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

Generering af nethindedegeneration for at undersøge cellulære reparationsstrategier i 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.

Forfatter Spotlight: Udforskning af nethindens regenereringsmekanismer i Xenopus-frøen 

Generering af retinale skademodeller i Xenopus haletudser

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  

Betinget stavcelleablation i xenopus haletudser ved hjælp af NTR-MTZ-systemet

Til at begynde med skal du bruge en transgen linje, der udtrykker GFP og nitroreduktase eller NTR under en stangfotoreceptorspecifik promotor. NTR omdanner prodrug metronidazol eller MTZ til et cytotoksisk middel til selektiv ablation af NTR-ekspressionsstænger. Til GFP-overvågning skal du bruge en ske til forsigtigt at fange en bedøvet haletudse og forsigtigt placere den på et fugtigt væv i en lille petriskål.Overhold GFP-fluorescensen under et fluorescerende stereomikroskop og tag fotografiske billeder af øjnene. Nedsænk forsigtigt petriskålen med haletudsen i en stor tank med opdrætsvand, indtil den er helt vækket. For individuel overvågning af retinal degeneration skal du placere hver haletudse i en lille beholder indeholdende 30 ml 10 millimolær MTZ eller transgene søskende i kontrolopløsningen.Til batchbehandling anbringes de transgene haletudser i en større beholder med en liter MTZ eller kontrolopløsning. Hæv haletudserne i en uge ved 20 grader Celsius i mørke for at undgå nedbrydning af MTZ. Efter syv dage skal du observere GFP under det fluorescerende stereomikroskop og tage fotografiske billeder af øjet.Et fald i fluorescensintensitet eller endda et virtuelt fravær af fluorescens indikerer nedbrydning af stænger. Øjnene på transgene haletudser behandlet med MTZ viste et fald i GFP-fluorescens sammenlignet med kontrollerne, hvilket yderligere bekræftes på retinale sektioner ved GFP-immunfarvning.

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 ...

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms  

Mekanisk retinal punktering i Xenopus haletudser til at studere retinale regenereringsmekanismer

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

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

Stangcelledegeneration i Xenopus haletudser af CRISPR/Cas9-medieret Rhodopsin Knockout

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections  

Retinal celledegeneration i Xenopus haletudser ved cytotoksiske intraokulære CoCl2-injektioner