During mammalian preimplantation development, the transition from morula to blastocyst is a critical biological event. This process involves polarization and initial specification of lineages, regulated by various transcription factors that have been extensively studied in mice. Our single-cell RNA sequencing analyses revealed that TEAD3 is specifically expressed in the trophectoderm cells of bovine preimplantation embryos, unlike in mice. The objective of this study is to determine the functional role of TEAD3 in bovine preimplantation development. While TEAD3 knockdown does not affect blastocyst formation in cattle, embryos fail to progress to the blastocyst stage when both TEAD3 and TEAD4, another member of the TEAD family, are disrupted using RNA interference and base editing techniques, respectively. This finding suggests a redundant role for TEAD3 and TEAD4 in preimplantation development in cattle. RNA sequencing analysis identified dysregulation of 215 genes, with 53 genes upregulated and 162 genes downregulated. Notably, we observed a reduction in the expression of trophectoderm-specifier genes KRT8, KRT18, and EZR, as well as HIPPO signaling pathway components. Immunofluorescence analysis further revealed that the protein expression levels of KRT8 and EZR were significantly decreased. Importantly, the initial expression of trophectoderm lineage-specific factors such as TFAP2C and GATA3, as well as the inner cell mass lineage-specific transcription factor OCT4, remained unaffected. This contrasts with the role of TEAD4 in directly regulating trophectoderm lineage specification in mice. Thus, our studies demonstrate that TEAD3 and TEAD4 play essential and redundant roles upstream of TE fate decisions during preimplantation development in cattle.
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