Studies On Porcine Early Development And The Mechanisms Of Trophoblast/Inner Cell Mass Differentiation

During pre-implantation development, the mammalian embryo develops from a zygote to a three dimensionally structured blastocyst; with trophectoderm(TE) and an inner cell mass(ICM). TE cells are differentiated cells with epithelial features, whereas ICM cells are undifferentiated pluripotent cells, which generate all of the embryonic body and part of the extra-embryonic tissues. Although the morphogenesis and molecular mechanisms of TE/ICM segregation in the mouse have been defined clearly, it is still unclear in other mammals. Recently, some results indicated that these early differentiation processes might be not conserved between mouse and pig, thus the model of mouse TE/ICM segregation could not take place of all the mammalian.In mouse, specification between TE and ICM lineages depends on the reciprocal inhibition between two lineage-specific transcription factors, caudal-type homeodomain transcription factor(CDX2) and POU-domain transcription factor(OCT4)(Niwa et al., 2005; Loh et al., 2006; Chen et al., 2008; Kim et al., 2008). CDX2 initiates expression at the morula stage with cellular variability, and then it gradually eliminated from inner cells. Finally, at the early blastocyst stage Cdx2 is specifically expressed in the TE and loses expression in the ICM(Dietrich and Hiiragi, 2007). In contrast OCT4 is highly expressed in all blastomeres until the mid-blastocyst stage, gradually losing expression in the TE. At the late blastocyst stage OCT4 could only be detected in the ICM(Dietrich and Hiiragi, 2007). In the mouse TE, OCT4 is directly repressed by CDX2 and, without OCT4, the pluripotent regulatory networks are not maintained(Niwa et al., 2005). In ICM cells, OCT4 cooperating with NANOG and SOX2 form a circuit to maintain the pluripotent regulatory network. It has been demonstrated that, in mouse embryo, CDX2 is regulated by the co-activator TEAD-YAP and YAP is the effector that regulated by Hippo and ROCK signalling.In pig, like the mouse, CDX2 expression can be detected specifically in TE cells, in contrast, OCT4 expression is maintained at high levels in both ICM and TE cells even at the late blastocyst stage after hatching. Such a long period of co-expression of OCT4 and CDX2 in TE cells indicates that CDX2 does not efficiently repress OCT4 expression during TE/ICM differentiation and OCT4 is not as sensitive to differentiation as that in mouse, or has altered function in the TE. Thus clearly in the pig, the molecular basis of TE/ICM specification and regulatory networks of these two lineages are ill defined and different from the mouse.In the pig, the progress and specific markers of TE/ICM specification are ill defined, this make it difficult to further study the molecular mechanisms of porcine TE/ICM specification. So we first try to selecte the marker of porcine TE and ICM. Immunofluorescence of CDX2 in porcine in-vivo and in-vitro embryos shows that CDX2 specific expresses in porcine TE cells and it initiates expression after the blastocyst stage, which is late than that in mouse embryo. Then we examine expression pattern of pluripotency factors, including SOX2, REX1, SALL4, ESG1, NANOG, TBX3, LIN28, KLF2 and KLF5, in porcine blastocysts. We found that SOX2 is a faithful pluripotent marker that anchored to the pluripotent cells including embryonic part cells, ICM cells and newly EPI cells along with developmental progress. Analysis of spatio-temporal distribution of SOX2 and the TE marker CDX2 revealed an exclusive expression pattern in D6 blastocysts. These results demonstrate SOX2 is the marker of pluripotency during porcine early development. Based on the results of in-vivo and in-vitro embryos, we propose a model of dynamic changes at the molecular level during porcine TE and ICM differentiation, which are different from mouse.It has been demonstrated that FBS could improve hatching and cell number of porcine in-vitro blastocyst. However, FBS is a mixture with unclear components and the influence of FBS on porcine early embryonic development on the molecular level is also unclear. This restricted the application of FBS on the culture of porcine in-vitro embryos. After have known the molecular basis of porcine TE/ICM differentiation, we further evaluate the influence of FBS on porcine TE/ICM differentiation by using CDX2 and SOX2 as TE and ICM marker, respectively. We found that FBS could increase hatching rate and cell number of porcine D7 blastocyst. Immunofluorescence of CDX2 and SOX2 shows that FBS could increase porcine ICM cell number and cell rate, what‘s more we find that FBS could improve the ablilty of ICM cells to undertake a further differentiation. These results demonstrated that FBS make porcine in-vitro embryos much more like in-vivo embryos.As we have demonstrated that CDX2 initates its expression in porcine embryo is later than that in mouse, we compare the expression of CDX2 between porcine and mouse embryo with similar cell number. The results show that CDX2 initates expression at porcine blastocyst stage, whereas it initates expression at mouse morula stage. So we further detect expression of CDX2 upstream regulator, YAP, And we find that nuclear location of YAP always co-happen with CDX2 expression. Then we use Y27632 to block ROCK signalling, which could further prevent YAP nuclear location. After adding Y27632 into the culture medium, we find that expression of CDX2 is prevented in D7 porcine blastocyst, thus expression of CDX2 in porcine embryo is also regulated by the ROCK-YAP signalling. As it has been demonstrated that ROCK signalling regulate YAP activity by maintaining the polarity of mouse blastomeres, we detect the polarization progress of porcine embryo by examining expression of polarity marker P-EZRIN. Compared to mouse, polarization of porcine blastomeres could be first detected at morula stage, which could be clearly detected at 8-cell satge in mouse. And only some of the outer blastomeres in porcine morula and blastocyst are polarized. These results demonstrate that the delay of porcine CDX2 initating expression may because of the different model and progression of polarization between porcine and mouse embryo. And this lay the foundation for us to further study the specific mechanism of porcine TE/ICM differentiation.As we have demonstrate a long period of co-expression of OCT4 and CDX2 in porcine TE cells. This indicates that the ineration between CDX2 and OCT4 is different from that in mouse, and maybe the molecular mechanism of TE/ICM differentiation is also different between these two species. So we try to study the interation of CDX2 and OCT4 during porcine early development. The results show that over expression of CDX2 can compromises porcine embryonic development, and this phenotype could be rescue by over expression of OCT4, thus Oct4 loss is the main reason for CDX2 overexpression induced developmental arrest at cleavage stage. However, we find that CDX2 overexpression didn’t decrease OCT4 mRNA and protein level. Thus CDX2 represses OCT4 at the post-translational level rather than the transcriptional level. Further suudy find that CDX2 might dysfunctionalize OCT4 protein through competeing binding sites on chromosome, so it could promote OCT4 exporting from the nuclear and degradation. This model is quite different from that in mouse.Taken tegother, these results show the specific TE/ICM differentiation molecular basis and progression during porcine eraly development, add the model of mammalian TE/ICM differentiation, and provide a theoretical basis for further study the mechanism of porcine TE/ICM differentiation.