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

Generazione della degenerazione retinica per studiare le strategie di riparazione cellulare in 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.

Autore in primo piano: Esplorazione dei meccanismi di rigenerazione retinica nella rana Xenopus 

Generazione di modelli di lesioni retiniche nei girini di Xenopus

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  

Ablazione condizionale dei bastoncelli nei girini di Xenopus utilizzando il sistema NTR-MTZ

Per iniziare, utilizzare una linea transgenica che esprima GFP e nitro reduttasi, o NTR, sotto un promotore specifico del fotorecettore dei bastoncelli. NTR converte il profarmaco metronidazolo, o MTZ, in un agente citotossico per l'ablazione selettiva dei bastoncelli che esprimono NTR. Per il monitoraggio della GFP, utilizzare un cucchiaio per afferrare delicatamente un girino anestetizzato e posizionarlo con cautela su un tessuto umido all'interno di una piccola capsula di Petri.Osservare la fluorescenza GFP con uno stereomicroscopio fluorescente e acquisire immagini fotografiche degli occhi. Immergere con cura la capsula di Petri con il girino in una grande vasca con acqua di allevamento fino a completo risveglio. Per il monitoraggio individuale della degenerazione retinica, mettere ogni girino in un piccolo contenitore contenente 30 millilitri di MTZ da 10 millimolari o fratelli transgenici nella soluzione di controllo.Per il trattamento in batch, mettere i girini transgenici in un contenitore più grande con un litro di MTZ o soluzione di controllo. Allevare i girini per una settimana a 20 gradi Celsius nell'oscurità per evitare il degrado di MTZ. Dopo sette giorni, osservare la GFP con lo stereomicroscopio fluorescente e acquisire immagini fotografiche dell'occhio.Una diminuzione dell'intensità della fluorescenza, o anche una virtuale assenza di fluorescenza, indica la degradazione dei bastoncelli. Gli occhi dei girini transgenici trattati con MTZ hanno mostrato una diminuzione della fluorescenza della GFP rispetto ai controlli, che è ulteriormente confermata sulle sezioni retiniche dall'immunocolorazione della 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 ...

Ricerca

Biologia dello sviluppo

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms  

Puntura meccanica della retina nei girini di Xenopus per studiare i meccanismi di rigenerazione retinica

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

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

Degenerazione delle cellule dei bastoncelli nei girini di Xenopus mediante knockout della rodopsina mediato da CRISPR/Cas9

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections  

Degenerazione delle cellule retiniche nei girini di Xenopus mediante iniezioni intraoculari citotossiche di CoCl2