Dual Oxidase Mutation Retards Mauthner-cell Axon Regeneration In Zebrafish

Oxygen metabolism balance is important for axon regeneration and myelin development.Up to now,most of relevant studies focused on whole mammalian or in vitro models to observe the anatomical and morphological differences after spinal cord or optic nerve injury,but in vivo imaging and single-cell axon regeneration are absent.Therefore,we used zebrafish to explore the dynamic changes in visualized axon regeneration and myelin development and underlie mechanisms under the imbalance of oxygen metabolism.Initially,reactive oxygen species(ROS)were considered as mitochondrial metabolic by-products which lead to consistently cell damage,aging and death.Until the discovery of NADPH oxidase(Nox)family,studies found that Nox-derived ROS were closely associated with axonal outgrowth,sensory axonal regeneration.In vitro studies show that Nox activity plays an important role in neuronal development and function.However,the specific roles that the single Nox isoform playing throughout nervous system development in vivo remains indistinct.In addition,zebrafish has the advantage of researching the effect of duox on neural regeneration and development because its genome has a single duox gene in contrast to the two duoxl and duox2 in rodents and humans.In this study,I obtained duox-/-homozygous mutant zebrafish line using CRISPRCas9 technology.With two-photon axotomy technology,results showed that duox-/homozygous mutant inhibited the unilateral Mauthner cell axon regeneration in zebrafish in vivo.To explain the mechanism,I compared the high-throughout transcription sequencing results between the duox-/-homozygous mutant fish and the wild-type(WT)fish and verified the results with quantitative real-time PCR(q-PCR).Results showed that mitochondrial functional related genes expression were decreased in duox-/-mutant fish.In addition,the analysis of mitochondrial ultrastructure morphology,dynamic and redox state showed that duox deficiency disrupted mitochondrial morphology and redox function,especially in terms of a slower speed of mitochondrial transport in Mauthner cells.Taken together,these results suggested that duox deficiency affected mitochondrial speed and motility in the early stages of regeneration,which may have induced the retard of Mauthner cell axon regeneration.The above is the study of the effects of Duox deficiency on axon regeneration,and the following is the study of the effects of hypoxia on the early development of oligodendrocyte progenitor cell(OPC)migration and myelin formation in zebrafish larvae.Referred the previous study,we set up a semi-automatic hypoxia device for larvae.Based on the device,the study observed the number of olig2+cells migrating to the dorsal spinal cord was decreased and the myelin process of oligodendrocytes was severely impeded in hypoxia.In addition,the thickness of myelin sheath was imaged with transmission electron microscope(TEM).Results showed that immature and unuseful myelin sheath under hypoxia.Experiments with Bmp2b receptor inhibitors and bmp2b mRNA remediation futher confirmed that hypoxia inhibited OPCs differentiation by inhibited expression of Bmp2b.In summary,imbalance of oxygen metabolism inhibits axon regeneration and oligodendrocyte development in larval zebrafish.Moreover,Duox-mediated ROS deficiency retards axon regeneration by mediating mitochondrial dynamic in axon transport.In contrast,mitochondria-mediated ROS production due to hypoxia inhibits the development of spinal oligodendrocytes by suppressing Bmp2b signaling.