Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
Graduate Program in Biophysics, Harvard University, Cambridge, MA, USA.
Methods Mol Biol. 2023;2626:219-232. doi: 10.1007/978-1-0716-2970-3_11.
Drosophila oogenesis is a powerful and tractable model for studies of cell and developmental biology due to the multitude of well-characterized events in both germline and somatic cells, the ease of genetic manipulation in fruit flies, and the large number of egg chambers produced by each fly. Recent improvements in live imaging and ex vivo culturing protocols have enabled researchers to conduct more detailed, longer-term studies of egg chamber development, enabling insights into fundamental biological processes. Here, we present a protocol for dissection, culturing, and imaging of late-stage egg chambers to study intercellular and directional cytoplasmic flow during "nurse cell dumping." This critical developmental process towards the latter stages of oogenesis (stages 10b/11) results in rapid growth of the oocyte and shrinkage of the nurse cells and is accompanied by dynamic changes in cell shape. We also describe a procedure to record high-time-resolution movies of the flow of unlabeled cytoplasmic contents within nurse cells and through cytoplasmic bridges in the nurse cell cluster using reflection microscopy, and we describe two ways to analyze data from nurse cell dumping.
果蝇卵子发生是研究细胞和发育生物学的强大而易于处理的模型，这是由于生殖细胞和体细胞中存在大量特征明确的事件、在果蝇中进行遗传操作的便利性，以及每只果蝇产生的大量卵室。最近，活体成像和离体培养方案的改进使研究人员能够对卵室发育进行更详细、更长期的研究，从而深入了解基本的生物学过程。在这里，我们介绍了一种用于晚期卵室的解剖、培养和成像的方案，以研究“滋养细胞倾倒”过程中的细胞间和定向细胞质流动。这个关键的发育过程发生在卵子发生的后期阶段（10b/11 期），导致卵母细胞的快速生长和滋养细胞的缩小，并伴随着细胞形状的动态变化。我们还描述了一种使用反射显微镜记录未标记的细胞质内容物在滋养细胞内以及通过滋养细胞簇中的细胞质桥的高速时间分辨率电影的过程，并且我们描述了两种分析滋养细胞倾倒数据的方法。
