Live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos.
Live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos.
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DOI：
10.3791/1726
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发表时间：
2010-02-03
期刊：
影响因子：
--
通讯作者：
Halloran, Mary
中科院分区：
文献类型：
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作者：
Andersen, Erica;Asuri, Namrata;Halloran, Mary
斑马鱼是体内发育过程中细胞行为成像的理想模型。斑马鱼胚胎是外部受精的，因此在发育的各个阶段都很容易获得。此外，它们的光学清晰度允许在完整胚胎的自然环境中对细胞和分子动力学进行高分辨率成像。我们正在使用实时成像方法来分析神经嵴细胞迁移和神经轴突生长和引导过程中的细胞行为。实时成像对于理解调节细胞运动过程的机制特别有用。为了可视化细胞运动的细节，如突出活动和分子动力学，标记单个细胞是有利的。在斑马鱼中，质粒DNA注射产生瞬时马赛克表达模式，与其他细胞标记方法相比具有明显的优势。例如，转基因细胞系通常标记整个细胞群，因此可能模糊单个细胞中细微突起（或分子分布变化）的可视化。此外，在单细胞阶段注射DNA比在后期注射染料侵入性更小，更精确。在这里，我们描述了一种方法来标记个体发育神经元或神经嵴细胞和成像他们的行为在体内。我们将质粒DNA注入到1细胞期胚胎中，导致嵌合转基因的表达。载体包含细胞特异性启动子，驱动感兴趣的基因表达在一个子集的感觉神经元或神经嵴细胞。我们提供了用膜靶向GFP或生物传感器探针标记细胞的例子，该探针允许在活细胞中可视化f -肌动蛋白。
The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons. Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages. Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells.