Name: dissection and mounting of drosophila pupal eye discs
Uploaded: 2014-11-09T15:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Dissection and Mounting of Drosophila Pupal Eye Discs at JoVE.com

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.

Bu prosedür 2 gün işlemdir. Gün 1 (2 saat + zamanı diseksiyon) 1. Pupa Göz Disk Diseksiyon <ol><li> Diseksiyon için bir pupa seçin. NOT: disseke pupa yaşı deneysel gereksinimlerine göre belirlenecektir. Hücre morfolojisi inceleyerek Ancak, bu sık sık videoda gösterilen pupa yaşı 25 ° C&#39;de puparium oluşumu (APF) sonra 42 saat sonra yapılır. Islatılmış bir fırça ile beyaz pupa (kabul 0 saat APF) toplayın ve 25 ° C&#39;de muhafaza yeni bir sinek gıda şişede (toplama zamana dayalı) kronolojik sırayla bunları düzenlemek. Mümkün olduğunca yakın pupa arasındaki zaman farkları tutmak için bu aynı kronolojik sırayla yaşlı pupa teşrih. </li><li> Fosfat tamponlu tuzlu su, bir damla halinde pupa aktarın bir silikon kesme plakası üzerinde (PBS, pH 7.4, tarifi için malzeme bakınız). </li><li> Forseps ve delgi ile pupa halinde üst tutun ve l açınKeskin forseps bir çift ile pupa davanın ower bölümü. Yeni oluşturulan deliğe kasasından pupa çıkarın. </li><li> Pupa makas, orta toraks, çapraz bir kesim olun. Enkaz kaldırmak veya PBS yeni bir damla pupa kafasını aktarın. Temiz pipet üfleme borusunu (malzeme ve detaylar için bakınız Şekil 1) kullanılarak ve gerektiğinde ilave PBS ekleyin. Dehidratasyonu önlemek için emin olun. </li><li> Güvenli kavrama forseps bir çift ile pupa kafa manikür üst. Blower tüp ucunu kullanarak, yavaşça beyin ve pupa halinde dışarı göz diskler dahil olmak üzere diğer dokuları, a&#39;ya için pupa halinde itin. Manikür içinde herhangi bir doku kapma önlemek için emin olun. </li><li> PBS ile dolu üfleme borusunu kullanarak, yavaşça açıkça ayırmak için baş davanın dışında vücut yağ dokusu ve diğer enkaz darbe ve beyin ve göz diskleri tanımlamak. NOT: pupa baş duruma bağlı beyin tutulması araştırmacı o kullanmanızı sağlayacakGöz diskleri veya beyni zarar vermeden dokusunun gelecek manipülasyon için bir &quot;sap&quot; olarak reklam durumda. Bununla birlikte, bu kafa kutusundan, göz ve beyin diskleri çıkarmak için ortaktır ve bu da genel prosedür ve sonuçları için zararlı değildir. Genellikle üfleme ve başarıyla onlar açıkça doku çevreleyen ayrı olduğu pupa kafa durumda ve bu yüzden içinde göz diskleri ve beyni çıkarmak için bastırıyor birden girişimleri sürüyor. Daha temiz ve daha ayrı bir göz disklere sabitleme gereksiz doku önlemeye yardımcı olacaktır çevreleyen dokudan göz diskleri alabilirsiniz. </li><li> Diseksiyon sonra derhal düzeltme çözeltiden 0.5 ml (malzeme bakınız) ile buz üzerinde 3 de cam tabak içine doku aktarın. Birkaç dakika sonra (bu göz disklerinin bir sonraki kümesi incelemek için gereken tipik süre) saptamak çözeltinin yüzeyinde doku Float. NOT: Bu diseksiyon, completel bu turda disseke ilk göz disk değilsey Fix çözeltisi içine bir önceki göz diski daldırın. (Örn, reaktif oksijen türleri, lizozomları) özel boyama tekniklerinin kullanılmasını ise bu tespitin önce yapılmalıdır. Bunu yapmak için, önce uygun lekeleme protokolü ile devam eden buzla soğutulmuş PBS içinde kesilen göz disklerini. </li><li> Tüm göz disklerin diseksiyonu ve suya batmaya ardından disseke, 30 dakika boyunca oda sıcaklığında bir rocker ve rock 3 de cam tabak taşıyın. </li></ol> 2. immün <ol><li> Dikkatlice Düzeltme çözüm kaldırmak ve% 0.3 Triton X-100 ile 0.5 ml PBS ilave (% 0.3 PBT, malzeme bakınız) ve 10 dakika boyunca bir rocker doku yıkayın. 4 kez (10 dk) bir toplam bu adım, üç kez daha tekrarlayın. Not: kuyudan solüsyonun dikkatli bir şekilde çıkarılması, doku hasarı ya da kaybını önlemek için bir tahlil mikroskobu altında gerçekleştirilebilir. Çözelti, bir pipet yardımıyla dikkatli bir şekilde aspire edilir; Biz ince tippe tercihd doku aspire şansını azaltmak için basık açılış olmuştur atılabilir transferi pipet. </li><li> 4. yıkamanın sonunda, yıkama kaldırmak ve oyuğuna (malzeme bakınız) 0.5 mi blok çözeltisi ekleyin ve 30 dakika kuluçkada bırakın. NOT: ihtiyaçlarınıza göre bu zaman çerçevesi uzatılabilir. </li><li> 30 dakika blok sürenin sonunda, bir mikro plaka içindeki yuvalara uygun dizi halinde (blok çözeltisi içinde seyreltilmiş): Primer antikor çözeltisi pipetle 10 ul. NOT: Bir mikro plakanın biri de pupa duruma bağlı göz diskler yaklaşık 4 çift veya bağlı beyinleri ile göz disklerin 8 izole çiftleri ağırlayacak. (: 500 seyreltme oranı: 1) ve anti-Kesim (1: 200 dilüsyon) ve temsili veriler için, bu protokolde kullanılan birincil antikorlar özel anti-fosfotirozin bulunmaktadır. </li><li> 3 de, cam çanak primer antikor çözeltisi ile doldurulmuş bir mikro oyuk plaka oyuklarına parçalara doku aktarın. </li><li> YerBir ıslatılmış kağıt havlu üzerinde mikro plaka gece boyunca 4 ° C&#39;de boş bir pipet ucu kutusu (veya diğer benzer kap) ve mağaza (dehidratasyon önlemek için). </li></ol> Gün 2 (4 hr + zamanı montaj) <ol start="6"><li> % 0.3 PBT (≈0.5 mi) ile yeni bir 3 de cam tabak içine mikro plaka doku aktarın ve dengesiz üzerinde 10 dakika süreyle doku yıkayın. 4 yıkama toplam bu adımı (10 dk), üç kez daha tekrarlayın </li><li> Son yıkama çıkarın ve oyuğuna Bloke çözeltisi 0.5 ml ilave ediniz. 30 dakika engelleyin. NOT: ihtiyaçlarınıza göre bu zaman çerçevesi uzatılabilir. </li><li> Bir kapak veya alüminyum ile ışıktan 3 de cam tabak Bloke çözeltisini çıkarın ve oyuğuna (blok çözeltisi içinde seyreltilmiş birincil antikoru ve floresan dalga boyu için uygun şekilde floresan ile etiketlenmiş antikorlar ile yapılan) sekonder antikor çözeltisi, 0.5 ml ilave ve koruma folyo ve oda sıcaklığında en az 2 saat süreyle inkübeSıcaklık. NOT: Tüm sonraki adımlar benzer ışığa maruz korunmalıdır. Bu inkübasyon birincil antikor inkübasyon benzer bir kurulumda, 4 ° C&#39;de gece boyunca uzatılabilir, ama aynı zamanda laboratuar tipi film veya antikor koruma isteniyorsa, bir mikro levha ile kaplanmış bir 3 de cam tabak içinde gerçekleştirilebilir. </li><li> Antikor İnkübasyonun sonunda, ikinci antikor çözeltisini çıkarın ve kuyuya% 0.3 PBT 0.5 ml ilave edin ve 10 dakika boyunca ROCKER&#39;da yıkayın. 4 yıkama toplam bu adımı (10 dk) tekrarlayın. Not: (: 1000 seyreltme 1) DAPI boyama isteniyorsa, birinci yıkama başlamadan önce, ilk yıkama programı için kullanılacak PBT 500 ul DAPI stok çözeltisi, 0.5 ul (Malzeme) ekleyin. </li></ol> 3. Montaj <ol><li> Son yıkama sonunda, br çıkarmak için bir araç olarak% 70 gliserol ya da PBS kullanılarak bir lam üzerine anten / göz diskler monteain ve sürgünün bir tarafında bir havuzda diğer istenmeyen dokular. </li><li> Dikkatle forseps arasındaki hidrostatik basıncı kullanarak dokuyu taşımak, ya da çok nazikçe bir forseps ucu ile ve slayt merkezine doğru bir sütunda göz diskleri sıraya. </li><li> Doku çevresindeki tüm yabancı gliserol veya PBS çıkarın (bir laboratuvar ıslanma eylemini mendil kullanarak veya forseps bunun için çalışmak). </li><li> Göz Disk kolonu bir kenarı boyunca montaj orta küçük bir miktar (≈10 ul) yerleştirin ve göz diskler üzerine montaj ortamı yaymak üzere doku üzerinde bir lamel. </li><li> 1-2 dk doku / montaj orta tamamen yaymak için izin için slayt bekletin. </li><li> Lamel kenarlarında herhangi bir ekstra montaj orta silin ve şeffaf tırnak cilası ile mühür. </li><li> Konfokal mikroskopi kullanılarak göz disklerin floresan görüntüler yakalayın. </li></ol>

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The authors have nothing to disclose.

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 should achieve the goal of making the forceps into a natural extension of the fingers and thumb, allowing for the discrete and subtle movements required in the dissection. 
If using PBS to mount in, always have extra PBS available, as the small amount of PBS quickly evaporates in the mounting process. Another common problem is the folding of the eye discs in the Fix solution, thus fixing the eye disc in that orientation. This can be avoided by floating the eye disc on the surface of the Fix solution for some time (typically the length of the next eye disc dissection) before submerging it completely in the Fix solution.
While other published techniques focus on the dissection of the pupal eye disc at the midpupal stage9, when dissection is easily accomplished, this technique provides a method that can be applied to any pupal developmental time, except in the case of the pharate adult or early white pupa. We feel our technique provides an alternative and complementary approach to already published methods for pupal eye disc dissection. 
This technique, accompanied with common and useful genetic manipulations like FLP-FRT analysis14, UAS-GAL expression of transgenes15 and others, will allow researchers to study the eye disc in further detail during any pupal time frame.

Drosophila melanogaster: gelişim ve hücre biyolojisi alanlarını büyük ölçüde model organizma tarafından etkilendi. Bu model çerçevesinde, göz diskin çalışmalar bilgisi ile ilgili sinyalizasyon, hücre biyolojisi ve diğer alanlarda büyük bir katkıda bulunmuştur. Geç üçüncü larva göz disk geniş olarak araştırılmış ve bu gelişim dönemlerinin bir dizi bir anlık verir gibi, kullanmak için güçlü bir model olmuştur, kendine özgü sinyal molekülleri ve süreçleri ile her morfojenetik karık göz disk boyunca ilerledikçe 8. Ancak, daha fazla geliştirme pupa fazına gelişim süreçleri anlamamız genişletmek için bir ihtiyaç vardır. Pupa göz disk 3-7 çalışmalar varken, bizim bilgi Üçüncü dönem göz disk üzerinde yapılmıştır işin genişliği yaklaşım değildir. Bu pupa göz diski diseksiyon büyük zorluk, kısmen, kaynaklanmaktadır. Bu nedenle, bir sunumdiseksiyon uygun yöntem büyük ölçüde bu alanda araştırma genişletmek olabilir. Kolayca özellikle orta-pupa döneminde çevresinde, disseke pupa göz disk gelişimi içinde aşamaları varken, diğer süreler çok daha zorlu incelemek için vardır. Bu protokol, evrensel olarak tüm pupa gelişim zaman dilimlerinde kullanılabilir pupa göz diskler kesme için, bir yöntemi temsil eder. Bu protokol midpupal zaman noktalarından göz diskleri incelemek için daha kolay ve daha hızlı bir yöntem gösterir, başka bir protokole 9 alternatif olarak kullanılabilir. Bu protokol başlangıçta çekildi ve Fonksiyonel Genomik (URCFG) pupa göz diseksiyon tekniği 10,11 UCLA Lisans Araştırma Konsorsiyumu ileri lisans öğrencilerine eğitim vermek için geliştirilmiştir. Birçok lisans öğrenci bu zorlu tekniği öğrenmek için bu videoyu ve yöntemini kullanmak için başardık.

<table><tbody><tr><td>Phosphate-buffered saline (PBS, pH 7.4)</td><td></td><td></td><td>80 g NaCl, 2g&amp;nbsp; KCl, 14.4 g Na&lt;sub&gt;2&lt;/sub&gt;HPO4, 2.4 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, Bring volume to 1 &amp;#956;l, adjust the pH to 7.4, autoclave or filter sterilize, dilute to 1x PBS with autoclaved ddH&lt;sub&gt;2&lt;/sub&gt;O before using.</td></tr><tr><td>Triton X-100</td><td>Promega</td><td>H5142</td><td>Caution: Irritant! Wear gloves.</td></tr><tr><td>0.3% PBT</td><td></td><td></td><td>1.5 ml of Triton X-100, 500 ml 1X PBS.</td></tr><tr><td>37% formaldehyde solution</td><td>Fisher Scientific</td><td>F75P1GAL</td><td>Caution: Toxic, probable human carcinogen! Wear gloves.&amp;nbsp;</td></tr><tr><td>Fix Solution</td><td></td><td></td><td>(&amp;asymp;4% Formaldehyde in PBS) 50 &amp;mu;l of 37% Formaldehyde, 450 &amp;mu;l 1x PBS, make fresh before use</td></tr><tr><td>Normal goat serum</td><td>Rockland antibodies &amp;amp; assays</td><td>B304</td><td>Aliquot in 1 ml volumes and store at -80 &amp;#176;C</td></tr><tr><td>Block Solution</td><td></td><td></td><td>10% NGS in PBT.&amp;nbsp; This can be made and stored at 4 &amp;deg;C for a few days prior to use.</td></tr><tr><td>DAPI stock solution</td><td>Life Technologies</td><td>D3571</td><td>For coutnerstaining nuclei. Prepare a 1 mg/ml solution with ddH&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>VectaShield Mounting Medium</td><td>Vector Labs</td><td>H-1000</td><td>Mounting medium</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td>For mounting. Prepare 70% dilution with ddH&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Nutating mixer</td><td>VWR</td><td>82007-202</td><td>Used to rock tissue in 3 well glass dish</td></tr><tr><td>SylGard 182 Silicone Elastomer Kit</td><td>Krayden</td><td>NC9897184</td><td>Used to make silicone dissection dish</td></tr><tr><td>Silicone dissecting dish</td><td></td><td></td><td>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 &amp;deg;C incubator.</td></tr><tr><td>3 well glass dish</td><td>Corning</td><td>7220-85</td><td>The 3 well variety of these are no longer available, this is the 9 well product.</td></tr><tr><td>72 well microwell minitray</td><td>Nunc</td><td>438733</td><td></td></tr><tr><td>Sharp forceps (Dumont #55)</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Vannas-type Micro Scissors, Straight, 5mm blade</td><td>Ted Pella</td><td>1346</td><td></td></tr><tr><td>100 mm Borosilicate glass capillaries</td><td>World Precision Instruments</td><td>1B100-4</td><td>Pull with needle puller to make fine point tip that allows a small stream of PBS to flow.</td></tr><tr><td>Disposable Transfer Pipets, Fine Tip</td><td>Samco Scientific</td><td>231</td><td></td></tr><tr><td>Tubing dimensions given are inner diameter (ID) x outer diameter (OD) x wall thickness in inches</td><td></td><td></td><td></td></tr><tr><td>PVC tubing (1/8 x 3/16 x 1/32)</td><td>Nalgene</td><td>8000-0010</td><td>Use these with pulled needle to assemble the blower tube as shown in Figure 2.</td></tr><tr><td>Tygon Silicone tubing (3/32 x 5/32 x 1/32)</td><td>Saint Gobain Performance Plastics</td><td>ABW00004</td><td></td></tr><tr><td>Tygon Silicone tubing (1/32 x 3/32 x 1/32)</td><td>Saint Gobain Performance Plastics</td><td>ABW00001</td><td></td></tr></tbody></table>

dissection and mounting of drosophila pupal eye discs

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.

Bu protokolün kullanımına ilişkin bir örnek olarak, sonuçlar midpupal (42 saat 25 ° C&#39;de APF) farklı antikorlar ile boyanmış göz diskler gösteren fosfotirosin kalıntıları karşı yönlendirilen bir antikor kullanılarak, Şekil 2&#39;de sunulmuştur, hücrelerin membran olabilir gözlenen (Şekil 2A). Bu midpupal aşamasından önce meydana nihai model verme işlemleri takiben pupa göz ommatidial hücrelerin düzenli bir şekilde tertip tanımlamak için kullanılabilir. Başka bir temsili görüntü koni hücrelerinin çekirdekleri Kesme transkripsiyon faktörü (Şekil 2B), karşı bir antikor kullanılarak tespit edilmiştir olabilir göstermektedir. <img alt="Şekil 1," fo:content-width="4.5in" src="/files/ftp_upload/52315/52315fig1highres.jpg" width="450" /> Şekil 1:. Diseksiyon işlemi kullanılan fan tüp Meclisi diyagramı th ilgili ayrıntılar için malzeme bölümüne bakınÜfleç borunun E ayrı bileşenler. <img alt="Şekil 2," src="/files/ftp_upload/52315/52315fig2highres.jpg" /> Şekil 2:. Midpupal göz disklerin immün 42 saat APF at) ommatidia (fosfotirozin) ve pupa göz diskler B) koni hücre çekirdeğinin (kes) apikal morfolojisi A görselleştirmek için bu protokolün kullanımını gösteren Temsilcisi immünoboyamasıdır. Ölçek çubukları 50 um =.

Watch this Scientific Journal Video about Dissection and Mounting of Drosophila Pupal Eye Discs at JoVE.com

Dissection and Mounting of Drosophila Pupal Eye Discs

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.

Diseksiyon ve montajı<em&gt; Drosophila</em&gt; Pupa Göz Diskler

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

dissection-and-mounting-of-drosophila-pupal-eye-discs

Nanyang Technological University

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JoVE Journal

Biology

Dissection and Mounting of Drosophila Pupal Eye Discs

12.1K Görüntüleme.  University of California at Los Angeles (UCLA). 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.

Video: Dissection and Mounting of Drosophila Pupal Eye Discs

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Canlı-görüntüleme<em&gt; Drosophila</em&gt; Pupa Göz

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Gelişim Biyolojisi

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

3. Instar Drosophila Larva hayali Disklerin Diseksiyon

Biyoloji

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

Uzun süreli Canlı Görüntüleme<em&gt; Drosophila</em&gt; Göz Disk

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Drosophila Göz hayali Disk Glial Hücre Göç Canlı Görüntüleme

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

Larva, Pupa ve Erişkin diseksiyonu ve İmmünohistokimya<em&gt; Drosophila</em&gt; Retinaları

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Nörobilim

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

Drosophila pupa Retina diseksiyon immünhistokimya, Batı analiz ve RNA izolasyon için

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

Göz hayali Disk Görselleştirme Yardım Roman RFP Reporter Drosophila</em

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

Yetişkin hazırlanması Drosophila Gözler İnce Kesit ve Mikroskobik Analizi

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

Diseksiyon ve hayali Disklerin immün itibaren<em&gt; Drosophila melanogaster</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Dissection of the Eye-Brain Complex from Fly Pupae: A Method to Isolate Retinal Tissue

Source: DeAngelis, M. W., Johnson, R. I. <a href="https://www.jove.com/video/59299/" target="_blank">Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation</a>. J. Vis. Exp.&#160;(2019).
This video describes how to dissect and isolate the eye-brain complex from Drosophila pupae. The featured protocol demonstrates the procedure yielding high-quality tissue, compatible with, for example, immunostaining, as well as, other gene expression experiments.

Sinek Pupalarından Göz-Beyin Kompleksinin Diseksiyonu: Retina Dokusunu İzole Etmek İçin Bir Yöntem

Source: DeAngelis, M. W., Johnson, R. I. Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation. J. Vis. ...

Diseksiyon ve montajı

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