Generation of Stem Cell-Based Mouse Embryo-Like Structures.

In this chapter, we detail the experimental protocol leading to the generation of stem cell-based mouse embryo-like structures termed "ETiX-embryoids." ETiX-embryoids are formed from combined embryonic stem cells, trophoblast stem cells, and embryonic stem cells transiently induced to express Gata4. Cells are seeded into AggreWell dishes where they form aggregates that develop to resemble post-implantation mouse embryos following 4 days of culture. ETiX-embryoids establish an anterior signaling center and undergo gastrulation over the following 2 days. By day 7, ETiX-embryoids undergo neurulation and form an anterior-posterior axis with head folds at one end and a tail bud on the other. On day 8, they develop a brain and form a heart-like structure and a gut tube.

Assembly of complete mouse embryo models from embryonic and induced stem cell types in vitro.

The interaction between embryonic and extraembryonic tissues is critical in natural mouse embryogenesis. Here, to enable such interaction in vitro, we describe a protocol to assemble a complete mouse embryo model using mouse embryonic stem cells and induced embryonic stem cells to express Cdx2 (or trophoblast stem cells) and Gata4 to reconstitute the epiblast, extraembryonic ectoderm and visceral endoderm lineages, respectively. The resulting complete embryo models recapitulate development from embryonic day 5.0 to 8.5, generating advanced embryonic and extraembryonic tissues that develop through gastrulation to initiate organogenesis to form a head and a beating heart structure as well as a yolk sac and chorion. Once the required stem cell lines are stably maintained in culture, the protocol requires 1 day to assemble complete embryo models and a further 8 days to culture them until headfold stages, although structures can be collected at earlier developmental stages as required. This protocol can be easily performed by researchers with experience in mouse stem cell culture, although they will benefit from knowledge of natural mouse embryos at early postimplantation stages.

spinDrop: a droplet microfluidic platform to maximise single-cell sequencing information content.

Droplet microfluidic methods have massively increased the throughput of single-cell sequencing campaigns. The benefit of scale-up is, however, accompanied by increased background noise when processing challenging samples and the overall RNA capture efficiency is lower. These drawbacks stem from the lack of strategies to enrich for high-quality material or specific cell types at the moment of cell encapsulation and the absence of implementable multi-step enzymatic processes that increase capture. Here we alleviate both bottlenecks using fluorescence-activated droplet sorting to enrich for droplets that contain single viable cells, intact nuclei, fixed cells or target cell types and use reagent addition to droplets by picoinjection to perform multi-step lysis and reverse transcription. Our methodology increases gene detection rates fivefold, while reducing background noise by up to half. We harness these properties to deliver a high-quality molecular atlas of mouse brain development, despite starting with highly damaged input material, and provide an atlas of nascent RNA transcription during mouse organogenesis. Our method is broadly applicable to other droplet-based workflows to deliver sensitive and accurate single-cell profiling at a reduced cost.

Behind the developing brains and beating hearts of stem cell-derived embryo models.

Studies over the past decade have shown how stem cells representing embryonic and extra-embryonic tissues of the mouse can self-assemble in the culture dish to recapitulate an astonishing part of early embryonic development. A systematic analysis has demonstrated how pluripotent embryonic stem cells can be induced to behave like the implanting epiblast; how they can interact with trophectoderm stem cells to form a patterned structure resembling the implanting embryo prior to gastrulation; and how the third stem cell type-extra-embryonic endoderm cells-can be incorporated to generate structures that undergo the cell movements and gene expression patterns of gastrulation. Moreover, such stem cell-derived embryo models can proceed to neurulation and establish progenitors for all parts of the brain and neural tube, somites, beating heart structures and gut tube. They develop within extra-embryonic yolk sacs that initiate haematopoiesis. Here we trace this journey of discovery.

Mouse embryo model derived exclusively from embryonic stem cells undergoes neurulation and heart development.

Several in vitro models have been developed to recapitulate mouse embryogenesis solely from embryonic stem cells (ESCs). Despite mimicking many aspects of early development, they fail to capture the interactions between embryonic and extraembryonic tissues. To overcome this difficulty, we have developed a mouse ESC-based in vitro model that reconstitutes the pluripotent ESC lineage and the two extraembryonic lineages of the post-implantation embryo by transcription-factor-mediated induction. This unified model recapitulates developmental events from embryonic day 5.5 to 8.5, including gastrulation; formation of the anterior-posterior axis, brain, and a beating heart structure; and the development of extraembryonic tissues, including yolk sac and chorion. Comparing single-cell RNA sequencing from individual structures with time-matched natural embryos identified remarkably similar transcriptional programs across lineages but also showed when and where the model diverges from the natural program. Our findings demonstrate an extraordinary plasticity of ESCs to self-organize and generate a whole-embryo-like structure.

Embryo model completes gastrulation to neurulation and organogenesis.

Embryonic stem (ES) cells can undergo many aspects of mammalian embryogenesis in vitro1-5, but their developmental potential is substantially extended by interactions with extraembryonic stem cells, including trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells and inducible XEN (iXEN) cells6-11. Here we assembled stem cell-derived embryos in vitro from mouse ES cells, TS cells and iXEN cells and showed that they recapitulate the development of whole natural mouse embryo in utero up to day 8.5 post-fertilization. Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells. This complete embryo model develops within an extraembryonic yolk sac that initiates blood island development. Notably, we demonstrate that the neurulating embryo model assembled from Pax6-knockout ES cells aggregated with wild-type TS cells and iXEN cells recapitulates the ventral domain expansion of the neural tube that occurs in natural, ubiquitous Pax6-knockout embryos. Thus, these complete embryoids are a powerful in vitro model for dissecting the roles of diverse cell lineages and genes in development. Our results demonstrate the self-organization ability of ES cells and two types of extraembryonic stem cells to reconstitute mammalian development through and beyond gastrulation to neurulation and early organogenesis.

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.

Basement membrane remodelling regulates mouse embryogenesis.

Tissue sculpting during development has been attributed mainly to cellular events through processes such as convergent extension or apical constriction1,2. However, recent work has revealed roles for basement membrane remodelling in global tissue morphogenesis3-5. Upon implantation, the epiblast and extraembryonic ectoderm of the mouse embryo become enveloped by a basement membrane. Signalling between the basement membrane and these tissues is critical for cell polarization and the ensuing morphogenesis6,7. However, the mechanical role of the basement membrane in post-implantation embryogenesis remains unknown. Here we demonstrate the importance of spatiotemporally regulated basement membrane remodelling during early embryonic development. Specifically, we show that Nodal signalling directs the generation and dynamic distribution of perforations in the basement membrane by regulating the expression of matrix metalloproteinases. This basement membrane remodelling facilitates embryo growth before gastrulation. The establishment of the anterior-posterior axis8,9 further regulates basement membrane remodelling by localizing Nodal signalling-and therefore the activity of matrix metalloproteinases and basement membrane perforations-to the posterior side of the embryo. Perforations on the posterior side are essential for primitive-streak extension during gastrulation by rendering the basement membrane of the prospective primitive streak more prone to breaching. Thus spatiotemporally regulated basement membrane remodelling contributes to the coordination of embryo growth, morphogenesis and gastrulation.

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.

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

Embryonic stem cells can be incorporated into the developing embryo and its germ line, but, when cultured alone, their ability to generate embryonic structures is restricted. They can interact with trophoblast stem cells to generate structures that break symmetry and specify mesoderm, but their development is limited as the epithelial-mesenchymal transition of gastrulation cannot occur. Here, we describe a system that allows assembly of mouse embryonic, trophoblast and extra-embryonic endoderm stem cells into structures that acquire the embryo's architecture with all distinct embryonic and extra-embryonic compartments. Strikingly, such embryo-like structures develop to undertake the epithelial-mesenchymal transition, leading to mesoderm and then definitive endoderm specification. Spatial transcriptomic analyses demonstrate that these morphological transformations are underpinned by gene expression patterns characteristic of gastrulating embryos. This demonstrates the remarkable ability of three stem cell types to self-assemble in vitro into gastrulating embryo-like structures undertaking spatio-temporal events of the gastrulating mammalian embryo.

A Smaug2-Based Translational Repression Complex Determines the Balance between Precursor Maintenance versus Differentiation during Mammalian Neurogenesis.

Here, we have asked about post-transcriptional mechanisms regulating murine developmental neurogenesis, focusing upon the RNA-binding proteins Smaug2 and Nanos1. We identify, in embryonic neural precursors of the murine cortex, a Smaug2 protein/nanos1 mRNA complex that is present in cytoplasmic granules with the translational repression proteins Dcp1 and 4E-T. We show that Smaug2 inhibits and Nanos1 promotes neurogenesis, with Smaug2 knockdown enhancing neurogenesis and depleting precursors, and Nanos1 knockdown inhibiting neurogenesis and maintaining precursors. Moreover, we show that Smaug2 likely regulates neurogenesis by silencing nanos1 mRNA. Specifically, Smaug2 knockdown inappropriately increases Nanos1 protein, and the Smaug2 knockdown-mediated neurogenesis is rescued by preventing this increase. Thus, Smaug2 and Nanos1 function as a bimodal translational repression switch to control neurogenesis, with Smaug2 acting in transcriptionally primed precursors to silence mRNAs important for neurogenesis, including nanos1 mRNA, and Nanos1 acting during the transition to neurons to repress the precursor state. SIGNIFICANCE STATEMENT: The mechanisms instructing neural stem cells to generate the appropriate progeny are still poorly understood. Here, we show that the RNA-binding proteins Smaug2 and Nanos1 are critical regulators of this balance and provide evidence supporting the idea that neural precursors are transcriptionally primed to generate neurons but translational regulation maintains these precursors in a stem cell state until the appropriate developmental time.

An asymmetrically localized Staufen2-dependent RNA complex regulates maintenance of mammalian neural stem cells.

The cellular mechanisms that regulate self-renewal versus differentiation of mammalian somatic tissue stem cells are still largely unknown. Here, we asked whether an RNA complex regulates this process in mammalian neural stem cells. We show that the RNA-binding protein Staufen2 (Stau2) is apically localized in radial glial precursors of the embryonic cortex, where it forms a complex with other RNA granule proteins including Pumilio2 (Pum2) and DDX1, and the mRNAs for ß-actin and mammalian prospero, prox1. Perturbation of this complex by functional knockdown of Stau2, Pum2, or DDX1 causes premature differentiation of radial glial precursors into neurons and mislocalization and misexpression of prox1 mRNA. Thus, a Stau2- and Pum2-dependent RNA complex directly regulates localization and, potentially, expression of target mRNAs like prox1 in mammalian neural stem cells, and in so doing regulates the balance of stem cell maintenance versus differentiation.

Quantification of character-impacting compounds in Ocimum basilicum and 'Pesto alla Genovese' with selected ion flow tube mass spectrometry.

Basil (Ocimum basilicum) is an important flavourant plant which constitutes the major ingredient of the pasta sauce 'Pesto alla Genovese'. The characteristic smell of basil stems mainly from a handful of terpenoids (methyl cinnamate, eucalyptol, linalool and estragole), the concentration of which varies according to basil cultivars. The simple and rapid analysis of the terpenoid constituents of basil would be useful as a means to optimise harvesting times and to act as a quality control process for basil-containing foodstuffs. Classical analytical techniques such as gas chromatography/mass spectrometry (GC/MS) are, however, slow, technically demanding and therefore less suitable for routine analysis. A new chemical ionisation technique which allows real-time quantification of traces gases, Selected Ion Flow Tube Mass Spectrometry (SIFT-MS), was therefore utilised to determine its usefulness for the assay of terpenoid concentrations in basil and pesto sauce headspace. Trace gas analysis was performed using the NO(+) precursor ion which minimised interference from other compounds. Character-impacting compound concentration was measured in basil headspace with good reproducibility and statistically significant differences were observed between cultivars. Quantification of linalool in pesto sauce headspace proved more difficult due to the presence of interfering compounds. This was resolved by careful selection of reaction product ions which allowed us to detect differences between various commercial brands of pesto. We conclude that SIFT-MS may be a valid tool for the fast and reproducible analysis of flavourant terpenoids in basil and basil-derived foodstuffs.

The reactions of a series of terpenoids with H(3) O(+) , NO(+) and O 2+ studied using selected ion flow tube mass spectrometry.

The reactions of H(3) O(+) , NO(+) and O 2+ with twelve terpenoids and one terpene, all of which occur naturally in plants and which possess important smell and flavourant properties, were characterized using Selected Ion Flow Tube Mass Spectrometry (SIFT-MS). The H(3) O(+) reactions resulted primarily in the formation of the proton transfer product and occasionally in a water elimination product. The NO(+) reactions instead generated the charge transfer product or NO(+) adducts, and occasionally alkyl fragments, or resulted in hydride abstraction. Reaction with O 2+ caused a higher fragmentation of the terpenoids with the molecular ion being the minor product of most reactions. Identification and quantification of each compound in complex mixtures are probably possible in most cases using the H(3) O(+) and/or NO(+) precursors while O 2+ may be useful for isomer discrimination. Our data suggests that SIFT-MS may be a useful tool for the rapid analysis of these compounds in plants and derived foodstuffs.