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 av näthinnedegeneration för att undersöka cellulära reparationsstrategier hos 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.

Författare i fokus: Utforska mekanismer för näthinneregenerering hos Xenopus-grodan 

Generering av modeller för näthinneskador hos Xenopus-grodyngel

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  

Villkorlig stavcellsablation i Xenopus-grodyngel med hjälp av NTR-MTZ-systemet

Till att börja med, använd en transgen linje som uttrycker GFP och nitroreduktas, eller NTR, under en stavfotoreceptorspecifik promotor. NTR omvandlar prodrogen metronidazol, eller MTZ, till ett cytotoxiskt medel för selektiv ablation av NTR-uttryckande stavar. För GFP-övervakning, använd en sked för att försiktigt fånga ett bedövat grodyngel och placera det försiktigt på en fuktig vävnad i en liten petriskål.Observera GFP-fluorescensen under ett fluorescerande stereomikroskop och ta fotografiska bilder av ögonen. Sänk försiktigt ner petriskålen med grodyngel i ett stort akvarium med odlingsvatten tills den är helt vaken. För individuell övervakning av näthinnedegeneration, placera varje grodyngel i en liten behållare som innehåller 30 milliliter 10 millimolar MTZ, eller transgena syskon i kontrolllösningen.För batchbehandling, placera de transgena grodynglen i en större behållare med en liter MTZ, eller kontrolllösning. Höj grodynglen i en vecka vid 20 grader Celsius i mörker för att undvika nedbrytning av MTZ. Efter sju dagar, observera GFP under det fluorescerande stereomikroskopet och ta fotografiska bilder av ögat.En minskning av fluorescensintensiteten, eller till och med en virtuell frånvaro av fluorescens, indikerar nedbrytningen av stavar. Ögonen hos transgena grodyngel som behandlats med MTZ visade en minskning av GFP-fluorescens jämfört med kontrollerna, vilket ytterligare bekräftas på näthinnesnitt genom GFP-immunfärgning.

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 punktion hos Xenopus grodyngel för att studera näthinneregenereringsmekanismer

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

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

Rod Cell Degeneration i Xenopus grodyngel genom CRISPR/Cas9-medierad rodopsinknockout

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections  

Retinal celldegeneration i Xenopus grodyngel genom cytotoxiska intraokulära CoCl 2-injektioner