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

Vi takker Reiko Kise og Tomotsune Ameku for deres tekniske støtte til dette arbejde. Vi er også taknemmelige for Kei Ito, Olga Alekseyenko, Akiko Koto, Masayuki Miura, Bloomington Drosophila Stock Centre, Kyoto Stock Center (DGRC), og Developmental Studies Hybridoma Bank for aktier og reagenser. Dette arbejde blev støttet af tilskud til YSN fra JSP&#39;er KAKENHI Grant Number 16K20945, The Naito Foundation, og Inoue Science Research Award; og af en bevilling til RN fra MEXT KAKENHI Grant Antal 16H04792.

BEMÆRK: generelle opbygning af protokoller er vist i figur 1. 1. Dissektion af Larver Ring kirtel (RG) BEMÆRK: I D. melanogaster, som tilhører cyclorrhaphous Diptera, PG er inden for en sammensat endokrint organ kaldet ringen kirtel (RG, figur 2D). Da det er umuligt at PG kirurgisk er adskilt fra andre typer af celler (omtalt senere), et praktisk mål er at isolere et intakt, ubeskadig...

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Vi studerede ecdysteroid biosyntese og dens reguleringsmekanisme i D. melanogaster, og udtænkt en protokol for dissektion og immunfarvning. Timingen af ecdysteroid biosyntese påvirkes af miljømæssige påvirkninger gennem neuronale input 33, så det er vigtigt at opretholde den innervation af de ecdysteroidogenic organer sammen med hjernen, VNC, og andre væv under dissektion. Som beskrevet ovenfor, D. melanogaster PG danner et kompleks endokrint ...

I flercellede organismer, er en gruppe af celler udstyret med en specialiseret funktion i deres biogene aktivitet, der er afgørende for hele kroppen. At opfylde deres opgaver, hvert væv eller organ udtrykker en række gener relateret til deres funktioner, og kommunikerer med andre væv til at organisere deres aktiviteter i forbindelse med udvikling. At karakterisere sådanne specialiserede cellulære funktioner og inter-organ-vekselvirkninger, skal vi angive en gruppe af celler sammen med andre typer af celler holdes intakt i flercellede arkitektur. Et eksempel på sådanne specialiserede organer er en steroidogeniske organ, hvor mange biosynteseenzymer medierer trinene konvertering fra cholesterol til aktive steroidhormoner 1. De fleste af disse enzymgener udtrykkes specifikt i steroidogene organer og biosyntesevejen er stramt reguleret af mange ydre stimuli via humorale input og neuronale input. Enkelt gangsyntetiserede, steroidhormoner udskilles i hæmolymfe og er målrettet mod mange væv og organer til regulering af ekspression af en række gener 2. Derfor virkningen af ​​et steroid hormon inducerer en systemisk reaktion på opretholde homøostase, vækst og reproduktion. For at undersøge funktionerne af steroidhormon biosyntese og pleiotropiske virkninger af steroidhormoner, kan Drosophila melanogaster anvendes som et egnet modelsystem. Under larvestadierne, insektet steroidhormon, ecdysteroid, biosyntetiseres i en specialiseret endokrine organ kaldet prothoracic kirtel (PG) 3. I PG, flere ecdysteroidogenic enzymer specifikt katalyserer de multiple omdannelsestrin fra cholesterol til ecdyson, som styrer molting og metamorfose på de passende udviklingsstadier 4. Derfor er en dynamisk ændring i ecdysteroid titer reguleretaf mange signalveje som reaktion på miljømæssige signaler. På den anden side, i det voksne stadium, ecdysteroid spiller væsentlige roller i fysiologi, herunder reproduktion, søvn, hukommelse og levetid 5, 6, 7, 8. Det er kendt, at ecdysteroid aktivt biosyntetiseres i ovariet, regulering af progressionen af oogenesen 6, 7, 8, 9, 10, 11. For nylig har vi rapporteret, at antallet af germlinie stamceller (GSCS) påvirkes af ecdysteroid og køn peptid signalering som respons på parring stimuli 12. Kraftfulde værktøjer D. melanogaster genetik og cellebiologi, herunder godt kommenteret genom information, binær genekspressionssystemer og transgene RNAi teknikker, har gjort det muligt at identificere gener essentielle for ecdysteroid biosyntese i PG og ovarie 13, 14, 15. Når ecdysteroidogenic gener er identificeret, kan den transkriptionelle regulering af disse gener og de dynamiske lokaliseringer af genprodukter undersøges i biosyntesevejen 16. Til dette formål, kvantitativt-revers transkription-PCR, RNA in situ hybridisering og immunohistologisk analyse udføres. Anvendelsen af ​​disse teknikker omfatter en udfordrende opgave; den omfattende dissektion af PG eller æggestokkene. Især PG af bananfluen er relativt mindre end for andre insekter (f.eks avlen og spyflue), så er man nødt til at praktisere den vitale færdigheder af frugt flyve dissektion for prøvetagning. Endvidere begge ecdysteroidogenic organer modtager innervations fra centralnervesystemet (CNS) 17, 18, 19, 20. Således for nøjagtige anatomiske analyser, de ecdysteroidogenic organer bør holdes intakt sammen med CNS og andre organer, for ikke at forstyrre deres neuronale forbindelser. Her giver vi protokoller for dissektion og visualisering af steroidogene organer i D. melanogaster. At lære dissektion teknikken er det centrale udgangspunkt for disse eksperimenter. Derudover kan man med held mærke de steroidogene organer samt deres interaktive organer med flere antistoffer og GAL4 driver linjer. At udnytte disse teknikker, materialer og genetik, kan man studere de omfattende mekanismer steroidhormon biosyntese.

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			<td>egg collection</td>
			<td></td>
			<td></td>
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			<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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			<td>collection cup</td>
			<td>HIKARI KAGAKU</td>
			<td></td>
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			<td>yeast paste</td>
			<td>Oriental dry yeast, Tokyo</td>
			<td></td>
			<td></td>
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			<td>100% grape juice</td>
			<td>Welch Food Inc.</td>
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			<td></td>
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			<td>rearing larvae</td>
			<td></td>
			<td></td>
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			<td>small vials (12ml, 40&#215;23.5 mm, PS)</td>
			<td>SARSTEDT</td>
			<td>58.487</td>
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			<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

I flercellede organismer, er en lille gruppe af celler udstyret med en specialiseret funktion i deres biogene aktivitet, inducere en systemisk reaktion på vækst og reproduktion. I insekter, larve prothoracic kirtel (PG) og de voksne kvindelige æggestok play væsentlige roller i biosyntese de vigtigste steroid hormoner kaldet ecdysteroid. Disse ecdysteroidogenic organer innerveret fra nervesystemet, hvorigennem timingen af ​​biosyntesen påvirkes af miljømæssige signaler. Her beskriver vi en protokol til visualisering ecdysteroidogenic organer og deres interaktive organer i larver og voksne i bananfluen Drosophila melanogaster, som giver en egnet modelsystem til at studere steroid hormon biosyntese og dens reguleringsmekanisme. Dygtige dissektion giver os mulighed for at opretholde holdninger ecdysteroidogenic organer og deres interaktive organer, herunder hjernen, den ventrale nerve ledning, og andre væv. Immunfarvning med enntibodies mod ecdysteroidogenic enzymer, sammen med transgene fluorescens-proteiner drevet af vævsspecifikke promotorer, er tilgængelige til at mærke ecdysteroidogenic celler. Desuden kan de innervationer af ecdysteroidogenic organer også mærkes med specifikke antistoffer eller en samling af GAL4 bilister i forskellige typer af neuroner. Derfor kan ecdysteroidogenic organer og deres neuronforbindelser visualiseres samtidigt af immunfarvning og transgene teknikker. Endelig beskriver vi, hvordan at visualisere kimcellelinje stamceller, hvis proliferation og vedligeholdelse styres af ecdysteroid. Denne fremgangsmåde bidrager til omfattende forståelse af steroidhormon biosyntese og dens neuronal reguleringsmekanisme.

Vi brugte de ovennævnte protokoller til at visualisere steroidogene organer og deres interaktive organer i D. melanogaster larver og voksne kvinder. Den samlede ordning af protokoller er vist i figur 1. RG, herunder PG (figur 2D), er mindre og mere gennemsigtige end hjernen og er placeret ved den forreste-dorsale side af hjernen (figur 2A-C og 3A-E). At mærke PG-celler har adskillige grupper genereret forskellige typer af antistoffer mod ecdysteroidogenic...

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

Vi beskriver en protokol for dissektion, fiksering og immunfarvning af steroidogene organer i Drosophila larver og voksne kvinder at studere steroidhormon biosyntese og dens reguleringsmekanisme. Foruden steroidogene organer, vi visualisere innervation steroidogene organer samt steroidogene målceller såsom germlinie stamceller.

Protokoller til visualisering steroidogeniske organer og deres interaktive organer med Immunfarvning i bananfluen<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

Analyse blodlegemer befolkninger i<em&gt; Drosophila melanogaster</em&gt; Larver

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

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Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions

Opdræt bananfluen<em&gt; Drosophila melanogaster</em&gt; Under Axeniske og gnotobiotiske Betingelser

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

Visualisere Interrenal steroidogeniske Tissue og dens Vaskulær mikromiljø i Zebrafisk

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

High-Resolution Episcopic Microscopy (HREM) - Simple og robuste protokoller til behandling og visualisering af organiske materialer

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

Dissektion og immunfluorescent farvning af champignon krop og fotoreceptor neuroner i voksen Drosophila melanogaster hjerner

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

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Hele-mount Clearing og farvning af Arabidopsis blomst organer og 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

Celle sammenlægning Assays til at evaluere bindingen af Drosophila Notch med Trans-ligander og dens hæmning af Cis-ligander

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

Tre-dimensionelle gengivelse og analyse af Immunolabeled, klarere menneskelige placenta villøs vaskulære netværk

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

Tre-dimensionelle Organotypic kulturer af vestibulære og auditive sanseorganer

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

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