Name: Dissection of Drosophila melanogaster Third Instar Larval Brain for Live-Cell Imaging
Uploaded: 2023-06-23T12:40:00
Description: Watch this Scientific Journal Video about Dissection of Drosophila melanogaster Third Instar Larval Brain for Live-Cell Imaging at JoVE.com

dissection of drosophila melanogaster third instar larval brain for live-cell imaging

来自 黑腹果蝇 第三龄幼虫的活脑外植体成像

Asymmetric cell division (ACD) is the process by which subcellular components such as RNA, proteins, and organelles are partitioned unequally between daughter cells1,2. This process is commonly seen in stem cells, which undergo ACD to give rise to daughter cells with different developmental fates. Drosophila NBs divide asymmetrically to produce one NB, which retains its stemness, and one ganglion mother cell (GMC). The GMC undergoes further divisions to produce differentiating neurons or glia3. Asymmetrically dividing NBs are abundant in the developing brains of third-instar larvae, which are readily observed via microscopy. At the third instar larval stage, there are roughly 100 NBs present in each central brain lobe3,4,5,6.
Asymmetric cell division is a highly dynamic process. Live-cell imaging protocols have been used to measure and quantify the dynamics of cell polarity7,8,9,10, spindle orientation11,12,13, the dynamics of the actomyosin cortex14,15,16,17,18, microtubule and centrosome biology19,20,21,22,23,24,25,26,27, and membrane10,28 and chromatin dynamics29. Qualitative and quantitative descriptions of ACD rely on robust methods and protocols to image dividing NBs in intact living brains. The following protocol outlines methods to prepare, dissect, and image third instar larval brains for live-cell imaging in vivo using two different mounting approaches. These methods are best suited for researchers interested in the spatiotemporal dynamics of stem cell divisions, as well as divisions in other brain cells, as they allow for short- and long-term observations of cellular events. Additionally, these techniques are readily accessible to newcomers to the field. We demonstrate the effectiveness and adaptability of this approach with larval brains expressing fluorescently tagged microtubule and cortical fusion proteins. We additionally discuss methods of analysis and considerations for application in other studies.

作者聚焦：研究不对称细胞分裂动力学：果蝇幼虫脑外植体实时成像的方案 

黑腹果蝇第三龄幼虫脑的活细胞成像

Our research focuses on studying the impact of asymmetric cell division or ACD, on stem cell proliferation and differentiation. We employ Drosophila neural stem cells or neuroblasts as a model to understand the mechanisms, regulations, and impacts of asymmetric cell division on development and neurogenesis. The recent developments in artificial intelligence have been making major strides in image quantification and analysis, which has significantly advanced the field of life cell imaging.Maintaining sample quality for a long-term movie is a long-standing challenge. While it's insightful to observe a sample for eight to 10 hours, logistical constraints, like the sample's imaging requirements and available imaging tools, make it difficult. Our protocol addresses this by demonstrating long-term imaging without impacting the sample's quality.Our technique could be easily adopted and applied to the field by newcomers. With some time and practice, one can feasibly generate short and long-term movies, which have the potential to yield insightful and meaningful data.

Watch this Scientific Journal Video about Dissection of Drosophila melanogaster Third Instar Larval Brain for Live-Cell Imaging at JoVE.com

Dissection of Drosophila melanogaster Third Instar Larval Brain for Live-Cell Imaging  

解剖黑腹果蝇第三龄幼虫脑进行活细胞成像

首先将约400微升的解剖和成像介质移液到三孔解剖培养皿的每个孔中。在解剖显微镜下工作，在解剖和成像介质中洗涤实验幼虫以去除食物残渣。在继续在解剖显微镜下工作的同时，用一组镊子用嘴钩握住幼虫。用另一组镊子，小心地从后侧切掉大约 1/3 的幼虫。接下来，在用一组镊子抓住嘴钩的同时，用另一组镊子轻轻地将角质层刷向口钩。同时，用镊子夹住口钩向内推，直到整个幼虫翻过来。倒置幼虫以暴露中枢神经系统和其他组织，同时保持它们与角质层的连接。定位中枢神经系统（CNS），以避免意外移除。使用镊子轻轻去除非中枢神经系统组织，仅保持中枢神经系统和大脑附着在角质层上。通过切断轴突连接将大脑从角质层释放出来，使用显微切割剪刀，在脑叶下方轻轻切割。然后，切断腹侧神经索下的连接。分批解剖幼虫，将解剖时间保持在20分钟以内。将解剖的大脑转移到包含解剖和成像介质的孔中。对于持续三个小时的成像，用脂肪体补充培养基。

Research

JoVE Journal

Developmental Biology

Dissection of Drosophila melanogaster Third Instar Larval Brain for Live-Cell Imaging

Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brains

Drosophila neural stem cells (neuroblasts, NBs hereafter) undergo asymmetric divisions, regenerating the self-renewing neuroblast, while also forming a ...

科研

发育生物学

﻿Staging and Collecting Drosophila melanogaster Third Instar Larvae

Staging and Collecting Drosophila melanogaster Third Instar Larvae  

分期和收集黑 腹果蝇 第三龄幼虫

Fat Body Collection from Drosophila melanogaster Third Instar Larvae

Fat Body Collection from Drosophila melanogaster Third Instar Larvae  

黑腹果蝇第三龄幼虫的脂肪体收集

Mounting and Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brain

Mounting and Live-Cell Imaging of Drosophila melanogaster Third Instar Larval Brain  

黑腹果蝇第三龄幼虫脑的安装和活细胞成像