nox2/cybb Deficiency Affects Zebrafish Retinotectal Connectivity

Abstract

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) have been linked to neuronal polarity, axonal outgrowth, cerebellar development, regeneration of sensory axons, and neuroplasticity. However, the specific roles that individual Nox isoforms play during nervous system development remain unclear. To address this problem, we investigated the role of Nox activity in the development of retinotectal connections in zebrafish embryos. Zebrafish broadly express four genes (, ,, and ) throughout the CNS during early development. Application of a pan-Nox inhibitor, celastrol, during the time of optic nerve (ON) outgrowth resulted in significant expansion of the ganglion cell layer (GCL), thinning of the ON, and a decrease in retinal axons reaching the optic tectum (OT). With the exception of GCL expansion, these effects were partially ameliorated by the addition of HO, a key ROS involved in Nox signaling. To address isoform-specific Nox functions, we used CRISPR/Cas9 to generate mutations in each zebrafish gene. We found that chimeric mutants displayed ON thinning and decreased OT innervation. Furthermore, homozygous mutants () showed significant GCL expansion and mistargeted retinal axons in the OT. Neurite outgrowth from cultured zebrafish retinal ganglion cells was reduced by Nox inhibitors, suggesting a cell-autonomous role for Nox in these neurons. Collectively, our results show that Nox2/Cybb is important for retinotectal development in zebrafish. Most isoforms of NADPH oxidase (Nox) only produce reactive oxygen species (ROS) when activated by an upstream signal, making them ideal candidates for ROS signaling. Nox enzymes are present in neurons and their activity has been shown to be important for neuronal development and function largely by studies. However, whether Nox is involved in the development of axons and formation of neuronal connections has remained unclear. Using mutant zebrafish embryos, this study shows that a specific Nox isoform, Nox2/Cybb, is important for the establishment of axonal connections between retinal ganglion cells and the optic tectum.

Hydrogen Peroxide and Redox Regulation of Developments

“…This distribution occurs in the embryonic tectum where the organization of the retinotectal projections is impaired by Pan NOX inhibition [ 61 ]. Nox2 invalidation using the CRISPR/Cas9 strategy induced the same phenotype [ 103 ], confirming Nox2 involvement in axon pathfinding during zebrafish development. Very recently, the group of E. Amaya proved that Duox activity was necessary for the development of zebrafish thyroid [ 104 ], and that ROS play a role in Xenopus mesoderm formation [ 59 ].…”

NOX5: Molecular biology and pathophysiology

“…However, most of those studies were performed in monocyte/macrophage leukaemia cell lines (Marzaioli et al., ), and the physiological role of neutrophil NOX5 is unclear. In the developing zebrafish, Nox5 is expressed throughout the CNS and, together with Nox2, it might be important in neurodevelopment and regeneration (Weaver et al., ; Weaver, Leung, & Suter, ). This is confirmed in human oligodendrocytes, which require NOX5 for differentiation and maturation (Accetta et al., ).…”

Hydrogen Peroxide and Redox Regulation of Development

“…by Pan NOX inhibition [66]. Nox2 invalidation using the CRISPR/Cas9 strategy induced the same phenotype [83], confirming NOX2 involvement in axon pathfinding during zebrafish development. A role for Nox enzymes was also evidenced ex vivo for the establishment of rat hippocampal neuron polarity in culture [84,85], the in vitro maturation of rat cerebellar granule neurons [86], and growth cone dynamics in…”