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 (GRNs) that coordinate transcription in space and time. While 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 murine Embryonic Stem cells (ESC) stimulated to undergo gastrulation-like events and elongation in vitro, are capable of organising a post-occipital pattern of neural, mesodermal and endodermal derivatives that mimic the embryonic spatial and temporal gene expression. The establishment of the three major body axes in such '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 Hox collinear transcriptional patterns along an extending anteriorposterior axis. These results reveal an unanticipated self-organising capacity for aggregated ESC and suggest that gastruloids may be used as a complementary system to study early developmental events in the mammalian embryo.Recent work on stem cell derived organoids has revealed a surprising autonomy in the development of particular tissues and organs 4,5 . When ca. 250 ESCs are aggregated, given a pulse of the Wnt agonist CHIR99021 (Chi) between 48 and 72h of culture, and returned to N2B27 medium ( Fig. 1a), a pole of T/Brachyury (Bra) expression, resembling the elongating embryonic tail bud emerges reproducibly 6 ( Fig. 1b, Extended data Fig. 1). The aggregates keep elongating up to 120h after aggregation (AA), when they display a 'rostral' cell-dense region and a polar extension towards a 'caudal' extremity reaching up to 500µm in size ( Fig. 1b). Shaking the culture allows to reach 850-1000 micron in length at 168hrs AA ( Fig. 1c,d). At these late stages, a Gata6-positive domain is detected at the opposite side of a Bra and Cdx2 expressing region, likely corresponding to the cardiac crescent, which delimits the embryonic post-occipital region 7 ( Fig. 1b-d, Extended data Fig.1, Supplementary Movies 1 and 2). In contrast, Sox1/Sox2 positive cells localised centrally, with the exception of the rostral-most portion ( Fig. 1c, d).To characterize the transcriptional programmes of these gastruloids, we carried out RNAseq on duplicated pools and compared their profiles with those of developing mouse embryos from E6.5 to E9.5. Since gastruloids display hallmarks of post-occipital embryos 6 ( Fig.1b-d) we excluded the anterior portion of E7.5-E9.5 embryos (Fig. 1e, top). Principal Component Analysis (PCA) showed reproducibility between samples and a clear clustering along PC1 corresponding to the temporal 3 order of samples ( Fig. 1e), while embryo samples segregated from gastruloids in PC2 only. The main (top 100) clust...

Waddington's epigenetic landscape is an abstract metaphor frequently used to represent the relationship between gene activity and cell fates during development. Over the past few years, it has become a useful framework for interpreting results from single-cell transcriptomics experiments. It has led to the proposal that, during fate transitions, cells experience smooth, continuous progressions of global transcriptional activity, which can be captured by (pseudo)temporal dynamics. Here, focusing strictly on the fate decision events, we suggest an alternative view: that fate transitions occur in a discontinuous, stochastic manner whereby signals modulate the probability of the transition events.

The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues ordered with reference to three orthogonal axes 1 . While model organisms have provided much insight into this process, we know very little about gastrulation in humans due to the difficulty of obtaining embryos at such early stages of development, as well as to the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo 2 . Here we show that human embryonic stem cells can be used to generate gastruloids: three dimensional multicellular aggregates that differentiate to derivatives of the three germ layers organised spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis and, using spatial transcriptomics, we show that they exhibit patterned gene expression. This includes a somitogenesis signature that suggests that 72 hour human gastruloids exhibit features of Carnegie Stage 9 embryos 3 . Our study represents a new, experimentally tractable model system to reveal and probe human-specific regulatory processes occurring during axial organisation in early development.The body plan of mammalian embryos emerges through interactions of sequential cell fate decisions and morphogenetic events, which have hitherto been difficult to observe in humans.Human Embryonic Stem Cells (hESCs) 4 have opened up opportunities for studying early fate decisions, and have hinted at the existence of regulatory mechanisms specific to humans 5,6 . But, in contrast to the embryo, where proportionate populations interact with one another to generate tissues and organs, differentiation in adherent culture is heterogeneous and favours a limited number of cell types 7 . Seeding hESCs on micropatterned surfaces yields coordinated patterns of gene expression, but without the axial organization characteristic of embryos 8 . However, when mouse ESCs are aggregated in suspension under defined conditions, they generate 'gastruloids': a three-dimensional, in vitro model of mammalian development, which exhibits an embryo-like spatiotemporal organization of gene expression 9,10 . We hypothesised that similar human gastruloids could be derived from hESCs. Generation of human gastruloidsWhen hESCs in 2D culture were treated with Chiron, a Wnt agonist, for one day before seeding defined numbers in low-adherence plates in the presence of Chiron, they formed compact, spherical aggregates within a few hours (Fig. 1a, Extended Data Fig. 1a-c). These aggregates progressively broke symmetry and formed elongated structures, with maximal elongation at 72-96h (Fig. 1a-d). On average, ~66% of aggregates from each experiment displayed an elongated morphology at 72h. Although some of the structures remained 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. and A.v.O.. This w...

Gastruloids are three-dimensional aggregates of embryonic stem cells (ESCs) that display key features of mammalian post-implantation development, including germ layer specification and axial organization 1-3. So far, the expression pattern of only a small number of genes in gastruloids has been explored with microscopy, but it is still unclear to what extent genome-wide expression patterns mimic those in embryos. Here, we compared mouse gastruloids with mouse embryos using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (tomo-seq). We

Cell fate transitions in mammalian stem cell systems have often been associated with transcriptional heterogeneity; however, existing data have failed to establish a functional or mechanistic link between the two phenomena. Experiments in unicellular organisms support the notion that transcriptional heterogeneity can be used to facilitate adaptability to environmental changes and have identified conserved chromatin‐associated factors that modulate levels of transcriptional noise. Herein, we show destabilization of pluripotency‐associated gene regulatory networks through increased transcriptional heterogeneity of mouse embryonic stem cells in which paradigmatic histone acetyl‐transferase, and candidate noise modulator, Kat2a (yeast orthologue Gcn5), have been inhibited. Functionally, network destabilization associates with reduced pluripotency and accelerated mesendodermal differentiation, with increased probability of transitions into lineage commitment. Thus, we show evidence of a relationship between transcriptional heterogeneity and cell fate transitions through manipulation of the histone acetylation landscape of mouse embryonic stem cells, suggesting a general principle that could be exploited in other normal and malignant stem cell fate transitions. stem cells 2018;36:1828–11