Dissection et de montage de<em> Drosophila</em> pupes disques yeux
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
Les domaines de la biologie du développement et de la cellule ont été fortement touchés par l'organisme modèle: Drosophila melanogaster. Dans ce modèle, les études du disque de l'œil ont contribué beaucoup de connaissances sur la signalisation, biologie cellulaire et d'autres domaines. Le troisième disque stade de l'œil larvaire fin a été largement étudié et est un modèle puissant à utiliser, car il donne un aperçu d'une série de périodes de développement, chacun avec ses propres molécules et des processus de signalisation unique, comme le sillon morphogénétique progresse à travers le disque de l'oeil 8. Cependant, il est nécessaire d'étendre notre compréhension des processus de développement dans la phase de chrysalide de développement. Bien qu'il y ait eu des études sur le disque d'œil pupe 3-7, notre connaissance ne se rapproche pas de l'ampleur du travail qui a été effectué sur le troisième disque stade de l'œil. Cela est dû, en partie, à la plus grande difficulté à disséquer le disque d'oeil chrysalide. En conséquence, une présentation de labonne méthode de dissection pourrait grandement augmenter la recherche dans ce domaine.
Bien qu'il existe des étapes dans le développement du disque de l'œil des pupes qui sont facilement disséqués, en particulier autour de la période mi-pupe, d'autres périodes sont beaucoup plus difficiles à disséquer. Ce protocole représente une méthode pour disséquer disques oculaires de nymphose qui peuvent être universellement utilisés pour tous les délais de développement des pupes. Ce protocole peut être utilisé comme une alternative à un autre protocole que 9 montre un procédé plus facile et plus rapide à disséquer les disques de l'oeil à partir des points de temps midpupal. Ce protocole a été filmé et mis au point pour la formation des étudiants avancés de premier cycle dans le cycle UCLA Consortium de recherche en génomique fonctionnelle (URCFG) 10,11 dans la technique de dissection de l'oeil chrysalide. Beaucoup d'étudiants de premier cycle ont pu utiliser cette vidéo et méthode pour apprendre cette technique difficile.
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 | |
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