解剖と実装<em>ショウジョウバエ</em>蛹アイディスク
Summary
The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.
Abstract
The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.
Introduction
キイロショウジョウバエ ：発達と細胞生物学の分野が大幅にモデル生物の影響を受けてきた。このモデルの中では、目のディスクの研究はシグナル伝達、細胞生物学やその他の分野に関する知識を大いに貢献してきました。後半に第三の幼虫齢目のディスクが広く研究し、それが発達期間の一連のスナップショットを与えるように、利用するための強力なモデルであるされている、独自のシグナリング分子、およびプロセスとのそれぞれが、形態形成溝は、目のディスクを横切って進むにつれて8。しかし、更なる発展の蛹の段階に発達過程の理解を拡大する必要性がある。蛹のアイディスク3-7の研究がなされてきた一方で、私たちの知識は3齢目のディスクに行われている仕事の幅広さに近づかない。これは、蛹のアイディスク解剖で大きな困難に一部起因している。したがって、プレゼンテーション解剖の適切な方法が大幅にこの分野の研究を展開することができます。
簡単に、特に中旬蛹期間を中心に、解剖された蛹のアイディスク開発中の段階がありますが、他の期間は、はるかに困難な解剖べきである。このプロトコルは、普遍的にすべての蛹の発育時間枠のために使用することができる蛹目ディスクを切開するための1つの方法を表している。このプロトコルは、midpupal時点から目ディスクを分析するための簡単かつ迅速に方法を示す別のプロトコル9の代替として使用することができる。このプロトコルは、もともと撮影さと機能ゲノミクス（URCFG）蛹眼の解剖の手法で10,11でUCLAの学部研究コンソーシアムで高度な学部学生を訓練するために開発されました。多くの学部学生がこのような厳しいテクニックを学ぶためにこのビデオと方法を利用することができました。
Protocol
Representative Results
Discussion
While it appears that the process is simple and easy to perform, in reality, this technique requires a great deal of practice to master. Routinely, we start students off by learning to dissect and mount third instar eye discs12, which are much easier to work with. This practice helps to develop an appropriate dissection position of the arms, hands and fingers13 so that manipulation of the forceps under the dissecting microscope is stable, easy and experienced. In essence, the practice period shou…
Declarações
The authors have nothing to disclose.
Acknowledgements
We appreciate and would like to thank the Howard Hughes Medical Institute for the HHMI Professor award to U.B. which made this project possible. We thank the college at the University of California, Los Angeles for providing facilities and teaching infrastructure support for this work. The work was also supported with funding from Midwestern University and a generous donation from the Charity Fidelity Gift Fund. We thank John VandenBrooks for comments on the manuscript and Krista Pearman for her technical assistance.
Materials
| Phosphate-buffered saline (PBS, pH 7.4) | 80g NaCl, 2g KCl, 14.4g Na2HPO4, 2.4g KH2PO4, Bring volume to 1 l, adjust the pH to 7.4, autoclave or filter sterilize, dilute to 1X PBS with autoclaved ddH2O before using. | ||
| Triton X-100 | Promega | H5142 | Caution: Irritant! Wear gloves. |
| 0.3% PBT | 1.5 ml of Triton X-100, 500 ml 1X PBS. | ||
| 37% formaldehyde solution | Fisher Scientific | F75P1GAL | Caution: Toxic, probable human carcinogen! Wear gloves. |
| Fix Solution | (≈4% Formaldehyde in PBS) 50 μl of 37% Formaldehyde, 450 μl 1XPBS, make fresh before use | ||
| Normal goat serum | Rockland antibodies & assays | B304 | Aliquot in 1 ml volumes and store at -80C |
| Block Solution | 10% NGS in PBT. This can be made and stored at 4 °C for a few days prior to use. | ||
| DAPI stock solution | Life Technologies | D3571 | For coutnerstaining nuclei. Prepare a 1 mg/ml solution with ddH2O. |
| VectaShield Mounting Medium | Vector Labs | H-1000 | Mounting medium |
| Glycerol | Sigma | G5516 | For mounting. Prepare 70% dilution with ddH2O. |
| Equipment | |||
| Nutating mixer | VWR | 82007-202 | Used to rock tissue in 3 well glass dish |
| SylGard 182 Silicone Elastomer Kit | Krayden | NC9897184 | Used to make silicone dissection dish |
| Silicone dissecting dish | Mix Sylgard elastomer kit (above) according to directions gently (to avoid bubbles). Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37 °C incubator. | ||
| 3 well glass dish | Corning | 7220-85 | The 3 well variety of these are no longer available, this is the 9 well product. |
| 72 well microwell minitray | Nunc | 438733 | |
| Sharp forceps (Dumont #55) | Fine Science Tools | 11255-20 | |
| Vannas-type Micro Scissors, Straight, 5mm blade | Ted Pella | 1346 | |
| 100 mm Borosilicate glass capillaries | World Precision Instruments | 1B100-4 | Pull with needle puller to make fine point tip that allows a small stream of PBS to flow. |
| Disposable Transfer Pipets, Fine Tip | Samco Scientific | 231 | |
| Tubing dimensions given are inner diameter (ID) x outer diameter (OD) x wall thickness in inches | |||
| PVC tubing (1/8 x 3/16 x 1/32) | Nalgene | 8000-0010 | Use these with pulled needle to assemble the blower tube as shown in Figure 2. |
| Tygon Silicone tubing (3/32 x 5/32 x 1/32) | Saint Gobain Performance Plastics | ABW00004 | |
| Tygon Silicone tubing (1/32 x 3/32 x 1/32) | Saint Gobain Performance Plastics | ABW00001 | |
Referências
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