Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

Summary

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

Abstract

An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]^mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Introduction

Unlike humans, zebrafish (Danio rerio) exhibit a robust regeneration response upon cell death of retinal neurons^1,2,3,4. Tumor necrosis factor α, a signaling molecule that is released from dying retinal neurons induces Müller glia residing in the basal Inner Nuclear Layer (INL) of the retina, to proliferate⁵ and produce neuronal progenitor cells that continue to proliferate before differentiating into the neuronal cell types that died^2,3

Protocol

Note: Zebrafish were raised and maintained in the Notre Dame Zebrafish facility in the Freimann Life Sciences Center. The methods described in this manuscript are approved by the University of Notre Dame Animal Care and Use Committee and are in compliance with the statement for the use of animals in vision research by the Association for Research in Vision and Ophthalmology.

1. Solutions

1. Prepare 70% ethanol to sterilize the tissue culture hood and any equipment/reagents that are transferred into the tissue culture hood.
2. Add 2 mL of 2-phenoxyethanol to 1 L of system water (1:500 2-phenoxyethanol).
3. Prepare 0.1 ....

Results

The isolation of the retina according to the procedure outlined in the schematic in Figure 1 allows the culturing of a flattened dorsal retina from light-damaged adult Tg[gfap:nGFP]^mi2004 zebrafish over a period of at least 24 h in a 5% CO₂/air environment. These flat-mounted retinal explants can be used to image focal planes at deep tissue levels. An example is Müller glia/neuronal progenitor cell nuclei labeled with GFP from the Müller gli.......

Discussion

Studies investigating the mechanisms governing regeneration of the damaged adult zebrafish retina predominantly used immunocytochemical methods^{5,25,26,27,28,29,30}. Establishing conditions to culture retinal explants and to perform live-cell imaging on phenomena, such as INM, provide us a tec.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dumont forceps #5	World precision instruments	14098
Dumont forceps #5, 45° angle	World precision instruments	14101
McPherson-Vannas scissors	World precision instruments	501233
Fluordishes	World precision instruments	FD35-100
Stereomicroscope	Nikon	SMZ-1B	similar type of dissection stereomicroscope will work
Biological Safety Cabinet class type A2	Labconco		equivalent type will work
tissue culture incubator	Thermoscientific	HEPA-class 100	equivalent type will work
Sylvania fluorescent lamps OSFP5835HOECO	Bulbtronics	31850
0.2 µm pore-size Acrodisc syringe filter	VWR	4192
10 mL Luer-lok syringe	VWR	BD309604
60 mL Luer-lok syringe	VWR	BD309653
NaHCO3	FischerScientific	S233-500
CaCl2	ThermoScientific	C79-500
MgCl2	EMD Millipore	5980
HBSS w/o Ca2+/Mg2+, w/o phenol red, Gibco	ThermoScientific	14175-095
MEM w/o phenol red, Gibco	ThermoScientific	5100-038
Horse serum, heat-inactivated	ThermoScientific	26050-070
penicillin/streptomycin	VWR	16777-164
Ultrapure low melting point agarose	ThermoScientific	16520-100
ethanol, absolute	ThermoScientific	BP2818-4
2-phenoxyethanol	Sigma	77699
Corning Cell-Tak cell and tissue adhesive	VWR	354240
refractive index liquid	Cargille Lab	1803Y
Nikon A1 multiphoton microscope equipped with a MaiTai infrared laser	Nikon		equivalent system will work
40X Apo long-distance water immersion objective (N.A. 1.15)
environmental chamber equipped with insert for 35 mm petridishes	Okolab		equivalent system will work
NIS analysis software	Nikon