Oct4 is well established as a core regulator of pluripotency, yet emerging evidence points to an additional role in lineage specification during the exit from the pluripotent state. Although Oct4 expression has been observed in early mesodermal progenitors, its precise function in this developmental context remains unclear. To investigate this, we employed embryoid bodies (EBs) as a model of spontaneous differentiation that recapitulates key aspects of early embryonic development in vitro. In accordance with previous studies, reporter assay revealed a distinct temporal pattern characterized by the strong, transient co-expression of Oct4 and the early mesoderm-specifying marker gene Brachyury within a narrow developmental window, consistent with the Oct4 role in early mesodermal progenitors. We further examined the consequences of the Oct4 loss at early stages of this differentiation. Conditional knockout of the Oct4 gene resulted in a significant reduction in EB size and accumulation of cells in the G0/G1 phase, indicating a critical requirement for Oct4 in maintaining cell proliferation. Despite this defect, cells retained the ability to initiate multilineage differentiation, albeit with reduced expression of Brachyury and elevated expression of endodermal markers FoxA2 and Sox17. Interestingly, the formation of beating cardiomyocyte-like structures was also diminished following Oct4 loss and could not be rescued by simply increasing cell numbers. Taken together, these findings highlight an important Oct4 function in mesodermal differentiation, mediated through the maintenance of proliferative capacity of early mesodermal progenitors.

Embryo implantation is accompanied by the naïve-to-primed pluripotency transition in epiblast cells, making them receptive to external differentiation signals. In addition to this developmental program switch, implantation suggests that an anabolic boost is required for this process, as the embryo-uterine connection begins supplying the requisite nutrients. In this study, we show that the DNA-binding protein Pcbp1 plays a key role in intensifying amino acid metabolism during the priming of pluripotent stem cells. Knockout of the Pcbp1 gene leads to embryo growth arrest a few days after implantation. By modeling the naïve-to-primed pluripotency transition in vitro , we observe reduced proliferation and induction of apoptosis in cells deficient for Pcbp1. Using multi-omics approaches, we uncover a crucial role for Pcbp1 in driving a transcriptional burst of numerous genes involved in the import and the de novo synthesis of essential and conditionally essential amino acids. Pcbp1 deficiency is consequently associated with a slowdown in protein biosynthesis, explaining the early lethal phenotype of knockout embryos. Our findings thus uncover the molecular mechanisms underlying anabolic changes during the naïve-to-primed pluripotency transition and highlight the essential role of Pcbp1 in this process, also pointing to its functions in highly proliferative cells. Graphical

Protein degradation systems, such as auxin-inducible degron (AID) and dTAG-mediated degradation, have become essential tools in molecular biology. Their primary advantage lies in the rapid and efficient elimination of proteins, which facilitates the study of short-term molecular perturbations. However, these techniques remain underutilized for investigating protein function during the dynamic process of embryonic stem cell (ESC) differentiation. Here, we employed these methods to unveil the role of Nanog during definitive endoderm specification. We demonstrate that dTAG-inducible degradation is significantly more efficient than the AID system for Nanog elimination. Using this approach, we show that Nanog is a context-dependent regulator; while it maintains pluripotency in undifferentiated ESCs, it promotes definitive endoderm maturation during spontaneous differentiation. Our results highlight the advantages of targeted protein degradation for studying developmental transitions and provide new insights into the multifaceted functions of Nanog.