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Gastruloids are aggregates of mouse embryonic stem cells that can be used to study key aspects of mammalian post-implantation development in vitro1–4. Gastruloids generated with previously published protocols do not generate somite-like structures4–6. Here, we describe a modified version of the gastruloids culture protocol5,6 that results in gastruloids that do generate somite-like structures in vitro (van den Brink et al., Nature, 2020)7. Under these conditions, about 50% of the gastruloids generated form structures with features that are characteristic of somites7.This protocol takes 6 days, with relatively little hands-on time. The protocol starts with the aggregation of the cultured cells. Then, the Wnt-agonist Chiron is added 2 days (48h) later. The medium of the aggregates is replaced 3 days (72h) after aggregation. To induce somite-formation, gastruloids are embedded in Matrigel 4 days (96h) after aggregation. After 5 days (120h) of culture, gastruloids resemble E8.5 mouse embryos. At this timepoint they can be fixed (fixative is added on day 5 and washed away on day 6 after overnight incubation in PFA) to prepare them for staining or microscopy experiments.

Gastruloids are three-dimensional aggregates of embryonic stem cells that display key features of mammalian development after implantation, including germ-layer specification and axial organization1-3. To date, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, and the extent to which genome-wide expression patterns in gastruloids mimic those in embryos is unclear. Here we compare mouse gastruloids with mouse embryos using single-cell RNA sequencing and spatial transcriptomics. We identify various embryonic cell types that were not previously known to be present in gastruloids, and show that key regulators of somitogenesis are expressed similarly between embryos and gastruloids. Using live imaging, we show that the somitogenesis clock is active in gastruloids and has dynamics that resemble those in vivo. Because gastruloids can be grown in large quantities, we performed a small screen that revealed how reduced FGF signalling induces a short-tail phenotype in embryos. Finally, we demonstrate that embedding in Matrigel induces gastruloids to generate somites with the correct rostral-caudal patterning, which appear sequentially in an anterior-to-posterior direction over time. This study thus shows the power of gastruloids as a model system for exploring development and somitogenesis in vitro in a high-throughput manner. This work was supported by an European Research Council Advanced grant (ERC-AdG 742225-IntScOmics), a Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) TOP award (NWO-CW 714.016.001) and the Foundation for Fundamental Research on Matter, financially supported by NWO (FOM-14NOISE01) to S.C.v.d.B., A.A., V.v.B., M.B., J.V. and A.v.O., a Biotechnology and Biological Sciences Research Council (no. BB/P003184/1), Newton Trust (INT16.24b) and Medical Research Council (MR/R017190/1) grant to A.M.A., a Newnham College Cambridge Junior Research Fellowship to N.M. and a studentship from the Engineering and Physical Sciences Research Council to P.B.-J. The Cambridge Stem Cell Institute is supported by core funding from the Wellcome Trust and Medical Research Council; J.N. was funded by the University of Cambridge and K.F.S. by core funding from the Hubrecht Institute. This work is part of the Oncode Institute, which is partly financed by the Dutch Cancer Society. We thank A. Ebbing and M. Betist for the robotized tomo-seq protocol; G. Keller for the Brachyury-GFP cell line; J. Collignon for the Nodal-YFP line; K. Hadjantonakis for the TCF/LEF-mCherry line; S. van den Brink and E. R. Maandag for the E14-IB10 cells; J. Kress and A. Aulehla for the LfngT2AVenus mouse ES cell line; I. Misteli Guerreiro, J. Peterson-Maduro and J. Hoeksma for suggestions for in situ hybridization experiments; W. Thomas, Y. el Azhar, J. Juksar and J. Beumer for reagents and inhibitors; A. de Graaff and A. Stokkermans for help with multiphoton microscopy and analysis of the microscopy data; D. A. Turner for microscopy panels that were used for tomo-seq validation; J. Korving for help with the somite-size measurements in embryos; the Hubrecht FACS facility and R. van der Linden for FACS experiments; Single Cell Discoveries for 10x Genomics scRNA-seq; the Utrecht Sequencing facility for sequencing; and P. Zeller, H. Viñas Gaza, M. Vaninsberghe, V. Bhardwaj and all members of the van Oudenaarden, Sonnen and Martinez Arias laboratories for discussions.

During vertebrate embryogenesis, the body axis elongates posteriorly through a self renewing population of precursor cells in the tail bud. The Caudal Lateral Epiblast (CLE) of the mammalian embryo harbours a stem cell/progenitor zone of bipotent progenitors that contribute to both the elongating spinal cord and the paraxial mesoderm of the embryo. These progenitors, called the Neural Mesodermal Progenitors (NMPs), are characterized by the coexpression of Sox2 and T. A number of in vitro studies have been able to produce NMP-like (NMP-l) cells from Pluripotent Stem Cells (PSCs). However, in contrast with embryonic NMPs these in vitro derived ones do not self renew. Here we use different protocols for NMP-l cells and find that, in addition to NMPs, all produce cells with the potential to differentiate into Lateral Plate and Intermediate Mesoderm precursors. We show that Epiblast Stem Cells (EpiSCs) are a better starting source to produce NMPs and show that a specific differentiation protocol yields cells with the potential to self renew in vitro and to make large contributions in xenotransplant assay to axial elongation in both neural and mesodermal tissue. These cells are derived from a population that resembles the Caudal Epiblast (CE) of the embryo at the time of the appearance of the node and we show that a balance between Nodal and BMP signalling is important to define this population. Our study suggests that at the end of gastrulation and associated with the node, a multipotential progenitor population emerges that will give rise to the spinal cord and the mesodermal populations posterior to the brain.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Neuromesodermal progenitors (NMps) are a population of bipotent progenitors that maintain competence to generate both spinal cord and paraxial mesoderm throughout the elongation of the posterior body axis. Recent studies have generated populations of NMp-like cells in culture and have been shown to differentiate to both neural and mesodermal cell fates when transplanted into either mouse or chick embryos. Here, we aim to compare the potential of mouse embryonic stem (ES) cell-derived progenitor populations to generate NMp behavior against both undifferentiated and differentiated populations. We define NMp behaviour as the ability of cells to i) contribute to a significant proportion of the anterior-posterior body axis, ii) enter into both posterior neural and somitic compartments and, iii) retain a proportion of the progenitor population within the posterior growth zone. We compare previously identified ES cell-derived NMp-like populations to undifferentiated mouse ES cells and find that they all display similar potentials to generate NMp behaviourin vivo. To assess whether this competence is lost upon further differentiation, we generated anterior and posterior embryonic cell types through the generation of 3D gastruloids and show that NMp competence is lost within the anterior (Brachyury negative) portion of the gastruloid. Taken together, this demonstrates that the chick caudo-lateral epiblast is a highly permissive environment for testing NMp competence and is therefore not suitable as a positive test of neuromesodermal progenitor identity(ie. specification). However, it does act as an appropriate system to test for the loss of NMp potential, and therefore offers insight as a functional test for the regulation of NMp competencein vivo.

The emergence of multiple axes is an essential element in the establishment of the mammalian body plan. This process takes place shortly after implantation of the embryo within the uterus and relies on the activity of gene regulatory networks that coordinate transcription in space and time. Whereas genetic approaches have revealed important aspects of these processes(1), a mechanistic understanding is hampered by the poor experimental accessibility of early post-implantation stages. Here we show that small aggregates of mouse embryonic stem cells (ESCs), when stimulated to undergo gastrulation-like events and elongation in vitro, can organize a post-occipital pattern of neural, mesodermal and endodermal derivatives that mimic embryonic spatial and temporal gene expression. The establishment of the three major body axes in these 'gastruloids'(2,3) suggests that the mechanisms involved are interdependent. Specifically, gastruloids display the hallmarks of axial gene regulatory systems as exemplified by the implementation of collinear Hox transcriptional patterns along an extending anteroposterior axis. These results reveal an unanticipated self-organizing capacity of aggregated ESCs and suggest that gastruloids could be used as a complementary system to study early developmental events in the mammalian embryo.

Establishment of the three body axes is a critical step during animal development. In mammals, genetic studies have shown that a combination of precisely deployed signals from extraembryonic tissues position the anteroposterior axis (AP) within the embryo and lead to the emergence of the dorsoventral (DV) and left-right (LR) axes. We have used Gastruloids, embryonic organoids, as a model system to understand this process and find that they are able to develop AP, DV and LR axes as well as to undergo axial elongation in a manner that mirror embryos. The Gastruloids can be grown for 160 hours and form derivatives from ectoderm, mesoderm and endoderm. We focus on the AP axis and show that in the Gastruloids this axis is registered in the expression of T/Bra at one pole that corresponds to the tip of the elongation. We find that localisation of T/Bra expression depends on the combined activities of Wnt/β-Catenin and Nodal/Smad2,3 signalling, and that BMP signalling is dispensable for this process. Furthermore, AP axis specification occurs in the absence of both extraembryonic tissues and of localised sources of signalling. Our experiments show that Nodal, together with Wnt/β-Catenin signalling, is essential for the expression of T/Bra but that Wnt signalling has a separable activity in the elongation of the axis. The results lead us to suggest that, in the embryo, the role of the extraembryonic tissues might not be to induce the axes but to bias an intrinsic ability of the embryo to break its initial symmetry and organise its axes.One sentence summaryCulture of aggregates of defined number of Embryonic Stem cells leads to self-organised embryo-like structures which, in the absence of localised signalling from extra embryonic tissues and under the autonomous influence of Wnt and Nodal signalling, develop the three main axes of the body.

Mouse embryonic stem cells (mESCs) are clonal populations derived from preimplantation mouse embryos that can be propagated in vitro and, when placed into blastocysts, contribute to all tissues of the embryo and integrate into the normal morphogenetic processes, i.e. they are pluripotent. However, although they can be steered to differentiate in vitro into all cell types of the organism, they cannot organise themselves into structures that resemble embryos. When aggregated into embryoid bodies they develop disorganised masses of different cell types with little spatial coherence. An exception to this rule is the emergence of retinas and anterior cortex-like structures under minimal culture conditions. These structures emerge from the cultures without any axial organisation. Here, we report that small aggregates of mESCs, of about 300 cells, self-organise into polarised structures that exhibit collective behaviours reminiscent of those that cells exhibit in early mouse embryos, including symmetry breaking, axial organisation, germ layer specification and cell behaviour, as well as axis elongation. The responses are signal specific and uncouple processes that in the embryo are tightly associated, such as specification of the anteroposterior axis and anterior neural development, or endoderm specification and axial elongation. We discuss the meaning and implications of these observations and the potential uses of these structures which, because of their behaviour, we suggest to call ‘gastruloids’.

Generation of asymmetry within the early embryo is a critical step in the establishment of the three body axes, providing a reference for the patterning of the organism. To study the establishment of asymmetry and the development of the anteroposterior axis (AP) in culture, we utilised our ‘Gastruloid’ model system. ‘Gastruloids’, highly reproducible embryonic organoids formed from aggregates of mouse embryonic stem cells, display symmetry-breaking, polarised gene expression and axial development, mirroring the processes on a time-scale similar to that of the mouse embyro. UsingGastruloidsformed from mouse ESCs containing reporters for Wnt, FGF and Nodal signalling, we were able to quantitatively assess the contribution of these signalling pathways to the establishment of asymmetry through single time-point and live-cell fluorescence microscopy.During the first 24-48h of culture, interactions between the Wnt/β-Catenin and Nodal/TGF/βsignalling pathways promote the initial symmetry-breaking event, manifested through polarisedBrachyury(T/Bra) expression. Neither BMP nor FGF signalling is required for the establishment of asymmetry, however Wnt signalling is essential for the amplification and stability of the initial patterning event. Additionally, low, endogenous levels of FGF (24-48h) has a role in the amplification of the established pattern at later time-points.Our results confirm thatGastruloidsbehave like epiblast cells in the embryo, leading us to translate the processes and signalling involved in pattern formation ofGastruloidsin culture to the development of the embryo, firmly establishingGastruloidsas a highly reproducible, robust model system for studying cell fate decisions and early pattern formation in culture.

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/β-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

We have developed a protocol improving current Embryoid Body (EB) culture which allows the study of self-organization, symmetry breaking, axial elongation and cell fate specification using aggregates of mouse embryonic stem cells (mESCs) in suspension culture. Small numbers of mESCs are aggregated in basal medium for 48 hr in non-tissue-culture-treated, U-bottomed 96-well plates, after which they are competent to respond to experimental signals. Following treatment, these aggregates begin to show signs of polarized gene expression and gradually alter their morphology from a spherical mass of cells to an elongated, well organized structure in the absence of external asymmetry cues. These structures are not only able to display markers of the three germ layers, but actively display gastrulation-like movements, evidenced by a directional dislodgement of individual cells from the aggregate, which crucially occurs at one region of the elongated structure. This protocol provides a detailed method for the reproducible formation of these aggregates, their stimulation with signals such as Wnt/β-Catenin activation and BMP inhibition and their analysis by single time-point or time-lapse fluorescent microscopy. In addition, we describe modifications to current whole-mount mouse embryo staining procedures for immunocytochemical analysis of specific markers within fixed aggregates. The changes in morphology, gene expression and length of the aggregates can be quantitatively measured, providing information on how signals can alter axial fates. It is envisaged that this system can be applied both to the study of early developmental events such as axial development and organization, and more broadly, the processes of self-organization and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo that are unobtainable from conventional adherent culture such as spinal cord and motor neurones.

The development of the central nervous system is known to result from two sequential events. First, an inductive event of the mesoderm on the overlying ectoderm that generates a neural plate that, after rolling into a neural tube, acts as the main source of neural progenitors. Second, the axial regionalization of the neural plate that will result in the specification of neurons with different anteroposterior identities. Although this description of the process applies with ease to amphibians and fish, it is more difficult to confirm in amniote embryos. Here, a specialized population of cells emerges at the end of gastrulation that, under the influence of Wnt and FGF signalling, expands and generates the spinal cord and the paraxial mesoderm. This population is known as the long-term neuromesodermal precursor (NMp). Here, we show that controlled increases of Wnt/β-catenin and FGF signalling during adherent culture differentiation of mouse embryonic stem cells (mESCs) generates a population with many of the properties of the NMp. A single-cell analysis of gene expression within this population reveals signatures that are characteristic of stem cell populations. Furthermore, when this activation is triggered in three-dimensional aggregates of mESCs, the population self-organizes macroscopically and undergoes growth and axial elongation that mimics some of the features of the embryonic spinal cord and paraxial mesoderm. We use both adherent and three-dimensional cultures of mESCs to probe the establishment and maintenance of NMps and their differentiation.

Dissociated mouse embryonic stem (ES) cells were cultured to form aggregates in small volumes of basal medium in U-bottomed, non tissue-culture-treated 96-well plates and subsequently maintained in suspension culture. After growth for 48 hours, the aggregates are competent to respond to ubiquitous experimental signals which result in their symmetry-breaking and generation of defined polarised structures by 96 hours. It is envisaged that this system can be applied both to the study of early developmental events and more broadly to the processes of self-organisation and cellular decision-making. It may also provide a suitable niche for the generation of cell types present in the embryo but unobtainable from conventional adherent culture.