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: The transgenic flies used in this study include the following: DE-cad::GFP 10.
1. Preparation of Apple Plate
<ol>
	<li>Prepare a mixture of 12.5 g agar, 125 mL 100% commercially available apple juice, 12.5 g glucose, and 375 mL H2O. Heat the mixture in a microwave and pour it into a 3 cm cell culture dish. Store the mixture at 4 &#176;C for future use.</li>
	<li>After preparing the apple plate, add a thin layer of kneaded yeast paste on top of it to allow the flies to lay egg...

<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...

Gastrulation is the first set of morphologically dynamic events that occur during embryonic development of multicellular animals such as Drosophila1,2. Interestingly, emerging evidence suggests that this process is regulated through the interplay between mechanical and molecular mechanisms3. Moreover, the epithelial to mesenchymal transition (EMT), which is a crucial process in gastrulation, is also implicated in human disease processes such as cancer metastasis4-8. As such, many genes that control apical constriction are also known to be key factors in the EMT observed in cancer metastasis9. Thus, apical constriction at the time of gastrulation is an excellent model to investigate the aforementioned regulatory mechanisms and to enhance our understanding of cancer metastasis. The advantage of this technique is that we can observe cell movement at the time of gastrulation in real-time and therefore, we will be able to screen genes involved in gastrulation as well as cancer metastasis.
Although relatively unknown, cell-to-cell adhesion is thought to play a central role in apical constriction1. Drosophila genetics is well suited for single cell level investigations exploring regulatory molecular mechanisms. This model will enable us to uncover the importance of apical constriction during gastrulation. Moreover, this method can be used to screen genes involved in cancer metastasis. Capturing live images of Drosophila gastrulation has further enabled us to understand in greater detail the molecular mechanisms governing tissue rearrangement. Herein, we provide a comprehensive description of a simple method to achieve this.

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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>
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		<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>
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			<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>
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		<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>
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			<td height="21" style="height:21px;width:268px;">DE-Cadherin knock-in flies</td>
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			<td>REF (10)</td>
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imaging of cell shape alteration and cell movement in drosophila gastrulation using de-cadherin reporter transgenic flies

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. This morphological alteration is also recognized as epithelial to mesenchymal transition (EMT). Dysregulation of EMT is associated with fibrosis and cancer metastasis. There is emerging evidence that EMT is controlled by a number of molecular mechanisms. As such, many key genes that control apical constriction are also known to be important factors in the EMT observed in cancer metastasis. Like EMT during Drosophila gastrulation, epithelial cells can be induced to change their shape and be reprogrammed to redirect cell fate towards various other cell types. Here we provide a robust imaging method of Drosophila gastrulation to assay the initiation of morphogenetic cellular movements and cell fate identification during this stage of embryonic development. Using this method, we identify cell rearrangement at the time of gastrulation and demonstrate the importance of apical constriction during gastrulation using GFP labeled DE-cadherin.

Here, we show the gastrulation events of the Drosophila embryo and a general overview of the embryo preparation procedure (Figure 1). Cell membranes are labeled using DE-cadherin::GFP and live imaging of cell movements is performed at the time of gastrulation in Drosophila (Figure 2). Since DE-cadherin GFP flies allow us to visualize cell adherence junctions, we are able to trace apical cell shape and movements using this system. More importantly...

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

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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Developmental Biology

7.0K 조회수.  Jahangirnagar University. 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, ...