Mannose controls mesoderm specification and symmetry breaking in mouse gastruloids.

Patterning and growth are fundamental features of embryonic development that must be tightly coordinated. To understand how metabolism impacts early mesoderm development, we used mouse embryonic stem-cell-derived gastruloids, that co-expressed glucose transporters with the mesodermal marker T/Bra. We found that the glucose mimic, 2-deoxy-D-glucose (2-DG), blocked T/Bra expression and abolished axial elongation in gastruloids. However, glucose removal did not phenocopy 2-DG treatment despite a decline in glycolytic intermediates. As 2-DG can also act as a competitive inhibitor of mannose in protein glycosylation, we added mannose together with 2-DG and found that it could rescue the mesoderm specification both in vivo and in vitro. We further showed that blocking production and intracellular recycling of mannose abrogated mesoderm specification. Proteomics analysis demonstrated that mannose reversed glycosylation of the Wnt pathway regulator, secreted frizzled receptor Frzb. Our study showed how mannose controls mesoderm specification in mouse gastruloids.

Modelling post-implantation human development to yolk sac blood emergence.

Implantation of the human embryo begins a critical developmental stage that comprises profound events including axis formation, gastrulation and the emergence of haematopoietic system1,2. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons3-5. Stem cell models of human embryo have emerged to help unlock the mysteries of this stage6-16. Here we present a genetically inducible stem cell-derived embryoid model of early post-implantation human embryogenesis that captures the reciprocal codevelopment of embryonic tissue and the extra-embryonic endoderm and mesoderm niche with early haematopoiesis. This model is produced from induced pluripotent stem cells and shows unanticipated self-organizing cellular programmes similar to those that occur in embryogenesis, including the formation of amniotic cavity and bilaminar disc morphologies as well as the generation of an anterior hypoblast pole and posterior domain. The extra-embryonic layer in these embryoids lacks trophoblast and shows advanced multilineage yolk sac tissue-like morphogenesis that harbours a process similar to distinct waves of haematopoiesis, including the emergence of erythroid-, megakaryocyte-, myeloid- and lymphoid-like cells. This model presents an easy-to-use, high-throughput, reproducible and scalable platform to probe multifaceted aspects of human development and blood formation at the early post-implantation stage. It will provide a tractable human-based model for drug testing and disease modelling.

What approaches are needed to understand human development and disease?

Researchers are leveraging what we have learned from model organisms to understand if the same principles arise in human physiology, development, and disease. In this collection of Voices, we asked researchers from different fields to discuss what tools and insights they are using to answer fundamental questions in human biology.

Modelling Human Post-Implantation Development via Extra-Embryonic Niche Engineering.

Implantation of the human embryo commences a critical developmental stage that comprises profound morphogenetic alteration of embryonic and extra-embryonic tissues, axis formation, and gastrulation events. Our mechanistic knowledge of this window of human life remains limited due to restricted access to in vivo samples for both technical and ethical reasons. Additionally, human stem cell models of early post-implantation development with both embryonic and extra-embryonic tissue morphogenesis are lacking. Here, we present iDiscoid, produced from human induced pluripotent stem cells via an engineered a synthetic gene circuit. iDiscoids exhibit reciprocal co-development of human embryonic tissue and engineered extra-embryonic niche in a model of human post-implantation. They exhibit unanticipated self-organization and tissue boundary formation that recapitulates yolk sac-like tissue specification with extra-embryonic mesoderm and hematopoietic characteristics, the formation of bilaminar disc-like embryonic morphology, the development of an amniotic-like cavity, and acquisition of an anterior-like hypoblast pole and posterior-like axis. iDiscoids offer an easy-to-use, high-throughput, reproducible, and scalable platform to probe multifaceted aspects of human early post-implantation development. Thus, they have the potential to provide a tractable human model for drug testing, developmental toxicology, and disease modeling.

Shifting early embryology paradigms: Applications of stem cell-based embryo models in bioengineering.

Technologies to reproduce specific aspects of early mammalian embryogenesis in vitro using stem cells have skyrocketed over the last several years. With these advances, we have gained new perspectives on how embryonic and extraembryonic cells self-organize to form the embryo. These reductionist approaches hold promise for the future implementation of precise environmental and genetic controls to understand variables affecting embryo development. Our review discusses recent progress in cellular models of early mammalian embryo development and bioengineering advancements that can be leveraged to study the embryo-maternal interface. We summarize current gaps in the field, emphasizing the importance of understanding how intercellular interactions at this interface contribute to reproductive and developmental health.

A Spatiotemporal Compartmentalization of Glucose Metabolism Guides Mammalian Gastrulation Progression.

Gastrulation is considered the sine qua non of embryogenesis, establishing a multidimensional structure and the spatial coordinates upon which all later developmental events transpire. At this time, the embryo adopts a heavy reliance on glucose metabolism to support rapidly accelerating changes in morphology, proliferation, and differentiation. However, it is currently unknown how this conserved metabolic shift maps onto the three-dimensional landscape of the growing embryo and whether it is spatially linked to the orchestrated cellular and molecular processes necessary for gastrulation. Here we identify that glucose is utilised during mouse gastrulation via distinct metabolic pathways to instruct local and global embryonic morphogenesis, in a cell type and stage-specific manner. Through detailed mechanistic studies and quantitative live imaging of mouse embryos, in parallel with tractable in vitro stem cell differentiation models and embryo-derived tissue explants, we discover that cell fate acquisition and the epithelial-to-mesenchymal transition (EMT) relies on the Hexosamine Biosynthetic Pathway (HBP) branch of glucose metabolism, while newly-formed mesoderm requires glycolysis for correct migration and lateral expansion. This regional and tissue-specific difference in glucose metabolism is coordinated with Fibroblast Growth Factor (FGF) activity, demonstrating that reciprocal crosstalk between metabolism and growth factor signalling is a prerequisite for gastrulation progression. We expect these studies to provide important insights into the function of metabolism in other developmental contexts and may help uncover mechanisms that underpin embryonic lethality, cancer, and congenital disease.

Self-patterning of human stem cells into post-implantation lineages.

Investigating human development is a substantial scientific challenge due to the technical and ethical limitations of working with embryonic samples. In the face of these difficulties, stem cells have provided an alternative to experimentally model inaccessible stages of human development in vitro1-13. Here we show that human pluripotent stem cells can be triggered to self-organize into three-dimensional structures that recapitulate some key spatiotemporal events of early human post-implantation embryonic development. Our system reproducibly captures spontaneous differentiation and co-development of embryonic epiblast-like and extra-embryonic hypoblast-like lineages, establishes key signalling hubs with secreted modulators and undergoes symmetry breaking-like events. Single-cell transcriptomics confirms differentiation into diverse cell states of the perigastrulating human embryo14,15 without establishing placental cell types, including signatures of post-implantation epiblast, amniotic ectoderm, primitive streak, mesoderm, early extra-embryonic endoderm, as well as initial yolk sac induction. Collectively, our system captures key features of human embryonic development spanning from Carnegie stage16 4-7, offering a reproducible, tractable and scalable experimental platform to understand the basic cellular and molecular mechanisms that underlie human development, including new opportunities to dissect congenital pathologies with high throughput.

Carnegie in 4D? Stem-cell-based models of human embryo development.

How cells build embryos is still a major mystery. Many unresolved questions require the study of the processes that pattern and shape the embryo in live specimens, in toto, across spatial and temporal scales. In mammalian embryogenesis, this remains a major challenge as the embryo develops in utero, precluding easy accessibility. For human embryos, technical, ethical and legal limitations further hamper the in-depth investigation of embryogenesis, especially beyond gastrulation stages. This has resulted in an over-reliance on model organisms, particularly mice, to understand mammalian development. However, recent efforts show critical differences between rodent and primate embryos, including timing, architecture and transcriptional regulation. Thus, a human-centric understanding of embryogenesis is much needed. To empower this, novel in vitro approaches, which coax human pluripotent stem cells to form embryonic organoids that model embryo development, are pivotal. Here, we summarize these emergent technologies that recapitulate aspects of human development "in a dish". We show how these technologies can provide insights into the molecular, cellular and morphogenetic processes that fuel the formation of a fully formed fetus, and discuss the potential of these platforms to revolutionize our understanding of human development in health and disease. Despite their clear promise, we caution against over-interpreting the extent to which these in vitro platforms model the natural embryo. In particular, we discuss how fate, form and function - a tightly coupled trinity in vivo, can be disconnected in vitro. Finally, we propose how careful benchmarking of existing models, in combination with rational protocol design based on an increased understanding of in vivo developmental dynamics and insights from mouse in vitro models of embryo development, will help guide the establishment of better models of human embryo development.

Human embryo polarization requires PLC signaling to mediate trophectoderm specification.

Apico-basal polarization of cells within the embryo is critical for the segregation of distinct lineages during mammalian development. Polarized cells become the trophectoderm (TE), which forms the placenta, and apolar cells become the inner cell mass (ICM), the founding population of the fetus. The cellular and molecular mechanisms leading to polarization of the human embryo and its timing during embryogenesis have remained unknown. Here, we show that human embryo polarization occurs in two steps: it begins with the apical enrichment of F-actin and is followed by the apical accumulation of the PAR complex. This two-step polarization process leads to the formation of an apical domain at the 8-16 cell stage. Using RNA interference, we show that apical domain formation requires Phospholipase C (PLC) signaling, specifically the enzymes PLCB1 and PLCE1, from the eight-cell stage onwards. Finally, we show that although expression of the critical TE differentiation marker GATA3 can be initiated independently of embryo polarization, downregulation of PLCB1 and PLCE1 decreases GATA3 expression through a reduction in the number of polarized cells. Therefore, apical domain formation reinforces a TE fate. The results we present here demonstrate how polarization is triggered to regulate the first lineage segregation in human embryos.

Reconstructing aspects of human embryogenesis with pluripotent stem cells.

Understanding human development is of fundamental biological and clinical importance. Despite its significance, mechanisms behind human embryogenesis remain largely unknown. Here, we attempt to model human early embryo development with expanded pluripotent stem cells (EPSCs) in 3-dimensions. We define a protocol that allows us to generate self-organizing cystic structures from human EPSCs that display some hallmarks of human early embryogenesis. These structures mimic polarization and cavitation characteristic of pre-implantation development leading to blastocyst morphology formation and the transition to post-implantation-like organization upon extended culture. Single-cell RNA sequencing of these structures reveals subsets of cells bearing some resemblance to epiblast, hypoblast and trophectoderm lineages. Nevertheless, significant divergences from natural blastocysts persist in some key markers, and signalling pathways point towards ways in which morphology and transcriptional-level cell identities may diverge in stem cell models of the embryo. Thus, this stem cell platform provides insights into the design of stem cell models of embryogenesis.

Unfolded protein response triggers differential apoptotic mechanisms in ovaries and early embryos exposed to maternal type 1 diabetes.

Diabetes mellitus (DM) has profound effects on the female mammalian reproductive system, and early embryonic development, reducing female reproductive outcomes and inducing developmental programming in utero. However, the underlying cellular and molecular mechanisms remain poorly defined. Accumulating evidence implicates endoplasmic reticulum (ER)-stress with maternal DM associated pathophysiology. Yet the direct pathologies and causal events leading to ovarian dysfunction and altered early embryonic development have not been determined. Here, using an in vivo mouse model of Type 1 DM and in vitro hyperglycaemia-exposure, we demonstrate the activation of ER-stress within adult ovarian tissue and pre-implantation embryos. In diabetic ovaries, we show that the unfolded protein response (UPR) triggers an apoptotic cascade by the co-activation of Caspase 12 and Cleaved Caspase 3 transducers. Whereas DM-exposed early embryos display differential ER-associated responses; by activating Chop in within embryonic precursors and Caspase 12 within placental precursors. Our results offer new insights for understanding the pathological effects of DM on mammalian ovarian function and early embryo development, providing new evidence of its mechanistic link with ER-stress in mice.

Machine learning-assisted high-content analysis of pluripotent stem cell-derived embryos in vitro.

Stem cell-based embryo models by cultured pluripotent and extra-embryonic lineage stem cells are novel platforms to model early postimplantation development. We showed that induced pluripotent stem cells (iPSCs) could form ITS (iPSCs and trophectoderm stem cells) and ITX (iPSCs, trophectoderm stem cells, and XEN cells) embryos, resembling the early gastrula embryo developed in vivo. To facilitate the efficient and unbiased analysis of the stem cell-based embryo model, we set up a machine learning workflow to extract multi-dimensional features and perform quantification of ITS embryos using 3D images collected from a high-content screening system. We found that different PSC lines differ in their ability to form embryo-like structures. Through high-content screening of small molecules and cytokines, we identified that BMP4 best promoted the morphogenesis of the ITS embryo. Our study established an innovative strategy to analyze stem cell-based embryo models and uncovered new roles of BMP4 in stem cell-based embryo models.

The dynamics of morphogenesis in stem cell-based embryology: Novel insights for symmetry breaking.

Breaking embryonic symmetry is an essential prerequisite to shape the initially symmetric embryo into a highly organized body plan that serves as the blueprint of the adult organism. This critical process is driven by morphogen signaling gradients that instruct anteroposterior axis specification. Despite its fundamental importance, what triggers symmetry breaking and how the signaling gradients are established in time and space in the mammalian embryo remain largely unknown. Stem cell-based in vitro models of embryogenesis offer an unprecedented opportunity to quantitatively dissect the multiple physical and molecular processes that shape the mammalian embryo. Here we review biochemical mechanisms governing early mammalian patterning in vivo and highlight recent advances to recreate this in vitro using stem cells. We discuss how the novel insights from these model systems extend previously proposed concepts to illuminate the extent to which embryonic cells have the intrinsic capability to generate specific, reproducible patterns during embryogenesis.

Inducible Stem-Cell-Derived Embryos Capture Mouse Morphogenetic Events In Vitro.

The development of mouse embryos can be partially recapitulated by combining embryonic stem cells (ESCs), trophoblast stem cells (TS), and extra-embryonic endoderm (XEN) stem cells to generate embryo-like structures called ETX embryos. Although ETX embryos transcriptionally capture the mouse gastrula, their ability to recapitulate complex morphogenic events such as gastrulation is limited, possibly due to the limited potential of XEN cells. To address this, we generated ESCs transiently expressing transcription factor Gata4, which drives the extra-embryonic endoderm fate, and combined them with ESCs and TS cells to generate induced ETX embryos (iETX embryos). We show that iETX embryos establish a robust anterior signaling center that migrates unilaterally to break embryo symmetry. Furthermore, iETX embryos gastrulate generating embryonic and extra-embryonic mesoderm and definitive endoderm. Our findings reveal that replacement of XEN cells with ESCs transiently expressing Gata4 endows iETX embryos with greater developmental potential, thus enabling the study of the establishment of anterior-posterior patterning and gastrulation in an in vitro system.

BMP signalling is required for extra-embryonic ectoderm development during pre-to-post-implantation transition of the mouse embryo.

At implantation, the mouse embryo undergoes a critical transformation which requires the precise spatiotemporal control of signalling pathways necessary for morphogenesis and developmental progression. The role played by such signalling pathways during this transition are largely unexplored, due to the inaccessibility of the embryo during the implantation when it becomes engulfed by uterine tissues. Genetic studies demonstrate that mutant embryos for BMPs die around gastrulation. Here we have aimed to dissect the role of BMPs during pre-to post-implantation transition by using a protocol permitting the development of the embryo beyond implantation stages in vitro and using stem cells to mimic post-implantation tissue organisation. By assessing both the canonical and non-canonical mechanisms of BMP, we show that the loss of canonical BMP activity compromises the extra-embryonic ectoderm development. Our analyses demonstrate that BMP signalling maintains stem cell populations within both embryonic/extra-embryonic tissues during pre-to post-implantation development. These results may provide insight into the role played by BMP signalling in controlling early embryogenesis.

Correction to: FoxO transcription factors regulate mouse preimplantation embryo development.

The original article unfortunately contained a mistake. There is a misplaced "1" in the article title and the correct title is shown above.

Self-Organization of Mouse Stem Cells into an Extended Potential Blastoid.

Mammalian blastocysts comprise three distinct cell lineages essential for development beyond implantation: the pluripotent epiblast, which generates the future embryo, and surrounding it the extra-embryonic primitive endoderm and the trophectoderm tissues. Embryonic stem cells can reintegrate into embryogenesis but contribute primarily to epiblast lineages. Here, we show that mouse embryonic stem cells cultured under extended pluripotent conditions (EPSCs) can be partnered with trophoblast stem cells to self-organize into blastocyst-like structures with all three embryonic and extra-embryonic lineages. Morphogenetic and transcriptome profiling analyses reveal that these blastocyst-like structures show distinct embryonic-abembryonic axes and primitive endoderm differentiation and can initiate the transition from the pre- to post-implantation egg cylinder morphology in vitro.

FoxO transcription factors 1 regulate mouse preimplantation embryo development.

PURPOSE: The aim of the present study is to investigate role of FoxO transcription factors in preimplantation embryo development by knocking down FoxO1, FoxO3, and FoxO4 genes and also to assess cell cycle arrest related proteins, p53 and p21, and apoptosis-related proteins, fas ligand (FASL), and cleaved caspase 3. METHODS: Knockdown of FoxOs using siRNA was confirmed utilizing RT-PCR and qRT-PCR in gene level and using immunofluorescence in protein level. Following knockdown of FoxO1, FoxO3, and FoxO4 in two-cell mouse embryos with or without resveratrol treatment; developmental competence of embryos and expression patterns of SIRT1, p53, p21, FASL, and CLEAVED CASPASE 3 proteins in embryos by immunofluorescence were assessed after 48 h. ROS levels were measured in knockdown embryos. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to determine resveratrol dose. RESULTS: Successful knockdown of FoxO genes in mouse embryos utilizing a non-invasive siRNA method was achieved. Significantly, knockdown of FoxO genes impaired preimplantation embryo development which cannot be prevented by resveratrol treatment. Immunofluorescence results showed that resveratrol could protect embryos from cell cycle arrest and apoptosis. FOXO proteins regulate apoptosis and cell cycle related proteins in mouse preimplantation embryos. Moreover, there might be an autofeedback mechanism where FOXO1, FOXO3, and FOXO4 regulate SIRT1 protein expression. CONCLUSIONS: These results suggest that FOXO transcription factors could contribute to mouse preimplantation embryo development, and it remains to investigate whether they have crucial roles in human preimplantation embryo and infertility.

Dehydroepiandrosterone supplementation attenuates ovarian ageing in a galactose-induced primary ovarian insufficiency rat model.

PURPOSE: Almost every female classic galactosemia patient develops primary ovarian insufficiency (POI). The unique pathophysiology of classic galactosemia, with a severely reduced follicle pool at an early age, requires a new therapeutic approach. This study evaluated the effect of dehydroepiandrosterone (DHEA) on ovarian tissue in a galactose-induced POI rat model. METHODS: Pregnant rats were fed with either a normal or a 35% galactose-containing diet from day 3 of conception continuing through weaning of the litters. Galactose-exposed female offspring were further divided into 5 groups on PND21. The first group received no application. Treatment groups were fed orally by gavage once daily with sesame oil (group 2), or DHEA at doses of 0.1 mg/kg (group 3), 1 mg/kg (group 4) or 10 mg/kg (group 5) until PND70. Fertility rates of mothers with galactosemia, body weights (BWs), and ovarian weights of the litters from PND21 to PND70 were recorded. Ovarian follicle count, immunohistochemistry for proliferation and apoptosis marker expressions and TUNEL for cell death assessment were performed in offspring ovaries. RESULTS: Decreased fertility, ovarian/body weights were observed under galactosemic conditions, together with decreased follicle number and increased atresia. Improved postnatal development, primordial follicle recruitment and follicular growth were observed after DHEA treatment. After DHEA treatment, the expression of Ki67 protein was found to be increased; elevated expression of cleaved-caspase-3 under galactosemia was found to be reduced. CONCLUSIONS: Our data suggests that DHEA treatment may be a potentially useful clinical therapy to improve ovarian ageing in women with POI-induced by galactosemia.

Publisher Correction: Self-assembly of embryonic and two extra-embryonic stem cell types into gastrulating embryo-like structures.

In the version of this Technical Report originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Report.