Summary

解剖和安装的<em>果蝇</em>蛹眼光盘

Published: November 09, 2014
doi:

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

果蝇 :发育和细胞生物学领域已经通过模式生物受到很大影响。在该模型中,眼睛光盘的研究已经作出了很大贡献有关知识信令,细胞生物学等领域。已故第三龄幼虫眼盘已被广泛研究,并是一个功能强大的模型利用,因为它给出了一系列发育阶段的快照,每个都有其自己的独特信号传导分子和过程,如在整个眼睛光盘的形态发生沟前进8。然而,有必要进一步扩大我们的发展过程的认识到发展的蛹阶段。虽然有研究蛹眼盘3-7,我们的知识不接近已经在三龄眼睛光盘已经完成的工作的广度。这是由于,在某种程度上,在解剖蛹眼睛光盘的更大的困难。因此,一个呈现的解剖适当的方法可以在这方面大大拓展研究。

虽然有在蛹眼睛光盘的发展阶段,很容易解剖,特别是围绕中期蛹期,其他时间段都更有挑战性解剖。该协议代表了一种方法解剖,可普遍用于所有蛹的发育时限蛹的眼睛光盘。这个协议可以用来作为一种替代另一种协议9,其示出了一种更简单和更快速的方法,从midpupal时间点解剖眼光盘。该协议最初拍摄,并在功能基因组学(URCFG)在蛹眼部解剖技术培训10,11先进的本科生在加州大学洛杉矶分校本科生研究协会开发的。许多本科学生能够利用这种视频和方法,了解这一具有挑战性的技术。

Protocol

这个过程是2天的过程。 第1天(2小时+解剖时) 1.蛹眼睛光盘解剖选择一个蛹解剖。 注:蛹被解剖的年龄将通过实验需要来确定。然而,如果检查细胞形态,这是经常做在puparium形成(APF)后42小时,在25℃,这是在视频中所示的蛹的年龄。收集白色蛹(视为0小时,APF)与润湿画笔并安排它们按时间顺序(基于收集时间)在保持于25℃的新飞食品?…

Representative Results

因为使用该协议的一个例子,结果示midpupal(42小时APF,在25℃)进行免疫染色用不同抗体眼光盘示于图2。通过使用针对磷酸酪氨酸残基的抗体,该细胞的膜可以是观察到的( 图2A)。这可以被用来确定网膜细胞的规则排列在蛹眼以下之前midpupal阶段中发生的最终图案化工艺。另一个代表性图像描绘锥体细胞的细胞核可以通过使用抗体对剪切转录因子( 图2B)来标识。 <…

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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Tea, J. S., Cespedes, A., Dawson, D., Banerjee, U., Call, G. B. Dissection and Mounting of Drosophila Pupal Eye Discs. J. Vis. Exp. (93), e52315, doi:10.3791/52315 (2014).

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