Work described herein was supported by the National Institutes of Health (RO1-EY29410 to J.M.G, and NIH CORE Grant P30-EY08098 to the Department of Ophthalmology); the UPMC Immune Transplant &#38; Therapy Center (to L.L.L. and J.M.G.); and the E. Ronald Salvitti Chair in Ophthalmology Research (to J.M.G.). Additional support was received from the Wiegand Fellowship in Ophthalmology (to L.L.L), the Eye &#38; Ear Foundation of Pittsburgh, and an unrestricted grant from Research to Prevent Blindness, New York, NY. Authors also wish to thank Amanda Platt for technical assistance and Dr. Hugh Hammer and the aquatics staff for excellent animal care support.

L.L.L. is the co-inventor on US Patent #9,458,428, which describes an expedited method to derive retinal pigment epithelium from human pluripotent stem cells; this is unrelated to the content herein. J.M.G. and G.B.F. have nothing to disclose.

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...

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 injuries or progressive degenerative disease is irreversible. In humans, RPE degenerative diseases (e.g.,&#160;age-related macular degeneration (AMD) and Stargardt disease) lead to permanent vision loss and, with few treatment options available, decreased patient&#160;quality of life. The limited ability for mammalian RPE to self-repair has created a knowledge gap in the field of RPE regenerative processes. Given the robust regenerative capacity of the zebrafish across many different tissue types, this protocol was developed to establish an in vivo vertebrate system to facilitate studies on intrinsically regenerating RPE and uncover mechanisms that drive that response. Using the ablation paradigm outlined here, the canonical Wnt signaling pathway3, the mTOR pathway4, and immune-related responses5 have been identified as critical mediators of RPE regeneration, likely with overlapping functions.
In this genetic ablation paradigm, Tg(rpe65a:nfsB-eGFP)3 zebrafish express the bacterial-derived nitroreductase (NTR/nfsB) gene6 fused to eGFP under control of the RPE enhancer element, rpe65a7. Ablation is achieved by adding the prodrug, metronidazole (MTZ), to system water housing zebrafish. Intracellular activation of MTZ by nitroreductase results in DNA crosslinking and apoptosis in NTR/nfsB-expressing cells8,9. This technology has been widely used in zebrafish to ablate cells of the retina10,11,12,13 and other tissues8. Together, these elements enable targeted expression (rpe65a) of an inducible cell ablation methodology (NTR/MTZ)8,9 and a fluorescent marker (eGFP) for visualization.
Other interesting in vivo models also exist that can be used to study the regenerative potential of the RPE14. These are broad and include RPE-to-retina transdifferentiation post-retinectomy in amphibians, in which RPE cells lost to retinal regrowth are replaced15,16; RPE restoration post-injury in the &#34;super healing&#34; MRL/MpJ mouse17; and exogenous stimulation of RPE proliferation in a rat model of spontaneous RPE and retinal degeneration18, among others. In vitro models, such as adult human RPE stem cells (RPESCs)19 have also been developed. These models are all valuable tools working to uncover the cellular processes related to RPE regeneration (e.g.,&#160;proliferation, differentiation, etc.); however, the zebrafish is unique in its capacity for intrinsic RPE repair post-ablation.
While the methodology here is written to focus on understanding the mechanisms driving RPE regeneration, the Tg(rpe65a:nfsB-eGFP) line and this genetic ablation protocol could be utilized to study other cellular processes such as RPE apoptosis, RPE degeneration, and the effect of RPE injury on adjacent retinal and vascular tissues. The ablation protocol can also be modified to include pharmacological manipulation, which is a convenient preliminary strategy to screen signaling pathways of interest. For example, blocking the canonical Wnt pathway using Inhibitor of Wnt Response-1 (IWR-1)20, has been shown to impair RPE regeneration3. This was repeated here to guide users through a pharmacological manipulation experiment and serve as proof-of-concept to validate a MATLAB script (RpEGEN) created to quantify RPE regeneration based on recovery of pigmentation. Like the transgenic line and ablation protocol, the RpEGEN scripts are adaptable and could be used to quantify other markers/cellular processes within the RPE.

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

nitroreductase/metronidazole-mediated ablation and a matlab platform (rpegen) for studying regeneration of the zebrafish retinal pigment epithelium

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&#160;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.

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...

Watch this Scientific Journal Video about Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium at JoVE.com

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

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.

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

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

nitroreductasemetronidazole-mediated-ablation-matlab-platform-rpegen

Research

JoVE Journal

Biology

2.5K Views.  University of Pittsburgh School of Medicine. De zebravis is uniek in zijn intrinsieke vermogen om veel verschillende weefseltypen te regenereren, waaronder de RPE. Dit protocol kan worden gebruikt om moleculaire routes te identificeren die RPE-regeneratie en RPE-ziektegerelateerde mechanismen aansturen. Veelzijdigheid is een belangrijk voordeel van deze methodiek.Naast het bestuderen van RPE-regeneratie, kan dit protocol worden gebruikt om RPE-degeneratieve processen en de effecten van RPE-schade op aangrenzende weefsels in het o...

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

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Developmental Biology

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

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller 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 M&#252;ller 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 M&#252;ller 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.

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.

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

Neuroscience

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial world. Animal models are of pivotal importance to study such diseases. In this regard the photoreceptor-specific toxin N-methyl-N-nitrosourea (MNU) has been widely used in rodents to pharmacologically induce retinal degeneration. Previously, we have established a MNU-induced retinal degeneration model in the zebrafish, another popular model system in visual research.
A fascinating difference to mammals is the persistent neurogenesis in the adult zebrafish retina and its regeneration after damage. To quantify this observation we have employed visual acuity measurements in the adult zebrafish. Thereby, the optokinetic reflex was used to follow functional changes in non-anesthetized fish. This was supplemented with histology as well as immunohistochemical staining for apoptosis (TUNEL) and proliferation (PCNA) to correlate the developing morphological changes.
In summary, apoptosis of photoreceptors occurs three days after MNU treatment, which is followed by a marked reduction of cells in the outer nuclear layer (ONL). Thereafter, proliferation of cells in the inner nuclear layer (INL) and ONL is observed. Herein, we reveal that not only a complete histological but also a functional regeneration occurs over a time course of 30 days. Now we illustrate the methods to quantify and follow up zebrafish retinal de- and regeneration using MNU in a video-format.

Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Herein we demonstrate quantification of retinal de- and regeneration and its impact on visual function using N-methyl-N-nitrosourea in the adult zebrafish. Loss of visual acuity and decreased photoreceptor numbers were followed by proliferation in the inner nuclear layer. Complete morphological and functional regeneration occurred 30 days after the initial treatment.

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial ...

Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the mode of action (MOA) of such molecules leverage bulk treatment of experimental specimens with an excess of a specific reactive species. In this approach, the high reactivity of electrophiles renders non-discriminate labeling of the proteome in a time- and context-dependent manner; redox-sensitive proteins and processes can also be indirectly and often irreversibly affected. Against such a backdrop of innumerable potential targets and indirect secondary effects, linking phenotype to specific target engagement remains a complex task. Zebrafish targeting reactive electrophiles and oxidants (Z-REX)-an on-demand reactive-electrophile delivery platform adapted for use in larval zebrafish-is designed to deliver electrophiles to a specific protein of interest (POI) in otherwise unperturbed live fish embryos. Key features of this technique include a low level of invasiveness, along with dosage-, chemotype-, and spatiotemporally-controlled precision electrophile delivery. Thus, in conjunction with a unique suite of controls, this technique sidesteps off-target effects and systemic toxicity, otherwise observed following uncontrolled bulk exposure of animals to reactive electrophiles and pleiotropic electrophilic drugs. Leveraging Z-REX, researchers can establish a foothold in the understanding of how individual stress responses and signaling outputs are altered as a result of specific reactive ligand engagement with a specific POI, under near-physiologic conditions in intact living animals.

Zebrafish targeting reactive electrophiles and oxidants (Z-REX) is a chemical biology-based method for the investigation of reactive small-molecule signaling. This technique can be applied to live fish of different developmental stages. Here, we couple standard assays in zebrafish with Z-REX for signaling pathway analysis.

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the ...

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

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