Functional Analysis Of Atg7 And Beclin1 During The Larval-to-Juvenil Transition And In Adult Male Liver Metabolism Of Zebrafish(Danio Rerio)

On the basis of the cell mechanisms,macroautophagy,a natural regulated pathway disassembles unnecessary or dysfunctional cellular cargo orchestrated by more than 36 autophagy related-genes.Among those genes are atg7 and beclin1 which have been proved to play an important roles in regulating post-natal development in some mammals however their functions during zebrafish development still unedited.During this research,we focus on the functional roles of atg7 and beclin1 in null and heterozygous zebrafish strains using CRISPR/Cas9 gene editing technology.Firstly,to investigate whether autophagy plays a role during the larval-to-juvenile transition,atg7 and beclin1 zebrafish mutant lines were generated.Herein,we observed that both atg7 and beclin1 F2 null embryos died during the larval-to-juvenile transition because they were unable to cope with the metabolic stress after yolk absorption and fail to activate autophagy in response to nutrient restriction.Meanwhile,dramatic defects in the intestinal architecture and hepatic metabolic disequilibrium were observed in these mutants even though refeeding them,suggesting that autophagy not only important during yolk transition but also within food plenty.Treatment with rapamycin,an activator of autophagy,could effectively extend the survival time of both atg7 and beclin1 null zebrafish through lowering the metabolic rate while it could not activate autophagy in mutants via the canonical pathway.Our findings provided molecular evidence for the physiological,histological,and metabolic changes that occur during the transition process from the larval-to-juvenile stage and the chief role of autophagy on the body metabolism during these turning milestones that gives a clue to resolve the problem of high mortality in aquaculture.On the other side,beclin1 heterozygous adult males showed higher mortality percentage compared with their WT and atg7 heterozygous siblings.At 12-month-old males of beclin1+/-appeared with enlarged belly and curved bodies and hepatic tissues were modified into diffused cords that increased by 16-month-old with solid necrosis in about 90%of the beclin1+/-zebrafish.Almost no mortality occurred in beclin1+/-males before 6-month-old,whereas 40%and 75%of beclin1+/-males died at 12 and 16 months respectively unlike their atg7+/-siblings indicating that beclin1 has an independent identity to affect the whole body health and survival rate.Interestingly,beclin1+/-has a specific strategy to induce hepatic malignancy and creates a suitable microenvironment for tumorigenesis by mediating the downstream signals of PI3K/AKT pathway that responsible for cell growth.Briefly,beclin1+/-suppresses both autophagy and apoptosis that led to elevation of mutated tP53,induction of inflammation and proliferation pathways,and activation of glycolysis and lipogenesis processes and all together increase the risk of hepatic malignancy.Our results support the evidence that autophagy process is not just important during the cell fasting or starvation but also during food plenty.Blocking of autophagy pathway by knocking-out led to death at early embryonic development even though providing the embryos with external food supply after maternal yolk depletion.Moreover,disturbing autophagy genes that involved in PI3K/AKT pathway(such as beclin1)by haplotype mutagenesis has a prolonged or chronic effects as it permits the larvae survival but causes liver defects upon elder age.