A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish

Abstract

A study of the role of hydrogen peroxide (H2O2) during the early events of wound responses in zebrafish larvae — a popular vertebrate model for inflammatory and regenerative responses to wounds — reveals a that tissue gradient of H2O2 forms in response to injury of the zebrafish tail fin. The gradient, made visible by the use of a genetically encoded fluorescent sensor protein, is created by the activity of dual oxidase (DUOX) and acts to attract leukocytes to the wound margin during the initial phase of inflammation. This is the first report of a role for H2O2 in signalling to leukocytes, in addition to its established role as an antiseptic. In animals, within minutes of wounding, leukocytes are recruited to the site of injury across distances of hundreds of micrometres. Early leukocyte recruitment after injury is now shown to be driven by the establishment of an H2O2 gradient from the epithelium to the vasculature in zebrafish. Barrier structures (for example, epithelia around tissues and plasma membranes around cells) are required for internal homeostasis and protection from pathogens. Wound detection and healing represent a dormant morphogenetic program that can be rapidly executed to restore barrier integrity and tissue homeostasis. In animals, initial steps include recruitment of leukocytes to the site of injury across distances of hundreds of micrometres within minutes of wounding. The spatial signals that direct this immediate tissue response are unknown. Owing to their fast diffusion and versatile biological activities, reactive oxygen species, including hydrogen peroxide (H2O2), are interesting candidates for wound-to-leukocyte signalling. Here we probe the role of H2O2 during the early events of wound responses in zebrafish larvae expressing a genetically encoded H2O2 sensor1. This reporter revealed a sustained rise in H2O2 concentration at the wound margin, starting ∼3 min after wounding and peaking at ∼20 min, which extended ∼100–200 μm into the tail-fin epithelium as a decreasing concentration gradient. Using pharmacological and genetic inhibition, we show that this gradient is created by dual oxidase (Duox), and that it is required for rapid recruitment of leukocytes to the wound. This is the first observation, to our knowledge, of a tissue-scale H2O2 pattern, and the first evidence that H2O2 signals to leukocytes in tissues, in addition to its known antiseptic role.