rod cell degeneration in xenopus tadpoles by crispr/cas9-mediated rhodopsin knockout

יצירת ניוון רשתית כדי לחקור אסטרטגיות תיקון תאים בקסנופוס

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.

מחבר זרקור: חקר מנגנוני התחדשות הרשתית בצפרדע הקסנופוס 

יצירת מודלים של פגיעות רשתית בראשנים של קסנופוס

My lab works on retinal regeneration. We are using the Xenopus frog as a model system. This amphibian is indeed quite fascinating because unlike mammals, like us, it can regenerate its retina very efficiently in case of injury.And we are studying the underlying mechanism because, in the future, it could be useful to trigger retinal regeneration in human patients afflicted with retinal neurodegenerative diseases. We have recently discovered that in addition to stem cells in the periphery of the retina and the retinal pigmented epithelium, neuroglial cells can also be recruited for retinal regeneration in case of injury. And so we are now studying the links between neuroinflammation and the regenerative capacity of these cells.Indeed, it seems that the neuroinflammatory niche is a key player in the modulation of the regeneration of the retina. To study the cellular and molecular mechanisms involved in retinal regeneration, we developed several retinal injury paradigms in Xenopus. The first is a mechanical retinal injury.The second is a transgenic line allowing for nitroreductase-mediated photoreceptor conditional ablation. The third is a retinitis pigmentosa model based on CRISPR/Cas9-mediated rhodopsin knockout, and, to finish, a cytotoxic model driven by intraocular injection of cobalt chloride or CoCl2. My host laboratory has showed that although Xenopus can regenerate its retina, the efficiency is highly variable and depends on the stages of the tadpole or on species, Xenopus laevis or tropicalis.This makes Xenopus a fantastic model for illustrating the molecular mechanisms that trigger or limit retinal regeneration.

Watch this Scientific Journal Video about Rod Cell Degeneration in Xenopus Tadpoles by CRISPR/Cas9-Mediated Rhodopsin Knockout at JoVE.com

Rod Cell Degeneration in Xenopus Tadpoles by CRISPR/Cas9-Mediated Rhodopsin Knockout  

ניוון תאי מוט בראשנים של קסנופוס על ידי נוקאאוט רודופסין בתיווך CRISPR/Cas9

כדי להתחיל, קח נימים מושחזים, השתמש בקצה מטעין מיקרו כדי לטעון 2 עד 10 מיקרוליטר של קומפלקס ריבונוקלאו פרוטאין CRISPR Cas9 או תמיסת בקרה. לאחר מכן, הניחו את הנימים המלאים לתוך המטפל במזרק מיקרו. נתקו את קצה הנימים בעזרת מלקחיים עדינים כדי להתאים את נפח הפליטה ל-10 ננוליטר.הניחו עוברים בשלב תא אחד על רשת המודבקת בצלחת פטרי בקוטר 100 מ"מ עם תמיסת פוליסוכרוז 3%. באמצעות מזרק מיקרו, ותחת מיקרוסקופ סטריאו, מזריקים 10 ננוליטר של התמיסה לאזור קליפת המוח, במיוחד ברמת קוטב בעלי החיים מתחת לקרום הציטופלזמי. לאחר 24 שעות, דמיינו פלואורסצאין, ליזין, דקסטרין תחת מיקרוסקופ סטריאו פלואורסצנטי, ובחרו עוברי rho crispant מוזרקים היטב.תיוג קספז 3 של רשתית ראשנים פריכים הראה כי חלק מהמוטות עוברים אפופטוזיס. צביעה היסטולוגית חשפה שימור עולמי של שכבות גרעיניות, אך קיצור חמור של מקטעים חיצוניים של קולטני אור. ניתוח צביעה חיסונית נוספת עם סמנים photoreceptor הראה ניוון של מקטעים חיצוניים מוט.

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Research

JoVE Journal

Developmental Biology

Rod Cell Degeneration in Xenopus Tadpoles by CRISPR/Cas9-Mediated Rhodopsin Knockout

96 Views.  Université Paris-Saclay. כדי להתחיל, קח נימים מושחזים, השתמש בקצה מטעין מיקרו כדי לטעון 2 עד 10 מיקרוליטר של קומפלקס ריבונוקלאו פרוטאין CRISPR Cas9 או תמיסת בקרה. לאחר מכן, הניחו את הנימים המלאים לתוך המטפל במזרק מיקרו. נתקו את קצה הנימים בעזרת מלקחיים עדינים כדי להתאים את נפח הפליטה ל-10 ננוליטר.הניחו ע...

Video: Rod Cell Degeneration in Xenopus Tadpoles by CRISPR/Cas9-Mediated Rhodopsin Knockout  

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.

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 ...

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms  

Begin by using a dip net to capture the tadpoles from their tanks and transfer them to a fresh tank filled with 0.005%benzocaine solution for anesthesia. Then wait for a few minutes to allow the tadpoles to stop reacting to stimuli. Now, capture the anesthetized tadpole with a spoon and carefully place it with its dorsal side up in the center of a small Petri dish lined with moist tissue.Then position the specimen under the stereo microscope. Using a 0.2 millimeter diameter pin attached to a pin holder, carefully insert the tool on the dorsal side of the right eye, piercing the retina, and passing through completely. Then rotate the Petri dish 180 degrees and puncture the left eye.Carefully immerse the Petri dish containing the lesioned tadpole into a large tank with one liter of rearing water. Monitor the tadpoles until they are fully awakened. Retinal sections of tadpoles subjected to mechanical injury showed the retinal lesion in all layers of the tissue while remaining limited to the puncture site.

ניקוב רשתית מכני בראשנים של קסנופוס לחקר מנגנוני התחדשות הרשתית

Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System

Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System  

To begin, use a transgenic line expressing GFP and nitro reductase, or NTR, under a rod photoreceptor specific promoter. NTR converts the prodrug metronidazole, or MTZ, into a cytotoxic agent for selective ablation of NTR expressing rods. For GFP monitoring, use a spoon to gently catch one anesthetized tadpole and cautiously place it onto a moist tissue within a small Petri dish.Observe the GFP fluorescence under a fluorescent stereo microscope and capture photographic images of the eyes. Carefully immerse the Petri dish with the tadpole into a large tank with rearing water until fully awakened. For individual monitoring of retinal degeneration, place each tadpole in a small container containing 30 milliliters of 10 millimolar MTZ, or transgenic siblings in the control solution.For batch treatment, place the transgenic tadpoles in a larger container with one liter of MTZ, or control solution. Raise the tadpoles for one week at 20 degrees Celsius under darkness to avoid degradation of MTZ. After seven days, observe the GFP under the fluorescent stereo microscope and capture photographic images of the eye.A decrease in fluorescence intensity, or even a virtual absence of fluorescence indicates the degradation of rods. The eyes of transgenic tadpoles treated with MTZ showed a decrease in GFP fluorescence compared to the controls, which is further confirmed on retinal sections by GFP immuno staining.

אבלציה מותנית של תאי מוט בראשנים של קסנופוס באמצעות מערכת NTR-MTZ

To begin, take a sharpened capillary, use a micro loader tip to load 2 to 10 microliters of the CRISPR Cas9 ribonucleoprotein complex or control solution. Afterward, place the filled capillary into the micro injector handler. Break off the capillary tip with fine forceps to adjust the ejection volume at 10 nanoliters.Position one cell stage embryos on a grid glued in a 100 millimeter Petri dish with 3%polysucrose solution. Using the micro injector, and under a stereo microscope, inject 10 nanoliter of the solution into the cortical region, specifically at the animal pole level under the cytoplasmic membrane. After 24 hours, visualize fluorescein, lysine, dextrin under a fluorescent stereo microscope, and select well-injected rho crispant embryos.Caspase 3 labeling of rho crispant tadpole retinas showed that some rods undergo apoptosis. Histological staining revealed global preservation of nuclear layers, but a severe shortening of photoreceptor outer segments. Further immuno staining analysis with photoreceptor markers showed the degeneration of rod outer segments.

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections  

Use a dip net to capture tadpoles from their tanks and transfer them into a 50 milliliter solution of 0.005%benzocaine. Allow them to remain in the solution until they no longer respond to stimuli. Use a spoon to gently pick up a tadpole and place it with its dorsal side facing up into a small Petri dish containing a damp tissue.Then position the specimen under the stereo microscope. To begin, take a sharpened capillary, use a micro loader tip to load 10 microliters of cobalt chloride solution. Afterward, place the filled capillary into the micro injector handler to break off the capillary tip to adjust the ejection volume at about 30 nanoliters.Using the micro injector and a stereo microscope, insert the capillary filled with cobalt chloride over the lens into the eye. Upon reaching the inside of the eye with the capillary tip, eject two drops of about 30 nanoliters per eye. After the right eye has been injected, turn the Petri dish to a 180 degree angle to inject the left eye.Transfer the Petri dish containing the injected tadpole to a large tank containing one liter of rearing water. Keep observing the tadpoles until they have fully awakened. Injection of 10 millimolar cobalt chloride led to specific cell death of cone photoreceptors, while 25 millimolar led to broad retinal cell death.Immuno staining revealed the absence of cones in 10 millimolar cobalt chloride injected retinas while rods were largely preserved. After injections of 25 millimolar cobalt chloride, both photoreceptors and bipolar cells were severely affected.