29 Unbalanced copper (Cu 2+ ) homeostasis is associated with neurological development defects and 30 diseases. However, the molecular mechanisms remain elusive. Here, central neural system (CNS) myelin 31 defects and down-regulated expression of Wnt/Notch signaling and their down-stream mediator hoxb5b 32 were observed in Cu 2+ stressed zebrafish larvae. Loss/knockdown-of-function of hoxb5b phenocopied 33 the myelin and axon defects observed in Cu 2+ stressed embryos. Meanwhile, activation of Wnt/Notch 34 signaling and ectopic expression of hoxb5b could rescue copper-induced myelin defects, suggesting 35 Wnt&Notch-hoxb5b axis mediated Cu 2+ induced myelin and axon defects. Additionally, whole genome 36 DNA methylation sequencing unveiled that a novel gene fam168b, similar to pou3f1/2, exhibited 37 significant promoter hypermethylation and reduced expression in Cu 2+ stressed embryos. The 38 hypermethylated locus in fam168b promoter acted pivotally in its transcription, and loss/knockdown of 39 fam168b/pou3f1 also induced myelin defects. Moreover, this study unveiled that fam168b/pou3f1 and 40 hoxb5b axis acted in a seesaw manner during fish embryogenesis, and demonstrated that copper induced 41 the down-regulated expression of the Wnt&Notch-hoxb5b axis dependent of the function of copper 42 transporter cox17, coupled with the promoter methylation of genes fam168b/pou3f1 and their subsequent 43 down-regulated expression dependent of the function of another transporter atp7b, making joint 44 contributions to myelin defects in embryos. Those data will shed some light on the linkage of unbalanced 45 copper homeostasis with specific gene promoter methylation and signaling transduction as well as the 46 resultant neurological development defects and diseases.47 48
Author summary 49In this study, we first unveiled that copper induced central neural system (CNS) myelin defects via 50 3 down-regulating Wnt/Notch-hoxb5b signaling, and parallel with hypermethylating promoters of genes 51 fam168b/pou3f2 and their subsequent down-regulated expression. Additionally, we unveiled that 52 fam168b/pou3f1 and hoxb5b axis acted in a seesaw manner during fish embryogenesis. Genetically, we 53 unveiled that copper was trafficked to mitochondrion via cox17 then led to the down-regulation of 54 Wnt&Notch-hoxb5b axis, and was trafficked to trans-Golgi network via atp7b to induce the 55 hypermethylation and the down-regulated expression of pou3f1/fam168b genes, making joint 56 contributions to myelin defects in embryos.