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

Gerando degeneração retiniana para investigar estratégias de reparo celular em 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 em destaque: explorando os mecanismos de regeneração da retina no sapo Xenopus 

Gerando modelos de lesão retiniana em girinos 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  

Ablação condicional de células de haste em girinos Xenopus usando o sistema NTR-MTZ

Para começar, use uma linha transgênica expressando GFP e nitro redutase, ou NTR, sob um promotor específico do fotorreceptor de bastonete. NTR converte a pró-droga metronidazol, ou MTZ, em um agente citotóxico para ablação seletiva de bastonetes que expressam NTR. Para o monitoramento de GFP, use uma colher para pegar suavemente um girino anestesiado e coloque-o cautelosamente em um tecido úmido dentro de uma pequena placa de Petri.Observe a fluorescência da GFP sob um estereomicroscópio fluorescente e capture imagens fotográficas dos olhos. Mergulhe cuidadosamente a placa de Petri com o girino em um grande tanque com água de criação até ficar totalmente acordado. Para monitoramento individual da degeneração retiniana, coloque cada girino em um pequeno recipiente contendo 30 mililitros de MTZ 10 milimolares ou irmãos transgênicos na solução controle.Para o tratamento em batelada, coloque os girinos transgênicos em um recipiente maior com um litro de MTZ, ou solução controle. Levante os girinos por uma semana a 20 graus Celsius sob a escuridão para evitar a degradação do MTZ. Após sete dias, observar a GFP sob estereomicroscópio fluorescente e capturar imagens fotográficas do olho.Uma diminuição na intensidade da fluorescência, ou mesmo uma virtual ausência de fluorescência, indica a degradação dos bastões. Os olhos de girinos transgênicos tratados com MTZ mostraram uma diminuição na fluorescência da GFP em comparação com os controles, o que é confirmado em cortes da retina pela imunomarcação de 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 ...

Biologia do Desenvolvimento

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms

Mechanical Retinal Puncture in Xenopus Tadpoles to Study Retinal Regeneration Mechanisms  

Punção Retiniana Mecânica em Girinos Xenopus para Estudo dos Mecanismos de Regeneração Retiniana

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

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

Degeneração de células bastonetes em girinos de Xenopus por knockout de rodopsina mediado por CRISPR/Cas9

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections

Retinal Cell Degeneration in Xenopus Tadpoles by Cytotoxic Intraocular CoCl2 Injections  

Degeneração Celular da Retina em Girinos de Xenopus por Injeções Intraoculares Citotóxicas de CoCl2