Technical challenges of studying early human development.

Recent years have seen exciting progress across human embryo research, including new methods for culturing embryos, transcriptional profiling of embryogenesis and gastrulation, mapping lineage trajectories, and experimenting on stem cell-based embryo models. These advances are beginning to define the dynamical principles of development across stages, tissues and organs, enabling a better understanding of human development before birth in health and disease, and potentially leading to improved treatments for infertility and developmental disorders. However, there are still significant roadblocks en route to this goal. Here, we highlight technical challenges to studying early human development and propose ways and means to overcome some of these constraints.

NANOGP1, a tandem duplicate of NANOG, exhibits partial functional conservation in human naïve pluripotent stem cells.

Gene duplication events can drive evolution by providing genetic material for new gene functions, and they create opportunities for diverse developmental strategies to emerge between species. To study the contribution of duplicated genes to human early development, we examined the evolution and function of NANOGP1, a tandem duplicate of the transcription factor NANOG. We found that NANOGP1 and NANOG have overlapping but distinct expression profiles, with high NANOGP1 expression restricted to early epiblast cells and naïve-state pluripotent stem cells. Sequence analysis and epitope-tagging revealed that NANOGP1 is protein coding with an intact homeobox domain. The duplication that created NANOGP1 occurred earlier in primate evolution than previously thought and has been retained only in great apes, whereas Old World monkeys have disabled the gene in different ways, including homeodomain point mutations. NANOGP1 is a strong inducer of naïve pluripotency; however, unlike NANOG, it is not required to maintain the undifferentiated status of human naïve pluripotent cells. By retaining expression, sequence and partial functional conservation with its ancestral copy, NANOGP1 exemplifies how gene duplication and subfunctionalisation can contribute to transcription factor activity in human pluripotency and development.

Multi-omics profiling of mouse gastrulation at single-cell resolution.

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes1-5. Global epigenetic reprogramming accompanies these changes6-8, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.

Species-specific regulation of XIST by the JPX/FTX orthologs.

X chromosome inactivation (XCI) is an essential process, yet it initiates with remarkable diversity in various mammalian species. XIST, the main trigger of XCI, is controlled in the mouse by an interplay of lncRNA genes (LRGs), some of which evolved concomitantly to XIST and have orthologues across all placental mammals. Here, we addressed the functional conservation of human orthologues of two such LRGs, FTX and JPX. By combining analysis of single-cell RNA-seq data from early human embryogenesis with various functional assays in matched human and mouse pluripotent stem- or differentiated post-XCI cells, we demonstrate major functional differences for these orthologues between species, independently of primary sequence conservation. While the function of FTX is not conserved in humans, JPX stands as a major regulator of XIST expression in both species. However, we show that different entities of JPX control the production of XIST at various steps depending on the species. Altogether, our study highlights the functional versatility of LRGs across evolution, and reveals that functional conservation of orthologous LRGs may involve diversified mechanisms of action. These findings represent a striking example of how the evolvability of LRGs can provide adaptative flexibility to constrained gene regulatory networks.

Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq.

Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3' ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3' ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells.

An open and decondensed chromatin organization is a defining property of pluripotency. Several epigenetic regulators have been implicated in maintaining an open chromatin organization, but how these processes are connected to the pluripotency network is unknown. Here, we identified a new role for the transcription factor NANOG as a key regulator connecting the pluripotency network with constitutive heterochromatin organization in mouse embryonic stem cells. Deletion of Nanog leads to chromatin compaction and the remodeling of heterochromatin domains. Forced expression of NANOG in epiblast stem cells is sufficient to decompact chromatin. NANOG associates with satellite repeats within heterochromatin domains, contributing to an architecture characterized by highly dispersed chromatin fibers, low levels of H3K9me3, and high major satellite transcription, and the strong transactivation domain of NANOG is required for this organization. The heterochromatin-associated protein SALL1 is a direct cofactor for NANOG, and loss of Sall1 recapitulates the Nanog-null phenotype, but the loss of Sall1 can be circumvented through direct recruitment of the NANOG transactivation domain to major satellites. These results establish a direct connection between the pluripotency network and chromatin organization and emphasize that maintaining an open heterochromatin architecture is a highly regulated process in embryonic stem cells.

The application of cell surface markers to demarcate distinct human pluripotent states.

Recent advances in human pluripotent stem cell (hPSC) research have uncovered different subpopulations within stem cell cultures and have captured a range of pluripotent states that hold distinct molecular and functional properties. At the two ends of the pluripotency spectrum are naïve and primed hPSC, whereby naïve hPSC grown in stringent conditions recapitulate features of the preimplantation human embryo, and the conventionally grown primed hPSC align closer to the early postimplantation embryo. Investigating these cell types will help to define the mechanisms that control early development and should provide new insights into stem cell properties such as cell identity, differentiation and reprogramming. Monitoring cell surface marker expression provides a valuable approach to resolve complex cell populations, to directly compare between cell types, and to isolate viable cells for functional experiments. This review discusses the discovery and applications of cell surface markers to study human pluripotent cell types with a particular focus on the transitions between naïve and primed states. Highlighted areas for future study include the potential functions for the identified cell surface proteins in pluripotency, the production of new high-quality monoclonal antibodies to naïve-specific protein epitopes and the use of cell surface markers to characterise subpopulations within pluripotent states.

Identifying Human Naïve Pluripotent Stem Cells - Evaluating State-Specific Reporter Lines and Cell-Surface Markers.

Recent reports that human pluripotent stem cells can be captured in a spectrum of states with variable properties has prompted a re-evaluation of how pluripotency is acquired and stabilised. The latest additions to the stem cell hierarchy open up opportunities for understanding human development, reprogramming, and cell state transitions more generally. Many of the new cell lines have been collectively termed 'naïve' human pluripotent stem cells to distinguish them from the conventional 'primed' cells. Here, several transcriptional and epigenetic hallmarks of human pluripotent states in the recently described cell lines are reviewed and evaluated. Methods to derive and identify human naïve pluripotent stem cells are also discussed, with a focus on the uses and future developments of state-specific reporter cell lines and cell-surface proteins. Finally, opportunities and uncertainties in naïve stem cell biology are highlighted, and the current limitations of human naïve pluripotent stem cells considered, particularly in the context of differentiation.

DNA methylation is dispensable for changes in global chromatin architecture but required for chromocentre formation in early stem cell differentiation.

Epiblast stem cells (EpiSCs), which are pluripotent cells isolated from early post-implantation mouse embryos (E5.5), show both similarities and differences compared to mouse embryonic stem cells (mESCs), isolated earlier from the inner cell mass (ICM) of the E3.5 embryo. Previously, we have observed that while chromatin is very dispersed in E3.5 ICM, compact chromatin domains and chromocentres appear in E5.5 epiblasts after embryo implantation. Given that the observed chromatin re-organization in E5.5 epiblasts coincides with an increase in DNA methylation, in this study, we aimed to examine the role of DNA methylation in chromatin re-organization during the in vitro conversion of ESCs to EpiSCs. The requirement for DNA methylation was determined by converting both wild-type and DNA methylation-deficient ESCs to EpiSCs, followed by structural analysis with electron spectroscopic imaging (ESI). We show that the chromatin re-organization which occurs in vivo can be re-capitulated in vitro during the ESC to EpiSC conversion. Indeed, after 7 days in EpiSC media, compact chromatin domains begin to appear throughout the nuclear volume, creating a chromatin organization similar to E5 epiblasts and embryo-derived EpiSCs. Our data demonstrate that DNA methylation is dispensable for this global chromatin re-organization but required for the compaction of pericentromeric chromatin into chromocentres.

Epigenetic status of human embryonic stem cells.

We examined the allele-specific expression of six imprinted genes and the methylation profiles of three imprinting control regions to assess the epigenetic status of human embryonic stem cells. We identified generally monoallelic gene expression and normal methylation patterns. During prolonged passage, one cell line became biallelic with respect to H19, but without loss of the gametic methylation imprint. These data argue for a substantial degree of epigenetic stability in human embryonic stem cells.

Gata3 regulates trophoblast development downstream of Tead4 and in parallel to Cdx2.

The mouse blastocyst and stem cells derived from its tissue lineages provide a unique genetic system for examining the establishment and loss of pluripotency. The transcription factor Cdx2 plays a central role by repressing pluripotency genes, such as Oct4, and promoting extraembryonic trophoblast fate at the blastocyst stage. However, genetic evidence has suggested that Cdx2 does not work alone in the trophoblast lineage. We have used bioinformatic and functional genomic strategies to identify the transcription factor Gata3 as a trophoblast factor. We show Gata3 to be capable of inducing trophoblast fate in embryonic stem cells and driving trophoblast differentiation in trophoblast stem cells. In addition, Cdx2 is not required for Gata3-induced expression of a subset of trophoblast genes in embryonic stem cells. We show that Gata3 is coexpressed with Cdx2 in the blastocyst, but this does not depend on Cdx2. In the embryo, expression of Gata3, like that of Cdx2, depends on Tead4, and the expression of both factors becomes restricted to trophoblast by a mechanism that does not initially rely on Oct4. These observations suggest that Gata3 and Cdx2 can act in parallel pathways downstream of Tead4 to induce the expression of common and independent targets in the trophoblast lineage, whereas Oct4 is required for continued repression of trophoblast fate in the embryonic lineage.

Derivation of pluripotent epiblast stem cells from mammalian embryos.

Although the first mouse embryonic stem (ES) cell lines were derived 25 years ago using feeder-layer-based blastocyst cultures, subsequent efforts to extend the approach to other mammals, including both laboratory and domestic species, have been relatively unsuccessful. The most notable exceptions were the derivation of non-human primate ES cell lines followed shortly thereafter by their derivation of human ES cells. Despite the apparent common origin and the similar pluripotency of mouse and human embryonic stem cells, recent studies have revealed that they use different signalling pathways to maintain their pluripotent status. Mouse ES cells depend on leukaemia inhibitory factor and bone morphogenetic protein, whereas their human counterparts rely on activin (INHBA)/nodal (NODAL) and fibroblast growth factor (FGF). Here we show that pluripotent stem cells can be derived from the late epiblast layer of post-implantation mouse and rat embryos using chemically defined, activin-containing culture medium that is sufficient for long-term maintenance of human embryonic stem cells. Our results demonstrate that activin/Nodal signalling has an evolutionarily conserved role in the derivation and the maintenance of pluripotency in these novel stem cells. Epiblast stem cells provide a valuable experimental system for determining whether distinctions between mouse and human embryonic stem cells reflect species differences or diverse temporal origins.

Distinct histone modifications in stem cell lines and tissue lineages from the early mouse embryo.

A unique property of the mammalian embryo is that stem cells can be derived from its early tissue lineages. These lineages will give rise to the fetus as well as essential extraembryonic tissues. Understanding how chromatin regulation participates in establishment of these lineages in the embryo and their derived stem cells provides insight that will critically inform our understanding of embryogenesis and stem cell biology. Here, we compare the genomewide location of active and repressive histone modifications in embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm stem cells from the mouse. Our results show that the active modification H3K4me3 has a similar role in the three stem cell types, but the repressive modification H3K27me3 varies in abundance and genomewide distribution. Thus, alternative mechanisms mediate transcriptional repression in stem cells from the embryo. In addition, using carrier chromatin immunoprecipitation we show that bivalent histone domains seen in embryonic stem cells exist in pluripotent cells of the early embryo. However, the epigenetic status of extraembryonic progenitor cells in the embryo did not entirely reflect the extraembryonic stem cell lines. These studies indicate that histone modification mechanisms may differ between early embryo lineages and emphasize the importance of examining in vivo and in vitro progenitor cells.

Epigenetic regulation of early human embryo development.

Studies of mammalian development have advanced our understanding of the genetic, epigenetic, and cellular processes that orchestrate embryogenesis and have uncovered new insights into the unique aspects of human embryogenesis. Recent studies have now produced the first epigenetic maps of early human embryogenesis, stimulating new ideas about epigenetic reprogramming, cell fate control, and the potential mechanisms underpinning developmental plasticity in human embryos. In this review, we discuss these new insights into the epigenetic regulation of early human development and the importance of these processes for safeguarding development. We also highlight unanswered questions and key challenges that remain to be addressed.

Dbx2, an Aging-Related Homeobox Gene, Inhibits the Proliferation of Adult Neural Progenitors.

In the adult mouse brain, the subventricular zone (SVZ) underlying the lateral ventricles harbours a population of quiescent neural stem cells, which can be activated (aNSCs) to initiate proliferation and generate a neurogenic lineage consisting of transit amplifying progenitors (TAPs), neuroblasts (NBs) and newborn neurons. This process is markedly reduced during aging. Recent studies suggest that the aged SVZ niche decreases the pool of proliferating neural/stem progenitor cells (NSPCs), and hence adult neurogenesis, by causing transcriptomic changes that promote NSC quiescence. The transcription factors that mediate these changes, however, remain unclear. We previously found that the homeobox gene Dbx2 is upregulated in NSPCs of the aged mouse SVZ and can inhibit the growth of NSPC cultures. Here, we further investigate its role as a candidate transcriptional regulator of neurogenic decline. We show that Dbx2 expression is downregulated by Epidermal Growth Factor receptor signaling, which promotes NSPC proliferation and decreases in the aged SVZ. By means of transgenic NSPC lines overexpressing Dbx2, we also show that this gene inhibits NSPC proliferation by hindering the G2/M transition. Furthermore, we exploit RNA sequencing of transgenic NSPCs to elucidate the transcriptomic networks modulated by Dbx2. Among the top hits, we report the downregulation of the molecular pathways implicated in cell cycle progression. Accordingly, we find that Dbx2 function is negatively correlated with the transcriptional signatures of proliferative NSPCs (aNSCs, TAPs and early NBs). These results point to Dbx2 as a transcription factor relaying the anti-neurogenic input of the aged niche to the NSPC transcriptome.

Epigenetic features of the mouse trophoblast.

Trophoblast cells are required for the growth and survival of the fetus during pregnancy, and failure to maintain appropriate trophoblast regulation is associated with placental insufficiencies and intrauterine growth restriction. Development of the trophoblast lineage is mediated by interactions between genetic and epigenetic factors. This review will focus on new insights that have been gained from analysis of mouse models into the epigenetic mechanisms that are required for the early establishment of the trophoblast lineage and for the development of specialized cell types of the fetal placenta. In particular, the importance of DNA methylation, 5-hydroxymethylcytosine and histone modifications in orchestrating trophoblast gene expression and functional outcome will be discussed. These insights are beginning to be extended towards human studies and initial results suggest that the causes and consequences of a variety of placental pathologies are related to epigenetic processes. Furthermore, the epigenetic landscape that regulates trophoblast cells seems to be particularly vulnerable to perturbation during development. This has major implications for diet and other environmental factors during pregnancy.

Induction of Human Naïve Pluripotency Using Chemical Resetting.

Human pluripotent stem cells exist in naïve and primed states that recapitulate the distinct molecular and cellular properties of pre- and post-implantation epiblast cells, respectively. Naïve pluripotent stem cells can be captured directly from blastocysts but, more commonly, the cells are reprogrammed from primed cells in a process called "resetting". Several methods to achieve resetting have been described. Chemical resetting of primed cells to a naïve pluripotent state is one such method and has come to the forefront as a simple, efficient, and transgene-free method to induce naïve pluripotency. The process involves the transient application of a histone deacetylase inhibitor to initiate resetting, followed by the emergence of nascent naïve pluripotent stem cells in supportive conditions, and finally the stabilization and expansion of naïve pluripotent stem cell cultures. Here, a detailed protocol is provided for chemical resetting starting from plating primed cells until a stable culture of naïve pluripotent stem cells is established.

Flow Cytometry Analysis of Cell-Surface Markers to Identify Human Naïve Pluripotent Stem Cells.

Cell-surface proteins provide excellent biomarkers to identify specific cell types and resolve heterogeneous cell populations. The analysis of cell-surface proteins by flow cytometry produces robust and quantitative information with single-cell resolution, and allows live target cells to be purified and characterized or re-cultured. Studies using antibody screens, proteomics, and candidate analysis have identified a comprehensive set of proteins that are expressed on the surface of naïve and primed human pluripotent stem cells. These findings have led to the development of suitable protein markers and antibodies to accurately distinguish between these two cell types. Here, a detailed protocol is provided that uses multi-color flow cytometry to analyze cell-surface protein expression in naïve and primed human pluripotent stem cells. This method enables the unambiguous identification of pluripotent cell types and the opportunity to sort target cells including during cell state transitions. The protocol can be combined to additionally investigate the expression of reporter genes and other informative features, such as DNA content.

Amniogenesis occurs in two independent waves in primates.

In primates, the amnion emerges through cavitation of the epiblast during implantation, whereas in other species it does so later at gastrulation by the folding of the ectoderm. How the mechanisms of amniogenesis diversified during evolution remains unknown. Unexpectedly, single-cell analysis of primate embryos uncovered two transcriptionally and temporally distinct amniogenesis waves. To study this, we employed the naive-to-primed transition of human pluripotent stem cells (hPSCs) to model peri-implantation epiblast development. Partially primed hPSCs transiently gained the ability to differentiate into cavitating epithelium that transcriptionally and morphologically matched the early amnion, whereas fully primed hPSCs produced cells resembling the late amnion instead, thus recapitulating the two independent differentiation waves. The early wave follows a trophectoderm-like pathway and encompasses cavitation, whereas the late wave resembles an ectoderm-like route during gastrulation. The discovery of two independent waves explains how amniogenesis through cavitation could emerge during evolution via duplication of the pre-existing trophectoderm program.