Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue
Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue
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DOI：
10.3791/60961
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发表时间：
2020-05-01
影响因子：
1.2
通讯作者：
Smyth, Jeremy T.
中科院分区：
文献类型：
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作者：
Karabasheva, Darya;Smyth, Jeremy T.
对活的、完整的组织和器官中细胞分裂进行实验分析，对于我们理解细胞分裂如何与发育、组织内稳态以及疾病过程相结合至关重要。正在进行减数分裂的果蝇精母细胞非常适合这种分析，因为（1）包含精母细胞的整个果蝇睾丸相对容易制备用于显微镜观察，（2）精母细胞体积较大，非常适合高分辨率成像，（3）强大的果蝇遗传工具可与体内分析相结合。在此，我们介绍一种易于操作的方案，用于从果蝇三龄幼虫和早期蛹制备整个睾丸。我们描述了如何在制备好的整个睾丸中识别减数分裂的精母细胞，以及如何通过延时显微镜对它们进行活体成像。还提供了固定和对整个睾丸进行免疫染色的方案。与使用成年睾丸进行精母细胞分析的现有方案相比，使用幼虫睾丸具有若干优势。最重要的是，幼虫睾丸比成年睾丸更小，细胞也不那么密集，这极大地有利于精母细胞的高分辨率成像。为了展示这些优势以及这些方案的应用，我们展示了通过延时共聚焦显微镜对单个精母细胞成像所得到的结果，即在细胞分裂过程中内质网相对于纺锤体微管的重新分布。这些方案可以与任意数量的荧光标记蛋白或细胞器标记物的表达相结合，也可以与基因突变及其他遗传工具相结合，使得这种方法在整个组织和器官的生理环境下对细胞分裂机制的分析特别有效。
Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes' large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.