Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Abstract

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 extrusion 1 . 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. Protocol Basic Workflow for the Visualization of Actin Dynamics During Cell Extrusion in the Epidermis of Developing ZebrafishThe 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 membrane 2 . The cells of the outer surface layer undergo apoptosis and are eliminated from the tissue by extrusion 3 (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 8 6 (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. In vitro transcribe the RNA from a linearized DNA template using the mMessage mMachine SP6 kit (Ambion) and purify the capped RNA using the RNeasy Mini kit (Qiagen). Dilute the RNA to˜60ng/ul using sterile water, make 3-5μl aliquots and store at -80°C until ready for injection. Note: Avoid repetitive freeze/thawing to prevent degradation of the RNA. 2. Make a mold to hold the embryos during injection by pouring 40mL of 2% agarose in E3 embryo medium (E3) into a 100 x 15mm culture plate and placing a microinjection mold (Adaptive Science Tools, # I-34) in this tray. Once the agarose has solidified, remove the mold to expose the troughs in the agarose. Store the plate at 4°C until ready fo...

Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish

“…7D-DЉ). This is reminiscent of the extrusion and death of surface epithelial cells in other systems (Eisenhoffer et al, 2012;Eisenhoffer and Rosenblatt, 2011). Consistent with the idea of cell extrusion, the number of cells per clone decreased significantly between 10 and 15 dpf (Fig.…”

Epidermal cell turnover across tight junctions based on Kelvin's tetrakaidecahedron cell shape

“…Further investigation on whether this characteristic interdigitation pattern is observed in the SC of other vertebrates, such as amphibians, reptiles and birds, may reveal the general applicability of the f-TKD model to cornified stratified epithelia. Other mechanisms could be involved in maintaining TJ barrier homeostasis in simple epithelia and non-cornified stratified epithelia, where apoptotic cells are extruded to the outside TJ barrier by adjacent cells that migrate into the basal side of the apoptotic cells and form multi-junctional TJs (Pentecost et al, 2006; Eisenhoffer and Rosenblatt, 2011). …”

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

“…The protocol presented here describes a straightforward method to conditionally induce preneoplastic-cell transformation in the superficial skin layer of zebrafish larvae, and allows for subsequent interaction analysis with innate immune cells by live imaging. The basic methodologies in our protocol follow wellestablished zebrafish techniques [9][10][11][12][13] , and our protocol can be easily adapted and modified.…”