Summary
Abstract
Introduction
Protocol
Representative Results
Discussion
Acknowledgements
Materials
References
Developmental Biology
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。
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。
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....
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。
....Name | Company | Catalog Number | Comments |
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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