Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah
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Eisenhoffer, G. T., Rosenblatt, J. Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish. J. Vis. Exp. (52), e2689, doi:10.3791/2689 (2011).
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.
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 inject RNA encoding red fluorescent protein fused to the calponin homology domain of utrophin, an actin binding protein (RFP-UtrCH) 4,5, into one-cell stage transgenic zebrafish expressing green fluorescent protein (GFP) under the stratified epithelia promoter cytokeratin 86 (Figure 2A). Although RFP-UtrCH is ubiquitously expressed in the animal after RNA injection, we focus on the superficial epidermal cells that express both RFP and GFP to follow actin dynamics specifically in the epidermis (Figure 2B). We then treat the larvae with G418 to induce apoptotic cell extrusion and image the epidermis and actin filaments using a spinning disc confocal microscope and acquire 4D datasets.
Prior to Starting the Experiment
1. Injection of RNA Encoding the Fluorescently Tagged Actin Binding Protein
2. Induction of Cell Extrusion in Zebrafish Larvae using G418
We have found that exposure to the aminoglycoside antibiotic G418, or Geneticin, causes apoptosis and extrusion of epidermal cells in developing zebrafish larvae3 by an unknown mechanism. Importantly, this treatment only works on larvae that are 4 days post fertilization and older. We find that ˜ 5-25 cells can be found extruding at any given time from the fin epithelial of G418 treated larval zebrafish. As the amount of apoptotic extruding cells can vary from fish to fish, we recommend mounting multiple fish for imaging.
3. Mounting Zebrafish Larvae for Imaging
4. Live Imaging of Actin Dynamics and Individual Epithelial Cell Behaviors During Cell Extrusion using a Spinning Disc Confocal Microscope
We have found that the entire process of apoptotic cell extrusion from the epidermis of developing zebrafish larvae takes approximately 20 minutes3. Here we demonstrate how to setup a time-lapse imaging experiment to collect a series of z planes through a single layer of the zebrafish epidermis to visualize the extrusion process. We have found that typical widefield fluorescent microscopes cause significant photobleaching of the actin filaments and do not allow for timelapse imaging. Therefore, to maximize our resolution and prevent photobleaching, we use a spinning disc confocal microscope. Below we describe how to set up the experiment using the Andor IQ software with a Nikon microscope, although the principles discussed can be applied to similar microscope set-ups.
5. Representative Results
Figure 1. Schematic of apoptotic cell extrusion. Actin filaments are shown in red, GFP positive epidermal cells are green.
Figure 2. Experimental Workflow. A. Schematic of the workflow for the experiment. B. Expression of RFP-UtrCH in the epidermis of a 4dpf CK:GFP zebrafish.
Figure 3. Still frames (z-projections) from a time-lapse movie that follows live actin dynamics during the process of epithelial cell extrusion. Arrows denote the ring of actin formed by neighboring cells which contracts and closes over the course of 2 minutes.
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 clear apoptotic cells, which we expect will lead to inflammatory responses or the accumulation of damaged cells, as seen in cancer. For example, our laboratory has previously shown that chemical inhibitors can be used in combination with G418 to alter the targeting of the actin filaments along the basolateral surface of the cell, which effects the direction a cell extrudes3. One limitation to the current method is that due to the stochastic and unpredictable nature of cell death, our datasets tend to focus on the later stages of extrusion. To study the formation of the multicellular actin ring and initiation of contraction, future experiments will apply techniques to induce apoptosis in a subset of fluorescently-labeled epithelial cells that are genetically targeted for cell death10. Combining this strategy with our method to image actin dynamics in the epidermis should facilitate studies of the early steps leading to apoptotic cell extrusion. Together, the information gained from these experiments will add significantly to our understanding of epithelial cell extrusion and its medical significance.
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).
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’s New Innovator Award 1 DP2 OD002056-01 to JR. GTE was supported by NIH Multidisciplinary Cancer Training Program Grant 5T32 CA03247-8.
|RNeasy Mini Kit||Reagent||Qiagen||74104|
|mMessage mMachine SP6 Kit||Reagent||Ambion||AM1340|
|Crossing Tanks||Tool||Thoren Aquatic Systems|
|The Pipet Pump||Tool||Bel Art Products||F3789|
|5 ¾ Pasteur Pipet||Tool||VWR international||14672-608|
|Microinjection Mold||Tool||Adaptive Science Tools||I-34|
|100x15mm Culture Plate||Tool||Fisher Scientific||08-757-12|
|Flamming/Brown Micropipet Puller||Tool||Sutter Instrument Co.||Model P97|
|Borosilicate Glass Capillaries with Filament||Tool||Sutter Instrument Co.||B1400-78-10||OD 1.0mm, ID 0.78mm, 10cm length|
|Electrode Storage Jar||Tool||World Precision Instruments, Inc.||E210|
|Phenol Red||Reagent||Sigma-Aldrich||P0290||0.5% in DPBS|
|Pressure-Controlled Microinjector and Micromanipulator||Tool||Harvard Apparatus||PLI-100|
|35x10mm Culture Dish||Tool||Cellstar||627-160|
|G418 (Geneticin)||Reagent||GIBCO, by Life Technologies||10131-035||50 mg/mL in dH20|
|Dumont #5 Forceps||Tool||Fine Science Tools||11253-20|
|12cm Pin Holder||Tool||Fine Science Tools||26016-12|
|Insert Pins||Tool||Fine Science Tools||26007-02 and-03|
|Glass Bottom Culture Dish with 1.5 coverglass||Tool||MatTek Corp.||P35G-1.5-10-C|
|Low Melt Agarose||Reagent||Fisher Scientific||BP1360-100|
|Dissecting Microscope||Microscope||Leica Microsystems||MZ6|
|Dissecting Microscope||Microscope||Nikon Instruments||SMZ645|
|Fluorescent Dissecting Microscope||Microscope||Olympus Corporation||S2X12|
|Spinning Disc Confocal Microscope||Microscope||Nikon Instruments||Eclipse Ti||Outfitted with a Yokagawa spinning disc head|
|CCD Camera||Camera||Andor||DV-885-VP||1002x1004x8 micron square pixels|