Name: Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System
Uploaded: 2023-10-13T12:00:00
Description: Watch this Scientific Journal Video about Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System at JoVE.com

conditional rod cell ablation in xenopus tadpoles using the ntr-mtz system

Génération de la dégénérescence rétinienne pour étudier les stratégies de réparation cellulaire chez le xénope

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.

Pleins feux sur l’auteur : Explorer les mécanismes de régénération rétinienne chez la grenouille xénope 

Génération de modèles de lésions rétiniennes chez les têtards Xénope

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 Conditional Rod Cell Ablation in Xenopus Tadpoles Using the NTR-MTZ System at JoVE.com

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

Ablation conditionnelle des cellules en bâtonnets chez les têtards de Xénope à l’aide du système NTR-MTZ

Pour commencer, utilisez une lignée transgénique exprimant la GFP et la nitroréductase, ou NTR, sous un promoteur spécifique des photorécepteurs en bâtonnets. Le NTR convertit le métronidazole, ou MTZ, en un agent cytotoxique pour l’ablation sélective des bâtonnets exprimant le NTR. Pour la surveillance de la GFP, utilisez une cuillère pour attraper doucement un têtard anesthésié et placez-le prudemment sur un tissu humide dans une petite boîte de Pétri.Observez la fluorescence GFP sous un stéréomicroscope fluorescent et capturez des images photographiques des yeux. Plongez soigneusement la boîte de Pétri avec le têtard dans un grand réservoir avec de l’eau d’élevage jusqu’à ce qu’il soit complètement réveillé. Pour une surveillance individuelle de la dégénérescence rétinienne, placez chaque têtard dans un petit récipient contenant 30 millilitres de 10 millimolaires MTZ, ou des frères et sœurs transgéniques dans la solution témoin.Pour le traitement par lots, placez les têtards transgéniques dans un récipient plus grand avec un litre de MTZ, ou solution témoin. Élevez les têtards pendant une semaine à 20 degrés Celsius dans l’obscurité pour éviter la dégradation de la ZMT. Après sept jours, observez la GFP sous le stéréomicroscope fluorescent et capturez des images photographiques de l’œil.Une diminution de l’intensité de la fluorescence, voire une quasi-absence de fluorescence indique la dégradation des bâtonnets. Les yeux des têtards transgéniques traités avec MTZ ont montré une diminution de la fluorescence GFP par rapport aux témoins, ce qui est confirmé sur les coupes rétiniennes par immunomarquage GFP.

Research

JoVE Journal

Developmental Biology

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

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 ...

Recherche

Biologie du développement

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms  

Ponction mécanique de la rétine chez les têtards de Xénope pour étudier les mécanismes de régénération de la rétine

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

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

Dégénérescence des cellules en bâtonnets chez les têtards de Xénope par knock-out de la rhodopsine médiée par CRISPR/Cas9

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections  

Dégénérescence des cellules rétiniennes chez les têtards de Xénope par injections intraoculaires cytotoxiques de CoCl2