Endoderm and mesoderm are both formed upon activation of Nodal signaling but how endoderm differentiates from mesoderm is still poorly explored. The sox-related gene casanova (sox32) acts downstream of the Nodal signal, is essential for endoderm development and requires the co-factor Pou2 (Pou5f1, Oct3, Oct4) in this process. Conversely, BMP signals have been shown to inhibit endoderm development by an as yet unexplained mechanism. In a search for Casanova regulators in zebrafish, we identified two of its binding partners as the transcription factors Pou2 and Vox, a member of the Vent group of proteins also involved in the patterning of the gastrula. In overexpression studies we show that vox and/or Vent group genes inhibit the capacity of Casanova to induce endoderm, even in the presence of its co-factor Pou2, and that Vox acts as a repressor in this process. We further show that vox, but not other members of the Vent group, is essential for defining the proper endodermal domain size at gastrulation. In this process, vox acts downstream of BMPs. Cell fate analysis further shows that Vox plays a key role downstream of BMP signals in regulating the capacity of Nodal to induce endoderm versus mesoderm by modulating the activity of the Casanova/Pou2 regulatory system.

BACKGROUND

The cellular signaling mechanisms and morphogenic movements involved in axis formation and gastrulation are well conserved between vertebrates. In nearly all described fish, gastrulation and the initial patterning of the embryonic axis occur concurrently with epiboly. However, annual killifish may be an exception to this norm. Annual killifish inhabit ephemeral ponds in South America and Africa and permanent populations persist by the production of stress-tolerant eggs. Early development of annual killifish is unique among vertebrates because their embryonic blastomeres disperse randomly across the yolk during epiboly and reaggregate several days later to form the embryo proper. In addition, annual killifish are able to arrest embryonic development in one to three stages, known as diapause I, II, and III. Little is known about how the highly conserved developmental signaling mechanisms associated with early vertebrate development may have shifted in order to promote the annual killifish phenotype. One of the most well-characterized and conserved transcription factors, oct4 (Pou5f1), may have a role in maintaining pluripotency. In contrast, BMP-antagonists such as chordin, noggin, and follistatin, have been previously shown to establish dorsal-ventral asymmetry during axis formation. Transcription factors from the SOXB1 group, such as sox2 and sox3, likely work to induce neural specification. Here, we determine the temporal expression of these developmental factors during embryonic development in the annual killifish Austrofundulus limnaeus using quantitative PCR and compare these patterns to other vertebrates.

RESULTS

Partial transcript sequences to oct4, sox2, sox3, chordin, noggin-1, noggin-2, and follistatin were cloned, sequenced, and identified in A. limnaeus. We found oct4, sox3, chordin, and noggin-1 transcripts to likely be maternally inherited. Expression of sox2, follistatin, and noggin-2 transcripts were highest in stages following a visible embryonic axis.

CONCLUSIONS

Our data suggest that embryonic cells acquire their germ layer identity following embryonic blastomere reaggregation in A. limnaeus. This process of cellular differentiation and axis formation may involve similar conserved signaling mechanisms to other vertebrates. We propose that the undifferentiated state is prolonged during blastomere dispersal, thus functioning as a developmental stress buffer prior to the establishment of embryonic asymmetry and positional identity among the embryonic cells.

This study investigated the effects on fertilized embryo development of somatic cytoplasm after its injection into intact mouse oocytes. Mature oocytes collected from female B6D2F1 mice were injected with cumulus cell cytoplasm of different volumes and from different mouse strains (B6D2F1, ICR, and C57BL/6), or with embryonic cytoplasm. After culture for 1 h, B6D2F1 sperm were injected into those oocytes by intracytoplasmic sperm injection (ICSI). The oocytes were examined for pre- and postimplantation developmental competence. Increases in the volume of the somatic cytoplasm from onefold to fourfold resulted in an impairment of blastocyst development and full-term development (28% and 7%, respectively, vs. 96% and 63%, respectively, in the control group; P < 0.01). An increase in the volume of somatic cytoplasm reduced the expression of POU5F1 (more commonly known as OCT4) in expanded blastocysts. The frequency of embryos that developed to the blastocyst stage did not differ when B6D2F1 or ICR somatic cytoplasm was injected, but injection of C57BL/6 somatic cytoplasm induced a two-cell block in embryo development. Injection of the cytoplasm from fertilized embryos did not reduce the frequency of embryos attaining full-term development. Interestingly, somatic cytoplasm significantly increased the placental weight of ICSI embryos, even the injection of onefold cytoplasm (0.20 +/- 0.02 [n = 32] vs. 0.12 +/- 0.02 in the control group [n = 87]; P < 0.01). These findings indicate that the injection of somatic cytoplasm into oocytes before ICSI causes a decrease in preimplantation development, clearly impairs full-term development, and causes placental overgrowth in fertilized embryos. To our knowledge, placental overgrowth phenotypes are only caused by interspecies hybridization and cloning, and in genetically modified mice. Here, we report for the first time that somatic cytoplasm causes abnormal placentas in fertilized embryos. This study suggests that somatic cell cytoplasmic material is one cause of the low rate of full-term development in cloned mammals.

Cloned mouse embryo development to blastocyst stage correlates positively with the expression level of Oct4 (Pou5f1) at the morula stage, as reported previously by our laboratory. However, whether this correlation is based on a cause-effect relationship has remained unclear. To address this question, we artificially increased the level of Oct4 prior and subsequent to somatic cell nuclear transfer, by microinjection of Oct4 mRNA into ooplasts and by transgenic Oct4 induction at the morula stage, respectively. We observed higher developmental rates of cloned embryos to blastocyst when higher levels of Oct4 were superimposed with the initial reprogramming events; whereas increasing Oct4 at later stages of preimplantation development did not have a significant effect on developmental rates. Our results show that supplemental Oct4 facilitates oocyte-mediated reprogramming only during the first cleavages, implying that the higher Oct4 level observed in developmentally competent cloned morulae is a readout of reprogramming events that successfully took place earlier.

The differentiation of embryonic stem cells (ESCs) into neurons and glial cells represents a promising cell-based therapy for neurodegenerative diseases. Because the rhesus macaque is physiologically and phylogenetically similar to humans, it is a clinically relevant animal model for ESC research. In this study, the pluripotency and neural differentiation potential of a rhesus monkey ESC line (ORMES6) was investigated. ORMES6 was derived from an in vitro produced blastocyst, which is the same way human ESCs have been derived. ORMES6 stably expressed the embryonic transcription factors POU5F1 (Oct4), Sox2 and NANOG. Stage-specific embryonic antigen 4 (SSEA 4) and the glycoproteins TRA-1-60 and TRA-1-81 were also expressed. The embryoid bodies (EBs) formed from ORMES6 ESCs spontaneously gave rise to cells of three germ layers. After exposure to basic fibroblast growth factor (bFGF) for 14-16 days, columnar rosette cells formed in the EB outgrowths. Sox2, microtubule-associated protein (MAP2), beta-tublinIII and glial fibrillary acidic protein (GFAP) genes and Nestin, FoxD3, Pax6 and beta-tublinIII antigens were expressed in the rosette cells. Oct4 and NANOG expression were remarkably down-regulated in these cells. After removal of bFGF from the medium, the rosette cells differentiated along neural lineages. The differentiated cells expressed MAP2, beta-tublinIII, Neuro D and GFAP genes. Most differentiated cells expressed early neuron-specific antigen beta-tublinIII (73+/-4.7%) and some expressed intermediate neuron antigen MAP2 (18+/-7.2%). However, some differentiated cells expressed the glial cell antigens A2B5 (7.17%+/-1.2%), GFAP (4.93+/-1.9%), S100 (7+/-3.5%) and O4 (0.27+/-0.2%). The rosette cells were transplanted into the striatum of immune-deficient NIHIII mice. The cells persisted for approximately 2 weeks and expressed Ki67, NeuN, MAP2 and GFAP. These results demonstrate that the rhesus monkey ESC line ORMES6 retains the pluripotent characteristics of ESCs and can be efficiently induced to differentiate along neural lineages.

Induced pluripotent stem (iPS) cells are a new alternative for the development of patient-specific stem cells, and the aim of this study was to determine whether differences exist between the cellular and molecular profiles of iPS cells, generated using lentiviral vectors, compared to ES cells. The lentiviral infection efficiency differed according to the method of cell culture (adherent cells: 0.085%; suspended cells: 0.785%). Six iPS cell lines exhibited typical ES cell morphology and marker expression, but varied in their in vitro/in vivo differentiation ability. Global gene transcription analysis revealed that core pluripotency genes were expressed at lower levels in iPS cell lines compared to D3-ES cells (Pou5f1: x1.6~2.2-fold, Sox2: x2.58~10.0-fold, Eras: x1.08~2.54-fold, Dppa5a: x1.04~1.41-fold), while other genes showed higher expression in iPS cells (Lin28: x1.43~2.33-fold; Dnmt3b: x1.33~2.64-fold). This pattern was repeated in a survey of specific functional groups of genes (surface markers, cell death, JAK-STAT and P13K-AKT signaling pathways, endothelial, cardiovascular, and neurogenesis genes). Among the iPS cell lines examined, only two showed similar characteristics to ES cells. These results demonstrated that, in addition to cellular characterization, the numerical evaluation of gene expression using DNA microarrays might help to identify the stem cell stability and pluripotency of iPS cells.

Maintenance of pluripotency in stem cells is tightly regulated among vertebrates. One of the key genes in this process is oct4, also referred to as pou5f1 in mammals and pou2 in teleosts. Pou5f1 evolved by duplication of pou2 early in the tetrapod lineage, but only monotremes and marsupials retained both genes. Either pou2 or pou5f1 was lost from the genomes of the other tetrapods that have been analyzed to date. Consequently, these two homologous genes are often designated oct4 in functional studies. In most vertebrates oct4 is expressed in pluripotent cells of the early embryo until the blastula stage, and later persist in germline stem cells until adulthood. The isolation and analysis of stem cells from embryo or adult individuals is hampered by the need for reliable markers that can identify and define the cell populations. Here, we report the faithful expression of EGFP under the control of endogenous pou2/oct4 promoters in transgenic medaka (Oryzias latipes). In vivo imaging in oct4-EGFP transgenic medaka reveals the temporal and spatial expression of pou2 in embryos and adults alike. We describe the temporal and spatial patterns of endogenous pou2 and oct4-EGFP expression in medaka with respect to germline and adult stem cells, and discuss applications of oct4-EGFP transgenic medaka in reproductive and stem cell biology.

Currently, our knowledge of early mammalian embryogenesis, stem cell differentiation and development is largely based on studies performed in mouse models. However, in important aspects, e.g. the timing of epigenetic reprogramming and embryonic genome activation, livestock species probably reflect far more closely the situation in men and other non-rodent mammals. A major challenge is the fact that in mammals, the development of individual zygotes is highly variable and vulnerable, and the outcome is uncertain. Valid indicators of the highly heterogeneous development and health status, and the actual developmental potential of individual oocytes, zygotes or embryos would be crucially important to tap the full power of holistic transcriptome and proteome analyses. Fluorescent reporter proteins opened new vistas for embryology and stem cell research: they can be used as reporters for the activity of gene promoters or tagged to functional proteins to study their intracellular localization in living cells, tissues and organisms. Fluorescent reporter genes may be used to microscopically observe key processes of early development. Thus, novel information related to developmental potential can be obtained from living embryos before processing them, e.g. for "-omic" studies. This review summarizes the main current reporter gene techniques and gene transfer approaches, which might be suitable for the investigation of early embryogenesis in livestock mammals. The potential of promoter reporter genes is exemplified by a bovine model system for quantitative monitoring of transcriptional reactivation of the so-called pluripotency gene POU5F1 in cloned bovine embryos.

The development of somatic cell nuclear transfer (SCNT) embryos critically depends on appropriate reprogramming and expression of pluripotency genes, such as Pou5f1/POU5F1 (previously known as Oct4/OCT4). To study POU5F1 transcription activation in living bovine SCNT embryos without interference by maternal POU5F1 mRNA, we generated chromosomally normal fetal fibroblast donor cells stably carrying a mouse Pou5f1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a single integration site without detectable EGFP expression. Morphologic and quantitative analyses of whole-mount SCNT embryos by confocal microscopy revealed robust initial activation of the Pou5f1 reporter gene during the fourth cell cycle. In Day 6 SCNT embryos EGFP expression levels were markedly higher than in Day 4 embryos but varied substantially between individual embryos, even at comparable cell numbers. Embryos with low EGFP levels had far more morphologically abnormal cell nuclei than those with high EGFP levels. Our data strongly suggest that bovine SCNT embryos consistently start activation of the POU5F1 promoter during the fourth cell cycle, whereas later in development the expression level substantially differs between individual embryos, which may be associated with developmental potential. In fibroblasts from phenotypically normal SCNT fetuses recovered on Day 34, the Pou5f1 reporter promoter was silent but was activated by a second round of SCNT. The restoration of pluripotency can be directly observed in living cells or SCNT embryos from such Pou5f1-EGFP transgenic fetuses, providing an attractive model for systematic investigation of epigenetic reprogramming in large mammals.

Analysis of gene expression changes during preimplantation development by quantitative reverse transcription-polymerase chain reaction (Q-PCR) requires appropriate internal standards. Ideally, such a gene should show a constant level of transcripts per embryo across all preimplantation stages from unfertilized eggs to blastocysts. By analysing the microarray-based gene expression profiles of preimplantation embryos, it was found that a conserved helix-loop-helix ubiquitous kinase gene (Chuk, also known as IkappaB kinase alpha, IKKalpha or IKK1) satisfied this criterion. To test the utility of this gene as an internal standard for Q-PCR, the expression levels of two known genes (Nalp5/Mater, Pou5f1/Oct3/Oct4) were normalized by Chuk and other housekeeping genes (Actb, Gapdh, Eef1a1, and H2afz) and demonstrated that the former was more consistent with the expression patterns obtained by a whole-mount in-situ hybridization than those reported previously with the latter. It is concluded that Chuk, unlike other commonly used normalization controls, is a reliable and suitable internal standard for measuring gene expression levels by Q-PCR in mouse oocytes and preimplantation embryos.

Miniature pigs share many similar characteristics such as anatomy, physiology and body size with humans and are expected to become important animal models for therapeutic cloning using embryonic stem cells (ESCs) derived by somatic cell nuclear transfer (SCNT). In the present study, we observed that miniature pig SCNT blastocysts possessed a lower total number of nuclei and a lower percentage of POU5F1-positive cells than those possessed by in vitro fertilized (IVF) blastocysts. To overcome these problems, we evaluated the applicability of aggregating miniature pig SCNT embryos at the four-cell stage. We showed that (i) aggregation of two or three miniature pig SCNT embryos at the four-cell stage improves the total number of nuclei and the percentage of POU5F1-positive cells in blastocysts, and (ii) IVF blastocysts with low cell numbers induced by the removal of two blastomeres at the four-cell stage did not exhibit a decrease in the percentage of POU5F1-positive cells. These results suggest that the aggregation of miniature pig SCNT embryos at the four-cell stage can be a useful technique for improving the quality of miniature pig SCNT blastocysts and indicating that improvement in the percentage of POU5F1-positive cells in aggregated SCNT embryos is not simply the consequence of increased cell numbers.

Klinefelter's syndrome is a male sex-chromosomal disorder (47,XXY), causing hypogonadism, cognitive and metabolic deficits. The majority of patients are infertile due to complete germ cell loss after puberty. As the depletion occurs during development, the possibilities to study the underlying causes in humans are limited. In this study, we used the 41,XX(Y*) mouse model to characterise the germ line postnatally. We examined marker expression of testicular cells focusing on the spermatogonial stem cells (SSCs) and found that the number of germ cells was approximately reduced fivefold at day 1pp in the 41,XX(Y*) mice, indicating the loss to start prenatally. Concurrently, immunohistochemical SSC markers LIN28A and PGP9.5 also showed decreased expression on day 1pp in the 41,XX(Y*) mice (48.5 and 38.9% of all germ cells were positive), which dropped to 7.8 and 7.3% on 3dpp, and were no longer detectable on days 5 and 10pp respectively. The differences in PCNA-positive proliferating cells in XY* and XX(Y*) mice dramatically increased towards day 10pp. The mRNA expression of the germ cell markers Lin28a (Lin28), Pou5f1 (Oct4), Utf1, Ddx4 (Vasa), Dazl, and Fapb1 (Sycp3) was reduced and the Lin28a regulating miRNAs were deregulated in the 41,XX(Y*) mice. We suggest a model for the course of germ cell loss starting during the intrauterine period. Neonatally, SSC marker expression by the already lowered number of spermatogonia is reduced and continues fading during the first postnatal week, indicating the surviving cells of the SSC population to be disturbed in their stem cell characteristics. Subsequently, the entire germ line is then generally lost when entering meiosis.

Murine embryonic stem cells (mESCs) inoculated at passage P13 with the mycoplasma species M. hominis, M. fermentans and M. orale and cultured over 20 passages showed reduced growth rate and viability (P < 0.0001) compared to control mESCs. Spectral karyotypic analysis of mycoplasma-infected mESCs showed a number of non-clonal chromosomal aberrations which increased with the duration of infection. The differentiation status of the infected mESCs was most affected at passage P13+6 where the infection was strongest and 46.3% of the mESCs expressed both POU5F1 and SSEA-1 markers whereas 84.8% of control mESCs expressed both markers. The percentage of germline chimeras from mycoplasma-infected mESCs was examined after blastocyst injection and embryo transfer to suitable recipients at different passages and, compared to the respective control group, was most affected at passage P13+5 (50% vs. 90%; P < 0.07). Further reductions were obtained at the same passage in the percentage of litters born (50% vs. 100%; P < 0.07) and in the percentage of pups born (22% vs. 45%; P < 0.001). Thirty three chimeras (39.8%) obtained from blastocyst injection with mycoplasma-infected mESCs showed reduced body weight (P < 0.0001), nasal discharge, osteoarthropathia, and cachexia. Flow cytometric analysis of plasma from chimeras produced with mycoplasma-infected mESCs revealed statistically significant differences in the proportions of T-cells and increased levels of IgG1 (P < 0.001), IgG2a (P < 0.05) and IgM (P < 0.05), anti-DNA antibodies (P < 0.05) and rheumatoid factor (P < 0.01). The present data indicate that mycoplasma contamination of mESCs affects various cell parameters, germline transmission, and postnatal development of the resulting chimeras.

The Sox17 is an important transcription factor for endodermal cells (Danio rerio). According to the predictions of the GRNs, based on perturbation experiments and literature search, the sox17 gene is engaged with two other regulatory genes, sox32 and pou5f1. Nodal signaling operated on several endoderm-specific transcription factors to determine the endoderm specification. In addition, endoderm specification requires the Fgf and Bmp signaling pathways to be repressed in the cells which will become endoderm. It is predicted that Nodal activates sox32 and works synergistically with Pou5f1 to activate sox17. Bmp represses the expression of sox17 on the ventral side and Fgf represses it on the dorsal side. The regulatory inputs of sox17 at the genomic sequence level are not known. Here, we have uncovered the relevant sox17 cis-regulatory elements, and examined the specific input predictions of the GRNs. We discovered three conserved modules, A, B, and C, with a synergistic effect among them. We revealed that the Pou5f1-binding element on the B module and the Sox32-binding element on the C module work synergistically. Furthermore, an evolutionarily non-conserved R module exhibits a repressive effect on both the ventral and dorsal side. We have directly demonstrated the structural and functional relationships of the genomic code at this key node of the endoderm GRNs in zebrafish development. This information provides new insight into the complexity of endoderm formation and serves as a valuable resource for the establishment of a complete endoderm gene regulatory network.

In this study, fibroblast cells were stably transfected with mouse POU5F1 promoter-driven enhanced green fluorescent protein (EGFP) to investigate the effect of S-adenosylhomocysteine (SAH), the reversible non-toxic inhibitor of DNA-methyltransferases (DNMTs), at different intervals post-fusion on in vitro development of cloned bovine embryos. Treatment with SAH for 12 hr resulted in 54.6 ± 7.7% blastocyst production, which was significantly greater than in vitro fertilized embryos (IVF: 37.2 ± 2.7%), cloned embryos treated with SAH for 72 hr (31.0 ± 7.6%), and control cloned embryos (34.6 ± 3.6%). The fluorescence intensities of the EGFP-POU5F1 reporter gene at all intervals of SAH treatment, except of 72 hr, were significantly higher than control somatic cell nuclear transfers (SCNT) embryos. The intensity of DNA-methylation in cloned embryos treated with SAH for 48 hr was similar to that of IVF embryos, and was significantly lower than the other SCNT groups. The levels of H3K9 acetylation in all SCNT groups were significantly lower than IVF embryos. Real-time PCR analysis of gene expression revealed significantly higher expression of POU5F1 in cloned versus IVF blastocysts. Neither embryo production method (SCNT vs. IVF) nor the SAH treatment interval affected expression of the BCL2 gene. Cloned embryos at all intervals of SAH treatment, except for 24 hr, had significantly increased VEGF transcript compared to IVF and control SCNT embryos. It was suggested that the time interval of DNMT inhibition may have important consequences on different in vitro features of bovine SCNT, and the improving effects of DNMT inhibition on developmental competency of cloned embryos are restricted to a specific period of time preceding de novo methylation.

During the peri-implantation development of the mouse embryo from the blastocyst through gastrulation, Pou5f1 (OCT-4) down-regulation is closely linked to the initial step of lineage allocation to extraembryonic and embryonic somatic tissues. Subsequently, differentiation of the lineage precursors is subject to inductive tissue interactions and intercellular signalling that regulate cell proliferation and the acquisition of lineage-specific morphological and molecular characteristics. A notable variation of this process of lineage specification is the persistence of Pou5f1 activity throughout the differentiation of the primordial germ cells, which may underpin their ability to produce pluripotent progeny either as stem cells (embryonic germ cells) in vitro or as gametes in vivo. Nevertheless, intercellular signalling still plays a critical role in the specification of the primordial germ cells. The findings that primordial germ cells can be induced from any epiblast cells and that they share common progenitors with other somatic cells provide compelling evidence for the absence of a pre-determined germ line in the mouse embryo.

Differentiation of certain cell types is followed by a downregulation of PARP1 expression. We show that the reduction in the abundance of PARP1 in hematopoietic progenitor cells and monocytes is tightly controlled by the cell cycle. The differentiation-associated cell cycle exit induces E2F1 replacement with E2F4 at the PARP1 promoter and the assembly of an E2F4-RBL2-HDAC1-BRM(SWI/SNF) repressor complex which deacetylates nucleosomes and compacts chromatin. In G1 arrested cells, PARP1 transcription is reduced by the recruitment of E2F1-RB1-HDAC1-EZH2(PRC2)-BRM/BRG1(SWI/SNF), which additionally trimethylates H3K27 and causes an even higher increase in nucleosome density. The re-establishment of an active chromatin structure by treating post-mitotic monocytes with the HDAC inhibitor and G1 arrested cells with a combination of HDAC and EZH2 inhibitors restores PARP1 expression completely but does not affect the interaction between the components of the repressor complex with chromatin. This suggests that RB1 and RBL2, as well as PRC2, SWI/SNF and HDAC1, do not interfere with the transcription machinery. Interestingly, reinstatement of PARP1 expression by the silencing of RBL2 or by the inhibition of HDACs in monocytes and by transfection with the PARP1 expression vector in differentiated THP-1 cells substantially increased transcription of pluripotency stem cell factors such as POU5F1, SOX2 and NANOG.

Pigs are valuable animal models in pre-clinical research due to their anatomical and similarity to human-beings. Little is known about porcine embryonic development and porcine pluripotent stem cells. Recently, porcine-induced pluripotent stem cells (piPSCs) have been generated with Oct4 (Pou5f1), Sox2, Klf4 and c-Myc (termed OSKM, 4 F). Here, we found two other factors (Tbx3 and Nr5α2, termed TN), with important roles in piPSCs induction. They could improve the generation of piPSCs by supplementing these two factors on the basis of OSKM (OSKMTN, 6 F) orientated to mouse ESCs-like. Surprisingly, Nr5α2 alone could induce piPSCs formation in the presence or absence of c-Myc. These results suggested that Tbx3 and Nr5α2 may have vital roles in Sus scrofa and proposed new insights into pig pluripotent stem cells.

Epithelial ovarian cancer (EOC) is the deadliest gynecologic cancer. Recently, the existence of ovarian cancer stem cells has been reported. Sox2, Nanog and Oct4 are key markers of "stemness". The objective of this study was to determine whether Sox2, Nanog, and Oct4 are associated with EOC and poor outcome. The expression of these markers was assessed by immunofluorescence staining and real-time RT-PCR in human EOC cell lines MDAH-2774 and SKOV-3, while the cancer genome atlas (TCGA) dataset was analyzed for associations with survival. Sox2, Nanog and Oct4 (POU5F1) were all detected by immunofluorescence staining and these results were confirmed by real-time RT-PCR. The TCGA dataset revealed a 26%, 9%, and 6% amplification of Sox2, Nanog and POU5F1, respectively. Additionally, K-M survival analyses showed a significant median overall survival difference (41 versus 48.3 months, P = .01) for Sox2 amplification, but not for Nanog (44.1 versus 36.2 months, P>> .05) and POU5F1 (43.5 versus 45.0 months, P>> .05). Our results suggest that Sox2 gene amplification significantly influences overall survival.

Despite significant advances achieved through gene targeting in mouse embryonic stem (ES) cells, this technology is presently only available in mice. Because the rat is a species of undeniable importance to biomedical research, attempts at derivation of rat ES cell lines have been ongoing for many years; however, the putative rat ES cell lines that have been reported to date have not yet displayed the ability to contribute in vivo to developing tissues following embryo injection. In contrast to previous studies, we describe herein the successful derivation and characterization of rat ES-like cell lines that not only express markers of undifferentiated cells, alkaline phosphatase (AP) activity and stage-specific embryonic antigen-1 (SSEA-1) cell surface antigen, but also retain expression of Oct4 (also known as Pou5f1) a homeodomain transcription factor and molecular marker of pluripotent cells. Notably, these rat ES-like cells, when injected into blastocysts transferred to pseudopregnant females, can contribute to developing extraembryonic tissues. This report demonstrates for the first time that rat ES-like cells can be derived efficiently, can express a panel of pluripotent cell markers, can be genetically modified in vitro and cryopreserved, and importantly, are capable of contributing to extraembryonic tissues in vivo.

We investigated the effects of zinc supplementation during the IVM of porcine oocytes. Nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, subsequent embryonic development, and gene expression were evaluated. Zinc concentrations in porcine plasma and follicular fluid were 0.82 and 0.84 μg/mL, respectively. Zinc was not detected in IVM medium. After treatment with various zinc concentrations (0.0, 0.4, 0.8, 1.2, and 1.6 μg/mL), no significant difference in IVM was observed among groups (85.7%, 88.7%, 90.4%, 90.3%, and 87.2%, respectively). The effects of different zinc concentrations on porcine oocyte intracellular GSH and ROS levels were examined in mature oocytes. Intracellular GSH levels were significantly higher in the 0.8-, 1.2-, and 1.6-μg/mL groups than in the control (P < 0.05). Intracellular ROS levels of oocytes matured with 0.8, 1.2, and 1.6 μg/mL were reduced significantly (P < 0.05) compared with the control and 0.4-μg/mL groups. The developmental competence of oocytes matured with different zinc concentrations was evaluated after parthenogenetic activation (PA) and in vitro fertilization (IVF). Oocytes treated with zinc during IVM showed no significant difference in cleavage rate after PA. Oocytes treated with 0.8 and 1.2 μg/mL zinc during IVM had significantly higher blastocyst formation rates after PA (41.5% and 41.1%, respectively) than the control (27.2%). IVF embryos showed similar results. The blastocyst formation rate was significantly higher (28.2%) in the 0.8-μg/mL group. TNFAIP2 and Bax were decreased in zinc-treated cumulus cells. Increased POU5F1 and decreased Bax transcript levels were observed in zinc-treated oocytes. POU5F1 and Bcl-2 transcript levels were significantly higher in zinc-treated IVF blastocysts. These results indicate that treatment with adequate zinc concentrations during IVM improved the developmental potential of porcine embryos by regulating the intracellular GSH concentration, the ROS level, and transcription factor expression.

BACKGROUND

Inflammatory bowel disease (IBD) is typically diagnosed at 20 to 40 years of age. However, very young versus elderly patients with IBD may have different mechanisms of disease that may affect prognosis and care.

OBJECTIVE

The purpose of this work was to identify single nucleotide polymorphisms associated with age of onset of Crohn's disease and ulcerative colitis.

METHODS

Patients were genotyped using a custom microarray chip containing 332 IBD-associated single nucleotide polymorphisms. Age at diagnosis as a continuous variable was assessed using linear regression. Patients were then subgrouped by age at diagnosis and compared by the Fisher exact test. Bonferroni correction was used in all of the analyses.

METHODS

This study was conducted at a tertiary academic hospital.

METHODS

Sixty patients with Crohn's disease and 26 with ulcerative colitis were ≤ 16 years old, 259 patients with Crohn's disease and 248 with ulcerative colitis were 17-60 years old, and 10 patients with Crohn's disease and 20 with ulcerative colitis were >60 years old at diagnosis and included in this study.

METHODS

Age at diagnosis and single nucleotide polymorphism correlations were measured in this study.

RESULTS

The NOD2 single nucleotide polymorphism rs2076756 was associated with younger age at Crohn's disease diagnosis (p = 0.0002). Patients with the AA/wild-type genotype were diagnosed at 31.9 ± 1.23 years, AG heterozygotes at 25.6 ± 0.99 years, and GG/at-risk allele homozygotes at 22.6 ± 1.32 years. Depending on age categories compared, single nucleotide polymorphisms in POU5F1, TNFSF15, and HLA DRB1*501 were associated with age of Crohn's disease diagnosis. No genetic associations were seen between ulcerative colitis and linear age at diagnosis; however, the G allele of the LAMB1 single nucleotide polymorphism rs886774 was found to be associated with ulcerative colitis diagnosed at ≤ 16 versus >17 years old (p = 0.008).

CONCLUSIONS

This study was limited to known IBD single nucleotide polymorphisms.

CONCLUSIONS

This analysis reaffirms the association between NOD2, a molecule of innate immunity, and early Crohn's disease onset. This is the first report of a possible association between early Crohn's disease and the POU5F1, TNFSF15, and HLA DRB1*501 genes. The LAMB1 gene, associated with mucosal basement membrane integrity, was associated with early onset ulcerative colitis and, thus, suggests a fundamentally different mechanism of early disease pathogenesis in ulcerative colitis versus Crohn's disease.

The iron chelator deferasirox (DFX) prevents complications related to transfusional iron overload in several haematological disorders characterized by marrow failure. It is also able to induce haematological responses in a percentage of treated patients, particularly in those affected by myelodysplastic syndromes. The underlying mechanisms responsible for this feature, however, are still poorly understood. In this study, we investigated the effect of DFX-treatment in human haematopoietic/progenitor stem cells, focussing on its impact on the redox balance, which proved to control the interplay between stemness maintenance, self-renewal and differentiation priming. Here we show, for the first time, that DFX treatment induces a significant diphenyleneiodonium-sensitive reactive oxygen species (ROS) production that leads to the activation of POU5F1 (OCT4), SOX2 and SOX17 gene expression, relevant in reprogramming processes, and the reduction of the haematopoietic regulatory proteins CTNNB1 (β-Catenin) and BMI1. These DFX-mediated events were accompanied by decreased CD34 expression, increased mitochondrial mass and up-regulation of the erythropoietic marker CD71 (TFRC) and were compound-specific, dissimilar to deferoxamine. Our findings would suggest a novel mechanism by which DFX, probably independently on its iron-chelating property but through ROS signalling activation, may influence key factors involved in self-renewal/differentiation of haematopoietic stem cells.

BACKGROUND

Tenm4 is a mouse homolog of the Drosophila gene Tenascin-m (Ten-m (Odd oz)), which functions in motor neuron routing. Recently, a genome-wide association analysis for bipolar disorder identified a new susceptibility locus at TENM4 increasing the importance of understanding Tenm4. A series of Tenm4 mouse alleles showing a broad range of phenotypes were isolated after ENU mutagenesis. Here, we examine the timing and features of gastrulation failure in a loss of function allele.

RESULTS

Embryonic mesoderm did not form in loss of function Tenm4m1/m1 mutant embryos. Genes normally expressed in embryonic mesoderm were not expressed in the mutant, the primitive streak did not form, and markers of the anteroposterior axis were not expressed or were mislocalized. The lack of embryonic mesoderm could not be attributed to poor proliferation of the epiblast, as normal numbers of dividing cells were observed. Epiblast cells maintained expression of Pou5f1 suggesting that they remain pluripotent, but they did not have the capacity to form any germ layer derivatives in teratomas, showing that the inability to induce mesoderm is cell autonomous. Misexpression of E-cadherin and N-cadherin suggest that the embryos did not undergo an epithelial-to-mesenchymal transition. In addition, Wnt signaling did not occur in the mutants, as assessed by the TOPGAL reporter assay, while a GSK3β inhibitor partially rescued the mutant embryos, and rescued TOPGAL reporter expression.

CONCLUSIONS

These data demonstrate that Tenm4 mutants fail to form a primitive streak and to induce embryonic mesoderm. Markers of anterior posterior patterning fail to be expressed or are mislocalized. Further, Tenm4 mutants lack the ability to differentiate in a cell autonomous manner. Together, our data suggest that embryos become impaired prior to E6.5 and as a result, Wnt signaling fails to occur; however, the involvement of other signaling pathways remains to be examined.