PATJ And MPDZ Regulate Trophectoderm Lineage Specification In The Mouse Preimplantation Embryo

Cell polarity and ICM/TE lineage specification of mouse preimplantation embryo are essential for subsequent development.However,in the ICM/TE specification event,the exact signal transduction mechanism of how cell polarity determines the fate of the lineage remains unclear.We studied the role of Patj/Mpdz in early embryo development to explore the relationship between cell polarity and lineage fate.Firstly,RT-PCR,real-time quantitative PCR and immunocytochemistry were used to detect the expression and localization patterns of CRB polarity protein complex in mouse preimplantation embryos.The results showed that CRB3 began to express in 2-cell embryos and increased in 8-cell embryos;PALS1 was expressed in different degrees at each stage of preimplantation embryos,but there was no significant difference;PATJ was moderately expressed from zygote to 4-cell embryos and increased in 8-cell embryos;MPDZ began to express in compact 8-cell embryos and expressed at a low level.From the view of protein localization,the components of CRB polatity protein complex did not show polarity localization before compact 8-cell embryos and evenly distributed in all blastomeres of the whole embryo.However,from compact 8-cell embryos,CRB polarity protein complexes began to show polarity distribution.CRB3 and PALS1 were located on the apical membrane in outer cells.PATJ/MPDZ was located on the TJ in outer cells.These express patterns suggest that the polarity distribution of CRB polarity protein complexes may be involved in the regulation of embryo development and lineage differentiation.Secondly,we specifically knockdown the expression of Patj/Mpdz gene by sh RNA.The results showed that knockdown of Patj gene could restrain blastocyst formation,but nearly half of the embryos could develop normally.At the same time,knockdown of Patj/Mpdz gene could restrain 72.7% of the embryos from developing into blastocysts.and even if they developed into blastocysts,these embryos could not from trophoblast outgrowth normally.These results suggest that knockdown the expression of Patj/Mpdz gene through sh RNA can effectively reduce blastocyst formation rate.Patj/Mpdz may play an important role in blastocyst formation.Thirdly,the mechanism of blastocyst development blockade by knockdown of Patj/Mpdz gene was further explored.CRB polarity protein complex,cytoskeleton,E-cadherin and ZO-1 were stained by immunocytochemistry and cytoskeleton staining.The results showed that the knockdown of Patj/Mpdz gene resulted in the disorder of ZO-1 localization,CRB polarity protein complex and phalloidin,but did not affect the localization of E-cadherin.It can be seen that knockdown Patj/Mpdz gene does not affect the normal compaction of embryo,but destroys the apical-basal polarity,cytoskeleton and TJ,which ultimately leads to the failure of blastocyst development.AbstractFinally,the localization of YAP was tracked by immunocytochemistry to indicate the regulation of Hippo pathway.The results showed that the knockdown of Patj/Mpdz gene resulted in the disorder of YAP localization.The phosphorylation of YAP in both outer and inner cells,which resulted in the weakening of the expression of CDX2,the specific transcription factor of TE,and eventually led to the loss of epithelialization characteristics in outer cells.To sum up,Patj/Mpdz regulates the differentiation of ICM/TE lineage in preimplantation embryos by stabilizing embryo polarity,cytoskeleton and TJ via Hippo pathway.