Copper Induces Zebrafish Central Neural System Myelin Defects: the Regulatory Mechanisms in Wnt/Notch-hoxb5b Signaling and Underlying DNA Methylation

Unbalanced copper (Cu2+) homeostasis is associated with neurological development defects and diseases. However, the molecular mechanisms remain elusive. Here, central neural system (CNS) myelin defects and down-regulated expression of Wnt/Notch signaling and their down-stream mediator hoxb5b were observed in Cu2+ stressed zebrafish larvae. Loss/knockdown-of-function of hoxb5b phenocopied the myelin and axon defects observed in Cu2+ stressed embryos. Meanwhile, activation of Wnt/Notch signaling and ectopic expression of hoxb5b could rescue copper-induced myelin defects, suggesting Wnt&Notch-hoxb5b axis mediated Cu2+ induced myelin and axon defects. Additionally, whole genome DNA methylation sequencing unveiled that a novel gene fam168b, similar to pou3f1/2, exhibited significant promoter hypermethylation and reduced expression in Cu2+ stressed embryos. The hypermethylated locus in fam168b promoter acted pivotally in its transcription, and loss/knockdown of fam168b/pou3f1 also induced myelin defects. Moreover, this study unveiled that fam168b/pou3f1 and hoxb5b axis acted in a seesaw manner during fish embryogenesis, and demonstrated that copper induced the down-regulated expression of the Wnt&Notch-hoxb5b axis dependent of the function of copper transporter cox17, coupled with the promoter methylation of genes fam168b/pou3f1 and their subsequent down-regulated expression dependent of the function of another transporter atp7b, making joint contributions to myelin defects in embryos. Those data will shed some light on the linkage of unbalanced copper homeostasis with specific gene promoter methylation and signaling transduction as well as the resultant neurological development defects and diseases. Author summary In this study, we first unveiled that copper induced central neural system (CNS) myelin defects via down-regulating Wnt/Notch-hoxb5b signaling, and parallel with hypermethylating promoters of genes fam168b/pou3f2 and their subsequent down-regulated expression. Additionally, we unveiled that fam168b/pou3f1 and hoxb5b axis acted in a seesaw manner during fish embryogenesis. Genetically, we unveiled that copper was trafficked to mitochondrion via cox17 then led to the down-regulation of Wnt&Notch-hoxb5b axis, and was trafficked to trans-Golgi network via atp7b to induce the hypermethylation and the down-regulated expression of pou3f1/fam168b genes, making joint contributions to myelin defects in embryos.


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Additionally, compared with the WT larvae, hoxb5b morphants or hoxb5b -/mutants exhibited 119 significantly decreased expression in the CNS myelin markers mbp and plp1a at 96 hpf (Figs 2C, 2D 120 S2A1, and S2A2), identical to that in Cu 2+ stressed larvae. The olig2 promoter driven fluoresce was 121 observed to be significantly down-regulated in hoxb5b-MO injected olig2:dsRED transgenic embryos, 122 compared with that in the control embryos at 48 hpf ( Fig 2E). Furthermore, the length of each axon was 123 significantly reduced in hoxb5b morphants (Fig 2E6), which also phenocopied the defects observed in 124 Cu 2+ stressed embryos.  The microarray data showed that the expressions of Wnt and Notch signaling genes were reduced 131 in Cu 2+ stressed embryos (Figs 3A, S2B1 and Table S9), and qRT-PCR assays confirmed the down-132 regulated expressions of Wnt signaling(27) and Notch signaling genes (Fig S2B2) in Cu 2+ stressed 133 embryos. It has been reported that both Wnt and Notch signaling specified the oligodendrocyte fate (1, 134 28-30), and hoxb5b, is downstream of these two signaling pathways (31,32). In this study, both Wnt 135 agonist BIO and NICD notch3 mRNA partially rescued hoxb5b expression in Cu 2+ stressed embryos 136 separately (Figs 3B, 3C). WISH and qRT-PCR analysis exhibited the expression of mbp and plp1a was 137 recovered to nearly normal level in Cu 2+ stressed embryos co-exposed with BIO (Figs 3D, 3E).

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Additionally, BIO significantly recovered the down-regulated fluorescence level and the length of the 139 fluorescent axon to nearly normal level in Cu 2+ stressed olig2:dsRED transgenic embryos (Fig 3F).

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Under stress conditions, epigenetic DNA methylation has been reported to function importantly in 146 disease process and intergenerational inheritance (33,34). Thus, the whole genome methylation level in 147 Cu 2+ stressed larvae was examined to unveil the potential epigenetic mechanisms underlying Cu 2+ 148 induced myelin and axon defects. It has been unveiled that Cu 2+     Transcriptional profiles in fam168a and fam168b morphants were investigated by KEGG pathway 183 (Figs 5C1, S4D1 and Table S10, S11) and cellular component GO analyses (Figs 5D1, S4E1 and Table   184 S12, S13). They showed enrichment in the nervous system and synapse for the down-regulated DEGs, 185 identical to transcriptional profiles in pou3f1 morphants (Figs S4D2, S4E2, Table S14 and S15).

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Additionally, 85 genes in the nervous system ( Fig 5C2) and 8 genes in synapse ( Fig 5D2) were down-187 regulated and overlapped in the three fam168a, fam168b, and pou3f1 morphants. Meanwhile, 104 genes 188 in the nervous system ( Fig S4D3) and 8 genes in synapse ( Fig S4E3) were down-regulated and

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Myelin specification was further tested in atp7b -/embryos after copper stimulation. When

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ER stress antagonist PBA was used to further study the role of copper-induced ER stresses in copper-240 induced down-regulated expression of mbp, hoxb5b, and fam168a. No significant recovery was observed 241 in the expression of the three genes in Cu 2+ stressed embryos after PBA co-treatment ( Fig S6G).

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Cu 2+ has been reported to induce dysfunctional locomotor in zebrafish larvae(22), but the underlying 244 mechanisms are still poorly understood. In this study, Cu 2+ was revealed to induce uncompacted and 245 thinner myelin in the spinal cord, which was consistent with the observations in epb41l2 mutants with 246 dysfunctional locomotor behaviors(36).

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It is reported that mbp, a widely used marker for myelin(37), expressed in both the CNS and PNS

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This not only suggested that copper induced changes in the promoter chromatin structure and the down-    Table S1.      Table S4 and 379 cloned into pCS2 vector for synthesizing mRNAs. 5' unidirectional deleted mutants of fam168b promoter, 18 including -1672, -1414, -1240, -927, -623, and -284, were amplified using the primers shown in Table   381 S5 and cloned separately into pCS2-GFP vector and pGL3 vector. All constructs were verified by 382 sequencing.   Table S6.  Table S7. Whole-mount in situ hybridization (WISH) was performed as described previously(50, 53,

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Whole genome bisulfite sequencing (WGBS) has been performed in the control and the Cu 2+ 417 stressed embryos at 96 hpf, and 57 differential methylated genes (DMGs) were unveiled (35). In this 418 study, the regions for differentially methylated loci of the candidate genes such as fam168bbetween the 419 control group and Cu 2+ stressed group were used for bisulfite PCR to validate the results of whole-420 genome bisulfite sequencing. The target fragments were amplified using specific primers (Table S8)