Study On The DNA Methylation Mechanisms In Copper Stress Induced Zebrafish Embryonic Developmental Defects

As an important metal trace element in organisms,copper plays an indispensable biological function in the body.Copper is a cofactor of various enzymes and cell structure component,and is closely related to the body’s skeletal development,hematopoiesis,nervous system and immune system.Excess copper in the water body has a serious negative impact on the growth and development of fish,resulting in the degradation of germplasm resources.In addition,excessive copper accumulates in the human body through bioconcentration,causing human health problems.However,there are still few studies on the DNA methylation molecular and genetic mechanisms of excess copper in inducing embryonic developmental defects and the resulting in degradation of fish genetical characters.In this study,the model organism zebrafish（Danio rerio）was used as the research subject,and bioinformatics analysis of the transcriptome and DNA methylome of acute Cu²⁺stressed embryos was carried out.Furthermore,the cellular and molecular mechanisms of acute Cu²⁺stress induced defects in angiogenesis and lymphangiogenesis in embryos,and the developmental defects of F1 offspring embryos and sperm-carrying methylation modifications in adult zebrafish under chronic copper stress were further investigated.The main results are as follows:1.Acute Cu²⁺stress led to altered transcriptional and DNA methylation levels in zebrafish embryosUsing Ag NPs as a reference,the transcriptomes and methylomes in zebrafish embryos under acute stress of Cu²⁺or Ag NPs were compared and analyzed,and we found the common differentially expressed genes（DEGs）of embryos in response to both Cu²⁺and Ag NPs stress were significantly enriched in ion-relevant GO items such as calcium binding,iron binding,and blood circulation,etc.In addition,12 of the 15differentially methylated genes（DMGs）shared by embryos in response to metal（including Cu）stress were ion-relevant genes,and one of them was the lymphangiogenic factor ccbe1.Moreover,there was a negative correlation between the transcriptional expression of DMGs and the changes of methylation modification.2.Acute Cu²⁺stress impaired angiogenesis and lymphangiogenesis during zebrafish embryogenesisBy identifying the developmental phenotype of the hematopoietic system of Cu²⁺stressed zebrafish embryos,this study found that blood circulation in the intersegmental vessels of acute Cu²⁺stressed zebrafish embryos was blocked,and the expressions of angiogenesis factors amot12,p ERK1/2,and foxm1 were down-regulated.Through the knockdown/knockout and overexpression of the foxm1gene,it was found that the amot12-p ERK1/2-foxm1-mmp2/9 regulatory axis responded to acute Cu²⁺stress and inhibited angiogenesis.Moreover,the functions and regulatory mechanisms of acute Cu²⁺stress inhibiting cell migration and angiogenesis are highly conserved from fish to mammals.Furthermore,in acute Cu²⁺stressed zebrafish embryos,the thoracic duct,an important structure of lymphatic vessels,was partially absent.The expression of the lymphangiogenic factor ccbe1 was down-regulated,and acute Cu²⁺stress led to hypermethylation of the E2F7/8 binding site in the ccbe1 promoter region,while the expression of E2F7/8 protein was not significantly changed.Meanwhile,Ch IP-q PCR results showed that the enrichment of E2F7/8 in the ccbe1 promoter region was reduced.In conclusion,acute Cu²⁺stress impairs angiogenesis and lymphangiogenesis by inhibiting the amot12-p ERK1/2-foxm1-mmp2/9 regulatory axis and altering the methylation modification on the ccbe1 promoter,respectively.3.Chronic Cu²⁺stress in adult zebrafish led to altered DNA methylation of F0sperm and defective embryonic development of F1 offspringThis study identified developmental characteristics of F1 offspring embryos of chronic Cu²⁺stressed adult zebrafish,found that the F1 offspring embryos of chronic Cu²⁺stressed adult fish had developmental defects such as dysfunctional locomotor,cerebellum and microphthalmia,and found that male sperm carried genetic factors that led to embryonic developmental defects in F1 offspring.Significant dysregulation of marker genes in the nervous system,eye/retina,and intestine,and a large number of enriched neural-related GO terms were unveiled in F1 offspring.Meanwhile,whole-genome methylation analysis of F0 sperms and their fertilized F1 embryos of adult fish under chronic Cu²⁺stress revealed that a large number of DMGs（differential methylated genes）were expressed in the brain/central nervous system,eye/retina,liver/intestine,respectively.Further functional analysis of DMGs pmpcb,crebl2 and tab2 in sperm revealed that the differentially methylated regions played an important role in the expressions of their related genes.Additionally,the methylation change of pmpcb,crebl2 and tab2 in F0 sperms passed on to their fertilized F1embryos,which reprogramed the development of nervous system,eye/retina,and liver/intestine,respectively.These data suggested that chronic Cu²⁺stress led to changes in the methylome and transcriptome of F0 sperm of adult fish,which then led to methylome and transcriptome reprogramming of their fertilized F1 embryos and induced developmental defects in F1 embryos.