The Role Of The Effector Protein YAP Of HIPPO Signaling Pathway In Porcine Early Embryonic Development

The first lineage differentiation of mammalian embryos produces two distinct cell populations,trophectoderm cells and inner cell mass cells.The differentiation of trophectoderm epithelium is a process of epithelial differentiation characterized by several cellular events,such as the polarization of blastomeres,establishment of tight junction,formation of Na+/K+pump and H2O channel.The differentiation of trophectoderm epithelium is an important basis for compaction,blastocyst formation and the first segregation of embryonic cell lineage.The HIIPO signaling pathway regulates gene expression in multiple biological processes through activating its downstream effector YAP.Previous studies indicated that HIIPO signaling pathway is involved in the regulation of the first segregation of lineage during the preimplantation development of mouse,porcine and bovine embryos.Furthermore,YAP protein is essential for the embryonic genome activation in mice and the blastocyst development in cattle.However,the role of the effector YAP of HIPPO signaling pathway in the trophectoderm differentiation of porcine early embryos remains to be yet unclear.In the present study,the dynamic expression of YAP mRNA and protein was characterized.The effect of YAP knockdown on the development of porcine preimplantation embryos were further examined.These results will elucidate the function and regulatory mechanisms of HIPPO signaling pathway in the development of porcine early embryos.First,to determine the dynamic expression of YAP in oocytes and embryos,real-time fluorescence quantitative PCR(RT-qPCR)and indirect immunofluorescence(IF)were used to detect the expression of YAP..Results revealed that YAP mRNA was highly expressed from GV oocytes to 4-cell embryos,but decreased significantly from 8-cell embryos to blastocysts.YAP protein was expressed throughout porcine early embryos and its levels is peaked at 2-cell to 4-cell stage.YAP protein localizes atboth the nucleus and cytoplasmbefore 8-cell stagewhile it mainly localizes in the nucleus of outer cell of the embryo.In addition,2-cell,4-cell and day 5 blastocyst were treated with RNA polymerase Ⅱ inhibitor α-amanitin,respectively,suggesting that YAP is a maternal factor.Second,to investigate the role of YAP in porcine early embryonic development,siRNAtargeting YAP was designed and then injected into MII oocytes by microinjection,and then the injected oocytes were parthenogenetic activated and cultured.The results showed that knockdown of maternal YAP resulted in the arrest of most embryos at 8-cell to morula(44.78%Vs.97.77%),and the blastocyst rate was significantly decreased(55.22±2.78%Vs.2.23 ± 1.03%)compared to control group.Furthermore,analysis of YAP knocked down blastocysts revealed that the total cell number did not change,but the number of trophectoderm cells decreased(42.20±2.63%Vs.23.80±6.76%),the number of inner cell mass cells increased(11.80±0.73%Vs.22.20 ±2.46%),the ratio of inner cell mass to trophectoderm increased(0.28±0.02%Vs.1.18±0.22%).In addition,the embryo is treated with Verteporfin(VP),an inhibitor of the interaction between YAP and TEAD4.The results showed that VP treatment significantly reduced the blastocyst rate(48.90±4.40%Vs.17.80±4.50%)and the number of trophectoderm cells(34.67±5.05%Vs.19.50±2.43%)but increasd the number of inner cell mass cells(9.17±0.70%Vs.19.83±2.02%)and the ratio of inner cell mass to trophectoderm cells(0.37±0.11%Vs.1.32±0.28%).Finally,to further explore the effect of YAP knockdown on embryo lineage differentiation,YAP knocked down morula were collected,and RT-qPCR technique was used to detect the expression of genes associated with lineage differentiation and tight junction.The results indicated that the expression of most lineage differentiation-related genesincluding CDX2,OCT4,SOX2 and TEAD4,was significantly down-regulated,and the expression of tight-junction related genes was also significantly decreased,especially CLDN4,CLDN6,TJP1,TJP2 and OCLN.In addition,the expression of AQP3,ATP1A1,ATP1B1,ATP1B3 and ROCK2 genes related to the establishment of cell polarization and cavity formation was also severely alterd.Taken together,maternal YAP not only regulates the first cell lineage separation in porcine early embryonic development,but also regulates the formation of tight junction,sodium and potassium pumps,and water ion channels in the differentiation of porcine trophectoderm cells.