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Neuroscience

手动排水的斑马鱼胚胎脑室

Published: December 16, 2012
doi:
10.3791/4243
,
1Department of Biology, Whitehead Institute of Biomedical Research,Massachusetts Institute of Technology

Summary

我们提出一个方法，收集脑脊液（CSF），并建立系统，缺乏的胚胎斑马鱼大脑脑室系统内脑脊液。 CSF的组成及其胚胎大脑发育过程中的要求，这使得进一步的检查。

Abstract

脑脊液（CSF）是一种富含蛋白质的脑室内所含的流体。这是目前早期脊椎动物的胚胎发育过程中，坚持在整个生命。成人CSF认为，以减轻大脑，排除废物，并进行分泌的分子1,2。在成人和年龄较大的胚胎中，大多数的CSF是由脉络丛，一系列的高度血管化的分泌位于脑室3-5相邻的区域。在斑马鱼，脉络丛完全形成受精后144小时（HPF）6。在此之前，在包括鼠标，一个显着量的胚胎CSF（eCSF）的斑马鱼和其他脊椎动物胚胎是本。这些数据和研究表明，在鸡的神经上皮分泌在开发的早期，可能是之前的主要来源eCSF发展脉络丛7。

eCSF包含了大约3倍以上的蛋白质吨韩成人CSF，这表明它可能具有重要作用，在发展8,9。研究表明，在小鸡和老鼠分泌因子eCSF，流体压力，或这些的组合，是非常重要的神经发生，基因表达，细胞增殖和细胞生存的神经上皮10-20中。人，大鼠，小鼠和小鸡eCSF的蛋白质组学分析，已经确定了许多蛋白质​​可能是必要的CSF功能。这些包括胞外基质成分，载脂蛋白，渗透压调节蛋白和蛋白参与细胞死亡和增殖21-24。然而，复杂的功能在很大程度上是未知的eCSF。

我们已经开发出一种方法，用于除去从斑马鱼脑室eCSF，从而允许识别eCSF组件，并在开发过程中进行分析的eCSF规定的。虽然可以采集更多eCSF，从其他脊椎动物系统W第i个较大的胚胎，eCSF可以收集到的斑马鱼发育的早期阶段，与遗传或环境条件下，导致脑室体积异常或形态。拆卸和回收的eCSF允许质谱分析，调查eCSF功能，并重新选择因素破入脑室分析它们的功能。因此，斑马鱼早期胚胎的可访问性可以在开发过程中的eCSF功能进行详细的分析。

Protocol

1。准备显微注射针和细胞电根据制造商的指示，填写Eppendorf公司CellTram油微量注射器装置与矿物油。 准备显微注射针拉毛细管萨特仪器针拔出器。 微操作机器人连接到Eppendorf公司CellTram上安装针。 小心打破针尖。为针尖大小均匀，用千分尺测量，或比较的参考针。 填写针的长度与油，使用CellTram，推动油价下跌的长度，小心，以避免任何气泡。 <p class="…

Representative Results

一个排水脑室的一个例子示出在图1B-C。作为他们缺乏eCSF的（ 图1B和C），脑室倒塌。可以看出在背侧的图像（ 图1B-C，以及图2A-D）的后脑神经上皮确实保留其特征的形态，而且似乎是开放，尽管缺乏eCSF可能是由于强大的铰接点。然而，横向次（ 图2A'-D'）表明已后脑脑室沥干，的存在下，一薄的柔性顶板上皮折叠腹侧一致。在?…

Discussion

使用这种技术的手动排水eCSF的从斑马鱼脑室用于确定eCSF在开发过程中的要求将是有益的。此外，这种技术将允许在胚胎发育过程中描述的eCSF蛋白更新。在此期间，不同的蛋白质的鉴定将能够进一步调查到CSF和大脑发育过程中的潜在作用的功能。在脊椎动物中，在eCSF（FGF2，IGF2，视黄酸和载脂蛋白）的一些因素有证明作用在神经上皮细胞存活，增殖和神经细胞13,17,20,23,27,28。然而，似乎有…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作是由美国国家精神卫生研究所和美国国家科学基金会的支持。特别感谢仁Gutzman博士，博士阿曼达迪金森和其他许多有益的讨论和建设性的批评的的西伯实验室成员，并奥利维尔Paugois专家鱼的饲养。

Materials

Name of Reagent Company Catalogue number
Eppendorf CellTram Oil Eppendorf 516 000.025
Mineral Oil Sigma M8410
Tricaine powder Sigma A5040
Capillary Tubes FHC Inc. 30-30-1
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Tags

Manual DrainageZebrafish Embryonic Brain VentriclesCerebrospinal FluidProtein-rich FluidVertebrate Embryonic DevelopmentChoroid PlexusHighly Vascularized Secretory RegionsNeuroepitheliumEmbryonic CSFECSFAdult CSFSecreted FactorsNeurogenesisGene ExpressionCell ProliferationCell SurvivalProteomic Analyses
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Chang, J. T., Sive, H. Manual Drainage of the Zebrafish Embryonic Brain Ventricles. J. Vis. Exp. (70), e4243, doi:10.3791/4243 (2012).
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