Targeted cell ablation in zebrafish using optogenetic transcriptional control

Abstract

Cell ablation is a powerful method for elucidating the contributions of individual cell populations to embryonic development and tissue regeneration. Targeted cell loss in whole organisms has been typically achieved through expression of a cytotoxic or prodrug-activating gene product in the cell type of interest. This approach depends on the availability of tissue-specific promoters, and it does not allow further spatial selectivity within the promoter-defined region(s).To address this limitation, we have developed ablative methods that combine genetically encoded toxins, the tissue specificity afforded by cis-regulatory elements, and the conditionality of optogenetics. Using this integrative approach, we have ablated cells in zebrafish embryos with spatial and temporal precision.

Rapid and robust optogenetic control of gene expression in Drosophila

“…Accordingly, there have been several attempts to develop optogenetic binary systems (de Mena et al, 2018). Some success has been reported with optogenetic Gal4 approaches in cell culture, zebrafish embryos, Drosophila, and mice (Liu et al, 2012;Wang et al, 2012;Pathak et al, 2017;de Mena and Rincon-Limas, 2020;Mruk et al, 2020;Yamada et al, 2020). These advances are significant, but they come with some limitations: (i) the long light exposure (in the order of hours) necessary to induce phenotypes or reporter expression (de Mena and Rincon-Limas, 2020; Mruk et al, 2020), likely incompatible with live imaging or the study of rapid biological processes; (ii) leakiness in the dark (Mruk et al, 2020;Pathak et al, 2017;Yamada et al, 2020); (iii) the lack of a fully transgenic optogenetic Gal4/UAS system (Liu et al, 2012;Pathak et al, 2017;Yamada et al, 2020); or (iv) the requirement of an exogenous chromophore, which is only readily administered to tissue explants (de Mena and Rincon-Limas, 2020).…”

Optogenetic Protein Cleavage in Zebrafish Embryos

“…Other systems for light induced cell ablation are based on optogenetically controlled transcriptional activation of nitroreductase with subsequent metronidazole addition, or mutant M2 proton channel expression originally from influenza. 4 The NBid-PhoCl-CBid system induces immediate caspase cascade activation and is easy to use since it only requires injection of a single mRNA.…”

“…3 Their transparency also makes them amenable to using light as an control tool that offers high spatial and temporal precision. Optogenetics has garnered many successful uses in zebrafish embryos, including control over gene expression, 4 cell migration, 5 protein localization, 6 cell signaling, 7 and cell ablation. 8 These tools have been used to study important biological processes due to the unparalleled spatiotemporal control afforded by light.…”

Non-neuromodulatory Optogenetic Tools in Zebrafish

“…The dimer then binds to UAS sites to induce expression of a gene of interest. The LightOn system was applied in zebrafish to gain spatiotemporal control over expression of two cell ablation systems, nitroreductase and the cytotoxic viral ion channel M2H37A (Mruk et al, 2019). GAVPO-mediated nitroreductase expression induced apoptosis in embryos when treated with metronidazole and irradiated with blue light.…”