在鸡胚神经诱导的含量为
Summary
神经诱导是在大脑形成的第一步。它是一种机制，使恒胜的节点（组织者），指示相邻的组织采用了神经的命运，即会引起神经系统。本视频演示了在鸡胚神经诱导实验。
Abstract
鸡胚是一个有价值的工具，在早期胚胎发育的研究。其透明度，可获得性和易于操纵，使其为研究神经系统的形成和最初的图案的理想工具。本视频演示了如何移植到主机组织者组织，恒胜小号节点的方法是，（在鸡胚的组织者）被嫁接到一个主机主管外胚层。主办移植指示覆NA已经组织采取通过神经诱导信号的神经命运。这一机制被称为神经诱导，并构成了脊椎动物的大脑和脊髓的形成初始步骤。这种方法实质上是公认的在小鸡的神经诱导分子的特性。本视频演示了在神经诱导实验的不同步骤，首先，德纳胚胎explanted和寄托一道菜。然后，主机胚胎准备为新的文化。移植是切除和移植到主机领域的透明带保证金。主机是培养18-22小时。大会是固定的，为进一步应用（例如原位杂交）处理。这种方法最初设计由沃丁顿<sup> 1,2</sup>和Gallera<sup> 3,4</sup>。
Protocol
一，原理概述： 本视频演示了在不同的步骤检测在鸡胚神经诱导。首先，主机的胚胎是explanted在新的文化[NI1]。然后，德纳胚胎是在生理盐水和恒胜的节点（在鸡的“组织者”）explanted与荧光染料DII标记[NI2]。恒胜的节点是从德纳胚胎切除[NI3]和主机的胚胎移植到[NI4，NI5]。主机…
Discussion
本视频演示了不同的步骤执行检测神经诱导，这种检测基本上是公认的在小鸡的神经诱导分子的特性，因此可以使用一个广泛的应用， 范围从胚胎 micromanipulations 1-4，; 6日至揭开新的信号级联反应，7,8，所有旨在了解初步形成大脑和神经系统的其余步骤。
Acknowledgements
DP是从国家药物滥用研究所露丝Kirschstein奖1F32 DA021977 – 01A1收件人。这项工作是支持的玛格丽特M. Alkek基金会RHF。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Eggs | Animal | Charles River Laboratories | Premium Fertile | Fertilized, HH3+ (14 hr) |
| Stereomicroscope | Microscope | Leica Microsystems | MZ9.5 or similar | |
| Hybridization Incubator | Equipment | Robbins Scientific | M1000 | Use with inverted Pyrex dish and 500 ml ddH2O beaker |
| Marsh Automatic Incubator | Equipment | Lyon | RX | |
| Pyrex dish | ||||
| Watchmaker’s glass 50mm | Tool | VWR | 66112-060 | |
| Glass rings | Tool | Physical Plant facility | cut 4 mm thick sections of glass tubing (27 mm outer diam, 25 mm inner diam). Do not fine polish. | |
| Curved Forceps (1) | Tool | Electron Microscopy Sciences | 72991-4C | |
| Forceps (2) | Tool | Fine Science Tools | 11002-13 | blunt ended using sharpening Stone and 100ul mineral oil |
| Fine scissors | Tool | Fine Science Tools | 14161-10 | |
| Plastic dishes | Tool | Falcon | 353001 | |
| Rubber Bulb | Tool | Electron Microscopy Sciences | 70980 | |
| DiI | Reagent | Invitrogen | D-282 | |
| Aspirator tube assembly | Tool | Sigma | A5177-5EA | |
| Microelectrode puller | Equipment | Sutter Instruments | Sutter Instruments P-97 Flaming/Brown Micropipette | |
| Pasteur Capillary Pipette | Tool | Electron Microscopy Sciences | 70950-12 | round edge under flame |
| Culture chamber | Tool | Pioneer Plastics | 030C | |
| Microcapillary tube | Tool | Sigma | P1049-1PAK | |
| Microdissecting knife | Tool | Fine Science Tools | 10056-12 | Use to puncture cavities prior to in situ hybridization |
| Minuten pins 0.2mm diam | Tool | Fine Science Tools | 26002-20 | Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C |
| Diethylpyrocarbonate (depc) | Reagent | Electron Microscopy Sciences | 15710 | |
| Sylgard 184 Silicon Elastomer Curing Agent and Base | Dow Corning | 0001986475 | Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C | |
| Diethylpyrocarbonate (depc) | Acros Organics | 10025025 | Add 1ml depc to 1l PBS; shake; autoclave | |
| 16% PFA | Electron Microscopy Sciences | 15710 |
References
- Waddington, C. H. Experiments on the development of chick and duck embryos, cultivated in vitro. Phil. Trans. R. Soc. Lond. B. 221, 179-230 (1932).
- Waddington, C. H. Induction by the primitive streak and its derivatives in the chick. J. Exp. Biol. 10, 38-46 (1933).
- Gallera, J. Excision et transplantation des différentes régions de la ligne primitive chez le poulet. C. R. Ass. Anat. 49, 632-639 (1964).
- Gallera, J. Primary induction in birds. Adv. Morph. 9. , 149-180 (1971).
- Serbedzija, G. N., Fraser, S. E., Bronner-Fraser, M. Pathways of neural crest cell migration in the mouse embryo as revealed by vital dye labeling. Development 108. , 605-612 (1990).
- Psychoyos, D., Stern, C. D. Restoration of the organizer after radical ablation of Hensen’s node and the anterior primitive streak in the chick embryo. Development 122. , 3263-3273 (1996).
- Joubin, K., Stern, C. D. Molecular interactions continuously define the organizer during the cell movements of gastrulation. Cell. 98, 559-571 (1999).
- Streit, A., Berliner, A. J., Papanayotou, C., Sirulnik, A., Stern, C. D. Initiation of neural induction by FGF signaling before gastrulation. Nature. , 406-474 (2000).
- Psychoyos, D., Finnell, R. Method for Culture of Early Chick Embryos ex vivo (New Culture. JoVE. 20, 10-3791 (2008).
Cite This Article
Psychoyos, D., Finnell, R. Assay for Neural Induction in the Chick Embryo. J. Vis. Exp. (24), e1027, doi:10.3791/1027 (2009).