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 isolate an intact, undamaged RG by dissection.
<ol>
	<li>Preparation of larvae at the appropriate developmental stages 
	NOTE: To synchronize the developmental stages of D. melanogaster larvae, it is necessary to collect eggs within a narrow time window and to collect larvae at the appropriate times.
	<ol>
		<li>Collect eggs for 2 h on a grape-juice agar plate at 25 &#176;C.</li>
		<li>Collect newly-hatched 1st instar larvae under a dissecting microscope with forceps and transfer them to a small vial containing mashed standard yeast/cornmeal fly food. 
		NOTE: The age of larvae is represented by &#34;hours after egg laying (hAEL)&#34;, &#34;hours after hatch (hAH)&#34;, or &#34;hours after L3 ecdysis (hAL3E)&#34;.</li>
		<li>At the appropriate time point, collect the staged larvae with a disposable plastic loop to avoid undesirable injury. To minimize a difference in developmental progression of the 3rd instar larvae, collect the 2nd instar larvae at 70 hAEL (48 hAH) and allow them to molt in 2 h intervals. Then, collect the 3rd instar larvae within 2 h after the L3 ecdysis; this procedure gives 0-2 hAL3E larvae.</li>
		<li>Transfer the staged larvae to a dish filled with phosphate buffered saline (PBS).</li>
		<li>Rinse the larvae with PBS to remove residual food from the body.</li>
	</ol>
	</li>
	<li>Coarse dissection of larvae under a dissecting microscope
	<ol>
		<li>Hold the mouth hook of a larva with forceps. Sever the larval body at the anterior one-third of the body length using&#160;micro scissors.</li>
		<li>Hold the cut end of the anterior larval body with one pair of forceps. Using the other pair of forceps, gently push the tip of the head toward the inside of the body; this procedure turns the larval body inside out. 
		NOTE: This step can be skipped for the dissection of 1st instar larvae.</li>
		<li>Repeat steps 1.2.1-1.2.2 with additional larvae for 5-10 min. 
		NOTE: The number of larvae should be equal to or less than 20 to save time for dissection.</li>
	</ol>
	</li>
	<li>Fillet dissection of larvae 
	NOTE: This method allows keeping the position of tissues to remain&#160;intact.
	<ol>
		<li>Leave larvae in water only for 1 h. The larvae are immobilized due to asphyxiation.</li>
		<li>Put a larva dorsal side up in a drop of PBS on a silicon-coated dish, with its dorsal side up. 
		NOTE: The dorsal side has 2 tracheal tubes running longitudinally.</li>
		<li>Under a dissecting microscope, insert an insect pin into the anterior tip of the larva using&#160;forceps. Stretch the larval body to the posterior side and put a&#160;second pin into the posterior tip of the larva. 
		NOTE:&#160;In addition to insect pins, a needle made from a tungsten wire can be useful21. A needle can be embedded in a pipette tip through heating for use as a dissecting needle.</li>
		<li>Under a dissecting microscope, make a small incision near the tail with micro scissors. From the incision, cut the dorsal cuticle longitudinally along the dorsal midline toward the head. Be careful not to damage tissues under the cuticle.</li>
		<li>Stretch the bodywall cuticle on each side and place 4 pins at each corner of the dissected bodywall.</li>
		<li>Remove a portion of anterior fat body with forceps, to expose the&#160;brain-RG complex be exposed on the surface. Remove a pair of salivary glands, if necessary.</li>
	</ol>
	</li>
	<li>Fixation and staining of tissue with immunohistochemistry
	<ol>
		<li>Put the anterior one-third of the&#160;larval bodies (step 1.2.2) into a 1.5 mL microtube filled with 500 &#181;L the fix solution (4% paraformaldehyde in PBS). Fix less than 20 samples at one time, otherwise PBS attached to the fixed samples may cause an unfavorable dilution of the fixative. For fillet dissection, remove a drop of PBS and then add a drop of fix solution. 
		NOTE: For fix solution, either 3.7% formaldehyde or 4% paraformaldehyde may be used.</li>
		<li>Incubate the dissected tissues in the fix solution for 30 min at room temperature (RT) or alternatively for 2 h at 4 &#176;C.</li>
		<li>Replace the fix solution with 500 &#181;L PBS and quickly rinse the samples 3 times. Wash the samples with 500 &#181;L 0.3% PBT (PBS + 0.3% octylphenol ethoxylate) for 15 min on a rocker at RT. For fillet dissection, remove the insect pins and transfer the samples into a 1.5 mL microtube. 
		NOTE: For increasing the permeability of antibodies into tissues, the samples are treated with 500 &#181;L 2.0% PBT for 1-2 h on a rocker at RT.</li>
		<li>Replace PBT with 500 &#181;L blocking solution (2% bovine serum albumin in PBT). Incubate the samples for 1.5 h on a rocker at RT.</li>
		<li>Replace the blocking solution with 50 &#181;L the primary antibody solution, i.e. antibodies diluted in the blocking solution.</li>
		<li>Incubate the samples overnight on a rocker at 4 &#176;C.</li>
		<li>Replace the primary antibody solution with 500 &#181;L 0.3% PBT and quickly rinse the samples 5 times. Wash the samples 3 times with 500 &#181;L 0.3% PBT for 15 min each on a rocker at RT.</li>
		<li>Replace 0.3% PBT with 50 &#181;L the secondary antibody solution (dye-conjugated antibodies diluted in the blocking solution). For nuclear staining, 0.5 &#181;L 4&#39;,6-diamidino-2-phenylindole (DAPI, 0.1 mg/mL stock solution) is added to 50 &#181;L&#160;solution. Incubate the samples on a rocker for 2 h at RT or alternatively at 4 &#176;C overnight. 
		NOTE: Keep the samples in the dark.</li>
		<li>Replace the antibody solution with 500 &#181;L 0.3% PBT and rinse 5 times. Wash the samples 3 times with 500 &#181;L 0.3% PBT for 15 min each at RT.</li>
	</ol>
	</li>
	<li>Fine dissection of the brain-RG complex containing the PG and mounting
	<ol>
		<li>Use a disposable pipet to transfer the immunostained samples to a dish filled with 0.3% PBT. 
		NOTE: To avoid inconvenient bubbles during dissection and mounting, 0.3% PBT can be replaced with PBS.</li>
		<li>Under a dissecting microscope, hold the cuticle or mouth hook with one pair of forceps. Using a 27 G needle attached to a 1 mL syringe as a &#34;knife&#34;, cut the anterior tip of the esophagus and eye discs to remove the brain-RG-eye disc complex from body cuticle.</li>
		<li>Separate the esophagus and gut from the brain-RG-eye disc complex with forceps. Since the esophagus passes through the brain above the ventral nerve cord (VNC), pull out the gut to the posterior side. 
		NOTE: To remove extra imaginal discs from the samples, cut the connections between the brain, eye discs, and leg discs with a needle knife.</li>
		<li>Repeat the steps 1.5.1-1.5.4 for other samples. 
		NOTE: To prevent tissue debris from sticking to the samples, transfer each completed sample to a different clean dish filled with 0.3% PBT or PBS.</li>
		<li>Transfer the samples to the center of a clean glass slide with a micropipette. 
		NOTE: For 2nd or 3rd instar larvae, the end of a micropipette tip should be truncated with a razor blade.</li>
		<li>Under a dissecting microscope, align individual samples with their dorsal side up by using forceps. 
		NOTE: The RG is located on the dorsal side of the brain (Figure 2A). This alignment facilitates the specification of individual samples during imaging.</li>
		<li>Tilt a glass slide and wipe off as much excess PBT as possible. Put a drop of mounting reagent on a side of the slide. Place the edge of a cover slip from the other side of the drop and put the cover slip on the samples slowly with forceps. 
		NOTE: This procedure prevents the samples from moving outside the cover slip.</li>
		<li>Store the samples at 4 &#176;C. Keep the samples in the dark.</li>
	</ol>
	</li>
</ol>
2. The Dissection of the Ovary in Adult Females
<ol>
	<li>Preparation of adult females
	<ol>
		<li>Feed female flies with standard yeast/cornmeal fly food for 3-4 days to fatten up the ovary. Maintain at 25 &#176;C.</li>
	</ol>
	</li>
	<li>Dissection of the adult female ovary
	<ol>
		<li>Anesthetize female flies with CO2 gas and cut off their heads.</li>
		<li>Transfer fly bodies to a 3 cm dish filled with PBS.</li>
		<li>Under a dissecting microscope, hold the thorax on the dorsal side using&#160;forceps. Grasp the abdominal segment A5 or A6 with the other forceps and peel away the&#160;abdominal cuticle toward the posterior side. Wipe off the sticky cuticle from the tip of the&#160;forceps.</li>
		<li>Pinch an oviduct and take out the ovary from the body.</li>
		<li>Comb and spread the tips of the ovary with forceps. 
		NOTE: This operation improves the efficiency of immunostaining.</li>
	</ol>
	</li>
	<li>Dissection of adult female brain, VNC and reproductive organs. 
	NOTE: This method allows the innervation of the ovary to be kept intact.
	<ol>
		<li>Anesthetize female flies with CO2 gas and transfer them to a silicon-coated dish filled with PBS.</li>
		<li>Under a dissecting microscope, hold the proboscis and peel away the head cuticle to expose the brain with forceps. Remove the trachea attached to the brain. 
		NOTE: The brain is a white and rounded structure. The brain connects to the VNC in the thorax.</li>
		<li>Hold the thorax on the dorsal side and cut off the legs and wings with one pair of forceps. Using the other pair of forceps, peel away the thorax ventral cuticle from the bases of the legs. Once the VNC is exposed, remove the residual dorsal cuticle and muscles attached to the VNC using&#160;forceps. Be careful not to damage the connection between the brain and the VNC.</li>
		<li>Use forceps to peel away the abdominal cuticle and&#160;expose the ovary and their interactive organs including neurons, the crop, the gut, the oviduct, the uterus, and the accessary gland.</li>
		<li>Remove the residual tissues including the trachea and the fat body using&#160;forceps.</li>
	</ol>
	</li>
	<li>Fixation and staining of tissue with immunohistochemistry
	<ol>
		<li>Transfer approximately 8-10 pairs of the ovaries to a 1.5 mL microtube filled with 250 &#181;L the fix solution (4% paraformaldehyde in PBS) and incubate the samples for 30 min at RT.</li>
		<li>Replace the fix solution with 500 &#181;L PBS and quickly rinse the samples 3 times.</li>
		<li>Wash the samples 2 times with 500 &#181;L 0.1% PBT for 5 min each at RT.</li>
		<li>Replace PBT with 50 &#181;L blocking solution. Incubate the samples for 1 h on a rocker at RT.</li>
		<li>Replace the blocking solution with 50 &#181;L the primary antibody solution.</li>
		<li>Incubate the samples overnight on a rocker at 4 &#176;C.</li>
		<li>Replace the&#160;primary antibody solution with 500 &#181;L 0.1% PBT. Wash the samples 3 times with 500 &#181;L 0.1% PBT for 15 min each on a rocker at RT.</li>
		<li>Replace 0.1% PBT with 50 &#181;L secondary antibody solution. For nuclear staining, add 0.5 &#181;L DAPI &#160;to 50 &#181;L&#160;the solution. Incubate the samples for 2 h on a rocker at RT.</li>
		<li>Replace the secondary antibody solution with 500 &#181;L 0.1% PBT. Wash the samples 3 times with 500 &#181;L 0.1% PBT for 15 min each on a rocker at RT. 
		NOTE: The washing procedure can be done at 4 &#176;C overnight. Keep the samples in the dark.</li>
	</ol>
	</li>
	<li>Mounting the samples onto glass slides
	<ol>
		<li>Transfer the samples to a glass slide with 0.1% PBT using a micropipette. 
		NOTE: To avoid inconvenient bubbles during dissection and mounting, 0.1% PBT can be replaced with PBS.</li>
		<li>For the ovary, separate the strings of the ovarioles from each other with forceps under a dissecting microscope. Do not injure the tips of the ovarioles containing the germaria. For the brain-VNC-reproductive organ complex, remove the residual cuticle and arrange the position on a glass slide.</li>
		<li>Wipe off as much excess PBT as possible and put a drop of mounting reagent in the center of the samples. Place a cover slip slowly using&#160;forceps.</li>
		<li>Store the samples at 4 &#176;C. Keep the samples in the dark.</li>
	</ol>
	</li>
</ol>
3. Imaging with a Confocal Laser Scanning Microscope
<ol>
	<li>Observe the samples under the microscope and bring the steroidogenic organs in the field of view. Once the view is fixed, switch the imaging system to the image acquisition mode. 
	NOTE: The 10-20X objective lenses are used to obtain the images of the brain-RG complex or the whole ovary. Alternatively, the 40-63X objective lenses (water or oil immersion) are used for visualizing the subcellular localization of proteins in GSCs or the neuronal innervations of the PG and the ovary.</li>
	<li>Set up the parameters of image acquisition on the attached software. Select the combination of fluorescence (e.g. GFP and RFP). By fast scanning procedure, adjust the laser power, the sensitivity of detectors (Gain/Offset), and the size of the pinhole. 
	NOTE: To acquire images at high quality, the scanning laser power and the sensitivity of detector (Gain) should be optimized for each sample. To outline the shape of the tissues, a transmitted-light image can be taken in addition to a fluorescent image.</li>
	<li>Take Z stacks of the images. During&#160;a continuous fast scan, move the focal plane with the focus drive of the microscope and set the starting position and the end position. The number of slices and&#160;the interval distance between slices are adjusted accordingly.</li>
	<li>Select the scan speed, the scan method, and the bidirectional scan mode.</li>
	<li>&#34;Start the real scans&#34;&#160;for image acquisition.</li>
	<li>Save the image with the&#160;format of the attached software. 
	NOTE: The original image files contain the information of image acquisition parameters and scales. Exported images can be processed using an image processing software on the computer22.</li>
</ol>

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+and+feedback+regulation+of+cyclic+steroid+production+in+Drosophila+melanogaster.">Forward and feedback regulation of cyclic steroid production in Drosophila melanogaster.</a> Development. 141 (20), 3955-3965 (2014).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118 (2), 401-415 (1993).</li><li>Rewitz, K. F., Yamanaka, N., Gilbert, L. I., O'Connor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Insect+Neuropeptide+PTTH+Activates+Receptor+Tyrosine+Kinase+Torso+to+Initiate+Metamorphosis.">The Insect Neuropeptide PTTH Activates Receptor Tyrosine Kinase Torso to Initiate Metamorphosis.</a> Science. 326 (5958), 1403-1405 (2009).</li><li>Li, H. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+GAL4+driver+resource+for+developmental+and+behavioral+studies+on+the+larval+CNS+of+Drosophila.">A GAL4 driver resource for developmental and behavioral studies on the larval CNS of Drosophila.</a> Cell Rep. 8 (3), 897-908 (2014).</li><li>Alekseyenko, O. V., Lee, C., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+manipulation+of+serotonergic+neurotransmission+affects+the+escalation+of+aggression+in+adult+male+Drosophila+melanogaster.">Targeted manipulation of serotonergic neurotransmission affects the escalation of aggression in adult male Drosophila melanogaster.</a> PLOS One. 5 (5), e10806 (2010).</li><li>Domanitskaya, E., Anllo, L., Sch&#252;pbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phantom,+a+cytochrome+P450+enzyme+essential+for+ecdysone+biosynthesis,+plays+a+critical+role+in+the+control+of+border+cell+migration+in+in+Drosophila.">Phantom, a cytochrome P450 enzyme essential for ecdysone biosynthesis, plays a critical role in the control of border cell migration in in Drosophila.</a> Dev. Biol. 386 (2), 408-418 (2014).</li><li>Song, X., Zhu, C. -. H., Doan, C., Xie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+stem+cells+anchored+by+adherens+junctions+in+the+Drosophila+ovary+niches.">Germline stem cells anchored by adherens junctions in the Drosophila ovary niches.</a> Science. 296 (5574), 1855-1857 (2002).</li><li>Niwa, Y. S., Niwa, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+control+of+steroid+hormone+biosynthesis+during+development+in+the+fruit+fly+Drosophila+melanogaster.">Neural control of steroid hormone biosynthesis during development in the fruit fly Drosophila melanogaster.</a> Genes Genet. Syst. 89 (1), 27-34 (2014).</li><li>Yoshiyama-Yanagawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+conserved+Rieske+oxygenase+DAF-36/Neverland+is+a+novel+cholesterol-metabolizing+enzyme.">The conserved Rieske oxygenase DAF-36/Neverland is a novel cholesterol-metabolizing enzyme.</a> J. Biol. Chem. 286 (29), 25756-25762 (2011).</li><li>Niwa, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-molting+glossy/shroud+encodes+a+short-chain+dehydrogenase/reductase+that+functions+in+the+&amp;#34;Black+Box&amp;#34;+of+the+ecdysteroid+biosynthesis+pathway.">Non-molting glossy/shroud encodes a short-chain dehydrogenase/reductase that functions in the &amp;#34;Black Box&amp;#34; of the ecdysteroid biosynthesis pathway.</a> Development. 137 (12), 1991-1999 (2010).</li><li>Komura-Kawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+Zinc+Finger+Transcription+Factor+Ouija+Board+Controls+Ecdysteroid+Biosynthesis+through+Specific+Regulation+of+spookier.">The Drosophila Zinc Finger Transcription Factor Ouija Board Controls Ecdysteroid Biosynthesis through Specific Regulation of spookier.</a> PLOS Genet. 11 (12), e1005712 (2015).</li><li>Yamanaka, N., Marqu&#233;s, G., O'Connor, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle-Mediated+Steroid+Hormone+Secretion+in+Drosophila+melanogaster.">Vesicle-Mediated Steroid Hormone Secretion in Drosophila melanogaster.</a> Cell. 163 (4), 907-919 (2015).</li><li>Riemensperger, T., Pech, U., Dipt, S., Fiala, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+calcium+imaging+in+the+nervous+system+of+Drosophila+melanogaster.">Optical calcium imaging in the nervous system of Drosophila melanogaster.</a> BBA-Gen. Subjects. 1820 (8), 1169-1178 (2012).</li><li>Owald, D., Lin, S., Waddell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light,+heat,+action:+neural+control+of+fruit+fly+behavior.">Light, heat, action: neural control of fruit fly behavior.</a> Phil. T. Roy. Soc. B. 370 (1677), 20140211 (2015).</li></ol>

No conflicts of interest declared.

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 endocrine organ RG with the corpora allata (CA) and the corpora cardiaca (CC) (Figure 2D). While the PG produces ecdysteroids, the CA and the CC produce juvenile hormones and adipokinetic hormone, respectively. This anatomical property has been an impediment to&#160;researchers studying&#160;ecdysteroidogenesis in D. melanogaster, as compared to other insects. However, in the past decades, fly genetics has allowed us to identify many ecdysteroidogenic genes that are specifically expressed in the PG4. These genes are also expressed in nurse cells or follicle cells of the ovary34,35. Now one can use a unique set of gene expression profiles in ecdysteroidogenic organs, making it possible to specify ecdysteroidogenic cells by immunostaining and transgenic techniques.
During dissection, it is essential to use fine forceps with sharp edges. Prior to dissection, the edges of the forceps can be sharpened with an Arkansas grinding stone to bring the edges together without any gaps. During dissection, debris or the residual tissues, such as the fat body, the gut, and the imaginal discs, should be removed as much as possible. In particular, pieces of the fat body easily stick to the tissue of interest. In addition, the mounting of the samples onto&#160;glass slides should also be performed carefully. A complex of tissues may be disarranged when a cover slip is placed on the samples in sticky mounting medium. Therefore, a sufficient number of samples must be dissected, and suitable samples must be chosen for imaging, such as those in which the RG or the ovary is on the uppermost side and the neuronal connections remain intact.
To perform&#160;successful immunostaining and to obtain the reproducible results, it is critical to maintain the same conditions for the sampling and handling procedures. The extent of immunostaining highly depends on the condition of individual animals at the time of sampling and fixation. For example, the age of the animals, the rearing temperatures, nutrient condition, and the dissection time should be carefully considered. To minimize handling errors of fixation in multiple experiments, we carefully keep almost equal number of samples to coordinate the dissection time and fixation reaction times. For the fixation solution, 4% paraformaldehyde or 3.7% formaldehyde is used. Because paraformaldehyde is quickly degraded over time, its powder is&#160;usually dissolved to make 10% stock solution in PBS, and aliquots are stored at -20 &#176;C. The aliquots are&#160;unfrozen to make&#160;the fresh 4% fix solution prior to every dissection. Moreover, the extensive wash steps of samples reduce the background non-specific staining.
In the step of image acquisition, we follow the manufacturer&#39;s protocol for confocal microscopy. The microscopy system provides a&#160;user-friendly interface for researchers or students, such&#160;that the optimal sets of excitation/emission filters are automatically selected when the types of fluorophore are specified. While the microscopy system is easy to use, one needs to optimize the parameters of image acquisition on each sample or each focal plane, by changing the detector gain, scan speed, scan numbers, and the pinhole size.
One of the limitations of this technique is that immunostaining largely depends on the quality of antibodies. While antibodies against Noppera-bo, Neverland, and Shroud were successfully generated, we failed to generate several antibodies against other ecdysteroidogenic gene products, such as Ouija board36. To overcome this issue, the knock-in technique can be applied to insert a HA-tag in the gene locus&#160;of ouija board&#160;using the&#160;CRISPR/Cas9 system. By using such transgenic techniques in&#160;combination with immunostaining, at least 3-4 proteins can be simultaneously visualized in the PG or the ovary. In contrast to protein localization, however, a method to visualize endogenous ecdysteroids by immunostaining has not been established. Once anti-ecdysteroid antibody is available for immunostaining, the subcellular localizations of ecdysteroid biosynthesis and the transport of ecdysteroid can be investigated in the future.
Another limitation is that the temporal dynamics of ecdysteroidogenesis cannot be examined in&#160;fixed tissues. Considering that the ecdysteroid titer is fluctuated along the developmental stages, the activity of ecdysteroidogenic organs and the projecting neurons should be temporally regulated in response to various environmental stimuli. A recent study monitored the Ca2+ dynamics of the PG cells using&#160;live imaging techniques37. For future applications, we are currently developing a protocol for the live imaging of PG or cultured ovaries along with their projecting neurons. With respect to existing methods, this new protocol aims to visualize the activity of neurons and their target organs simultaneously. Once the activity of neurons can be monitored with the Ca2+ indicator GCaMP or Cameleon38, it can be manipulated with optogenetic or thermogenetic approaches at the appropriate times39. These methods contribute to a comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

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 enzymes (i.e., Neverland23, Spookier24, Shroud20, Phantom25, Disembodied25, and Shadow26). Among them, anti-Shroud (Sro) antibody is reliably used to label the PG by immunostaining (Figure 2C, 2E, and 3C). Alternatively, the binary gene expression system, GAL4/UAS system27, can be used to express fluorescent protein genes in PG cells. Through the promoter analysis of the ecdysteroidogenic gene phantom (phm), the PG-specific promoter can induce gene expression exclusively in the PG28. Therefore, the PG cells can be specifically visualized by expressing GFP or RFP under the control of phm-GAL4#22 (Figure 3D).&#160;
To visualize the neuronal connection between the PG and the brain, a group of neurons can be labeled with mCD8::GFP under the control of various GAL4 drivers and antibodies (Figure 2C, 2E, 3D, and 3E). The FlyLight database of GAL4 line collections is optimized for labeling neurons29. Among them, GMR45C06-GAL4 labels PG-projecting neurons (Figure 2C and E). The immunostaining of GFP-expressing larvae with anti-GFP antibody is effective in enhancing GFP signals. Furthermore, TRH-GAL4&#62;mCD8::GFP larvae show the stomatogastric nervous system, in which serotonergic neurons project to not only the PG, but also to the proventriculus (PV, insect foregut) and the pharyngeal muscles (PM) (Figure 3D and E)20,30. Therefore, it is critical to maintain the position of the RG, the brain, and the other surrounding tissues during fillet dissections (Figure 3).
In adult females, ovarian ecdysteroid affects many aspects of oogenesis, such as GSC proliferation, cyst differentiation, egg chamber growth, and stress response11. As in the PG, ecdysteroidogenic enzymes in the ovary are also visualized with the specific antibodies described above12,31. As a downstream event upon which ecdysteroids act, the number of GSCs in the germarium is our focus (Figure 4). Although the germarium is an assembly of multiple cell types, GSCs are specified by immunostaining with two GSC marker antibodies, 1B1 and DE-cadherin32. To visualize the innervation of the ovary, the ovary is dissected along with the brain, VNC, gut, crop, uterus, and spermatheca (Figure 5). Neurons are visualized by mCD8::GFP under the control of nSyb-GAL4, a pan-neuronal driver (Figure 5B and D). Muscles around the ovary, uterus, and gut are stained with dye-conjugated phalloidin.&#160;
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55519/55519fig1.jpg" /> 
Figure 1: The overall scheme of protocols. Two distinct dissection methods are applicable to&#160;larvae and adult females, depending on the purpose of the experiments. The mounting methods are also devised according to sample conditions. Fixation, staining, and imaging techniques are basically common to all samples. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55519/55519fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55519/55519fig2.jpg" /> 
Figure 2: Visualization of the PG and the PG-projecting Neurons. (A and B) The brain-ring gland (RG) complex of the wild-type 3rd instar larva. In (B), the PG is outlined by dotted lines. The filamentous structure between the brain and the RG is the trachea. (C-E) The innervation of the PG was visualized with mCD8::GFP driven by GMR45C06-GAL4 in FlyLight collection. The PG and GFP-positive neurons were labeled with anti-Sro antibody (magenta) and anti-GFP antibody (green), respectively. The fluorescent image is merged with the transmitted-light image to specify the outline of tissues. In (D), the PG, the corpora allata (CA) and the corpora cardiaca (CC) are illustrated. The PG-projecting neurons are more prominent in the high-power image with 40X objective lens (arrows in E) than the low-power image with 10X objective lens (C). Scale bar = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55519/55519fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55519/55519fig3.jpg" /> 
Figure 3: The Stomatogastric Nervous System Projects to the PG, the PV, and the PM. (A and B) The fillet dissection of a&#160;wild-type 3rd instar larva. The positions of the PG, brain (Br), ventral nerve cord (VNC), eye discs (ED), wing discs (WD), pharyngeal muscle (PM), and proventriculus (PV) remain intact. (C) The PG was exclusively labeled with anti-Sro antibody (magenta). (D) The PG was labeled with turboRFP driven by phm-GAL4#22. Serotonergic neurons were labeled with anti-5HT antibody (green). (E) The stomatogastric nervous system was visualized with&#160;mCD8::GFP driven by TRH-GAL4. Serotonergic neurons project to the PG, the PM, and the PV (arrows). The Scale bar = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55519/55519fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55519/55519fig4.jpg" /> 
Figure 4: The Germaria of Wild-type Flies Containing One, Two, or Three GSCs. (A) An overview of the female reproductive organ. An ovary is composed of 16-20 ovarioles that are strings of egg chambers. The germarium, where GSCs are located, is at the tip of each ovariole. (B-D) One, two, or three GSCs are located in each germarium&#160;(white dotted circles) of the wild-type females. GSCs are stained with 1B1 antibody (green), which labels a spherical structure called the spectrosome (arrows) and a membranous cytoskeletal structure known as the fusome. The cell boundaries are visualized with anti-DE-cadherin antibody (magenta). The Scale bar = 10 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55519/55519fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55519/55519fig5.jpg" /> 
Figure 5: The Female Reproductive Organs and their Interactive Organs. (A) The ovary (Ov), gut (Gut), crop (Cr), brain (Br), ventral nerve cord (VNC), and spermatheca (Sp) were dissected under the microscope. (B-D) The innervation of the ovary was visualized with mCD8::GFP driven by nSyb-GAL4. GFP-positive neurons extend from the VNC (arrow), projecting to the surface of the ovaries (arrowheads). The ovaries are outlined by dotted lines. The samples were stained with anti-GFP antibody (green) and dye-conjugated phalloidin (magenta). Phalloidin associates with the filamentous actin of muscles around the ovaries (Ov) and uterus (Ut). The illustration is shown in C. The Scale bar = 200 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55519/55519fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

protocols-for-visualizing-steroidogenic-organs-their-interactive

Nanyang Technological University

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JoVE Journal

Developmental Biology

10.3K Views.  University of Tsukuba. 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.

Video: Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly 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 ...

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-&#946;-Hydroxysteroid dehydrogenase /&#916;5-4 isomerase (3&#946;-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3&#946;-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3&#946;-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

The interrenal gland in zebrafish is the teleostean counterpart of the mammalian adrenal gland. This protocol introduces how to perform the 3-&#946;-hydroxysteroid dehydrogenase (&#916;5-4 isomerase; 3&#946;-Hsd) enzymatic activity assay, which detects differentiated steroidogenic cells in the developing zebrafish.

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

High-resolution Episcopic Microscopy (HREM) - Simple and Robust Protocols for Processing and Visualizing Organic Materials

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging organic materials with volumes up to 5 x 5 x 7 mm3 in typical numeric resolutions between 1 x 1 x 1 and 5 x 5 x 5 &#181;m3. Specimens are embedded in methacrylate resin and sectioned on a microtome. After each section an image of the block surface is captured with a digital video camera that sits on the phototube connected to the compound microscope head. The optical axis passes through a green fluorescent protein (GFP) filter cube and is aligned with a position, at which the bock holder arm comes to rest after each section. In this way, a series of inherently aligned digital images, displaying subsequent block surfaces are produced. Loading such an image series in three-dimensional (3D) visualization software facilitates the immediate conversion to digital volume data, which permit virtual sectioning in various orthogonal and oblique planes and the creation of volume and surface rendered computer models. We present three simple, tissue specific protocols for processing various groups of organic specimens, including mouse, chick, quail, frog and zebra fish embryos, human biopsy material, uncoated paper and skin replacement material.

High-resolution Episcopic Microscopy HREM - Simple and Robust Protocols for Processing and Visualizing Organic Materials

We provide simple and robust protocols for processing biopsy material of various species, embryos of biomedical model organisms and samples of other organic tissues in order to permit digital volume data generation with the high-resolution episcopic microscopy method.

We provide simple protocols for generating digital volume data with the high-resolution episcopic microscopy (HREM) method. HREM is capable of imaging ...

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 ...

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, especially, in plant reproductive biology. However, plant morphological, anatomical, and ultrastructural analyses traditionally involve time-consuming embedding and sectioning procedures for bright field, scanning, and electron microscopy. Recent progress in confocal fluorescence microscopy, state-of-the-art 3-D computer-aided microscopic analyses, and the continuous refinement of molecular techniques to be used on minimally processed whole-mount specimens, has led to an increased demand for developing efficient and minimal sample processing techniques. In this protocol, we describe techniques for properly dissecting Arabidopsis flowers and siliques, basic clearing techniques, and some staining procedures for whole-mount observations of reproductive structures.

Whole-mount Clearing and Staining of Arabidopsis Flower Organs and Siliques

In this protocol, we describe techniques for the proper dissection of Arabidopsis flowers and siliques, some basic clearing techniques, and selected staining procedures for whole-mount observations of reproductive structures.

Due to its formidable tools for molecular genetic studies, Arabidopsis thaliana is one of the most prominent model species in plant biology and, ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...