Igf2bp3 Regulates Early Embryonic Development In Zebrafish Through Forming Liquid-liquid Phase Separation With Buc

In the field of fish breeding,the quality of eggs depends on the nutrients stored in the mother’s body,including various proteins and maternal mRNA.During early embryo development,there is a key process called maternal-to-zygotic transition(MZT),which regulates organismal growth and development and involves the degradation of maternal RNA and activation of zygotic genes.As an important part of this process,the degradation of maternal mRNA regulates the smooth development of early embryos.The mechanism of maternal mRNA degradation requires further study and is an urgent scientific problem in embryo development.Due to the long period of sexual maturity in economic fish,this study explores the molecular mechanisms of early embryonic development using transgenic and gene editing technologies with zebrafish(Danio rerio)as experimental materials.Previous studies have shown that igf2bp3 is involved in the maternal-zygotic transition process,maintaining the stability of maternal mRNA and regulating early embryonic development.In vivo observations have shown that igf2bp3 interacts with buc and colocalizes at the cleavage furrow of early embryos.To further explore the functions of igf2bp3 and buc in early embryonic development,we combined transgenic and gene editing technologies.1.Buc transgenic is accompanied by delayed embryonic development and slow degradation of parental genes;buc mutants exhibit abnormal early embryonic development and rapid degradation of parental genes.To study the effect of overexpression of igf2bp3 and buc on early embryonic development,our laboratory constructed igf2bp3 and buc transgenic strains.Phenotypic observations showed that buc transgenic is accompanied by delayed embryonic development and slow degradation of parental genes.To investigate the effect of igf2bp3 and buc deficiency on early embryonic development,our laboratory constructed igf2bp3 and buc mutants.Phenotypic observations showed that buc mutants exhibit severe developmental abnormalities in early embryo developmental abnormalities in early embryos and rapid degradation of parental genes.Interestingly,buc mutants and buc transgenic embryos showed the same maternal mRNA expression as igf2bp3 mutants and igf2bp3 transgenic embryos.2.Studies on the Buc protein show that Buc-FL and BucΔC proteins separate from each other in vivo,and the generation of the separate domain results in changes in parental gene expression.In this study,the structural domains of the Buc protein were predicted using a prediction website,and the results showed that there is a Pr D-like structural domain in Buc that can undergo phase separation.Therefore,according to the prediction results,the protein was truncated accordingly,and the truncated front part of the protein is called Buc△C protein,the truncated rear part of the protein is called Buc△N protein,and the full-length protein with the entire structural domain is called Buc-FL protein.Injection of Buc-FL-GFP mRNA and Buc△C-GFP mRNA resulted in delayed early embryonic development and accelerated degradation of maternal mRNA.Injection of GFP mRNA and Buc△N-GFP mRNA did not result in abnormal early embryonic development or maternal mRNA degradation.In addition,it was found that Buc-FL and Buc△C proteins exist in a phase-separated state in vitro,and the generation of the separate domain results in changes in parental gene expression.3.The impact of the absence of RNA binding protein Igf2bp3 on the aggregation and flow properties of Buc,and the resulting abnormal early embryonic development due to the lack of this regulatory interaction.To understand the role of Igf2bp3 in the phase separation of Buc protein,this study was conducted in wild-type and igf2bp3 mutant embryos to examine the in vivo changes of endogenous Buc protein and exogenous Buc full-length and truncated proteins under different conditions during early embryonic development stages.Immunofluorescence assays were performed on wild-type and igf2bp3 mutant embryos at different developmental stages,and the changes of exogenous proteins in vivo were observed through microinjection of mRNA.These studies revealed that Buc protein aggregates in igf2bp3 mutant embryos were more dispersed and had smaller fluorescent protein regions compared to wild-type embryos.Phenotypic and maternal mRNA changes were also observed in igf2bp3 mutants.Therefore,it was concluded that deletion of Igf2bp3 affects the flow and aggregation properties of Buc protein,and the lack of this regulatory interaction leads to abnormal early embryonic development.In this study,we used transgenic and gene editing technologies to explore the mechanisms of early Buc regulation from the perspective of phase separation and demonstrated that Igf2bp3 regulates early zebrafish embryonic development by affecting Buc phase separation.This study contributes to improving embryo quality and to the development of aquaculture and genetic breeding.