Project description:Stem cell models that replicate the gastrulation process in human embryos have been created, but they lack the essential extraembryonic cells needed for early embryonic development and patterning. Here, we introduce a robust and efficient method that prompts human extended pluripotent stem (EPS) cells to self-organize into embryo-like structures, called peri-gastruloids, which encompass both embryonic (epiblast) and extraembryonic (hypoblast) tissues. These peri-gastruloids simulate critical stages of human peri-gastrulation development, such as forming amniotic and yolk sac cavities, developing bilaminar and trilaminar embryonic discs, specifying primordial germ cells, initiating gastrulation, and early neurulation. Single-cell RNA sequencing unveiled transcriptomic characteristics of these human peri-gastruloids, which closely resemble the primary peri-gastrulation cell types found in human and non-human primates. Our results emphasize the remarkable self-organizing ability of EPS cells to generate advanced human embryo-like structures. This peri-gastruloid platform allows for further exploration beyond gastrulation and may potentially aid in the development of human fetal tissues for use in regenerative medicine.
Project description:We analyzed scRNA-seq data in human pluripotent stem cells derived peri-gastrulation trilaminar embryonic disc (PTED) human embryo models with trilaminar embryonic disc-, amnion- and yolk sac-like structures.
Project description:Establishment of the mammalian body plan occurs shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. While methods for in vitro culture of pre- and peri-implantation mouse embryos are routinely utilized, approaches for robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. We develop herein highly stable ex utero post-implantation mouse embryo culture platforms, that enable appropriate development of embryos before gastrulation (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in 3D rotating bottles settings, while extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of novel static and rotating bottle culture protocols. Histological, molecular, and single cell RNA-seq analysis validate that the ex utero developed embryos recapitulate precisely in utero development. This culture system is amenable to introducing a variety of embryonic perturbations and micro-manipulations that can be followed ex utero for up to 6 days. Establishment of a system to robustly grow normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool to investigate post-implantation embryogenesis, eliminating the uterine barrier to mechanistically interrogate morphogenesis and tissue specification in mammals.
Project description:Our understanding of human early development is severely hampered by limited access to embryonic tissues. Due to their close evolutionary relationship with humans, non-human primates (NHPs) are often used as surrogates to understand human development but currently suffer from a lack of in vivo datasets, especially from gastrulation to early organogenesis during which the major embryonic cell types are dynamically specified. To fill this gap, we have collected six Carnegie stage (CS) 8-CS11 cynomolgus monkey embryos and performed in-depth transcriptome analyses of 56,636 single cells. Our analyses reveal transcriptomic features of major peri-gastrulation cell types, which help shed light on morphogenetic events including primitive streak (PS) development, somitogenesis, gut tube formation, neural tube patterning, and neural crest regionalization in primates. In addition, comparative analyses with mouse embryos and human embryoids uncover conserved and divergent features of peri-gastrulation development across species, e.g. species-specific dependency on Hippo signaling during presomitic mesoderm differentiation, and provide an initial assessment of relevant stem cell models of human early organogenesis. This comprehensive single-cell transcriptome atlas not only fills the knowledge gap in the NHP research field but also serves as an invaluable resource for understanding human embryogenesis and developmental disorders.
Project description:Micropatterned human pluripotent stem cells (hPSCs) treated with BMP4, known as a 2D gastruloid, are among the most widely used stem cell models for human gastrulation. Due to its simplicity and reproducibility, this system is ideal for high throughput quantitative studies of tissue patterning and has led to many insights into the mechanisms of mammalian gastrulation. However, 2D gastruloids have only been studied up to 48h. Here we extended this system to 96h.
Project description:The bilaminar disc of early pig embryos closely mirrors that of humans making it a powerful model for studying gastrulation. Given the difficulties obtaining embryos in non-rodents, early cell-fate decisions during mammalian gastrulation remain ill-understood. Here we present a single-cell transcriptomic atlas of pig gastrulation and early organogenesis. We uncover the dynamics of cell fate emergence during pig peri-gastrulation and reveal conserved and species-specific transcriptional programs across different mammals. Combined with investigations in embryos and embryonic stem cells, we elucidate the spatial, molecular, and temporal events during definitive endoderm (DE) formation. We show that early FOXA2+ epiblast progenitors become DE without undergoing epithelial-to-mesenchymal transition, contrasting later emerging FOXA2/TBXT+ anterior primitive streak, which form node/notochord progenitors. We demonstrate that DE fate is driven by hypoblast-derived NODAL signalling, which is extinguished upon DE differentiation. These findings highlight the interplay between temporal and topological signalling during early cell fate decisions during mammalian gastrulation.
Project description:Centromeres in budding yeast are surrounded by ~10-kb DNA loops generated by the cohesin complex. These peri-centromeric loops are shown to play a role in faithful chromosome segregation. It remains not fully understood how the dynamics of the peri-centromeric loops are controlled. Here we found that gene deletion of two cohesin regulators, Wpl1 and Eco1, resulted in larger peri-centromeric loops that connected the centromeres and genome regions up to ~ 300 kb distant in G2/M-arrested cells. Deletion of Wpl1 and Eco1 synergistically contributed to the loop size enlargement. ChIP-seq revealed that cohesin and its ATPase activator, Scc2, were colocalized at the anchor sites of the extended peri-centromeric loops, specifically in the double-deletion mutant Δwpl1 Δeco1. Consistently, acute depletion of Scc2 in the G2/M phase resulted in the disappearance of the extended loops. Given that Scc2 is bound prominently only at the centromeres in wild-type cells, our results suggest that Wpl1 and Eco1 jointly promote the dissociation of Scc2 from cohesin and inhibit excessive expansion of peri-centromeric DNA loop size. Notably, we found that nuclear division after benomyl block and release was significantly delayed in the Δwpl1 Δeco1 double-deletion mutant, and this delay was rescued by Scc2 depletion in the G2/M phase. This indicates that cohesin regulators cooperatively regulate the size of the peri-centromeric DNA loops to ensure faithful chromosome segregation.
Project description:Centromeres in budding yeast are surrounded by ~10-kb DNA loops generated by the cohesin complex. These peri-centromeric loops are shown to play a role in faithful chromosome segregation. It remains not fully understood how the dynamics of the peri-centromeric loops are controlled. Here we found that gene deletion of two cohesin regulators, Wpl1 and Eco1, resulted in larger peri-centromeric loops that connected the centromeres and genome regions up to ~ 300 kb distant in G2/M-arrested cells. Deletion of Wpl1 and Eco1 synergistically contributed to the loop size enlargement. ChIP-seq revealed that cohesin and its ATPase activator, Scc2, were colocalized at the anchor sites of the extended peri-centromeric loops, specifically in the double-deletion mutant Δwpl1 Δeco1. Consistently, acute depletion of Scc2 in the G2/M phase resulted in the disappearance of the extended loops. Given that Scc2 is bound prominently only at the centromeres in wild-type cells, our results suggest that Wpl1 and Eco1 jointly promote the dissociation of Scc2 from cohesin and inhibit excessive expansion of peri-centromeric DNA loop size. Notably, we found that nuclear division after benomyl block and release was significantly delayed in the Δwpl1 Δeco1 double-deletion mutant, and this delay was rescued by Scc2 depletion in the G2/M phase. This indicates that cohesin regulators cooperatively regulate the size of the peri-centromeric DNA loops to ensure faithful chromosome segregation.