The Regulation Mechanism Of Tdgf1 In Lineage Specification Of Preimplantation Embryos And Maintenance Of MESCs Pluripotency

Preimplantation embryos undergo zygotic genome activation and lineage specification resulting in three distinct cell types in the late blastocyst.Embryonic stem cells(ESCs)derived from the inner cell mass of blastocysts have important values in the field of cell therapy,drug discovery,genes editing,and animal breeding.The molecular mechanisms underlying the preimplantation embryonic development are largely unknown in pigs and eattle due to limited methods and embryonic eells.This is one of the reasons for not obtained ESCs with the potency of germ line transmission in pigs and cattle.Therefore,it is essential to reveal the similarities and differences of the regulation mechanisms in preimplantation embryonic development between pigs,cattle and mice,which are helpful to set up human diseases and development models,and to provide theoretical basis for animal breeding,embryonic engineering and ESCs isolation.This study firstly compared the development of preimplantation embryos in mice,pigs and cattle at the single-cell level,Subsequently,we explored the regulation mechanisms of Tdgf1,which showed similar expression patterns,in lineage specification of mouse preimplantation embryos and maintenance of mouse ESCs pluripotency.Here,we sought to analyze an extensive set of regulators at the single-cell level to define the events involved in the development of the blastocyst and to compare the similarities and differences in mouse,porcine and bovine early embryonic development.Using a quantitative microfluidics approach in single cells,we firstly analyzed mRNA levels of 96 genes known to function in early embryonic development and maintenance of stem cell pluripotency in parallel in total of 1247 individual cells from mouse,porcine and bovine preimplantation embryos.The developmental transitions can be distinguished by distinctive gene expression profiles in mice,pigs and cattle,and we identified different genes expressed in the early and Iate developmental stages.The trophoblast(TE)and inner cell mass(ICM)lineages can be segregated in mouse,porcine and bovine early blastocysts.The TE,epiblast(EPI)and primitive endodern(PE)lineages can be segregated in mouse and bovine,but not in porcine late blastocysts based on the expression patterns of lineage-specific genes.Lineage-specific genes were highly expressed in mouse and bovine,but not in porcine morulae.Tdgf7 and Prdm14 were highly expressed in EPI cells of mice and cattle.These results indicated that the similarities and differences existed in the preimplantation embryonic development of mice,pigs and cattle.To investigate the function of Tdgf1,which was highly expressed in mouse and bovine EPI cells,further studies were carried out in mouse preimplantation embryos and mouse ESCs.Exogenous TDGF1 protein treated from the 4-cell stage can promote the formation of PE cell lineage.Tdgfl knockdown by siRNA cytoplasmic injection can promote EPI and inhibit PE cell lineage differentiation.Transcriptome sequencing analysis of Tdgf1 knockdown embryos revealed that the expression of EPI cell lineage marker genes increased,while the PE cell lineage-specific genes decreased.Moreover,a BMP signaling receptor,Bmpr2,and activators of MAPK signaling pathway were downregulated.At the same time,the expression of genes that inhibit the GI/S phase transition increased,and the expression of genes relating to the ATP synthesis and the electron transport chain in mitochondrial respiratory decreased.After Tdgf1 knockdown by shRNA interference,the morphology of ESCs clones changed from solid and compact to flat and loose.Cell cycle analysis showed that the proportion of Gl phase increased,while the proportion of G2 phase decreased.The ATP production and mitochondrial membrane potential of ESCs also decreased.Mass spectromelry and Co-IP revealed that TDGF1 can bind to SLC25AI,which is a citric acid carrier,and NDUFA4,which is a subunit of complex ? of the mitochondrial electron transport chain.These results suggested that Tdgf2 influenced the differentiation of EPI and PE cell lineages in mouse preimplantation enbryos,and was necessary for maintenance of pluripotency,cell cycle and mitochondrial function in ESCs.In summary,this study analyzed and compared the expression patterms of an extensive set of regulators at the single-cell level in mouse,bovine and porcine preimplantation embryos.We further explored the regulation mechnisms of Tdgf1,which is highly expressed in EPI cells,in lineage specification of mouse preimplantation embryos and maintenance of ESCs pluripotency.This study enriched the research on the regulation mechanisms of porcine and bovine preimplantation embryos,and deepened the understanding of Tdgf1 function,and also provided a theoretical basis for isolating ESCs in large animals.