协议的可视化类固醇机关及其互动器官在果蝇免疫染色<em>果蝇</em
Summary
我们描述了解剖，固定，并在果蝇幼虫和成年女性类固醇合成器官，研究类固醇激素的生物合成及其调控机制的免疫染色的协议。除了类固醇合成器官，我们可以形象地类固醇合成器官的神经支配和类固醇合成的靶细胞，如生殖系干细胞。
Abstract
在多细胞生物体中，一小群细胞被赋予了在其生物活性的专门功能，诱导生长繁殖全身反应。在昆虫幼虫前胸腺（PG）和生物合成的主要类固醇激素称为蜕皮类的成年女性卵巢发挥重要作用。这些ecdysteroidogenic器官来自神经系统，通过它合成的时间是由环境因素影响的支配。在这里，我们描述了可视化ecdysteroidogenic机关和幼虫它们之间的互动器官和果实蝇成虫果蝇 ，这为研究类固醇激素的生物合成及其调控机制的合适模型系统的协议。熟练的解剖，我们保持着ecdysteroidogenic器官的位置和它们互动的器官，包括大脑，腹神经索和其他组织。用免疫染色针对ecdysteroidogenic酶ntibodies，通过组织特异性启动子的转基因驱动的荧光蛋白质一起，是可用来标记ecdysteroidogenic细胞。此外，ecdysteroidogenic器官的支配，也可以通过特异性抗体或多种类型的神经元GAL4驱动程序的集合标记。因此，ecdysteroidogenic机关及其神经元连接可以同时通过免疫组化和转基因技术，可视化。最后，我们将介绍如何可视生殖干细胞，其增殖和维护由蜕皮类控制。这种方法有助于类固醇激素的合成及其神经调节机制的全面了解。
Introduction
在多细胞生物，一组单元被赋予了在其生物活性的特殊功能是整个人体不可缺少的。为了履行自己的使命，每一个组织或器官表达了一系列有关其功能的基因，并与其他组织进行通信，以协调在发展的背景下开展活动。为了描述这种专门的细胞功能和脏器间的相互作用，我们需要与其他类型的细胞被保持完整的多架构沿着指定一组细胞。
这种专门的器官的一个例子是类固醇合成器官，许多生物合成酶介导的转化步骤从胆固醇活性类固醇激素1。大多数这些酶的基因的类固醇在器官特异性表达，并且所述生物合成途径被紧紧地通过经由体液输入和神经元的输入许多外部刺激调节。一旦合成类固醇激素分泌到血淋巴和靶向许多组织和器官，用于调节各种基因2的表达。因此，类固醇激素的作用诱导全身性反应保持动态平衡，生长和繁殖。
为了研究类固醇激素的生物合成的功能和类固醇激素的多效性的动作， 果蝇可被用作一个合适的模型系统。在幼虫期，昆虫类固醇激素，蜕皮甾类，在专门的内分泌器官生物合成称为前胸腺（PG）3。在PG，几个ecdysteroidogenic酶特异性催化多个转换步骤从胆固醇蜕皮激素，其控制在适当的发育阶段4蜕皮和变态。因此，在蜕皮甾体滴度的动态变化被调节通过响应于环境暗示许多信号传导途径。在另一方面，在成人阶段，蜕皮甾类起着生理学重要作用，包括复制，睡眠，记忆和寿命5，6，7，8。已知的是，蜕皮甾类正在积极生物合成在卵巢，调节卵子发生6，7，8，9，10，11的进展。最近，我们报道了生殖系干细胞（GSC中）的数目由蜕皮激素和性别肽信令响应于配合影响刺激12。
D.功能强大的工具果蝇遗传学和细胞生物学，包括注释良好的基因组信息，二进制基因表达系统和转基因的RNAi技术，使我们能够鉴定基因必需在PG和卵巢13，14，15到蜕皮甾类生物合成。一旦ecdysteroidogenic基因鉴定，这些基因和基因产物的动态本地化的转录调控可以在生物合成途径16进行检查。为了这个目的，定量反转录PCR，RNA 原位杂交，免疫组织学和分析中进行。这些技术的应用包括一项艰巨的任务;在PG或卵巢的精细解剖。特别地，果蝇的PG比其他昆虫的相对较小（ 例如 ，蚕和吹飞），因此人们需要能够实践水果的至关重要的技能飞清扫采样。此外，无论是ecdysteroidogenic机关收到支配期从中枢神经系统（CNS）17，18，19，20。因此，准确的解剖分析，在ecdysteroidogenic机关应保持与中枢神经系统和其他器官，不破坏他们的神经连接沿完好无损。
在这里，我们提供的解剖和D中类固醇合成器官的可视化协议。 果蝇。学习解剖技术是这些实验的关键起点。此外，可以成功地标记类固醇合成器官以及它们之间的互动与器官的几种抗体和GAL4驱动程序行。以这些技术，材料和遗传学的优势，可以研究类固醇激素合成的综合机制。
Protocol
注：协议的总体方案示于图1。 1.幼虫环压盖的解剖（RG） 注：在D中。 黑腹果蝇 ，属于cyclorrhaphous双翅目，在PG是内复合内分泌器官称为环腺（RG， 图2D）。既然是不可行的是，PG被手术从其它类型的细胞（稍后讨论）的分离，一个实际的目标是通过解剖分离完整的，未损坏的RG。 幼…
Representative Results
我们使用了上述协议，以可视化类固醇合成器官和果蝇幼虫和成年女性的互动器官。协议的总体方案示于图1。 的RG，包括PG（ 图2D），是比大脑更小和更透明的，并且位于大脑（ 图2A-C和3A-E）的前侧-背侧。以标记PG细胞，几组都产生各种类型的针对ecdysteroidogenic酶的抗体（ 即 ，梦幻岛23，Spookier 24，…
Discussion
我们研究了蜕皮类固醇的生物合成和果蝇及其调控机制，并制定了解剖和免疫染色的协议。蜕皮甾类生物合成的定时是通过神经元输入33受环境线索，所以它是用脑，VNC，和解剖过程中其他组织沿着保持ecdysteroidogenic器官的神经支配必不可少的。
如上所述， 黑腹果蝇 PG形成与语料库allata（CA）和所述语料库cardiaca（CC）（ ?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢赖科·凯泽和Tomotsune Ameku他们为这项工作提供技术支持。我们也感谢凯·托，奥尔加·亚历克西科，明子江东，马赛基·米拉，布鲁明顿果蝇库存中心，京都库存中心（DGRC），以及发展研究杂交瘤细胞银行股票和试剂。这项工作是由日本学术振兴会KAKENHI授权号16K20945，内藤基金会，和井上学研究奖授予YSN支持;并通过文部科学省从格兰特KAKENHI数16H04792一笔赠款，RN。
Materials
| egg collection | |||
| tissue culture dish (55 mm) | AS ONE | 1-8549-02 | for grape-juice agar plates |
| collection cup | HIKARI KAGAKU | ||
| yeast paste | Oriental dry yeast, Tokyo | ||
| 100% grape juice | Welch Food Inc. | ||
| rearing larvae | |||
| small vials (12ml, 40×23.5 mm, PS) | SARSTEDT | 58.487 | |
| disposable loop | AS ONE | 6-488-01 | |
| standard fly food | the recepi us on the website of Blooington stock center. | ||
| dissection | |||
| dissecting microscope | Carl Zeiss | Stemi 2000-C | |
| dissecting microscope | Leica | S8 AP0 | |
| tissue culture dish (35 x 10 mm, non-treated) | IWAKI | 1000-035 | |
| Sylgard | TORAY | coarting silicon inside dishes | |
| Terumo needle (27G, 0.40 x 19 mm) | TERUMO | NN-2719S | A "knife" to cut the tissue |
| Terumo syringe, 1ml | TERUMO | SS-01T | |
| forceps, Inox, #5 | Dumont, Switzerland | ||
| insect pin (0.18 mm in diameter) | Shiga Brand | for fillet dissection | |
| micro scissors | NATSUME SEISAKUSHO CO LTD. | MB-50-10 | |
| fixation | |||
| ultrapure water | Merck Millipore | ||
| phosphate buffered saline (PBS) | |||
| Formaldehyde | Nacalai tesque | 16222-65 | |
| Paraformaldehyde | Nacalai tesque | 02890-45 | |
| Triton-X100 | Nacalai tesque | 35501-15 | |
| microtubes (1.5 ml) | INA OPTIKA | CF-0150 | |
| Incubation | |||
| As one swist mixer TM-300 (rocker) | As one | TM-300 | rocker |
| Bovine Serum Albumin | SIGMA | 9048-46-8 | |
| primary antibody | |||
| anti-Sro (guinea pig), 1:1000 | |||
| anti-GFP (rabbit), 1:1000 | Molecular Probes | A6455 | Shimada-Niwa ans Niwa, 2014 |
| anti-GFP (mouse mAb, GF200), 1:100 | Nakarai tesque | 04363-66 | |
| anti-5HT (rabbit), 1:500 | SIGMA | S5545 | |
| anti-Hts 1B1 (mouse) | Developmental Studies Hybridoma Bank (DSHB) | 1B1 | |
| anti-DE-cadherin (rat), 1:20 | DSHB | DCAD2 | |
| anti-nc82 (mouse), 1:50 | DSHB | nc82 | |
| secondary antibody | |||
| Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate | Life Technologies | A-11008 | |
| Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate | Life Technologies | A-11001 | |
| Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 546 conjugate | Life Technologies | A-11081 | |
| Goat anti-Guinea Pig IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate | Life Technologies | A-21435 | |
| Alexa Fluor 546 dye-conjugated phalloidin | Life Technologies | A-22283 | |
| Mounting reagents | |||
| Micro slide glass | Matsunami Glass Ind.,Ltd. | SS7213 | |
| Square microscope cover glass | Matsunami Glass Ind.,Ltd. | C218181 | |
| FluorSave reagent (Mounting reagent) | Calbiochem | 345789 | |
| Transfer pipette 1 ml (Disposable dropper) | WATSON | 5660-222-1S | |
| imaging | |||
| LSM700 laser scanning microscope system | Carl Zeiss | inverted Axio Observer. Z1 SP left | |
| image processing | |||
| LSM700 ZEN | Carl Zeiss | It is a special user interface based on the 64 bit Microsoft Windows7 operating system | |
| ImageJ | |||
| Fly stocks | |||
| w; GMR45C06-GAL4 | from Bloomington Drosophila Stock Center. (#46260) | ||
| UAS–GFP; UAS–mCD8::GFP | gifts from K. Ito, The University of Tokyo. | ||
| w[1118] | |||
| w; phantom-GAL4#22/UAS-turboRFP | |||
| w; UAS-mCD8::GFP; TRH-GAL4 | see in Ref29, Alekseyenko, O. V, Lee, C. & Kravitz, E. A.(2010) | ||
| w; UAS-mCD8::GFP | from Bloomington Drosophila Stock Center. (#32188) | ||
| yw;; nSyb-GAL4 | from Bloomington Drosophila Stock Center. (#51941) |
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Citar este artigo
Imura, E., Yoshinari, Y., Shimada-Niwa, Y., Niwa, R. Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster. J. Vis. Exp. (122), e55519, doi:10.3791/55519 (2017).