Name: imaging of cell shape alteration and cell movement in drosophila gastrulation using de-cadherin reporter transgenic flies
Uploaded: 2016-12-29T14:00:00.000+00:00
Duration: 4 min 42 s
Description: Watch this Scientific Journal Video about Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies at JoVE.com

This study was supported by the Astellas Foundation for Research on Metabolic Disorders (HT), Takeda Science Foundation (HT), and MEXT-Supported Program for the Strategic Research Foundation at Private Universities (HT).

ملاحظة: الذباب المعدلة وراثيا المستخدمة في هذه الدراسة ما يلي: DE-نذل :: GFP 10. 1. إعداد أبل لوحة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحضير خليط من 12.5 غ أجار، 125 مل 100٪ المتاحة تجاريا عصير التفاح، 12.5 غرام جلوكوز، و 375 مل H 2 O. حرارة المزيج في الميكروويف وسكبه في 3 سم طبق ثقافة الخلية. تخزين الخليط في 4 درجات مئوية لاستخدامها في المستقبل. </li><li style=";text-align:right;direction:rtl"> بعد إعداد لوحة التفاح، إضافة طبقة رقيقة من معجون معجون الخميرة على أعلى من ذلك للسماح للالذباب لوضع البيض. </li></ol> 2. الأجنة إعداد وبروتوكول تصوير لايف <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع حوالي 50 الذباب (25 ذكور و 25 إناث) في زجاجة للتزاوج ووضع البيض في وقت لاحق بين عشية وضحاها على لوحة التفاح المشمولة في خميرة تعجن. تعيين لوحة عصير التفاح في فم الزجاجة. إبقاء زجاجة رأسا على عقب في حاضنة. التفاف الزجاجات البلاستيكية الأسهم ذبابة الفاكهة بورق الألمنيوم للمطالبة عشرالبريد الذباب لوضع المزيد من البيض. </li><li style=";text-align:right;direction:rtl"> في صباح اليوم التالي، تحل محل القديمة لوحة عصير التفاح مع واحدة جديدة. </li><li style=";text-align:right;direction:rtl"> اسمحوا الذباب تضع البيض لمدة 3 إلى 4 ساعات بواسطة التفاف زجاجة مع رقائق الألومنيوم. </li><li style=";text-align:right;direction:rtl"> بعد ثلاث إلى أربع ساعات، وجمع الأجنة في مصفاة الجنين من لوحة عصير التفاح باستخدام فرشاة أو القطن سيئة وغسلها مع برنامج تلفزيوني. غسل الأجنة 2-3 مرات مع برنامج تلفزيوني في أطباق ثقافة 12-جيدا. </li><li style=";text-align:right;direction:rtl"> Dechorionate الأجنة مع التبييض 50٪ لمدة 5 دقائق في أطباق ثقافة 12-جيدا. </li><li style=";text-align:right;direction:rtl"> بعد عملية dechorionation، وغسل الأجنة 2-3 مرات مع برنامج تلفزيوني. </li><li style=";text-align:right;direction:rtl"> نقل الأجنة إلى صحن ثقافة الخلية 3 سم تحتوي على برنامج تلفزيوني ثم اختر المرحلة 5 الأجنة تحت المجهر استنادا إلى مستوى من الشفافية في حدودها. ماصة للأجنة نظموا باستخدام 200 ميكرولتر ماصة. </li><li style=";text-align:right;direction:rtl"> بعد ذلك، وضع اثنين من الأجنة المختارة على ساترة الزجاج، وإزالة الزائد برنامج تلفزيوني مع المناديل الورقية الملتوية ناعما. توجيه الأجنة الجانب الظهري حتى علىساترة باستخدام المناديل الورقية الملتوية ناعما. بعد ذلك، ونعلق الأجنة إلى ساترة الزجاج مع الشحوم السيليكون باستخدام إبرة رفيعة والمناديل الورقية الملتوية. </li><li style=";text-align:right;direction:rtl"> إضافة قطرة صغيرة من زيت الهالوكربون 700 على الأجنة ووضع ساترة تحتوي على أجنة رأسا على عقب على الشريحة بادئة، وترك بعض المساحة في أسفل الشريحة. </li><li style=";text-align:right;direction:rtl"> فحص الأجنة باستخدام نظام متحد البؤر التصوير، والأرجون / 488 ليزر، وهدف النفط الغمر (63X). ملاحظة: تحديد الجنين في هذه المرحلة التنموية المناسبة مهمة لالتقاط الصور لهذا الحدث تكون المعيدة. في ظل هذه الظروف، ومراقبة تقريبا جميع الأجنة تتطور عادة ما يصل إلى ما لا يقل عن مرحلة 14. الجنين تحليلها يمكن تربيتها مواصلة تطوير كشخص بالغ. </li></ol>

<ol>
	<li>Haruta, T., Warrior, R., Yonemura, S., Oda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+proximal+half+of+the+Drosophila+E-cadherin+extracellular+region+is+dispensable+for+many+cadherin-dependent+events+but+required+for+ventral+furrow+formation.">The proximal half of the Drosophila E-cadherin extracellular region is dispensable for many cadherin-dependent events but required for ventral furrow formation.</a> Genes Cells. 15 (3), 193-208 (2010).</li><li>Oda, H., Takeichi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution:+structural+and+functional+diversity+of+cadherin+at+the+adherens+junction.">Evolution: structural and functional diversity of cadherin at the adherens junction.</a> J. Cell Biol. 193 (7), 1137-1146 (2011).</li><li>Fernandez-Sanchez, M. E., Serman, F., Ahmadi, P., Farge, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+induction+in+embryonic+development+and+tumor+growth+integrative+cues+through+molecular+to+multicellular+interplay+and+evolutionary+perspectives.">Mechanical induction in embryonic development and tumor growth integrative cues through molecular to multicellular interplay and evolutionary perspectives.</a> Methods Cell Biol. 98, 295-321 (2010).</li><li>Alderton, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+Epithelial+to+mesenchymal+and+back+again.">Metastasis: Epithelial to mesenchymal and back again.</a> Nat. Rev. Cancer. 13 (1), 3 (2013).</li><li>Fabregat, I., Malfettone, A., Soukupova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Insights+into+the+Crossroads+between+EMT+and+Stemness+in+the+Context+of+Cancer.">New Insights into the Crossroads between EMT and Stemness in the Context of Cancer.</a> J. Clin. Med. 5 (3), (2016).</li><li>Serrano-Gomez, S. J., Maziveyi, M., Alahari, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+epithelial-mesenchymal+transition+through+epigenetic+and+post-translational+modifications.">Regulation of epithelial-mesenchymal transition through epigenetic and post-translational modifications.</a> Mol. Cancer. 15, (2016).</li><li>Yu, A. Q., Ding, Y., Li, C. L., Yang, Y., Yan, S. R., Li, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TALEN-induced+disruption+of+Nanog+expression+results+in+reduced+proliferation,+invasiveness+and+migration,+increased+chemosensitivity+and+reversal+of+EMT+in+HepG2+cells.">TALEN-induced disruption of Nanog expression results in reduced proliferation, invasiveness and migration, increased chemosensitivity and reversal of EMT in HepG2 cells.</a> Oncol. Rep. 35 (3), 1657-1663 (2016).</li><li>Zheng, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-to-mesenchymal+transition+is+dispensable+for+metastasis+but+induces+chemoresistance+in+pancreatic+cancer.">Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer.</a> Nature. 527 (7579), 525-530 (2015).</li><li>Kalluri, R., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basics+of+epithelial-mesenchymal+transition.">The basics of epithelial-mesenchymal transition.</a> J. Clin. Invest. 119 (6), 1420-1428 (2009).</li><li>Huang, J., Zhou, W., Dong, W., Watson, A. M., Hong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed,+efficient,+and+versatile+modifications+of+the+Drosophila+genome+by+genomic+engineering.">Directed, efficient, and versatile modifications of the Drosophila genome by genomic engineering.</a> Proc. Natl. Acad. Sci. U.S.A. 106 (20), 8284-8289 (2009).</li></ol>

The authors have nothing to disclose.

Although we have previously reported a similar procedure to capture live images of the gastrulation process in Drosophilla1, the method we describe here is detailed and easy to trace endogenous cadherin expression and thus is quite useful for genetic screening of key factors involved in gastrulation. To maximize success with this imaging procedure, it is essential to use an indented slide. Mechanical pressure sometimes causes embryonic death. Therefore, it is also important to handle the embryos as ge...

تكون المعيدة هي أول مجموعة من الفعاليات الحيوية شكليا التي تحدث أثناء التطور الجنيني من الحيوانات متعددة الخلايا مثل ذبابة الفاكهة 1،2. ومن المثير للاهتمام، شواهد تشير إلى أن هذه العملية ينظم من خلال التفاعل بين الآليات الميكانيكية والجزيئية 3. وعلاوة على ذلك، هو تورط الظهارية للانتقال الوسيطة (EMT)، وهي عملية حاسمة في تكون المعيدة، وأيضا في عمليات الأمراض البشرية مثل سرطان خبيث 4-8. على هذا النحو، فإن العديد من الجينات التي تتحكم في انقباض قمي ومن المعروف أيضا أن تكون العوامل الرئيسية في EMT لوحظ في سرطان خبيث 9. وهكذا، انقباض قمي في وقت تكون المعيدة هو نموذج ممتاز للتحقيق في الآليات التنظيمية المشار إليها وتعزيز فهمنا لورم خبيث من السرطان. والاستفادة من هذه التقنية هو أن نتمكن من مراقبة حركة الخلية في وقت تكون المعيدة في الوقت الحقيقي، وبالتالي، سنكونقادرة على فحص الجينات المسؤولة عن تكون المعيدة وكذلك الانبثاث سرطان. على الرغم من أن غير معروف نسبيا، ويعتقد التصاق الخلية الى خلية للعب دور مركزي في انقباض القمي 1. علم الوراثة ذبابة الفاكهة هي مناسبة تماما للتحقيقات واحدة على مستوى الخلية استكشاف الآليات الجزيئية التنظيمية. وهذا النموذج سوف تمكننا من كشف أهمية انقباض قمي خلال المعيدة. وعلاوة على ذلك، وهذه الطريقة يمكن أن تستخدم لفحص الجينات المسؤولة في الانبثاث السرطان. والتقاط الصور الحية من ذبابة الفاكهة تكون المعيدة مزيد مكنتنا من فهم بمزيد من التفصيل الآليات الجزيئية التي تحكم إعادة ترتيب الأنسجة. هنا، ونحن نقدم وصفا شاملا لطريقة بسيطة لتحقيق ذلك.

<table><tbody><tr><td>Halocarbon oil 700</td><td>Sigma</td><td>MKBH 5726</td><td></td></tr><tr><td>Vacuum grease Silicone</td><td>Beckman</td><td>335148</td><td></td></tr><tr><td>Glass coverslip&amp;nbsp;</td><td>Matsunami glass</td><td>Thickness No1</td><td>24 - 36 mm</td></tr><tr><td>Embryo stariner</td><td>Corning</td><td>Corning3477</td><td></td></tr><tr><td>Plastic Drosophilla Stock Bottles</td><td>Hitec</td><td>MKC-100</td><td></td></tr><tr><td>DE-Cadherin knock-in flies</td><td></td><td></td><td>REF (10)</td></tr></tbody></table>

imaging of cell shape alteration and cell movement in drosophila gastrulation using de-cadherin reporter transgenic flies

تكون المعيدة هي أول مجموعة من الفعاليات الحيوية شكليا التي تحدث أثناء التطور الجنيني من الحيوانات متعددة الخلايا مثل ذبابة الفاكهة. يتم التعرف على هذا التغيير الصرفي أيضا الظهارية للانتقال الوسيطة (EMT). ويرتبط ديسريغولاتيون من EMT يعانون من التليف والانبثاث السرطان. وهناك أدلة الناشئة التي EMT مسيطر عليها من قبل عدد من الآليات الجزيئية. على هذا النحو، فإن العديد من الجينات الرئيسية التي تتحكم في انقباض قمي ومن المعروف أيضا أن يكون من العوامل الهامة في EMT لوحظ في الانبثاث السرطان. مثل EMT خلال ذبابة الفاكهة تكون المعيدة، الخلايا الظهارية يمكن أن يتسبب في تغيير شكلها وإعادة برمجتها لإعادة توجيه مصير خلية تجاه مختلف أنواع الخلايا الأخرى. هنا نقدم طريقة التصوير قوية من ذبابة الفاكهة تكون المعيدة لفحص بدء الحركات الخلوية التخلق وتحديد مصير الخلايا خلال هذه المرحلة من التطور الجنيني. باستخدام هذه الطريقة، أن نحدد جالذراع إعادة ترتيب في وقت تكون المعيدة وإظهار أهمية انقباض قمي خلال المعيدة باستخدام GFP المسمى DE-كادهيرين.

هنا، نقدم لك مجموعة من الأحداث تكون المعيدة للجنين ذبابة الفاكهة ولمحة عامة عن إجراءات إعداد الجنين (الشكل 1). وصفت أغشية الخلايا باستخدام DE-كادهيرين :: GFP وأن يتم التصوير المباشر لحركات الخلية في وقت تكون المعيدة في ذبابة الفاكهة (ا...

Watch this Scientific Journal Video about Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies at JoVE.com

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Herein we describe a procedure to capture live images of Drosophila gastrulation. This has enabled us to better understand the apical constriction involved in early development and further analyze mechanisms governing cellular movements during tissue structure modification.

التصوير من التعديلات خلية الشكل والحركة الخلية في<em&gt; ذبابة الفاكهة</em&gt; المعيدة عن طريق DE-كادهيرين مراسل الذباب المعدلة وراثيا

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. ...

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Research

JoVE Journal

Developmental Biology

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

7.0K Views.  Jahangirnagar University. Herein we describe a procedure to capture live images of Drosophila gastrulation. This has enabled us to better understand the apical constriction involved in early development and further analyze mechanisms governing cellular movements during tissue structure modification.

Video: Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

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.

يعيش التصوير لل<em&gt; ذبابة الفاكهة</em&gt; العذراء العين

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

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

ال<em&gt; ذبابة الفاكهة</em&gt; تخيلي ورم القرص نموذج: التصور والكمي للالتعبير الجيني وغزو الورم عن طريق الفسيفساء الوراثية

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Cancer Research

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.

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.

التصوير تستقيم من<em&gt; ذبابة الفاكهة</em&gt; غرف البيض

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

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

باستخدام رقائق الموائع الدقيقة للتصوير لايف ودراسة الردود الإصابات في<em&gt; ذبابة الفاكهة</em&gt; يرقات

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Bioengineering

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.

يعيش التصوير للهجرة الخلايا الدبقية في القرص العين تخيلي ذبابة الفاكهة

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

Biology

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted epithelial morphogen. In this assay, Fog is used as an agonist to activate Rho through a signaling cascade that includes a G-protein-coupled receptor (Mist), a G&#945;12/13 protein (Concertina/Cta), and a PDZ-domain-containing guanine nucleotide exchange factor (RhoGEF2). Fog signaling results in the rapid and dramatic reorganization of the actin cytoskeleton to form a contractile purse string. Soluble Fog is collected from a stable cell line and applied ectopically to S2R+ cells, leading to morphological changes like apical constriction, a process observed during developmental processes such as gastrulation. This assay is amenable to high-throughput screening and, using RNAi, can facilitate the identification of additional genes involved in this pathway.

A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells

Here we describe a contractility assay using Drosophila S2R+ cells. The application of an exogenous ligand, folded gastrulation (Fog), leads to the activation of the Fog signaling pathway and cellular contractility. This assay can be used to investigate the regulation of cellular contractility proteins in the Fog signaling pathway.

تحليل يستند إلى الخلية للتحقيق غير العضلات الميوسين الثاني Contractility عبر gastrulation مطوية إشارات الطريق في المورفولوجية S2R + الخلايا

We have developed a cell-based assay using Drosophila cells that recapitulates apical constriction initiated by folded gastrulation (Fog), a secreted ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

يعيش تصوير ذبابة الفاكهة السوداء البطن هجرات كرية دموية الجنينية

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

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.

التصوير والتحليل لاتجاهات الأنسجة وديناميات النمو في الظهارة الهوائية النامية Drosophila خلال مراحل البوبال

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

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

تشريح وتصوير حي للغدد التناسلية ذبابة الفاكهة الجنينية المتأخرة

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

التصوير تغيير الشكل في الخلية الحية ذبابة الفاكهة الأجنة

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

التصوير من التعديلات خلية الشكل والحركة الخلية في

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