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

Erzeugung von Netzhautdegeneration zur Untersuchung zellulärer Reparaturstrategien bei 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. 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.

Autor im Rampenlicht: Erforschung der Regenerationsmechanismen der Netzhaut beim Xenopus-Frosch 

Generierung von Netzhautverletzungsmodellen bei Xenopus-Kaulquappen

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  

Bedingte Stäbchenzellablation bei Xenopus-Kaulquappen mit dem NTR-MTZ-System

Verwenden Sie zunächst eine transgene Linie, die GFP und Nitroreduktase (NTR) unter einem Stäbchen-Photorezeptor-spezifischen Promotor exprimiert. NTR wandelt das Prodrug Metronidazol oder MTZ in ein zytotoxisches Mittel für die selektive Ablation von NTR-exprimierenden Stäbchen um. Für die GFP-Überwachung wird eine betäubte Kaulquappe vorsichtig mit einem Löffel aufgefangen und vorsichtig auf ein feuchtes Gewebe in einer kleinen Petrischale gelegt.Beobachten Sie die GFP-Fluoreszenz unter einem fluoreszierenden Stereomikroskop und nehmen Sie fotografische Bilder der Augen auf. Tauche die Petrischale mit der Kaulquappe vorsichtig in ein großes Becken mit Aufzuchtwasser, bis sie vollständig erwacht ist. Zur individuellen Überwachung der Netzhautdegeneration wird jede Kaulquappe in einen kleinen Behälter gegeben, der 30 Milliliter 10 Millimolar MTZ oder transgene Geschwister in der Kontrolllösung enthält.Für die Chargenbehandlung geben Sie die transgenen Kaulquappen in einen größeren Behälter mit einem Liter MTZ oder Kontrolllösung. Ziehen Sie die Kaulquappen eine Woche lang bei 20 Grad Celsius in der Dunkelheit auf, um einen Abbau von MTZ zu vermeiden. Beobachten Sie nach sieben Tagen das GFP unter dem fluoreszierenden Stereomikroskop und nehmen Sie fotografische Bilder des Auges auf.Eine Abnahme der Fluoreszenzintensität oder sogar eine nahezu fehlende Fluoreszenz deutet auf den Abbau von Stäbchen hin. Die Augen von transgenen Kaulquappen, die mit MTZ behandelt wurden, zeigten im Vergleich zu den Kontrollen eine Abnahme der GFP-Fluoreszenz, was an Netzhautschnitten durch GFP-Immunfärbung weiter bestätigt wird.

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 ...

Forschung

Entwicklungsbiologie

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms  

Mechanische Netzhautpunktion bei Xenopus-Kaulquappen zur Untersuchung der Regenerationsmechanismen der Netzhaut

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

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

Stäbchenzelldegeneration in Xenopus-Kaulquappen durch CRISPR/Cas9-vermittelten Rhodopsin-Knockout

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections  

Retinale Zelldegeneration bei Xenopus-Kaulquappen durch zytotoxische intraokulare CoCl 2-Injektionen