Name: imaging of cell shape alteration and cell movement in drosophila gastrulation using de-cadherin reporter transgenic flies
Uploaded: 2016-12-29T14:00:00
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).

NOTE: Les mouches transgéniques utilisées dans cette étude sont les suivants: DE-cad :: GFP 10. 1. Préparation d&#39;Apple Plate <ol><li> Préparer un mélange de 12,5 g d&#39; agar, 125 mL 100% disponible dans le commerce de jus de pomme, 12,5 g de glucose et 375 ml H 2 O. Chauffer le mélange dans un micro - ondes et versez - le dans une boîte de culture de cellules de 3 cm. Stocker le mélange à 4 ° C pour une utilisation future. </li><li> Après la préparation de la p...

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	<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>

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...

Gastrulation est le premier ensemble d&#39;événements morphologiquement dynamiques qui se produisent au cours du développement embryonnaire des animaux pluricellulaires tels que la drosophile 1,2. Il est intéressant de nouvelles preuves suggèrent que ce processus est régi par l&#39;interaction entre les mécanismes mécaniques et moléculaires 3. En outre, la transition épithélio - mésenchymateuse (EMT), qui est un processus crucial dans la gastrulation, est également impliquée dans des processus pathologiques humains tels que les métastases du cancer 8/4. En tant que tels, de nombreux gènes qui contrôlent la constriction apicale sont également connus pour être des facteurs clés dans l&#39;OGD observée dans les métastases du cancer 9. Ainsi, constriction apicale au moment de la gastrulation est un excellent modèle pour étudier les mécanismes de régulation précités et d&#39;améliorer notre compréhension des métastases du cancer. L&#39;avantage de cette technique est que nous pouvons observer le mouvement des cellules au moment de la gastrulation en temps réel et, par conséquent, nous seronsen mesure de dépister les gènes impliqués dans la gastrulation, ainsi que les métastases du cancer. Bien que relativement inconnu, l&#39; adhésion de cellule à cellule est pensé pour jouer un rôle central dans la constriction apicale 1. Génétique de la Drosophile est bien adapté pour les enquêtes individuelles au niveau cellulaire explorant les mécanismes moléculaires de régulation. Ce modèle nous permettra de découvrir l&#39;importance de la constriction apicale pendant la gastrulation. En outre, cette méthode peut être utilisée pour cribler des gènes impliqués dans la métastase du cancer. Capture d&#39; images en direct de la drosophile gastrulation nous a en outre permis de comprendre plus en détail les mécanismes moléculaires qui régissent le réarrangement des tissus. Ici, nous fournissons une description complète d&#39;une méthode simple pour y parvenir.

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			<td height="54" style="height:54px;width:268px;">Halocarbon oil 700</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:129px;">MKBH 5726</td>
			<td style="width:133px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:268px;">Vacuum grease Silicone</td>
			<td style="width:164px;">Beckman</td>
			<td style="width:129px;">335148</td>
			<td style="width:133px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:268px;">Glass coverslip&#160;</td>
			<td style="width:164px;">Matsunami glass</td>
			<td style="width:129px;">Thickness No1</td>
			<td style="width:133px;">24-36mm</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:268px;">Embryo stariner</td>
			<td style="width:164px;">Corning</td>
			<td style="width:129px;">Corning3477</td>
			<td style="width:133px;"></td>
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		<tr height="38">
			<td height="38" style="height:38px;width:268px;">Plastic Drosophilla Stock Bottles</td>
			<td style="width:164px;">Hitec</td>
			<td style="width:129px;">MKC-100</td>
			<td style="width:133px;"></td>
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			<td height="21" style="height:21px;width:268px;">DE-Cadherin knock-in flies</td>
			<td></td>
			<td></td>
			<td>REF (10)</td>
		</tr>
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imaging of cell shape alteration and cell movement in drosophila gastrulation using de-cadherin reporter transgenic flies

Gastrulation est le premier ensemble d&#39;événements morphologiquement dynamiques qui se produisent pendant le développement embryonnaire des animaux pluricellulaires tels que la drosophile. Cette altération morphologique est également reconnu comme transition épithélio mésenchymateuse (EMT). Dysrégulation de EMT est associée à la fibrose et les métastases du cancer. Il existe des preuves émergentes que EMT est contrôlé par un certain nombre de mécanismes moléculaires. En tant que tels, de nombreux gènes clés qui contrôlent la constriction apicale sont également connus pour être des facteurs importants dans l&#39;OGD observée dans les métastases du cancer. Comme EMT pendant la drosophile gastrulation, les cellules épithéliales peuvent être amenés à changer leur forme et être reprogrammées pour rediriger le destin des cellules vers d&#39;autres types de cellules. Ici , nous fournissons une méthode d&#39;imagerie robuste de Drosophila gastrulation pour doser l&#39;initiation des mouvements cellulaires morphogénétiques et l&#39; identification du destin cellulaire au cours de ce stade de développement embryonnaire. En utilisant cette méthode, nous identifions cell réarrangement au moment de la gastrulation et de démontrer l&#39;importance de la constriction apicale pendant la gastrulation utilisant GFP marquées DE-cadhérine.

Ici, nous montrons les événements gastrulation de l&#39;embryon de drosophile et un aperçu général de la procédure de préparation de l&#39; embryon (Figure 1). Les membranes cellulaires sont marquées en utilisant le document DE-cadhérine :: GFP et l&#39; imagerie en temps réel des mouvements de cellules est effectuée au moment de la gastrulation chez la drosophile (figure 2). Depuis DE-cadhérine GFP mouches nous permettent de visuali...

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.

Imaging of Cell Forme Altération et Mouvement cellulaire dans<em&gt; Drosophila</em&gt; Gastrulation Utilisation DE-cadhérine Reporter mouches transgéniques

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

The overall goal of this procedure, is to better understand the cellular shape and movement, during Drosophila gastrulation. Here we provide a robustic measuring method of Drosophila gastrulation, to assay the initiation of morphogenetic cellular movement, during this stage of embryonic development. The main advantage of this technique is that, cell rearrangement at the time of gastrulation, can be identified along with apical constriction, during gastrulation.To begin the experiment, prepare a mixture of 375 milliliters of water. 125 milliliters 100%commercially available apple juice. 12.5 grams of agar.12.5 grams of glucose. Next, heat the mixture in a microwave, until the agar gets melted down. And pour it into a three centimeter cell culture dish.Store the mixture at 4 degrees celsius, for future use. After preparing the apple plate, add a thin layer of kneaded yeast paste on top of it, to allow the flies to lay eggs. Place 25 male and 25 female flies in a bottle on an apple plate covered in kneaded yeast.Wrap the plastic Drosophilas stock bottles with aluminum foil to prompt the flies to lay more eggs. Keep the bottle upside down in an incubator overnight. The next morning, replace the apple juice plate with a fresh one.Then wrap the bottle with aluminum foil and allow the flies to lay eggs for three to four hours. After the flies have laid eggs, loosen the eggs from the plate with a cotton bud. Collect the embryos in a strainer, and wash them with phosphate buffered saline, or PBS.Wash the embryos three more times with PBS, in 12-well culture dishes. Dechorionate the embryos with 50%bleach, in 12-well culture dishes. Following the dechorionation process, wash the embryos three more times with PBS.Transfer the embryos to a three centimeter cell culture dish containing PBS. And select stage five embryos under the microscope, based on the level of transparency at their borders. Using a 200 microliter pipette tip, collect the staged embryos.Next, place two selected embryos on a glass cover slip, and remove excess PBS with tissue paper. With a finely twisted tissue paper, orient the embryos dorsal side up to the cover slip. Then attach the embryos to the glass cover slip with silicone grease using a fine needle.Add a small drop of hollow carbon oil 700 on the embryos, and place the coverslip containing the embryos upside down on the indented slide. Leaving some space at the bottom of the slide. Then stick the cover slip to the slide.Finally, examine the embryos using a confocal imaging system, an argon 488 laser, and an oil-immersion objective. Time-lapse images were captured from live imaging, using DE-cadherin GFP of cell movement over 500 seconds, to analyze the apical areas of individual cells, of Drosophila during gastrulation. At 500 seconds, apical constriction, and invagination are clearly visible.By using our method. We can further understand, the molecular mechanisms, of Drosophila gastrulation, as well as, epithelial to mesenchymal transition, involved in cancer metastasis.

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

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