Summary

Dissektion og Montering af<em> Drosophila</em> puppe Eye Discs

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

Områderne for udviklingsmæssige og cellebiologi er blevet stærkt påvirket af modelorganismen: Drosophila melanogaster. Inden for denne model, har undersøgelser i øjet skive bidraget med en stor viden om signal-, cellebiologi og andre områder. Den sene tredje larve instar øje disk er undersøgt grundigt, og er en kraftfuld model til at udnytte, da det giver et øjebliksbillede af en række udviklingsmæssige perioder, hver med sine egne unikke signalmolekyler og processer, som det morfogenetiske fure skrider hen over øjet disken 8. Der er imidlertid et behov for yderligere at udvide vores forståelse af udviklingsprocesser i puppe udviklingsfase. Mens der har været undersøgelser af puppe øje skiven 3-7, er vores viden ikke nærme bredden af arbejde, der er udført på tredje stadie øje disk. Dette skyldes til dels den større vanskeligheder med at dissekere den puppe øje disken. Derfor er en præsentation afrette metode til dissektion kunne i høj grad udvide forskningen på dette område.

Mens der er stadier inden puppe udvikling øje disc, som er let dissekeret, især omkring midten af ​​puppe periode, andre perioder er langt mere udfordrende at dissekere. Denne protokol udgør en metode til at dissekere puppe øje diske, der kan bruges alment til alle puppe udviklingsmæssige tidsrammer. Denne protokol kan anvendes som et alternativ til en anden protokol 9, som viser en lettere og hurtigere metode til at dissekere øjet diske fra midpupal tidspunkter. Denne protokol blev oprindeligt filmet og udviklet til at træne avancerede bachelorstuderende i UCLA Undergraduate Research Consortium i Functional Genomics (URCFG) 10,11 i teknikken med puppe øje dissektion. Mange studerende var i stand til at udnytte denne video og metode til at lære denne udfordrende teknik.

Protocol

Denne procedure er en 2 dages procedure. 1 dag (2 timer + dissekere tid) 1. puppe Eye Disc Dissection Vælg en puppe til dissektion. BEMÆRK: alder puppe til dissekeres vil blive bestemt af de eksperimentelle behov. Men hvis behandlingen cellemorfologi, dette sker ofte ved 42 timer efter puparium formation (APF) ved 25 ° C, hvilket er en alder af pupperne vist i videoen. Saml hvid pupper (betragtet 0 HR APF) med en fugtet pensel og arrangere dem i kr…

Representative Results

Som et eksempel på anvendelsen af ​​denne protokol resultater illustrerer midpupal (42 timer APF ved 25 ° C) øjet diske immunofarvede med forskellige antistoffer er vist i Figur 2. Ved hjælp af et antistof rettet mod phosphotyrosinresterne kan membranen af ​​cellerne være observeret (figur 2A). Dette kan anvendes til at identificere den regelmæssige arrangement af ommatidial celler i puppe øje efter de endelige mønsterdannende processer, der foregår før midpupal fase. En anden repræse…

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…

Disclosures

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

References

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Cite This Article
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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