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
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
Representative Results
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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