The authors are very thankful to Miriam Osterfield for sharing her advice on the in vitro life imaging of egg chambers using an approach adopted from the Celeste Berg laboratory4.&#160;We also thank to Sebastian Tosi for the Fiji script that enables cell segmentation.

NOTE: The following protocol provides instructions on how to analyze actomyosin at the local cellular and the tissue scale in Drosophila egg chambers. The local-scale approach allows users to analyze detailed actomyosin behavior in up to 15 cells per egg chamber and requires the acquisition of TLMs for a short period of time (5&#8211;10 min) by using high-speed imaging and an inverted confocal microscope. In contrast, the tissue scale provides users with actomyosin information in 50&#8211;100 ce...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=bullwinkle+is+required+for+epithelial+morphogenesis+during+Drosophila+oogenesis.">bullwinkle is required for epithelial morphogenesis during Drosophila oogenesis.</a> Developmental Biology. 267, 320-341 (2004).</li><li>Cetera, M., 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+rotation+promotes+the+global+alignment+of+contractile+actin+bundles+during+Drosophila+egg+chamber+elongation.">Epithelial rotation promotes the global alignment of contractile actin bundles during Drosophila egg chamber elongation.</a> Nature Communications. 5, 5511 (2014).</li><li>Viktorinova, I., Henry, I., Tomancak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+rotation+is+preceded+by+planar+symmetry+breaking+of+actomyosin+and+protects+epithelial+tissue+from+cell+deformations.">Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations.</a> PLOS Genetics. 13, e1007107 (2017).</li><li>Rauzi, M., Lenne, P. F., Lecuit, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+polarized+actomyosin+contractile+flows+control+epithelial+junction+remodelling.">Planar polarized actomyosin contractile flows control epithelial junction remodelling.</a> Nature. 468, 1110-1114 (2010).</li><li>Spracklen, A. J., Fagan, T. N., Lovander, K. E., Tootle, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pros+and+cons+of+common+actin+labeling+tools+for+visualizing+actin+dynamics+during+Drosophila+oogenesis.">The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.</a> Developmental Biology. 393, 209-226 (2014).</li><li>. Surface manager Available from: <a target="_blank" href="https://git.mpi-cbg.de/tomancaklab/surface_manager">https://git.mpi-cbg.de/tomancaklab/surface_manager</a> (2018)</li><li>. Actomyosin dynamics: Required Software and dependencies Available from: <a target="_blank" href="https://git.mpi-cbg.de/scicomp/viktorinova_et_al_actomyosin_dynamics/blob/master/Software/Software_installation.md#surface-manager-and-ellipsoid-surface-projection-plugins">https://git.mpi-cbg.de/scicomp/viktorinova_et_al_actomyosin_dynamics/blob/master/Software/Software_installation.md#surface-manager-and-ellipsoid-surface-projection-plugins</a> (2018)</li><li>. 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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+the+actin+and+microtubule+cytoskeletons+in+the+morphogenesis+of+epidermal+hairs+during+wing+development+in+Drosophila.">Distinct roles for the actin and microtubule cytoskeletons in the morphogenesis of epidermal hairs during wing development in Drosophila.</a> Mechanisms of Development. 70, 181-192 (1998).</li><li>Heemskerk, I., Streichan, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+cartography:+compressing+bio-image+data+by+dimensional+reduction.">Tissue cartography: compressing bio-image data by dimensional reduction.</a> Nature Methods. 12, 1139-1142 (2015).</li><li>Chen, D. Y., Lipari, K. R., Dehghan, Y., Streichan, S. J., Bilder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symmetry+Breaking+in+an+Edgeless+Epithelium+by+Fat2-Regulated+Microtubule+Polarity.">Symmetry Breaking in an Edgeless Epithelium by Fat2-Regulated Microtubule Polarity.</a> Cell Reports. 15, 1125-1133 (2016).</li><li>Reynaud, E. G., Peychl, J., Huisken, J., Tomancak, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guide+to+light-sheet+microscopy+for+adventurous+biologists.">Guide to light-sheet microscopy for adventurous biologists.</a> Nature Methods. 12, 30-34 (2015).</li></ol>

The authors have nothing to disclose.

Critical steps and troubleshooting for the dissection and culturing of egg chambers
If too many flies are placed into a small vial, the fly food can turn muddy after 2&#8211;3 days due to extensive amounts of feeding larvae and adult flies getting trapped in the fly food. In such a case, flip the rest of these flies into a new vial with fresh food and downsize their number. In particular, exclude females that were stuck in the food.
The Schneider mi...

The continual development of novel imaging and software technologies with applications in the life sciences has provided an enormous impact on understanding the basic principles of life. One of the main challenges is the reliable visualization of developmental processes in combination with their live imaging in various tissues. Tissues are parts of organs and bodies and, as such, the majority are not easily accessible for imaging. Therefore, protocols that allow their dissection and in vitro culturing have been developed in order to visualize biological events that sufficiently reflect the in vivo situation within a living body.
Over the past decades, the culturing and live imaging of Drosophila egg chambers, acinar-like structures resembling primitive organs, has contributed immensely to the understanding of the basic principles of primitive organ development1,2,3. Currently, there are several culturing protocols available, and their usage depends on acquisition time, cell type to be imaged, and their accessibility (e.g., inner germline vs. outer somatic line)4.
A common feature in all these culturing protocols is the need for the immobilization of analyzed egg chambers that display a high contractile activity in liquid media. The contractile activity of egg chambers is caused mainly by the muscle sheet that covers a long string of connected egg chambers5,6,7. Therefore, to achieve proper immobilization of young egg chambers, various approaches have been developed, involving covering egg chambers with coverslips6,8,9 or flexible blankets4,10 or embedding them in low-melting-point agarose3,11. These approaches are popular as they also allow the imaging of a larger visual plane due to the subtle flattening of the circumferential surface of the egg chambers.
However, recently, it has been shown that young egg chambers (stage 1-8) rotate around their anterior-posterior axis6 and that this tissue motion is powered by a fine actomyosin network close to the circumferential surface of these young egg chambers12. Therefore, artificial alteration of the cellular surface caused by a subtle flattening of this tissue may have a negative impact on the behavior of the force-generating actomyosin machinery. The counterpoint is that if the egg chamber tissue is not flattened, microscopic imaging of proteins at the circumferential surface of egg chambers becomes even more limited by the decreased size of the acquisition plane.
Therefore, we have combined protocols from Prasad et al.9 and the lab of Celeste Berg4,10 and further modified them so that no coverslip/flexible blanket/agarose is used in the developed method. Drosophila egg chambers are freely cultured in media and the protocol presented here applies only inverted microscopy. There are two parts to the protocol. The first part is focused on the analysis of actomyosin signals at the local cellular scale (up to 15 cells) within egg chambers. In the second part, we focus on overcoming the limitations associated with a small acquisition plane caused by the free culturing of egg chambers. In this regard, we have developed a novel Fiji-based computational method with a semi-interactive graphical user interface that selectively extracts and unfolds defined layers of a circumferential tissue surface. This is followed by a protocol that describes how to analyze actomyosin at the tissue scale (i.e., &#62;50 cells). As the selective extraction of a defined thin layer of curved epithelial tissues has not been easily possible using a classical z-stack projection (Figure 1), this easy-to-use method serves as an important prerequisite to comprehensively understand&#160;the behavior of a thin (&#60;1 &#181;m) actomyosin network at the tissue scale in Drosophila egg chambers.
In addition, to facilitate the protocol, we provide example time-lapse movies (TLMs) and sample files of fluorescently tagged nonmuscle conventional Myosin II behavior (see Supplementary File 3). Myosin&#160;II is a motor protein and represents the active contractile part of the actomyosin machinery. In order to image Myosin II, we use Drosophila transgenic lines that contain a modified regulatory light chain of Myosin II called MRLC::GFP (see Table of Materials for details)12,13. In order to visualize cell membranes, the protocol is based on commercial dyes (see Table of Materials). This protocol is suitable not only for the analysis of small subcellular MRLC::GFP signals12 but also for any similar-sized subcellular particles around &#177;300 &#181;M, such as those observed with Life-Act::GFP12,14.
Although both these protocols are presented using in vitro cultured Drosophila egg chambers, the acquisition of actomyosin signals can also be performed using other tissues upon the optimization of the culturing media and depending on the availability of either fluorescently tagged proteins with corresponding commercial dyes or, for example, mRNA microinjections to obtain transient gene expression profiles. Similarly, the Fiji-based protocol for the extraction of a thin layer from a circumferential surface can be applied more generally to ellipsoid and organ-like tissues.

<table>
	<tbody>
		<tr>
			<td>Schneider Medium</td>
			<td>Gibco</td>
			<td>21720-024</td>
			<td>[+]-Glutamine</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>&#160;[+] 10.000 Units/ml Penicillin [+] 10.000 &#181;g/ml Streptomycin</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>&#160;F135</td>
			<td>Heat inactivated</td>
		</tr>
		<tr>
			<td>Insulin Solution Human</td>
			<td>Sigma</td>
			<td>&#160;I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Falcon tubes</td>
			<td>Eppendorf</td>
			<td></td>
			<td>sterile</td>
		</tr>
		<tr>
			<td>Millex-GV filter</td>
			<td>Millipore</td>
			<td>SLGV033NS</td>
			<td>33 mm</td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td>35 mm Petri dish, 14 mm Microwell, No. 1.5 cover glass</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>A. Dumont &#38; Fils</td>
			<td></td>
			<td>&#160;#55</td>
		</tr>
		<tr>
			<td>CellMask Deep Red (cell membrane dye)</td>
			<td>Invitrogen</td>
			<td>C10046</td>
			<td></td>
		</tr>
		<tr>
			<td>FM4-64 (cell membrane dye)</td>
			<td>ThermoFisher/Invitrogen</td>
			<td>T13320</td>
			<td></td>
		</tr>
		<tr>
			<td>Depression dissection slide</td>
			<td>Fisher Scientific</td>
			<td>12-560B</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopic equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Steromicroscope</td>
			<td>e.g. Zeiss</td>
			<td></td>
			<td>Stemi SV 6</td>
		</tr>
		<tr>
			<td>Inverted confocal microscope</td>
			<td>e.g. Zeiss/Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disc microscope</td>
			<td>e.g. Zeiss/Andor</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopic glass/cover glass</td>
			<td>e.g. Thermo Scientific/Menzel Glas</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cactus tool</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wironit needle holder</td>
			<td>Hammacher</td>
			<td>9160020</td>
			<td></td>
		</tr>
		<tr>
			<td>Cactus spine</td>
			<td>Echinocactus grusonii/barrel cactus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drosophila stocks used in the manuscript</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AX3/AX3; sqh-MRLC::GFP/sqh-MRLC::GFP</td>
			<td></td>
			<td></td>
			<td>For detail see Rauzi et al.: Planar polarized actomyosin contractile flows control epithelial junction remodeling. Nature 2010, Vol. 468, pg. 1110-1115.</td>
		</tr>
		<tr>
			<td>AX3/AX3; sqh-MRLC::GFP/sqh-MRLC::GFP; fat258D/fat103C</td>
			<td></td>
			<td></td>
			<td>For detail see Viktorinova et al.: Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations. PLoS Genetics 2017, Vol. 13, Issue 11, pg. e1007107.</td>
		</tr>
	</tbody>
</table>

analysis of actomyosin dynamics at local cellular and tissue scales using time-lapse movies of cultured drosophila egg chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

This protocol enables scientists to investigate the behavior of actomyosin networks in epithelial tissues. This is only possible when a detailed analysis of actomyosin behavior at the local cellular scale (a few cells) is combined with a similar analysis at the tissue scale (many cells). However, epithelial tissues are often curved and the extraction of a thin layer of these tissues was previously not easily possible, as shown in Drosophila egg chambers (Figu...

Watch this Scientific Journal Video about Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers at JoVE.com

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Our protocol helps scientists to analyze subcellular actomyosin network in individual epithelial cells and also allows them to perform similar analysis at multicellular scale in curved epithelial tissues. Our protocol is a nice example of a Fiji application. It has an intuitive graphical user interface, and it doesn't require any knowledge of scripting.Therefore, it is highly suitable also for novice users in the field. Although our method was developed for Drosophila egg chambers, it is also applicable outside Drosophila epithelia. We encourage scientists to test our protocol in their model systems.Use the accompanied test files to get familiar with this protocol. It will help you to decide which part of this protocol is suitable for your individual research question. This part of the protocol allows users to segment epithelial cells in time-lapse movies and subsequently analyze actomyosin pulses in individual cells over time using the Surface Manager.To begin, open Fiji, and import the macro TissueCellSegmentMovie. ijm as described in the accompanying text protocol. Then, open a bleach-corrected TIFF file, and split the channels of the bleach-corrected file by going to the menu bar, selecting Image, followed by Color, and selecting Split Channels.Run the uploaded script on the active cell membrane channel of the selected time-lapse movie by pressing the icon Run in the open script. In order to get a nice cell mask, set the Gaussian blur to 2.500 and the cell detection sensitivity to minus one, and click OK.Then, set the estimated noise tolerance between 10 and 20 for time-lapse movies acquired with the 63x objective, and click OK.A generated cell mask appears in the analyzed time-lapse movie, a new window called ParticleStack appears, and a little window called Action Required also appears. From the cell mask on the time-lapse movie, focus only on cells in the center, and select those that can provide complete and well-defined outlines throughout the time-lapse movie.When the selected cells in the center nicely correspond to the real cell membranes, save ParticleStack as a TIFF file. Open the corresponding ParticleStack TIFF file in Fiji. With the Surface Manager plugin installed in Fiji, open the plugin.In Surface Manager, click on the Read outline image button, and set the Jaccard index to 60%Loading the outlines may take several minutes depending on the number of cells. Once loaded, each cell will be assigned an S number and appear in the left part of the Surface Manager window. Click on the selected S number so that the imported cell outline from ParticleStack1.tiff appears on the time-lapse movie. Check each imported S number for cell outline quality throughout the movie, and remove unwanted cells that display incorrect cell outlines. To remove an unwanted cell, highlight the cell outline and click on the Delete button.Use the Brush tool to correct cell outlines. When finished, save the corrected cells as an RoiSet. zip file.To identify whether actomyosin pulses are present in the analyzed tissue and to understand the detailed behavior, as well as the directionality of actomyosin signals, begin by opening Fiji. Open a time-lapse movie, load the saved ParticleStack1. tiff cell mask and the bleach-corrected time-lapse movie, then load the Surface Manager plugin and the corresponding region of interest from the RoiSet.zip file. Next, in Surface Manager, activate a channel with signals of interest. Click the Statistics button to obtain the window called Average gray value Slice by Slice.The mean and median intensities of the actomyosin signals will be displayed over time in arbitrary units, as well as other parameters related to the cell's area and shape. Save the values as a spreadsheet file by going to the Statistics window, clicking on File, and selecting Save As.Similarly, generate a cell mask, and load it into Surface Manager in order to analyze actomyosin pulses at the tissue scale. This part of the protocol allows users to selectively extract a thin layer of actomyosin in the curved epithelial tissue over time.These steps are user-friendly and based on an intuitive graphical interface. Open Fiji, and ensure that the Ellipsoid Surface Projection plugin is installed. Then, open a time-lapse movie, and export it as an XML/HDF5 file by clicking on Plugins, going to BigDataViewer, and selecting Export Current Image as XML/HDF5 file.Next, open the exported file in Ellipsoid Surface Projection by going to Plugins, clicking on BigDataViewer, followed by Ellipsoid Surface Projection, and selecting XML file. This will open a new window with sagittal views of the egg chamber, and a dialog to guide the user through processing will appear. The dialog window, called Ovaries Projection, has several tabs.In the first dialog tab, called bounding box, define the x and y borders of an egg chamber in the time-lapse movie together with the z width of the bounding box, and press set. The selected parts of the egg chamber are highlighted in pink. Next, define the size of signal blobs in the dialog tab called find blobs in the Ovaries Projection window.Define the sigma and the minimal peak value. Then, press compute to identify the actomyosin signals, which will appear as green blobs. Retry this step with different parameters until around 100 spots are found.Identifying the actomyosin signals is a crucial step. It depends on the signal quality and the correct size of the detected blobs. If there are no visible green spots in the image, then the next step will not work.To design the ellipsoid, continue with the tab called fit ellipsoid. Set random samples to 10, 000, the outside/inside cutoff distance to one to 10, and then click compute. After this, the tab called projection will automatically open and allows for the definition of the surface extraction.In the projection tab, set up a minimum and maximum projection distance as the width of the desired ellipsoid. The minimal and maximal projection distance must fit so the pink defined ellipsoid region includes the entire outside layer of the egg chamber. Then, define the slice distance as one.Make sure that the pink region of the ellipsoid with the defined with on the egg chamber is visible before pressing compute. It helps to evaluate the quality of a new ellipsoid fit. Next, set an output width of the ellipsoid to greater than or equal to 800 and a height that is greater than or equal to 400.Then, set the duration of the surface extraction, and choose either a spherical or a cylindrical projection. If required, flip Z and align Y.Press compute to obtain a surface extraction for both channels in new windows called image. Adjust the brightness and the contrast of the obtained image windows to be able to see the projected actomyosin signals by clicking on Image, going to Adjust, and selecting Brightness/Contrast.If the surface extraction looks good, save both channels separately as TIFF files. Additionally, save the Log window file for future reference as to how the surface of this particular egg chamber was extracted. Then, merge the channels with a preferred color code in Fiji, and save the results as TIFF files.This protocol enables scientists to investigate the behavior of actomyosin networks in epithelial tissues. This is only possible when a detailed analysis of actomyosin behavior at the local cellular scale is combined with a similar analysis at the tissue scale. Shown here are representative examples of dynamic myosin II behavior at the local cellular scale and at the tissue scale for control and fat2 mutant Drosophila egg chambers.On the basal single plane, the green modified regulatory light chain of myosin II signals move perpendicular to the anterior-posterior axis of control egg chambers. This polarity is lost in fat2 mutant egg chambers and leads to anisotropic myosin II pulses, oscillations At the tissue level, the green modified regulatory light chain of myosin II in the control sample generates the synchronized force required to promote epithelial rotation. In contrast, the follicle epithelium of a fat2 mutant egg chamber pulse strongly at the basal side and fails to generate the synchronized force required to promote epithelial rotation.In a thin apical region of the Drosophila egg chamber, the fat2 mutant also presents with altered dynamic behavior of the green modified regulatory light chain of myosin II.After watching this video, you should have a good idea of how to analyze a actomyosin network at local and tissue scale in Drosophila egg chambers using Fiji. For further practical tips and troubleshooting, please see the discussion part after the protocol. If you have any questions related to Fiji or our plugins, see the respective webpages or contact us.

analysis-actomyosin-dynamics-at-local-cellular-tissue-scales-using

Research

JoVE Journal

Developmental Biology

7.3K vues.  Max Planck Institute of Molecular Cell Biology and Genetics. Notre protocole aide les scientifiques à analyser le réseau d’actomyosine subcellulaire dans les cellules épithéliales individuelles et leur permet également d’effectuer une analyse similaire à l’échelle multicellulaire dans les tissus épithéliales incurvés. Notre protocole est un bel exemple d’une application fidjienne. Il dispose d’une interface utilisateur graphique intuitive, et il ne nécessite aucune connaissance du script.Par conséquent, il est très appropr...