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The interplay of signalling input and downstream transcriptional activity is the key molecular attribute driving the differentiation of germ layer tissue and the specification of cell lineages within each germ layer during gastrulation. This review delves into the current understanding of signalling and transcriptional control of lineage development in the germ layers of mouse embryo and non-human primate embryos during gastrulation and highlights the inter-species conservation and divergence of the cellular and molecular mechanisms of germ layer development in the human embryo., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The preimplantation mammalian embryo has the potential to self-organize, allowing the formation of a correctly patterned embryo despite experimental perturbation. To better understand the mechanisms controlling the developmental plasticity of the early mouse embryo, we used chimaeras composed of an embryonic day (E)3.5 or E4.5 inner cell mass (ICM) and cleaving 8-cell embryo. We revealed that the restricted potential of the ICM can be compensated for by uncommitted 8-cell embryo-derived blastomeres, thus leading to the formation of a normal chimaeric blastocyst that can undergo full development. However, whether such chimaeras maintain developmental competence depends on the presence or specific orientation of the polarized primitive endoderm layer in the ICM component. We also demonstrated that downregulated FGFR1 and FGFR2 expression in 8-cell embryos disturbs intercellular interactions between both components and results in an inverse proportion of primitive endoderm and epiblast within the resulting ICM and abnormal embryo development. This finding suggests that FGF signalling is a key part of the regulatory mechanism that assigns cells to a given lineage and ensures the proper composition of the blastocyst, which is a prerequisite for its successful implantation in the uterus and for further development.

The epiblast of vertebrate embryos is comprised of neural and non-neural ectoderm, with the border territory at their intersection harboring neural crest and cranial placode progenitors. Here, we a generate single-cell atlas of the developing chick epiblast from late gastrulation through early neurulation stages to define transcriptional changes in the emerging 'neural plate border' as well as other regions of the epiblast. Focusing on the border territory, the results reveal gradual establishment of heterogeneous neural plate border signatures, including novel genes that we validate by fluorescent in situ hybridization. Developmental trajectory analysis infers that segregation of neural plate border lineages only commences at early neurulation, rather than at gastrulation as previously predicted. We find that cells expressing the prospective neural crest marker Pax7 contribute to multiple lineages, and a subset of premigratory neural crest cells shares a transcriptional signature with their border precursors. Together, our results suggest that cells at the neural plate border remain heterogeneous until early neurulation, at which time progenitors become progressively allocated toward defined neural crest and placode lineages. The data also can be mined to reveal changes throughout the developing epiblast., Competing Interests: RW, ML, TS No competing interests declared, MB Senior editor, eLife, (© 2022, Williams et al.)

Parthenogenetic embryos have been widely studied as an effective tool related to paternal and maternal imprinting genes and reproductive problems for a long time. In this study, we established a parthenogenetic epiblast-like stem cell line through culturing parthenogenetic diploid blastocysts in a chemically defined medium containing activin A and bFGF named paAFSCs. The paAFSCs expressed pluripotent marker genes and germ-layer-related genes, as well as being alkaline-phosphatase-positive, which is similar to epiblast stem cells (EpiSCs). We previously showed that advanced embryonic stem cells (ASCs) represent hypermethylated naive pluripotent embryonic stem cells (ESCs). Here, we converted paAFSCs to ASCs by replacing bFGF with bone morphogenetic protein 4 (BMP4), CHIR99021, and leukemia inhibitory factor (LIF) in a culture medium, and we obtained parthenogenetic advanced stem cells (paASCs). The paASCs showed similar morphology with ESCs and also displayed a stronger developmental potential than paAFSCs in vivo by producing chimaeras. Our study demonstrates that maternal genes could support parthenogenetic EpiSCs derived from blastocysts and also have the potential to convert primed state paAFSCs to naive state paASCs.

During early mammalian embryo development, a small number of cells make robust fate decisions at particular spatial locations in a tight time window to form inner cell mass (ICM), and later epiblast (Epi) and primitive endoderm (PE). While recent single-cell transcriptomics data allows scrutinization of heterogeneity of individual cells, consistent spatial and temporal mechanisms the early embryo utilize to robustly form the Epi/PE layers from ICM remain elusive. Here we build a multiscale three-dimensional model for mammalian embryo to recapitulate the observed patterning process from zygote to late blastocyst. By integrating the spatiotemporal information reconstructed from multiple single-cell transcriptomic datasets, the data-informed modeling analysis suggests two major processes critical to the formation of Epi/PE layers: a selective cell-cell adhesion mechanism (via EphA4/EphrinB2) for fate-location coordination and a temporal attenuation mechanism of cell signaling (via Fgf). Spatial imaging data and distinct subsets of single-cell gene expression data are then used to validate the predictions. Together, our study provides a multiscale framework that incorporates single-cell gene expression datasets to analyze gene regulations, cell-cell communications, and physical interactions among cells in complex geometries at single-cell resolution, with direct application to late-stage development of embryogenesis., Competing Interests: The authors have declared that no competing interests exist.

Mammalian cells stably maintain high levels of DNA methylation despite expressing both positive (DNMT3A/B) and negative (TET1-3) regulators. Here, we analyzed the independent and combined effects of these regulators on the DNA methylation landscape using a panel of knockout human embryonic stem cell (ESC) lines. The greatest impact on global methylation levels was observed in DNMT3-deficient cells, including reproducible focal demethylation at thousands of normally methylated loci. Demethylation depends on TET expression and occurs only when both DNMT3s are absent. Dynamic loci are enriched for hydroxymethylcytosine and overlap with subsets of putative somatic enhancers that are methylated in ESCs and can be activated upon differentiation. We observe similar dynamics in mouse ESCs that were less frequent in epiblast stem cells (EpiSCs) and scarce in somatic tissues, suggesting a conserved pluripotency-linked mechanism. Taken together, our data reveal tightly regulated competition between DNMT3s and TETs at thousands of somatic regulatory sequences within pluripotent cells.

Heterochronic blood sharing rejuvenates old tissues, and most of the studies on how this works focus on young plasma, its fractions, and a few youthful systemic candidates. However, it was not formally established that young blood is necessary for this multi-tissue rejuvenation. Here, using our recently developed small animal blood exchange process, we replaced half of the plasma in mice with saline containing 5% albumin (terming it a "neutral" age blood exchange, NBE) thus diluting the plasma factors and replenishing the albumin that would be diminished if only saline was used. Our data demonstrate that a single NBE suffices to meet or exceed the rejuvenative effects of enhancing muscle repair, reducing liver adiposity and fibrosis, and increasing hippocampal neurogenesis in old mice, all the key outcomes seen after blood heterochronicity. Comparative proteomic analysis on serum from NBE, and from a similar human clinical procedure of therapeutic plasma exchange (TPE), revealed a molecular re-setting of the systemic signaling milieu, interestingly, elevating the levels of some proteins, which broadly coordinate tissue maintenance and repair and promote immune responses. Moreover, a single TPE yielded functional blood rejuvenation, abrogating the typical old serum inhibition of progenitor cell proliferation. Ectopically added albumin does not seem to be the sole determinant of such rejuvenation, and levels of albumin do not decrease with age nor are increased by NBE/TPE. A model of action (supported by a large body of published data) is that significant dilution of autoregulatory proteins that crosstalk to multiple signaling pathways (with their own feedback loops) would, through changes in gene expression, have long-lasting molecular and functional effects that are consistent with our observations. This work improves our understanding of the systemic paradigms of multi-tissue rejuvenation and suggest a novel and immediate use of the FDA approved TPE for improving the health and resilience of older people.

To explain the evolutionary origin of vertebrate teeth from odontodes, it has been proposed that competent epithelium spread into the oropharyngeal cavity via the mouth and other possible channels such as the gill slits [Huysseune et al., 2009, J. Anat. 214, 465-476]. Whether tooth formation deep inside the pharynx in extant vertebrates continues to require external epithelia has not been addressed so far. Using zebrafish we have previously demonstrated that cells derived from the periderm penetrate the oropharyngeal cavity via the mouth and via the endodermal pouches and connect to periderm-like cells that subsequently cover the entire endoderm-derived pharyngeal epithelium [Rosa et al., 2019, Sci. Rep. 9, 10082]. We now provide conclusive evidence that the epithelial component of pharyngeal teeth in zebrafish (the enamel organ) is derived from medial endoderm, as hitherto assumed based on position deep in the pharynx. Yet, dental morphogenesis starts only after the corresponding endodermal pouch (pouch 6) has made contact with the skin ectoderm, and only after periderm-like cells have covered the prospective tooth-forming endodermal epithelium. Manipulation of signaling pathways shown to adversely affect tooth development indicates they act downstream of these events. We demonstrate that pouch-ectoderm contact and the presence of a periderm-like layer are both required, but not sufficient, for tooth initiation in the pharynx. We conclude that the earliest interactions to generate pharyngeal teeth encompass those between different epithelial populations (skin ectoderm, endoderm, and periderm-like cells in zebrafish), in addition to the epithelial-mesenchymal interactions that govern the formation of all vertebrate teeth., Competing Interests: The authors declare no competing interest.

Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals.

Gastrulation entails specification and formation of three embryonic germ layers-ectoderm, mesoderm and endoderm-thereby establishing the basis for the future body plan. In zebrafish embryos, germ layer specification occurs during blastula and early gastrula stages (Ho & Kimmel, 1993), a period when the main morphogenetic movements underlying gastrulation are initiated. Hence, the signals driving progenitor cell fate specification, such as Nodal ligands from the TGF-β family, also play key roles in regulating germ layer progenitor cell segregation (Carmany-Rampey & Schier, 2001; David & Rosa, 2001; Feldman et al., 2000; Gritsman et al., 1999; Keller et al., 2008). In this review, we summarize and discuss the main signaling pathways involved in germ layer progenitor cell fate specification and segregation, specifically focusing on recent advances in understanding the interplay between mesoderm and endoderm specification and the internalization movements at the onset of zebrafish gastrulation., (© 2020 Elsevier Inc. All rights reserved.)

Gastrulation is a central process in mammalian development in which a spatiotemporally coordinated series of events driven by cross-talk between adjacent embryonic and extra-embryonic tissues results in stereotypical morphogenetic cell behaviors, massive cell proliferation and the acquisition of distinct cell identities. Gastrulation provides the blueprint of the body plan of the embryo, as well as generating extra-embryonic cell types of the embryo to make a connection with its mother. Gastrulation involves the specification of mesoderm and definitive endoderm from pluripotent epiblast, concomitant with a highly ordered elongation of tissue along the anterior-posterior (AP) axis. Interestingly, cells with an endoderm identity arise twice during mouse development. Cells with a primitive endoderm identity are specified in the preimplantation blastocyst, and which at gastrulation intercalate with the emergent definitive endoderm to form a mosaic tissue, referred to as the gut endoderm. The gut endoderm gives rise to the gut tube, which will subsequently become patterned along its AP axis into domains possessing unique visceral organ identities, such as thyroid, lung, liver and pancreas. In this way, proper endoderm development is essential for vital organismal functions, including the absorption of nutrients, gas exchange, detoxification and glucose homeostasis., (© 2020 Elsevier Inc. All rights reserved.)

Gastrulation is the period of development when the three germ layers, mesoderm, endoderm and ectoderm, are not only formed, but also shaped into a rudimentary body plan. An elongated anteroposterior (AP) axis is a key feature of all vertebrate body plans, and it forms during gastrulation through the highly conserved morphogenetic mechanism of convergence & extension (C&E). As the name suggests, this process requires that cells within each germ layer converge toward the dorsal midline to narrow the tissue in the mediolateral (ML) dimension and concomitantly extend it in the AP dimension. In a number of vertebrate species, C&E is driven primarily by mediolateral intercalation behavior (MIB), during which cells elongate, align, and extend protrusions in the ML direction and interdigitate between their neighbors. MIB is only one of many complex cellular mechanisms that contributes to C&E in zebrafish embryos, however, where a combination of individual cell migration, collective migration, random walk, radial intercalation, epiboly movements, and MIB all act together to shape the nascent germ layers. Each of these diverse cell movements is driven by a distinct suite of dynamic cellular properties/activities, such as actin-rich protrusions, myosin contractility, and blebbing. Here, we discuss the spatiotemporal patterns of cellular behaviors underlying C&E gastrulation movements within each germ layer of zebrafish embryos. These behaviors must be coordinated with the embryonic axes, and we highlight the roles of Planar Cell Polarity (PCP) in orienting and BMP signaling in patterning C&E cell behaviors with respect to the AP and dorsoventral axes. Finally, we address the role of GPCR signaling, extracellular matrix, and mechanical signals in coordination of C&E movements between adjacent germ layers., (© 2020 Elsevier Inc. All rights reserved.)

Embryonic stem cells (ESCs) exist in at least two states that transcriptionally resemble different stages of embryonic development. Naïve ESCs resemble peri-implantation stages and primed ESCs the pre-gastrulation epiblast. In mouse, primed ESCs give rise to definitive endoderm in response to the pathways downstream of Nodal and Wnt signalling. However, when these pathways are activated in naïve ESCs, they differentiate to a cell type resembling early primitive endoderm (PrE), the blastocyst-stage progenitor of the extra-embryonic endoderm. Here, we apply this context dependency to human ESCs, showing that activation of Nodal and Wnt signalling drives the differentiation of naïve pluripotent cells toward extra-embryonic PrE, or hypoblast, and these can be expanded as an in vitro model for naïve extra-embryonic endoderm (nEnd). Consistent with observations made in mouse, human PrE differentiation is dependent on FGF signalling in vitro , and we show that, by inhibiting FGF receptor signalling, we can simplify naïve pluripotent culture conditions, such that the inhibitor requirements closer resemble those used in mouse. The expandable nEnd cultures reported here represent stable extra-embryonic endoderm, or human hypoblast, cell lines.This article has an associated 'The people behind the papers' interview., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The first lineage specification of pluripotent mouse epiblast segregates neuroectoderm (NE) from mesoderm and definitive endoderm (ME) by mechanisms that are not well understood. Here we demonstrate that the induction of ME gene programs critically relies on the T-box transcription factors Eomesodermin (also known as Eomes) and Brachyury, which concomitantly repress pluripotency and NE gene programs. Cells deficient in these T-box transcription factors retain pluripotency and differentiate to NE lineages despite the presence of ME-inducing signals transforming growth factor β (TGF-β)/Nodal and Wnt. Pluripotency and NE gene networks are additionally repressed by ME factors downstream of T-box factor induction, demonstrating a redundancy in program regulation to safeguard mutually exclusive lineage specification. Analyses of chromatin revealed that accessibility of ME enhancers depends on T-box factor binding, whereas NE enhancers are accessible and already activation primed at pluripotency. This asymmetry of the chromatin landscape thus explains the default differentiation of pluripotent cells to NE in the absence of ME induction that depends on activating and repressive functions of Eomes and Brachyury.

A switch from E- to N-cadherin regulates the transition from pluripotency to neural identity, but the mechanism by which cadherins regulate differentiation was previously unknown. Here, we show that the acquisition of N-cadherin stabilises neural identity by dampening anti-neural signals. We use quantitative image analysis to show that N-cadherin promotes neural differentiation independently of its effects on cell cohesiveness. We reveal that cadherin switching diminishes the level of nuclear β-catenin, and that N-cadherin also dampens FGF activity and consequently stabilises neural fate. Finally, we compare the timing of cadherin switching and differentiation in vivo and in vitro , and find that this process becomes dysregulated during in vitro differentiation. We propose that N-cadherin helps to propagate a stable neural identity throughout the emerging neuroepithelium, and that dysregulation of this process contributes to asynchronous differentiation in culture., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Understanding how cell identity transitions occur and whether there are multiple paths between the same beginning and end states are questions of wide interest. Here we show that acquisition of naive pluripotency can follow transcriptionally and mechanistically distinct routes. Starting from post-implantation epiblast stem cells (EpiSCs), one route advances through a mesodermal state prior to naive pluripotency induction, whereas another transiently resembles the early inner cell mass and correspondingly gains greater developmental potency. These routes utilize distinct signaling networks and transcription factors but subsequently converge on the same naive endpoint, showing surprising flexibility in mechanisms underlying identity transitions and suggesting that naive pluripotency is a multidimensional attractor state. These route differences are reconciled by precise expression of Oct4 as a unifying, essential, and sufficient feature. We propose that fine-tuned regulation of this "transition factor" underpins multidimensional access to naive pluripotency, offering a conceptual framework for understanding cell identity transitions., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Our current knowledge of the mechanisms leading to human primordial germ cell (PGC) specification stems solely from differentiation experiments starting from human pluripotent stem cells. However, information regarding the origin of PGCs in vivo remains obscure. Here we apply an improved system for extended in vitro culture of human embryos to investigate the presence of PGC-like cells (PGCLCs) 12 days post fertilization (dpf). Good quality blastocysts (n = 141) were plated at 6 dpf and maintained in hypoxia, in medium supplemented with Activin A until 12 dpf. We primarily reveal that 12 dpf outgrowths recapitulate human peri-implantation events and demonstrate that blastocyst quality significantly impacts both embryo viability at 12 dpf, as well as the presence of POU5F1+ cells within viable outgrowths. Moreover, detailed examination of 12 dpf blastocyst outgrowths revealed a population of POU5F1+, SOX2- and SOX17+ cells that may correspond to PGCLCs, alongside POU5F1+ epiblast-like cells and GATA6+ endoderm-like cells. Our findings suggest that, in human, PGC precursors may become specified within the epiblast and migrate either transiently to the extra-embryonic mesoderm or directly to the dorsal part of the yolk sac endoderm around 12 dpf. This is a descriptive analysis and as such the conclusion that POU5F1+ and SOX17+ cells represent bona fide PGCs can only be considered as preliminary. In the future, other PGC markers may be used to further validate the observed cell populations. Overall, our findings provide insights into the origin of the human germline and may serve as a foundation to further unravel the molecular mechanisms governing PGC specification in human., (© The Author(s) 2019. Published by Oxford University Press.)

Clinical applications of human induced pluripotent stem cells (hiPSCs) are expected, but hiPSC lines vary in their differentiation propensity. For efficient selection of hiPSC lines suitable for differentiation into desired cell lineages, here we identify SALL3 as a marker to predict differentiation propensity. SALL3 expression in hiPSCs correlates positively with ectoderm differentiation capacity and negatively with mesoderm/endoderm differentiation capacity. Without affecting self-renewal of hiPSCs, SALL3 knockdown inhibits ectoderm differentiation and conversely enhances mesodermal/endodermal differentiation. Similarly, loss- and gain-of-function studies reveal that SALL3 inversely regulates the differentiation of hiPSCs into cardiomyocytes and neural cells. Mechanistically, SALL3 modulates DNMT3B function and DNA methyltransferase activity, and influences gene body methylation of Wnt signaling-related genes in hiPSCs. These findings suggest that SALL3 switches the differentiation propensity of hiPSCs toward distinct cell lineages by changing the epigenetic profile and serves as a marker for evaluating the hiPSC differentiation propensity.

Although pluripotent stem cells can generate various types of differentiated cells, it is unclear why lineage-committed stem/progenitor cells derived from pluripotent stem cells are decelerated and why the differentiation-resistant propensity of embryonic stem cell (ESC)/induced pluripotent stem cell (iPSC)-derived cells is predominant compared with the in vivo equivalents derived from embryonic/adult tissues. In this study, we demonstrated that iPSCs reprogrammed and maintained with three chemical inhibitors of the fibroblast growth factor 4-mitogen-activated protein kinase cascade and GSK3β (3i) could be differentiated into all three germ layers more efficiently than the iPSCs reprogrammed without the 3i chemicals, even though they were maintained with 3i chemicals once they were reprogrammed. Although the iPSCs reprogrammed with 3i had increased numbers of Zscan4-positive cells, the Zscan4-positive cells among iPSCs that were reprogrammed without 3i did not have an accelerated differentiation ability. These observations suggest that 3i exposure during the reprogramming period determines the accelerated differentiation/maturation potentials of iPSCs that are stably maintained at the distinct state., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Upon virus infection, pluripotent stem cells neither induce nor respond to canonical type I interferons (IFN-I). To better understand this biology, we characterized induced pluripotent stem cells (iPSCs) as well as their differentiated parental or rederived counterparts. We confirmed that only iPSCs failed to respond to viral RNA, IFN-I, or viral infection. This lack of response could be phenocopied in fibroblasts with the expression of a reprogramming factor which repressed the capacity to induce canonical antiviral pathways. To ascertain the consequences of restoring the antiviral response in the context of pluripotency, we engineered a system to engage these defenses in iPSCs. Inducible expression of a recombinant virus-activated transcription factor resulted in the successful reconstitution of antiviral defenses through the direct up-regulation of IFN-I-stimulated genes. Induction of the antiviral signature in iPSCs, even for a short duration, resulted in the dysregulation of genes associated with all three germ layers despite maintaining pluripotency markers. Trilineage differentiation of these same cells showed that engagement of the antiviral defenses compromised ectoderm and endoderm formation and dysregulated the development of mesodermal sublineages. In all, these data suggest that the temporal induction of the antiviral response primes iPSCs away from pluripotency and induces numerous aberrant gene products upon differentiation. Together these results suggest that the IFN-I system and pluripotency may be incompatible with each other and thus explain why stem cells do not utilize the canonical antiviral system., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The propensity for differentiation varies substantially across human pluripotent stem cell (hPSC) lines, greatly restricting the use of hPSCs for cell replacement therapy or disease modeling. Here, we investigate the underlying mechanisms and demonstrate that activation of the retinoblastoma (Rb) pathway in a transient manner is important for differentiation. In prior work, we demonstrated that pre-treating hPSCs with dimethylsulfoxide (DMSO) before directed differentiation enhanced differentiation potential across all three germ layers. Here, we show that exposure to DMSO improves the efficiency of hPSC differentiation through Rb and by repressing downstream E2F-target genes. While transient inactivation of the Rb family members (including Rb, p107, and p130) suppresses DMSO's capacity to enhance differentiation across all germ layers, transient expression of a constitutively active (non-phosphorylatable) form of Rb increases the differentiation efficiency similar to DMSO. Inhibition of downstream targets of Rb, such as E2F signaling, also promotes differentiation of hPSCs. More generally, we demonstrate that the duration of Rb activation plays an important role in regulating differentiation capacity., Competing Interests: The authors have declared that no competing interests exist.

Somatic cell nuclear transfer (SCNT) or the forced expression of transcription factors can be used to generate autologous pluripotent stem cells (PSCs). Although transcriptomic and epigenomic comparisons of isogenic human NT-embryonic stem cells (NT-ESCs) and induced PSCs (iPSCs) in the undifferentiated state have been reported, their functional similarities and differentiation potentials have not been fully elucidated. Our study showed that NT-ESCs and iPSCs derived from the same donors generally displayed similar in vitro commitment capacity toward three germ layer lineages as well as proliferative activity and clonogenic capacity. However, the maturation capacity of NT-ESC-derived hematopoietic progenitors was significantly greater than the corresponding capacity of isogenic iPSC-derived progenitors. Additionally, donor-dependent variations in hematopoietic specification and commitment capacity were observed. Transcriptome and methylome analyses in undifferentiated NT-ESCs and iPSCs revealed a set of genes that may influence variations in hematopoietic commitment and maturation between PSC lines derived using different reprogramming methods. Here, we suggest that genetically identical iPSCs and NT-ESCs could be functionally unequal due to differential transcription and methylation levels acquired during reprogramming. Our proof-of-concept study indicates that reprogramming mechanisms and genetic background could contribute to diverse functionalities between PSCs.

In mice, primordial germ cells (PGCs), the precursors of eggs and sperm, originate from pregastrulation postimplantation embryos. By contrast, the origin of human PGCs (hPGCs) has been less clear and has been difficult to study because of the technical and ethical constraints that limit direct studies on human embryos. In recent years, however, in vitro simulation models using human pluripotent stem cells, together with surrogate non-rodent mammalian embryos, have provided insights and experimental approaches to address this issue. Here, we review these studies, which suggest that the posterior epiblast and/or the nascent amnion in pregastrulation human embryos is a likely source of hPGCs, and that a different gene regulatory network controls PGCs in humans compared with in the mouse. Such studies on the origins and mechanisms of hPGC specification prompt further consideration of the somatic cell fate decisions that occur during early human development., (© 2018. Published by The Company of Biologists Ltd.)

Discovered in chick embryos by Wilhelm His in 1868 and named the neural crest by Arthur Milnes Marshall in 1879, the neural crest cells that arise from the neural folds have since been shown to differentiate into almost two dozen vertebrate cell types and to have played major roles in the evolution of such vertebrate features as bone, jaws, teeth, visceral (pharyngeal) arches, and sense organs. I discuss the discovery that ectodermal neural crest gave rise to mesenchyme and the controversy generated by that finding; the germ layer theory maintained that only mesoderm could give rise to mesenchyme. A second topic of discussion is germ layers (including the neural crest) as emergent levels of organization in animal development and evolution that facilitated major developmental and evolutionary change. The third topic is gene networks, gene co-option, and the evolution of gene-signaling pathways as key to developmental and evolutionary transitions associated with the origin and evolution of the neural crest and neural crest cells., (© 2018 Wiley Periodicals, Inc.)

Recent studies have suggested a role for the Cyclin Dependent Kinase-2 Associated Protein 1 (CDK2AP1) in stem cell differentiation and self-renewal. In studies with mouse embryonic stem cells (mESCs) derived from generated mice embryos with targeted deletion of the Cdk2ap1 gene, CDK2AP1 was shown to be required for epigenetic silencing of Oct4 during differentiation, with deletion resulting in persistent self-renewal and reduced differentiation potential. Differentiation capacity was restored in these cells following the introduction of a non-phosphorylatible form of the retinoblastoma protein (pRb) or exogenous Cdk2ap1. In this study, we investigated the role of CDK2AP1 in human embryonic stem cells (hESCs). Using a shRNA to reduce its expression in hESCs, we found that CDK2AP1 knockdown resulted in a significant reduction in the expression of the pluripotency genes, OCT4 and NANOG. We also found that CDK2AP1 knockdown increased the number of embryoid bodies (EBs) formed when differentiation was induced. In addition, the generated EBs had significantly higher expression of markers of all three germ layers, indicating that CDK2AP1 knockdown enhanced differentiation. CDK2AP1 knockdown also resulted in reduced proliferation and reduced the percentage of cells in the S phase and increased cells in the G2/M phase of the cell cycle. Further investigation revealed that a higher level of p53 protein was present in the CDK2AP1 knockdown hESCs. In hESCs in which p53 and CDK2AP1 were simultaneously downregulated, OCT4 and NANOG expression was not affected and percentage of cells in the S phase of the cell cycle was not reduced. Taken together, our results indicate that the knockdown of CDK2AP1 in hESCs results in increased p53 and enhances differentiation and favors it over a self-renewal fate.

Due to high infertility ratio nowadays, it is essential to explore efficient ways of enhancing mammalian reproductivity, in particular female reproductivity. Using female Oct4-GFP embryonic stem cells, we mimic the in vivo development procedure to induce ES cells into epiblast cell-like cells (EpiLCs) and then primordial germ cell-like cells (PGCLCs). GFP positive PGCLCs that showed typical PGC markers and epigenetic modification were efficiently obtained. Further transplantation of the GFP positive PGCLC and native ovary cell mixture into ovary of infertile mice revealed that both MVH and GFP positive cells could be developed in ovary, but no later developmental stage germ cells were observed. This study suggested that Oct4-GFP ES cells may be only suitable for tracing early germ cell development., (© 2018 International Federation for Cell Biology.)

Background: Miguel Fernández was an Argentinian zoologist who published the first account of obligate polyembryony in armadillos. His contribution is here discussed in relation to his contemporaries, Newman and Patterson, and more recent work., Findings: Fernandez worked on the mulita (Dasypus hybridus). He was able to get early stages before twinning occurred and show it was preceded by inversion of the germ layers. By the primitive streak stage there were separate embryonic shields and partition of the amnion. There was, however, a single exocoelom and all embryos were enclosed in a common set of membranes comprising chorion towards the attachment site in the uterine fundus and inverted yolk sac on the opposite face. He showed that monozygotic twinning did not occur in another armadillo, the peludo (Chaetophractus villosus)., Conclusions: Fernández's work represented a major breakthrough in understanding how twinning occurred in armadillos. His work and that of others is of intrinsic interest to zoologists and has a direct bearing on the origin of monozygotic twins and birth defects in humans., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Inner cell Mass (ICM) specification into epiblast (Epi) and primitive endoderm (PrE) is an asynchronous and progressive process taking place between E3.0 to E3.75 under the control of the Fibroblast Growth Factor (FGF)/Extracellular signal-Regulated Kinase (ERK) signaling pathway. Here, we have analyzed in details the kinetics of specification and found that ICM cell responsiveness to the up and down regulation of FGF signaling activity are temporally distinct. We also showed that PrE progenitors are generated later than Epi progenitors. We further demonstrated that, during this late phase of specification, a 4 hours period of FGF/ERK inhibition prior E3.75 is sufficient to convert ICM cells into Epi. Finally, we showed that ICM conversion into Epi in response to inhibition during this short time window requires both transcription and proteasome degradation. Collectively, our data give new insights into the timing and mechanisms involved in the process of ICM specification.

Gastrulation initiates with the formation of the primitive streak, during which, cells of the epiblast delaminate to form the mesoderm and definitive endoderm. At this stage, the pluripotent cell population of the epiblast undergoes very rapid proliferation and extensive epigenetic programming. Here we show that Fam208a, a new epigenetic modifier, is essential for early post-implantation development. We show that Fam208a mutation leads to impaired primitive streak elongation and delayed epithelial-to-mesenchymal transition. Fam208a mutant epiblasts had increased expression of p53 pathway genes as well as several pluripotency-associated long non-coding RNAs. Fam208a mutants exhibited an increase in p53-driven apoptosis and complete removal of p53 could partially rescue their gastrulation block. This data demonstrates a new in vivo function of Fam208a in maintaining epiblast fitness, establishing it as an important factor at the onset of gastrulation when cells are exiting pluripotency.

In cattle early gastrulation-stage embryos (Stage 5), four tissues can be discerned: (i) the top layer of the embryonic disc consisting of embryonic ectoderm (EmE); (ii) the bottom layer of the disc consisting of mesoderm, endoderm and visceral hypoblast (MEH); (iii) the trophoblast (TB); and (iv) the parietal hypoblast. We performed microsurgery followed by RNA-seq to analyse the transcriptome of these four tissues as well as a developmentally earlier pre-gastrulation embryonic disc. The cattle EmE transcriptome was similar at Stages 4 and 5, characterised by the OCT4/SOX2/NANOG pluripotency network. Expression of genes associated with primordial germ cells suggest their presence in the EmE tissue at these stages. Anterior visceral hypoblast genes were transcribed in the Stage 4 disc, but no longer by Stage 5. The Stage 5 MEH layer was equally similar to mouse embryonic and extraembryonic visceral endoderm. Our data suggest that the first mesoderm to invaginate in cattle embryos is fated to become extraembryonic. TGFβ, FGF, VEGF, PDGFA, IGF2, IHH and WNT signals and receptors were expressed, however the representative members of the FGF families differed from that seen in equivalent tissues of mouse embryos. The TB transcriptome was unique and differed significantly from that of mice. FGF signalling in the TB may be autocrine with both FGFR2 and FGF2 expressed. Our data revealed a range of potential inter-tissue interactions, highlighted significant differences in early development between mice and cattle and yielded insight into the developmental events occurring at the start of gastrulation.

The process of spatial rearrangement of cells of the inner cell mass (ICM) that are destined to become hypoblast is not well understood. The observation that the chemokine (C-C motif) ligand 24 (CCL24) and several other genes involved in chemokine signaling are expressed more in the ICM than in the trophectoderm of the bovine embryo resulted in the hypothesis that CCL24 participates in spatial organization of the ICM. Temporally, expression of CCL24 in the bovine embryo occurs coincidently with blastocyst formation: transcript abundance was low until the late morula stage, peaked in the blastocyst at Day 7 of development and declined by Day 9. Treatment of embryos with two separate antagonists of C-C motif chemokine receptor 3 (the prototypical receptor for CCL24) decreased the percent of GATA6+ cells (hypoblast precursors) that were located in the outside of the ICM. Similarly, injection of zygotes with a CCL24-specific morpholino decreased the percent of GATA6+ cells in the outside of the ICM. In conclusion, CCL24 assists in spatial arrangement of the ICM in the bovine embryo. This experiment points to new functions of chemokine signaling in the bovine embryo and is consistent with the idea that cell migration is involved in the spatial organization of hypoblast cells in the blastocyst., (© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please journals.permissions@oup.com.)

The regulative capability of single cells to give rise to all primary embryonic lineages is termed pluripotency. Observations of fluctuating gene expression and phenotypic heterogeneity in vitro have fostered a conception of pluripotency as an intrinsically metastable and precarious state. However, in the embryo and in defined culture environments the properties of pluripotent cells change in an orderly sequence. Two phases of pluripotency, called naïve and primed, have previously been described. In this Hypothesis article, a third phase, called formative pluripotency, is proposed to exist as part of a developmental continuum between the naïve and primed phases. The formative phase is hypothesised to be enabling for the execution of pluripotency, entailing remodelling of transcriptional, epigenetic, signalling and metabolic networks to constitute multi-lineage competence and responsiveness to specification cues., (© 2017. Published by The Company of Biologists Ltd.)

Evidence has accumulated that adult hematopoietic tissues and other organs contain a population of dormant stem cells (SCs) that are more primitive than other, already restricted, monopotent tissue-committed SCs (TCSCs). These observations raise several questions, such as the developmental origin of these cells, their true pluripotent or multipotent nature, which surface markers they express, how they can be efficiently isolated from adult tissues, and what role they play in the adult organism. The phenotype of these cells and expression of some genes characteristic of embryonic SCs, epiblast SCs, and primordial germ cells suggests their early-embryonic deposition in developing tissues as precursors of adult SCs. In this review, we will critically discuss all these questions and the concept that small dormant SCs related to migratory primordial germ cells, described as very small embryonic-like SCs, are deposited during embryogenesis in bone marrow and other organs as a backup population for adult tissue-committed SCs and are involved in several processes related to tissue or organ rejuvenation, aging, and cancerogenesis. The most recent results on successful ex vivo expansion of human very small embryonic-like SC in chemically defined media free from feeder-layer cells open up new and exciting possibilities for their application in regenerative medicine., Competing Interests: – None to report, (© 2017 American Heart Association, Inc.)

Background: Cellular reprogramming is a stressful process, which requires cells to engulf somatic features and produce and maintain stemness machineries. Autophagy is a process to degrade unwanted proteins and is required for the derivation of induced pluripotent stem cells (iPSCs). However, the role of autophagy during iPSC maintenance remains undefined., Methods: Human iPSCs were investigated by microscopy, immunofluorescence, and immunoblotting to detect autophagy machinery. Cells were treated with rapamycin to activate autophagy and with bafilomycin to block autophagy during iPSC maintenance. High concentrations of rapamycin treatment unexpectedly resulted in spontaneous formation of round floating spheres of uniform size, which were analyzed for differentiation into three germ layers. Mass spectrometry was deployed to reveal altered protein expression and pathways associated with rapamycin treatment., Results: We demonstrate that human iPSCs express high basal levels of autophagy, including key components of APMKα, ULK1/2, BECLIN-1, ATG13, ATG101, ATG12, ATG3, ATG5, and LC3B. Block of autophagy by bafilomycin induces iPSC death and rapamycin attenuates the bafilomycin effect. Rapamycin treatment upregulates autophagy in iPSCs in a dose/time-dependent manner. High concentration of rapamycin reduces NANOG expression and induces spontaneous formation of round and uniformly sized embryoid bodies (EBs) with accelerated differentiation into three germ layers. Mass spectrometry analysis identifies actin cytoskeleton and adherens junctions as the major targets of rapamycin in mediating iPSC detachment and differentiation., Conclusions: High levels of basal autophagy activity are present during iPSC derivation and maintenance. Rapamycin alters expression of actin cytoskeleton and adherens junctions, induces uniform EB formation, and accelerates differentiation. IPSCs are sensitive to enzyme dissociation and require a lengthy differentiation time. The shape and size of EBs also play a role in the heterogeneity of end cell products. This research therefore highlights the potential of rapamycin in producing uniform EBs and in shortening iPSC differentiation duration.

During mouse preimplantation embryo development, the classically described second cell-fate decision involves the specification and segregation, in blastocyst inner cell mass (ICM), of primitive endoderm (PrE) from pluripotent epiblast (EPI). The active role of fibroblast growth factor (Fgf) signalling during PrE differentiation, particularly in the context of Erk1/2 pathway activation, is well described. However, we report that p38 family mitogen-activated protein kinases (namely p38α/Mapk14 and p38β/Mapk11; referred to as p38-Mapk14/11) also participate in PrE formation. Specifically, functional p38-Mapk14/11 are required, during early-blastocyst maturation, to assist uncommitted ICM cells, expressing both EPI and earlier PrE markers, to fully commit to PrE differentiation. Moreover, functional activation of p38-Mapk14/11 is, as reported for Erk1/2, under the control of Fgf-receptor signalling, plus active Tak1 kinase (involved in non-canonical bone morphogenetic protein (Bmp)-receptor-mediated PrE differentiation). However, we demonstrate that the critical window of p38-Mapk14/11 activation precedes the E3.75 timepoint (defined by the initiation of the classical 'salt and pepper' expression pattern of mutually exclusive EPI and PrE markers), whereas appropriate lineage maturation is still achievable when Erk1/2 activity (via Mek1/2 inhibition) is limited to a period after E3.75. We propose that active p38-Mapk14/11 act as enablers, and Erk1/2 as drivers, of PrE differentiation during ICM lineage specification and segregation., (© 2016 The Authors.)

Mesoderm represents the evolutionary youngest germ layer and forms numerous novel tissues in bilaterian animals. Despite the established conservation of the gene regulatory networks that drive mesoderm differentiation (e.g. myogenesis), mechanisms of mesoderm specification are highly variable in distant model species. Thus, broader phylogenetic sampling is required to reveal common features of mesoderm formation across bilaterians. Here we focus on a representative of Spiralia, the marine annelid Alitta virens, whose mesoderm development is still poorly investigated on the molecular level. We characterize three novel early mesodermal markers for A. virens - Twist, Mox, and Evx - which are differentially expressed within the mesodermal lineages. The Twist mRNA is ubiquitously distributed in the fertilized egg and exhibits specific expression in endomesodermal- and ectomesodermal-founder cells at gastrulation. Twist is expressed around the blastopore and later in a segmental metameric pattern. We consider this expression to be ancestral, and in support of the enterocoelic hypothesis of mesoderm evolution. We also revealed an early pattern of the MAPK activation in A. virens that is different from the previously reported pattern in spiralians. Inhibition of the MAPK pathway by U0126 disrupts the metameric Twist and Mox expression, indicating an early requirement of the MAPK cascade for proper morphogenesis of endomesodermal tissues., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Upon its implantation in the uterus of the mother in mammals, the embryo is composed by three morphologically distinct tissues: the Epiblast (Epi), the Trophectoderm (TE) and the Primitive Endoderm (PrE). Both Epi and PrE are formed from the same cell homogeneous population called the Inner Cell Mass (ICM). Based on our studies, we discuss in this review what molecular interactions are necessary for the specification of these two lineages. For this, we have combined a biological approach with mathematical modeling. We have shown the central role of the gene regulation group composed by NANOG, FGF4, GATA6 and FGFR2 for Epi/PrE cell specification., (© 2016 médecine/sciences – Inserm.)

During embryonic development, tissues deform by a succession and combination of morphogenetic processes. Tissue compaction is the morphogenetic process by which a tissue adopts a tighter structure. Recent studies characterized the respective roles of cells' adhesive and contractile properties in tissue compaction. In this review, we formalize the mechanical and molecular principles of tissue compaction and we analyze through the prism of this framework several morphogenetic events: the compaction of the early mouse embryo, the formation of the fly retina, the segmentation of somites and the separation of germ layers during gastrulation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Innate pluripotency of mouse embryos transits from naive to primed state as the inner cell mass differentiates into epiblast. In vitro, their counterparts are embryonic (ESCs) and epiblast stem cells (EpiSCs), respectively. Activation of the FGF signaling cascade results in mouse ESCs differentiating into mEpiSCs, indicative of its requirement in the shift between these states. However, only mouse ESCs correspond to the naive state; ESCs from other mammals and from chick show primed state characteristics. Thus, the significance of the naive state is unclear. In this study, we use zebra finch as a model for comparative ESC studies. The finch blastoderm has mESC-like properties, while chick blastoderm exhibits EpiSC features. In the absence of FGF signaling, finch cells retained expression of pluripotent markers, which were lost in cells from chick or aged finch epiblasts. Our data suggest that the naive state of pluripotency is evolutionarily conserved among amniotes.

Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve pluripotent state to a primed epiblast state. Addition of inhibitors of GSK3β and MEK (so-called 2i conditions) pushes ESC cultures toward a more homogeneous naïve pluripotent state, but the molecular underpinnings of this naïve transition are not completely understood. Here, we demonstrate that DAZL, an RNA-binding protein known to play a key role in germ-cell development, marks a subpopulation of ESCs that is actively transitioning toward naïve pluripotency. Moreover, DAZL plays an essential role in the active reprogramming of cytosine methylation. We demonstrate that DAZL associates with mRNA of Tet1, a catalyst of 5-hydroxylation of methyl-cytosine, and enhances Tet1 mRNA translation. Overexpression of DAZL in heterogeneous ESC cultures results in elevated TET1 protein levels as well as increased global hydroxymethylation. Conversely, null mutation of Dazl severely stunts 2i-mediated TET1 induction and hydroxymethylation. Our results provide insight into the regulation of the acquisition of naïve pluripotency and demonstrate that DAZL enhances TET1-mediated cytosine hydroxymethylation in ESCs that are actively reprogramming to a pluripotent ground state., (© 2015 The Authors.)

The epiblast is a single cell-layered epithelium which generates through gastrulation all tissues in an amniote embryo proper. Specification of the epiblast as a cell lineage in early development is coupled with that of the trophoblast and hypoblast, two lineages dedicated to forming extramebryonic tissues. The complex relationship between molecular specification and morphogenetic segregation of these three lineages is not well understood. In this review I will compare the ontogeny of epithelial epiblast in different amniote groups and emphasize the diversity in cell biological mechanisms employed by each group to reach this conserved epithelial structure as the pre-requisite for gastrulation. The limitations of associating cell fate with cell shape and position will also be discussed. In most amniote groups, bi-potential precursors for the epiblast and hypoblast, similar to the inner cell mass in the eutherian mammals, are not associated with an apolar, inside location in the blastocyst. Conversely, a blastocyst cell with epithelial morphology and superficial location is not indicative of its trophoblast fate. The polar trophoblast is absent in all amniotes except for the eutherian mammals. In the avian, reptilian and eutherian groups, epithelialization of the epiblast occurs after its fate specification and involves a mesenchymal-to-epithelial transition (MET) process, whereas in the monotremes and marsupials, pre-epiblast cells adopt an epithelial morphology prior to their commitment to the epiblast fate. The conservation of an epithelialized epiblast is viewed as an adaptation to evolutionary constraints placed on pre-gastrulation ectoderm in the ancestral amniote. The relationship between epiblast MET and epiblast pluripontency will also be discussed. Whether such an MET/epithelialization process is advantageous for the self-renewal and/or differentiation of human epiblast stem cells in vitro is unclear., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The nervous system of vertebrates is derived from an early embryonic region referred to as the neural plate. In the chick embryo, the neural plate is populated by neural stem cells specified from the epiblast shortly after the onset of gastrulation. Accompanying the formation of the plate, chondroitin sulfate glycosaminoglycans are expressed in the basal extracellular matrix. We describe in vitro experiments measuring cell adhesion of epiblast cells during the formation of the neural plate. Our findings may suggest that neural stem cells are set apart from non-neural epiblast by changes in relative cell-cell and cell-substrate adhesion. Specifically, changes in cell adhesion separating neural stem cells from the non-neural epiblast may be augmented by the presence of exogenous chondroitin-6-sulfate in the epiblast basal lamina at the time neural progenitors are specified in the epiblast.

Specification of the epiblast and primitive endoderm is one of the earliest differentiation steps during embryogenesis. In vitro tracking of pluripotency markers in ESCs suggests that epiblast specification may be plastic; however, live imaging of blastocysts, as detailed in a recent paper from Xenopoulos et al (2015), showed that, unlike in ESCs, fate commitment in vivo is largely irreversible., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Shortly after implantation the embryonic lineage transforms from a coherent ball of cells into polarized cup shaped epithelium. Recently we elucidated a previously unknown apoptosis-independent morphogenic event that reorganizes the pluripotent lineage. Polarization cues from the surrounding basement membrane rearrange the epiblast into a polarized rosette-like structure, where subsequently a central lumen is established. Thus, we provided a new model revising the current concept of apoptosis-dependent epiblast morphogenesis. Cell death however has to be tightly regulated during embryogenesis to ensure developmental success. Here, we follow the stages of early mouse development and take a glimpse at the critical signaling and morphogenic events that determine cells destiny and reshape the embryonic lineage., (© 2015 The Authors. Bioessays published by WILEY Periodicals, Inc.)

Recently, Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) has been developed for whole genome amplification of an individual cell, relying on quasilinear instead of exponential amplification to achieve high coverage. Here we adapt MALBAC for single-cell transcriptome amplification, which gives consistently high detection efficiency, accuracy and reproducibility. With this newly developed technique, we successfully amplified and sequenced single cells from 3 germ layers from mouse embryos in the early gastrulation stage, and examined the epithelial-mesenchymal transition (EMT) program among cells in the mesoderm layer on a single-cell level.

During early development, modulations in the expression of Nodal, a TGFβ family member, determine the specification of embryonic and extra-embryonic cell identities. Nodal has been extensively studied in the mouse, but aspects of its early expression remain unaccounted for. We identified a conserved hotspot for the binding of pluripotency factors at the Nodal locus and called this sequence "highly bound element" (HBE). Luciferase-based assays, the analysis of fluorescent HBE reporter transgenes, and a conditional mutation of HBE allowed us to establish that HBE behaves as an enhancer, is activated ahead of other Nodal enhancers in the epiblast, and is essential to Nodal expression in embryonic stem cells (ESCs) and in the mouse embryo. We also showed that HBE enhancer activity is critically dependent on its interaction with the pluripotency factor Oct4 and on Activin/Nodal signaling. Use of an in vitro model of epiblast maturation, relying on the differentiation of ESCs into epiblast stem cells (EpiSCs), revealed that this process entails a shift in the regulation of Nodal expression from an HBE-driven phase to an ASE-driven phase, ASE being another autoregulatory Nodal enhancer. Deletion of HBE in ESCs or in EpiSCs allowed us to show that HBE, although not necessary for Nodal expression in EpiSCs, is required in differentiating ESCs to activate the differentiation-promoting ASE and therefore controls this regulatory shift. Our findings clarify how early Nodal expression is regulated and suggest how this regulation can promote the specification of extra-embryonic precusors without inducing premature differentiation of epiblast cells. More generally, they open new perspectives on how pluripotency factors achieve their function., Competing Interests: The authors have declared that no competing interests exist.

The embryonic stem cell (ESC) derived from the inner cell mass is viewed as the core pluripotent cell (PC) type from which all other cell types emanate. This familiar perspective derives from an embryological time line in which PCs are ordered according to their time of appearance. However, this schema does not take into account their potential for interconversion, thereby excluding this critical quality of PCs. The persistence of bona fide pluripotent adult stem cells has garnered increasing attention in recent years. Adult pluripotent spermatogonial germ stem cells (aSGSCs) arise from primordial germ cells (pGCs) that emerge from the epiblast during gastrulation. Adult definitive neural stem cells (dNSCs) arise clonally from pluripotent embryonic primitive neural stem cells (pNSCs), which can also be derived clonally from ESCs. To test for stem cell-type convertibility, we employed differentiation in the clonal lineage from ESCs to pNSCs to dNSCs, and revealed the relationships and lineage positioning among various PC populations, including spermatogonial germ cells (aSGSCs), epiblast-derived stem cells (Epi-SCs) and the bFGF, Activin, and BIO-derived stem cell (FAB-SC). Adult, murine aSGSCs assumed a 'pseudo-ESC' state in vitro, and then differentiated into dNSCs, but not pNSCs. Similarly, Epi-SCs and FAB-SCs only gave rise to dNSCs and not to pNSCs. The results of these experiments suggest a new pluripotency lineage model describing the relationship(s) among PCs that better reflects the transitions between these cell types in vitro.

During the course of preimplantation development, the mammalian embryo develops from a single totipotent cell into a blastocyst that is composed of three distinct cell types. Two waves of lineage specification events take place, setting aside a pluripotent cell population, the epiblast, from extraembryonic tissues. The epiblast that will form the somatic cells and germ line of the adult organism remains pluripotent until gastrulation, which commences shortly after the embryo implants. The epiblast's remarkable property of pluripotency has been harnessed by researchers for decades through derivation of embryonic stem cells and epiblast stem cells. Both types of cells can self-renew indefinitely and still retain the ability of germ layer differentiation. However, a central conundrum to the field of stem cell biology is the extent to which these in vitro cultured cells represent their in vivo tissue of origin. In this review we discuss the development of in vivo pluripotency, and compare and contrast the role of signaling pathways and downstream transcription factors in embryo-derived stem cell types and their in vivo equivalent lineage counterparts., (© 2014 Elsevier Inc. All rights reserved.)

During embryonic development, cells or groups of cells migrate from their locations of origin to assume their correct anatomical positions. Intercellular adhesion plays an active and instructive role in orchestrating this process. Precisely how adhesion provides spatial positioning information is a subject of intense interest. In the 1960s, Steinberg proposed the differential adhesion hypothesis (DAH) to explain how differences in the intensity of cell adhesion could give rise to predictable spatial interactions between different cell types. The DAH is grounded in the same set of physical principles governing the interaction of immiscible fluids and thus provides a rigorous conceptual framework connecting the chemistry of cell adhesion to the physics underlying cell and tissue segregation. Testing the DAH required the development of methods to measure intercellular cohesion and of assays to accurately assess relative spatial position between cells. The DAH has been experimentally verified and computationally simulated. Moreover, evidence concerning the role of differential adhesion in a number of morphodynamic events is accumulating. It is clear that differential adhesion is a major driving force in various aspects of embryonic development, but recent studies have also advanced the concept that other factors, such as cortical tension and elasticity, may also be involved in fine tuning, or even driving the process. It is likely that an interplay between adhesion and these other factors co-operate to generate the forces required for tissue self-organization., (Copyright © 2013 Wiley Periodicals, Inc.)