Imprinting And Expression Regulation Analysis Of Qpct And Aqp1 Gene In Mice Placenta Development

Qpct(glutaminyl-peptide cyclotransferase)and Aqp1(Aquaporin-1)are located on mouse chromosome 17 and chromosome 6.The glutamyl cyclase encoded by Qpct is particularly important in the formation of ?-amyloid protein,the excessive accumulation of which is the cause of Alzheimer's disease.It was indicated that Qpct had an important inhibition effect on cancers.The protein encoded by Aqp1 is one of the aquaporin family,which function is transport water.Thus it is extremely important in the growth progress of organism.So far,the imprinting statuses and functions in embryonic development of Qpct and Aqp1 have not been reported.Therefore,the imprinting statuses of Qpct and Aqp1 in embryo and placenta development were identified by SNP that discriminated the two parental alleles.To reveal the potential functions of Qpct and Aqp1 in embryo and placenta development,their expression patterns and regulation mechanisms were studied.In addition,the function of Aqp1 was investigated further in the embryonic and placental development by Aqp1 knock-out mouse model.It aimed to reveal the important functions of Aqp1 in the mouse embryonic development.Imprinting statuses of Qpct and Aqp1 predicted by bioinformatics were analyzed firstly.By c DNA collection and SNP loci sequencing in mouse intraembryonic and extraembryonic tissues,it was detected that SNP loci of Qpct and Aqp1 were single-peak and maternally expressed only in mouse placentae.However,biallelic expressions of the SNP loci were detected in the embryonic tissues.Thus,Qpct and Aqp1 were placenta-specific maternally expressed imprinted gene in mouse.Then,spatiotemporal expression patterns and imprinting regulation mechanisms of Qpct were studied in mice.The expression characteristics of Qpct in embryos and placentae were analyzed by real-time PCR,in situ hybridization(section/whole-mount)and immunohistochemistry.The real-time PCR showed that Qpct expression in the blastocysts was relatively high and it increased gradually during the mid-late embryonic stage(E8.5-E15.5).Besides,during the mid-late placental stage(E12.5-E18.5),the expression of Qpct first rose then decreased and it was highest at E15.5.In situ hybridization indicated that Qpct was highly expressed in the brain,neural tube and otic vesicle at the mid embryonic stage.And it was expressed extensively at later embryonic stage,especially highly in brain,lung and liver.Meanwhile,immunohistochemistry showed that strong expression signals of Qpct were detected in decidua and the labyrinthine layer during the mid-late placental stage(E12.5-E18.5).According to the analysis,a Cp G island existed in promoter of Qpct.Methylation analysis and Ch IP experiment showed that the imprinting of Qpct was regulated by histone H3K4me3 instead of methylation of Cp G island which regulated Qpct expression in main embryo tissues.Last,spatiotemporal expression patterns,expression regulation mechanisms and functions of Aqp1 were studied in mice.The real-time PCR of Aqp1 suggested that Aqp1 expression increased gradually during the mid-late embryonic stage(E8.5-E15.5)and achieved the highest at E15.5.The expression of Aqp1 was higher in lung and liver while lower in brain and kidney at E15.5.Immunohistochemistry indicated Aqp1 was mainly expressed in decidua and the labyrinthine layer of E15.5 placenta.In bioinformatics databases,Cp G islands were not found in promoter of Aqp1,while high density CG sites were in promoter(region A)and exon 1(region B)of Aqp1.The results of methylation analysis indicated that the region A and region B regulated Aqp1 expression in embryo and placenta.By Aqp1 knock-out mouse model,the functions of Aqp1 were explored in embryonic and placental development.The results revealed that in Aqp1-/+ and Aqp1-/-mice,the placentae were enlaged,and embryonic and placental weight and the area ratios of the JZ and labyrinthine layer increased.The number and branching of blood vessels also increased in the labyrinthine layer.It was demonstrated that Aqp1 had a specific inhibitory role in the embryonic development by the regulation of the nutrients transportation from placenta during mouse pregnancy.In summary,the two identified novel placenta-specific imprinted genes Qpct and Aqp1 were ubiquitously expressed in the mouse embryonic and placental development.The modification of histone H3K4me3 resulted in the maternal expression and paternal silencing of Qpct.The methylation of the promoter(region A)and exon 1(region B)of Aqp1 regulated its expression in embryo and placenta.Simultaneously,Aqp1 had a significant inhibited role on the development of mouse embryo and placenta during pregnancy.