Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Summary

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.

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 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.

Protocol

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 ....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
Tricaine	Reagent	Sigma-Aldrich	A-5040
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