H2O2-dependent redox homeostasis in a zebrafish model of endocrine disease

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Redox reactions are fundamental reactions in biology. Reactive oxygen species (ROS) arising from these reactions must be tightly controlled to prevent damage to nucleic acids, proteins and lipids. Excessive ROS production is a feature of a number of diseases, including endocrine and metabolic diseases. However, ROS also serve signaling or regulatory functions in the organism, notably via reversible modifications of thiols in cysteine residues of proteins. It is well documented that such reversible modifications change protein conformation, localization and activity. Nevertheless, regulation of only a few of these thiol switches has been well characterized so far. In addition, knowledge on the spatiotemporal distribution of ROS in cells and tissues of multicellular organisms is limited. This information is needed to map sites and processes where thiol switches normally operate and to allow us a better grasp of causal relationships between shifts in ROS homeostasis and (patho)physiological processes.Here, we wish to use the zebrafish model system for the imaging of in vivo redox events across embryonic development and under pathological conditions mimicking endocrine and metabolic diseases. To this end, we will use zebrafish transgenic lines expressing engineered thiol-switch biosensors, such as roGFP2-Orp1 and the recently developed more sensitive roGFP2-Tsa2deltaCR, for the monitoring of H2O2 dynamics across cytosol and mitochondria of embryonic tissues during development. Preliminary data indicate the presence of migrating cells in the embryo that show flashes of increased H2O2 levels lasting for minutes to hours. By combining the sensor lines with lines carrying cell specific fluorescent markers, we aim to identify the nature of these cells. FACS of cells showing different redox signal levels followed by next generation sequencing analysis of their transcriptomes will give hints as to the regulatory and functional changes linked with the flashes, which will be further studied by manipulating redox levels via chemical treatment or genetic manipulation.To examine redox changes in an endocrine disease model, we will introduce the sensor lines into rx3 strong mutants, a zebrafish model of adrenal insufficiency in which we have previously described numerous glucocorticoid dependent changes in transcriptional and metabolic dynamics. We will also examine embryos treated with glucocorticoids to create an excess of glucocorticoid signalling, as observed in Cushings syndrome. The data will reveal the consequences of both a lack and an excess of glucocorticoids on ROS levels and dynamics in various tissues. This information will be particularly valuable given the scarcity of reports on redox changes in human patients suffering from disorders of the glucocorticoid system. Importantly, the project will provide tools and concepts for the study of redox biology in a model organism highly amenable to in vivo drug screening approaches.