fat body collection from drosophila melanogaster third instar larvae

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

Watch this Scientific Journal Video about Fat Body Collection from Drosophila melanogaster Third Instar Larvae at JoVE.com

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

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。顕微鏡下で、10匹の十分に給餌された野生型の幼虫を解剖鉗子でそっと保持し、一番下の解剖媒体に出し入れして洗浄し、すべての食物粒子を取り除きます。解剖顕微鏡下で作業を続けながら、きれいな幼虫を真ん中の井戸に移します。1セット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、幼虫のキューティクルの片側を破裂させて脂肪体をこぼします。鉗子を使用して、各幼虫からできるだけ多くの脂肪体を収集し、室温で400マイクロリットルの解剖培地を含む一番上のウェルに移します。

fat-body-collection-from-drosophila-melanogaster-third-instar-larvae

Research

JoVE Journal

Developmental Biology

Fat Body Collection from Drosophila melanogaster Third Instar Larvae

1.1K ビュー. 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。顕微鏡下で、10匹の十分に給餌された野生型の幼虫を解剖鉗子でそっと保持し、一番下の解剖媒体に出し入れして洗浄し、すべての食物粒子を取り除きます。解剖顕微鏡下で作業を続けながら、きれいな幼虫を真ん中の井戸に移します。1セットのピン...

ビデオ: Fat Body Collection from Drosophila melanogaster Third Instar Larvae  

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 differentiating ganglion mother cell (GMC), which will undergo one additional division to give rise to two neurons or glia. Studies in NBs have uncovered the molecular mechanisms underlying cell polarity, spindle orientation, neural stem cell self-renewal, and differentiation. These asymmetric cell divisions are readily observable via live-cell imaging, making larval NBs ideally suited for investigating the spatiotemporal dynamics of asymmetric cell division in living tissue. When properly dissected and imaged in nutrient-supplemented medium, NBs in explant brains robustly divide for 12-20 h. Previously described methods are technically difficult and may be challenging to those new to the field. Here, a protocol is described for the preparation, dissection, mounting, and imaging of live third-instar larval brain explants using fat body supplements. Potential problems are also discussed, and examples are provided for how this technique can be used.

Here, we discuss a workflow to prepare, dissect, mount, and image live explant brains from Drosophila melanogaster third instar larvae to observe the cellular and subcellular dynamics under physiological conditions.

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