Name: Generating Retinal Injury Models in Xenopus Tadpoles (Video) | JoVE Journal
Uploaded: 2023-10-13T12:00:00.000+00:00
Duration: 9 min 29 s
Description: Learn retinal injury models in Xenopus laevis tadpoles to study regeneration mechanisms and Muller glia recruitment. This method helps researchers explore neuroinflammation's role in retinal repair.

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5 minutes

This research was supported by grants to M.P. from the Association Retina France, Fondation de France, FMR (Fondation Maladies Rares), BBS (Association du syndrome de Bardet-Biedl), and UNADEV (Union Nationale des Aveugles et D&#233;ficients Visuels) in partnership with ITMO NNP (Institut Th&#233;matique Multi-Organisme Neurosciences, sciences cognitives, neurologie, psychiatrie) / AVIESAN (Alliance Nationale pour les sciences de la vie et de la sant&#233;).

Generating Retinal Degeneration to Investigate Cellular Repair Strategies in Xenopus

<ol><li>Goldman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((M%C3%BCller+glial+cell+reprogramming+and+retina+regeneration%5BTitle%5D)+AND+%22Nature+reviews.+Neuroscience%22%5BJournal%5D)">Müller glial cell reprogramming and retina regeneration.</a> Nature reviews. Neuroscience. 15 (7), 431-442 (2014).</li>
<li>Hamon, A., Roger, J. E., Yang, X. -J., Perron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((M%C3%BCller+glial+cell-dependent+regeneration+of+the+neural+retina%3A+An+overview+across+vertebrate+model+systems%5BTitle%5D)+AND+%22Developmental+Dynamics%22%5BJournal%5D)">Müller glial cell-dependent regeneration of the neural retina: An overview across vertebrate model systems.</a> Developmental Dynamics. 245 (7), 727-738 (2016).</li>
<li>García-García, D., Locker, M., Perron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Update+on+M%C3%BCller+glia+regenerative+potential+for+retinal+repair%5BTitle%5D)+AND+%22Current+Opinion+in+Genetics+%26+Development%22%5BJournal%5D)">Update on Müller glia regenerative potential for retinal repair.</a> Current Opinion in Genetics & Development. 64, 52-59 (2020).</li>
<li>Todd, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+stimulation+of+retinal+regeneration+from+M%C3%BCller+glia+in+adult+mice+using+combinations+of+proneural+bHLH+transcription+factors%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Efficient stimulation of retinal regeneration from Müller glia in adult mice using combinations of proneural bHLH transcription factors.</a> Cell Reports. 37 (3), 109857(2021).</li>
<li>Hoang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+regulatory+networks+controlling+vertebrate+retinal+regeneration%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Gene regulatory networks controlling vertebrate retinal regeneration.</a> Science. 370 (6519), (2020).</li>
<li>Langhe, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((M%C3%BCller+glial+cell+reactivation+in+Xenopus+models+of+retinal+degeneration%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Müller glial cell reactivation in Xenopus models of retinal degeneration.</a> Glia. 65 (8), 1333-1349 (2017).</li>
<li>Chesneau, A., Bronchain, O., Perron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conditional+chemogenetic+ablation+of+photoreceptor+cells+in+Xenopus+retina%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Conditional chemogenetic ablation of photoreceptor cells in Xenopus retina.</a> Methods in Molecular Biology. 1865, 133-146 (2018).</li>
<li>Martinez-De Luna, R. I., Zuber, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rod-specific+ablation+using+the+nitroreductase%2Fmetronidazole+system+to+investigate+regeneration+in+Xenopus%5BTitle%5D)+AND+%22Cold+Spring+Harbor+protocols%22%5BJournal%5D)">Rod-specific ablation using the nitroreductase/metronidazole system to investigate regeneration in Xenopus.</a> Cold Spring Harbor protocols. 2018 (12), (2018).</li>
<li>Zahn, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Normal+Table+of+Xenopus+development%3A+a+new+graphical+resource%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Normal Table of Xenopus development: a new graphical resource.</a> Development. 149 (14), (2022).</li>
<li>McNamara, S., Wlizla, M., Horb, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Husbandry%2C+general+care%2C+and+transportation+of+Xenopus+laevis+and+Xenopus+tropicalis%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Husbandry, general care, and transportation of Xenopus laevis and Xenopus tropicalis.</a> Methods in Molecular Biology. 1865, 1-17 (2018).</li>
<li>Parain, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRISPR%2FCas9-mediated+models+of+retinitis+pigmentosa+reveal+differential+proliferative+response+of+M%C3%BCller+cells+between+Xenopus+laevis+and+Xenopus+tropicalis%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">CRISPR/Cas9-mediated models of retinitis pigmentosa reveal differential proliferative response of Müller cells between Xenopus laevis and Xenopus tropicalis.</a> Cells. 11 (5), 807(2022).</li>
<li>Wlizla, M., McNamara, S., Horb, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+and+care+of+Xenopus+laevis+and+Xenopus+tropicalis+embryos%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Generation and care of Xenopus laevis and Xenopus tropicalis embryos.</a> Methods in Molecular Biology. 1865, 19-32 (2018).</li>
<li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments+JoVE%22%5BJournal%5D)">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> Journal of Visualized Experiments JoVE. (27), (2009).</li>
<li>Parain, K., Chesneau, A., Locker, M., Borday, C., Perron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regeneration+from+three+cellular+sources+and+ectopic+mini-retina+formation+upon+neurotoxic+retinal+degeneration+in+Xenopus%5BTitle%5D)+AND+%22bioRxiv%22%5BJournal%5D)">Regeneration from three cellular sources and ectopic mini-retina formation upon neurotoxic retinal degeneration in Xenopus.</a> bioRxiv. , (2023).</li>
<li>Vergara, M. N., Del Rio-Tsonis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retinal+regeneration+in+the+Xenopus+laevis+tadpole%3A+a+new+model+system%5BTitle%5D)+AND+%22Molecular+Vision%22%5BJournal%5D)">Retinal regeneration in the Xenopus laevis tadpole: a new model system.</a> Molecular Vision. 15, 1000-1013 (2009).</li>
<li>Lee, D. C., Hamm, L. M., Moritz, O. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Xenopus+laevis+tadpoles+can+regenerate+neural+retina+lost+after+physical+excision+but+cannot+regenerate+photoreceptors+lost+through+targeted+ablation%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Xenopus laevis tadpoles can regenerate neural retina lost after physical excision but cannot regenerate photoreceptors lost through targeted ablation.</a> Investigative Ophthalmology & Visual Science. 54 (3), 1859-1867 (2013).</li>
<li>Martinez-De Luna, R. I., Kelly, L. E., El-Hodiri, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+retinal+homeobox+%28Rx%29+gene+is+necessary+for+retinal+regeneration%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">The retinal homeobox (Rx) gene is necessary for retinal regeneration.</a> Developmental Biology. 353 (1), 10-18 (2011).</li>
<li>Choi, R. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cone+degeneration+following+rod+ablation+in+a+reversible+model+of+retinal+degeneration%5BTitle%5D)+AND+%22Investigative+Ophthalmology+%26+Visual+Science%22%5BJournal%5D)">Cone degeneration following rod ablation in a reversible model of retinal degeneration.</a> Investigative Ophthalmology & Visual Science. 52 (1), 364-373 (2011).</li>
</ol>

The authors have no conflicts of interest to disclose.

Advantages and disadvantages of various retinal injury paradigms in Xenopus tadpoles
Mechanical retinal injury 
Various surgical injuries of the neural retina have been developed in Xenopus tadpoles. The neural retina may either be entirely removed15,16 or only partly excised16,17. The mechanical injury presented here doe...

Millions of people worldwide are afflicted with various retinal degenerative diseases leading to blindness, such as retinitis pigmentosa, diabetic retinopathy, or age-related macular degeneration (AMD). To date, these conditions remain largely untreatable. Current therapeutic approaches under evaluation include gene therapy, cell or tissue transplantations, neuroprotective treatments, optogenetics, and prosthetic devices. Another emerging strategy is based on self-regeneration through the activation of endogenous cells with stem cell potential. M&#252;ller glial cells, the major glial cell type of the retina, are among cellular sources of interest in this context. Upon injury, they can dedifferentiate, proliferate, and generate neurons1,2,3. Although this process is very effective in zebrafish or Xenopus, it is largely inefficient in mammals.
Nonetheless, it has been shown that appropriate treatments with mitogenic proteins or overexpression of various factors can induce mammalian M&#252;ller glia cell-cycle re-entry and, in some cases, trigger their subsequent&#160;neurogenesis commitment1,2,3,4,5. This remains, however, largely insufficient for treatments. Hence, increasing our knowledge of the molecular mechanisms underlying regeneration is necessary to identify molecules able to efficiently turn M&#252;ller stem-like cell properties into new cellular therapeutic strategies.
With this aim, we developed several injury paradigms in Xenopus that trigger retinal cell degeneration. Here, we present (1) a mechanical retinal injury that is not cell type-specific, (2) a conditional and reversible cell ablation model using the NTR-MTZ system that targets rod cells, (3) a CRISPR/Cas9-mediated rhodopsin knockout, a model of retinitis pigmentosa that triggers progressive rod cell degeneration, and (4) a CoCl2-induced cytotoxic model that according to the dose can specifically target cones or lead to broader retinal cell degeneration. We highlight the particularities, advantages, and disadvantages of each paradigm.

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generating retinal injury models in xenopus tadpoles

Generating Retinal Injury Models in Xenopus Tadpoles

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair recently emerged as particularly appealing. A cellular source of interest for retinal repair is the M&#252;ller glial cell, which harbors stem cell potential and an extraordinary regenerative capacity in anamniotes. This potential is, however, very limited in mammals. Studying the molecular mechanisms underlying retinal regeneration in animal models with regenerative capabilities should provide insights into how to unlock the latent ability of mammalian M&#252;ller cells to regenerate the retina. This is a key step for the development of therapeutic strategies in regenerative medicine. To this aim, we developed several retinal injury paradigms in Xenopus: a mechanical retinal injury, a transgenic line allowing for nitroreductase-mediated photoreceptor conditional ablation, a retinitis pigmentosa model based on CRISPR/Cas9-mediated rhodopsin knockout, and a cytotoxic model driven by intraocular CoCl2 injections. Highlighting their advantages and disadvantages, we describe here this series of protocols that generate various degenerative conditions and allow the study of retinal regeneration in Xenopus.

Mechanical retinal injury 
Retinal sections of tadpoles subjected to the mechanical injury described in protocol section 1 show that the retinal lesion encompasses all layers of the tissue while remaining limited to the puncture site (Figure 2A,B).
Conditional rod cell ablation using the NTR-MTZ system 
The eyes of anesthetized Tg(rho:GFP-NTR) transgenic tadpoles treated with MTZ treatment, as descr...

We have developed several protocols to induce retinal damage or retinal degeneration in Xenopus laevis tadpoles. These models offer the possibility of studying retinal regeneration mechanisms.

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair ...

generating-retinal-injury-models-in-xenopus-tadpoles

Research

JoVE Journal

Developmental Biology

A method to conditionally knockdown a target protein’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’s role in the regenerating or intact retina.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The video demonstrates a method for culturing primary retinal ganglion cells from zebrafish embryos. Early-stage embryos undergo dissection to isolate the eyes, which are subsequently mechanically dissociated into a suspension of retinal ganglion cells. This cell suspension is cultured on coverslips coated with adhesive proteins to facilitate axon development.

Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

This video demonstrates a procedure to induce retinal degeneration in adult zebrafish using laser injury. The laser targets photoreceptor cells in the retina, triggering cell death and a degenerative process. This damage activates Müller glia (MG) cells, leading to their dedifferentiation into stem cell progenitors that aid in retinal regeneration.

Modeling Retinal Degeneration and Regeneration in Zebrafish Using a Focal Laser Injury

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.

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

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

This protocol describes a method to dissect, experimentally manipulate and culture whole retinal explants from chicken embryos. The explant cultures are useful when high success rate, efficacy and reproducibility are needed to test the effects of plasmids for electroporation and/or reagent substances, i.e., enzymatic inhibitors.

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

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.

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

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

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

Learn retinal injury models in Xenopus laevis tadpoles to study regeneration mechanisms and Muller glia recruitment. This method helps researchers explore neuroinflammation's role in retinal repair.

Generating Retinal Injury Models in Xenopus Tadpoles

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