We thank members of the Rosenblatt laboratory for scientific discussions, suggestions, and comments. We would also like to thank Mary Halloran who kindly provided the plasmid encoding RFP-UtrCH and David Grunwald who provided the CK:GFP transgenic zebrafish. Thanks also to Gretchen King and the staff of the Centralized Zebrafish Resource at the University of Utah for excellent maintenance and care of the zebrafish. This work was supported by NIH-NIGMS NIH Director&#x2019;s New Innovator Award 1 DP2 OD002056-01 to JR. GTE was supported by NIH Multidisciplinary Cancer Training Program Grant 5T32 CA03247-8.

Basic Workflow for the Visualization of Actin Dynamics During Cell Extrusion in the Epidermis of Developing Zebrafish 
The epidermis of the developing zebrafish is comprised of two distinct layers, the surface layer (or periderm) and a basal layer of cells 
that contact the basement membrane2. The cells of the outer surface layer undergo apoptosis and are eliminated from the tissue by
 extrusion3 (Figure 1). To visualize this process in real time, we inje...

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	<li>Rosenblatt, J., Raff, M. C., Cramer, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+epithelial+cell+destined+for+apoptosis+signals+its+neighbors+to+extrude+it+by+an+actin-+and+myosin-dependent+mechanism.">An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism.</a> Curr Biol. 11, 1847-1857 (2001).</li><li>Le Guellec, D., Morvan-Dubois, G., Sire, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+development+in+bony+fish+with+particular+emphasis+on+collagen+deposition+in+the+dermis+of+the+zebrafish+(Danio+rerio).">Skin development in bony fish with particular emphasis on collagen deposition in the dermis of the zebrafish (Danio rerio).</a> Int J Dev Biol. 48, 217-231 (2004).</li><li>Slattum, G., McGee, K. M., Rosenblatt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P115+RhoGEF+and+microtubules+decide+the+direction+apoptotic+cells+extrude+from+an+epithelium.">P115 RhoGEF and microtubules decide the direction apoptotic cells extrude from an epithelium.</a> J Cell Biol. 186, 693-702 (2009).</li><li>Burkel, B. M., Dassow, G. v. o. n., Bement, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+fluorescent+probes+for+actin+filaments+based+on+the+actin-binding+domain+of+utrophin.">Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin.</a> Cell Motil Cytoskeleton. 64, 822-832 (2007).</li><li>Berndt, J. D., Clay, M. R., Langenberg, T., Halloran, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-kinase+and+myosin+II+affect+dynamic+neural+crest+cell+behaviors+during+epithelial+to+mesenchymal+transition+in+vivo.">Rho-kinase and myosin II affect dynamic neural crest cell behaviors during epithelial to mesenchymal transition in vivo.</a> Dev Biol. 324, 236-244 (2008).</li><li>Gong, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein+expression+in+germ-line+transmitted+transgenic+zebrafish+under+a+stratified+epithelial+promoter+from+keratin8.">fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8.</a> Dev Dyn. 223, 204-215 (2002).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book, A Guide for the Laboratory Use of Zebrafish (Danio rerio). , (2000).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203, 253-310 (1995).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos.">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. , (2009).</li><li>Curado, S., Stainier, D. Y., Anderson, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitroreductase-mediated+cell/tissue+ablation+in+zebrafish:+a+spatially+and+temporally+controlled+ablation+method+with+applications+in+developmental+and+regeneration+studies.">Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies.</a> Nat Protoc. 3, 948-954 (2008).</li></ol>

Animal Welfare Assurance
All procedures performed in this protocol using the zebrafish, Danio rerio, have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Utah (Animal Welfare Assurance #10-07017).

The protocol described here demonstrates a simple and straightforward method to visualize the process of extrusion in a live epithelial tissue. This type of experiment allows us to examine subtleties of actin dynamics not previously appreciated in fixed tissue analyses, and therefore, complements common immunofluorescent methods. We can use this protocol in combination with chemical inhibitors or genetic mutants to better analyze the extrusion process and study disease states associated with the failure to remove and c...

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 <th>Type</th>
 <th>Company</th>
 <th>Catalog Number</th>
 <th>Comments</th>
 </tr>
 <tr>
 <td>RNeasy Mini Kit</td>
 <td>Reagent</td>
 <td>Qiagen</td>
 <td>74104</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>mMessage mMachine SP6 Kit</td>
 <td>Reagent</td>
 <td>Ambion</td>
 <td>AM1340</td>
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 <tr>
 <td>Crossing Tanks</td>
 <td>Tool</td>
 <td>Thoren Aquatic Systems</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
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 <tr>
 <td>The Pipet Pump</td>
 <td>Tool</td>
 <td>Bel Art Products</td>
 <td>F3789</td>
 <td>&nbsp;</td>
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 <td>5 &frac34; Pasteur Pipet </td>
 <td>Tool</td>
 <td>VWR</td>
 <td>14672-608</td>
 <td>&nbsp;</td>
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 <tr>
 <td>Microinjection Mold</td>
 <td>Tool</td>
 <td>Adaptive Science Tools</td>
 <td>I-34</td>
 <td>&nbsp;</td>
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 <tr>
 <td>100x15mm Culture Plate</td>
 <td>Tool</td>
 <td>Fisher</td>
 <td>08-757-12</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Flamming/Brown Micropipet Puller</td>
 <td>Tool</td>
 <td>Sutter Instrument Company</td>
 <td>Model P97</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Borosilicate Glass Capillaries with Filament </td>
 <td>Tool</td>
 <td>Sutter Instrument Company</td>
 <td>B1400-78-10</td>
 <td>OD 1.0mm, ID 0.78mm, 10cm length</td>
 </tr>
 <tr>
 <td>Electrode Storage Jar</td>
 <td>Tool</td>
 <td>World Precision Instruments</td>
 <td>E210</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phenol Red</td>
 <td>Reagent</td>
 <td>Sigma</td>
 <td>P0290</td>
 <td>0.5% in DPBS</td>
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 <tr>
 <td>Pressure-Controlled Microinjector and Micromanipulator</td>
 <td>Tool</td>
 <td>Harvard Apparatus</td>
 <td>PLI-100</td>
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 <td>Tool</td>
 <td>Cellstar</td>
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 <td>10131-035</td>
 <td>50 mg/mL in dH20</td>
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 <td>Dumont #5 Forceps</td>
 <td>Tool</td>
 <td>Fine Science Tools</td>
 <td>11253-20</td>
 <td>&nbsp;</td>
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 <td>12cm Pin Holder </td>
 <td>Tool</td>
 <td>Fine Science Tools</td>
 <td>26016-12</td>
 <td>&nbsp;</td>
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 <td>Insert Pins</td>
 <td>Tool</td>
 <td>Fine Science Tools</td>
 <td>26007-02 and-03</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass Bottom Culture Dish with 1.5 coverglass</td>
 <td>Tool</td>
 <td>MatTek</td>
 <td>P35G-1.5-10-C</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Low Melt Agarose</td>
 <td>Reagent</td>
 <td>Fisher</td>
 <td>BP1360-100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tricaine</td>
 <td>Reagent</td>
 <td>Sigma</td>
 <td>A-5040</td>
 <td>&nbsp;</td>
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 <tr>
 <td>Dissecting Microscope</td>
 <td>Microscope</td>
 <td>Leica</td>
 <td>MZ6</td>
 <td>&nbsp;</td>
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 <tr>
 <td>Dissecting Microscope</td>
 <td>Microscope</td>
 <td>Nikon</td>
 <td>SMZ645</td>
 <td>&nbsp;</td>
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 <tr>
 <td>Fluorescent Dissecting Microscope</td>
 <td>Microscope</td>
 <td>Olympus</td>
 <td>S2X12</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Spinning Disc Confocal Microscope</td>
 <td>Microscope</td>
 <td>Nikon</td>
 <td>Eclipse Ti</td>
 <td>Outfitted with a Yokagawa spinning disc head</td>
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 <td>CCD Camera</td>
 <td>Camera</td>
 <td>Andor</td>
 <td>DV-885-VP</td>
 <td>1002x1004x8 micron square pixels</td>
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live imaging of cell extrusion from the epidermis of developing zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Watch this Scientific Journal Video about Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish at JoVE.com

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

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Biology

14.5K ビュー.  University of Utah. 死細胞は、バリア機能を中断することなく、隣接する細胞の協調した収縮によって上皮組織から押し出される。ゼブラフィッシュの開発の光学的透明度は、上皮の生体内で押し出しを可視化するための優れたシステムを提供します。ここでは、携帯電話の解像度で幼生ゼブラフィッシュの表皮で押し出しを誘発し、イメージする方法を説明します。