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.

<table><tbody><tr><td>1,2-Propanediol (propyl&amp;egrave;ne glycol)</td><td>Sigma-Aldrich</td><td>398039</td><td></td></tr><tr><td>Absolute ethanol &amp;ge;99.8%</td><td>VWR chemicals</td><td>20821-365</td><td></td></tr><tr><td>Anti-Cleaved Caspase 3 antibody (rabbit)</td><td>Cell signaling</td><td>9661S</td><td>Dilution 1/300</td></tr><tr><td>Anti-GFP antibody (chicken)</td><td>Aveslabs</td><td>GFP-1020</td><td>Dilution 1/500</td></tr><tr><td>Anti-M-Opsin antibody (rabbit)</td><td>Sigma-Aldrich</td><td>AB5405</td><td>Dilution 1/500</td></tr><tr><td>Anti-mouse secondary antibody, Alexa Fluor 594 (goat)</td><td>Invitrogen Thermo Scientific</td><td>A11005</td><td>Dilution 1/1,000</td></tr><tr><td>Anti-Otx2 antibody (rabbit)</td><td>Abcam</td><td>Ab183951</td><td>Dilution 1/100</td></tr><tr><td>Anti-rabbit secondary antibody, Alexa Fluor 488 (goat)</td><td>Invitrogen Thermo Scientific</td><td>A11008</td><td>Dilution 1/1,000</td></tr><tr><td>Anti-rabbit secondary antibody, Alexa Fluor 594 (goat)</td><td>Invitrogen Thermo Scientific</td><td>A11012</td><td>Dilution 1/1,000</td></tr><tr><td>Anti-Recoverin antibody (rabbit)</td><td>Sigma-Aldrich</td><td>AB5585</td><td>Dilution 1/500</td></tr><tr><td>Anti-Rhodopsin antibody (mouse)</td><td>Sigma-Aldrich</td><td>MABN15</td><td>Dilution 1/1,000</td></tr><tr><td>Anti-S-Opsin antibody (rabbit)</td><td>Sigma-Aldrich</td><td>AB5407</td><td>Dilution 1/500</td></tr><tr><td>Apoptotis detection kit (Dead end fluorimetric TUNEL system)</td><td>Promega</td><td>G3250</td><td></td></tr><tr><td>Benzocaine&amp;nbsp;</td><td>Sigma-Aldrich</td><td>E1501</td><td>Stock solution 10%</td></tr><tr><td>bisBenzimide H 33258 (Hoechst)</td><td>Sigma-Aldrich</td><td>B2883</td><td>Stock solution 10 mg/mL</td></tr><tr><td>Butanol-1 &amp;ge;99.5%</td><td>VWR chemicals</td><td>20810.298</td><td></td></tr><tr><td>Calcium chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;, 2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich (Supelco)</td><td>1.02382</td><td>Use at 0.1 M</td></tr><tr><td>Cas9 (EnGen Spy Cas9 NLS)</td><td>New England Biolabs</td><td>M0646T</td><td></td></tr><tr><td>Clark Capillary Glass model GC100TF-10</td><td>Warner Instruments (Harvard Apparatus)</td><td>30-0038</td><td></td></tr><tr><td>Cobalt(II) chloride hexahydrate (CoCl&lt;sub&gt;2&lt;/sub&gt;, 6H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>C8661</td><td>Stock solution 100 mM</td></tr><tr><td>Coverslip 24 x 60 mm</td><td>VWR</td><td>631-1575</td><td></td></tr><tr><td>Dako REAL ab diluent&amp;nbsp;</td><td>Agilent</td><td>S202230-2</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418</td><td></td></tr><tr><td>Electronic Rotary Microtome</td><td>Thermo Scientific</td><td>Microm HM 340E&amp;nbsp;</td><td></td></tr><tr><td>Eosin 1% aqueous</td><td>RAL Diagnostics</td><td>312740</td><td></td></tr><tr><td>Fluorescein lysine dextran&amp;nbsp;&amp;nbsp;</td><td>Invitrogen Thermo Scientific</td><td>D1822</td><td></td></tr><tr><td>Fluorescent stereomicroscope</td><td>Olympus</td><td>SZX 200</td><td></td></tr><tr><td>Gentamycin</td><td>Euromedex</td><td>EU0410-B</td><td></td></tr><tr><td>Glycerin albumin acc. Mallory</td><td>Diapath</td><td>E0012</td><td>Use at 3% in water</td></tr><tr><td>Hematoxylin (Mayer&#039;s Hemalun)</td><td>RAL Diagnostics</td><td>320550</td><td></td></tr><tr><td>HEPES potassium salt</td><td>Sigma-Aldrich</td><td>H0527</td><td></td></tr><tr><td>Human chorionic gonadotropin hormone</td><td>MSD Animal Health</td><td>Chorulon 1500</td><td></td></tr><tr><td>Hydrochloric acid fuming, 37% (HCl)</td><td>Sigma-Aldrich (SAFC)</td><td>1.00314</td><td></td></tr><tr><td>L-Cysteine hydrochloride monohydrate</td><td>Sigma-Aldrich</td><td>C7880</td><td>Use at 2% in 0.1x MBS (pH 7.8 - 8.0)</td></tr><tr><td>Magnesium Sulfate Heptahydrate (MgSO&lt;sub&gt;4&lt;/sub&gt;, 7H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich (Supelco)</td><td>1.05886</td><td></td></tr><tr><td>Metronidazole&amp;nbsp;</td><td>Sigma-Aldrich (Supelco)</td><td>M3761</td><td>Use at 10 mM</td></tr><tr><td>Microloader tips</td><td>Eppendorf</td><td>5242956003</td><td></td></tr><tr><td>Micropipette puller (P-97 Flaming/Brown)</td><td>Sutter Instrument Co.</td><td>Model P-97</td><td>Program : Heat 700 / Pull 100 / Vel 75 / Time 90 / Unlocked p = 500</td></tr><tr><td>Mounting medium to preserve fluorescence, FluorSave Reagent</td><td>Millipore</td><td>345789</td><td></td></tr><tr><td>Mounting medium, Eukitt</td><td>Chem-Lab</td><td>CL04.0503.0500</td><td></td></tr><tr><td>MX35 Ultra Microtome blade</td><td>Epredia</td><td>3053835</td><td></td></tr><tr><td>Needle Agani 25 G x 5/8&#039;&#039;</td><td>Terumo</td><td>AN*2516R1</td><td></td></tr><tr><td>Nickel Plated Pin Holder</td><td>Fine Science Tools</td><td>26016-12</td><td></td></tr><tr><td>Nylon filtration tissue (sifting fabric) NITEX, mesh opening 1,000 &amp;micro;m</td><td>Sefar</td><td>06-1000/44</td><td></td></tr><tr><td>Paraffin histowax without DMSO</td><td>Histolab</td><td>00403</td><td></td></tr><tr><td>Paraformaldehyde solution (32%)</td><td>Electron Microscopy Sciences</td><td>EM-15714-S</td><td>Use at 4% in 1x PBS pH 7.4</td></tr><tr><td>Peel-A-Way Disposable Embedding Molds</td><td>Epredia</td><td>2219</td><td></td></tr><tr><td>Pestle</td><td>VWR</td><td>431-0094</td><td></td></tr><tr><td>Petri Dish 100 mm</td><td>Corning Gosselin</td><td>SB93-101</td><td></td></tr><tr><td>Petri Dish 55 mm</td><td>Corning Gosselin</td><td>BP53-06</td><td></td></tr><tr><td>Phosphate Buffer Saline Solution (PBS) 10x</td><td>Euromedex</td><td>ET330-A</td><td></td></tr><tr><td>PicoSpritzer Microinjection system</td><td>Parker Instrumentation Products</td><td>PicoSpritzer III</td><td></td></tr><tr><td>Pins&amp;nbsp;</td><td>Fine Science Tools</td><td>26002-20</td><td></td></tr><tr><td>Polysucrose (Ficoll PM 400 )</td><td>Sigma-Aldrich</td><td>F4375</td><td>Use at 3% in 0.1x MBS</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>Powdered fry food : sera Micron Nature</td><td>sera</td><td>45475 (00720)</td><td></td></tr><tr><td>Scissors dissection</td><td>Fine Science Tools</td><td>14090-09</td><td></td></tr><tr><td>Slide Superfrost&amp;nbsp;</td><td>&amp;nbsp;KNITTEL Glass</td><td>VS11171076FKA&amp;nbsp;</td><td></td></tr><tr><td>Slide warmer</td><td>Kunz instruments</td><td>HP-3</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Sodium citrate trisodium salt dihydrate (C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;5&lt;/sub&gt;Na&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;, 2H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>VWR chemicals</td><td>27833.294</td><td></td></tr><tr><td>Sodium hydrogen carbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich (Supelco)</td><td>1.06329</td><td></td></tr><tr><td>Sodium hydroxide 30% aqueous solution (NaOH)</td><td>VWR chemicals</td><td>28217-292</td><td></td></tr><tr><td>Stereomicroscope</td><td>Zeiss</td><td>Stemi 2000</td><td></td></tr><tr><td>Syringes Omnifix-F Solo Single-use Syringes 1 mL</td><td>B-BRAUN</td><td>9161406V</td><td></td></tr><tr><td>trans-activating crRNA (tracrRNA)</td><td>Integrated DNA Technologies</td><td>1072533</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X-100</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P9416</td><td></td></tr><tr><td>X-Cite 200DC Fluorescence Illuminator</td><td>X-Cite&amp;nbsp;</td><td>200DC</td><td></td></tr><tr><td>Xylene &amp;ge;98.5%&amp;nbsp;</td><td>VWR chemicals</td><td>28975-325</td><td></td></tr></tbody></table>

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 d...

Watch this Scientific Journal Video about Exploring Retinal Regeneration Mechanisms in the Xenopus Frog at JoVE.com

Author Spotlight: Exploring Retinal Regeneration Mechanisms in the Xenopus Frog 

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.

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 ...

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

Research

JoVE Journal

Developmental Biology

1.4K Views.  Université Paris-Saclay. 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.

Video: Author Spotlight: Exploring Retinal Regeneration Mechanisms in the Xenopus Frog 

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

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

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

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.

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Neuroscience

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Medicine

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, ...

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

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

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

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.

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

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.

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

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

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

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.

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

Biology

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.

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.

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

Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos

Source: Terzi, A., et al., <a href="https://app.jove.com/v/62165">ROS Live Cell Imaging During Neuronal Development.</a> J. Vis. Exp. (2021)
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.

1. Preparation of solutions

	E2 media (1x)
	
		Prepare 100x E2A (500 mL), 500x E2B (100 mL) and 500x E2C (100 mL) solutions by combining all components ...

Generating Retinal Injury Models in Xenopus Tadpoles

Summary

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

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

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

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

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

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

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

Generation of Primary Retinal Ganglion Cell Cultures From Zebrafish Embryos