Mechanisms of Oct4 in the entry to, maintenance of, and exit from pluripotency

Pluripotency is defined as the capacity to give rise to all cell types of the embryo proper. It arises in the early mammalian embryo but is lost after a short period of time as cells differentiate and become committed to different lineages. Prior to implantation, mouse epiblast cells enter the pluripotent naïve state, which can be captured in vitro in the form of embryonic stem cells. These cells are characterized by a capacity for indefinite self-renewal, and the ability to re-enter normal development upon being returned to the naïve epiblast. A complex transcription factor network promotes this state. Overexpression of one of many of these factors leads to stabilisation of the naïve state, with enhanced self-renewal and reduced spontaneous differentiation. However, the transcription factor Oct4 must be maintained within a tight window of expression; depletion results in extraembryonic differentiation, while overexpression also results in exit from pluripotency. Oct4 was identified as a protein expressed in the early embryo and in germ cells, and was subsequently discovered to be essential for the establishment of the naïve epiblast. In vitro studies determined that loss of Oct4 in ESCs induced trophoblast differentiation. Meanwhile, overexpression of Oct4 led to differentiation of ESCs, and constitutive expression of Oct4 was not sufficient to replace any of the extrinsic factors required for ESC self-renewal. Despite these dramatic phenotypes, the essential role of Oct4 remains unclear, further complicated by the finding that a reduced level of Oct4 promotes self-renewal at the expense of differentiation capacity. In this work, I generated a novel Oct4 fusion protein capable of rapid inducible degradation in order to study the immediate responses to removal of Oct4. This system utilizes the auxin responsive degradation domain of the Arabidopsis thaliana IAA17 protein to recruit a transgenic F-box protein Tir1 on addition of the small molecule auxin to the culture medium. Subsequent ubiquitination by the endogenous SCF complex leads to rapid proteolytic degradation of the Oct4 fusion protein, resulting in loss of detectable protein in as little as two hours. This system allows the study of immediate responses to loss of Oct4 in contrast to conventional depletion systems in which Oct4 levels decay over a protracted period making it difficult to disentangle direct and indirect effects. I established that several pluripotency-associated genes require Oct4 for their transcription. RNA levels of these factors decrease rapidly on depletion of Oct4, prior to significant changes in the expression of other key pluripotency factors such as Nanog and before protein levels of other factors can change dramatically. Furthermore, I established that the presence of Oct4 antagonises chromatin binding by a naïve transcription factor, revealing a possible mechanism by which increased Oct4 levels are detrimental to the naïve state. Together, these findings may be sufficient to explain the simultaneous requirement for, and antagonistic activity of, Oct4 in naïve pluripotent cells. I also found that an ESC level of Oct4 facilitates cell identity transitions. Cells constitutively expressing Oct4 and differentiated in vivo could be reverted to naïve pluripotency in vitro through the application of defined naïve pluripotency growth conditions. Additionally, in the course of these experiments I examined the phenotypic abnormalities that occur in mouse embryonic development under continuous expression of Oct4. In keeping with previous work, we observed abnormalities in limb development and in the skin. We also observed exencephaly in a number of embryos. In conventional exit from pluripotency, female cells must inactive an X chromosome in order to balance X-linked gene expression between males and females. This is achieved via expression of Xist from a single X chromosome, where it orchestrates chromosome-wide silencing. I show that Oct4 plays an important role in the regulation of Xist during the exit from pluripotency. Normally, Oct4 expression persists after the downregulation of most naïve transcription factors during early differentiation. I propose that this allows Oct4 to antagonise expression of the Xist, and thus ensure proper control over X chromosome inactivation. Together this work focuses on the dual roles of Oct4 in regulating both pluripotency and differentiation. I address the contradictory phenotypes relating to altered expression of Oct4, and establish a unifying theory to explain them. I put forward evidence that Oct4 promotes cell fate transitions by regulating naïve transcription factors. I propose that the environment plays a key role in determining cell identity, while Oct4 acts to maintain plasticity by preventing cells from being trapped within otherwise stable states.This work was funded by an MRC DTA PhD studentship in Stem Cell Biology & Medicine

Auxin-degron system identifies immediate mechanisms of OCT4.

The pluripotency factor OCT4 is essential for the maintenance of naive pluripotent stem cells in vitro and in vivo. However, the specific role of OCT4 in this process remains unknown. Here, we developed a rapid protein-level OCT4 depletion system that demonstrates that the immediate downstream response to loss of OCT4 is reduced expression of key pluripotency factors. Our data show a requirement for OCT4 for the efficient transcription of several key pluripotency factors and suggest that expression of trophectoderm markers is a subsequent event. In addition, we find that NANOG is able to bind to the genome in the absence of OCT4, and this binding is in fact enhanced. Globally, however, the active enhancer-associated histone mark H3K27ac is depleted. Our work establishes that, while OCT4 is required for the maintenance of the naive transcription factor network, at a normal embryonic stem cell levels it antagonizes this network through inhibition of NANOG binding

Exit from Naive Pluripotency Induces a Transient X Chromosome Inactivation-like State in Males.

A hallmark of naive pluripotency is the presence of two active X chromosomes in females. It is not clear whether prevention of X chromosome inactivation (XCI) is mediated by gene networks that preserve the naive state. Here, we show that robust naive pluripotent stem cell (nPSC) self-renewal represses expression of Xist, the master regulator of XCI. We found that nPSCs accumulate Xist on the male X chromosome and on both female X chromosomes as they become NANOG negative at the onset of differentiation. This is accompanied by the appearance of a repressive chromatin signature and partial X-linked gene silencing, suggesting a transient and rapid XCI-like state in male nPSCs. In the embryo, Xist is transiently expressed in males and in females from both X chromosomes at the onset of naive epiblast differentiation. In conclusion, we propose that XCI initiation is gender independent and triggered by destabilization of naive identity, suggesting that gender-specific mechanisms follow, rather than precede, XCI initiation.This study was supported by a Wellcome Trust Fellowship (WT101861) to J.C.R.S., who is a Wellcome Trust Senior Research Fellow. E.J.S. is the recipient of a Ph.D. fellowship from the Portuguese Foundation for Sciences and Technology, FCT (SFRH/BD/52197/2013). H.T.S. and L.E.B. are recipients of an MRC Ph.D. studentship

OCT4 induces embryonic pluripotency via STAT3 signaling and metabolic mechanisms.

OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 establishes and protects the pluripotent lineage in the embryo, we used comparative single-cell transcriptomics and quantitative immunofluorescence on control and OCT4 null blastocyst inner cell masses at two developmental stages. Surprisingly, activation of most pluripotency-associated transcription factors in the early mouse embryo occurs independently of OCT4, with the exception of the JAK/STAT signaling machinery. Concurrently, OCT4 null inner cell masses ectopically activate a subset of trophectoderm-associated genes. Inspection of metabolic pathways implicates the regulation of rate-limiting glycolytic enzymes by OCT4, consistent with a role in sustaining glycolysis. Furthermore, up-regulation of the lysosomal pathway was specifically detected in OCT4 null embryos. This finding implicates a requirement for OCT4 in the production of normal trophectoderm. Collectively, our findings uncover regulation of cellular metabolism and biophysical properties as mechanisms by which OCT4 instructs pluripotency.This work was supported by the University of Cambridge, BBSRC project grant RG74277, BB/R018588/1 and MR/R017735/1 to HS and LB respectively, MRC PhD studentship for AK and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute

The development of sentence-interpretation strategies in monolingual German-learning children with and without specific language impairment

Previous research on sentence comprehension conducted with German-learning children has concentrated on the role of case marking and word order in typically developing children. This paper compares, the performance of German-learning children with language impairment (age 4-6 years) and without language impairment (aged 2-6, 8-9 years) in two experiments that systematically vary the cues animacy, case marking; word-order, and subject-verb agreement. The two experiments differ with regard to the choice of case marking: in the first it is distinct but in the second it is neutralized. The theoretical framework is the competition model developed by Bates and Mac Whinney and their collaborators, a variant of the parallel distributed processing models. It is hypothesized that children of either population first appreciate the cue animacy that can be processed locally, that is, "on the spot," before they turn to more distributed cues leading ultimately up to subject-verb agreement, which presupposes the comparison of various constituents before an interpretation can be established. Thus agreement is more "costly" in processing than animacy or the (more) local cue initial NP. In experiment I with unambiguous case markers it is shown that the typically developing children proceed from animacy to the nominative (predominantly in coalition with the initial NP) to agreement, while in the second experiment with ambiguous case markers these children turn from animacy to the initial NP and then to agreement. The impaired children also progress from local to distributed cues. Yet, in contrast to the control group, they do not acknowledge the nominative in coalition with the initial NP in the first experiment but only in support of agreement. However, although they do not seem to appreciate distinct case markers to any large extent in the first experiment, they are irritated if such distinctions are lacking: in experiment II all impaired children turn to. animacy (some in coalition with the initial NP and/or particular word orders). In the discussion, the relationship between short-term memory and processing as well as the relationship between production and comprehension of case markers and agreement are addressed. Further research is needed to explore in more detail "cue costs" in sentence comprehension

The Maine Annex, vol. 1, no. 8

The Maine Annex, published by the students of the University of Maine at the Brunswick Campus, was launched January 10, 1947. Editors introduced the publication as the product of a group of progressive students attending the Brunswick Campus. The goal of the publication, according to editors, was to tell the story of our life on this campus. The four-page, tabloid-sized paper included display advertising from area businesses. Borrowing from the University of Maine, Orono campus, the Brunswick campus sponsored the election of a student Mayor as part of the spring dance escapades. Promotion of dance night activities includes the promise of plentiful women attending

The Maine Annex, vol. 1, no. 7

The Maine Annex, published by the students of the University of Maine at the Brunswick Campus, was launched January 10, 1947. Editors introduced the publication as the product of a group of progressive students attending the Brunswick Campus. The goal of the publication, according to editors, was to tell the story of our life on this campus. The four-page, tabloid-sized paper included display advertising from area businesses. Front-page interests in this issue revolve around sports, women, and the creation of a student dance band, and attracting women to campus by staging dances

The Maine Annex, vol. 1, no. 10

The Maine Annex, published by the students of the University of Maine at the Brunswick Campus, was launched January 10, 1947. Editors introduced the publication as the product of a group of progressive students attending the Brunswick Campus. The goal of the publication, according to editors, was to tell the story of our life on this campus. The four-page, tabloid-sized paper included display advertising from area businesses. The start of a funding drive to build the Memorial Union on the University of Maine, Orono campus, is covered in this issue of The Maine Annex. The Union is to be dedicated to 175 UMaine Alumni war dead. Following World War II, the federal G.I. Bill enabled approximately 2.3 million, predominantly white male Veterans to receive a post-secondary education. To accommodate increased enrollment, in 1946 the University of Maine established the Brunswick Campus at the former Brunswick Naval Air Station. The remote campus operated until spring 1949, when Veteran registrations waned

The Maine Annex, vol. 1, no. 11

The Maine Annex, published by the students of the University of Maine at the Brunswick Campus, was launched January 10, 1947. Editors introduced the publication as the product of a group of progressive students attending the Brunswick Campus. The goal of the publication, according to editors, was to tell the story of our life on this campus. The four-page, tabloid-sized paper included display advertising from area businesses. Following World War II, the federal G.I. Bill enabled approximately 2.3 million, predominantly white male Veterans to receive a post-secondary education. To accommodate increased enrollment, in 1946 the University of Maine established the Brunswick Campus at the former Brunswick Naval Air Station. The remote campus operated until spring 1949, when Veteran registrations waned. Page two discussions in this issue orbit the issue of G.I. Subsistence, from James McNiff\u27s article examining the discrepancy between the increased cost of living in the U.S. and the inadequate amount of money allocated through the Veterans subsistence allowance to commentary about the persistently, poor quality of food provided to students by the University in exchange for their money

The Maine Annex, vol. 1, no. 9

The Maine Annex, published by the students of the University of Maine at the Brunswick Campus, was launched January 10, 1947. Editors introduced the publication as the product of a group of progressive students attending the Brunswick Campus. The goal of the publication, according to editors, was to tell the story of our life on this campus. The four-page, tabloid-sized paper included display advertising from area businesses. Coverage in this issue emphasizes Veterans issues, including a story about the distribution of one million surplus textbooks from the Army and Navy special training programs to Veterans attending institutions nationwide