Molecular Mechanisms Of Copper-Induced Developmental Defects In Retina/Intestine/Vessel In Zebrafish

Copper considered one of the most prominent and essential trace elements such exerts numerous critical physiological and biochemical functions that related to growth and development of the organisms;including activation of various enzymes,synthesis of ceruloplasmin,and biosynthesis of collagen.Therefore,the disorder of copper homeostasis is linked with many diseases and developmental defects,such as anemia and peripheral neuropathy.Copper(including Cu NPs and Cu²⁺)is widely used in many fields resulting in serious environmental pollution which will adversely affect fish and human health.Many studies have evaluated the effects of copper on aquatic organisms.For instance,our previous studies have revealed that copper cause malformation of zebrafish embryos,including shorter body length,smaller eyes,and impaired motor function.However,there are still few studies on the mechanisms involved in copper-induced embryonic developmental defects.In this study,we investigated the further effects of copper on the development of retina,intestine,and vessel in zebrafish embryos.H&E staining,transmission electron microscopy（TEM）,DEGs analysis,whole mount in situ hybridization（WISH）,real-time PCR（RT-PCR）,immunofluorescence（IF）,western blot（WB）and other research methods were applied in our study.Herein,we could explore the developmental defects of zebrafish retina,intestine,and vessel under copper stress and the molecular mechanism of copper regulation of these developmental defects.Meanwhile,two copper transport mutants were constructed(cox17^-/-and atp7a^-/-)to study the potential genetic and biological regulatory mechanisms of copper-induced zebrafish retinal and intestinal malformations.Our study provides a theoretical basis for defects and diseases development under the conditions of copper stress and copper homeostasis.The main findings of this paper are as following:1. Copper causes development defects in zebrafish retina and intestine by inducing oxidative stress and endoplasmic reticulum stressThrough phenotypic observation,H&E staining,and TEM,we found out that copper-stressed zebrafish embryos had a significant small eye deformities and intestinal malformations;RNA-seq revealed that copper stress could down-regulate the expression of retinal and intestinal related genes;WISH,RT-PCR and IF results revealed that copper had a significant effects on the expression of intestine and retina three-layer cell markers,especially cones/rod cell marker;Moreover,by detecting the cell division and apoptosis,it was found that copper caused apoptosis of retinal cells without affecting cell division,revealing that defects were mainly caused by apoptosis.Moreover,through TEM observation of zebrafish retinal and intestinal cells,we found that copper damaged the structure of mitochondria and ER.Furthermore,WISH,RT-PCR and IF showed that oxidative stress and ER stress marker were up-regulated in copper-stressed embryos.We also revealed that retinal and intestinal defects caused by copper stress could be significantly restored by the addition of ER stress antagonists（PBA）and antioxidants（GSH and NAC）;these results suggest that copper causes embryonic development defects through ER stress and oxidative stress.2. Deficiency of COX17 and ATP7A affects retinal and intestinal developmental defects caused by copperTEM revealed that the retina of cox17^-/-mutant and the copper-stressed cox17^-/-possessed normal mitochondria and ER structures,whereas only slightly swollen mitochondria were observed in the cox17^-/-mutant intestinal cells.Both mitochondria and ER produced significant damage after copper stress in intestinal cells;WISH,RT-PCR,and IF found that compared with wild-type embryos,oxidative stress and endoplasmic reticulum stress levels in embryos of cox17^-/-mutants had no obvious differentiation.Furthermore,there was no significant change and only slightly up-regulated endoplasmic reticulum stress was observed in the copper-stressed cox17^-/-mutant.Through WISH and RT-PCR,we found that copper could only down-regulated the expression of intestinal marker gene without altering the expression of the retinal marker gene in cox17^-/-mutant.All results indicated that copper stressed retinal and intestinal defects resulted from the ER oxidative stress.Moreover,TEM revealed that the atp7a^-/-mutant and the copper-stressed atp7a^-/-mutant retina possessed normal mitochondrial and ER structures,while severe swelling and endometrial ablation mitochondria were observed in atp7a^-/-mutant intestinal cells and more severe swelling of the mitochondria but a structurally intestinal ER after copper stress.WISH and RT-PCR showed a significant increase in the oxidative stress and endoplasmic reticulum stress in atp7a^-/-mutant embryos compared to wild-type embryos,but the expression of the stress marker in the copper-stressed atp7a^-/-mutant embryos was down-regulated.Moreover,significant oxidative stress was observed in the copper-stressed intestine.This indicates that copper causes retinal defects requiring complete ATP7A transport function while oxidative stress produced by copper accumulation in the atp7a^-/-mutant intestinal tract is the main cause of developmental defects.3. Copper induces zebrafish vascular development defects via down-regulating foxm1expressionThrough vascular phenotype observation of zebrafish,we found that copper-stressed zebrafish embryos had an interstitial vascular blood flow obstruction;microarray analysis,RT-PCR and WISH results indicated that copper could down-regulate the expression of angiogenesis-related genes in embryos.By detecting the migration and proliferation of copper-stressed human venous endothelial cell lines（HUVECs）,we found that copper stress affected the migration and proliferation of HUVECs,suggesting that copper might cause vascular sprouting by inhibiting the migration and proliferation of endothelial cells.RT-PCR and WISH detection of vascular budding target genes validated this conclusion.Next,we observed the foxm1 MO zebrafish embryos,and the zebrafish embryos of foxm1MO had the same significant interstitial vascular blood flow obstruction.RT-PCR and WISH also detected the down-regulated expression of angiogenic genes.Copper may induce developmental defects in zebrafish blood vessels by down-regulating the expression of foxm1.