POUV is a relatively newly emerged class of POU transcription factors present in jawed vertebrates (Gnathostomata). The function of POUV-class proteins is inextricably linked to zygotic genome activation (ZGA). A large body of evidence now extends the role of these proteins to subsequent developmental stages. While some functions resemble those of other POU-class proteins and are related to neuroectoderm development, others have emerged de novo . The most notable of the latter functions is pluripotency control by Oct4 in mammals. In this review, we focus on these de novo functions in the best-studied species harbouring POUV proteins—zebrafish, Xenopus (anamniotes) and mammals (amniotes). Despite the broad diversity of their biological functions in vertebrates, POUV proteins exert a common feature related to their role in safeguarding the undifferentiated state of cells. Here we summarize numerous pieces of evidence for these specific functions of the POUV-class proteins and recap available loss-of-function data.

Embryonic stem cells (ESCs) are remarkable for the high activity level of ubiquitin–proteasome system—the molecular machinery of protein degradation in the cell. Various forms of the proteasome complexes comprising different subunits and interacting regulators are responsible for the substrate selectivity and degradation. Immunoproteasomes are amongst these forms which play an important role in antigen presentation; however, a body of recent evidence suggests their functions in pluripotent stem cells. Previous studies have established three consecutive phases of pluripotency, featured by epiblast cells and their cultured counterparts: naïve, formative, and primed phase. In this work, we report that immunoproteasomes and their chaperone co-regulators are suppressed in the naïve state but are readily upregulated in the formative phase of the pluripotency continuum, featured by epiblast-like cells (EpiLCs). Our data lay ground for the further investigation of the biological functions of immunoproteasome in the regulation of proteostasis during early mammalian development.

KH-domain PolyC-binding proteins perform many functions ranging from transcription regulation to alternative splicing. There are several evidences of their important role in embryogenesis and in embryonic stem cells (ESCs). ESCs are cultured counterparts of epiblast cells before implantation, and both cell types are in naïve state of pluripotency. We have previously shown that members of the KH-domain family—hnRNP-K, Pcbp1 and Pcbp2 proteins—are able to bind to the PolyC-sites within regulatory elements of the Pou5f1 gene that encodes the key pluripotency gatekeeper Oct4. In addition, early lethal phenotype of Pcbp1^–/– mouse embryos has been described in literature. In this study, we set to assess role of Pcbp1 in ESCs. To this end, we have obtained null-Pcbp1 ESCs. Contrary to previously observed by us lethal phenotype of hnRNP-K in ESCs, Pcbp1^–/– ESCs were viable, continued to express Oct4 and retained overall pluripotent properties. Therefore, the obtained data reveal no critical role of Pcbp1 in self-renewal of naïve pluripotent stem cells. Further studies should address whether this is the case during the transition of these cells to the primed state of pluripotency, as well as during the exit from this state due course of differentiation into the three germ layers.

Embryonic stem cells (ESCs) hold great promise for regenerative medicine thanks to their ability to self-renew and differentiate into somatic cells and the germline. ESCs correspond to pluripotent epiblast — the tissue from which the following three germ layers originate during embryonic gastrulation: the ectoderm, mesoderm, and endoderm. Importantly, ESCs can be induced to differentiate toward various cell types by varying culture conditions, which can be exploited for in vitro modeling of developmental processes such as gastrulation. The classical model of gastrulation postulates that mesoderm and endoderm specification is made possible through the FGF-, BMP-, Wnt-, and Nodal-signaling gradients. Hence, it can be expected that one of these signals should direct ESC differentiation towards specific germ layers. However, ESC specification appears to be more complicated, and the same signal can be interpreted differently depending on the readout. In this research, using chemically defined culture conditions, homogeneous naïve ESCs as a starting cell population, and the Foxa2 gene-driven EGFP reporter tool, we established a robust model of definitive endoderm (DE) specification. This in vitro model features formative pluripotency as an intermediate state acquired by the epiblast in vivo shortly after implantation. Despite the initially homogeneous state of the cells in the model and high Activin concentration during endodermal specification, there remains a cell subpopulation that does not reach the endodermal state. This simple model developed by us can be used to study the origins of cellular heterogeneity during germ layer specification.

Embryo implantation is accompanied by the naïve-to-primed pluripotency state transition in epiblast cells-the process involving proliferation and anabolic boost, needed for extensive embryo growth. Here, we show that Pcbp1 knockout leads to embryo growth arrest shortly after implantation. By modeling the naïve-to-primed pluripotency transition in vitro, we observe impaired proliferation and induction of apoptosis in cells deficient for PCBP1. Using multi-omics approaches, we reveal 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, which explains the early lethality of the 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 critical process.