The birth of embryonic pluripotency.

Formation of a eutherian mammal requires concurrent establishment of embryonic and extraembryonic lineages. The functions of the trophectoderm and primitive endoderm are to enable implantation in the maternal uterus, axis specification and delivery of nutrients. The pluripotent epiblast represents the founding cell population of the embryo proper, which is protected from ectopic and premature differentiation until it is required to respond to inductive cues to form the fetus. While positional information plays a major role in specifying the trophoblast lineage, segregation of primitive endoderm from epiblast depends upon gradual acquisition of transcriptional identity, directed but not initiated by fibroblast growth factor (FGF) signalling. Following early cleavage divisions and formation of the blastocyst, cells of the inner cell mass lose totipotency. Developing epiblast cells transiently attain the state of naive pluripotency and competence to self-renew in vitro as embryonic stem cells and in vivo by means of diapause. This property is lost after implantation as the epiblast epithelializes and becomes primed in preparation for gastrulation and subsequent organogenesis.We wish to thank the MRC, Wellcome Trust and University of Cambridge for our funding.This is the final published version. It first appeared at http://rstb.royalsocietypublishing.org/content/369/1657/20130541

Origin and function of the yolk sac in primate embryogenesis

Funder: Wellcome Trust (Wellcome); doi: https://doi.org/10.13039/100004440Abstract: Human embryogenesis is hallmarked by two phases of yolk sac development. The primate hypoblast gives rise to a transient primary yolk sac, which is rapidly superseded by a secondary yolk sac during gastrulation. Moreover, primate embryos form extraembryonic mesoderm prior to gastrulation, in contrast to mouse. The function of the primary yolk sac and the origin of extraembryonic mesoderm remain unclear. Here, we hypothesise that the hypoblast-derived primary yolk sac serves as a source for early extraembryonic mesoderm, which is supplemented with mesoderm from the gastrulating embryo. We discuss the intricate relationship between the yolk sac and the primate embryo and highlight the pivotal role of the yolk sac as a multifunctional hub for haematopoiesis, germ cell development and nutritional supply

Building a stem cell-based primate uterus.

Funder: Wellcome TrustThe uterus is the organ for embryo implantation and fetal development. Most current models of the uterus are centred around capturing its function during later stages of pregnancy to increase the survival in pre-term births. However, in vitro models focusing on the uterine tissue itself would allow modelling of pathologies including endometriosis and uterine cancers, and open new avenues to investigate embryo implantation and human development. Motivated by these key questions, we discuss how stem cell-based uteri may be engineered from constituent cell parts, either as advanced self-organising cultures, or by controlled assembly through microfluidic and print-based technologies

Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human pre-implantation epiblast.

Single-cell profiling techniques create opportunities to delineate cell fate progression in mammalian development. Recent studies have provided transcriptome data from human pre-implantation embryos, in total comprising nearly 2000 individual cells. Interpretation of these data is confounded by biological factors, such as variable embryo staging and cell-type ambiguity, as well as technical challenges in the collective analysis of datasets produced with different sample preparation and sequencing protocols. Here, we address these issues to assemble a complete gene expression time course spanning human pre-implantation embryogenesis. We identify key transcriptional features over developmental time and elucidate lineage-specific regulatory networks. We resolve post-hoc cell-type assignment in the blastocyst, and define robust transcriptional prototypes that capture epiblast and primitive endoderm lineages. Examination of human pluripotent stem cell transcriptomes in this framework identifies culture conditions that sustain a naïve state pertaining to the inner cell mass. Our approach thus clarifies understanding both of lineage segregation in the early human embryo and of in vitro stem cell identity, and provides an analytical resource for comparative molecular embryology.This work was supported by UK Biotechnology and Biological Sciences Research Council (BBSRC) research grant RG53615, UK Medical Research Council (MRC) programme grant G1001028, and institutional funding from the MRC and Wellcome Trust. AS is an MRC Professor

Lineage-Specific Profiling Delineates the Emergence and Progression of Naive Pluripotency in Mammalian Embryogenesis.

Naive pluripotency is manifest in the preimplantation mammalian embryo. Here we determine transcriptome dynamics of mouse development from the eight-cell stage to postimplantation using lineage-specific RNA sequencing. This method combines high sensitivity and reporter-based fate assignment to acquire the full spectrum of gene expression from discrete embryonic cell types. We define expression modules indicative of developmental state and temporal regulatory patterns marking the establishment and dissolution of naive pluripotency in vivo. Analysis of embryonic stem cells and diapaused embryos reveals near-complete conservation of the core transcriptional circuitry operative in the preimplantation epiblast. Comparison to inner cell masses of marmoset primate blastocysts identifies a similar complement of pluripotency factors but use of alternative signaling pathways. Embryo culture experiments further indicate that marmoset embryos utilize WNT signaling during early lineage segregation, unlike rodents. These findings support a conserved transcription factor foundation for naive pluripotency while revealing species-specific regulatory features of lineage segregation.We thank Peter Humphreys for assistance with imaging, and Samuel Jameson and staff for mouse husbandry. We are grateful to Charis Drummer, Ayako Sedohara, Akiko Shimada, Yuko Yamada, Ryo Oiwa, and Takeshi Kuge for technical support with marmoset embryo recovery. Illumina sequencing was provided by Bettina Haase and Dinko Pavlinic at the EMBL Genomics Core Facility. This work was supported by funding from the Wellcome Trust, the Genome Biology Unit of the European Molecular Biology Laboratory, BBSRC grants BB/G015678/1 and BB/M004023/1, an MRC Centenary Award, and the Louis Jeantet Foundation. A.S. is a Medical Research Council Professor.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.devcel.2015.10.01

Agarose microgel culture delineates lumenogenesis in naive and primed human pluripotent stem cells.

Human periimplantation development requires the transformation of the naive pluripotent epiblast into a polarized epithelium. Lumenogenesis plays a critical role in this process, as the epiblast undergoes rosette formation and lumen expansion to form the amniotic cavity. Here, we present a high-throughput in vitro model for epiblast morphogenesis. We established a microfluidic workflow to encapsulate human pluripotent stem cells (hPSCs) into monodisperse agarose microgels. Strikingly, hPSCs self-organized into polarized epiblast spheroids that could be maintained in self-renewing and differentiating conditions. Encapsulated primed hPSCs required Rho-associated kinase inhibition, in contrast to naive hPSCs. We applied microgel suspension culture to examine the lumen-forming capacity of hPSCs and reveal an increase in lumenogenesis during the naive-to-primed transition. Finally, we demonstrate the feasibility of co-encapsulating cell types across different lineages and species. Our work provides a foundation for stem cell-based embryo models to interrogate the critical components of human epiblast self-organization and morphogenesis

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

Origin and segregation of the human germline

Acknowledgements This work was supported by the Wellcome Investigator Awards in Science (2094)75/Z/17/Z (to MA Surani), the Wellcome Investigator Awards in Science 096738/Z/11/Z (to MA Surani), the BBSRC research grant G103986 (to MA Surani), the Croucher Postdoctoral Research Fellowship (to WWC Tang), the Wellcome 4-Yr PhD Programme in Stem Cell Biology & Medicine (2038)31/Z/16/Z (to A Castillo-Venzor) and the Cambridge Commonwealth European and International Trust (to A Castillo-Venzor), the Isaac Newton Trust (to WWC Tang), the Butterfield Awards of Great Britain Sasakawa Foundation (to T Kobayashi and MA Surani), and the Astellas Foundation for Research on Metabolic Disorders (to T Kobayashi). The marmoset embryo research is generously supported by the Wellcome Trust (WT RG89228, WT RG9242), the Centre for Trophoblast Research, the Isaac Newton Trust, and JSPS KAKENHI 15H02360, 19H05759. TE Boroviak was supported by a Wellcome Sir Henry Dale Fellowship. JC Marioni acknowledges core support from EMBL and from Cancer Research UK (C9545/A29580), which supports MD Morgan. We would like to thank Roger Barker and Xiaoling He for providing human embryonic tissues and Charles Bradshaw for bioinformatics support. We also thank The Weizmann Institute of Science for the WIS2 human PSC line and the Genomics Core Facility of CRUK Cambridge Institute for sequencing services. We thank members of the Surani laboratory for insightful comments and critical reading of the manuscript.Peer reviewedPublisher PD

Metabolic control of DNA methylation in naive pluripotent cells.

Naive epiblast and embryonic stem cells (ESCs) give rise to all cells of adults. Such developmental plasticity is associated with genome hypomethylation. Here, we show that LIF-Stat3 signaling induces genomic hypomethylation via metabolic reconfiguration. Stat3-/- ESCs show decreased α-ketoglutarate production from glutamine, leading to increased Dnmt3a and Dnmt3b expression and DNA methylation. Notably, genome methylation is dynamically controlled through modulation of α-ketoglutarate availability or Stat3 activation in mitochondria. Alpha-ketoglutarate links metabolism to the epigenome by reducing the expression of Otx2 and its targets Dnmt3a and Dnmt3b. Genetic inactivation of Otx2 or Dnmt3a and Dnmt3b results in genomic hypomethylation even in the absence of active LIF-Stat3. Stat3-/- ESCs show increased methylation at imprinting control regions and altered expression of cognate transcripts. Single-cell analyses of Stat3-/- embryos confirmed the dysregulated expression of Otx2, Dnmt3a and Dnmt3b as well as imprinted genes. Several cancers display Stat3 overactivation and abnormal DNA methylation; therefore, the molecular module that we describe might be exploited under pathological conditions

Metabolic control of DNA methylation in naive pluripotent cells.

Naive epiblast and embryonic stem cells (ESCs) give rise to all cells of adults. Such developmental plasticity is associated with genome hypomethylation. Here, we show that LIF-Stat3 signaling induces genomic hypomethylation via metabolic reconfiguration. Stat3-/- ESCs show decreased α-ketoglutarate production from glutamine, leading to increased Dnmt3a and Dnmt3b expression and DNA methylation. Notably, genome methylation is dynamically controlled through modulation of α-ketoglutarate availability or Stat3 activation in mitochondria. Alpha-ketoglutarate links metabolism to the epigenome by reducing the expression of Otx2 and its targets Dnmt3a and Dnmt3b. Genetic inactivation of Otx2 or Dnmt3a and Dnmt3b results in genomic hypomethylation even in the absence of active LIF-Stat3. Stat3-/- ESCs show increased methylation at imprinting control regions and altered expression of cognate transcripts. Single-cell analyses of Stat3-/- embryos confirmed the dysregulated expression of Otx2, Dnmt3a and Dnmt3b as well as imprinted genes. Several cancers display Stat3 overactivation and abnormal DNA methylation; therefore, the molecular module that we describe might be exploited under pathological conditions