THE ROLE OF NADPH OXIDASE 2 IN AXON GUIDANCE DURING ZEBRAFISH VISUAL SYSTEM DEVELOPMENT

Reactive oxygen species (ROS) are critical for maintaining cellular homeostasis and function when produced in physiological ranges. Important sources of cellular ROS include NADPH oxidases (Nox), which are evolutionarily conserved multi-subunit transmembrane proteins. Nox-mediated ROS regulate a variety of biological processes including stem cell proliferation and differentiation, calcium signaling, cell migration, and immunity. ROS participate in intracellular signaling by introducing post-translational modifications to proteins and thereby altering their functions. The central nervous system (CNS) expresses different Nox isoforms during both development and adulthood. There is now emerging evidence that Nox-derived ROS also control neuronal development and pathfinding. Our lab has recently shown that retinal ganglion cells (RGCs) from nox2 mutant zebrafish exhibit pathfinding errors. However, whether Nox could act downstream of receptors for axonal growth and guidance cues is presently unknown. To investigate this question, we conducted a detailed characterization of the zebrafish nox2 mutants that were previously established in our group. Abnormal axon projections were found throughout the CNS of the nox2 mutant zebrafish. Anterior commissural axons failed proper fasciculation, and aberrant axon projections were detected in the dorsal longitudinal fascicle of the spinal cord. We showed that the major brain regions are intact and that the early development of CNS is not significantly altered in nox2 mutants. Hence, the axonal deficits in nox2 mutants are not due to general developmental problems, and Nox2 plays a role in axonal pathfinding and targeting. Next, we investigated whether Nox2 could act downstream of slit2/Robo2-mediated guidance during RGC pathfinding. We found that slit2-mediated RGC growth cone collapse was abolished in nox2 mutants in vitro. Further, ROS biosensor imaging showed that slit2 treatment increased growth cone hydrogen peroxide levels via mechanisms through Nox2 activation. Finally, we investigated the possible relationship between slit2/Robo2 and Nox2 signaling in vivo. Astray/nox2 double heterozygous mutant larvae exhibited decreased tectal area as opposed to individual heterozygous mutants, suggesting both Nox2 and Robo2 are required for the establishment of retinotectal connections. Our results suggest that Nox2 is part of a signal transduction pathway downstream of slit2/Robo2 interaction regulating axonal guidance cell-autonomously in developing zebrafish retinal neurons.