High-throughput total RNA sequencing in single cells using VASA-seq.

Most methods for single-cell transcriptome sequencing amplify the termini of polyadenylated transcripts, capturing only a small fraction of the total cellular transcriptome. This precludes the detection of many long non-coding, short non-coding and non-polyadenylated protein-coding transcripts and hinders alternative splicing analysis. We, therefore, developed VASA-seq to detect the total transcriptome in single cells, which is enabled by fragmenting and tailing all RNA molecules subsequent to cell lysis. The method is compatible with both plate-based formats and droplet microfluidics. We applied VASA-seq to more than 30,000 single cells in the developing mouse embryo during gastrulation and early organogenesis. Analyzing the dynamics of the total single-cell transcriptome, we discovered cell type markers, many based on non-coding RNA, and performed in vivo cell cycle analysis via detection of non-polyadenylated histone genes. RNA velocity characterization was improved, accurately retracing blood maturation trajectories. Moreover, our VASA-seq data provide a comprehensive analysis of alternative splicing during mammalian development, which highlighted substantial rearrangements during blood development and heart morphogenesis.

Gastruloids: Pluripotent stem cell models of mammalian gastrulation and embryo engineering.

Gastrulation is a fundamental and critical process of animal development whereby the mass of cells that results from the proliferation of the zygote transforms itself into a recognizable outline of an organism. The last few years have seen the emergence of a number of experimental models of early mammalian embryogenesis based on Embryonic Stem (ES) cells. One of this is the Gastruloid model. Gastruloids are aggregates of defined numbers of ES cells that, under defined culture conditions, undergo controlled proliferation, symmetry breaking, and the specification of all three germ layers characteristic of vertebrate embryos, and their derivatives. However, they lack brain structures and, surprisingly, reveal a disconnect between cell type specific gene expression and tissue morphogenesis, for example during somitogenesis. Gastruloids have been derived from mouse and human ES cells and several variations of the original model have emerged that reveal a hereto unknown modularity of mammalian embryos. We discuss the organization and development of gastruloids in the context of the embryonic stages that they represent, pointing out similarities and differences between the two. We also point out their potential as a reproducible, scalable and searchable experimental system and highlight some questions posed by the current menagerie of gastruloids.

Establishment of the vertebrate body plan: Rethinking gastrulation through stem cell models of early embryogenesis.

A striking property of vertebrate embryos is the emergence of a conserved body plan across a wide range of organisms through the process of gastrulation. As the body plan unfolds, gene regulatory networks (GRNs) and multicellular interactions (cell regulatory networks, CRNs) combine to generate a conserved set of morphogenetic events that lead to the phylotypic stage. Interrogation of these multilevel interactions requires manipulation of the mechanical environment, which is difficult in vivo. We review recent studies of stem cell models of early embryogenesis from different species showing that, independent of species origin, cells in culture form similar structures. The main difference between embryos and in vitro models is the boundary conditions of the multicellular ensembles. We discuss these observations and suggest that the mechanical and geometric boundary conditions of different embryos before gastrulation hide a morphogenetic ground state that is revealed in the stem-cell-based models of embryo development.

In vitro teratogenicity testing using a 3D, embryo-like gastruloid system.

Pharmaceuticals intended for use in patients of childbearing potential need to be tested for teratogenicity before marketing. Several pharmaceutical companies use animal-free in vitro models which allow a more rapid selection of lead compounds and contribute to 3Rs principles ('replace, reduce and refine') by streamlining the selection of promising compounds submitted to further regulatory studies in animals. Currently available in vitro models typically rely on adherent monolayer cultures or disorganized 3D structures, both of which lack the spatiotemporal and morphological context of the developing embryo. A newly developed 3D 'gastruloid' model has the potential to achieve a more reliable prediction of teratogenicity by providing a robust recapitulation of gastrulation-like events alongside morphological coordination at relatively high-throughput. In this first proof-of-concept study, we used both mouse and human gastruloids to examine a panel of seven reference compounds, with associated in vivo data and known teratogenic risk, to quantitatively assess in vitro teratogenicity. We observed several gross morphological effects, including significantly reduced elongation or decreased size of the gastruloids, upon exposure to several of the reference compounds. We also observed aberrant gene expression using fluorescent reporters, including SOX2, BRA, and SOX17, suggestive of multi-lineage differentiation defects and disrupted axial patterning. Finally, we saw that gastruloids recapitulated some of the known in vivo species-specific susceptibilities between their mouse and human counterparts. We therefore suggest that gastruloids represent a powerful tool for teratogenicity assessment by enabling relevant physiological recapitulation of early embryonic development, demonstrating their use as a novel in vitro teratogenic model system.

Bioengineered embryoids mimic post-implantation development in vitro.

The difficulty of studying post-implantation development in mammals has sparked a flurry of activity to develop in vitro models, termed embryoids, based on self-organizing pluripotent stem cells. Previous approaches to derive embryoids either lack the physiological morphology and signaling interactions, or are unconducive to model post-gastrulation development. Here, we report a bioengineering-inspired approach aimed at addressing this gap. We employ a high-throughput cell aggregation approach to simultaneously coax mouse embryonic stem cells into hundreds of uniform epiblast-like aggregates in a solid matrix-free manner. When co-cultured with mouse trophoblast stem cell aggregates, the resulting hybrid structures initiate gastrulation-like events and undergo axial morphogenesis to yield structures, termed EpiTS embryoids, with a pronounced anterior development, including brain-like regions. We identify the presence of an epithelium in EPI aggregates as the major determinant for the axial morphogenesis and anterior development seen in EpiTS embryoids. Our results demonstrate the potential of EpiTS embryoids to study peri-gastrulation development in vitro.

Axis Specification in Zebrafish Is Robust to Cell Mixing and Reveals a Regulation of Pattern Formation by Morphogenesis.

A fundamental question in developmental biology is how the early embryo establishes the spatial coordinate system that is later important for the organization of the embryonic body plan. Although we know a lot about the signaling and gene-regulatory networks required for this process, much less is understood about how these can operate to pattern tissues in the context of the extensive cell movements that drive gastrulation. In zebrafish, germ layer specification depends on the inheritance of maternal mRNAs [1-3], cortical rotation to generate a dorsal pole of ß-catenin activity [4-8], and the release of Nodal signals from the yolk syncytial layer (YSL) [9-12]. To determine whether germ layer specification is robust to altered cell-to-cell positioning, we separated embryonic cells from the yolk and allowed them to develop as spherical aggregates. These aggregates break symmetry autonomously to form elongated structures with an anterior-posterior pattern. Both forced reaggregation and endogenous cell mixing reveals how robust early axis specification is to spatial disruption of maternal pre-patterning. During these movements, a pole of Nodal signaling emerges that is required for explant elongation via the planar cell polarity (PCP) pathway. Blocking of PCP-dependent elongation disrupts the shaping of opposing poles of BMP and Wnt/TCF activity and the anterior-posterior patterning of neural tissue. These results lead us to suggest that embryo elongation plays a causal role in timing the exposure of cells to changes in BMP and Wnt signal activity during zebrafish gastrulation. VIDEO ABSTRACT.

Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids.

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 processes1, 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 antero-posterior 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.

A Sprouty4 reporter to monitor FGF/ERK signaling activity in ESCs and mice.

The FGF/ERK signaling pathway is highly conserved throughout evolution and plays fundamental roles during embryonic development and in adult organisms. While a plethora of expression data exists for ligands, receptors and pathway regulators, we know little about the spatial organization or dynamics of signaling in individual cells within populations. To this end we developed a transcriptional readout of FGF/ERK activity by targeting a histone H2B-linked Venus fluorophore to the endogenous locus of Spry4, an early pathway target, and generated Spry4H2B-Venus embryonic stem cells (ESCs) and a derivative mouse line. The Spry4H2B-Venus reporter was heterogeneously expressed within ESC cultures and responded to FGF/ERK signaling manipulation. In vivo, the Spry4H2B-Venus reporter recapitulated the expression pattern of Spry4 and localized to sites of known FGF/ERK activity including the inner cell mass of the pre-implantation embryo and the limb buds, somites and isthmus of the post-implantation embryo. Additionally, we observed highly localized reporter expression within adult organs. Genetic and chemical disruption of FGF/ERK signaling, in vivo in pre- and post-implantation embryos, abrogated Venus expression establishing the reporter as an accurate signaling readout. This tool will provide new insights into the dynamics of the FGF/ERK signaling pathway during mammalian development.

Anteroposterior polarity and elongation in the absence of extra-embryonic tissues and of spatially localised signalling in gastruloids: mammalian embryonic organoids.

The establishment of the anteroposterior (AP) axis is a crucial step during animal embryo development. In mammals, genetic studies have shown that this process relies on signals spatiotemporally deployed in the extra-embryonic tissues that locate the position of the head and the onset of gastrulation, marked by T/Brachyury (T/Bra) at the posterior of the embryo. Here, we use gastruloids, mESC-based organoids, as a model system with which to study this process. We find that gastruloids localise T/Bra expression to one end and undergo elongation similar to the posterior region of the embryo, suggesting that they develop an AP axis. This process relies on precisely timed interactions between Wnt/ß-catenin and Nodal signalling, whereas BMP signalling is dispensable. Additionally, polarised T/Bra expression occurs in the absence of extra-embryonic tissues or localised sources of signals. We suggest that the role of extra-embryonic tissues in the mammalian embryo might not be to induce the axes but to bias an intrinsic ability of the embryo to initially break symmetry. Furthermore, we suggest that Wnt signalling has a separable activity involved in the elongation of the axis.

Transition states and cell fate decisions in epigenetic landscapes.

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.

Single-Cell Approaches: Pandora's Box of Developmental Mechanisms.

Single-cell approaches are providing a new lexicon of developmental cell biology by revealing heterogeneities in seemingly uniform cellular populations. By bridging scales, single-cell approaches should, in principle, galvanize our understanding of how individual cells adopt distinct fates as they build complex tissues.

Wnt/ß-catenin signalling and the dynamics of fate decisions in early mouse embryos and embryonic stem (ES) cells.

Wnt/ß-catenin signalling is a widespread cell signalling pathway with multiple roles during vertebrate development. In mouse embryonic stem (mES) cells, there is a dual role for ß-catenin: it promotes differentiation when activated as part of the Wnt/ß-catenin signalling pathway, and promotes stable pluripotency independently of signalling. Although mES cells resemble the preimplantation epiblast progenitors, the first requirement for Wnt/ß-catenin signalling during mouse development has been reported at implantation [1,2]. The relationship between ß-catenin and pluripotency and that of mES cells with epiblast progenitors suggests that ß-catenin might have a functional role during preimplantation development. Here we summarize the expression and function of Wnt/ß-catenin signalling elements during the early stages of mouse development and consider the reasons why the requirement in ES cells do not reflect the embryo.

Determining protein subcellular localization in mammalian cell culture with biochemical fractionation and iTRAQ 8-plex quantification.

Protein subcellular localization is a fundamental feature of posttranslational functional regulation. Traditional microscopy based approaches to study protein localization are typically of limited throughput, and dependent on the availability of antibodies with high specificity and sensitivity, or fluorescent fusion proteins. In this chapter we describe how Localization of Organelle Proteins by Isotope Tagging (LOPIT), a mass spectrometry based workflow coupling biochemical fractionation and iTRAQ™ 8-plex quantification, can be applied for the high-throughput characterization of protein localization in a mammalian cell culture line.

Contractile and mechanical properties of epithelia with perturbed actomyosin dynamics.

Mechanics has an important role during morphogenesis, both in the generation of forces driving cell shape changes and in determining the effective material properties of cells and tissues. Drosophila dorsal closure has emerged as a reference model system for investigating the interplay between tissue mechanics and cellular activity. During dorsal closure, the amnioserosa generates one of the major forces that drive closure through the apical contraction of its constituent cells. We combined quantitation of live data, genetic and mechanical perturbation and cell biology, to investigate how mechanical properties and contraction rate emerge from cytoskeletal activity. We found that a decrease in Myosin phosphorylation induces a fluidization of amnioserosa cells which become more compliant. Conversely, an increase in Myosin phosphorylation and an increase in actin linear polymerization induce a solidification of cells. Contrary to expectation, these two perturbations have an opposite effect on the strain rate of cells during DC. While an increase in actin polymerization increases the contraction rate of amnioserosa cells, an increase in Myosin phosphorylation gives rise to cells that contract very slowly. The quantification of how the perturbation induced by laser ablation decays throughout the tissue revealed that the tissue in these two mutant backgrounds reacts very differently. We suggest that the differences in the strain rate of cells in situations where Myosin activity or actin polymerization is increased arise from changes in how the contractile forces are transmitted and coordinated across the tissue through ECadherin-mediated adhesion. Altogether, our results show that there is an optimal level of Myosin activity to generate efficient contraction and suggest that the architecture of the actin cytoskeleton and the dynamics of adhesion complexes are important parameters for the emergence of coordinated activity throughout the tissue.

A membrane-associated ß-catenin/Oct4 complex correlates with ground-state pluripotency in mouse embryonic stem cells.

The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/ß-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of ß-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that ß-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of ß-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/ß-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that ß-catenin, but not its transcriptional activity, is central to pluripotency acting through a ß-catenin/Oct4 complex.

Wnt-Notch signalling: an integrated mechanism regulating transitions between cell states.

The activity of Wnt and Notch signalling is central to many cell fate decisions during development and to the maintenance and differentiation of stem cell populations in homeostasis. While classical views refer to these pathways as independent signal transduction devices that co-operate in different systems, recent work has revealed intricate connections between their components. These observations suggest that rather than operating as two separate pathways, elements of Wnt and Notch signalling configure an integrated molecular device whose main function is to regulate transitions between cell states in development and homeostasis. Here, we propose a general framework for the structure and function of the interactions between these signalling systems that is focused on the notion of 'transition states', i.e. intermediates that arise during cell fate decision processes. These intermediates act as checkpoints in cell fate decision processes and are characterised by the mixed molecular identities of the states involved in these processes.

Modulation of the ligand-independent traffic of Notch by Axin and Apc contributes to the activation of Armadillo in Drosophila.

There is increasing evidence for close functional interactions between Wnt and Notch signalling. In many instances, these are mediated by convergence of the signalling events on common transcriptional targets, but there are other instances that cannot be accounted for in this manner. Studies in Drosophila have revealed that an activated form of Armadillo, the effector of Wnt signalling, interacts with, and is modulated by, the Notch receptor. Specifically, the ligand-independent traffic of Notch serves to set up a threshold for the amount of this form of Armadillo and therefore for Wnt signalling. In the current model of Wnt signalling, a complex assembled around Axin and Apc allows GSK3 (Shaggy) to phosphorylate Armadillo and target it for degradation. However, genetic experiments suggest that the loss of function of any of these three elements does not have the same effect as elevating the activity of ß-catenin. Here, we show that Axin and Apc, but not GSK3, modulate the ligand-independent traffic of Notch. This finding helps to explain unexpected differences in the phenotypes obtained by different ways of activating Armadillo function and provides further support for the notion that Wnt and Notch signalling form a single functional module.

Wingless modulates the ligand independent traffic of Notch through Dishevelled.

Here we investigate the structural and functional basis of the interactions between Notch and Wingless signalling in Drosophila. Using yeast-two-hybrid and pull-down assays we show that Notch can bind directly a form of Dishevelled that is stabilized upon Wingless signalling. Moreover, we show that the mechanism by which Wingless signalling is able to downregulate Notch is by promoting its ligand-independent traffic to a compartment where it is degraded and that this activity depends on Dishevelled.

A role of receptor Notch in ligand cis-inhibition in Drosophila.

Notch and its ligands mediate short-range cell interactions that play a conserved role in inducing cell fate specification. Several regulatory mechanisms have been described to ensure robust polarized signaling from signal-sending to signal-receiving cells. High levels of ligand expression activate Notch in nearby cells and exert a cell-autonomous dominant-negative effect on Notch activity. This regulatory process is called cis-inhibition and helps to restrict Notch activation to signal-receiving cells. By combining genetic mosaics in the Drosophila wing primordium with cell culture assays, we present evidence here that Notch promotes the clearance of Serrate ligand from the cell surface and exerts an inhibitory effect on the activity of Serrate expressed in the same cell. These regulatory mechanisms are independent of Notch-mediated transcription and are executed by the extracellular domain of Notch. We show that this process is required to block Serrate-mediated activation of Notch in the signal-sending cell population and helps to restrict Notch activation to the signal-receiving cells. Altogether, our results, in concert with previous results on ligand-mediated Notch cis-inhibition, indicate that mutual inhibition between ligand and receptor in signal-sending cells helps to block Notch activity in these cells and to restrict receptor activation in signal-receiving cells.