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Using a transgenic zebrafish line harboring a heat-inducible dominant-interference pou5f3 gene (en-pou5f3), we reported that this PouV gene is involved in isthmus development at the midbrain-hindbrain boundary (MHB), which patterns the midbrain and cerebellum. Importantly, the functions of pou5f3 reportedly differ before and after the end of gastrulation. In the present study, we examined in detail the effects of en-pou5f3 induction on isthmus development during embryogenesis. When en-pou5f3 was induced around the end of gastrulation (bud stage), the isthmus was abrogated or deformed by the end of somitogenesis (24 hours post-fertilization). At this stage, the expression of MHB markers -- such as pax2a, fgf8a, wnt1, and gbx2 -- was absent in embryos lacking the isthmus structure, whereas it was present, although severely distorted, in embryos with a deformed isthmus. We further found that, after en-pou5f3 induction at late gastrulation, pax2a, fgf8a, and wnt1 were immediately and irreversibly downregulated, whereas the expression of en2a and gbx2 was reduced only weakly and slowly. Induction of en-pou5f3 at early somite stages also immediately downregulated MHB genes, particularly pax2a, but their expression was restored later. Overall, the data suggested that pou5f3 directly upregulates at least pax2a and possibly fgf8a and wnt1, which function in parallel in establishing the MHB, and that the role of pou5f3 dynamically changes around the end of gastrulation. We next examined the transcriptional regulation of pax2a using both in vitro and in vivo reporter analyses; the results showed that two upstream 1.0-kb regions with sequences conserved among vertebrates specifically drove transcription at the MHB. These reporter analyses confirmed that development of the isthmic organizer is regulated by PouV through direct regulation of pax2/pax2a in vertebrate embryos., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Early embryonic development is a finely orchestrated process that requires precise regulation of gene expression coordinated with morphogenetic events. TATA-box binding protein-associated factors (TAFs), integral components of transcription initiation coactivators like TFIID and SAGA, play a crucial role in this intricate process. Here we show that disruptions in TAF5, TAF12 and TAF13 individually lead to embryonic lethality in the mouse, resulting in overlapping yet distinct phenotypes. Taf5 and Taf12 mutant embryos exhibited a failure to implant post-blastocyst formation, and Taf5 mutants have aberrant lineage specification within the inner cell mass. In contrast, Taf13 mutant embryos successfully implant and form egg-cylinder stages but fail to initiate gastrulation. Strikingly, we observed a depletion of pluripotency factors in TAF13-deficient embryos, including OCT4, NANOG and SOX2, highlighting an indispensable role of TAF13 in maintaining pluripotency. Transcriptomic analysis revealed distinct gene targets affected by the loss of TAF5, TAF12 and TAF13. Thus, we propose that TAF5, TAF12 and TAF13 convey locus specificity to the TFIID complex throughout the mouse genome., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Pioneer transcription factors (TFs), such as OCT4 and SOX2, play crucial roles in pluripotency regulation. However, the master TF-governed pluripotency regulatory circuitry was largely inferred from cultured cells. In this work, we investigated SOX2 binding from embryonic day 3.5 (E3.5) to E7.5 in the mouse. In E3.5 inner cell mass (ICM), SOX2 regulates the ICM-trophectoderm program but is dispensable for opening global enhancers. Instead, SOX2 occupies preaccessible enhancers in part opened by early-stage expressing TFs TFAP2C and NR5A2. SOX2 then widely redistributes when cells adopt naive and formative pluripotency by opening enhancers or poising them for rapid future activation. Hence, multifaceted pioneer TF-enhancer interaction underpins pluripotency progression in embryos, including a distinctive state in E3.5 ICM that bridges totipotency and pluripotency.

Objectives: Curiosity about the role of OCT4, a core transcription factor that maintains inner cell mass (ICM) formation during preimplantation embryogenesis and the pluripotent state in embryonic development, has long been an issue. OCT4 has a species-specific expression pattern in mammalian preimplantation embryogenesis and is known to play an essential role in ICM formation. However, there is a need to study new roles for OCT4-related pluripotency networks and second-cell fate decisions., Materials and Methods: To determine the functions of OCT4 in lineage specification and embryo proliferation, loss- and gain-of-function studies were performed on porcine parthenotes using microinjection. Then, we performed immunocytochemistry and quantitative real-time polymerase chain reaction (PCR) to examine the association of OCT4 with other lineage markers and its effect on downstream genes., Results: In OCT4-targeted late blastocysts, SOX2, NANOG, and SOX17 positive cells were decreased, and the total cell number of blastocysts was also decreased. According to real-time PCR analysis, NANOG, SOX17, and CDK4 were decreased in OCT4-targeted blastocysts, but trophoblast-related genes were increased. In OCT4-overexpressing blastocysts, SOX2 and NANOG positive cells increased, while SOX17 positive cells decreased, and while total cell number of blastocysts increased. As a result of real-time PCR analysis, the expression of SOX2, NANOG, and CDK4 was increased, but the expression of SOX17 was decreased., Conclusion: Taken together, our results demonstrated that OCT4 leads pluripotency in porcine blastocysts and also plays an important role in ICM formation, secondary cell fate decision, and cell proliferation., (© 2022 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

The emergence of the first three lineages during development is orchestrated by a network of transcription factors, which are best characterized in mice. However, the role and regulation of these factors are not completely conserved in other mammals, including human and cattle. Here, we establish a gene inactivation system with a robust efficiency by introducing premature codon with cytosine base editors in bovine early embryos. By using this approach, we have determined the functional consequences of three critical lineage-specific genes (SOX2, OCT4 and CDX2) in bovine embryos. In particular, SOX2 knockout results in a failure of the establishment of pluripotency in blastocysts. Indeed, OCT4 level is significantly reduced and NANOG barely detectable. Furthermore, the formation of primitive endoderm is compromised with few SOX17 positive cells. RNA-seq analysis of single blastocysts (day 7.5) reveals dysregulation of 2074 genes, among which 90% are up-regulated in SOX2-null blastocysts. Intriguingly, more than a dozen lineage-specific genes, including OCT4 and NANOG, are down-regulated. Moreover, SOX2 level is sustained in the trophectoderm in absence of CDX2. However, OCT4 knockout does not affect the expression of SOX2. Overall, we propose that SOX2 is indispensable for OCT4 and NANOG expression and CDX2 represses the expression of SOX2 in the trophectoderm in cattle, which are all in sharp contrast with results in mice., Competing Interests: The authors have declared that no competing interests exist.

Pluripotency markers Pou5f1 and Nanog are core transcription factors regulating early embryonic development and maintaining the pluripotency and self-renewal of stem cells. Pou5f1 and Nanog also play important roles in germ cell development and gametogenesis. In this study, Pou5f1 ( EcPou5f1 ) and Nanog ( EcNanog ) were cloned from orange-spotted grouper, Epinephelus coioides . The full-length cDNAs of EcPou5f1 and EcNanog were 2790 and 1820 bp, and encoded 475 and 432 amino acids, respectively. EcPou5f1 exhibited a specific expression in gonads, whereas EcNanog was expressed highly in gonads and weakly in some somatic tissues. In situ hybridization analyses showed that the mRNA signals of EcNanog and EcPou5f1 were exclusively restricted to germ cells in gonads. Likewise, immunohistofluorescence staining revealed that EcNanog protein was limited to germ cells. Moreover, both EcPou5f1 and EcNanog mRNAs were discovered to be co-localized with Vasa mRNA, a well-known germ cell maker, in male and female germ cells. These results implied that EcPou5f1 and EcNanog could be also regarded as reliable germ cell marker genes. Therefore, the findings of this study would pave the way for elucidating the mechanism whereby EcPou5f1 and EcNanog regulate germ cell development and gametogenesis in grouper fish, and even in other protogynous hermaphroditic species.

As pluripotent human embryonic stem cells progress toward one germ layer fate, they lose the ability to adopt alternative fates. Using a low-dimensional reaction coordinate to monitor progression toward ectoderm, we show that a differentiating stem cell's probability of adopting a mesendodermal fate given appropriate signals falls sharply at a point along the ectoderm trajectory. We use this reaction coordinate to prospectively isolate and profile differentiating cells based on their mesendoderm competence and analyze their RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) profiles to identify transcription factors that control the cell's mesendoderm competence. By modulating these key transcription factors, we can expand or contract the window of competence to adopt the mesendodermal fate along the ectodermal differentiation trajectory. The ability of the underlying gene regulatory network to modulate competence is essential for understanding human development and controlling the fate choices of stem cells in vitro., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The pluripotency gene regulatory network of porcine induced pluripotent stem cells(piPSCs), especially in epigenetics, remains elusive. To determine the biological function of epigenetics, we cultured piPSCs in different culture conditions. We found that activation of pluripotent gene- and pluripotency-related pathways requires the erasure of H3K9 methylation modification which was further influenced by mouse embryonic fibroblast (MEF) served feeder. By dissecting the dynamic change of H3K9 methylation during loss of pluripotency, we demonstrated that the H3K9 demethylases KDM3A and KDM3B regulated global H3K9me2/me3 level and that their co-depletion led to the collapse of the pluripotency gene regulatory network. Immunoprecipitation-mass spectrometry (IP-MS) provided evidence that KDM3A and KDM3B formed a complex to perform H3K9 demethylation. The genome-wide regulation analysis revealed that OCT4 (O) and SOX2 (S), the core pluripotency transcriptional activators, maintained the pluripotent state of piPSCs depending on the H3K9 hypomethylation. Further investigation revealed that O/S cooperating with histone demethylase complex containing KDM3A and KDM3B promoted pluripotency genes expression to maintain the pluripotent state of piPSCs. Together, these data offer a unique insight into the epigenetic pluripotency network of piPSCs., (© 2021 Federation of American Societies for Experimental Biology.)

The pluripotency of embryonic stem cells (ESCs) is controlled by a multilayer regulatory network, of which the key factors include core pluripotency genes Oct4, Sox2 and Nanog , and multiple microRNAs (miRNAs). Recently, long noncoding RNAs (lncRNAs) have been discovered as a class of new regulators for ESCs, and some lncRNAs could function as competing endogenous RNAs (ceRNAs) to regulate mRNAs by competitively binding to miRNAs. Here, we identify mmu-miR-139-5p as a new regulator for Nanog by targeting Nanog 3' untranslated region (UTR) to repress Nanog expression in mouse ESCs and embryos. Such regulation could be released by an ESC-specifically expressed ceRNA named lnc-NAP . The expression of lnc-NAP is activated by OCT4, SOX2, as well as NANOG through promoter binding. Downregulation of lnc-NAP reduces Nanog abundance, which leads to decreased pluripotency of mouse ESCs and embryonic lethality. These results reveal lnc-NAP as a new regulator for Nanog in mouse ESCs, and uncover a feed-forward regulatory loop of Nanog through the participation of lnc-NAP .

In vitro induction of human primordial germ cell-like cells (hPGCLCs) provides an ideal platform to recapitulate hPGC development. However, the detailed molecular mechanisms regulating the induction of hPGCLCs remain largely uncharacterized. Here, we profiled the chromatin accessibility and transcriptome dynamics throughout the process of hPGCLC induction. Genetic ablation of SOX15 indicated the crucial roles of SOX15 in the maintenance of hPGCLCs. Mechanistically, SOX15 exerted its roles via suppressing somatic gene expression and sustaining latent pluripotency. Notably, ETV5, a downstream regulator of SOX15, was also uncovered to be essential for hPGCLC maintenance. Finally, a stepwise switch of OCT4/SOX2, OCT4/SOX17, and OCT4/SOX15 binding motifs were found to be enriched in closed-to-open regions of human embryonic stem cells, and early- and late-stage hPGCLCs, respectively. Collectively, our data characterized the chromatin accessibility and transcriptome landscapes throughout hPGCLC induction and defined the SOX15-mediated regulatory networks underlying this process., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Differentiation of the embryonic stem cells (ESCs) is regulated by a variety of different signaling pathways. Genetic depletion of murine Pelota gene (Pelo) leads to early embryonic lethality. Here, we aimed at determining the embryonic stage and deciphering the dysregulated signaling pathways affected upon Pelo deletion. We found that development of PELO-null embryos is perturbed between the embryonic days E4.5 and E5.5, at which first differentiation process of ESCs takes place. Molecular analysis revealed enhanced activity of phosphoinositide 3-kinase-protein kinase B/ AKT (PI3K-PKB/AKT) signaling, but nuclear accumulation of forkhead box O1 (FOXO1), and upregulation of the pluripotency-related gene, Oct4, in mutant ESCs cultured under differentiation condition. Despite increased levels of nuclear β-catenin in PELO-null ESCs as a result of decreased activity of glycogen synthase kinase-3β, the activity of the canonical wingless (Wnt)/β-catenin/T-cell factor (TCF) was significantly attenuated as judged by the promoter reporter assay, downregulated Wnt/β-catenin target genes, and impaired cell proliferation. Interestingly, we demonstrated an increased binding of β-catenin to FOXO1 in PELO-mutant ESCs cultured under differentiation condition that could explain, on one side, the nuclear accumulation of FOXO1 protein and hence persistent pluripotency of PELO-mutant ESCs, and on the other side, the dysregulated transcriptional activity of β-catenin/TCF and therefore attenuated PELO-null ESC self-renewal. Taken together, our results strongly suggest that PELO deletion averts ESC differentiation through promoting FOXO1/β-catenin binding with subsequent dysregulation of FOXO1 and canonical β-catenin/TCF signaling pathways., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Folate, a cofactor for the supply of one-carbon groups, is required by epigenetic processes to regulate cell lineage determination during development. The intake of folic acid (FA), the synthetic form of folate, has increased significantly over the past decade, but the effects of high periconceptional FA intake on cell lineage determination in the early embryo remains unknown. Here, we investigated the effect of maternal high FA (HFA) intake on blastocyst development and expression of key regulatory genes. C57BL/6 adult female mice were fed either Control diet (1 mg FA) for 4 weeks before conception and during the preimplantation period (Con-Con); Control diet for 4 weeks preconception, followed by HFA (5 mg FA) diet during preimplantation (Con-HFA); or HFA diet for 4 weeks preconception and during preimplantation (HFA-HFA). At E3.5, blastocyst cell number, protein, and mRNA expression were measured. In HFA-HFA blastocysts, trophectoderm cell numbers and expression of CDX2, Oct-4, and Nanog were reduced compared with Con-Con blastocysts; Con-HFA blastocysts showed lower CDX2 and Oct-4 expression than Con-Con blastocysts. These findings suggest periconceptional HFA intake induces changes in key regulators of embryo morphogenesis with potential implications for subsequent development., (© 2021 Wiley Periodicals LLC.)

Polycomb group (PcG) proteins exist in distinct multi-protein complexes and play a central role in silencing developmental genes, yet the underlying mechanisms remain elusive. Here, we show that deficiency of retinoblastoma binding protein 4 (RBBP4), a component of the Polycomb repressive complex 2 (PRC2), in embryonic stem cells (ESCs) leads to spontaneous differentiation into mesendodermal lineages. We further show that Rbbp4 and core PRC2 share an important number of common genomic targets, encoding regulators involved in early germ layer specification. Moreover, we find that Rbbp4 is absolutely essential for genomic targeting of PRC2 to a subset of developmental genes. Interestingly, we demonstrate that Rbbp4 is necessary for sustaining the expression of Oct4 and Sox2 and that the forced co-expression of Oct4 and Sox2 fully rescues the pluripotency of Rbbp4-null ESCs. Therefore, our study indicates that Rbbp4 links maintenance of the pluripotency regulatory network with repression of mesendoderm lineages., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein syntheses at appropriate timings are important for promoting diverse biological processes and are controlled at the levels of transcription and translation. Pou5f1/Oct4 is a transcription factor that is essential for vertebrate embryonic development. However, the precise timings when the mRNA and protein of Pou5f1/Oct4 are expressed during oogenesis and early stages of embryogenesis remain unclear. We analyzed the expression patterns of mRNA and protein of Pou5f1/Oct4 in mouse oocytes and embryos by using a highly sensitive in situ hybridization method and a monoclonal antibody specific to Pou5f1/Oct4, respectively. Pou5f1/Oct4 mRNA was detected in growing oocytes from the primary follicle stage to the fully grown GV stage during oogenesis. In contrast, Pou5f1/Oct4 protein was undetectable during oogenesis, oocyte maturation and the first cleavage stage but subsequently became detectable in the nuclei of early 2-cell-stage embryos. Pou5f1/Oct4 protein at this stage was synthesized from maternal mRNAs stored in oocytes. The amount of Pou5f1/Oct4 mRNA in the polysomal fraction was small in GV-stage oocytes but was significantly increased in fertilized eggs. Taken together, our results indicate that the synthesis of Pou5f1/Oct4 protein during oogenesis and early stages of embryogenesis is controlled at the level of translation and suggest that precise control of the amount of this protein by translational regulation is important for oocyte development and early embryonic development.

Somatic cells may be reprogrammed to pluripotent state by ectopic expression of certain transcription factors; namely, OCT4, SOX2, KLF4 and c-MYC. However, the molecular and cellular mechanisms are not adequately understood, especially for human embryonic development. Studies during the last five years implicated importance of OCT4 in human zygotic genome activation (ZGA), patterns of OCT4 protein folding and role of specialized sequences in binding to DNA for modulation of gene expression during development. Epigenetic modulation of OCT4 gene and post translational modifications of OCT4 protein activity in the context of multiple cancers are important issues. A consensus is emerging that chromatin organization and epigenetic landscape play crucial roles for the interactions of transcription factors, including OCT4 with the promoters and/or regulatory sequences of genes associated with human embryonic development (ZGA through lineage specification) and that when the epigenome niche is deregulated OCT4 helps in cancer progression, and how OCT4 silencing in somatic cells of adult organisms may impact ageing., Competing Interests: Declaration of Competing Interest I declare that there is no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Unlike in mice, the function of pluripotent markers in early embryonic development of domestic animals remains to be elucidated and this may account for the failure to establish embryonic stem cell lines for these species. To study the functions of the OCT-4 protein which has important actions in maintenance of pluripotent and self-renewal processes during early embryonic development, there was induced reduction in relative abundance of OCT-4 mRNA transcript during goat early embryonic development by using RNA interference techniques. The injection of OCT-4 siRNA into goat IVF presumptive zygotes resulted in a decrease in the relative abundance of OCT-4 mRNA transcript; however, there was development of these embryos to the blastocyst stage at the same rate as there was in the control group. The blastocysts from the treated groups had a similar number of TE, ICM, and total cells compared to those from the control group. Although there was a greater relative abundance of NANOG, REX1, and CDX2 mRNA transcript in the embryos injected with siRNA at the 8-16 cell stage, the relative transcript abundances were similar for the control and treatment groups at the blastocyst stage. The relative abundance of SOX2 mRNA transcript was similar for the treatment and control group. It, therefore, is concluded that inhibition of abundances of OCT-4 mRNA transcript to about 20 % of that of the untreated control group did not affect blastocyst formation rate in goats. The functions of OCT-4 in maintaining ICM and TE integrity, however, remains to be assessed., Competing Interests: Declaration of Competing Interest There is no conflict of interest in this study., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mouse embryonic stem cells (ESCs) sporadically transit into an early embryonic-like state characterized by the expression of 2-cell (2C) stage-restricted transcripts. Here, we identify a maternal factor-negative elongation factor A (NELFA)-whose heterogeneous expression in mouse ESCs is coupled to 2C gene upregulation and expanded developmental potential in vivo. We show that NELFA partners with Top2a in an interaction specific to the 2C-like state, and that it drives the expression of Dux-a key 2C regulator. Accordingly, loss of NELFA and/or Top2a suppressed Dux activation. Further characterization of 2C-like cells uncovered reduced glycolytic activity; remarkably, mere chemical suppression of glycolysis was sufficient to promote a 2C-like fate, obviating the need for genetic manipulation. Global chromatin state analysis on NELFA-induced cells revealed decommissioning of ESC-specific enhancers, suggesting ESC-state impediments to 2C reversion. Our study positions NELFA as one of the earliest drivers of the 2C-like state and illuminates factors and processes that govern this transition.

In many organisms, early embryonic development is driven by maternally provided factors until the controlled onset of transcription during zygotic genome activation. The regulation of chromatin accessibility and its relationship to gene activity during this transition remain poorly understood. Here, we generated chromatin accessibility maps with ATAC-seq from genome activation until the onset of lineage specification. During this period, chromatin accessibility increases at regulatory elements. This increase is independent of RNA polymerase II-mediated transcription, with the exception of the hypertranscribed miR-430 locus. Instead, accessibility often precedes the transcription of associated genes. Loss of the maternal transcription factors Pou5f3, Sox19b, and Nanog, which are known to be required for zebrafish genome activation, results in decreased accessibility at regulatory elements. Importantly, the accessibility of regulatory regions, especially when established by Pou5f3, Sox19b and Nanog, is predictive for future transcription. Our results show that the maternally provided transcription factors Pou5f3, Sox19b, and Nanog open up chromatin and prime genes for activity during zygotic genome activation in zebrafish., Competing Interests: The authors have declared that no competing interests exist.

We examined the allelic expression and positioning of two pluripotency-associated genes, OCT4 and SOX2, and two housekeeping genes, ACTB and TUBA, in 4- and 8-cell porcine embryos utilizing RNA and DNA fluorescence in situ hybridization (FISH) in single blastomeres. The proportion of blastomeres expressing SOX2 bi-allelically increased from 45% at the 4-cell stage to 60% at the 8-cell stage. Moreover, in 8-cell embryos, SOX2 was expressed bi-allelically in significantly more blastomeres than was the case for OCT4, and this was associated with a tendency for SOX2 alleles to move toward the nuclear interior during 4- to 8-cell transition. However, the radial location of OCT4 alleles did not change significantly during this transition. The locations of active and inactive alleles based on DNA and RNA FISH signals were also calculated. Inactive OCT4 alleles were located in very close proximity to the nuclear membrane, whereas active OCT4 alleles were more centrally disposed in the nucleus. Nevertheless, the nuclear location of active and inactive SOX2 alleles did not change in either 4- or 8-cell blastomeres. Our RNA and DNA FISH data provide novel information on the allelic expression patterns and positioning of pluripotency-associated genes, OCT4 and SOX2, during embryonic genome activation in pigs., (© 2020 The Authors. Animal Science Journal published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Animal Science.)

The scaffold protein Symplekin (Sympk) is involved in cytoplasmic RNA polyadenylation, transcriptional modulation, and the regulation of epithelial differentiation and proliferation via tight junctions. It is highly expressed in embryonic stem cells (ESCs), in which its role remains unknown. In this study, we found Sympk overexpression in mouse ESCs significantly increased colony formation, and Sympk deletion via CRISPR/Cas9 decreased colony formation. Sympk promoted ESC growth and its overexpression sustained ESC pluripotency, as assessed by teratoma and chimeric mouse formation. Genomic stability was preserved in these cells after long-term passage. The domain of unknown function 3453 (DUF3453) in Sympk was required for its interaction with the key pluripotent factor Oct4, and its depletion led to impaired colony formation. Sympk activated proliferation-related genes and suppressed differentiation-related genes. Our results indicate that Sympk interacts with Oct4 to promote self-renewal and pluripotency in ESCs and preserves genome integrity; accordingly, it has potential value for stem cell therapies. Stem Cells 2019;37:743-753., (©AlphaMed Press 2019.)

The transcription factor BTB and CNC homology 1 (Bach1) is expressed in the embryos of mice, but whether Bach1 regulates the self-renewal and early differentiation of human embryonic stem cells (hESCs) is unknown. We report that the deubiquitinase ubiquitin-specific processing protease 7 (Usp7) is a direct target of Bach1, that Bach1 interacts with Nanog, Sox2, and Oct4, and that Bach1 facilitates their deubiquitination and stabilization via the recruitment of Usp7, thereby maintaining stem cell identity and self-renewal. Bach1 also interacts with polycomb repressive complex 2 (PRC2) and represses mesendodermal gene expression by recruiting PRC2 to the genes' promoters. The loss of Bach1 in hESCs promotes differentiation toward the mesendodermal germ layers by reducing the occupancy of EZH2 and H3K27me3 in mesendodermal gene promoters and by activating the Wnt/β-catenin and Nodal/Smad2/3 signaling pathways. Our study shows that Bach1 is a key determinant of pluripotency, self-renewal, and lineage specification in hESCs.

Krüppel-like factor 4 (Klf4) is a zinc finger protein transcription factor of the KLF family and plays an important role in various important biological process, including cell proliferation, differentiation, embryonic development, and cancer formation. In this study, the full length Klf4 cDNA sequence of Japanese flounder (Paralichthys olivaceus) was analyzed. Phylogenetic, gene structure, and synteny analysis revealed that PoKlf4 was conserved to that of teleost. Transcript of PoKlf4 showed that its expression was high during early embryonic development but reduced when gastrulation began, thus indicating its possible role in embryonic development. Tissue expression pattern showed that PoKlf4 was expressed higher in ovaries than in testes. ISH revealed that PoKlf4 was also expressed in spermatogonium, oocytes, and oogonia and in high levels in epithelial and lamellar epithelial cells of gills. Functional characterization of PoKlf4 gene promoter provided valuable information in understanding the molecular regulatory mechanisms of PoKlf4 gene. PoKlf4 could up-regulate Nanog, a gene that prevents ES cells from differentiating and being up-regulated by Pou5f3 and Stat3 genes that could maintain ES cell self-renewal. These results suggested that PoKlf4 maintains the pluripotency of ES cells. This study provides valuable information in exploring the regulatory network of PoKlf4 and lays the foundation for further research on the function of this gene., (Copyright © 2018. Published by Elsevier B.V.)

Human in vitro fertilization (IVF) embryos usually have developmental block at 4-8 cell stages, in which only 30%-50% percent of IVF embryos develop to blastocyst stage. This is the main cause for low efficiency and repeated treatment failure in human fertility therapy. Transcription factor CDX2 is one of the key genes in human early embryonic development, which decides the differentiation from blastomere to trophoblastic lineage. However, the relationship between the developmental block of human IVF embryos and CDX2 is poorly understood. In this study, we showed that CDX2 is crucial for human IVF embryonic development. q-PCR analysis confirmed that the level of CDX2 in developmental block embryos was dramatically decreased. Immunofluorescence analysis further demonstrated that CDX2 protein level in developmental block embryos was significantly decreased compared with that in control embryos. We also addressed whether re-expression of CDX2 could rescue developmental block defects in human IVF developmental block embryos. As expected, these developmental block defects could be partially rescued by overexpression of CDX2 protein. In conclusion, these findings suggest that CDX2 plays an important role in the process of human early embryonic development.

The role of stem cells in tissue maintenance is appreciated and hierarchical models of stem cell self-renewal and differentiation often proposed. Stem cell activity in the male germline is restricted to undifferentiated A-type spermatogonia (A undiff ); however, only a fraction of this population act as stem cells in undisturbed testis and A undiff hierarchy remains contentious. Through newly developed compound reporter mice, here we define molecular signatures of self-renewing and differentiation-primed adult A undiff fractions and dissect A undiff heterogeneity by single-cell analysis. We uncover an unappreciated population within the self-renewing A undiff fraction marked by expression of embryonic patterning genes and homeodomain transcription factor PDX1. Importantly, we find that PDX1 marks a population with potent stem cell capacity unique to mature, homeostatic testis and demonstrate dynamic interconversion between PDX1+ and PDX1- A undiff states upon transplant and culture. We conclude that A undiff exist in a series of dynamic cell states with distinct function and provide evidence that stability of such states is dictated by niche-derived cues.

Ten-eleven translocation 3 (TET3) mediates active DNA demethylation of paternal genomes during mouse embryonic development. However, the mechanism of DNA demethylation in goat embryos remains unknown. In addition, aberrant DNA methylation reprogramming prevalently occurs in embryos cloned by somatic cell nuclear transfer (SCNT). In this study, we reported that TET3 is a key factor in DNA demethylation in goat pre-implantation embryos. Knockdown of Tet3 hindered DNA demethylation at the two- to four-cell stage in goat embryos and decreased Nanog expression in blastocysts. Overexpression of Tet3 in somatic cells can initiate DNA demethylation, reduce 5-methylcytosine level, increase 5-hydroxymethylcytosine level and promote the expression of key pluripotency genes. After SCNT, overexpression of Tet3 in donor cells corrected abnormal DNA hypermethylation of cloned embryos and significantly enhanced in vitro and in vivo developmental rate (P < 0.05). We conclude that overexpression of Tet3 in donor cells significantly improves goat SCNT efficiency., (© 2018 Federation of European Biochemical Societies.)

POU5F1 is a transcription factor and master regulator of cell pluripotency with indispensable roles in early embryo development and cell lineage specification. The role of embryonic POU5F1 in blastocyst formation and cell lineage specification differs between mammalian species but remains completely unknown in cattle. The CRISPR/Cas9 system was utilized for targeted disruption of the POU5F1 gene by direct injection into zygotes. Disruption of the bovine POU5F1 locus prevented blastocyst formation and was associated with embryonic arrest at the morula stage. POU5F1 knockout morulas developed at a similar rate as control embryos and presented a similar number of blastomeres by day 5 of development. Initiation of SOX2 expression by day 5 of development was not affected by lack of POU5F1. On the other hand, CDX2 expression was aberrant in embryos lacking POU5F1. Notably, the phenotype observed in bovine POU5F1 knockout embryos reveals conserved functions associated with loss of human embryonic POU5F1 that differ from Pou5f1- null mice. The similarity observed in transcriptional regulation of early embryo development between cattle and humans combined with highly efficient gene editing techniques make the bovine a valuable model for human embryo biology with expanded applications in agriculture and assisted reproductive technologies.

Mouse embryonic stem cells (mESCs) exist in a naive, primed and ground state of pluripotency. While comparative analyses of these pluripotency states have been reported, the mESCs utilized originated from various genetic backgrounds and were derived in different laboratories. mESC derivation in conventional LIF + serum culture conditions is strain dependent, with different genetic backgrounds potentially affecting subsequent stem cell characteristics. In the present study, we performed a comprehensive characterization of naive, primed and ground state mESCs originating from the same genetic background within our laboratory, by comparing their transcriptional profiles. We showed unique transcriptional profiles for naive, primed and ground state mESCs. While naive and ground state mESCs have more similar but not identical profiles, primed state mESCs show a very distinct profile. We further demonstrate that the differentiation propensity of mESCs to specific germ layers is highly dependent on their respective state of pluripotency.

Zebrafish pou5f3 (previously named pou2), a close homologue of mouse Oct4, encodes a PouV-family transcription factor. pou5f3 has been implicated in diverse aspects of developmental regulation during embryogenesis. In the present study, we addressed the molecular function of Pou5f3 as a transcriptional regulator and the mechanism by which pou5f3 expression is transcriptionally regulated. We examined the influence of effector genes on the expression of the luciferase gene under the control of the upstream 2.1-kb regulatory DNA of pou5f3 (Luc-2.2) in HEK293T and P19 cells. We first confirmed that Pou5f3 functions as a transcriptional activator both in cultured cells and embryos, which confirmed autoregulation of pou5f3 in embryos. It was further shown that Luc-2.2 was activated synergistically by pou5f3 and sox3, which is similar to the co-operative activity of Oct4 and Sox2 in mice, although synergy between pou5f3 and sox2 was less obvious in this zebrafish system. The effects of pou5f3 deletion constructs on the regulation of Luc-2.2 expression revealed different roles for the three subregions of the N-terminal region in Pou5f3 in terms of its regulatory functions and co-operativity with Sox3. Electrophoretic mobility shift assays confirmed that Pou5f3 and Sox3 proteins specifically bind to adjacent sites in the 2.1-kb DNA and that there is an interaction between the two proteins. The synergy with sox3 was unique to pou5f3-the other POU factor genes examined did not show such synergy in Luc-2.2 regulation. Finally, functional interaction was observed between pou5f3 and sox3 in embryos in terms of the regulation of dorsoventral patterning and convergent extension movement. These findings together demonstrate co-operative functions of pou5f3 and sox3, which are frequently coexpressed in early embryos, in the regulation of early development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Oxidative stress generates reactive oxygen species (ROS) that can promote or inhibit cardiac differentiation of stem cells dependent on the intensity of stimuli as well as cellular context in redox and differentiation status. In the current study, we confirmed that suitable intensity of hydrogen peroxide at the formation stage of embryoid bodies (EBs) effectively favored the formation of spontaneously beating cardiomyocytes from P19 embryonal carcinoma cells. Mechanistic studies implicated that extrinsic ROS enhanced the Caspase-mediated degradation of Oct4 and Nanog, two factors that governing pluripotent property. Further experiments suggested that a cohort of Nanog together with histone deacetylase 4 (Hdac4) played a critical role in establishing and maintaining the silent transcriptional status of Gata4 and Nkx2.5 in undifferentiated cells. Thus, an impulse of hydrogen peroxide depleted Nanog and Hdac4 via a caspase-dependent manner to ameliorate the repression on Gata4 and Nkx2.5 promoters, thereby generating a persistent activation on cardiac differentiation program. Meanwhile, we found that excessive ROS-activated JNK cascade to facilitate the ubiquitination and subsequent degradation of Gata4 protein. Overall, our results indicate that suitable ROS promotes the activation of Gata4 in transcription, while excessive ROS targets Gata4 protein for proteasome-dependent degradation. Gata4 is an important modulator balancing the promoting and inhibitory effects of oxidative stress on differentiation program of cardiomyogenesis.

Octamer-binding transcription factor 4 (Oct4) is a crucial pluripotent transcription factor in controlling pluripotency of embryonic stem cells (ESCs), formation of primordial germ cells (PGCs), early embryonic development, and gonadal germ cells. To understand the roles of Oct4 in the large yellow croaker (Larimichthys crocea) (Lc-Oct4), the full-length cDNA of Oct4 was cloned and analyzed. Lc-Oct4 includes a 104-bp 5' untranslated region (UTR), a 567-bp 3' UTR and a 1431-bp coding region encoding a protein of 476 amino acids (aa) with a predicted molecular mass (Mm) of 52.40 kDa and an isoelectric point (PI) of 6.34. Quantitative real time PCR (qRT-PCR) in tissues showed that Lc-Oct4 was only expressed in ovary. During ovarian development, the expression level in 635 days post hatching (635-dph) ovary was about 6.3-fold higher than in 270-dph and 1000-dph ovary. The in situ hybridization analysis showed that Lc-Oct4 had a high expression in the different developmental stages of oocytes with the highest level in stages II (later) and III oocytes. Lc-Oct4 was also expressed in spermatogonia and primary spermatocytes. Furthermore, the qRT-PCR analysis of Lc-Oct4 in different developmental embryos revealed that the expression from high to low was multiple-cell stage > mid-blastula stage = mid-gastrula stage, and the expression in multiple-cell stage was at less 1.6-fold higher than in other stages. Accordingly, the whole mount in situ hybridization (WISH) in embryos demonstrated that Lc-Oct4 was expressed only in early embryonic development from 2-cell stage to early-gastrula stage with the peak at 16-cell stage, but not detected in PGCs area. In conclusion, Lc-Oct4 participated in the oogenesis and early embryonic development in large yellow croaker. Overall, this study provided better understanding of Lc-Oct4 gene and lay the foundation for its function research in the large yellow croaker., (Copyright © 2017. Published by Elsevier B.V.)

Long noncoding RNAs (lncRNAs) have been implicated in diverse biological processes, including embryonic stem cell (ESC) maintenance. However, their functional mechanisms remain largely undefined. Here, we show that the lncRNA Panct1 regulates the transient recruitment of a putative X-chromosome-encoded protein A830080D01Rik, hereafter referred to as transient octamer binding factor 1 (TOBF1), to genomic sites resembling the canonical Oct-Sox motif. TOBF1 physically interacts with Panct1 and exhibits a cell-cycle-specific punctate localization in ESCs. At the chromatin level, this correlates with its recruitment to promoters of pluripotency genes. Strikingly, mutating an octamer-like motif in Panct1 RNA abrogates the strength of TOBF1 localization and recruitment to its targets. Taken together, our data reveal a tightly controlled spatial and temporal pattern of lncRNA-mediated gene regulation in a cell-cycle-dependent manner and suggest that lncRNAs might function as barcodes for identifying genomic addresses for maintaining cellular states., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Embryonic stem cells (ESCs) are derived from the preimplantation embryo and can differentiate into virtually any other cell type (termed pluripotency), which is governed by lineage specific transcriptions factors (TFs) binding to cis regulatory elements (CREs) to mediate changes in gene expression. The reliance on transcriptional regulation to maintain pluripotency makes ESCs a valuable model to study the role of distal CREs such as enhancers in modulating gene expression to affect cell fate decisions. This review will highlight recent advance on transcriptional enhancers, focusing on studies performed in ESCs. In addition, we argue that the Nanog locus, which encodes for an ESC-critical TF, is particularly informative because it contains multiple co-regulated genes and enhancers in close proximity to one another. The unique landscape at Nanog permits the study of ongoing questions including whether multiple enhancers function additively versus synergistically, determinants of gene specificity, and cell-to-cell variability in gene expression., (© 2017 WILEY Periodicals, Inc.)

Embryonic stem (ES) cell pluripotency is governed by OCT4-centric transcriptional networks. Conventional ES cells can be derived and maintained in vitro with media containing the cytokine leukemia inhibitory factor (LIF), which propagates the pluripotent state by activating STAT3 signaling, and simultaneous inhibition of glycogen synthase kinase-3 (GSK3) and MAP kinase/ERK kinase signaling. However, it is unclear whether overexpression of OCT4 is sufficient to overcome LIF-dependence. Here, we show that inducible expression of OCT4 (iOCT4) supports long-term LIF-independent self-renewal of ES cells cultured in media containing fetal bovine serum (FBS) and a glycogen synthase kinase-3 (GSK3) inhibitor, and in serum-free media. Global expression analysis revealed that LIF-independent iOCT4 ES cells and control ES cells exhibit similar transcriptional programs relative to epiblast stem cells (EpiSCs) and differentiated cells. Epigenomic profiling also demonstrated similar patterns of histone modifications between LIF-independent iOCT4 and control ES cells. Moreover, LIF-independent iOCT4 ES cells retain the capacity to differentiate in vitro and in vivo upon downregulation of OCT4 expression. These findings indicate that OCT4 expression is sufficient to sustain intrinsic signaling in a LIF-independent manner to promote ES cell pluripotency and self-renewal.

Pluripotent stem cell-derived human primordial germ cell-like cells (hPGCLCs) provide important opportunities to study primordial germ cells (PGCs). We robustly produced CD38 + hPGCLCs [∼43% of FACS-sorted embryoid body (EB) cells] from primed-state induced pluripotent stem cells (iPSCs) after a 72-hour transient incubation in the four chemical inhibitors (4i)-naïve reprogramming medium and showed transcriptional consistency of our hPGCLCs with hPGCLCs generated in previous studies using various and distinct protocols. Both CD38 + hPGCLCs and CD38 - EB cells significantly expressed PRDM1 and TFAP2C , although PRDM1 mRNA in CD38 - cells lacked the 3'-UTR harboring miRNA binding sites regulating mRNA stability. Genes up-regulated in hPGCLCs were enriched for cell migration genes, and their promoters were enriched for the binding motifs of TFAP2 (which was identified in promoters of T , NANOS3 , and SOX17 ) and the RREB-1 cell adhesion regulator. In EBs, hPGCLCs were identified exclusively in the outermost surface monolayer as dispersed cells or cell aggregates with strong and specific expression of POU5F1/OCT4 protein. Time-lapse live cell imaging revealed active migration of hPGCLCs on Matrigel. Whereas all hPGCLCs strongly expressed the CXCR4 chemotaxis receptor, its ligand CXCL12/SDF1 was not significantly expressed in the whole EBs. Exposure of hPGCLCs to CXCL12/SDF1 induced cell migration genes and antiapoptosis genes. Thus, our study shows that transcriptionally consistent hPGCLCs can be readily produced from hiPSCs after transition of their pluripotency from the primed state using various methods and that hPGCLCs resemble the early-stage PGCs randomly migrating in the midline region of human embryos before initiation of the CXCL12/SDF1-guided chemotaxis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

Embryonic stem cells (ESCs) are regulated by pluripotency-related transcription factors in concert with chromatin regulators. To identify additional stem cell regulators, we screened a library of endogenously labeled fluorescent fusion proteins in mouse ESCs for fluorescence loss during differentiation. We identified SET, which displayed a rapid isoform shift during early differentiation from the predominant isoform in ESCs, SETα, to the primary isoform in differentiated cells, SETβ, through alternative promoters. SETα is selectively bound and regulated by pluripotency factors. SET depletion causes proliferation slowdown and perturbed neuronal differentiation in vitro and developmental arrest in vivo, and photobleaching methods demonstrate SET's role in maintaining a dynamic chromatin state in ESCs. This work identifies an important regulator of pluripotency and early differentiation, which is controlled by alternative promoter usage., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite their fundamental biological and clinical importance, the molecular mechanisms that regulate the first cell fate decisions in the human embryo are not well understood. Here we use CRISPR-Cas9-mediated genome editing to investigate the function of the pluripotency transcription factor OCT4 during human embryogenesis. We identified an efficient OCT4-targeting guide RNA using an inducible human embryonic stem cell-based system and microinjection of mouse zygotes. Using these refined methods, we efficiently and specifically targeted the gene encoding OCT4 (POU5F1) in diploid human zygotes and found that blastocyst development was compromised. Transcriptomics analysis revealed that, in POU5F1-null cells, gene expression was downregulated not only for extra-embryonic trophectoderm genes, such as CDX2, but also for regulators of the pluripotent epiblast, including NANOG. By contrast, Pou5f1-null mouse embryos maintained the expression of orthologous genes, and blastocyst development was established, but maintenance was compromised. We conclude that CRISPR-Cas9-mediated genome editing is a powerful method for investigating gene function in the context of human development.

Despite of the importance of copper (Cu) during pregnancy, the roles of Cu-binding proteins during early embryonic development are unknown. The Cu chaperone ATOX1 was recently suggested to have additional functions related to transcription and cancer. When we analyzed single-cell RNA transcript data from early mouse embryos, Atox1 transcript levels increased dramatically at the 8-cell stage and, at 16- and 32-cell embryo stages, matched those of Oct4 which expresses a transcription factor essential for pluripotency in the inner cell mass. To explore this, we probed Atox1 expression during the first week of development of mouse embryos. ATOX1 appeared ubiquitously expressed throughout the cells until compaction; in subsequent embryo stages, ATOX1 relocalized to cytoplasmic perinuclear domains in the inner cell mass. Silencing of Oct4 did not affect Atox1 expression, but silencing of Atox1 at the 2-cell stage strongly diminished Oct4 expression in 16-cell embryos., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Transcription factors play crucial roles in patterning posterior neuroectoderm. Previously, zinc finger transcription factor znfl1 was reported to be expressed in the posterior neuroectoderm of zebrafish embryos. However, its roles remain unknown. Here, we report that there are 13 copies of znfl1 in the zebrafish genome, and all the paralogues share highly identical protein sequences and cDNA sequences. When znfl1s are knocked down using a morpholino to inhibit their translation or dCas9-Eve to inhibit their transcription, the zebrafish gastrula displays reduced expression of hoxb1b , the marker gene for the posterior neuroectoderm. Further analyses reveal that diminishing znfl1s produces the decreased expressions of pou5f3 , whereas overexpression of pou5f3 effectively rescues the reduced expression of hoxb1b in the posterior neuroectoderm. Additionally, knocking down znfl1s causes the reduced expression of sall4 , a direct regulator of pou5f3 , in the posterior neuroectoderm, and overexpression of sall4 rescues the expression of pou5f3 in the knockdown embryos. In contrast, knocking down either pou5f3 or sall4 does not affect the expressions of znfl1s Taken together, our results demonstrate that zebrafish znfl1s control the expression of hoxb1b in the posterior neuroectoderm by acting upstream of pou5f3 and sall4 ., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

HIV-1 Tat-interacting protein of 110 kDa, Tip110, plays important roles in multiple biological processes. In this study, we aimed to characterize the function of Tip110 in embryonic development. Transgenic mice lacking expression of a functional Tip110 gene (Tip110 -/- ) died post-implantation, and Tip110 -/- embryos exhibited developmental arrest between 8.5 and 9.5 days post coitum. However, in vitro cultures of Tip110 -/- embryos showed that Tip110 loss did not impair embryo growth from the zygote to the blastocyst. Extended in vitro cultures of Tip110 -/- blastocysts showed that Tip110 loss impaired both blastocyst outgrowth and self-renewal and survival of blastocyst-derived embryonic stem cells. Microarray analysis of Tip110 -/- embryonic stem cells revealed that Tip110 loss altered differentiation, pluripotency, and cycling of embryonic stem cells and was associated with downregulation of several major stem cell factors including Nanog, Oct4, and Sox2 through a complex network of signaling pathways. Taken together, these findings document for the first time the lethal effects of complete loss of Tip110 on mammalian embryonic development and suggest that Tip110 is an important regulator of not only embryonic development but also stem cell factors. Stem Cells 2017;35:1674-1686., (© 2017 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

To obtain insight into the transcription factor (TF)-dependent regulation of epiblast stem cells (EpiSCs), we performed ChIP-seq analysis of the genomic binding regions of five major TFs. Analysis of in vivo biotinylated ZIC2, OTX2, SOX2, POU5F1 and POU3F1 binding in EpiSCs identified several new features. (1) Megabase-scale genomic domains rich in ZIC2 peaks and genes alternate with those rich in POU3F1 but sparse in genes, reflecting the clustering of regulatory regions that act at short and long-range, which involve binding of ZIC2 and POU3F1, respectively. (2) The enhancers bound by ZIC2 and OTX2 prominently regulate TF genes in EpiSCs. (3) The binding sites for SOX2 and POU5F1 in mouse embryonic stem cells (ESCs) and EpiSCs are divergent, reflecting the shift in the major acting TFs from SOX2/POU5F1 in ESCs to OTX2/ZIC2 in EpiSCs. (4) This shift in the major acting TFs appears to be primed by binding of ZIC2 in ESCs at relevant genomic positions that later function as enhancers following the disengagement of SOX2/POU5F1 from major regulatory functions and subsequent binding by OTX2. These new insights into EpiSC gene regulatory networks gained from this study are highly relevant to early stage embryogenesis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Embryonic stem cells co-express Oct4 and Oct1, a related protein with similar DNA-binding specificity. To study the role of Oct1 in ESC pluripotency and transcriptional control, we constructed germline and inducible-conditional Oct1-deficient ESC lines. ESCs lacking Oct1 show normal appearance, self-renewal and growth but manifest defects upon differentiation. They fail to form beating cardiomyocytes, generate neurons poorly, form small, poorly differentiated teratomas, and cannot generate chimeric mice. Upon RA-mediated differentiation, Oct1-deficient cells induce lineage-appropriate developmentally poised genes poorly while lineage-inappropriate genes, including extra-embryonic genes, are aberrantly expressed. In ESCs, Oct1 co-occupies a specific set of targets with Oct4, but does not occupy differentially expressed developmental targets. Instead, Oct1 occupies these targets as cells differentiate and Oct4 declines. These results identify a dynamic interplay between Oct1 and Oct4, in particular during the critical window immediately after loss of pluripotency when cells make the earliest developmental fate decisions.

In the present study, we assessed whether absence of paternal genome imparts any differential response in embryos to chemical stress such as ammonia. Parthenogenesis was induced in MII stage oocytes using 10 mM SrCl 2 in M16 medium. Parthenotes and normally fertilized embryos at 2 cell stage were exposed to different concentrations of ammonia and cultured till blastocyst. Exposure of ammonia to normally fertilized embryos resulted in significant decrease in the developmental potential (p < 0.0001) and blastocyst quality (p < 0.001). Whereas, in parthenotes, even though lower concentrations of ammonia did not have any effect, at 200 μM concentration the blastocyst rate was two times higher than control. The baseline apoptotic index was higher in parthenotes compared to normally fertilized embryos, which further increased after ammonium exposure (p < 0.001). Unlike in normally fertilized embryos ammonia exposure altered the mitochondrial distribution pattern and lead to increased expression of Oct4, Nanog and Na + /K + ion exchange channel, while the cytochrome C expression was downregulated. This indicates that haploidy and/or absence of paternal factors in the embryo results in differential tolerance to stress induced by ammonia., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Transition from symmetric to asymmetric cell division requires precise coordination of differential gene expression. We show that embryonic stem cells (ESCs) mainly express DIDO3 and that their differentiation after leukemia inhibitory factor withdrawal requires DIDO1 expression. C-terminal truncation of DIDO3 (Dido3ΔCT) impedes ESC differentiation while retaining self-renewal; small hairpin RNA-Dido1 ESCs have the same phenotype. Dido3ΔCT ESC differentiation is rescued by ectopic expression of DIDO3, which binds the Dido locus via H3K4me3 and RNA POL II and induces DIDO1 expression. DIDO1, which is exported to cytoplasm, associates with, and is N-terminally phosphorylated by PKCiota. It binds the E3 ubiquitin ligase WWP2, which contributes to cell fate by OCT4 degradation, to allow expression of primitive endoderm (PE) markers. PE formation also depends on phosphorylated DIDO3 localization to centrosomes, which ensures their correct positioning for PE cell polarization. We propose that DIDO isoforms act as a switchboard that regulates genetic programs for ESC transition from pluripotency maintenance to promotion of differentiation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Abnormal epigenetic modifications are considered a main contributing factor to low cloning efficiency. In the present study, we explored the effects of quisinostat, a novel histone deacetylase inhibitor, on blastocyst formation rate in porcine somatic-cell nuclear transfer (SCNT) embryos, on acetylation of histone H3 lysine 9 (AcH3K9), and on expression of POU5F1 protein and apoptosis-related genes BAX and BCL2. Our results showed that treatment with 10 nM quisinostat for 24 hr significantly improved the development of reconstructed embryos compared to the untreated group (19.0 ± 1.6% vs. 10.2 ± 0.9%; p < 0.05). Quisinostat-treated SCNT embryos also possessed significantly increased AcH3K9 at the pseudo-pronuclear stage (p < 0.05), as well as improved immunostaining intensity for POU5F1 at the blastocyst stage (p < 0.05). While no statistical difference in BAX expression was observed, BCL2 transcript abundance was significantly different in the quisinostat-treated compared to the untreated control group. Of the 457 quisinostat-treated cloned embryos transferred into three surrogates, six fetuses developed from the one sow that became pregnant. These findings suggested that quisinostat can regulate gene expression and epigenetic modification, facilitating nuclear reprogramming, and subsequently improving the developmental competence of pig SCNT embryos and blastocyst quality., (© 2017 Wiley Periodicals, Inc.)

Objective: In the current study we aimed to evaluate the effect of embryo transfer on gene expression during pre-implantation development and its consequences on implantation rate, offspring rate at birth and embryonic and fetal losses in the rabbit model., Study Design: The mRNA expressions of 8 candidate genes were compared between 6-day-old in vivo-produced embryos (non-manipulated embryos) to those of 6-day-old embryos previously recovery at the third day of development and transferred into recipient rabbit females (manipulated embryos). Furthermore, we compared between both experimental groups the implantation rate and offspring rate at birth and embryonic and fetal losses., Results: Differences in transcript abundance of OCT4, C1qTNF1, EMP1 and TNFAIP6 were observed in transferred embryos. In addition, lower implantation and offspring rates at birth were obtained in transferred embryos than in the control group. In addition, embryonic losses were significantly higher in the transferred group than in the control. However, fetal losses were similar between groups., Conclusion: The findings of the current study show that embryo transfer manipulation influenced mRNA expression of late blastocysts prior to implantation, resulting in higher gestational losses as a consequence of faulty embryonic implantation., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Translational control of gene expression plays a key role during the early phases of embryonic development. Here we describe a transcriptional regulator of mouse embryonic stem cells (mESCs), Yin-yang 2 (YY2), that is controlled by the translation inhibitors, Eukaryotic initiation factor 4E-binding proteins (4E-BPs). YY2 plays a critical role in regulating mESC functions through control of key pluripotency factors, including Octamer-binding protein 4 (Oct4) and Estrogen-related receptor-β (Esrrb). Importantly, overexpression of YY2 directs the differentiation of mESCs into cardiovascular lineages. We show that the splicing regulator Polypyrimidine tract-binding protein 1 (PTBP1) promotes the retention of an intron in the 5'-UTR of Yy2 mRNA that confers sensitivity to 4E-BP-mediated translational suppression. Thus, we conclude that YY2 is a major regulator of mESC self-renewal and lineage commitment and document a multilayer regulatory mechanism that controls its expression., Competing Interests: The authors declare no conflict of interest.

The embryonic basal ganglia generates multiple projection neurons and interneuron subtypes from distinct progenitor domains. Combinatorial interactions of transcription factors and chromatin are thought to regulate gene expression. In the medial ganglionic eminence, the NKX2-1 transcription factor controls regional identity and, with LHX6, is necessary to specify pallidal projection neurons and forebrain interneurons. Here, we dissected the molecular functions of NKX2-1 by defining its chromosomal binding, regulation of gene expression, and epigenetic state. NKX2-1 binding at distal regulatory elements led to a repressed epigenetic state and transcriptional repression in the ventricular zone. Conversely, NKX2-1 is required to establish a permissive chromatin state and transcriptional activation in the sub-ventricular and mantle zones. Moreover, combinatorial binding of NKX2-1 and LHX6 promotes transcriptionally permissive chromatin and activates genes expressed in cortical migrating interneurons. Our integrated approach provides a foundation for elucidating transcriptional networks guiding the development of the MGE and its descendants., (Published by Elsevier Inc.)

POU5F1 (OCT4) is a crucial transcription factor for induction and maintenance of cellular pluripotency, as well as survival of germ cells in mammals. However, the homologues of POU5F1 in teleost fish, including zebrafish and medaka, now named Pou5f3, exhibit considerable differences in expression pattern and pluripotency-maintaining activity. To what extent the POU5F1 homologues are conserved in vertebrates has been unclear. In this study, we report that the POU5F1 homologue from the Nile tilapia (Oreochromis niloticus), OnPou5f3, displays an expression pattern and biological activity somewhat different from those in zebrafish or medaka. The expression of Onpou5f3 at both mRNA and protein levels was abundant in early development embryos until blastula stages, barely detectable as proceeding, and then displayed a transiently strong expression domain in the brain region during neurula stages similar to zebrafish but not medaka. Afterward, OnPou5f3 appeared as germline-restricted (including primordial germ cells and female and male gonad germ cells) expression just like medaka. Notably, OnPou5f3 depletion through morpholino oligos caused blastula blockage or lethality and failure of survival and proliferation of blastula cell-derived cells. These findings indicate that equivalent POU5F1-like expression and activity of Pou5f3 might be conserved accompanying with species-specific expression pattern during evolution. Our study provides insight into the evolutionary conservation of the POU5F1 homologues across vertebrates.

Considerable progress has been made in understanding the roles of Sox2 and Oct4 in embryonic stem cells and mammalian embryogenesis. Specifically, significant progress has been made in answering three questions about the functions of Sox2 and Oct4, which are the focus of this review. 1) Are the first or second cell lineage decisions during embryogenesis controlled by Oct4 and/or Sox2? 2) Do the levels of Oct4 and Sox2 need to be maintained within narrow limits to promote normal development and to sustain the self-renewal of pluripotent stem cells? 3) Do Oct4 and Sox2 work closely together or is the primary role of Sox2 in pluripotent cells to ensure the expression of Oct4? Although significant progress has been made in answering these questions, additional studies are needed to resolve several important remaining issues. Nonetheless, the preponderance of the evidence suggests there is considerable crosstalk between Sox2 and Oct4, and further suggests Sox2 and Oct4 function as molecular rheostats and utilize negative feedback loops to carefully balance their expression and other critical genes during embryogenesis. This article is part of a Special Issue entitled: The Oct transcription factor family, edited by Dr. Dean Tantin., (Copyright © 2016 Elsevier B.V. All rights reserved.)