在完整的感觉器官前体细胞的活细胞成像果蝇

Neuroscience
 

Summary

在这段视频中,我们描述了完好的感觉器官祖细胞和表皮细胞不对称分裂的活细胞成像的方法

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Zitserman, D., Roegiers, F. Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae. J. Vis. Exp. (51), e2706, doi:10.3791/2706 (2011).

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Abstract

绿色荧光蛋白(GFP)的发现以来,一直存在着革命性的变化作为理解基本的生物机制工具使用中的活细胞成像。显着进展表现得尤为明显, 果蝇的突变体和转基因株系,其广泛的工具包提供了一个方便的模型来研究发育和细胞生物学机制的进化保守。我们有兴趣了解的机制,成人外周血在果蝇的神经系统(PNS)的控制细胞命运规范 。鬃,包括头部,胸部,腹部,腿部和成人飞的翅膀个别mechanosensory机关,并已研究为了解一流的依赖细胞命运的决定的机制的模型系统。感觉器官易制毒化学(专科)的microchaetes(或小刷毛)的细胞,是分布在整个蛹胸部的上皮细胞,并在pupariation发病后12小时指定。规范后,SOP的细胞开始分裂,分离麻木,在有丝分裂过程中的一个子细胞的细胞命运的决定因素。 Notch信号通路作为细胞自治抑制剂麻木的功能。

在这里,我们表现出遵循的完整的蛹胸部内的蛋白质动力学的SOP细胞和它的后代的使用结合的方法组织特定的Gal4的司机和GFP标记的融合蛋白1,2。这项技术在固定组织或培养的优势外植体,因为它允许我们遵循整个开发的终端器官的生长和分化的神经前体规格器官。因此,我们可以直接关联细胞行为的变化,在终末分化的变化。此外,我们还可以与repressible细胞标记(MARCM)系统的实时成像技术结合起来,以评估在有丝分裂的SOP标签蛋白突变或野生型条件下的动态马赛克分析。使用这种技术,我们和其他人透露不对称细胞分裂的调节和控制(例子包括引用1-6,7,8)的SOP细胞活化的Notch信号,新的见解。

Protocol

Discussion

Disclosures

没有利益冲突的声明。

Acknowledgements

Materials

Name Company Catalog Number Comments
Scotch double stick tape
Glass slide
18mmX18mm coverslip
Forceps
Whatman paper
Silicone vacuum grease Dow Corning
5 ml syringe
Pipetter
Confocal or epifluorescence microscope

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References

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