Mapping subcellular H2O2 dynamics reveals tissue specific redox patterns in Drosophila

Redox signalling regulates development, tissue homeostasis, and organismal health. Hydrogen peroxide (H2O2) is a major signalling form of reactive oxygen species (ROS) that modulates protein activity through oxidation of redox-sensitive cysteines that is reversed by cellular reducing systems. Since H2O2 production, scavenging and reduction are spatially restricted, signalling specificity is strongly influenced by subcellular localisation. However, subcellular H2O2 dynamics in animal tissues remain poorly understood. To address this, we generated and validated Drosophila melanogaster lines expressing the ultrasensitive, ultrafast ratiometric H2O2 biosensor HyPer7 targeted to mitochondria, nucleus, cytosol, or plasma membrane. With its highly conserved metabolic and signalling pathways, tractable lifespan, and powerful genetic toolkit, Drosophila is an ideal model for studying redox biology. These new 'FlyPer' lines enable tissue-specific HyPer7 expression and high-resolution measurement of subcellular, in vivo H2O2 dynamics throughout the lifespan. Using FlyPer, we detected compartment-specific H2O2 dynamics during oxidative stress, ageing, wing disc development and embryogenesis, uncovering unexpected patterns of spatially and temporally regulated oxidation throughout the organism. Together, these findings establish FlyPer as a valuable toolkit for in vivo redox biology and suggest that compartmentalised redox dynamics are a fundamental yet still poorly understood layer of developmental programming.