Dissection The Role Of MiR-202 In Zebrafish Embryogenesis

MiR-202 is a member of the let-7 family which associate with cell differentiation,it is specific to and highly conserved in vertebrata,and play an important role in many physiological and pathological processes.In recent years,research about miR-202mainly focused on two major aspects,one is that as a tumor suppressor,it was deregulated in multiple cancerous transformations;and the other is that it is predicted to be involved in gonad development.Although it has been reported that miR-202played a role in regulating cell proliferation and differentiation,its role in embryo development is unknown.The early embryogenesis is a complicated process including cell proliferation,cell differentiation,cellular movement,germ layer formation and organogenesis.It needs the cooperation between multiple signaling pathways to regulate a normal embryonic development.Zebrafish as a model organism of vertebrates has many advantages in investigating the embryogenesis,such as its high fecundity,shorter life cycle,external fertilization and development and transparent embryo.During the early development of zebrafish,the mid blastula transition and the epiboly stage which initiate coordinated cell movement and start the expression zygotic gene are especially important,but the mechanism of its regulation remains unclear.In previous study,we found that inhibition of miR-202-3p by anatogmir injection in zebrafish embryos led to developmental processes halted and finally death at the late blastula transition stage.We further deleted the miR-202 locus by the CRISPR-cas9system.While the miR-202 homozygotes showed same termination of embryogenesis in the embryos injected with miR-202-3p antisense.We preliminary confirmed that miR-202 is required for zebrafish embryonic development.Based on previous study,we further explored the specific function of miR-202 in zebrafish embryogenesis and its mechanism.We found miR-202-3p is in low abundance in early stages but present in zebrafish oocyte at high level.Zygotic expression of miR-202-3p at the 3.5 hpf coincided with the timing of the occurrence of the abnormal phenotype when mir-203-3p was reduced by deletion.By observing the phenotype and genotyping the F2 offspring of miR-202 heterozygote zebrafish,from the embryo to adult zebrafish,we found that miR-202 homozygotes present drastic developmental abnormalities like disordered blastomere or developmental delay,especially in 3.5hpf the mid blastula transition to gastrula,such embryos eventually dissolved in the next few hours.While the miR-202 heterozygotes develop without detectable defects.There are no homozygotes exist in all F2 adult zebrafish,proved that homozygous miR-202 was lethal in developmental stages.To explain the onset of lethal phenotype of miR-202 homozygotes,we conducted transcriptome comparison and proteome comparison among the homozygous and wildtype embryos collected at 3.5 hpf.Transcriptome analysis showed that over 400genes were identified to be differentially expressed?DEGs?between the miR-202-/-embryos and the miR-202+/-or miR-202+/+embryos,according to the RNA changes at normal and abnormal conditions at the stage from 0hpf to 3.5hpf,we divided the DEGs into three dysregulation cases:insufficient degradation?ID?,overexpression?OE?and insufficient induction?II?in the miR-202-/-embryos.Such DEGs within which the pathways involving apoptosis,ribosome biogenesis,oxidative phosphorylation,cellular interaction,multiple signaling pathways are enriched.Proteome comparison showed ribosome proteins were reduced in the miR-202-/-embryos.We conclude that deletion of miR-202 pre-mature turned on critical gene and shifted multiple developmental pathways in the blastula stage.Further investigation showed a premature apoptosis of blastomere,lower rate of protein synthesis and week cell adhesion of miR-202^-/-embryos in the blastula stage.We propose that abnormal upregulation of apoptosis related genes in the blastomere by miR-202 deletion leads to the onset of apoptosis,which reduced ribosome activity and the rate of protein synthesis,additional,with the induce of ROS and weaken the cell adhesion,finally lead to the stagnant of embryo development.This work suggested that miR-202 is essential and involved in multiple signaling pathways important for zebrafish embryogenesis.We preliminary predicted the regulatory function of miR-202 in zebrafish early development.This research is supportive to achieve more information of molecular mechanism of miR-202.