Summary

分析<em>在体内</em>细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Published: April 29, 2016
doi:

Summary

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Abstract

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Introduction

在多细胞生物体,细胞迁移都为在那里它确保细胞的组织成组织和器官,胚胎的发育和成年生活中,在那里它需要一部分组织稳态(伤口愈合)和免疫是至关重要的。除了这些生理功能,细胞迁移也是参与多种病理情况,包括,尤其是癌症转移。

细胞迁移已在体外进行了分析了几十年,提供的分子机制确保在平坦的表面细胞运动的全面理解。 体内然而,细胞通过更复杂的环境面对。它清楚地出现在过去的几年中,一个生物体内迁移可能由外部线索的影响,如细胞外基质中,相邻引导迁移细胞或分泌的趋化因子,并且驱动细胞迁移可能与已描述改变机制<EM>体外1,2。确保在体内细胞迁移的机制已经很少受到关注,到目前为止,主要是增加的技术难度,因为,相对于体外研究, 在体内特别是细胞迁移的分析需要对迁移细胞直接的光学连接,技术标签在独特的细胞为了看到其动力学和形态学,以及增益或功能丧失方法来测试候选基因的作用。迄今,携带这些特征仅几个模型系统已被用于在体细胞迁移3解剖。

我们最近使用准脊索前板的迁移早期斑马鱼胚胎作为一种新的方便的模型系统,以评估在体内细胞迁移4,5-控制候选基因的功能。准脊索前板(也称为前内胚层)是在GAST发作形成一组单元rulation对胚胎的背侧。在原肠胚形成该组共同迁移朝向胚胎6-8的动物极,以形成脊索前板,一个mesendodermal增厚,前方脊索和神经板底层。的脊索前板的前部将产生孵化腺体,而它的后部可能有助于头部中胚层9。由于外部发展和鱼胚胎的光学清晰度,细胞迁移,可直接和容易地在该结构中观察到。

细胞移植是一种非常有效的技术,它允许快速和方便地创建镶嵌胚10。表达在分离的细胞中的标记移植的细胞的结果的荧光骨架标记物,形态和动力学,其中可以容易地观察到。这个组合损失或功能增益接近许可证一罐的细胞自治功能分析didate基因。

所提出的协议描述了我们如何在体内 5控制细胞迁移和肌动蛋白动力学评估了TORC2 SIN1组件的功能。但是,如在结果中提到和进一步讨论的,它可用于分析任何候选基因的潜在含义在体内控制细胞迁移。

Protocol

注意: 图1呈现了协议的轮廓。 1.注射和移植针的制备方法注意:针可以在任何时间来制备和储存。让他们在培养皿中,在橡皮泥带。密封封口膜防尘保护的菜。 对于注射针,拉玻璃毛细管(外径1.0mm时,直径0.58毫米里面,没有灯丝(见材料清单))用微量拉马(见材料清单)。喜欢短而细的针(约5mm锥形部…

Representative Results

使用所提出的技术来分析SIN1,Tor的络合物2(TORC2)的核心组件中的一个的作用,在体内细胞迁移的控制。利用细胞移植允许分离的细胞和细胞自治效果分析的标签。电影S1示出了移植脊索前板祖细胞的迁移。与肌动蛋白ABP140标签允许富含肌动蛋白胞质突起的方便的可视化。我们测量了它们的频率和方向。野生型细胞产生的动物极的方向取向频繁大胞质突起, 即</…

Discussion

这个协议提供了一个简单的方法在体内 ,通过组合使用细胞移植实时成像嵌合体胚胎的创建来研究细胞迁移的候选基因的作用。

马赛克胚胎的创建

研究细胞的动态要求其轮廓的可视化分析细胞质扩展。或不同标记 – – 环境,从而提供良好的视觉对比这可以通过标签在其它方面未标记的细胞分离来实现。

一种简单的方法,以随…

Divulgations

The authors have nothing to disclose.

Acknowledgements

We thank F. Bouallague and the IBENS animal facility for excellent zebrafish care. Research reported in this publication was supported by the Fondation ARC pour la recherche sur le cancer, grants N° SFI20111203770 and N° PJA 20131200143.

Materials

Glass capillaries (outside diameter 1.0 mm, inside diameter 0.58 mm) Harvard Apparatus 300085 standard thickness
Glass capillaries (outside diameter 1.0 mm, inside diameter 0.78 mm) Harvard Apparatus 300085 thin-walled
Penicillin-Streptomycin Sigma-Aldrich P4333 10 000 units penicillin and 10 mg streptomycin per ml
fine tweezers Dumont Fine Science Tools 11254-20 5F
glass bottom dishes MatTek P35G-0-10-C
Air transjector Eppendorf 5246
Micro-forge Narishige MF-900
Microgrinder Narishige EG-44
Micromanipulator (for injection) Narishige MN-151
Micromanipulator (for cell transplantation) Leica Leica Micromanipulator
Hammilton Syringe Narishige IM-9B
Micropipette puller David Kopf Instruments Model 720
Transplantation mold Adapative Science Tools PT-1
Needle holder Narishige HI-7
Tube connector Narishige CI-1
PTFE tubing Narishige CT-1

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Giger, F. A., Dumortier, J. G., David, N. B. Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos. J. Vis. Exp. (110), e53792, doi:10.3791/53792 (2016).

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