners. Then, using forceps, remove the anterior fat body and salivary glands, thus exposing the brain-ring gland complex on the surface.Now, aspirate the PBS, and immerse the preparation in fixative solution. After 30 minutes, use forceps to remove the insect pins and transfer the preparation into a microtube filled with 0.3%PBT. Then, proceed with immunostaining.After immunostaining the larvae, use a disposable pipet to transfer the samples to a dish filled with 0.3%PBT. Under a dissecting microscope, grip the cuticle or mouth hook with one pair of forceps. Then, use a 27 gauge needle attached to a one milliliter syringe, like a knife, to remove the brain-ring gland eye disc complex from the body cuticle, by cutting away the anterior tip of the esophagus and eye discs.Next, using forceps, carefully separate the esophagus and gut from the brain-ring gland eye disc complex. Pull the gut out to the posterior side, since the esophagus passes through the brain above the ventral nerve cord. Finally, to isolate the brain-ring gland complex, carefully cut the connections between the brain disc, eye discs, and leg discs, with a needle knife.It is quite essential to use fine forceps with sharp edges during dissection. I'd strongly recommend that you sharpen forceps with a piece of grinding stone to bring their edges together without any gaps. To transfer the samples, use a micro pipet with a wide bore tip.Deposit the samples onto the center of the glass slide. Then, use forceps to position the samples onto their ventral sides, so the ring gland, which is located on the dorsal side of the brain, can be imaged. Next, tilt the slide and wipe off as much of the excess PBT as possible.Then, put a drop of mounting reagent near the samples and use forceps to slowly lower a cover slip over the reagent, and then over the samples. To dissect the adult female ovary, anesthetize flies with carbon dioxide gas. Then, cut off their heads and transfer their bodies to a three centimeter dish filled with PBS.Next, under a dissecting microscope, grip the dorsal side of the thorax with forceps, and take hold of abdominal segment A-5 or A-6 with a second pair of forceps. Then, peel away the abdominal cuticle towards the posterior side. Before proceeding, clean the sticky cuticle off the forceps.Next, pinch an ovaduct, and isolate the ovary. Then comb out and spread the tips of the ovary using forceps. Once spread, immerse the ovary in fixative solution for 30 minutes, to prepare them for staining.To make a preparation that maintains the innervation of the ovary, transfer anesthetized female flies to a silicone-coated dish filled with PBS. Under the dissection microscope, hold the proboscis, and peel away the head cuticle to expose the brain. From the brain, remove the attached trachea, leaving the brain connected to the ventral nerve cord.Then, hold the thorax by the dorsal side, and remove the legs and wings. Next, peel away the ventral cuticle of the thorax, beginning at the base of each leg. Once the VNC is exposed, very carefully remove the residual dorsal cuticle and muscles attached to the VNC, using forceps.Do not damage the connections between the brain and the VNC. Next, using forceps, peel away the abdominal cuticle to expose the ovaries and their interactive organs, which includes the prop, the gut, the ovaduct, the uterus, and the accessory gland. Finally, remove any residual tissues, including the trachea, and the fat body.Then, immerse the samples in fixative solution for 30 minutes, to prepare them for immunostaining. After immunostaining, transfer the samples to a glass slide with 0.1%PBT using a micro pipet. Then, view the slide with a microscope, and separate the strings of the ovarioles from each other using forceps.Do not injure the tips of the ovarioles during this process. They contain the germaria. When preparing the brain to reproductive organ complex, remove any residual cuticle, and spread the structures out onto the slide.Next, wipe off most of the excess PBT, and then apply a drop of mounting reagent to the sample. Next, slowly place a cover slip onto the slide using forceps and store the slide at four degrees celsius in the dark. Later, observe the samples under the microscope, and bring the steroidogenic organs into the field of view.Once the view is fixed, switch the imaging system to the image acquisition mode. During the larval stages, the prothoracic gland was labeled with antibodies against ecdysteroidogenic enzymes, including the anti-shroud antibody. To visualize the neuronal connection between the prothoracic gland and the brain, the brain-ring gland complex was dissected.The filet dissection method shows the stomatogastric nervous system, in which a group of serotonergic neurons project to the prothoracic gland, the proventriculus, and the pharyngeal muscles. In adult females, ovarian ecdysteroid affects many aspects of ovogenesis, such as GSC proliferation. The GSCs are located in the germarium at the tip of each ovariole in the ovary.Within the germarium, the GSCs were specifically labeled using two antibodies, 1B1, and DE-cadherin. To visualize the innervation of the ovary, the ovary was dissected, along with the brain, VNC, gut, prop, uterus, and spermatheca. Neurons were visualized by mCD8:GFP under the control of the n-Synaptobrevin GAL4 driver.Additionally, muscles'structure were stained using diconjugated phalloidin. Although dissection of the steroidogenic organs can at first feel difficult, use this movie to learn the cutting point of the tissues to more easily isolate the organ without injury. We hope that our protocol is informative, not only for beginners, but also experienced researchers.Once mastered, you can apply this procedure to your research, and brush up to fit with your needs. We hope this protocol will help the comprehensive understanding of steroid hormone biosynthesis. More detailed step-by-step protocols are available in the manuscript.Please check it out.

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JoVE Journal

Developmental Biology

10.3K Views.  University of Tsukuba. The overall goal of this procedure is to visualize the steroidogenic organs and their interactive organs in larvae or adult females of the fruit fly, Drosophila melanogaster. This method can help understand insect steroid hormone biosynthesis, and the neuronal regulatory mechanism underlying mating and metamorphosis. The main advantage of this technique is that the innervation of steroidogenic organs and the relative portion of their interactive organs remains intact.Generally, individ...