Summary

Disseksjon og montering av<em> Drosophila</em> pupal 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

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.

Protocol

Denne fremgangsmåten er en 2 dagers prosedyre. Dag 1 (2 timer + dissekere tid) 1. pupal Eye Disc Dissection Velg en puppe for disseksjon. MERK: I en alder av puppe å bli dissekert vil bli bestemt av de eksperimentelle behov. Imidlertid, hvis undersøke cellemorfologi, er dette ofte gjort ved 42 timer etter at dannelsen puparium (APF) ved 25 ° C, som er en alder av puppe vist i videoen. Samle hvite puppe (regnes 0 hr APF) med en fuktet pensel og arr…

Representative Results

Som et eksempel på bruk av denne protokollen, som illustrerer resultatene midpupal (APF 42 timer ved 25 ° C) øye immunostained plater med forskjellige antistoffer er presentert i figur 2. Ved å bruke et antistoff rettet mot fosfotyrosin-rester, kan membranen være av cellene observerte (Figur 2A). Dette kan brukes til å identifisere det faste arrangement av ommatidial celler i pupal øyet etter den endelige mønstringsprosesser som forekommer før den midpupal stadium. Et annet representativt bilde…

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…

Divulgazioni

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

Riferimenti

  1. Ready, D. F., Hanson, T. E., Benzer, S. Development of the Drosophila retina, a neurocrystalline lattice. Dev Biol. 53 (2), 217-240 (1976).
  2. Wolff, T., Ready, D. F. The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave. Development. 113 (3), 841-850 (1991).
  3. Carthew, R. W. Pattern formation in the Drosophila eye. Curr Opin Genet Dev. 17 (4), 309-313 (2007).
  4. Bao, S., Cagan, R. Preferential adhesion mediated by Hibris and Roughest regulates morphogenesis and patterning in the Drosophila eye. Dev cell. 8 (6), 925-935 (2005).
  5. Grzeschik, N. A., Knust, E. IrreC/rst-mediated cell sorting during Drosophila pupal eye development depends on proper localisation of DE-cadherin. Development. 132 (9), 2035-2045 (2005).
  6. Sprecher, S. G., Desplan, C. Switch of rhodopsin expression in terminally differentiated Drosophila sensory neurons. Nature. 454 (7203), 533-537 (2008).
  7. Zuker, C. S., Cowman, A. F., Rubin, G. M. Isolation and structure of a rhodopsin gene from D. melanogaster. Cell. 40 (4), 851-858 (1985).
  8. Voas, M. G., Rebay, I. Signal integration during development: insights from the Drosophila eye. Dev Dyn. 229 (1), 162-175 (2004).
  9. Hsiao, H. Y., et al. Dissection and immunohistochemistry of larval, pupal and adult Drosophila retinas. J Vis Exp. , 4347 (2012).
  10. Call, G. B., et al. Genomewide clonal analysis of lethal mutations in the Drosophila melanogaster eye: comparison of the X chromosome and autosomes. Genetica. 177 (2), 689-697 (2007).
  11. Chen, J., et al. Discovery-based science education: functional genomic dissection in Drosophila by undergraduate researchers. PLoS Biol. 3 (2), 59 (2005).
  12. Wolff, T. Dissection techniques for pupal and larval Drosophila eyes. CSH Protoc. 2007, (2007).
  13. Williamson, W. R., Hiesinger, P. R. Preparation of developing and adult Drosophila brains and retinae for live imaging. J Vis Exp. 37, 1936 (2010).
  14. Xu, T., Rubin, G. M. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development. 117 (4), 1223-1237 (1993).
  15. Duffy, J. B. GAL4 system in Drosophila: a fly geneticist’s Swiss army knife. Genesis. 34 (1-2), 1-15 (2002).
check_url/it/52315?article_type=t

Play Video

Citazione di questo articolo
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).

View Video