The Role Of Pcgf5a On The Early Embryo Development

Background and ObjectivePolycomb Group(PcG)proteins are the important type of epigenetic regulatory factor that play regulatory role in many biological processes.They form two polymers,PRC1 and PRC2,which can catalyze histone ubiquitination and methylation.As the core component of PRC 1,Pcgf proteins are widely involved in the development of nervous system,X chromosome inactivation,tumorigenesis and germ layer differentiation and regulate multiple development-related signal pathways.In mice and humans,the Pcgf family includes six members of Pcgfl-6,while zebrafishs contain six Pcgf genes(Pcgfl,4a,4b,5a,5b and 6).In zebrafish,Pcgf5a is expressed maternally and the expression gradually increases from embryo fertilization to 24hpf and gradually localizes expression in the brain and nervous system regions.In mice,the expression of Pcgf5 also increases during the differentiation of P19 cells into neural stem cells induced by Retinoic Acid(RA).Above results suggest that the Pcgf5a and Pcgf5 could occupy an important position in the early development of nervous system.In this study,Pcgf5a and Pcgf5 was knocked down and overexpressed respectively to explore its role in early neural development,reveal its regulatory role in vertebrate embryonic development and improve the understanding of early neural induction process.Research results can provide direction and basis for the prevention and treatment of related diseases.MethodsPcgf5a morpholine antisense oligonucleotides(MO)was microinjected to repress the expression of Pcgf5a and possible phenotypes were examined in Pcgf5a-deficient embryos.The whole mount in situ hybridization,qRT-PCR and acridine orange staining were used to detect the regulation of Pcgf5a on the early embryo development.In vitro,RA-induced neural differentiation of P19 cells was used as a model to simulate the early neural induction process.The technologies of knockdown,overexpression,qPCR and Western Blot were used to further verify the role of Pcgf5 in the early neural induction process and to explore its mechanism.1.The expression patterns of Pcgf5a and Pcgf5b in the early development of zebrafish embryosFrom 1 cell stage to 24hpf stage,the expression of Pcgf5a increased,while the expression of Pcgf5b decreased significantly.Therefore,Pcgf5a was selected as the research object of the follow-up experiment.2.The regulation of Pcgf5a on the early development of zebrafish embryosAfter Pcgf5a knockdown,the development of zebrafish embryonic neuroectodermal layer was affected.The brain size became smaller,the eyes developed retarded,the tail was curled and the body axis was shortened.In addition,the expressions of neural development-related genes(Sox2,Sox3,Foxgl and Pax6a)and eyes development transcription factors(Pax2a,Vsx2 and Rx1)were significantly reduced.3.The regulations of Pcgf5a on zebrafish embryonic cell proliferation and apoptosisThe results of in situ hybridization of Pcna and acridine orange staining revealed that the inhibition of Pcgf5a decreased the ability of cell proliferation in the nervous system region and promoted apoptosis of the brain and tail.4.The role of Pcgf5 in the process of RA-induced neural differentiation of P19 cellsCorresponding to the zebrafish experiment,the neural induction process of P19 cells was inhibited after Pcgf5 knockdown.qPCR results showed that the expression of key genes related to the embryonic pluripotency(Oct4 and Nanog)increased,while the expression of Pax6 and Sox2 decreased.Western Blot results also conformed the alterations of Oct4,Pax6 and Sox2 proteins.Meanwhile,the overexpression experiment of Pcgf5 promotes the neural differentiation of P19 cells.5.The effects of Pcgf5 on the three germ layers differentiation of P19 cellsThe Pcgf proteins are involved in regulating the differentiation of the three germ layers.qPCR results showed that the key gene related to the embryonic(Bra)was regulated by Pcgf5,but the alterations of the endoderm-related genes were not obvious.6.Exploration of the mechanism of Pcgf5 in the early neural induction processThe Notch signaling pathway plays an important role in maintaining the proliferation of neural stem cells.Western Blot results showed that Pcgf5 can positively regulate the expression of Notchl protein and promote the proliferation of neural stem cells.As the core component of the epigenetic regulation complex,Pcgf proteins can regulate target genes through epigenetic modification of histones.Further experiments found that the expression level of H3K18ac was decreased and the level of H3K4me3 was increased after Pcgf5 knockdown.Conclusions1.Pcgf5a affects the morphogenesis of nervous system and eyes and participates in the process of body axis formation.2.Pcgf5a regulates the proliferation and apoptosis of zebrafish embryonic cells.3.Pcgf5 is involved in the neural induction and mesoderm differentiation of P19 cells.4.Pcgf5 participates in the regulation of expression levels of H3K18ac and H3K4me3 and promotes the proliferation of neural stem cells.