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Our understanding of how human embryos develop before gastrulation, including spatial self-organization and cell type ontogeny, remains limited by available two-dimensional technological platforms 1 , 2 that do not recapitulate the in vivo conditions 3 – 5 . Here we report a three-dimensional (3D) blastocyst-culture system that enables human blastocyst development up to the primitive streak anlage stage. These 3D embryos mimic developmental landmarks and 3D architectures in vivo, including the embryonic disc, amnion, basement membrane, primary and primate unique secondary yolk sac, formation of anterior–posterior polarity and primitive streak anlage. Using single-cell transcriptome profiling, we delineate ontology and regulatory networks that underlie the segregation of epiblast, primitive endoderm and trophoblast. Compared with epiblasts, the amniotic epithelium shows unique and characteristic phenotypes. After implantation, specific pathways and transcription factors trigger the differentiation of cytotrophoblasts, extravillous cytotrophoblasts and syncytiotrophoblasts. Epiblasts undergo a transition to pluripotency upon implantation, and the transcriptome of these cells is maintained until the generation of the primitive streak anlage. These developmental processes are driven by different pluripotency factors. Together, findings from our 3D-culture approach help to determine the molecular and morphogenetic developmental landscape that occurs during human embryogenesis. A 3D culture system to model human embryonic development, together with single-cell transcriptome profiling, provides insights into the molecular developmental landscape during human post-implantation embryogenesis.

The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation 1 . Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking 1 , 2 . Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs) 3 . Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions) 4 . Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13–14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior–posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development. The culture of genetically unmodified human naive embryonic stem cells in specific growth conditions gives rise to structures that recapitulate those of post-implantation human embryos up to 13–14 days after fertilization.

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. Why do human embryos need a yolk sac and how does it form? This Perspective by Thorsten Boroviak and Connor Ross explores the development and function of the yolk sac in primate embryogenesis.

The earliest blood vessels in mammalian embryos are formed when endothelial cells differentiate from angioblasts and coalesce into tubular networks. Thereafter, the endothelium is thought to expand solely by proliferation of pre-existing endothelial cells. Here we show that a complementary source of endothelial cells is recruited into pre-existing vasculature after differentiation from the earliest precursors of erythrocytes, megakaryocytes and macrophages, the erythro-myeloid progenitors (EMPs) that are born in the yolk sac. A first wave of EMPs contributes endothelial cells to the yolk sac endothelium, and a second wave of EMPs colonizes the embryo and contributes endothelial cells to intraembryonic endothelium in multiple organs, where they persist into adulthood. By demonstrating that EMPs constitute a hitherto unrecognized source of endothelial cells, we reveal that embryonic blood vascular endothelium expands in a dual mechanism that involves both the proliferation of pre-existing endothelial cells and the incorporation of endothelial cells derived from haematopoietic precursors. New blood vessel endothelial cells arise from differentiation of erythro-myeloid progenitor cells to complement the proliferation of pre-existing endothelial cells.

Macrophages are the first cells of the nascent immune system to emerge during embryonic development. In mice, embryonic macrophages infiltrate developing organs, where they differentiate symbiotically into tissue-resident macrophages (TRMs) 1 . However, our understanding of the origins and specialization of macrophages in human embryos is limited. Here we isolated CD45 + haematopoietic cells from human embryos at Carnegie stages 11 to 23 and subjected them to transcriptomic profiling by single-cell RNA sequencing, followed by functional characterization of a population of CD45 + CD34 + CD44 + yolk sac-derived myeloid-biased progenitors (YSMPs) by single-cell culture. We also mapped macrophage heterogeneity across multiple anatomical sites and identified diverse subsets, including various types of embryonic TRM (in the head, liver, lung and skin). We further traced the specification trajectories of TRMs from either yolk sac-derived primitive macrophages or YSMP-derived embryonic liver monocytes using both transcriptomic and developmental staging information, with a focus on microglia. Finally, we evaluated the molecular similarities between embryonic TRMs and their adult counterparts. Our data represent a comprehensive characterization of the spatiotemporal dynamics of early macrophage development during human embryogenesis, providing a reference for future studies of the development and function of human TRMs. Single-cell RNA sequencing of haematopoietic cells from human embryos at different developmental stages sheds light on the development and specification of macrophages in different tissues.

It is well established that haematopoietic stem and progenitor cells (HSPCs) are generated from a transient subset of specialized endothelial cells termed haemogenic, present in the yolk sac, placenta and aorta, through an endothelial-to-haematopoietic transition (EHT). HSPC generation via EHT is thought to be restricted to the early stages of development. By using experimental embryology and genetic approaches in birds and mice, respectively, we document here the discovery of a bone marrow haemogenic endothelium in the late fetus/young adult. These cells are capable of de novo producing a cohort of HSPCs in situ that harbour a very specific molecular signature close to that of aortic endothelial cells undergoing EHT or their immediate progenies, i.e., recently emerged HSPCs. Taken together, our results reveal that HSPCs can be generated de novo past embryonic stages. Understanding the molecular events controlling this production will be critical for devising innovative therapies. Yvernogeau, Gautier, Petit et al. demonstrate the existence of a haemogenic endothelium capable of de novo haematopoietic stem and progenitor cell generation in the forming bone marrow of chicken and mouse fetuses and newborns.

Pluripotent stem cell (PSC)-based therapies are currently in clinical trials. However, one of the main safety concerns includes the potential for cancer formation of the PSC-derived products. Currently, the teratoma in vivo assay is accepted by regulatory agencies for identifying whether PSCs have the potential to become malignant. Yolk sac elements (YSE) are one of the elements that could arise from PSC. Whereas the other malignant element, embryonal carcinoma, is thoroughly studied, this is not the case for YSE. Therefore, more research is needed to assess the nature of YSE. We propose that it is imperative to include the formation of YSE in the safety assessment of PSC due to their close resemblance to the clinical entity of yolk sac tumor (YST), a human malignant germ cell tumor (hGCT). In this review, we extrapolate knowledge from YST to better understand YSE derived from PSC. We demonstrate that both share a similar morphology and that the same characteristic immunohistochemical markers can be used for their identification. We discuss the risk these tumors pose, thereby touching upon genetic abnormalities and gene expression that characterize them, as well as possible disease mechanisms. Integrating the molecular and immunohistochemical markers identified in this review into future research will help to better address the potential malignancy associated with PSC.

Macrophages serve multiple functions including immune regulation, morphogenesis, tissue homeostasis and healing reactions. The current paradigm holds that mammary gland macrophages first arise postnatally during the prepubertal period from the bone marrow-derived monocytes. Here we delineate the origins of tissue-resident mammary gland macrophages using high-dimension phenotypic analyses, cell-fate mapping experiments, gene-deficient mice lacking selective macrophage subtypes, and antibody-based depletion strategies. We show that tissue-resident macrophages are found in mammary glands already before birth, and that the yolk sac-derived and fetal liver-derived macrophages outnumber the adult-derived macrophages in the mammary gland also in the adulthood. In addition, fetal-derived mammary gland macrophages have a characteristic phenotype, display preferential periductal and perivascular localization, and are highly active in scavenging. These findings identify fetal-derived macrophages as the predominant leukocyte type in the adult mammary gland stroma, and reveal previously unknown complexity of macrophage biology in the breast. Tissue-resident macrophages are highly specialized phagocytes that serve multiple functions. Here, using high-dimension analyses and fate-mapping experiments, the authors show that fetal liver-derived macrophages dominate the mammary gland in neonatal and adult, and display characteristic phenotypes and functions.

Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac 1 . Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human 2 – 5 and cow 6 . Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical–basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos. Comparative analysis of human, cow and mouse embryos shows that a mechanism involving atypical protein kinase C initiates the trophectoderm program during the morula stage in these three species.

Dyslipidemias are a major cause of morbidity and mortality in the world, particularly in developed nations. Investigating lipid and lipoprotein metabolism in experimentally tractable animal models is a critical step toward understanding and treating human dyslipidemias. The zebrafish, a well-established embryological model, is emerging as a notable system for studies in lipid metabolism. Here, we describe the value of the lecithotrophic, or yolk metabolizing, stages of the zebrafish as a model for studying lipid metabolism and lipoprotein transport. We demonstrate methods to assay yolk lipid metabolism in embryonic and larval zebrafish. These methods promote efficient uptake of yolk-delivered labeled fatty acids into the circulation. Using a genetic model for abetalipoproteinemia, we show that uptake of labeled fatty acids into the circulation is dependent on lipoprotein production. Further, we examine the metabolic fate of exogenously delivered fatty acids by assaying their incorporation into complex lipids. Moreover, we demonstrate that this technique is amenable to genetic and pharmacologic studies.