Role of OCT4 during preimplantation development of bovine embryos

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Evidence in literature suggests substantial differences in preimplantation development of bovine embryos to the extensively studied mouse model. Differences include the interaction of OCT4 and CDX2 in the trophectoderm as well as the role of FGF/MAPK signaling during the second lineage differentiation of the hypoblast. Enhanced knowledge of the mechanisms and their regulation involved in the first lineage differentiations during preimplantation development in bovine, as a model complementary to mouse, would increase our overall understanding of conserved and species-specific mechanisms of early lineage specification and selective maintenance of pluripotency during mammalian preimplantation embryo development. With OCT4 being involved in both regulation of pluripotency and differentiation, deciphering its function in bovine is of special interest. Therefore, using adult fibroblasts with a CRISPR/Cas9 induced knockout of OCT4 and somatic cell nuclear transfer (SCNT), we aim to elucidate the role of OCT4 during bovine preimplantation development. We specifically hypothesize, that the involvement of OCT4 during the second lineage differentiation differs between bovine embryos and published mechanisms in the mouse. Furthermore, we suggest that embryos from somatic cell nuclear transfer reflect the principle mechanisms of early lineage development observed in fertilized embryos and that loss of OCT4 in one of the two aggregation partners in embryonic chimeras induces bias in cell lineage allocation. Our work program will include immunofluorescence on markers specific to the cell lineages in the early embryo, i.e. GATA6 for hypoblast, NANOG for epiblast and CDX2 for trophectoderm, and transcriptome analysis by RNA sequencing. To confirm findings from embryos produced via SCNT, we want to produce OCT4 KO embryos through direct injection of CRISPR/Cas9 into in vitro fertilized zygotes. Additionally, we aim to elucidate the second lineage differentiation and the role of OCT4 during by transfer of embryos to recipient cows at day 7 and their recovery at day 12. This is to our knowledge the first study on the function of OCT4 during bovine preimplantation development using a reverse genetics approach and the first comprehensive investigation of the second lineage differentiation in bovine embryos.