PD-1 agonism by anti-CD80 inhibits T cell activation and alleviates autoimmunity.

Targeted blockade of the checkpoint molecule programmed cell death 1 (PD-1) can activate tumor-specific T cells to destroy tumors, whereas targeted potentiation of PD-1 is expected to suppress autoreactive T cells and alleviate autoimmune diseases. However, the development of methods to potentiate PD-1 remains challenging. Here we succeeded in eliciting PD-1 function by targeting the cis-PD-L1-CD80 duplex, formed by binding of CD80 to the PD-1 ligand PD-L1, that attenuates PD-L1-PD-1 binding and abrogates PD-1 function. By generating anti-CD80 antibodies that detach CD80 from the cis-PD-L1-CD80 duplex and enable PD-L1 to engage PD-1 in the presence of CD80, we demonstrate that the targeted dissociation of cis-PD-L1-CD80 duplex elicits PD-1 function in the condition where PD-1 function is otherwise restricted. We demonstrate using murine models that the removal of PD-1 restriction is effective in alleviating autoimmune disease symptoms. Our findings establish a method to potentiate PD-1 function and propose the removal of restraining mechanisms as an efficient strategy to potentiate the function of inhibitory molecules.

Binding of LAG-3 to stable peptide-MHC class II limits T cell function and suppresses autoimmunity and anti-cancer immunity.

Lymphocyte activation gene-3 (LAG-3) is a potent inhibitory co-receptor; yet, its functional ligand remains elusive, with distinct potential ligands identified. Here, we investigated the relative contribution of potential ligands, stable peptide-MHC class II complexes (pMHCII) and fibrinogen-like protein 1 (FGL1), to LAG-3 activity in vitro and in vivo. Binding of LAG-3 to stable pMHCII but not to FGL1 induced T cell suppression in vitro. Consistently, LAG-3 mutants lacking FGL1-binding capacity but not those lacking stable pMHCII-binding capacity retained suppressive activity in vitro. Accordingly, targeted disruption of stable pMHCII- but not FGL1-binding capacity of LAG-3 in NOD mice recapitulated diabetes exacerbation by LAG-3 deficiency. Additionally, the loss of stable pMHCII-binding capacity of LAG-3 augmented anti-cancer immunity comparably with LAG-3 deficiency in C57BL/6 mice. These results identify stable pMHCII as a functional ligand of LAG-3 both in autoimmunity and anti-cancer immunity. Thus, stable pMHCII-LAG-3 interaction is a potential therapeutic target in human diseases.

An inducible germ cell ablation chicken model for high-grade germline chimeras.

Chicken embryos are a powerful and widely used animal model in developmental biology studies. Since the development of CRISPR technology, gene-edited chickens have been generated by transferring primordial germ cells (PGCs) into recipients after genetic modifications. However, low inheritance caused by competition between host germ cells and the transferred cells is a common complication and greatly reduces production efficiency. Here, we generated a gene-edited chicken, in which germ cells can be ablated in a drug-dependent manner, as recipients for gene-edited PGC transfer. We used the nitroreductase/metronidazole (NTR/Mtz) system for cell ablation, in which nitroreductase produces cytotoxic alkylating agents from administered metronidazole, causing cell apoptosis. The chicken Vasa homolog (CVH) gene locus was used to drive the expression of the nitroreductase gene in a germ cell-specific manner. In addition, a fluorescent protein gene, mCherry, was also placed in the CVH locus to visualize the PGCs. We named this system 'germ cell-specific autonomous removal induction' (gSAMURAI). gSAMURAI chickens will be an ideal recipient to produce offspring derived from transplanted exogenous germ cells.

PD-1 preferentially inhibits the activation of low-affinity T cells.

Anti-PD-1 therapies can activate tumor-specific T cells to destroy tumors. However, whether and how T cells with different antigen specificity and affinity are differentially regulated by PD-1 remain vaguely understood. Upon antigen stimulation, a variety of genes is induced in T cells. Recently, we found that T cell receptor (TCR) signal strength required for the induction of genes varies across different genes and PD-1 preferentially inhibits the induction of genes that require stronger TCR signal. As each T cell has its own response characteristics, inducibility of genes likely differs across different T cells. Accordingly, the inhibitory effects of PD-1 are also expected to differ across different T cells. In the current study, we investigated whether and how factors that modulate T cell responsiveness to antigenic stimuli influence PD-1 function. By analyzing TCRs with different affinities to peptide-MHC complexes (pMHC) and pMHCs with different affinities to TCR, we demonstrated that PD-1 inhibits the expression of TCR-inducible genes efficiently when TCR:pMHC affinity is low. In contrast, affinities of peptides to MHC and MHC expression levels did not affect PD-1 sensitivity of TCR-inducible genes although they markedly altered the dose responsiveness of T cells by changing the efficiency of pMHC formation, suggesting that the strength of individual TCR signal is the key determinant of PD-1 sensitivity. Accordingly, we observed a preferential expansion of T cells with low-affinity to tumor-antigen in PD-1-deficient mice upon inoculation of tumor cells. These results demonstrate that PD-1 imposes qualitative control of T cell responses by preferentially suppressing low-affinity T cells.

The nephric mesenchyme lineage of intermediate mesoderm is derived from Tbx6-expressing derivatives of neuro-mesodermal progenitors via BMP-dependent Osr1 function.

In vertebrate embryos, the kidney primordium metanephros is formed from two distinct cell lineages, Wolffian duct and metanephric mesenchyme, which were classically grouped as intermediate mesoderm. Whereas the reciprocal interactions between these two cell populations in kidney development have been studied extensively, the mechanisms generating them remain elusive. Here, we show that the mouse cell lineage that forms nephric mesenchyme develops as a subpopulation of Tbx6-expressing mesodermal precursor derivatives of neuro-mesodermal progenitors (NMPs) under the condition of bone morphogenetic protein (BMP)-signal-dependent Osr1 expression. The Osr1-expressing nephric mesenchyme precursors were confirmed as descendants of NMPs because they were labeled by Sox2 N1 enhancer-EGFP. In Tbx6 mutant embryos, nephric mesenchyme changed its fate into neural tissues, which reflected its NMP origin. In Osr1 mutant embryos, the specific region of the Tbx6-expressing mesoderm precursor, which normally expresses Osr1 and develops into the nephric mesenchyme, instead expressed the somite marker FoxC2. BMP signaling activated Osr1 expression in a region of TBX6-expressing mesoderm and elicited nephric mesenchyme development. This study suggested a new model of cell lineage segregation during gastrulation.

Sox2 gene regulation via the D1 enhancer in embryonic neural tube and neural crest by the combined action of SOX2 and ZIC2.

The transcription factor (TF) SOX2 regulates various stem cells and tissue progenitors via functional interactions with cell type-specific partner TFs that co-bind to enhancer sequences. Neural progenitors are the major embryonic tissues where SOX2 assumes central regulatory roles. In order to characterize the partner TFs of SOX2 in neural progenitors, we investigated the regulation of the D1 enhancer of the Sox2 gene, which is activated in the embryonic neural tube (NT) and neural crest (NC), using chicken embryo electroporation. We identified essential TF binding sites for a SOX, and two ZIC TFs in the activation of the D1 enhancer. By comparison of dorso-ventral and antero-posterior patterns of D1 enhancer activation, and the effect of mutations on the enhancer activation patterns with TF expression patterns, we determined SOX2 and ZIC2 as the major D1 enhancer-activating TFs. Binding of these TFs to the D1 enhancer sequence was confirmed by chromatin immunoprecipitation analysis. The combination of SOX2 and ZIC2 TFs activated the enhancer in both the NT and NC. These results indicate that SOX2 and ZIC2, which have been known to play major regulatory roles in neural progenitors, do functionally cooperate. In addition, the recently demonstrated SOX2 expression during the NC development is accounted for at least partly by the D1 enhancer activity. Deletion of the D1 enhancer sequence from the mouse genome, however, did not affect the mouse development, indicating functional redundancies of other enhancers.

Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B- and Hirschsprung disease-associated missense mutations of the RET gene.

Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (RetM919T , RetN396K and RetY792F ) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. RetM919T/+ mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in RetM919T/- mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B- and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity.

Hoxa13 regulates expression of common Hox target genes involved in cartilage development to coordinate the expansion of the autopodal anlage.

To elucidate the role of Hox genes in limb cartilage development, we identified the target genes of HOXA11 and HOXA13 by ChIP-Seq. The ChIP DNA fragment contained evolutionarily conserved sequences and multiple highly conserved HOX binding sites. A substantial portion of the HOXA11 ChIP fragment overlapped with the HOXA13 ChIP fragment indicating that both factors share common targets. Deletion of the target regions neighboring Bmp2 or Tshz2 reduced their expression in the autopod suggesting that they function as the limb bud-specific enhancers. We identified the Hox downstream genes as exhibiting expression changes in the Hoxa13 knock out (KO) and Hoxd11-13 deletion double mutant (Hox13 dKO) autopod by Genechip analysis. The Hox downstream genes neighboring the ChIP fragment were defined as the direct targets of Hox. We analyzed the spatial expression pattern of the Hox target genes that encode two different categories of transcription factors during autopod development and Hox13dKO limb bud. (a) Bcl11a, encoding a repressor of cartilage differentiation, was expressed in the E11.5 autopod and was substantially reduced in the Hox13dKO. (b) The transcription factors Aff3, Bnc2, Nfib and Runx1t1 were expressed in the zeugopodal cartilage but not in the autopod due to the repressive or relatively weak transcriptional activity of Hox13 at E11.5. Interestingly, the expression of these genes was later observed in the autopodal cartilage at E12.5. These results indicate that Hox13 transiently suspends the cartilage differentiation in the autopodal anlage via multiple pathways until establishing the paddle-shaped structure required to generate five digits.

Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse.

The CRISPR/Cas9 system is a powerful tool for elucidating the roles of genes in a wide variety of organisms including mice. To obtain genetically modified embryos or mice by this method, Cas9 mRNA and sgRNA are usually introduced into zygotes by microinjection or electroporation. However, most mutants generated with this method are genetically mosaic, composed of several types of cells carrying different mutations, which complicates phenotype analysis in founder embryos or mice. To simplify the analysis and to elucidate the roles of genes involved in developmental processes, a method for producing non-mosaic mutants is needed. Here, we established a method for generating non-mosaic mouse mutant embryos. We introduced Cas9 protein and sgRNA into in vitro fertilized (IVF) zygotes by electroporation, which enabled the genome editing to occur before the first replication of the mouse genome. As a result, all of the cells in the mutant carried the same set of mutations. This method solves the problem of mosaicism/allele complexity in founder mutant embryos or mice generated by the CRIPSR/Cas9 system.

R26-WntVis reporter mice showing graded response to Wnt signal levels.

The canonical Wnt signaling pathway plays a major role in the regulation of embryogenesis and organogenesis, where signal strength-dependent cellular responses are of particular importance. To assess Wnt signal levels in individual cells, and to circumvent the integration site-dependent bias shown in previous Wnt reporter lines, we constructed a new Wnt signal reporter mouse line R26-WntVis. Heptameric TCF/LEF1 binding sequences were combined with a viral minimal promoter to confer a graded response to the reporter depending on Wnt signal strengths. The histone H2B-EGFP fusion protein was chosen as the fluorescent reporter to facilitate single-cell resolution analyses. This WntVis reporter gene was then inserted into the ROSA26 locus in an orientation opposite to that of the endogenous gene. The R26-WntVis allele was introduced into Wnt3a(-/-) and Wnt3a(vt/-) mutant mouse embryos and compared with wild-type embryos to assess its performance. The R26-WntVis reporter was activated in known Wnt-dependent tissues and responded in a graded fashion to signal intensity. This analysis also indicated that the major Wnt activity early in embryogenesis switched from Wnt3 to Wnt3a around E7.5. The R26-WntVis mouse line will be widely useful for the study of Wnt signal-dependent processes.

Tbx6-dependent Sox2 regulation determines neural or mesodermal fate in axial stem cells.

The classical view of neural plate development held that it arises from the ectoderm, after its separation from the mesodermal and endodermal lineages. However, recent cell-lineage-tracing experiments indicate that the caudal neural plate and paraxial mesoderm are generated from common bipotential axial stem cells originating from the caudal lateral epiblast. Tbx6 null mutant mouse embryos which produce ectopic neural tubes at the expense of paraxial mesoderm must provide a clue to the regulatory mechanism underlying this neural versus mesodermal fate choice. Here we demonstrate that Tbx6-dependent regulation of Sox2 determines the fate of axial stem cells. In wild-type embryos, enhancer N1 of the neural primordial gene Sox2 is activated in the caudal lateral epiblast, and the cells staying in the superficial layer sustain N1 activity and activate Sox2 expression in the neural plate. In contrast, the cells destined to become mesoderm activate Tbx6 and turn off enhancer N1 before migrating into the paraxial mesoderm compartment. In Tbx6 mutant embryos, however, enhancer N1 activity persists in the paraxial mesoderm compartment, eliciting ectopic Sox2 activation and transforming the paraxial mesoderm into neural tubes. An enhancer-N1-specific deletion mutation introduced into Tbx6 mutant embryos prevented this Sox2 activation in the mesodermal compartment and subsequent development of ectopic neural tubes, indicating that Tbx6 regulates Sox2 via enhancer N1. Tbx6-dependent repression of Wnt3a in the paraxial mesodermal compartment is implicated in this regulatory process. Paraxial mesoderm-specific misexpression of a Sox2 transgene in wild-type embryos resulted in ectopic neural tube development. Thus, Tbx6 represses Sox2 by inactivating enhancer N1 to inhibit neural development, and this is an essential step for the specification of paraxial mesoderm from the axial stem cells.

Sensitivity of the meiotic stage to hyperthermia during in vitro maturation of porcine oocytes.

The present study was conducted to clarify whether the meiotic stage of porcine oocytes has the highest sensitivity to hyperthermia during in vitro maturation by evaluating meiotic competence and DNA damage. Oocytes were exposed to 41 °C for 12 h at various intervals during 48 h of maturation culture. When the oocytes were exposed to 41 °C from 12 to 24 h of the maturation culture, the proportion of oocytes reaching metaphase II (MII) decreased as compared to the control oocytes cultured at 38.5 °C (P < 0.05). Moreover, the proportions of DNA fragmentation in all oocytes exposed to 41 °C in each culture period after 12 h from the start of maturation culture were significantly higher (P < 0.05) than for the control oocytes. When the meiotic stage of oocytes cultured at 38.5 °C between 12 and 24 h was examined, the majority of oocytes remained at the germinal vesicle (GV) stage at 12 h and approximately half of the oocytes reached metaphase I (MI) at 24 h. These results indicate that the meiotic stage of porcine oocytes having the highest sensitivity to hyperthermia during in vitro maturation is a transition period from the GV stage to the MI stage.

Axial level-dependent molecular and cellular mechanisms underlying the genesis of the embryonic neural plate.

The transcription factor gene Sox2, centrally involved in neural primordial regulation, is activated by many enhancers. During the early stages of embryonic development, Sox2 is regulated by the enhancers N2 and N1 in the anterior neural plate (ANP) and posterior neural plate (PNP), respectively. This differential use of the enhancers reflects distinct regulatory mechanisms underlying the genesis of ANP and PNP. The ANP develops directly from the epiblast, triggered by nodal signal inhibition, and via the combined action of TFs SOX2, OTX2, POU3F1, and ZIC2, which promotes the the ANP development and inhibits other cell lineages. In contrast, the PNP is derived from neuromesodermal bipotential axial stem cells that develop into the neural plate when Sox2 is activated by the N1 enhancer, whereas they develop into the paraxial mesoderm when the N1 enhancer is repressed by the action of TBX6. The axial stem cells are maintained by the activity of WNT3a and T (Brachyury). However, at axial levels more anterior to the 8th somites (cervical levels), the development of both the neural plate and somite proceeds in the absence of WNT3a, T, or TBX6. These observations indicate that distinct molecular and cellular mechanisms determine neural plate genesis based on the axial level, and contradict the classical concept of the term "neural induction," which assumes a pan-neural plate mechanism.

CHARACTERISTICS AND FERTILITY OF SUMATRAN TIGER SPERMATOZOA CRYOPRESERVED WITH DIFFERENT SUGARS.

BACKGROUND: Cryopreservation of semen is one of the most important methods for the preservation of endangered tigers. OBJECTIVE: This study evaluated the effects of sugar supplementation on the cryosurvival of spermatozoa from Sumatran tigers (Panthera tigris sumatrae). MATERIALS AND METHODS: The post-thaw characteristics and fertility of spermatozoa cryopreserved with different sugars (glucose, lactose, and trehalose) were evaluated using heterologous in-vitro fertilisation with cat oocytes. RESULTS: All parameters of post-thaw spermatozoa significantly decreased as compared with those of fresh spermatozoa. The index of sperm motility for semen cryopreserved with lactose was significantly higher than that for semen cryopreserved with trehalose. The percentage of total fertilisation for tiger spermatozoa cryopreserved with trehalose was significantly lower than that for control cat spermatozoa. CONCLUSION: Our findings indicated that supplementation with lactose or glycerol as the main sugar in the egg yolk extender resulted in a better motility and fertility potential for post-thawed spermatozoa.

The Origin and Regulation of Neuromesodermal Progenitors (NMPs) in Embryos.

Neuromesodermal progenitors (NMPs), serving as the common origin of neural and paraxial mesodermal development in a large part of the trunk, have recently gained significant attention because of their critical importance in the understanding of embryonic organogenesis and the design of in vitro models of organogenesis. However, the nature of NMPs at many essential points remains only vaguely understood or even incorrectly assumed. Here, we discuss the nature of NMPs, focusing on their dynamic migratory behavior during embryogenesis and the mechanisms underlying their neural vs. mesodermal fate choice. The discussion points include the following: (1) How the sinus rhomboidals is organized; the tissue where the neural or mesodermal fate choice of NMPs occurs. (2) NMPs originating from the broad posterior epiblast are associated with Sox2 N1 enhancer activity. (3) Tbx6-dependent Sox2 repression occurs during NMP-derived paraxial mesoderm development. (4) The nephric mesenchyme, a component of the intermediate mesoderm, was newly identified as an NMP derivative. (5) The transition of embryonic tissue development from tissue-specific progenitors in the anterior part to that from NMPs occurs at the forelimb bud axial level. (6) The coexpression of Sox2 and Bra in NMPs is conditional and is not a hallmark of NMPs. (7) The ability of the NMP pool to sustain axial embryo growth depends on Wnt3a signaling in the NMP population. Current in vitro models of NMPs are also critically reviewed.

Tumor endothelial cell-derived Sfrp1 supports the maintenance of cancer stem cells via Wnt signaling.

Cancer stem cells (CSCs), which are critical targets for cancer therapy as they are involved in drug resistance to anticancer drugs, and metastasis, are maintained by angiocrine factors produced by particular niches that form within tumor tissue. Secreted frizzled-related protein 1 (Sfrp1) is an extracellular protein that modulates Wnt signaling. However, the cells that produce Sfrp1 in the tumor environment and its function remain unclear. We aimed to elucidate angiocrine factors related to CSC maintenance, focusing on Sfrp1. Although Sfrp1 is a Wnt pathway-related factor, its impact on tumor tissues remains unknown. We investigated the localization of Sfrp1 in tumors and found that it is expressed in some tumor vessels. Analysis of mice lacking Sfrp1 showed that tumor growth was suppressed in Sfrp1-deficient tumor tissues. Flow cytometry analysis indicated that CSCs were maintained in the early tumor growth phase in the Sfrp1 knockout (KO) mouse model of tumor-bearing cancer. However, tumor growth was inhibited in the late tumor growth phase because of the inability to maintain CSCs. Real-time PCR results from tumors of Sfrp1 KO mice showed that the expression of Wnt signaling target genes significantly decreased in the late stage of tumor growth. This suggests that Sfrp1, an angiocrine factor produced by the tumor vascular niche, is involved in Wnt signaling-mediated mechanisms in tumor tissues.

Functional analysis of a first hindlimb positioning enhancer via Gdf11 expression.

During the early development of tetrapods, including humans, the embryonic body elongates caudally once the anterior-posterior axis is established. During this process, region-specific vertebral morphogenesis occurs, with the determination of limb positioning along the anterior-posterior axis. We previously reported that Gdf11 functions as an anatomical integration system that determines the positioning of hindlimbs and sacral vertebrae where Gdf11 is expressed. However, the molecular mechanisms underlying induction of Gdf11 expression remain unclear. In this study, we searched for non-coding regions near the Gdf11 locus that were conserved across species to elucidate the regulatory mechanisms of Gdf11 expression. We identified an enhancer of the Gdf11 gene in intron 1 and named it highly conserved region (HCR). In HCR knockout mice, the expression level of endogenous Gdf11 was decreased, and the position of the sacral-hindlimb unit was shifted posteriorly. We also searched for factors upstream of Gdf11 based on the predicted transcription factor binding sites within the HCR. We found that inhibition of FGF signaling increased endogenous Gdf11 expression, suggesting that FGF signaling negatively regulates Gdf11 expression. However, FGF signaling does not regulate HCR activity. Our results suggest that there are species-specific Gdf11 enhancers other than HCR and that FGF signaling regulates Gdf11 expression independent of HCR.

VDR is an essential regulator of hair follicle regression through the progression of cell death.

Vitamin D receptor (VDR) is essential for hair follicle homeostasis as its deficiency induces hair loss, although the mechanism involved remains unknown. Our research shows that, in Vdr-knockout mice, the hair cycle is halted during the catagen stage, preceding alopecia. In addition, in Vdr-knockout hair follicles, epithelial strands that normally regress during the catagen phase persist as "surviving epithelial strands." Single-cell RNA sequencing analysis suggests that these surviving epithelial strands are formed by cells in the lower part of the hair follicle. These findings emphasize the importance of the regression phase in hair follicle regeneration and establish VDR as a regulator of the catagen stage.

Intercellular exchange of Wnt ligands reduces cell population heterogeneity during embryogenesis.

Wnt signaling is required to maintain bipotent progenitors for neural and paraxial mesoderm cells, the neuromesodermal progenitor (NMP) cells that reside in the epiblast and tailbud. Since epiblast/tailbud cells receive Wnt ligands produced by one another, this exchange may average out the heterogeneity of Wnt signaling levels among these cells. Here, we examined this possibility by replacing endogenous Wnt3a with a receptor-fused form that activates signaling in producing cells, but not in neighboring cells. Mutant mouse embryos show a unique phenotype in which maintenance of many NMP cells is impaired, although some cells persist for long periods. The epiblast cell population of these embryos increases heterogeneity in Wnt signaling levels as embryogenesis progresses and are sensitive to retinoic acid, an endogenous antagonist of NMP maintenance. Thus, mutual intercellular exchange of Wnt ligands in the epiblast cell population reduces heterogeneity and achieves robustness to environmental stress.

The Pou5f1/Pou3f-dependent but SoxB-independent regulation of conserved enhancer N2 initiates Sox2 expression during epiblast to neural plate stages in vertebrates.

The transcription factor Sox2 is a core component of the pluripotency control circuits in the early embryo, and later controls many aspects of neural development. Here, we demonstrate that Sox2 expression in the epiblast (mouse blastoderm) and anterior neural plate (ANP) is determined by the upstream enhancer N2. The mouse enhancer N2 exhibits strong activity in mouse ES cells, epiblast and ANP, and is regulated correctly in chicken and zebrafish embryos. Targeted deletion of this enhancer in mouse embryos caused a large reduction of Sox2 expression to 10% of that of wild-type levels in epiblast and ANP. However, this was tolerated by mouse embryo, probably due to functional compensation by Sox3. The activity of enhancer N2 depends on phylogenetically conserved bipartite POU factor-binding motifs in a 73-bp core sequence that function synergistically, but this activation does not involve Sox2. The major POU factor expressed at the epiblastic stage is Pou5f1 (Oct3/4), while those in the anterior neural plate are Pou3f factors (Oct6, Brn2 etc.). These factors are gradually exchanged during the transition from epiblast to ANP stages in mouse embryos and epiblast stem cells (EpiSC). Consistently, enhancer N2 activity changes from full Pou5f1 dependence to Pou3f dependence during the development of neural plate cells (NPC) from EpiSC, as assessed by specific POU factor knockdown in these cells. Zebrafish mutant embryos completely devoid of Pou5f1 activity failed to activate enhancer N2 and to express Sox2 in the blastoderm and ANP, and these defects were rescued by exogenous supply of pou5f1. Previously, Pou5f1-Sox2 synergism-dependent Sox2 activation through enhancer SRR2 in ES cells has been highlighted, but this mechanism is limited to ES cells and amniotes. In contrast, the enhancer N2-mediated, POU factor-dependent activation of Sox2, without involvement of Sox2, is a phylogenetically conserved core mechanism that functions in gene regulatory networks at early embryonic stages.