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J. S. Mumm, J. Ariga, S. Walker, S. Berl, M. Parsons, E. Schroeter, M. Saxena; Targeted Ablation & Regeneration of an Inner Retinal Neuron Subtype in Zebrafish. Invest. Ophthalmol. Vis. Sci. 2009;50(13):136.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate whether the loss of a discrete subpopulation of inner retinal neurons is sufficient to induce a regenerative response in zebrafish.
We use an inducible ablation strategy that can be targeted to individual cellular subtypes to establish cell-specific regenerative paradigms in zebrafish. This methodology is based on transgenically targeted (cell-specific) expression of the bacterial enzyme, nitroreductase (NTR). NTR functions to convert otherwise innocuous "pro-drug" substrates into cytotoxins, thereby rendering NTR-expressing cells susceptible to pro-drug induced death. By co-expressing NTR with a fluorescent reporter, the regenerative process can be monitored over time in living fish - loss of fluorescence equating to cellular degeneration while subsequent gains in fluorescence indicate cells have regenerated. After cell ablation has been observed, pro-drugs are removed to determine whether the targeted cell type can be replaced.
Prior studies suggested that photoreceptor loss is required to stimulate retinal repair. However, recent data suggests that the fish retina can regenerate after minimal photoreceptor damage. Here we report that targeted ablation of a specific bipolar cell subtype is followed by their rapid regeneration in larval zebrafish. Immunolabeling has revealed a population of inner nuclear layer cells which begin to proliferate soon after bipolar cell degeneration can be detected. Data will be presented concerning efforts to define cellular and molecular mechanisms of bipolar cell regeneration. Quantitative high-throughput methods for detecting fluorescent reporters in living fish are being developed to facilitate these analyses, as well as proposed screens to identify regeneration-deficient mutants and discover regeneration-promoting compounds.
Application of targeted ablation strategies in zebrafish provides a means to investigate cell-type specific retinal regeneration programs and may facilitate the development of therapeutic strategies for blinding diseases in humans.
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