Name: protocols for visualizing steroidogenic organs and their interactive organs with immunostaining in the fruit fly drosophila melanogaster
Uploaded: 2017-04-14T14:00:00
Description: 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

Wij danken Reiko Kise en Tomotsune Ameku voor hun technische ondersteuning voor dit werk. We zijn ook dankbaar voor Kei Ito, Olga Alekseyenko, Akiko Koto, Masayuki Miura, de Bloomington Drosophila Stock Center, KYOTO Stock Center (DGRC) en de Developmental Studies Hybridoma Bank voor aandelen en reagentia. Dit werk werd ondersteund door subsidies aan YSN van JSPS KAKENHI Grant Number 16K20945, The Naito Foundation, en Inoue Science Research Award; en door een subsidie ​​aan RN uit MEXT KAKENHI Grant Number 16H04792.

OPMERKING: De opzet van protocollen is getoond in figuur 1. 1. Dissectie van de larvale ringwartel (RG) NB: In D. melanogaster, die behoort tot cyclorrhaphous Diptera, de PG in een samengesteld endocrien orgaan genaamd de ring pakkingbus (RG, Figuur 2D). Aangezien het niet haalbaar dat de PG operatief wordt gescheiden van andere typen cellen (later besproken), een praktisch doel om een ?...

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We bestudeerden ecdysteroid biosynthese en haar reguleringsmechanisme in D. melanogaster, en bedacht een protocol voor dissectie en immunokleuring. De timing van ecdysteroïdrespons biosynthese wordt beïnvloed door omgevingsfactoren tot neuronale ingangen 33, dus is het essentieel om de innervatie van de organen ecdysteroidogenic houden met de hersenen, VNC en andere weefsels tijdens dissectie. Zoals hierboven is beschreven, de D. melanogaster PG co...

In meercellige organismen, is een groep cellen begiftigd met een gespecialiseerde functie in hun biogene activiteit die essentieel is voor het hele lichaam. Hun taken te vervullen, elk weefsel of orgaan tot expressie brengt een reeks van genen betrokken bij hun functies en communiceert met andere weefsels hun activiteiten in het kader van de ontwikkeling orkestreren. Om dergelijke gespecialiseerde celfuncties en inter-orgaan interacties te karakteriseren, moeten we een groep cellen specificeren bij andere typen cellen die in de meercellige architectuur intact. Een voorbeeld van dergelijke speciale organen een steroïdogene orgaan, waar veel biosynthetische enzymen mediëren de omzetting stappen uit cholesterol actieve steroïde hormonen 1. De meeste van deze enzymen genen specifiek tot expressie in steroïdogene organen en de biosyntheseroute wordt strak gereguleerd door verschillende externe stimuli via humorale ingangen en neuronale ingangen. Een keergesynthetiseerd, steroïde hormonen worden uitgescheiden in de hemolymfe en zijn gericht op vele weefsels en organen voor het reguleren van de expressie van diverse genen 2. Derhalve is de werking van een steroïdhormoon induceert een systemische reactie op homeostase, groei en voortplanting te behouden. De functies van steroïdhormoon biosynthese en pleiotrope werkingen van steroïde hormonen te onderzoeken, kan Drosophila melanogaster worden gebruikt als een geschikt modelsysteem. Tijdens de larvale stadia, het insect steroïdhormoon, ecdysteroid wordt gebiosynthetiseerd in een gespecialiseerde endocrien orgaan genoemd prothoracale gland (PG) 3. In de PG verschillende ecdysteroidogenic enzymen specifiek katalyseren de omzetting meerdere stappen van cholesterol tot ecdyson, welke controles rui en metamorfose op het juiste ontwikkelingsstadia 4. Daarom wordt een dynamische verandering in ecdysteroid titer geregelddoor velen signaalwegen in reactie op omgevingsfactoren. Aan de andere kant, in het volwassen stadium, ecdysteroid speelt een essentiële rol in de fysiologie, met inbegrip van reproductie, slaap, het geheugen en de levensduur 5, 6, 7, 8. Het is bekend dat ecdysteroid actief gebiosynthetiseerd in de eierstok, reguleren de progressie van oogenesis 6, 7, 8, 9, 10, 11. Onlangs hebben we gemeld dat het aantal kiemlijn stamcellen (GSC) wordt beïnvloed door ecdysteroid en geslacht peptide signalering in respons op stimuli paring 12. Krachtige instrumenten van D. melanogaster genetica en celbiologie, waaronder goed geannoteerde genoominformatie, binaire genexpressiesystemen en transgene RNAi technieken hebben het mogelijk om genen te identificeren essentiële biosynthese ecdysteroid de PG en eierstok 13, 14, 15. Zodra de ecdysteroidogenic genen zijn geïdentificeerd, kunnen de transcriptionele regulatie van deze genen en het dynamische lokalisaties van genproducten in de biosyntheseroute 16 onderzocht. Daartoe kwantitatieve reverse transcriptie-PCR, RNA in situ hybridisatie en immunohistologische analyse worden uitgevoerd. De toepassing van deze technieken is voorzien van een uitdagende taak; de uitgebreide dissectie van de PG of de eierstok. Met name de PG van de fruitvlieg relatief kleiner is dan die van andere insecten (zoals de zijderups en klap vliegen), dus moet oefenen de vitale vaardigheid van fruitvlieg dissectie voor bemonstering. Bovendien zijn zowel ecdysteroidogenic organen ontvangen innervaties van het centrale zenuwstelsel (CNS) 17, 18, 19, 20. Zo accurate anatomische analyse, de ecdysteroidogenic organen komen intact naast het centraal zenuwstelsel en andere organen, de neuronenverbindingen niet te verstoren gehouden. Hier bieden we protocollen voor de dissectie en visualisatie van steroidogenic organen in D. melanogaster. Het leren van de dissectie techniek is het belangrijk uitgangspunt voor deze experimenten. Daarnaast kan men met succes het etiket van de steroidogenic organen evenals hun interactieve organen met een aantal antilichamen en GAL4 driver lijnen. Door gebruik te maken van deze technieken, materialen en genetica, kan men de uitgebreide mechanismen van steroïde hormoon biosynthese bestuderen.

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			<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>
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		<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 meercellige organismen, wordt een kleine groep cellen begiftigd met een gespecialiseerde functie in hun biogene activiteit induceren van een systemische reactie op groei en reproductie. In insecten, het larvale prothoracale klier (PG) en de volwassen vrouwelijke eierstok spelen een essentiële rol in de biosynthese van de belangrijkste steroïde hormonen genaamd ecdysteroïden. Deze ecdysteroidogenic organen geïnnerveerd door het zenuwstelsel, waar de timing van biosynthese wordt beïnvloed door omgevingsfactoren. Hier beschrijven we een protocol voor het visualiseren ecdysteroidogenic organen en hun interactieve organen in larven en volwassenen van de fruitvlieg Drosophila melanogaster, die een geschikt model voor het bestuderen steroïde hormonen biosynthese en haar reguleringsmechanisme biedt. Bekwame dissectie laat ons toe om de posities van ecdysteroidogenic organen en hun interactieve organen, inclusief de hersenen, de ventrale zenuw koord, en andere weefsels te behouden. Immunokleuring met eenntibodies tegen ecdysteroidogenic enzymen, naast transgene fluorescentie eiwitten aangestuurd door weefselspecifieke promoters zijn beschikbaar voor ecdysteroidogenic cellen te merken. Bovendien is de innervatie van de ecdysteroidogenic organen kan ook worden gelabeld met specifieke antilichamen of een verzameling van GAL4 drivers in verschillende soorten neuronen. Daarom kan de ecdysteroidogenic organen en hun neuronale verbindingen tegelijkertijd worden gevisualiseerd door immunokleuring en transgene technieken. Tot slot beschrijven we hoe kiemlijn stamcellen, waarvan de proliferatie en onderhoud worden gecontroleerd door ecdysteroïden te visualiseren. Deze methode draagt ​​bij aan beter begrip van steroïde hormonen biosynthese en de neuronale reguleringsmechanisme.

We gebruikten de bovenstaande protocollen om steroidogenic organen en hun interactieve organen in D. melanogaster larven en volwassen vrouwen te visualiseren. De opzet van protocollen is getoond in figuur 1. RG, inclusief PG (figuur 2D), kleiner en transparanter dan de hersenen en bevindt zich aan de voorste-dorsale zijde van de hersenen (Figuur 2A-C en 3A-E). De PG cellen te merken, hebben verschillende groepen verschillende typen antilichamen tegen ecdyst...

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 beschrijven een protocol voor dissectie, fixatie en immunokleuring van steroidogenic organen in Drosophila larven en volwassen vrouwtjes steroïde hormonen biosynthese en haar reguleringsmechanisme bestuderen. Naast steroïdogene organen visualiseren we de innervatie van steroidogenic organen en steroidogene doelcellen zoals kiemlijn stamcellen.

Protocollen voor het visualiseren steroidogenic organen en hun Interactive Organen met Immunokleuring in de fruitvlieg<em&gt; Drosophila melanogaster</em

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

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Developmental Biology

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

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Testen Bloedcel Populaties van de<em&gt; Drosophila melanogaster</em&gt; Larva

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

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

Het grootbrengen de fruitvlieg<em&gt; Drosophila melanogaster</em&gt; Onder Axenische en gnotobiotische voorwaarden

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

Het visualiseren van de Interrenal steroïdogene Tissue en haar Vasculaire Microenvironment in zebravis

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

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

Episcopische Microscopie met hoge resolutie (HREM) - Eenvoudige en robuuste protocollen voor het verwerken en visualiseren van organische materialen

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

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

Dissectie en immunefluorescentie kleuring van Mushroom lichaam en fotoreceptor neuronen in de hersenen van de volwassen Drosophila melanogaster

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

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

Geheel-mount Clearing en kleuring van Arabidopsis bloem organen en Siliques

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

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

Cel aggregatie testen om te evalueren van de Binding van de Drosophila inkeping met Trans-liganden en haar remming door Cis-liganden

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

Driedimensionale weergave en analyse van Immunolabeled, verduidelijkt menselijke placenta Villous vasculaire netwerken

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

Driedimensionale Organotypic culturen van vestibulaire en auditieve zintuigen

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

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

Visualiseren van het knooppunt en de Notochordal plaat In Gastrulating muis embryo's met behulp van Scanning elektronen microscopie en hele Mount immunofluorescentie

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...