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

Drosophila melanogaster Third Instar Larvaeのライブ脳外植片のイメージング

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.

著者スポットライト:非対称細胞分裂ダイナミクスの調査:ショウジョウバエ幼虫の脳外植片のライブイメージングのためのプロトコル 

ショウジョウバエメラノガスター第3齢幼虫脳の生細胞イメージング

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  

ショウ ジョウバエメラノガスター 第3齢幼虫脳の解剖による生細胞イメージング

まず、約400マイクロリットルの解剖およびイメージング媒体を3ウェル解剖皿の各ウェルにピペッティングします。解剖顕微鏡下で作業し、実験用幼虫を解剖およびイメージング媒体で洗浄して、食物残渣を除去する。解剖顕微鏡下で作業を続けながら、1組のピンセットを使用してマウスフックで幼虫を保持します。別のピンセットで、幼虫の約1/3を後側から慎重に切り取ります。次に、1組のピンセットでマウスフックで幼虫を持ちながら、もう1組のピンセットを使ってキューティクルをマウスフックに向かって優しくブラッシングします。同時に、幼虫全体が裏返しになるまで、マウスフックを保持しているピンセットで内側に押します。幼虫を反転させて、キューティクルへの接続を維持しながら中枢神経系や他の組織を露出させます。誤って除去しないように、中枢神経系(CNS)を見つけます。ピンセットを使用してCNS以外の組織をそっと取り除き、CNSと脳のみをキューティクルに付着させます。軸索接続を切断してキューティクルから脳を解放するには、マイクロダイセクションハサミを使用して、脳葉の下をそっと切ります。次に、腹側神経索の下の接続を切断します。幼虫をバッチで解剖して、解剖時間を20分未満に保ちます。解剖した脳を解剖および画像化媒体を含むウェルに移します。3時間以上続くイメージングの場合は、培地に脂肪体を補給します。

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 ...

発生生物学

﻿Staging and Collecting Drosophila melanogaster Third Instar Larvae

Staging and Collecting Drosophila melanogaster Third Instar Larvae  

ショウジョウバエメラノガスター第3齢幼虫の病期分類と収集

Fat Body Collection from Drosophila melanogaster Third Instar Larvae

Fat Body Collection from Drosophila melanogaster Third Instar Larvae  

ショウジョウバエメラノガスター第3齢幼虫からの脂肪体コレクション

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

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

ショウジョウバエメラノガスター第3齢幼虫脳のマウントと生細胞イメージング