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.
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.
Feltene utviklings og cellebiologi har vært sterkt påvirket av modellorganisme: Drosophila melanogaster. I denne modellen, har studier av øyets platen bidratt en stor del av kunnskap om signalisering, cellebiologi og andre områder. Den avdøde tredje larve instar øye plate har blitt studert mye og er en kraftig modell til å utnytte, da det gir et øyeblikksbilde av en rekke utviklingsmessige perioder, hver med sine egne unike signalmolekyler og prosesser, som morfogenetiske fure utvikler seg over øyet plate 8. Det er imidlertid et behov for ytterligere å øke vår forståelse av utviklingsprosesser inn i pupal fase. Mens det har vært studier på pupal øye plate 3-7, ikke vår kunnskap ikke nærme bredden i arbeidet som har blitt utført på den tredje stadium øye plate. Dette skyldes delvis til større problemer med å dissekere pupal øye plate. Derfor er en presentasjon avriktig metode for disseksjon kan i stor grad utvide forskningen på dette området.
Mens det er stadier innenfor pupal øye plate utvikling som er lett dissekert, spesielt rundt midten pupal periode, andre tidsperioder er mye mer utfordrende å dissekere. Denne protokollen representerer en metode for å dissekere pupal øye plater som kan være universelt brukes for alle pupal utviklingstidsrammer. Denne protokollen kan brukes som et alternativ til en annen protokoll 9 som viser en enklere og hurtigere fremgangsmåte for å dissekere øye plater fra de midpupal tidspunkter. Denne protokollen ble opprinnelig filmet og utviklet for opplæring av avanserte lavere grads studenter i UCLA Graduate Forskning Consortium i Funksjonell genom (URCFG) 10,11 i teknikken av pupal øye disseksjon. Mange studentene var i stand til å utnytte denne videoen og metoden for å lære dette utfordrende teknikk.
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…
The authors have nothing to disclose.
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.
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 |