We appreciate assistance from members of the fly community, particularly Dr. Denise Montell, Dr. Lynn Cooley, and Dr. Pernille Rorth, for reagents. We thank Flybase, the Bloomington Drosophila Stock Center, and Developmental Studies Hybridoma Bank for information and providing fly stocks and antibodies, respectively. LM is supported by the Department of Education&#160;Grant, Graduate Assistance in the Areas of National Need (GAANN) training fellowship (P200A120017) and by a NIGMS Initiative for Maximizing Student Development Grant (2 R25-GM55036). A portion of the microscopy work was supported by NSF MRI grant DBI-0722569 and the Keith R. Porter Core Imaging Facility. Research was supported in part by a NSF CAREER Award (1054422) and a Basil O&#8217;Connor Starter Scholar Award from the March of Dimes, both awarded to MSG.

Ovarioles 1. Diseksiyon <ol><li> Ilave aktif kuru maya ile taze sinek gıda flakon yaklaşık onbeş 2-4 gün eski erkek sineklerin ve birkaç erkek aktarın. Şişelerde için fiş olarak pamuk kullanın, ve yüksek nem tutmak için pamuk su birkaç damla ekleyin. </li><li> 14-16 saat için 25 ° C inkübatör yerleştirin flakon aşaması 8-10 yumurta odaları sayısını en üst düzeye çıkarmak için. Not: Kuluçka süresi sıcaklığa ve istenilen aşamasına bağlı olarak değişmektedir. </li><li> Bir sonraki gün için gerektiği gibi diseksiyon medya hazırlayın, 0.1M KPO 4 ve NP40 çözümleri (Malzeme). </li><li> CO 2 kullanarak sinek anestezisi ve bir diseksiyon mikroskop altında yerleştirin. Pipetleyin cam depresyon slayt her boşluğuna diseksiyon medya birkaç damla. </li><li> Bir kadın sinek aşağı kanatları ile masa üstü ve yönlendirmek için yaklaşık 30 ° açıyla her el forseps tutun. Senin baskın olmayan el ile, a onu kavrayarak, forseps kullanarak kadın pick upToraks yakın karın, bir nterior ve depresyon slayt (Şekil 1A içerlek) diseksiyon ortama onu daldırın. </li><li> Forseps diğer çift, karın ventral posterior Exoskeleton kavramak ve delip. Posterior sonunda yumurtalıkları tut, sonra karın çekip çıkarın ve depresyon slayt diğer tarafında taze ortama yerleştirin. (Şekil 1A ek). Gut ayrı çekerek kaçının. Karkas atın. </li><li> Tüm yumurtalıkların disseke kadar diğer kadın sinekler tekrarlayın. Erkek atın. </li><li> Forseps bir çift, (büyük yumurta odaları yakınında) arka ucunda bir yumurtalık tutun ve diğer elinizle en ön yumurta odaları (germarium) birini çimdik forseps bir çift kullanın. (Şekil 1A). </li><li> Yavaş ve istikrarlı, yumurtalık kılıfın dışında bir ovaryol zinciri çekin. İstenen tüm yumurta odaları r kadar tek tek ovarioles teşrih devamemoved. </li><li> Tekrar tüm disseke yumurtalıklar için 1,7-1,8 adımları. </li><li> Tamamlandığında, plastik transfer pipet kullanarak 0.6ml tüp ovarioles aktarın. </li><li> Ovarioles, tüpün dibine çöker sonra sabitleme ve boyama adımlara devam edelim. </li></ol> Ovarioles 2. Immunofluorescent (IF) Boyama <ol><li> Dikkatle bir pipet ile ovarioles gelen diseksiyon ortamı çıkarın. Herhangi bir ovarioles veya yumurta odaları kaldırmak için emin olun. Ovarioles ve diseksiyon medya yaklaşık 50 ul bırakın. </li><li> Bir tüp içinde 0.1 M KPO 4 Tampon 150 ul% 16 paraformaldehit 50 ul ekle ve bunları çözmek için yumurta odalarına solüsyonunu ilave edin. 10 dakika süre ile sallanan kuluçkalayın. DİKKAT: paraformaldehit toksiktir. </li><li> Fiksatif çıkarın ve 0.5 ml NP40 yıkama tamponu ile iki kez yıkayın. </li><li> 15 dakika oda sıcaklığında her biri için iki kez yıkanır. </li><li> Sonraki adımlar için standart IF protokol 15 bakın. <ol><li> Kısaca, tespit sonrasında, eklemekUygun seyreltmede primer antikorlar ile 4 ° C&#39;de O / N inkübe edin. (: 200 seyreltmeler 1) sonra ikincil antikorlar ekleyin, NP-40 birkaç kez yıkayın. </li><li> NP40 birkaç kez yıkayın, sonra DAPI (1: 1000) ekleyin, 10 dakika boyunca, daha sonra yıkama tekrarlayın. Protokol tamsa sonra, yumurta odalarına PBS içinde% 50 gliserol, 200 ul ve 2 saat boyunca oda sıcaklığında bekletin. </li><li> , Çözüm çıkarın PBS içinde% 70 gliserol ile değiştirin, ve folyo ile kaplayın. Montaj için hazır olana kadar 4 ᵒC de örnek yerleştirin. Bu arada, adım 3 ile devam edin. </li></ol></li></ol> Gliserin Jelly Slaytlar 3. Ön Hazırlıklar <ol><li> Jöle gliserin, bir spatula ile en az 4 ml ölçün ve yarım işareti cam hücre kültürü tüp doldurun. 30 dakika boyunca 55 ° C&#39;de bir su banyosu içinde gliserin jöle eritin. </li><li> İki mikroskop slaytlar üzerine stok çözeltisinden gliserol küçük bir damla, yaklaşık 300 ul, dökün. Bir dokuyu kullanarak, ince la gliserol yayıldıyer&#39;de slaytlar her karşısında. Bu temel sağlar ve adım 5.6 de gliserin jöle kolay çıkarılması için izin verir. </li><li> Filtrelenmiş 1.000 ul pipet kapalı ucu kesin. Kullanım kesme pipet yavaş yavaş her mikroskop lamı sıcak gliserin jöle 1000-1500 ul aktarmak için. Kabarcıklarını önlemek için özen gösterin. </li><li> Gliserin jöle slaytlar ayarlamak için oda sıcaklığında 5 dakika bekletin. </li><li> 60 mm x 15 mm petri her gliserin jöle slayt yerleştirin ve plastik wrap ile kaplayın. 4 ° CO / N Place. İsteğe bağlı olarak, mağaza 4 ° C&#39;de en fazla 5 gün jöle slaytlar gliserin. </li></ol> 4. Yumurta Chambers Montaj <ol><li> Adım 2.5, bir gliserin jöle slayt genelinde% 70 gliserol yumurta odaları pipet birkaç 3 ul damla örnekler kullanma. Tüm yumurta odaları bu slaytta kadar pipetlenmesi 3 ul damla devam edin. Her damla, en az on yumurta odaları içerdiğinden emin olun. Bu arada, 55 ° C su banyosu içinde gliserin jöle kültür tüpü ısı. </li><li> Bir diseksiyon mikroskop altında yumurta odaları ile gliserin jöle slayt yerleştirin. Yumurta bölmelerin tek bir damla görüş alanı (Şekil 1B) olacak şekilde büyütme ayarlayın. </li><li> İstenilen sahne yumurta odasını pick up, bir kaşık gibi 30 iğne kullanma. Ne zaman bir bıçak gibi iğne kullanılarak bir ovaryol zinciri gerekli, ayrı istenen yumurta odaları. </li><li> Görüntüleme engel olabilir enkaz önlemek için, sol (Şekil 1E -1) bakan anterior, ikinci gliserin jöle slayt yumurta odasına aktarın. En az yedi yumurta oda, bir sütun (Şekil 1C) dizilmiş kalmayana kadar yineleyin. </li><li> İstenen tüm yumurta odaları ikinci gliserin jöle slayt transfer kadar yedi yeni sütunlar oluşturur. </li><li> Doku ve büküm küçük bir parça koparın. Hafifçe her birine dokunarak yumurta odalarından fazla PBS-Gliserol emmek için bunu kullanın. Yumurta odalarına kaldırmak değil dikkat edin. </li><li> Ayırmakfiltrelenmiş 200 ul pipet ucu. Yumurta odalarının tüm sütunları kapsayacak şekilde jöle gliserin 150 ul aktarmak için bunu kullanın. NOT: sütunları karşılamak için gerekli gliserin jöle miktarı yumurta odası aşamaları ve sütun sayısına bağlıdır. Miktarı ayarlanabilir. </li><li> Gliserin jöle üst tabakası tamamen (Şekil 1D) katılaşmış kadar monte yumurta odaları oda sıcaklığında 5 dakika bekletin. </li><li> Place 60 mm x 15 mm petri monte yumurta odaları slayt ve plastik wrap ile kaplayın. 4 ° CO / N Mağaza (photobleaching hakkında endişe varsa karanlıkta yer) gliserin jöle katmanları bir araya katılaşmaya izin. </li></ol> Görüntüleme Yumurta Odaları 5. hazırlanması <ol><li> Bir diseksiyon mikroskobu altında monte yumurta odaları slayt yerleştirin. Yumurta bölmelerin sadece bir sütun görüş alanında olacak şekilde büyütme ayarlayın. </li><li> 45 ° açı minyatür neşter kullanarak, bir straig kesti(yumurta odaları arka tarafına yakın) yumurta odaları ilk sütunun sağ tarafı boyunca ht hattı. (Şekil 1D-e) </li><li> Sonraki, sütundaki son yumurta odasının üstündeki ilk yumurta odasının üstünde ve altında kesti. Bu noktada, yumurta bölmelerin kolon üç tarafı kesilmelidir. </li><li> Yumurta odalarına (Şekil 1E -3) ön tarafına mümkün olduğunca yakın bir microknife, sütunun sol tarafındaki dilim, kullanma. </li><li> Kesim sütun üzerinde soğuk 1X PBT 100 ul pipet. </li><li> Slaytta kalanı bırakarak, yumurta odaları ve atın kesme sütunu üç tarafı etrafında aşırı gliserin jöle kaldırmak için bir yuvarlak kenarlı spatula kullanın. </li><li> Yumurta odalarının ön tarafında yapılan kesim spatula yerleştirin ve birincil gliserin jöle bloğundan sütun ayrı. </li><li> Örnekleri için ya ters (5.8.1) veya dik (5.8.2) mikroskopi hazırlayın. <ol><li> Ters Mikroskop için: Devir tYumurta odalarının ön tarafı aşağı bakacak şekilde bir lamel yumurta odalarından o sütun. Gliserin jöle tüm sütunlar kaldırıldı ve monte edilene kadar tekrarlayın 5,2-5,8 adımları. Kurumasını önlemek için yumurta odaların her blok üzerine,% 50 PBS-Gliserin birkaç damla ekleyin. Doğrudan lamel Görüntü yumurta odaları. </li><li> Dik Mikroskop için: ön tarafı yukarı bakacak şekilde bir mikroskop lamı yumurta odalarının sütun aktarın. Gliserin jöle tüm sütunlar kaldırıldı ve mikroskop slaytlar üzerine monte edilene kadar tekrarlayın 5,2-5,9 adımları. # 1 ½ lamel ile yumurta odaları ve kapak blok, her blok üzerine% 50 PBS-Gliserin birkaç damla ekleyin. </li></ol></li></ol>

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The authors have no competing financial interests.

Burada bir son perspektif yumurta odaları gelişmekte, montaj için bir yöntem tarif ve görüntü küçük. Görüntüleme yumurta odaları için ortak teknikler yanal görünümler için optimize edilmiş ve floresan antikorlar ile boyanmış zaman öncelikle iç-dış folikül hücreleri hassas görselleştirme izin vardır. Birden fazla odak düzlemleri görüntüleme Z-yığınlarının veya 3-D rekonstrüksiyonlar yardımcıları kullanımı, ama yine de eliptik yumurta odaları (Şekil 2B) kutu...

Drosophila melanogaster Çalışma olayların geniş bir genetik yönetmelik seyircilerden büyük anlayışlar sağladı. Oogenez doku desen, hücre polarite değişiklikleri, hücre döngüsü anahtarlama ve translasyonel yönetmelik 1,2,3,4 dahil olmak üzere birçok farklı gelişim süreçleri, araştırmak için bir uysal bir yol sağlar, çünkü özellikle, yumurta gelişimi üzerine kapsamlı bir araştırma olmuştur. Oogenez sırasında önemli bir morfojenik olay (5 gözden) sınır hücreleri denilen hücreler bir dizi özellikleri, motilite edinimi ve göç. Hücre göçü beri hayvan morfojenezinin önemli bir özelliğidir ve bu sürecin genetik düzenleme iyi korunduğu için, sinekler belirlenen mekanizmaları diğer bağlamlarda önemli olması muhtemeldir. Böylece, biz sınır hücre migrasyonu moleküler kontrolü araştırıyor. Bizim çalışmalar için, biz yumurta geliştirme ön direkleri gözlemlemek için yeni bir yöntem geliştirdik,Sınır hücreleri ortaya yerlerde, detaylı olarak nasıl geliştiğini incelemek için. Yumurtalık içinde, çeşitli gelişim aşamalarında yumurta odaları ince kılıf (Şekil 1A) &#39;de muhafaza edilir ovarioles adı zincirleri boyunca mevcuttur. Her yumurta bir yumurta odası oluşturmak için devam edecektir. Oogenesiz sırasında, çok sayıda, farklı hücre tipleri, bir koordine bir şekilde geliştirilmesi gerekmektedir. D. melanogaster yumurta bölmesi tek katmanlı foliküler epitel 6 ile çevrili bir oosit, aşağıdakileri içeren 16 germ hat hücrelerine oluşur. Kutup hücreleri denilen özelleşmiş hücrelerin az sayıda, epitel anterior ve posterior kutuplarda ortaya çıkar. 6-8 sınır hücreleri kutup hücreleri 5,7 ile uyarılan yumurta odasının ön epiteli, köken. Orta-oogenez (evre 9), sınır hücreleri komşularından ayırmak ve yumurta odasının 5,7 olan posterior oosit ulaşmak için hemşire hücreleri arasındaki göç. Bu hareket yerine getirmesi gerekirSınır hücreleri bu kolektif hücre göçü bir tür yapma, olmayan iki hareketli kutup hücreleri çevreleyen bir kümede kalırken. Sınır hücre kümesinin Başarılı göç döllenme için gerekli olan yumurta kabuğu micropyle, düzgün gelişimini sağlar. ön kutup hücreleri sinyal iletim dizinini aktive ederek hücre sınır kaderi talimat. Polar hücreler oosit gelişimi 8,9 aşamasında 8 sırasında folikül hücreleri komşu, bir transmembran reseptörü, Kubbesiz (DOME) bağlanan bir sitokin, eşleşmemiş (UPD), salgılarlar. UPD bağlanması Janus tirozin kinaz (JAK) transkripsiyon (STAT) 8,10,11 arasında sinyal ileticisi ve Activator fosforile olur. STAT sonra transkripsiyonu aktive etmek çekirdeğine geçer. Yavaş Sınır Hücreler (SLBO) STAT doğrudan bir transkripsiyonel hedef ve sınır hücre göçü 12 için gerekli olan bir transkripsiyon faktörüdür. Yumurta odaları Yanal manzarası t gösterirşapka STAT etkinliği ön epiteli 8,11,13 boyunca bir degrade düzenlenmiştir. Kutup hücrelere yakın folikül hücreler bu nedenle sınır hücreleri haline ve komşu germ-line doku istila, aktif STAT en yüksek seviyeleri var. Sınır hücreleri içinde belirtilen ve epitel ayırmak nasıl anlamak için, biz doku nasıl düzenlendiğini gözlemlemek gerekir. Biz bir ön-perspektif yumurta odaları görmek, biz kutup hücreleri çevreleyen folikül hücreleri STAT aktivitesinin radyal simetri beklenebilir. Bir son bakış da daha doğru önce ve farklı odak düzlemlerinde hücreleri karşılaştırma daha Ayırma sırasında zar proteinleri ve hücre-hücre arayüzleri farklılıklar gösterir. Yumurta odaları dikdörtgen ve sap hücreleri birbirine bağlı olduğundan, onlar zor ön mimarisi gözlemlemek için yapım, yanal slaytlar üzerine yerleşmek. Böylece, yumurta odasının kutuplarda hücreler hakkında çok bilgi vardırLateral manzaralı olayla edilmiştir. Bazı bilgiler Photobleaching optik bölümlerde, ışık saçılması, algoritmik 3-D rekonstrüksiyon ve Z-ekseninde çözünürlük yoksul limitler aracılığıyla elde edilebilmesine rağmen süper çözünürlüklü mikroskopi 14 gibi pahalı tekniklerin yokluğunda bu bilgilerin daha az detaylı ve güvenilir hale . Bölüm-tabanlı görüntüleme diğer şekillerde (örneğin, elektron mikroskobu veya mikrotom kesit) eserler için olasılığını artırarak, dehidrasyon dahil dokuların, geniş manipülasyon gerektirir. Böylece, biz görüntü D. yeni bir yöntem geliştirdi dik iken melanogaster yumurta odaları. Bu yöntem zaten (inceleme altında Temsilcisi sonuçları ve Manning ve arkadaşları, bakınız) hareketli hücreler kaderde nasıl durulaştırmada yararlı kanıtlanmış ve daha geniş değerli oogenezisin diğer yönleri çalışmalarda olması muhtemeldir.

<table><tbody><tr><td>0.1 M Potassium phosphate Buffer (KPO&lt;sub&gt;4&lt;/sub&gt; Buffer)</td><td></td><td></td><td>Add 3.1 g of NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;H&lt;sub&gt;2&lt;/sub&gt;O and 10.9 g of Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (anhydrous) to distilled H&lt;sub&gt;2&lt;/sub&gt;O to make a volume of 1 L. The pH of the final solution will be 7.4. This buffer can be stored for up to 1 month at 4&amp;deg;C.</td></tr><tr><td>16% Paraformaldehyde aqueous solution, EM grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td>methanol-free to preserve GFP fluorescence</td></tr><tr><td>30G x 1/2 inch needle&amp;nbsp;</td><td>VWR</td><td>BD305106</td><td>Regular bevel</td></tr><tr><td>Active Dry Yeast</td><td>Genesee Scientific</td><td>62-103</td><td>To fatten female flies</td></tr><tr><td>Bovine Serum Albumin</td><td>PAA-cell culture company</td><td>A15-701</td><td>Used in NP40 Wash Buffer</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11295-10</td><td>Dumostar alloy, biologie tip; sharp tips are essential for ovariole dissection</td></tr><tr><td>DAPI (4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride)</td><td>Sigma Aldrich</td><td>D9542</td><td>5 mg/ml stock solution&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16140-071</td><td>Added to supplement dissection medium (10%)&amp;nbsp;</td></tr><tr><td>Glass culture tube</td><td>VWR</td><td>47729-576</td><td>14 ml</td></tr><tr><td>Glass Depression Slides</td><td>VWR</td><td>470019-020</td><td>1.2 mm Thick, Double Cavity</td></tr><tr><td>Glycerin Jelly&amp;nbsp;</td><td>Electron Microsocpy Sciences</td><td>17998-10</td><td>Mounting media</td></tr><tr><td>Glycerol</td><td>IBI Scientific</td><td>IB15760</td><td>70% in PBS</td></tr><tr><td>IGEPAL CA-630</td><td>Sigma Aldrich</td><td>I3021-500ML</td><td>(interchangable for Nonidet P-40)&amp;nbsp; Used in NP40 Wash Buffer&amp;nbsp;</td></tr><tr><td>Leica Fluorescent Stereoscope&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Leica SP5 Confocal Microscope</td><td>Leica Microsystems</td><td></td><td>40x/0.55NA dry objective&amp;nbsp;</td></tr><tr><td>Micro spatula&amp;nbsp;</td><td>VWR</td><td>82027-518</td><td>Stainless steel</td></tr><tr><td>Microscope Slide</td><td>VWR</td><td>16004-368</td><td>75x25x1 mm</td></tr><tr><td>NP40 Wash Buffer</td><td></td><td></td><td>50 mM TRIS-HCl, 150 mM NaCl, 0.5% Ipegal, 1 mg/ml BSA, and 0.02% sodium azide</td></tr><tr><td>Penicillin-Streptomycin-Glutamine</td><td>Life Technologies</td><td>10378-016</td><td>Added to supplement dissection medium (0.6X)</td></tr><tr><td>Petridish- Polysterine, sterile</td><td>VWR</td><td>82050-548</td><td>60 W x 15 H mm</td></tr><tr><td>Phosphate Buffer Saline</td><td>Sigma Aldrich</td><td>P3813-10PAK</td><td>10 packs of Powder</td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma Aldrich</td><td>P3786-500G</td><td>Used in 0.1 M KPO&lt;sub&gt;4&lt;/sub&gt; Buffer&amp;nbsp;</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P9791-500G</td><td>Used in 0.1 M KPO&lt;sub&gt;4&lt;/sub&gt; Buffer&amp;nbsp;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Schneider&amp;rsquo;s Insect Medium</td><td>Life Technologies</td><td>&lt;br/&gt; 11720018</td><td>With L-glutamine and sodium bicarbonate</td></tr><tr><td>Sodium azide</td><td>Sigma Aldrich</td><td>S2002-25G</td><td>Used in fluorescent antibody staining</td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>S3014-500G</td><td>Used in NP40 Wash Buffer</td></tr><tr><td>TRIS-HCl</td><td>IBI Scientific</td><td>IB70144</td><td>1 M TRIS-HCl, pH 7.4</td></tr><tr><td>Volocity 3D Image Analysis Software</td><td>PerkinElmer</td><td></td><td>For processing confocal Z-stacks</td></tr></tbody></table>

upright imaging of drosophila egg chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

dik görüntüleme yöntemi bize aşamada 8. anterior foliküler epitel hücrelerinin doğrudan organizasyonu görmek için izin bir folikül hücre kaderi için genel işaretleyici, Gözler Yok (EYA) proteini, yanı sıra nükleer DNA işaretleyici DAPI, hatta ifade gösterdi hücrelerin bu alan boyunca ve tüm hücreler içindeki boyama yoğunlukları (Şekil 2B &quot;) ile görülebilir gösterdi. Sitokin UPD yanıt olarak ayarlanır proteinler Bununla birlikte, değişken şekilleri ve sentezleme se...

Watch this Scientific Journal Video about Upright Imaging of Drosophila Egg Chambers at JoVE.com

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

Dik Görüntüleme<em&gt; Drosophila</em&gt; Yumurta Odaları

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

upright-imaging-of-drosophila-egg-chambers

Research

JoVE Journal

Developmental Biology

Upright Imaging of Drosophila Egg Chambers

7.6K Görüntüleme.  University of Maryland Baltimore County. The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

Video: Upright Imaging of Drosophila Egg Chambers

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

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

Görüntüleme Kalsiyum<em&gt; Drosophila</em&gt; Yumurta Aktifleştirme de

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Mezoskopik Floresans Tomografi için In-vivo Görüntüleme Geliştirme Drosophila</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Biyoloji

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Pupal Evrelerinde Gelişen Drosophila Epitelinde Doku Oryantasyonu ve Büyüme Dinamiğinin Görüntülenmesi ve Analizi

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

Mikro bilgisayarlı Tomografi kullanılarak Drosophila melanogaster'ın Tüm Hayvan Görüntülemesi

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.

The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

Canlı Görüntüleme Kullanarak Oksidatif Hasarın Drosophila Yumurta Odaları Üzerindeki Etkilerini Görselleştirme

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

Nuclear Migration in the Drosophila Oocyte

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools. During Drosophila mid-oogenesis, the nucleus migrates from a central position within the oocyte to adopt an asymmetric position mediated by microtubule-generated forces. This migration and positioning of the nucleus are necessary to determine the polarity axes of the embryo and the subsequent adult fly. One characteristic of this migration is that it occurs in three dimensions (3D), creating a necessity for live imaging. Thus, to study the mechanisms that regulate nuclear migration, we have developed a protocol to culture the dissected egg chambers and perform live imaging for 12 h by time-lapse acquisitions using spinning-disk confocal microscopy. Overall, our conditions allow us to preserve Drosophila egg chambers alive for a long period of time, thereby enabling the completion of nuclear migration to be visualized in a large number of samples in 3D.

Nuclear Migration in the Drosophila Oocyte

In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

Drosophila Oosyte'de Nükleer Göç

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

VE Dik Görüntüleme Drosophila Embriyolar

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Anastasis Vivo içinde CaspaseTracker Biyoalgılayıcı tarafından tespit

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

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Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

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Mezoskopik Floresans Tomografi için In-vivo Görüntüleme Geliştirme Drosophila