Extraembryonic Cell Types Research Articles

A single-cell atlas of pig gastrulation as a resource for comparative embryology

Cell-fate decisions during mammalian gastrulation are poorly understood outside of rodent embryos. The embryonic disc of pig embryos mirrors humans, making them a useful proxy for studying gastrulation. Here we present a single-cell transcriptomic atlas of pig gastrulation, revealing cell-fate emergence dynamics, as well as conserved and divergent gene programs governing early porcine, primate, and murine development. We highlight heterochronicity in extraembryonic cell-types, despite the broad conservation of cell-type-specific transcriptional programs. We apply these findings in combination with functional investigations, to outline conserved spatial, molecular, and temporal events during definitive endoderm specification. We find early FOXA2 + /TBXT- embryonic disc cells directly form definitive endoderm, contrasting later-emerging FOXA2/TBXT+ node/notochord progenitors. Unlike mesoderm, none of these progenitors undergo epithelial-to-mesenchymal transition. Endoderm/Node fate hinges on balanced WNT and hypoblast-derived NODAL, which is extinguished upon endodermal differentiation. These findings emphasise the interplay between temporal and topological signalling in fate determination during gastrulation.

Smad4 is essential for epiblast scaling and morphogenesis after implantation, but nonessential before implantation.

Bone morphogenic protein (BMP) signaling plays an essential and highly conserved role in embryo axial patterning in animal species. However, in mammalian embryos, which develop inside the mother, early development includes a preimplantation stage, which does not occur in externally developing embryos. During preimplantation, the epiblast is segregated from extra-embryonic lineages that enable implantation and development in utero. Yet, the requirement for BMP signaling is imprecisely defined in mouse early embryos. Here, we show that, in contrast to previous reports, BMP signaling (SMAD1/5/9 phosphorylation) is not detectable until implantation when it is detected in the primitive endoderm - an extra-embryonic lineage. Moreover, preimplantation development appears to be normal following deletion of maternal and zygotic Smad4, an essential effector of canonical BMP signaling. In fact, mice lacking maternal Smad4 are viable. Finally, we uncover a new requirement for zygotic Smad4 in epiblast scaling and cavitation immediately after implantation, via a mechanism involving FGFR/ERK attenuation. Altogether, our results demonstrate no role for BMP4/SMAD4 in the first lineage decisions during mouse development. Rather, multi-pathway signaling among embryonic and extra-embryonic cell types drives epiblast morphogenesis postimplantation.

Quiescence enables unrestricted cell fate in naive embryonic stem cells

Quiescence in stem cells is traditionally considered as a state of inactive dormancy or with poised potential. Naive mouse embryonic stem cells (ESCs) can enter quiescence spontaneously or upon inhibition of MYC or fatty acid oxidation, mimicking embryonic diapause in vivo. The molecular underpinning and developmental potential of quiescent ESCs (qESCs) are relatively unexplored. Here we show that qESCs possess an expanded or unrestricted cell fate, capable of generating both embryonic and extraembryonic cell types (e.g., trophoblast stem cells). These cells have a divergent metabolic landscape comparing to the cycling ESCs, with a notable decrease of the one-carbon metabolite S-adenosylmethionine. The metabolic changes are accompanied by a global reduction of H3K27me3, an increase of chromatin accessibility, as well as the de-repression of endogenous retrovirus MERVL and trophoblast master regulators. Depletion of methionine adenosyltransferase Mat2a or deletion of Eed in the polycomb repressive complex 2 results in removal of the developmental constraints towards the extraembryonic lineages. Our findings suggest that quiescent ESCs are not dormant but rather undergo an active transition towards an unrestricted cell fate.

The regulation of totipotency transcription: Perspective from in vitro and in vivo totipotency.

Totipotency represents the highest developmental potency. By definition, totipotent stem cells are capable of giving rise to all embryonic and extraembryonic cell types. In mammalian embryos, totipotency occurs around the zygotic genome activation period, which is around the 2-cell stage in mouse embryo or the 4-to 8-cell stage in human embryo. Currently, with the development of in vitro totipotent-like models and the advances in small-scale genomic methods, an in-depth mechanistic understanding of the totipotency state and regulation was enabled. In this review, we explored and summarized the current views about totipotency from various angles, including genetic and epigenetic aspects. This will hopefully formulate a panoramic view of totipotency from the available research works until now. It can also help delineate the scaffold and formulate new hypotheses on totipotency for future research works.

A gastruloid model of the interaction between embryonic and extra-embryonic cell types

Stem-cell derived in vitro systems, such as organoids or embryoids, hold great potential for modeling in vivo development. Full control over their initial composition, scalability, and easily measurable dynamics make those systems useful for studying specific developmental processes in isolation. Here we report the formation of gastruloids consisting of mouse embryonic stem cells (mESCs) and extraembryonic endoderm (XEN) cells. These XEN-enhanced gastruloids (XEGs) exhibit the formation of neural epithelia, which are absent in gastruloids derived from mESCs only. By single-cell RNA-seq, imaging, and differentiation experiments, we demonstrate the neural characteristics of the epithelial tissue. We further show that the mESCs induce the differentiation of the XEN cells to a visceral endoderm-like state. Finally, we demonstrate that local inhibition of WNT signaling and production of a basement membrane by the XEN cells underlie the formation of the neuroepithelial tissue. In summary, we establish XEGs to explore heterotypic cellular interactions and their developmental consequences in vitro.

Reconstitution of Human Peri-Implantation Embryogenesis With Integrated Stem Cell-Based Embryo Models

Studies of cultured human embryos have provided insights into human peri-implantation embryogenesis but are limited by ethical and technical constraints. Here we assembled a 3D integrated embryo model (IEM) using naive human embryonic stem cells (hESCs) and hESC-derived proliferating trophoblasts. By comparing phenotypic cell fate and comprehensive single-cell transcriptome of IEMs with 3D-cultured human embryos, we reveal that IEMs with embryonic and extra-embryonic cell types mimic developmental landmarks and 3D morphological features of natural human post­­­ ­­-implantation embryos, including pluripotent state transitions, epithelial-mesenchymal transition, anterior-posterior axis formation, amniotic cavity, yolk sac, bilaminar embryonic disc, extra-embryonic mesenchyme, primitive streak and primordial germ cells. Using IEMs, we unravel composition and gene expression patterns of embryonic lineages, and that trophoblasts secret WNT, BMP and TGFβ/Activin signals to instruct post-implantation embryogenesis . Our findings show that IEMs provide a scalable and tractable model for decoding regulatory mechanisms of human peri-implantation embryogenesis.

Human embryo research, stem cell-derived embryo models and invitro gametogenesis: Considerations leading to the revised ISSCR guidelines.

SummaryThe ISSCR Guidelines for Stem Cell Research and Clinical Translation were last revised in 2016. Since then, rapid progress has been made in research areas related to in vitro culture of human embryos, creation of stem cell-based embryo models, and in vitro gametogenesis. Therefore, a working group of international experts was convened to review the oversight process and provide an update to the guidelines. This report captures the discussion and summarizes the major recommendations made by this working group, with a specific emphasis on updating the categories of review and engagement with the specialized scientific and ethical oversight process.

NLRP7 plays a functional role in regulating BMP4 signaling during differentiation of patient-derived trophoblasts

Complete hydatidiform mole (HM) is a gestational trophoblastic disease resulting in hyperproliferation of trophoblast cells and absence of embryo development. Mutations in the maternal-effect gene NLRP7 are the major cause of familial recurrent complete HM. Here, we established an in vitro model of HM using patient-specific induced pluripotent stem cells (iPSCs) derived trophoblasts harboring NLRP7 mutations. Using whole transcriptome profiling during trophoblast differentiation, we showed that impaired NLRP7 expression results in precocious downregulation of pluripotency factors, activation of trophoblast lineage markers, and promotes maturation of differentiated extraembryonic cell types such as syncytiotrophoblasts. Interestingly, we found that these phenotypes are dependent on BMP4 signaling and BMP pathway inhibition corrected the excessive trophoblast differentiation of patient-derived iPSCs. Our human iPSC model of a genetic placental disease recapitulates aspects of trophoblast biology, highlights the broad utility of iPSC-derived trophoblasts for modeling human placental diseases and identifies NLRP7 as an essential modulator of key developmental cell fate regulators.

Guts and gastrulation: Emergence and convergence of endoderm in the mouse embryo.

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.

Concise Review: Mesenchymal Stem Cells Derived from Human Pluripotent Cells, an Unlimited and Quality-Controllable Source for Therapeutic Applications.

Despite the long discrepancy over their definition, heterogeneity, and functions, mesenchymal stem cells (MSCs) have proved to be a key player in tissue repair and homeostasis. Generally, somatic tissue-derived MSCs (st-MSCs) are subject to quality variations related to donated samples and biosafety concern for transmission of potential pathogens from the donors. In contrast, human pluripotent stem cells (hPSCs) are unlimited in supply, clear in the biological background, and convenient for quality control, genetic modification, and scale-up production. We, and others, have shown that hPSCs can differentiate in two dimensions or three dimensions to MSCs (ps-MSCs) via embryonic (mesoderm and neural crest) or extraembryonic (trophoblast) cell types under serum-containing or xeno-free and defined conditions. Compared to st-MSCs, ps-MSCs appear less mature, proliferate faster, express lower levels of inflammatory cytokines, and respond less to traditional protocols for st-MSC differentiation to other cell types, especially adipocytes. Nevertheless, ps-MSCs are capable of immune modulation and treatment of an increasing number of animal disease models via mitochondria transfer, paracrine, exosomes, and direct differentiation, and can be potentially used as a universal and endless therapy for clinical application. This review summarizes the progress on ps-MSCs and discusses perspectives and challenges for their potential translation to the clinic. Stem Cells 2019;37:572-581.

Self-Assembly of Embryonic and Two Extraembryonic Stem Cell Types Into Gastrulating Embryo-like Structures

(Abstracted from Nature Cell Biology 2018;20:979–989) Three stem cell types found in the natural embryo differentiate to become 3 distinct tissues: the embryonic tissue or epiblast, the trophectoderm, and the primitive endoderm. Signaling interactions between each of these tissue types are important for morphogenic pathways causing the cell rearrangements of gastrulation and establishing anterior and posterior identity.

Experimental Analysis of Imprinted Mouse X-Chromosome Inactivation.

X-chromosome inactivation is a dosage compensation mechanism that equalizes X-linked gene expression between male and female mammals through the transcriptional silencing of most genes on one of the two X-chromosomes in females. With a few key exceptions, once the X-chromosome is inactivated replicated copies of that X-chromosome are maintained as inactive in all descendant cells. X-inactivation is therefore a paradigm of epigenetic inheritance. Imprinted X-inactivation is a specialized form of X-inactivation that results in the silencing of the paternally derived X-chromosome. Due to its parent-of-origin-specific pattern of inactivation, imprinted X-inactivation is a model of mitotic as well as meiotic, i.e., transgenerational, epigenetic inheritance. All cells of the early mouse embryo undergo imprinted X-inactivation, a pattern that is subsequently maintained in extraembryonic cell types in vivo and in vitro. Here, we describe both high- and low-throughput approaches to interrogate imprinted X-inactivation in the mouse embryo as well in cultured extraembryonic stem cells.

Capturing and Interconverting Embryonic Cell Fates in a Dish.

Cells of the early embryo are totipotent because they will differentiate to produce the fetus and its surrounding extraembryonic tissues. By contrast, embryonic stem (ES) cells are considered to be merely pluripotent because they lack the ability to efficiently produce extraembryonic cell types. The relatively limited developmental potential of ES cells can be explained by the observation that ES cells are derived from the embryo after its cells have already begun to specialize and lose totipotency. Meanwhile, at the time that pluripotent ES cell progenitors are specified, so are the multipotent progenitors of two extraembryonic stem cell types: trophoblast stem (TS) cells and extraembryonic endoderm stem (XEN) cells. Notably, all three embryo-derived stem cell types are capable of either self-renewing or differentiating in a lineage-appropriate manner. These three types of embryo-derived stem cell serve as paradigms for defining the genes and pathways that define and maintain unique stem cell identities. Remarkably, some of the mechanisms that maintain the specific developmental potential of each stem cell line do so by preventing conversion to another stem cell fate. This chapter highlights noteworthy studies that have identified the genes and pathways that normally limit the interconversion of stem cell identities.

Our First Choice: Cellular and Genetic Underpinnings of Trophectoderm Identity and Differentiation in the Mammalian Embryo.

The trophectoderm (TE) is the first cell population to appear in the mammalian preimplantation embryo, as the result of the differentiation of totipotent blastomeres located on the outer surface of the late morula. Trophectodermal cells arrange in a monolayer covering the expanding blastocyst and acquire an epithelial phenotype with distinct apicobasal polarity and a basal lamina placed toward the blastocyst interior. During later development through the periimplantation and gastrulation stages, the TE gives rise to extraembryonic membranes and cell types that will eventually form most of the fetal placenta, the specialized organ through which the embryo obtains maternal nourishment necessary for subsequent exponential growth. The specification of the TE is controlled by the combination of morphological cues arising from cell polarity with differential activity of signaling pathways such as Hippo and Notch, and the restriction to outer cells of lineage specifiers such as CDX2. This is possibly the first symmetry-breaking decision undertaken by the uncommitted cells produced by a handful of mitosis divisions from the newly fertilized zygote. Understanding how this cell lineage is specified will therefore provide unique information about development, differentiation, and how the interplay between cellular morphology and signaling and regulatory factors results in a correctly 3D-patterned embryo.

Rlim-Dependent and -Independent Pathways for X Chromosome Inactivation in Female ESCs

During female mouse embryogenesis, two forms of Xchromosome inactivation (XCI) ensure dosage compensation from sex chromosomes. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp), and this pattern is maintained in extraembryonic cell types. Epiblast cells, which give rise to the embryo proper, reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI depend on the long non-coding RNA Xist. The ubiquitin ligase RLIM is required for iXCI invivo and occupies a central role in current models of rXCI. Here, we demonstrate the existence of Rlim-dependent and Rlim-independent pathways for rXCI in differentiating female ESCs. Upon uncoupling these pathways, we find more efficient Rlim-independent XCI in ESCs cultured under physiological oxygen conditions. Our results revise current models of rXCI and suggest that caution must be taken when comparing XCI studies in ESCs and mice.

Deficiency of microRNA miR-34a expands cell fate potential in pluripotent stem cells

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) efficiently generate all embryonic cell lineages but rarely generate extraembryonic cell types. We found that microRNA miR-34a deficiency expands the developmental potential of mouse pluripotent stem cells, yielding both embryonic and extraembryonic lineages and strongly inducing MuERV-L (MERVL) endogenous retroviruses, similar to what is seen with features of totipotent two-cell blastomeres. miR-34a restricts the acquisition of expanded cell fate potential in pluripotent stem cells, and it represses MERVL expression through transcriptional regulation, at least in part by targeting the transcription factor Gata2. Our studies reveal a complex molecular network that defines and restricts pluripotent developmental potential in cultured ESCs and iPSCs.

Pathway to a phenocopy: Heat stress effects in early embryogenesis.

Heat shocks applied at the onset of gastrulation in early Drosophila embryos frequently lead to phenocopies of U-shaped mutants-having characteristic failures in the late morphogenetic processes of germband retraction and dorsal closure. The pathway from nonspecific heat stress to phenocopied abnormalities is unknown. Drosophila embryos subjected to 30-min, 38 °C heat shocks at gastrulation appear to recover and restart morphogenesis. Post-heat-shock development appears normal, albeit slower, until a large fraction of embryos develop amnioserosa holes (diameters > 100 µm). These holes are positively correlated with terminal U-shaped phenocopies. They initiate between amnioserosa cells and open over tens of minutes by evading normal wound healing responses. They are not caused by tissue-wide increases in mechanical stress or decreases in cell-cell adhesion, but instead appear to initiate from isolated apoptosis of amnioserosa cells. The pathway from heat shock to U-shaped phenocopies involves the opening of one or more large holes in the amnioserosa that compromise its structural integrity and lead to failures in morphogenetic processes that rely on amnioserosa-generated tensile forces. The proposed mechanism by which heat shock leads to hole initiation and expansion is heterochonicity, i.e., disruption of morphogenetic coordination between embryonic and extra-embryonic cell types.

Transcriptome analysis of primary bovine extra-embryonic cultured cells.

The dataset described in this article pertains to the article by Hue et al. (2015) entitled “Primary bovine extra-embryonic cultured cells: A new resource for the study of in vivo peri-implanting phenotypes and mesoderm formation” [1]. In mammals, extra-embryonic tissues are essential to support not only embryo patterning but also embryo survival, especially in late implanting species. These tissues are composed of three cell types: trophoblast (bTCs), endoderm (bXECs) and mesoderm (bXMCs). Until now, it is unclear how these cells interact. In this study, we have established primary cell cultures of extra-embryonic tissues from bovine embryos collected at day-18 after artificial insemination. We used our homemade bovine 10K array (GPL7417) to analyze the gene expression profiles of these primary extra-embryonic cultured cells compared to the corresponding cells from in vivo micro-dissected embryos. Here, we described the experimental design, the isolation of bovine extra-embryonic cell types as well as the microarray expression analysis. The dataset has been deposited in Gene Expression Omnibus (GEO) (accession number GSE52967). Finally, these primary cell cultures were a powerful tool to start studying their cellular properties, and will further allow in vitro studies on cellular interactions among extra-embryonic tissues, and potentially between extra-embryonic vs embryonic tissues.

HIPPO pathway members restrict SOX2 to the inner cell mass where it promotes ICM fates in the mouse blastocyst.

Pluripotent epiblast (EPI) cells, present in the inner cell mass (ICM) of the mouse blastocyst, are progenitors of both embryonic stem (ES) cells and the fetus. Discovering how pluripotency genes regulate cell fate decisions in the blastocyst provides a valuable way to understand how pluripotency is normally established. EPI cells are specified by two consecutive cell fate decisions. The first decision segregates ICM from trophectoderm (TE), an extraembryonic cell type. The second decision subdivides ICM into EPI and primitive endoderm (PE), another extraembryonic cell type. Here, we investigate the roles and regulation of the pluripotency gene Sox2 during blastocyst formation. First, we investigate the regulation of Sox2 patterning and show that SOX2 is restricted to ICM progenitors prior to blastocyst formation by members of the HIPPO pathway, independent of CDX2, the TE transcription factor that restricts Oct4 and Nanog to the ICM. Second, we investigate the requirement for Sox2 in cell fate specification during blastocyst formation. We show that neither maternal (M) nor zygotic (Z) Sox2 is required for blastocyst formation, nor for initial expression of the pluripotency genes Oct4 or Nanog in the ICM. Rather, Z Sox2 initially promotes development of the primitive endoderm (PE) non cell-autonomously via FGF4, and then later maintains expression of pluripotency genes in the ICM. The significance of these observations is that 1) ICM and TE genes are spatially patterned in parallel prior to blastocyst formation and 2) both the roles and regulation of Sox2 in the blastocyst are unique compared to other pluripotency factors such as Oct4 or Nanog.

BMP4 regulation of human trophoblast development.

Since the derivation of human embryonic stem cells, and the subsequent generation of induced pluripotent stem cells, there has been much excitement about the ability to model and evaluate human organ development in vitro. The finding that these cells, when treated with BMP4, are able to generate the extraembryonic cell type, trophoblast, which is the predominant functional epithelium in the placenta, has not been widely accepted. This review evaluates this model, providing comparison to early known events during placentation in both human and mouse and addresses specific challenges. Keeping in mind the ultimate goal of understanding human placental development and pregnancy disorders, our aim here is two-fold: to distinguish gaps in our knowledge arising from mis- or over-interpretation of data, and to recognize the limitations of both mouse and human models, but to work within those limitations towards the ultimate goal.