Meeting Abstract
Reactive oxygen species (ROS) can produce major physiological challenge for organisms (oxidative stress) but also serve as important cellular signals. Recent development of genetically encoded redox indicators (GERIs) has provided unprecedented capability to visualize the oxidative state of cells and tissues in vivo, with the potential to provide insight into the role of ROS signaling in diverse developmental and physiological processes. In many species, cellular redox is known to play an important role in stem-cell maintenance, wound healing, and regeneration. In addition, the oxidation state of certain proteins is a ubiquitous feature of circadian rhythms, but the extent to which cellular or subcellular redox state may fall under circadian regulation is unknown. The cnidarian Nematostella vectensis is naturally exposed to drastic changes in salinity, temperature, UV exposure, and anoxia, however, variation in its cellular redox state has not been described. As a tool for probing ROS signaling in N. vectensis under diverse conditions, we synthesized capped mRNA encoding roGFP2 conjugated to either a glutaredoxin (Grx1) or the yeast Orp1. Within these constructs, roGFP2 exhibits a change in excitation spectrum in response to changes in glutathione redox state (Grx1) or hydrogen peroxide concentration (Orp1). As a ratiometric indicator, roGFP2 is well-suited to in vivo imaging because results are not biased by variation in basal expression levels. We injected N. vectensis zygotes with each of these constructs and observed strong transient expression that persisted throughout embryonic and larval development into the juvenile polyp stage. We determined that these protein constructs are responsive to changes in redox state by imaging embryos before and after hydrogen peroxide exposure. We will use these tools to understand diverse features of N. vectensis biology, with a particular focus on development and circadian physiology.
