Name: Exploring Retinal Regeneration Mechanisms in the Xenopus Frog (Video) | JoVE
Uploaded: 2023-10-13T12:00:00
Description: Watch this Scientific Journal Video about Exploring Retinal Regeneration Mechanisms in the Xenopus Frog at JoVE.com

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;).

Animal care and experimentation were conducted in accordance with institutional guidelines, under the institutional license A91272108. The study protocols were approved by the institutional animal care committee CEEA #59 and received authorization by the Direction D&#233;partementale de la Protection des Populations under the reference number APAFIS #32589-2021072719047904 v4 and APAFIS #21474-2019071210549691 v2. See the Table of Materials for details related to all materials, instruments, and reagents ...

Generating Retinal Degeneration to Investigate Cellular Repair Strategies in Xenopus

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,2-Propanediol (propyl&#232;ne glycol)</td>
			<td>Sigma-Aldrich</td>
			<td>398039</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol &#8805;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&#160;</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 &#8805;99.5%</td>
			<td>VWR chemicals</td>
			<td>20810.298</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2, 2H2O)</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 (CoCl2, 6H2O)</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&#160;</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&#160;</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&#160;&#160;</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&#39;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 (MgSO4, 7H2O)</td>
			<td>Sigma-Aldrich (Supelco)</td>
			<td>1.05886</td>
			<td></td>
		</tr>
		<tr>
			<td>Metronidazole&#160;</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&#39;&#39;</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 &#181;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&#160;</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&#160;</td>
			<td>&#160;KNITTEL Glass</td>
			<td>VS11171076FKA&#160;</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 (C6H5Na3O7, 2H2O)</td>
			<td>VWR chemicals</td>
			<td>27833.294</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate (NaHCO3)</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>
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			<td>trans-activating crRNA (tracrRNA)</td>
			<td>Integrated DNA Technologies</td>
			<td>1072533</td>
			<td></td>
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			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X-100</td>
			<td></td>
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			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td></td>
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			<td>X-Cite 200DC Fluorescence Illuminator</td>
			<td>X-Cite&#160;</td>
			<td>200DC</td>
			<td></td>
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			<td>Xylene &#8805;98.5%&#160;</td>
			<td>VWR chemicals</td>
			<td>28975-325</td>
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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.

Generating Retinal Injury Models in Xenopus Tadpoles

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

Exploring Retinal Regeneration Mechanisms in the Xenopus Frog (Video) | JoVE

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 ...