Epiblast Development Research Articles

Elucidation of the pluripotent potential of bovine embryonic lineages facilitates the establishment of formative stem cell lines.

The establishment of epiblast-derived pluripotent stem cells (PSCs) from cattle, which are important domestic animals that provide humans with milk and meat while also serving as bioreactors for producing valuable proteins, poses a challenge due to the unclear molecular signaling required for embryonic epiblast development and maintenance of PSC self-renewal. Here, we selected six key stages of bovine embryo development (E5, E6, E7, E10, E12, and E14) to track changes in pluripotency and the dependence on signaling pathways via modified single-cell transcription sequencing technology. The remarkable similarity of the gene expression patterns between cattle and pigs during embryonic lineage development contributed to the successful establishment of bovine epiblast stem cells (bEpiSCs) using 3i/LAF (WNTi, GSK3βi, SRCi, LIF, Activin A, and FGF2) culture system. The generated bEpiSCs exhibited consistent expression patterns of formative epiblast pluripotency genes and maintained clonal morphology, normal karyotypes, and proliferative capacity for more than 112 passages. Moreover, these cells exhibited high-efficiency teratoma formation as well as the ability to differentiate into various cell lineages. The potential of bEpiSCs for myogenic differentiation, primordial germ cell like cells (PGCLCs) induction, and as donor cells for cellnuclear transfer was also assessed, indicating their promise in advancing cell-cultured meat production, gene editing, and animal breeding.

A single-cell transcriptome atlas of human euploid and aneuploid blastocysts.

Aneuploidy is frequently detected in early human embryos as a major cause of early pregnancy failure. However, how aneuploidy affects cellular function remains elusive. Here, we profiled the transcriptomes of 14,908 single cells from 203 human euploid and aneuploid blastocysts involving autosomal and sex chromosomes. Nearly all of the blastocysts contained four lineages. In aneuploid chromosomes, 19.5% ± 1.2% of the expressed genes showed a dosage effect, and 90 dosage-sensitive domains were identified. Aneuploidy leads to prevalent genome-wide transcriptome alterations. Common effects, including apoptosis, were identified, especially in monosomies, partially explaining the lower cell numbers in autosomal monosomies. We further identified lineage-specific effects causing unstable epiblast development in aneuploidies, which was accompanied by the downregulation of TGF-β and FGF signaling, which resulted in insufficient trophectoderm maturation. Our work provides crucial insights into the molecular basis of human aneuploid blastocysts and may shed light on the cellular interaction during blastocyst development.

Nutrient uptake strategies shift during mouse epiblast development.

Nutrient uptake strategies shift during mouse epiblast development.

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.

Pre-hatching exposure to N2B27 medium improves post-hatching development of bovine embryos in vitro

Ungulate embryos undergo critical cell differentiation and proliferation events around and after blastocyst hatching. Failures in these processes lead to early pregnancy losses, which generate an important economic impact on farming. Conventional embryo culture media, such as SOF, are unable to support embryo development beyond hatching. In contrast, N2B27 medium supports early post-hatching development, evidencing a swift in embryonic nutritional requirements during this developmental window. Here, we investigate if earlier exposure to N2B27 could improve embryo development after hatching. Embryo culture in N2B27 from day (D) 5, 6 or 7 significantly enhanced complete hypoblast migration (>45 vs. ∼24%) and epiblast development into an embryonic disc (ED)-like structure at D12 (>40 vs. 23%), compared to embryos cultured in SOF up to D9. Culture in N2B27 from D5 significantly increased epiblast and hypoblast cell number in D8 blastocysts, but post-hatching embryos cultured in N2B27 from D5 or 6 frequently showed a disorganized distribution of epiblast cells. In conclusion, bovine embryo culture in N2B27 from D7 onwards improves subsequent post-hatching development. This improved fully in vitro system will be very useful to functionally explore cell differentiation mechanisms and the bases of early pregnancy failures without requiring animal experimentation.

Transforming growth factor beta (TGFβ) pathway is essential for hypoblast and epiblast development in ovine post-hatching embryos

The developmental failures occurring between blastocyst hatching and implantation in farm ungulates are a major cause of pregnancy losses. At the expanded blastocyst stage, three cell lineages emerge in the embryo: trophoblast, hypoblast and epiblast, the latter being the most vulnerable during post-hatching development. Transforming growth factor beta (TGFβ) signaling pathway is involved in hypoblast and epiblast development; however, previous in vitro functional studies are limited to the expanded blastocyst stage. In this study, we have analyzed the effect of TGFβ inhibition with 10, 20 or 40 μM SB431542 during ovine post-hatching developmental period using a recently developed culture system able to recapitulate major developmental landmarks. We have found a negative effect of TGFβ inhibition on hypoblast and epiblast development that could be partially reverted by Rho-associated protein kinase (ROCK) inhibitor Y-27632. Our findings provide new insights into the molecular networks regulating embryo development beyond the expanded blastocyst and could help to elucidate the causes of early pregnancy losses in farm ungulates.

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.

Maternal Ezh1/2 deficiency in oocyte delays H3K27me2/3 restoration and impairs epiblast development responsible for embryonic sub-lethality in mouse.

How maternal Ezh1 and Ezh2 function in H3K27 methylation in vivo in pre-implantation embryos and during embryonic development is not clear. Here, we have deleted Ezh1 and Ezh2 alone or simultaneously from mouse oocytes. H3K27me3 was absent in oocytes without Ezh2 alone, while both H3K27me2 and H3K27me3 were absent in Ezh1/Ezh2 (Ezh1/2) double knockout (KO) oocytes. The effects of Ezh1/2 maternal KO were inherited in zygotes and early embryos, in which restoration of H3K27me3 and H3K27me2 was delayed by the loss of Ezh2 alone or of both Ezh1 and Ezh2. However, the ablation of both Ezh1 and Ezh2, but not Ezh1 or Ezh2 alone, led to significantly decreased litter size due to growth retardation post-implantation. Maternal Ezh1/2 deficiency caused compromised H3K27me3 and pluripotent epiblast cells in late blastocysts, followed by defective embryonic development. By using RNA-seq, we examined crucial developmental genes in maternal Ezh1/2 KO embryos and identified 80 putatively imprinted genes. Maternal Ezh1/2-H3K27 methylation is inherited in offspring embryos and has a critical effect on fetal and placental development. Thus, this work sheds light on maternal epigenetic modifications during embryonic development.

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.

Generation and characterization of stable pig pregastrulation epiblast stem cell lines

Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

Cross-species single-cell transcriptomic analysis reveals pre-gastrulation developmental differences among pigs, monkeys, and humans

Interspecies blastocyst complementation enables organ-specific enrichment of xenogeneic pluripotent stem cell (PSC) derivatives, which raises an intriguing possibility to generate functional human tissues/organs in an animal host. However, differences in embryo development between human and host species may constitute the barrier for efficient chimera formation. Here, to understand these differences we constructed a complete single-cell landscape of early embryonic development of pig, which is considered one of the best host species for human organ generation, and systematically compared its epiblast development with that of human and monkey. Our results identified a developmental coordinate of pluripotency spectrum among pigs, humans and monkeys, and revealed species-specific differences in: (1) pluripotency progression; (2) metabolic transition; (3) epigenetic and transcriptional regulations of pluripotency; (4) cell surface proteins; and (5) trophectoderm development. These differences may prevent proper recognition and communication between donor human cells and host pig embryos, resulting in low integration and survival of human cells. These results offer new insights into evolutionary conserved and divergent processes during mammalian development and may be helpful for developing effective strategies to overcome low human–pig chimerism, thereby enabling the generation of functional human organs in pigs in the future.

45 Embryonic disc development invitro in ovine embryos

Embryonic mortality during the second week of pregnancy has an important economic impact on farming. At this time, the embryo undergoes critical developmental events that cannot be recapitulated invitro, limiting our understanding of these pregnancy losses. After the blastocyst stage, the hypoblast migrates to cover the inner surface of the embryo and the epiblast forms a flat embryonic disc (ED) that initiates gastrulation. The aim of this study was to develop an invitro culture system to support sheep embryo development after the blastocyst stage. Day 6/7 invitro-produced blastocysts were cultured over agarose gels to prevent attachment and allocated to different media: synthetic oviductal fluid with 10% fetal bovine serum (SOF-FBS, n=52), an invitro culture medium (hIVC, n=35) supporting ED formation in human embryos (Deglincerti et al. 2016 Nature 533, 251-254; https://doi.org/10.1038/nature17948), and chemically defined N2B27 medium (n=38) supporting ED formation in bovine embryos (Ramos-Ibeas et al. 2020 Reproduction 160, 579-589, https://doi.org/10.1530/REP-20-0243). At Day 14, survival and embryo area were recorded, the abundance of transcripts encoding interferon Tau (TP1) and metabolic enzymes was analysed by RT-qPCR, and the development of epiblast and hypoblast was assessed by immunostaining for SOX2 and SOX17. Embryo survival and size and the percentage of embryos achieving complete hypoblast migration were significantly reduced in SOF-FBS (Chi-squared test and one-way ANOVA; P<0.05). Only N2B27 medium supported epiblast survival in 11/28 embryos. TP1 expression increased at Day 14 in all culture conditions and metabolism-related genes revealed a shift from anaerobic glycolysis to oxidative phosphorylation after culture in hIVC and N2B27. Next, to promote epiblast development, we allocated blastocysts to N2B27 medium supplemented with activin A (n=45), rho-associated protein kinase (ROCK) inhibitor (ROCKi, n=42), or insulin growth factor 1 (IGF1, n=29). IGF1 reduced significantly the percentage of embryos showing an ED-like structure, whereas activin A supplementation significantly increased epiblast survival (Chi-squared test; P<0.05) and SOX2+ cell number was higher in embryos cultured with ROCK inhibitor. When we combined activin A and ROCKi supplementation (N2B27+A+R, n=151), SOX2+ cell number and the percentage of embryos showing an ED-like structure increased significantly (165.1±53.1 and 31/35 embryos with SOX2+ epiblast cells; ∼89%) compared with N2B27 alone (49.4±12.7 and 5/11; ∼45%) (one-way ANOVA and Chi-squared test; P<0.05). Moreover, 18/31 (∼58%) ED-like structures developed in N2B27+A+R lost the Rauber’s layer (polar trophoblast), and BRACHYURY expression, denoting the onset of gastrulation, was observed in 3/14. In conclusion, we have developed a culture system that supports complete hypoblast migration and ED development invitro, which represents a valuable tool to explore early embryo mortality in livestock species without the need for experimental animals.

Wnt/Beta-catenin/Esrrb signalling controls the tissue-scale reorganization and maintenance of the pluripotent lineage during murine embryonic diapause

The epiblast, which provides the foundation of the future body, is actively reshaped during early embryogenesis, but the reshaping mechanisms are poorly understood. Here, using a 3D in vitro model of early epiblast development, we identify the canonical Wnt/β-catenin pathway and its central downstream factor Esrrb as the key signalling cascade regulating the tissue-scale organization of the murine pluripotent lineage. Although in vivo the Wnt/β-catenin/Esrrb circuit is dispensable for embryonic development before implantation, autocrine Wnt activity controls the morphogenesis and long-term maintenance of the epiblast when development is put on hold during diapause. During this phase, the progressive changes in the epiblast architecture and Wnt signalling response show that diapause is not a stasis but instead is a dynamic process with underlying mechanisms that can appear redundant during transient embryogenesis.

MCRS1 is essential for epiblast development during early mouse embryogenesis.

Microspherule protein 1 (MCRS1, also known as MSP58) is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to MCRS1 in vitro, mammalian MCRS1 has not been studied in vivo. Here we report that MCRS1 is essential during early murine development. Mcrs1 mutant embryos exhibit normal morphology at the blastocyst stage but cannot be recovered at gastrulation, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts do not form a typical inner cell mass (ICM) colony, the source of embryonic stem cells (ESCs). Surprisingly, cell death and histone H4 acetylation analysis reveal that apoptosis and global H4 acetylation are normal in mutant blastocysts. However, analysis of lineage specification reveals that while the trophoblast and primitive endoderm are properly specified, the epiblast lineage is compromised and exhibits a severe reduction in cell number. In summary, our study demonstrates the indispensable role of MCRS1 in epiblast development during early mammalian embryogenesis.

Embryonic Cul4b is important for epiblast growth and location of primitive streak layer cells.

Cul4b-null (Cul4bΔ/Y) mice undergo growth arrest and degeneration during the early embryonic stages and die at E9.5. The pathogenic causes of this lethality remain incompletely characterized. However, it has been hypothesized that the loss of Cul4b function in extraembryonic tissues plays a key role. In this study, we investigated possible causes of death for Cul4b-null embryos, particularly in regard to the role of embryonic Cul4b. First, we show that the loss of embryonic Cul4b affects the growth of the inner cell mass in vitro and delays epiblast development during the gastrulation period at E6.5~E7.5 in vivo, as highlighted by the absence of the epiblastic transcription factor Brachyury from E6.5~E7.5. Additionally, at E7.5, strong and laterally expanded expression of Eomes and Fgf8 signaling was detected. Sectioning of these embryos showed disorganized primitive streak layer cells. Second, we observed that Mash2-expressing cells were present in the extraembryonic tissues of Cul4b-deficient embryos at E6.5 but were absent at E7.5. In addition, the loss of Cul4b resulted in decreased expression of cyclin proteins, which are required for the cell cycle transition from G1 to S. Taken together, these observations suggest that the embryonic expression of Cul4b is important for epiblast growth during E6.5~E7.5, and the loss of Cul4b results in either delayed growth of the epiblast or defective localization of primitive streak layer cells. As a result, the signaling activity mediated by the epiblast for subsequent ectoplacental cone development is affected, with the potential to induce growth retardation and lethality in Cul4bΔ/Y embryos.

Principles of early human development and germ cell program from conserved model systems.

Human primordial germ cells (hPGCs), the precursors of sperm and eggs, originate during week 2-3 of early postimplantation development1. Using in vitro models of hPGC induction2–4, recent studies suggest striking mechanistic differences in specification of human and mouse PGCs5. This may partly be due to the divergence in their pluripotency networks, and early postimplantation development6–8. Since early human embryos are inaccessible for direct studies, we considered alternatives, including porcine embryos that, as in humans, develop as bilaminar embryonic discs. Here we show that porcine PGCs (pPGCs) originate from the posterior pre-primitive streak competent epiblast by sequential upregulation of SOX17 and BLIMP1 in response to WNT and BMP signalling. Together with human and monkey in vitro models simulating peri-gastrulation development, we show conserved principles for epiblast development for competency for PGC fate, followed by initiation of the epigenetic programme9–11, regulated by a balanced SOX17–BLIMP1 gene dosage. Our combinatorial approach using human, porcine and monkey in vivo and in vitro models, provides synthetic insights on early human development.

Establishment of Functional Genomics Pipeline in Mouse Epiblast-Like Tissue by Combining Transcriptomic Analysis and Gene Knockdown/Knockin/Knockout, Using RNA Interference and CRISPR/Cas9.

The epiblast (foremost embryonic ectoderm) generates all three germ layers and therefore has crucial roles in the formation of all mammalian body cells. However, regulation of epiblast gene expression is poorly understood because of the difficulty of manipulating epiblast tissues in vivo. In the present study, using the self-organizing properties of mouse embryonic stem cell (ESC), we generated and characterized epiblast-like tissue in three-dimensional culture. We identified significant genome-wide gene expression changes in this epiblast-like tissue by transcriptomic analysis. In addition, we identified the particular significance of the Erk/Mapk and integrin-linked kinase pathways, and genes related to ectoderm/epithelial formation, using the bioinformatics resources IPA and DAVID. Here, we focused on Fgf5, which ranked in the top 10 among the discovered genes. To develop a functional analysis of Fgf5, we created an efficient method combining CRISPR/Cas9-mediated genome engineering and RNA interference (RNAi). Notably, we show one-step generation of various Fgf5 reporter lines including heterozygous and homozygous knockins (the GET method). For time- and dose-dependent depletion of fgf5 over the course of development, we generated an ESC line harboring Tol2 transposon-mediated integration of an inducible short hairpin RNA interference system (pdiRNAi). Our findings raised the possibility that Fgf/Erk signaling and apicobasal epithelial integrity are important factors in epiblast development. In addition, our methods provide a framework for a broad array of applications in the areas of mammalian genetics and molecular biology to understand development and to improve future therapeutics.

Smad2 and Smad3 have differential sensitivity in relaying TGFβ signaling and inversely regulate early lineage specification.

The transforming growth factor beta (TGFβ) related signaling is one of the most important signaling pathways regulating early developmental events. Smad2 and Smad3 are structurally similar and it is mostly considered that they are equally important in mediating TGFβ signals. Here, we show that Smad3 is an insensitive TGFβ transducer as compared with Smad2. Smad3 preferentially localizes within the nucleus and is thus sequestered from membrane signaling. The ability of Smad3 in oligomerization with Smad4 upon agonist stimulation is also impaired given its unique linker region. Smad2 mediated TGFβ signaling plays a crucial role in epiblast development and patterning of three germ layers. However, signaling unrelated nuclear localized Smad3 is dispensable for TGFβ signaling-mediated epiblast specification, but important for early neural development, an event blocked by TGFβ/Smad2 signaling. Both Smad2 and Smad3 bind to the conserved Smads binding element (SBE), but they show nonoverlapped target gene binding specificity and differential transcriptional activity. We conclude that Smad2 and Smad3 possess differential sensitivities in relaying TGFβ signaling and have distinct roles in regulating early developmental events.

Out-of-frame start codons prevent translation of truncated nucleo-cytosolic cathepsin L in vivo

The lysosomal protease cathepsin L has been reported to cleave various functionally important cytosolic or nuclear proteins. To explain nucleo-cytosolic localization of cathepsin L, it has been hypothesized that skipping of the first start codon during translation initiation results in an N-terminally truncated protein lacking the endoplasmic reticulum-import signal. Here we demonstrate that out-of-frame AUGs prevent translation of truncated cathepsin L in cell culture as well as in a new knock-in mouse model. We further evaluate potential roles of nuclear cathepsin L during early embryonic development. Our analysis reveals normal epiblast development of cathepsin L-deficient embryos, but uncovers a pronounced lysosomal storage phenotype in the extra-embryonic tissue of the visceral endoderm. In conclusion, the phenotypes of cathepsin L deficiency can be fully assigned to lack of canonically targeted cathepsin L, while the biogenesis and functionality of nucleo-cytosolic cathepsin L remain elusive.

Pluripotency in the embryo and in culture.

Specific cells within the early mammalian embryo have the capacity to generate all somatic lineages plus the germline. This property of pluripotency is confined to the epiblast, a transient tissue that persists for only a few days. In vitro, however, pluripotency can be maintained indefinitely through derivation of stem cell lines. Pluripotent stem cells established from the newly formed epiblast are known as embryonic stem cells (ESCs), whereas those generated from later stages are called postimplantation epiblast stem cells (EpiSCs). These different classes of pluripotent stem cell have distinct culture requirements and gene expression programs, likely reflecting the dynamic development of the epiblast in the embryo. In this chapter we review current understanding of how the epiblast forms and relate this to the properties of derivative stem cells. We discuss whether ESCs and EpiSCs are true counterparts of different phases of epiblast development or are culture-generated phenomena. We also consider the proposition that early epiblast cells and ESCs may represent a naïve ground state without any prespecification of lineage choice, whereas later epiblasts and EpiSCs may be primed in favor of particular fates.