Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

Summary

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

Abstract

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Introduction

The methodology described herein details a protocol to genetically ablate the retinal pigment epithelium (RPE) utilizing larval zebrafish. The RPE extends over the back of the eye and resides between the stratified layers of the neural retina and the layer of vasculature constituting the choroid. Trophic support, absorption of phototoxic light, and maintenance of visual cycle proteins are only some of the critical functions the RPE performs that are essential for sustaining the health and integrity of these adjacent tissues¹. Damage to mammalian RPE is reparable when lesions are small²; however, damage suffered by larger....

Protocol

All methodologies outlined herein are compliant with the Institutional Animal Care and Use Committee (IACUC) of the University of Pittsburgh.

1. Preparation prior to zebrafish embryo collection

1. Set embryo incubator to 28.5°C.
2. Prepare a 25x stock solution of the melanogenesis inhibitor, N-phenylthiourea (PTU)^21,22. This stock solution is scaled from a common recipe²² and 1x is equal to 0.003% weight per volume (% w/v) (e.g., 0.003 g of PTU powder into 100 mL of liquid solvent).
   1. To make a lar....

Results

Inhibiting the canonical Wnt signaling pathway is known to significantly impair zebrafish RPE regeneration using the genetic ablation paradigm (rpe65a:nfsB-eGFP) and pharmacological manipulation methodology (IWR-1) described in the protocol³. This experiment was repeated here to validate an automated method for quantifying zebrafish RPE regeneration based on pigmentation. The results summarized below encompassed all steps of the protocol, from the day of fertilization (0 dpf) to quantific.......

Discussion

This protocol describes methodology to genetically ablate the RPE and study mechanisms of degeneration and regeneration in larval-aged zebrafish. This protocol has also been successfully performed in adult zebrafish³ but with less extensive characterization, which is why larvae are the focus here. Critical aspects of this part of the protocol (steps 1-4) include: 1) adding 1.5x PTU to embryos prior to the onset of melanogenesis, 2) dechorionating PTU-treated embryos on 2-3 dpf, 3) careful screenin.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lab Material/Equipment
2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI)	Millipore Sigma	D9542
6-well plates	Fisher Scientific	07-200-83
Conical Polypropylene Centrifuge Tubes	Fisher Scientific	05-539-13	Catalog number is for 50 mL tubes
Diamond tip scribing pen	Fisher Scientific	50-254-51	Manufactured by Electron Microscopy Sciences, items similar to this part number are adequate
Dimethyl sulfoxide (DMSO) ≥99.7 %	Fisher Scientific	BP231	Check instiutional chemical waste disposal requirements
Embryo incubator (large)	Fisher Scientific	3720A
Embryo incubator (mini/tabletop)	Labnet	I5110A
Fluorescence stereo microscope	Zeiss	Axio Zoom.V16	Or similar, with 488 nm excitation laser/filter
Glass Pasteur pipette	Fisher Scientific	13-678-4	Manufactured by Corning, non-sterile
InSolution Wnt Antagonist I, IWR-1-endo	Millipore Sigma	5.04462	Manufactured by Calbiochem; 25 mM in DMSO; check instiutional chemical waste disposal requirements
Methylene blue (powder)	Fisher Scientific	BP117-100	Also available as a premade aqeuous solution
Metronidazole (MTZ)	Millipore Sigma	M3761	Check instiutional chemical waste disposal requirements
N-phenylthiourea (PTU)	Millipore Sigma	P7629	Check instiutional chemical waste disposal requirements
Paraformaldehyde (16 % w/v) methanol free	Fisher Scientific	AA433689M	Chemical waste, proper disposal required
Petri dishes	Fisher Scientific	FB0875712	10 cm diameter
Phosphate buffered saline (powder packets)	Millipore Sigma	P3813	Used to make 10 X PBS stock
Pronase	Millipore Sigma	PRON-RO
Shaking incubator	Benchmark	H2010	Used for incubating MTZ for 1 hour at 37 degrees Celcius
Stereo microscope	Leica	S9i	Or similar, with transmitted light illumination
Student Dumont #5 forceps	Fine Science Tools	91150-20	Fine-tipped forceps for manual dechorionation
Tabletop rotator/shaker	Scilogex	SK-D1807-E
Transfer pipette	Millipore Sigma	Z135003	3.2 mL bulb draw, non-sterile
Tricaine methanesulfonate (MS-222)	Pentair	TRS1, TRS2, TRS5	Also available from Fisher Scientific (NC0342409)
VECTASHIELD Antifade Mounting Medium with DAPI	Vector Laboratories	H-1200
Software Material
FIJI (Fiji is Just ImageJ)	FIJI (Fiji is Just ImageJ)	https://imagej.net/software/fiji/	Version: 2.0.0-rc-69/1.52p; Build: 269a0ad53f; Plugin needed: Bio-Formats
GRAMM examples and how-tos	MathWorks	https://www.mathworks.com/matlabcentral/fileexchange/54465-gramm-complete-data-visualization-toolbox-ggplot2-r-like.
MATLAB	MathWorks	https://www.mathworks.com/products/get-matlab.html	Toolboxes needed to run RpEGEN: Image Processing Toolbox, Curve Fitting Toolbox, Statistics and Machine Learning Toolbox
MATLAB support	MathWorks	https://www.mathworks.com/support.html