Mechanisms Of Copper-Induced Zebrafish Myelin And Axon Defects

Copper?Cu?is an essential catalytic and structural cofactor in many enzymes,and is involved in a variety of vital biological processes.Unbalanced copper homeostasis causes dysfunctional locomotor behavior in fishes,and is associated with neurological development defects and diseases.However,the underlying molecular mechanisms are still limited.In this study,dysfunctional movement and neural defects were observed in Cu²⁺stressed zebrafish embryos.Microarray data revealed that copper down-regulated nervous system genes include myelin and glial cell,axon guidance.Myelin and axon are essential for communication between neuron and a gland or muscle cell.Moreover,defects of myelin and axon are common pathological features in neurodegenerative diseases.Therefore,the detailed molecular characters of CNS myelin and axon defects were detected by microarray,WISH and q RT-PCR analysis.By microarray and the whole genome methylation sequencing analysis,we identified the crucial regulators in copper induced myelin and axon defects.Subsequently,loss/knockdown-and gain-of function experiments were performed to address the role of them in copper induced myelin and axon defects.Furthermore,cox17^-/-,atp7a^-/-,and atp7b^-/-copper transport mutants were used to verify the correlation of copper trafficking deficiency in specific organelle and the occurrence of developmental myelin defects in copper stressed embryos.This study revealed the molecular characteristics and potential regulatory mechanisms of myelin and axon defects in copper stressed zebrafish embryos.The main results are as follows:1. Cu²⁺induces CNS myelin and axon defects in zebrafishCopper induced delayed hatching,dysfunctional movement,and myelin and axon defects in zebrafish.By transmission electron microscopy?TEM?detection,Cu²⁺stressed larvae exhibited significantly thinner and uncompacted myelin in the spinal cord.In addition,significantly reduced expression of mbp and olig2 in the spinal cord were observed in Cu²⁺stressed embryos by WISH and q RT-PCR.Those data indicate copper induced CNS myelin and axon defects.2. Cu²⁺induces CNS myelin and axon defects by down-regulating Wnt&Notch-hoxb5b regulatory axis in zebrafish embryosMicroarray and WISH data showed that hoxb5b specifically exhibited significantly reduced expression in Cu²⁺stressed embryos.By loss/knockdown-and gain-of function,combined with RNA-seq analysis,we investigated the crucial role of hoxb5b in Cu²⁺induced myelin and axon defects.Firstly,knockdown of hoxb5b embryos exhibited similar phenotype defects with Cu²⁺stressed embryos.WISH and q RT-PCR analysis data revealed that loss/knockdown-of-function of hoxb5b phenocopied the myelin and axon defects which observed in Cu²⁺stressed embryos.Moreover,ectopic expression of hoxb5b partially rescued delayed hatching,and the myelin and axon defects in Cu²⁺stressed embryos.In addition,microarray and q RT-PCR data showed that Wnt and Notch signaling were reduced in Cu²⁺stressed embryos.Activation of Wnt and Notch signaling rescued hoxb5b expression,and myelin and axon in Cu²⁺stressed embryos,suggesting Wnt&Notch-hoxb5b axis mediated Cu²⁺induced myelin and axon defects.3. DNA methylation of fam168b/pou3f1 and their down-regulated expression partially contributes to Cu²⁺induced CNS myelin defects in zebrafish embryosThe whole genome methylation level was examined to unveil the potentially epigenetic mechanisms underlying Cu²⁺induced myelin and axon defects.Bisulfite sequencing data revealed 26 hyper methylated genes in Cu²⁺stressed embryos.Among them,genes pou3f1,pou3f2 and fam168b,which were associated with myelin and axon,were hyper methylated in the promotor domain,but down-regulated in transcript level.WISH data revealed that fam168a and fam168b exhibited similar expression patterns during embryogenesis,and loss/knockdown-of-function of fam168a or fam168b both induced similar developmental defects,transcriptional profiles and gene expression patterns,as observed in pou3f1 morphants.Additionally,loss/knockdown of fam168b,fam168a,or pou3f1 induced myelin and axon defects,similar to their defects in Cu²⁺stressed larvae.And ectopic expression of fam168a/fam168b/pou3f1 partially rescued the myelin and axon defects in Cu²⁺stressed embryos,suggesting hypermethylation of fam168b/fam168a/pou3f1 and their down-regulated transcription contributed to copper induced CNS myelin defects.4. Hypermethylation of fam168b promoter and its transcriptional regulation in Cu²⁺stressed zebrafish embryosAnalysis of promoter activity revealed that deletion of the hypermethylated locus in fam168b promotor induced significantly down-regulated transcriptional activity,suggesting the hypermethylated locus are required and pivotal for fam168b transcriptional activation.Additionally,significantly reduced expression of fam168b was observed in Cu²⁺stressed larvae from 24 hpf till to 96 hpf,and its promotor exhibited significantly hypermethylation from 96 hpf,suggesting regional methylation could be a secondary consequence of changes in transcriptional complex and chromosome DNA structure5. The crosstalk between hoxb5b regulator and hypermethylated genes of fam168a/fam168b/pou3f1WISH and q RT-PCR were used to test the crosstalk between the two mediators,Wnt&Notch-hoxb5b and fam168a/fam168b/pou3f1 transcriptional factors.The significantly increased expression of pou3f1,fam168a,and fam168b was observed in both hoxb5b morphants and hoxb5b-/-mutants.Meanwhile,hoxb5b exhibited significantly increased expression in pou3f1,fam168a,or fam168b morphants,and combined down-regulating two signaling induced CNS myelin defects,indicating fam168b/fam168a/pou3f1and hoxb5b axis acted in a seesaw manner during fish embryogenesis,and copper induced myelin defects.6. Cox17 and atp7b mediate Wnt&Notch-hoxb5b axis and hypermethylated genes of pou3f1/fam168a/fam168b in Cu²⁺stressed zebrafish embryosCopper transport cox17^-/-,atp7b^-/-,and atp7a^-/-mutants were applied in this study.WISH and q RT-PCR data revealed that pou3f1,fam168a,or fam168b all exhibited significantly reduced expression in cox17^-/-larvae after copper stimulation,and significantly down-regulated expression of pou3f1 but no changed expression of fam168a and fam168b was observed in Cu²⁺stressed atp7b^-/-larvae.Additionally,hoxb5b exhibited significantly reduced expression in Cu²⁺stressed atp7b^-/-mutants but not in Cu²⁺stressed cox17^-/-mutants,Those data demonstrated that the epigenetic methylation of pou3f1/fam168a/fam168b in Cu²⁺stressed cox17^-/-embryos,but the down-regulated Wnt&Notch-hoxb5b axis in Cu²⁺stressed atp7b^-/-respectively mediated the down-regulated expression of myelin genes in Cu²⁺stressed mutants.