The Localization And Function Of PI4KB In Mouse Embryonic Development

Recombinant phosphatidylinositol 4-Kinase beta（PI4KB）is a member of the phosphatidylinositol 4 kinase（PI4K）family,primarily localized in the Golgi.PI4KB mediates PI phosphorylation to form PI4P through its kinase structural domain,which regulates nonvesicular cholesterol and ceramide transport,protein glycosylation,vesicle transport and cytoplasmic division.PI4P also acts as a precursor for PI（4,5）P₂ and PI（3,4,5）P₃,which are involved in regulating cellular pathways such as mediating normal lysosome outgrowth and efflux.PI4KB binds with ACBD3 and viral 3A proteins to co-mediate viral protein synthesis.PI4KB also binds with PI4KB-interacting proteins to enhance the stability of PI4KB proteins and mediate the transport of substances.Currently,most of the research on PI4KB has focused on its binding with the ACBD3 protein and viral proteins to mediate viral replication.Embryonic development is the beginning of life for multicellular organisms.For mammals,it begins with the zygote,which undergoes early oogenesis to form morula.Then the cells begin to differentiate to form the blastocyst,which eventually develops into a complete individual after the embryo has bedded down and undergone continuous division and differentiation.PI4KB is involved in embryonic development,and some studies have reported abnormal development of multiple organs in Pi4kb phomozygous(Pi4kb^-/-)zebrafish and death at 5-14 dpf.In a human family PI4KB gene mutation analysis,it was found that family members who carry the PI4KB mutation gene had abnormal inner ear development.Therefore,PI4KB may have an important role in mammalian embryonic development,and deletion of PI4KB may have a lethal effect on embryonic development.Mice are model organisms for the study of mammalian life activities.In the process of breeding Pi4kb heterozygous(Pi4kb^+/-)mice,we found that Pi4kb^-/-mice were created,so PI4KB deletion causes mice to die during embryonic development.However,the lethal time of Pi4kb^-/-embryos and the function of PI4KB in embryonic development are currently unknown.Therefore,the whole genome amplification,embryo immunofluorescence,microinjection,and inhibitor treatment were used to study the lethal time of Pi4kb^-/-embryos and the localization and function of PI4KB in mouse embryonic development.Firstly,this study investigated the lethality time of Pi4kb^-/-mouse embryos by whole genome amplification and genotype identification of mouse tissues.It was found that Pi4kb^-/-embryos survived through the hatching blastocyst stage and the surviving Pi4kb^-/-embryos were not different with their littermates Pi4kb^+/-and Pi4kb-not knocked out(Pi4kb^+/+)embryo,morphologically.It was found that Pi4kb^-/-embryos survived through the hatching blastocyst stage,and the surviving Pi4kb^-/-embryos were not significantly different from their littermates,Pi4kb^+/-and Pi4kb-not knocked out(Pi4kb^+/+)embryos,morphologically.E 9.5 Pi4kb^+/-mice still have a similarly high proportion of empty ovules as E 8.5 mates.At this point,the diameter of the embryo bundle in which the empty ovules are located is significantly smaller than that of the bundle with E 9.5 embryos.All surviving embryos of E 8.5 and E 9.5 were identified as Pi4kb^+/-and Pi4kb^+/+embryos.Therefore,it is suspected that Pi4kb^-/-embryos died before E 8.5 and ovule development ceased after death.E 5.5-E 8.5 immunohistochemical results for the embryo package PI4KB showed darker colouration in the epiblast starting at E 7.5 and darker colouration in the neural tube at E 8.5.Therefore,it is presumed that E 8.5 empty ovules were filled with absorbed Pi4kb^-/-embryos and that the embryos were lethal around E 7.5.Secondly,we examined the m RNA expression level and localization of PI4KB during preimplantation embryonic development by immunofluorescence,q PCR,and microinjection.It was found that the expression of PI4KB showed dynamic changes in each period of the preimplantation embryo,with less expression during the MⅡand fertilized egg periods,hardly any expression during the two-cell period due to the degradation of maternal m RNA.During the four-cell period,there was a significant increase in expression,with two expression peaks in the four-cell and mulberry embryos.In preimplantation embryos,PI4KB fluorescence signals from zygote to the morula are clustered and granular in the perinuclear and cytoplasmic areas and distributed at the cell membrane cortex;at blastocyst stage,PI4KB is clustered on the perinuclear side;at cytokinesis,PI4KB is clustered around the chromatin;at cytoplasmic division,PI4KB is localized in the perinuclear area,between the two cells and at the plasma membrane of the division furrow,and PI4KB is partially co-localized with the Golgi marker molecule TGN46.Therefore,it is hypothesized that PI4KB may be involved in Golgi-related functions.We used PI4KB inhibitors to treat embryos and found abnormal cytoplasmic division of embryonic cells during two-to four-cell development,the appearance of multinucleated embryonic cells and a decrease in the developmental rate of mulberry to blastocyst embryos.Therefore,it is hypothesized that PI4KB may be involved in the stable contraction of the contractile ring during cytoplasmic division and replenishment of the plasma membrane at the division furrow as well as Golgi-mediated vesicle transport during the development of morula.In summary,this study found that the mouse embryos with Pi4kb knockouts were lethal around E 7.5.PI4KB is involved in the regulation of cytoplasmic division and blastocyst development through the regulation of Golgi-associated functions during preimplantation embryonic development.However,the cause of embryonic lethality needs to be confirmed by further research.This study lays the foundation for the elucidation of the mechanism of PI4KB in embryonic development and has important theoretical and practical implications for the problem of fetal abortion caused by PI4KB protein deficiency.