Disección y montaje de<em> Drosophila</em> Pupal discos Eye
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
Los campos de la biología celular y del desarrollo se han visto afectados en gran medida por el organismo modelo: Drosophila melanogaster. Dentro de este modelo, los estudios sobre el ojo disco han contribuido en gran medida de los conocimientos relativos a la señalización, la biología celular y otras áreas. El tercer disco ojo estadio larval tarde se ha estudiado ampliamente y es un modelo de gran alcance de utilizar, ya que da una instantánea de una serie de períodos de desarrollo, cada uno con sus propias moléculas y procesos de señalización único, como la morfogénesis surco progresa a través del disco ojo 8. Sin embargo, hay una necesidad de ampliar aún más nuestra comprensión de los procesos de desarrollo en la fase de pupa de desarrollo. Si bien se han realizado estudios sobre el disco de ojo de pupa 3-7, nuestro conocimiento no se acerca a la amplitud del trabajo que se ha realizado en el tercer disco de ojo estadio. Esto es debido, en parte, a la mayor dificultad en la disección de ojo disco de pupa. Por lo tanto, una presentación de lamétodo apropiado de disección podría ampliar en gran medida la investigación en esta área.
Mientras que hay etapas en el desarrollo del disco ojo de pupa que son fácilmente disecados, especialmente en torno al período de mediados de pupa, otros períodos de tiempo son mucho más difícil de diseccionar. Este protocolo representa un método para la disección de los discos de los ojos de pupas que se pueden utilizar de forma universal para todos los marcos de tiempo de desarrollo de pupas. Este protocolo se puede utilizar como una alternativa a otro protocolo 9 que muestra un método más fácil y más rápido para diseccionar ojo discos a partir de los puntos de tiempo midpupal. Este protocolo fue filmado y desarrollado para capacitar a los estudiantes avanzados de pregrado en el Consorcio de Investigación de Pregrado de la UCLA en Genómica Funcional (URCFG) 10,11 en la técnica de disección del ojo de pupa originalmente. Muchos estudiantes universitarios fueron capaces de utilizar el vídeo y el método para aprender esta técnica difícil.
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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