Age-related and species-specific methylation changes in the protein-coding marmoset sperm epigenome.

The sperm epigenome is thought to affect the developmental programming of the resulting embryo, influencing health and disease in later life. Age-related methylation changes in the sperm of old fathers may mediate the increased risks for reproductive and offspring medical problems. The impact of paternal age on sperm methylation has been extensively studied in humans and, to a lesser extent, in rodents and cattle. Here, we performed a comparative analysis of paternal age effects on protein-coding genes in the human and marmoset sperm methylomes. The marmoset has gained growing importance as a non-human primate model of aging and age-related diseases. Using reduced representation bisulfite sequencing, we identified age-related differentially methylated transcription start site (ageTSS) regions in 204 marmoset and 27 human genes. The direction of methylation changes was the opposite, increasing with age in marmosets and decreasing in humans. None of the identified ageTSS was differentially methylated in both species. Although the average methylation levels of all TSS regions were highly correlated between marmosets and humans, with the majority of TSS being hypomethylated in sperm, more than 300 protein-coding genes were endowed with species-specifically (hypo)methylated TSS. Several genes of the glycosphingolipid (GSL) biosynthesis pathway, which plays a role in embryonic stem cell differentiation and regulation of development, were hypomethylated (<5%) in human and fully methylated (>95%) in marmoset sperm. The expression levels and patterns of defined sets of GSL genes differed considerably between human and marmoset pre-implantation embryo stages and blastocyst tissues, respectively.

Plakoglobin is a mechanoresponsive regulator of naive pluripotency.

Biomechanical cues are instrumental in guiding embryonic development and cell differentiation. Understanding how these physical stimuli translate into transcriptional programs will provide insight into mechanisms underlying mammalian pre-implantation development. Here, we explore this type of regulation by exerting microenvironmental control over mouse embryonic stem cells. Microfluidic encapsulation of mouse embryonic stem cells in agarose microgels stabilizes the naive pluripotency network and specifically induces expression of Plakoglobin (Jup), a vertebrate homolog of ß-catenin. Overexpression of Plakoglobin is sufficient to fully re-establish the naive pluripotency gene regulatory network under metastable pluripotency conditions, as confirmed by single-cell transcriptome profiling. Finally, we find that, in the epiblast, Plakoglobin was exclusively expressed at the blastocyst stage in human and mouse embryos - further strengthening the link between Plakoglobin and naive pluripotency in vivo. Our work reveals Plakoglobin as a mechanosensitive regulator of naive pluripotency and provides a paradigm to interrogate the effects of volumetric confinement on cell-fate transitions.

Origin and segregation of the human germline.

Human germline-soma segregation occurs during weeks 2-3 in gastrulating embryos. Although direct studies are hindered, here, we investigate the dynamics of human primordial germ cell (PGCs) specification using in vitro models with temporally resolved single-cell transcriptomics and in-depth characterisation using in vivo datasets from human and nonhuman primates, including a 3D marmoset reference atlas. We elucidate the molecular signature for the transient gain of competence for germ cell fate during peri-implantation epiblast development. Furthermore, we show that both the PGCs and amnion arise from transcriptionally similar TFAP2A-positive progenitors at the posterior end of the embryo. Notably, genetic loss of function experiments shows that TFAP2A is crucial for initiating the PGC fate without detectably affecting the amnion and is subsequently replaced by TFAP2C as an essential component of the genetic network for PGC fate. Accordingly, amniotic cells continue to emerge from the progenitors in the posterior epiblast, but importantly, this is also a source of nascent PGCs.

Evolutionary divergence of embryo implantation in primates.

Implantation of the conceptus into the uterus is absolutely essential for successful embryo development. In humans, our understanding of this process has remained rudimentary owing to the inaccessibility of early implantation stages. Non-human primates recapitulate many aspects of human embryo development and provide crucial insights into trophoblast development, uterine receptivity and embryo invasion. Moreover, primate species exhibit a variety of implantation strategies and differ in embryo invasion depths. This review examines conservation and divergence of the key processes required for embryo implantation in different primates and in comparison with the canonical rodent model. We discuss trophectoderm compartmentalization, endometrial remodelling and embryo adhesion and invasion. Finally, we propose that studying the mechanism controlling invasion depth between different primate species may provide new insights and treatment strategies for placentation disorders in humans. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.

Microgel culture and spatial identity mapping elucidate the signalling requirements for primate epiblast and amnion formation.

The early specification and rapid growth of extraembryonic membranes are distinctive hallmarks of primate embryogenesis. These complex tasks are resolved through an intricate combination of signals controlling the induction of extraembryonic lineages and, at the same time, safeguarding the pluripotent epiblast. Here, we delineate the signals orchestrating primate epiblast and amnion identity. We encapsulated marmoset pluripotent stem cells into agarose microgels and identified culture conditions for the development of epiblast- and amnion-spheroids. Spatial identity mapping authenticated spheroids generated in vitro by comparison with marmoset embryos in vivo. We leveraged the microgel system to functionally interrogate the signalling environment of the post-implantation primate embryo. Single-cell profiling of the resulting spheroids demonstrated that activin/nodal signalling is required for embryonic lineage identity. BMP4 promoted amnion formation and maturation, which was counteracted by FGF signalling. Our combination of microgel culture, single-cell profiling and spatial identity mapping provides a powerful approach to decipher the essential cues for embryonic and extraembryonic lineage formation in primate embryogenesis.

An integrated atlas of human placental development delineates essential regulators of trophoblast stem cells.

The trophoblast lineage safeguards fetal development by mediating embryo implantation, immune tolerance, nutritional supply and gas exchange. Human trophoblast stem cells (hTSCs) provide a platform to study lineage specification of placental tissues; however, the regulatory network controlling self-renewal remains elusive. Here, we present a single-cell atlas of human trophoblast development from zygote to mid-gestation together with single-cell profiling of hTSCs. We determine the transcriptional networks of trophoblast lineages in vivo and leverage probabilistic modelling to identify a role for MAPK signalling in trophoblast differentiation. Placenta- and blastoid-derived hTSCs consistently map between late trophectoderm and early cytotrophoblast, in contrast to blastoid-trophoblast, which correspond to trophectoderm. We functionally assess the requirement of the predicted cytotrophoblast network in an siRNA-screen and reveal 15 essential regulators for hTSC self-renewal, including MAZ, NFE2L3, TFAP2C, NR2F2 and CTNNB1. Our human trophoblast atlas provides a powerful analytical resource to delineate trophoblast cell fate acquisition, to elucidate transcription factors required for hTSC self-renewal and to gauge the developmental stage of in vitro cultured cells.

A human embryo model cracks symmetry breaking.

In this issue of Cell Stem Cell, Simunovic et al. (2022) establish embryoids by combining embryonic and extraembryonic components derived from human pluripotent stem cells. The embryoids resemble human embryos cultured to post-implantation stages in vitro with regard to morphology, symmetry breaking, and the formation of primitive streak-like cell types.

Spatial profiling of early primate gastrulation in utero.

Gastrulation controls the emergence of cellular diversity and axis patterning in the early embryo. In mammals, this transformation is orchestrated by dynamic signalling centres at the interface of embryonic and extraembryonic tissues1-3. Elucidating the molecular framework of axis formation in vivo is fundamental for our understanding of human development4-6 and to advance stem-cell-based regenerative approaches7. Here we illuminate early gastrulation of marmoset embryos in utero using spatial transcriptomics and stem-cell-based embryo models. Gaussian process regression-based 3D transcriptomes delineate the emergence of the anterior visceral endoderm, which is hallmarked by conserved (HHEX, LEFTY2, LHX1) and primate-specific (POSTN, SDC4, FZD5) factors. WNT signalling spatially coordinates the formation of the primitive streak in the embryonic disc and is counteracted by SFRP1 and SFRP2 to sustain pluripotency in the anterior domain. Amnion specification occurs at the boundaries of the embryonic disc through ID1, ID2 and ID3 in response to BMP signalling, providing a developmental rationale for amnion differentiation of primate pluripotent stem cells (PSCs). Spatial identity mapping demonstrates that primed marmoset PSCs exhibit the highest similarity to the anterior embryonic disc, whereas naive PSCs resemble the preimplantation epiblast. Our 3D transcriptome models reveal the molecular code of lineage specification in the primate embryo and provide an in vivo reference to decipher human development.

A hexa-species transcriptome atlas of mammalian embryogenesis delineates metabolic regulation across three different implantation modes.

Mammalian embryogenesis relies on glycolysis and oxidative phosphorylation to balance the generation of biomass with energy production. However, the dynamics of metabolic regulation in the postimplantation embryo in vivo have remained elusive due to the inaccessibility of the implanted conceptus for biochemical studies. To address this issue, we compiled single-cell embryo profiling data in six mammalian species and determined their metabolic dynamics through glycolysis and oxidative phosphorylation associated gene expression. Strikingly, we identify a conserved switch from bivalent respiration in the late blastocyst towards a glycolytic metabolism in early gastrulation stages across species, which is independent of embryo implantation. Extraembryonic lineages followed the dynamics of the embryonic lineage, except visceral endoderm. Finally, we demonstrate that in vitro primate embryo culture substantially impacts metabolic gene regulation by comparison to in vivo samples. Our work reveals a conserved metabolic programme despite different implantation modes and highlights the need to optimise postimplantation embryo culture protocols.

Building a stem cell-based primate uterus.

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

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

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

OCT4 induces embryonic pluripotency via STAT3 signaling and metabolic mechanisms.

OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 establishes and protects the pluripotent lineage in the embryo, we used comparative single-cell transcriptomics and quantitative immunofluorescence on control and OCT4 null blastocyst inner cell masses at two developmental stages. Surprisingly, activation of most pluripotency-associated transcription factors in the early mouse embryo occurs independently of OCT4, with the exception of the JAK/STAT signaling machinery. Concurrently, OCT4 null inner cell masses ectopically activate a subset of trophectoderm-associated genes. Inspection of metabolic pathways implicates the regulation of rate-limiting glycolytic enzymes by OCT4, consistent with a role in sustaining glycolysis. Furthermore, up-regulation of the lysosomal pathway was specifically detected in OCT4 null embryos. This finding implicates a requirement for OCT4 in the production of normal trophectoderm. Collectively, our findings uncover regulation of cellular metabolism and biophysical properties as mechanisms by which OCT4 instructs pluripotency.

Origin and function of the yolk sac in primate embryogenesis.

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