Self-organization of the in vitro attached human embryo

Implantation of the blastocyst is a developmental milestone in mammalian embryonic development. At this time, a coordinated program of lineage diversification, cell-fate specification, and morphogenetic movements establishes the generation of extra-embryonic tissues and the embryo proper, and determines the conditions for successful pregnancy and gastrulation. Despite its basic and clinical importance, this process remains mysterious in humans. Here we report the use of a novel in vitro system1,2 to study the post-implantation development of the human embryo. We unveil the self-organizing abilities and autonomy of in vitro attached human embryos. We find human-specific molecular signatures of early cell lineage, timing, and architecture. Embryos display key landmarks of normal development, including epiblast expansion, lineage segregation, bi-laminar disc formation, amniotic and yolk sac cavitation, and trophoblast diversification. Our findings highlight the species-specificity of these developmental events and provide a new understanding of early human embryonic development beyond the blastocyst stage. In addition, our study establishes a new model system relevant to early human pregnancy loss. Finally, our work will also assist in the rational design of differentiation protocols of human embryonic stem cells to specific cell types for disease modelling and cell replacement therapy

Hypoxia induces a transcriptional early primitive streak signature in pluripotent cells enhancing spontaneous elongation and lineage representation in gastruloids

The cellular microenvironment, together with intrinsic regulators, shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and appear to benefit from hypoxic culture in vitro. Yet, how hypoxia influences stem cell transcriptional networks and lineage choices remain poorly understood. Here, we investigated the molecular effects of acute and prolonged hypoxia on embryonic and extra-embryonic stem cells as well as the functional impact on differentiation potential. We find a temporal and cell type-specific transcriptional response including an early primitive streak signature in hypoxic embryonic stem cells mediated by HIF1α. Using a 3D gastruloid differentiation model, we show that hypoxia-induced T expression enables symmetry breaking and axial elongation in the absence of exogenous WNT activation. When combined with exogenous WNT activation, hypoxia enhances lineage representation in gastruloids, as demonstrated by highly enriched signatures of gut endoderm, notochord, neuromesodermal progenitors and somites. Our findings directly link the microenvironment to stem cell function and provide a rationale supportive of applying physiological conditions in models of embryo development

Sequential formation and resolution of multiple rosettes drive embryo remodelling after implantation

The morphogenetic remodelling of embryo architecture after implantation culminates in pro-amniotic cavity formation. Despite its key importance, how this transformation occurs remains unknown. Here, we apply high-resolution imaging of embryos developing in vivo and in vitro, spatial RNA sequencing and 3D trophoblast stem cell models to determine the sequence and mechanisms of these remodelling events. We show that cavitation of the embryonic tissue is followed by folding of extra-embryonic tissue to mediate the formation of a second extra-embryonic cavity. Concomitantly, at the boundary between embryonic and extra-embryonic tissues, a hybrid 3D rosette forms. Resolution of this rosette enables the embryonic cavity to invade the extra-embryonic tissue. Subsequently, β1-integrin signalling mediates the formation of multiple extra-embryonic 3D rosettes. Podocalyxin exocytosis leads to their polarized resolution, permitting the extension of embryonic and extra-embryonic cavities and their fusion into a unified pro-amniotic cavity. These morphogenetic transformations of embryogenesis reveal a previously unappreciated mechanism for lumen expansion and fusionThe M.Z.G lab is supported by grants from the European Research Council (669198) and the Welcome Trust (098287/Z/12/Z) and the EU Horizon 2020 Marie Sklodowska-Curie actions (ImageInLife,721537). C.K is supported by BBSRC Doctoral training studentship

In vitro generation of mouse polarized embryo-like structures from embryonic and trophoblast stem cells

Mammalian embryogenesis requires the coordination of embryonic and extra-embryonic tissues to enable implantation into the uterus and post-implantation development to establish the body plan. Mouse embryonic stem cells (ESCs) are a useful tool for studying pluripotent embryonic tissue in vitro. However, they cannot undertake correct embryogenesis alone. Many attempts to model the early embryo in vitro involve the aggregation of ESCs into spheroids of variable size and cell number that undertake germ-layer specification but fail to recapitulate the characteristic architecture and arrangement of tissues of the early embryo. Here, we describe a protocol to generate the first embryo-like structures by directing the assembly of mouse ESCs and extra-embryonic trophoblast stem cells (TSCs) in a 3D extracellular matrix (ECM) into structures we call ‘polarized embryo-like structures’. By establishing the medium and culture conditions needed to support the growth of both stem cell types simultaneously, embryonic architecture is generated within 4 d of co-culture. This protocol can be performed by those proficient in standard ESC culture techniques and can be used in developmental studies to investigate the interactions between embryonic and extra-embryonic tissues during mammalian development.We are grateful to the Wellcome Trust for the Senior Research fellowship (grant no. 098287/Z/12/Z) and for a European Research Council grant (code: 669198) awarded to M.Z.-G. to fund this work. We are also grateful for the BBSRC DTP studentship that supports S.E.H. and to the Scientific and Technological Research Council of Turkey, which supports B.S.

Micropattern differentiation of mouse pluripotent stem cells recapitulates embryo regionalized cell fate patterning.

During gastrulation epiblast cells exit pluripotency as they specify and spatially arrange the three germ layers of the embryo. Similarly, human pluripotent stem cells (PSCs) undergo spatially organized fate specification on micropatterned surfaces. Since in vivo validation is not possible for the human, we developed a mouse PSC micropattern system and, with direct comparisons to mouse embryos, reveal the robust specification of distinct regional identities. BMP, WNT, ACTIVIN and FGF directed mouse epiblast-like cells to undergo an epithelial-to-mesenchymal transition and radially pattern posterior mesoderm fates. Conversely, WNT, ACTIVIN and FGF patterned anterior identities, including definitive endoderm. By contrast, epiblast stem cells, a developmentally advanced state, only specified anterior identities, but without patterning. The mouse micropattern system offers a robust scalable method to generate regionalized cell types present in vivo, resolve how signals promote distinct identities and generate patterns, and compare mechanisms operating in vivo and in vitro and across species

Self-organization of the human embryo in the absence of maternal tissues.

Remodelling of the human embryo at implantation is indispensable for successful pregnancy. Yet it has remained mysterious because of the experimental hurdles that beset the study of this developmental phase. Here, we establish an in vitro system to culture human embryos through implantation stages in the absence of maternal tissues and reveal the key events of early human morphogenesis. These include segregation of the pluripotent embryonic and extra-embryonic lineages, and morphogenetic rearrangements leading to generation of a bilaminar disc, formation of a pro-amniotic cavity within the embryonic lineage, appearance of the prospective yolk sac, and trophoblast differentiation. Using human embryos and human pluripotent stem cells, we show that the reorganization of the embryonic lineage is mediated by cellular polarization leading to cavity formation. Together, our results indicate that the critical remodelling events at this stage of human development are embryo-autonomous, highlighting the remarkable and unanticipated self-organizing properties of human embryos.This work was supported by the Wellcome Trust grant to M.Z- G. Work in Dr. K.K.N lab was supported by The Francis Crick Institute, which receives its core funding from Cancer Research UK, the Medical Research Council and the Wellcome Trust. Dr. M.N.S. was initially supported by a Ramon Areces Spanish Foundation Fellowship, and subsequently by an EMBO Postdoctoral Fellowship. Dr. S.V was supported by a Post Doc Pool Grant from the Finnish Cultural Foundation. Dr. GR was supported by a Newton Fellowship.This is the author accepted manuscript. It is currently under an indefinite embargo pending publication by Nature Publishing Group

A Cdh1^HA Knock-in Allele Rescues the Cdh1^-/- Phenotype but Shows Essential Cdh1 Function During Placentation

The specific roles of classical cadherins at key morphogenetic events during development are still not fully understood. As part of a project to study cadherin function during early mammalian development, we generated mice carrying an HA-epitope tagged Cdh1 (E-cadherin) cDNA knocked into the Cdh1 locus, similar to the previously described mouse mutants in which we forced Cdh2 (N-cadherin) expression in the Cdh1 expression domain. As expected and in contrast to Cdh1Cdh2/Cdh2 and Cdh1-/-, our Cdh1HA/HA mutant embryos form proper trophectoderm, implant and undergo both gastrulation and neurulation. However, Cdh1HA/HA mice display an unexpected phenotype at embryonic day 10.5. Cdh1HA/HA embryos are smaller, paler and suffer from an insufficient nutrient supply. We detected a reduced expression of Cdh1HA specifically in the extraembryonic ectoderm and in the labyrinth layer, whereas expression in the embryo proper was normal. With this approach, we show for the first time that Cdh1 is essential for the correct formation of the placenta. Placentas without Cdh1 expression are impaired and incapable of establishing a proper connection between the embryonic and the maternal blood vessels for efficient nutrient and oxygen transport