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 recently emerged as particularly appealing. A cellular source of interest for retinal repair is the Müller 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 Müller 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.
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. Müller 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 Müller glia cell-cycle re-entry and, in some cases, trigger their subsequent 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 Müller 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.
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 Départementale 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 used in these protocols.
1. Mechanical retinal injury
NOTE: The injury paradigm protocol described here consists of a mechanical retinal puncture of a tadpole from stage 45 onwards. It, therefore, does not target any particular retinal cell type but damages the whole thickness of the retina at the puncture site.
2. Conditional rod cell ablation using the NTR-MTZ system
The protocol aims to induce specific rod cell ablation in the Xenopus Tg(rho:GFP-NTR) transgenic line6, available at the TEFOR Paris-Saclay's zootechnics service, which hosts the French Xenopus resource center. This chemogenetic system uses the capacity of nitroreductase (NTR) enzyme to convert the prodrug metronidazole (MTZ) into a cytotoxic DNA cross-linking agent, to specifically ablate NTR-expressing rods (Figure 1B). Two detailed protocols to make Tg(rho:GFP-NTR) or Tg(rho:NTR) transgenic animals and induce degeneration have been published previously 7,8. Here, we simply detail the induction of retinal degeneration and how to monitor the degeneration process in vivo.
3. Rod cell degeneration by CRISPR/Cas9-mediated rhodopsin knockout
NOTE: This protocol is established to generate a model of retinitis pigmentosa in Xenopus laevis by inducing specific rod cell degeneration using CRISPR/Cas9-mediated rhodopsin knockout. The % of insertion-deletion (indels) in F0 is provided in11 and is ~75%. Such a CRISPR/Cas9-mediated rhodopsin knockout can also be performed in Xenopus tropicalis tadpoles 11.
4. Retinal cell degeneration by cytotoxic intraocular CoCl 2 injections
NOTE: This protocol is established to induce retinal cell degeneration by intraocular injections of cobalt chloride (CoCl2) in Xenopus laevis tadpoles. According to the dose, it can trigger a cone-specific degeneration or a broader degeneration14. We use this protocol in Xenopus laevis tadpoles aged from stage 48 onwards. It can also be used in Xenopus tropicalis tadpoles.
5. Staining to assess retinal damage or retinal degeneration
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 described in protocol section 2, were analyzed under the stereomicroscope (Figure 3A,B). The decrease in GFP fluorescence reveals the progressive targeted rod cell ablation compared to the controls (Figure 3B), confirmed on retinal sections by GFP immunostaining, as described in protocol section 5 (Figure 3C).
Rod cell degeneration by CRISPR/Cas9-mediated rhodopsin knockout
Retinas of rho crispant tadpoles, obtained as described in protocol section 3, were analyzed as described in protocol section 5 (Figure 4A). Caspase 3 and Rhodopsin labeling highlights that some rods undergo apoptosis (Figure 4B). H&E staining reveals global preservation of nuclear layers but a severe shortening of photoreceptor outer segments (Figure 4C). Further immunostaining analysis with photoreceptor markers show the degeneration of rods and subsequent cone defects (Figure 4D and not shown). The phenotype begins to be visible from stage 40 onward, and the outer segments of rods progressively disappear until they are absent from the central retina from stage 47.
Retinal cell degeneration by cytotoxic intraocular CoCl2 injections
Retinas of tadpoles subjected to CoCl2 intraocular injections, as described in protocol section 4, were analyzed by immunostaining and TUNEL assay, as described in protocol section 5 (Figure 5A). This reveals that 10 mM CoCl2 injection leads to specific cell death of cone photoreceptors (Figure 5B,C) while 25 mM leads to broad retinal cell death (Figure 5E). Immunostaining analysis, as described in protocol section 5, further revealed the absence of cones in 10 mM CoCl2-injected retinas (Figure 5D) and a severe loss of both bipolar cells and photoreceptors following 25 mM CoCl2 injections (Figure 5F).
Figure 1: Illustrations of some experimental procedures. (A) A 0.2 mm diameter pin attached to a pin holder is used to puncture the retina. The diagrams of the dorsal, lateral, and frontal views of a tadpole illustrate where the pin (in red) is inserted. (B) Schematic representation of the transgenic model of conditional rod cell ablation. The rhodopsin promoter drives the expression of a GFP-Nitroreductase fusion protein in rod cells. When added to the Xenopus rearing water, NTR converts metronidazole to a cytotoxin, specifically ablating GFP-NTR rods. (C) The head of a Tg(rho:GFP-NTR) transgenic tadpole is shown in white light or under fluorescence. The GFP staining in the eye allows the sorting of transgenic tadpoles. (D) One-cell stage embryos on a nylon grid under a stereomicroscope ready to be injected with a Picospritzer microinjection system. (E) Embryos at the neurula stage (schematic in the inset) after injection at the one-cell stage of the CRISPR-Cas9 ribonucleoprotein complex containing fluorescein lysine dextran. Well-injected fluorescent embryos (green arrows) can be distinguished from uninjected ones (white arrows). (F) Image showing the spoon used to transfer the anesthetized tadpoles. (G) Anesthetized tadpoles in a Petri dish over a wet tissue. The capillary inserted in the microinjector allows for intraocular injections of the CoCl2 solution. (H) Image showing the transfer of a tadpole into a 1 L tank where the animal can be monitored until awakening. Scale bars = 2 mm (C), 1 mm (E). Abbreviations: GFP = green fluorescent protein; NTR = nitroreductase; MTZ = metronidazole; NF = Nieuwkoop and Faber stage. Please click here to view a larger version of this figure.
Figure 2: Mechanical retinal injury. (A) Outline of the experimental procedure used in (B). Xenopus laevis tadpole eyes are punctured and analyzed on retinal sections 7 days post injury. The dotted box indicates the imaged area. (B) Cell nuclei are counterstained with Hoechst. Three different retinas are shown. The arrows point to the injured site that crosses all retinal layers. Scale bar = 50 µm. Abbreviations: dpi = days post injury; GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer. Please click here to view a larger version of this figure.
Figure 3: Conditional rod cell ablation using the NTR-MTZ system. (A) Outline of the experimental procedure used in (B,C). Tg(rho:GFP-NTR) transgenic Xenopus laevis tadpoles are treated with MTZ for 7-9 days. The progressive degeneration of the GFP-labeled rods can be monitored in real-time under a fluorescence stereomicroscope as illustrated at 7 days in (B). Rod cell death can also be analyzed on retinal sections as illustrated after 14 days in (C), the dotted box indicating the imaged area. (B) After 7 days of treatment, the GFP intensity in the retina decreases with time in transgenic MTZ-treated tadpoles compared to controls. (C) The decrease in GFP fluorescence is confirmed on retinal sections at 14 days by GFP immunostaining. Cell nuclei are counterstained in blue with Hoechst. Scale bars = 500 µm (B), 50 µm (C). Abbreviations: GFP = green fluorescent protein; NTR = nitroreductase; MTZ = metronidazole; L = lens; R = retina; GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer; OS = outer segments. Please click here to view a larger version of this figure.
Figure 4: Rod degeneration in Xenopus laevis rho crispants. (A) Outline of the experimental procedure used in (B-D). One-cell stage embryos are injected with the CRISPR-Cas9 ribonucleoprotein complex (gRNA duplex/Cas9 protein) containing the fluorescent lysin dextran. At the neurula stage, fluorescent injected embryos can be sorted and allowed to develop until various stages to perform (B) cleaved Caspase 3 immunostaining for cell death analysis, or (C) hematoxylin and eosin staining for anatomical analysis, or (D) immunostaining with rod markers for photoreceptor cell degeneration analysis. The dotted box indicates the imaged area. (B) Double labeling of cleaved Caspase 3 (a marker of apoptotic cells) and Rhodopsin (a marker of rod outer segments) on retinal sections of control or rho crispants Xenopus laevis embryos at stage 39 shows an absence of dying cells in the controls and that dying cells in rho crispants are rod photoreceptors. (C) H&E staining on retinal sections of rho crispants Xenopus laevis tadpoles at stage 48 reveals the decreased size of photoreceptors' outer segments compared to controls. (D) Recoverin (a marker of rod inner segments) and Rhodopsin co-immunostaining on retinal sections of rho crispants Xenopus laevis tadpoles at stage 48 reveals a severe degeneration compared to controls. Cell nuclei are counterstained in blue with Hoechst. Scale bars = 50 µm. Abbreviations: gRNA = guide RNA; H&E = hematoxylin and eosin; GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer; OS = outer segments. Please click here to view a larger version of this figure.
Figure 5: Retinal cell degeneration by cytotoxic intraocular CoCl2 injections. (A) Outline of the experimental procedure used in (B-F). The dotted box indicates the imaged area. A solution of 10 mM or 25 mM CoCl2 is intraocularly injected into the tadpole eye. Cell apoptosis is analyzed by a TUNEL assay 2 days post injury (dpi) (B,C,E), while retinal cell degeneration is analyzed 7 to 14 dpi by immunostaining with various markers (D,F). (B–D) Retinal sections of tadpoles injected with a saline solution (Controls) or with a 10 mM CoCl2 solution at stages 51-54. (B) Cell death analysis at 2 dpi reveals the presence of dying cells following CoCl2 injection mainly in the photoreceptor nuclear layer. (C) Coupling cell death staining with S/M Opsin (a marker of cones) immunostaining reveals that these dying cells are mainly cones; arrows point to double-labeled cells. (D) S/M Opsin (a marker of cones) and Rhodopsin (a marker of rods) co-immunostaining reveals a severe decrease of cone cell labeling in CoCl2 injected retinas compared to controls at 14 dpi. (E,F) Retinal sections of tadpoles injected with a 25 mM CoCl2 solution at stages 51-54. (E) Cell death analysis at 2 dpi reveals the presence of dying cells in both the photoreceptor and the inner nuclear layers. (F) Otx2 (a marker of both photoreceptors and bipolar cells) immunostaining reveals a severe decrease of the staining in both layers in CoCl2-injected retinas compared to controls at 7 dpi, indicating CoCl2-induced cell death of both photoreceptors and bipolar cells. Cell nuclei are counterstained in blue with Hoechst. Scale bars: 25 µm. Abbreviations: ONL = Outer Nuclear Layer; INL = Inner Nuclear Layer; GCL = Ganglion Cell Layer. Please click here to view a larger version of this figure.
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 does not involve any retinal excision but a simple eye puncture that we previously developed in Xenopus tadpoles6. Given the manual procedure of such a mechanical retinal injury model, the phenotype may vary significantly from one tadpole to another. Moreover, the replicability and the extent of the damage may also vary depending on who is performing the experiment. We, therefore, recommend that the same person perform all the injury procedures.
Conditional rod cell ablation using the NTR-MTZ system
The NTR-MTZ system has been used to ablate rod cells in the transgenic Xenopus retina6,7,8,18. The duration of the MTZ treatment varies significantly from one study to another, from 2 to 7 days. This may reflect different activities of the NTR depending on the chromosomal insertion in the different transgenic lines. Moreover, we noticed some variability in the response of Tg(rho:GFP-NTR) tadpoles for a given time of MTZ treatment, with some experiencing severe rod degeneration while others exhibiting very little damage. Extending the MTZ treatment from 7 to 9-10 days with this transgenic line seems to reduce such variability. However, caution should be exercised given the potentially toxic effects of MTZ. The obvious advantages of this model are that it allows for conditional and reversible rod cell ablation and for live monitoring of rod degeneration.
Rod cell degeneration by CRISPR/Cas9-mediated rhodopsin knockout
The CRISPR-Cas9 rho gene editing model of retinitis pigmentosa leads to rod cell degeneration in Xenopus tadpole11. However, in contrast to the NTR-MTZ model, rod cell ablation is constitutive and not reversible. Interestingly, the high efficiency of this approach (we now regularly obtain >90% of tadpoles exhibiting severe rod degeneration) allows working on this model on the F0 generation. We were initially working with sgRNA but noticed variability from different batches of RNA preparations. We recently found that ordering synthetic crRNA and tracrRNA and preparing gRNA duplex as described in this protocol provides more reliable results.
Retinal cell degeneration by cytotoxic intraocular CoCl2 injections
This model has several advantages over the other models described here. It allows not only to induce the ablation of retinal cell types conditionally but also to modulate the severity of the damage14. Moreover, it represents to our knowledge the only model of specific cone degeneration in Xenopus. Finally, it generates low variability in the degenerative phenotype.
Efficacy of the four retinal lesion paradigms
To take into account the potential variability of phenotypes from one batch of tadpoles to another, it is recommended to check the efficacy of retinal degeneration on 8-10 tadpoles. In the case of mechanical injury, the damage must be analyzed on the days following injury, as the lesion is no longer visible a week later. In the NTR-MTZ model, rod degeneration is clearly visible 1 week after the end of MTZ treatment. For rho crispants, as rod cell death begins when the photoreceptors fully differentiate, we recommend analyzing degenerative efficiency from stage 45 onwards. In the CoCl2 model, cell death occurs within a week of injection. With regard to the expected % survival for each method, we would like to note that CRISPR-Cas9 ribonucleoprotein complex injections lead to higher mortality rates in the first 48 h than conventional RNA injections (no growth or survival problems persist after these first 48 h), and therefore, more embryos should be injected as necessary. In contrast, other procedures (mechanical injuries, MTZ treatment, or intraocular CoCl2 injections) do not pose any survival problems.
Potential applications of these injury paradigms to study retinal regeneration
One potential application of this series of retinal injury models is the study of retinal regeneration. Different sources of retinal stem or stem-like cells can be recruited in a pathological context, including CMZ cells, Müller glial cells, and RPE cells. We have reported that the mechanical injury model, the NRT/MTZ model, and the CRISPR/Cas9 model, are all attractive models for studying Müller cell activation upon injury6,11. Interestingly, in the CoCl2 model, all three cellular sources can be recruited14, providing a novel model for studying the mechanisms underlying the recruitment and reprogramming of different retinal cell types.
The authors have nothing to disclose.
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 Déficients Visuels) in partnership with ITMO NNP (Institut Thématique Multi-Organisme Neurosciences, sciences cognitives, neurologie, psychiatrie) / AVIESAN (Alliance Nationale pour les sciences de la vie et de la santé).
1,2-Propanediol (propylène glycol) | Sigma-Aldrich | 398039 | |
Absolute ethanol ≥99.8% | VWR chemicals | 20821-365 | |
Anti-Cleaved Caspase 3 antibody (rabbit) | Cell signaling | 9661S | Dilution 1/300 |
Anti-GFP antibody (chicken) | Aveslabs | GFP-1020 | Dilution 1/500 |
Anti-M-Opsin antibody (rabbit) | Sigma-Aldrich | AB5405 | Dilution 1/500 |
Anti-mouse secondary antibody, Alexa Fluor 594 (goat) | Invitrogen Thermo Scientific | A11005 | Dilution 1/1,000 |
Anti-Otx2 antibody (rabbit) | Abcam | Ab183951 | Dilution 1/100 |
Anti-rabbit secondary antibody, Alexa Fluor 488 (goat) | Invitrogen Thermo Scientific | A11008 | Dilution 1/1,000 |
Anti-rabbit secondary antibody, Alexa Fluor 594 (goat) | Invitrogen Thermo Scientific | A11012 | Dilution 1/1,000 |
Anti-Recoverin antibody (rabbit) | Sigma-Aldrich | AB5585 | Dilution 1/500 |
Anti-Rhodopsin antibody (mouse) | Sigma-Aldrich | MABN15 | Dilution 1/1,000 |
Anti-S-Opsin antibody (rabbit) | Sigma-Aldrich | AB5407 | Dilution 1/500 |
Apoptotis detection kit (Dead end fluorimetric TUNEL system) | Promega | G3250 | |
Benzocaine | Sigma-Aldrich | E1501 | Stock solution 10% |
bisBenzimide H 33258 (Hoechst) | Sigma-Aldrich | B2883 | Stock solution 10 mg/mL |
Butanol-1 ≥99.5% | VWR chemicals | 20810.298 | |
Calcium chloride dihydrate (CaCl2, 2H2O) | Sigma-Aldrich (Supelco) | 1.02382 | Use at 0.1 M |
Cas9 (EnGen Spy Cas9 NLS) | New England Biolabs | M0646T | |
Clark Capillary Glass model GC100TF-10 | Warner Instruments (Harvard Apparatus) | 30-0038 | |
Cobalt(II) chloride hexahydrate (CoCl2, 6H2O) | Sigma-Aldrich | C8661 | Stock solution 100 mM |
Coverslip 24 x 60 mm | VWR | 631-1575 | |
Dako REAL ab diluent | Agilent | S202230-2 | |
Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D8418 | |
Electronic Rotary Microtome | Thermo Scientific | Microm HM 340E | |
Eosin 1% aqueous | RAL Diagnostics | 312740 | |
Fluorescein lysine dextran | Invitrogen Thermo Scientific | D1822 | |
Fluorescent stereomicroscope | Olympus | SZX 200 | |
Gentamycin | Euromedex | EU0410-B | |
Glycerin albumin acc. Mallory | Diapath | E0012 | Use at 3% in water |
Hematoxylin (Mayer's Hemalun) | RAL Diagnostics | 320550 | |
HEPES potassium salt | Sigma-Aldrich | H0527 | |
Human chorionic gonadotropin hormone | MSD Animal Health | Chorulon 1500 | |
Hydrochloric acid fuming, 37% (HCl) | Sigma-Aldrich (SAFC) | 1.00314 | |
L-Cysteine hydrochloride monohydrate | Sigma-Aldrich | C7880 | Use at 2% in 0.1x MBS (pH 7.8 – 8.0) |
Magnesium Sulfate Heptahydrate (MgSO4, 7H2O) | Sigma-Aldrich (Supelco) | 1.05886 | |
Metronidazole | Sigma-Aldrich (Supelco) | M3761 | Use at 10 mM |
Microloader tips | Eppendorf | 5242956003 | |
Micropipette puller (P-97 Flaming/Brown) | Sutter Instrument Co. | Model P-97 | Program : Heat 700 / Pull 100 / Vel 75 / Time 90 / Unlocked p = 500 |
Mounting medium to preserve fluorescence, FluorSave Reagent | Millipore | 345789 | |
Mounting medium, Eukitt | Chem-Lab | CL04.0503.0500 | |
MX35 Ultra Microtome blade | Epredia | 3053835 | |
Needle Agani 25 G x 5/8'' | Terumo | AN*2516R1 | |
Nickel Plated Pin Holder | Fine Science Tools | 26016-12 | |
Nylon filtration tissue (sifting fabric) NITEX, mesh opening 1,000 µm | Sefar | 06-1000/44 | |
Paraffin histowax without DMSO | Histolab | 00403 | |
Paraformaldehyde solution (32%) | Electron Microscopy Sciences | EM-15714-S | Use at 4% in 1x PBS pH 7.4 |
Peel-A-Way Disposable Embedding Molds | Epredia | 2219 | |
Pestle | VWR | 431-0094 | |
Petri Dish 100 mm | Corning Gosselin | SB93-101 | |
Petri Dish 55 mm | Corning Gosselin | BP53-06 | |
Phosphate Buffer Saline Solution (PBS) 10x | Euromedex | ET330-A | |
PicoSpritzer Microinjection system | Parker Instrumentation Products | PicoSpritzer III | |
Pins | Fine Science Tools | 26002-20 | |
Polysucrose (Ficoll PM 400 ) | Sigma-Aldrich | F4375 | Use at 3% in 0.1x MBS |
Potassium chloride (KCl) | Sigma-Aldrich | P3911 | |
Powdered fry food : sera Micron Nature | sera | 45475 (00720) | |
Scissors dissection | Fine Science Tools | 14090-09 | |
Slide Superfrost | KNITTEL Glass | VS11171076FKA | |
Slide warmer | Kunz instruments | HP-3 | |
Sodium chloride (NaCl) | Sigma-Aldrich | S7653 | |
Sodium citrate trisodium salt dihydrate (C6H5Na3O7, 2H2O) | VWR chemicals | 27833.294 | |
Sodium hydrogen carbonate (NaHCO3) | Sigma-Aldrich (Supelco) | 1.06329 | |
Sodium hydroxide 30% aqueous solution (NaOH) | VWR chemicals | 28217-292 | |
Stereomicroscope | Zeiss | Stemi 2000 | |
Syringes Omnifix-F Solo Single-use Syringes 1 mL | B-BRAUN | 9161406V | |
trans-activating crRNA (tracrRNA) | Integrated DNA Technologies | 1072533 | |
Triton X-100 | Sigma-Aldrich | X-100 | |
Tween-20 | Sigma-Aldrich | P9416 | |
X-Cite 200DC Fluorescence Illuminator | X-Cite | 200DC | |
Xylene ≥98.5% | VWR chemicals | 28975-325 |