Human neural tube morphogenesis in vitro by geometric constraints.

Understanding human organ formation is a scientific challenge with far-reaching medical implications1,2. Three-dimensional stem-cell cultures have provided insights into human cell differentiation3,4. However, current approaches use scaffold-free stem-cell aggregates, which develop non-reproducible tissue shapes and variable cell-fate patterns. This limits their capacity to recapitulate organ formation. Here we present a chip-based culture system that enables self-organization of micropatterned stem cells into precise three-dimensional cell-fate patterns and organ shapes. We use this system to recreate neural tube folding from human stem cells in a dish. Upon neural induction5,6, neural ectoderm folds into a millimetre-long neural tube covered with non-neural ectoderm. Folding occurs at 90% fidelity, and anatomically resembles the developing human neural tube. We find that neural and non-neural ectoderm are necessary and sufficient for folding morphogenesis. We identify two mechanisms drive folding: (1) apical contraction of neural ectoderm, and (2) basal adhesion mediated via extracellular matrix synthesis by non-neural ectoderm. Targeting these two mechanisms using drugs leads to morphological defects similar to neural tube defects. Finally, we show that neural tissue width determines neural tube shape, suggesting that morphology along the anterior-posterior axis depends on neural ectoderm geometry in addition to molecular gradients7. Our approach provides a new route to the study of human organ morphogenesis in health and disease.

Mesenchymal-epithelial transition regulates initiation of pluripotency exit before gastrulation.

The pluripotent epiblast gives rise to all tissues and organs in the adult body. Its differentiation starts at gastrulation, when the epiblast generates mesoderm and endoderm germ layers through epithelial-mesenchymal transition (EMT). Although gastrulation EMT coincides with loss of epiblast pluripotency, pluripotent cells in development and in vitro can adopt either mesenchymal or epithelial morphology. The relationship between epiblast cellular morphology and its pluripotency is not well understood. Here, using chicken epiblast and mammalian pluripotency stem cell (PSC) models, we show that PSCs undergo a mesenchymal-epithelial transition (MET) prior to EMT-associated pluripotency loss. Epiblast MET and its subsequent EMT are two distinct processes. The former, a partial MET, is associated with reversible initiation of pluripotency exit, whereas the latter, a full EMT, is associated with complete and irreversible pluripotency loss. We provide evidence that integrin-mediated cell-matrix interaction is a key player in pluripotency exit regulation. We propose that epiblast partial MET is an evolutionarily conserved process among all amniotic vertebrates and that epiblast pluripotency is restricted to an intermediate cellular state residing between the fully mesenchymal and fully epithelial states.

cytoNet: Spatiotemporal network analysis of cell communities.

We introduce cytoNet, a cloud-based tool to characterize cell populations from microscopy images. cytoNet quantifies spatial topology and functional relationships in cell communities using principles of network science. Capturing multicellular dynamics through graph features, cytoNet also evaluates the effect of cell-cell interactions on individual cell phenotypes. We demonstrate cytoNet's capabilities in four case studies: 1) characterizing the temporal dynamics of neural progenitor cell communities during neural differentiation, 2) identifying communities of pain-sensing neurons in vivo, 3) capturing the effect of cell community on endothelial cell morphology, and 4) investigating the effect of laminin α4 on perivascular niches in adipose tissue. The analytical framework introduced here can be used to study the dynamics of complex cell communities in a quantitative manner, leading to a deeper understanding of environmental effects on cellular behavior. The versatile, cloud-based format of cytoNet makes the image analysis framework accessible to researchers across domains.

A novel self-organizing embryonic stem cell system reveals signaling logic underlying the patterning of human ectoderm.

During development, the ectoderm is patterned by a combination of BMP and WNT signaling. Research in model organisms has provided substantial insight into this process; however, there are currently no systems in which to study ectodermal patterning in humans. Further, the complexity of neural plate border specification has made it difficult to transition from discovering the genes involved to deeper mechanistic understanding. Here, we develop an in vitro model of human ectodermal patterning, in which human embryonic stem cells self-organize to form robust and quantitatively reproducible patterns corresponding to the complete medial-lateral axis of the embryonic ectoderm. Using this platform, we show that the duration of endogenous WNT signaling is a crucial control parameter, and that cells sense relative levels of BMP and WNT signaling in making fate decisions. These insights allowed us to develop an improved protocol for placodal differentiation. Thus, our platform is a powerful tool for studying human ectoderm patterning and for improving directed differentiation protocols.This article has an associated 'The people behind the papers' interview.

Stem-cell-based embryo models for fundamental research and translation.

Despite its importance, understanding the early phases of human development has been limited by availability of human samples. The recent emergence of stem-cell-derived embryo models, a new field aiming to use stem cells to construct in vitro models to recapitulate snapshots of the development of the mammalian conceptus, opens up exciting opportunities to promote fundamental understanding of human development and advance reproductive and regenerative medicine. This Review provides a summary of the current knowledge of early mammalian development, using mouse and human conceptuses as models, and emphasizes their similarities and critical differences. We then highlight existing embryo models that mimic different aspects of mouse and human development. We further discuss bioengineering tools used for controlling multicellular interactions and self-organization critical for the development of these models. We conclude with a discussion of the important next steps and exciting future opportunities of stem-cell-derived embryo models for fundamental discovery and translation.

Dissecting the dynamics of signaling events in the BMP, WNT, and NODAL cascade during self-organized fate patterning in human gastruloids.

During gastrulation, the pluripotent epiblast self-organizes into the 3 germ layers-endoderm, mesoderm and ectoderm, which eventually form the entire embryo. Decades of research in the mouse embryo have revealed that a signaling cascade involving the Bone Morphogenic Protein (BMP), WNT, and NODAL pathways is necessary for gastrulation. In vivo, WNT and NODAL ligands are expressed near the site of gastrulation in the posterior of the embryo, and knockout of these ligands leads to a failure to gastrulate. These data have led to the prevailing view that a signaling gradient in WNT and NODAL underlies patterning during gastrulation; however, the activities of these pathways in space and time have never been directly observed. In this study, we quantify BMP, WNT, and NODAL signaling dynamics in an in vitro model of human gastrulation. Our data suggest that BMP signaling initiates waves of WNT and NODAL signaling activity that move toward the colony center at a constant rate. Using a simple mathematical model, we show that this wave-like behavior is inconsistent with a reaction-diffusion-based Turing system, indicating that there is no stable signaling gradient of WNT/NODAL. Instead, the final signaling state is homogeneous, and spatial differences arise only from boundary effects. We further show that the durations of WNT and NODAL signaling control mesoderm differentiation, while the duration of BMP signaling controls differentiation of CDX2-positive extra-embryonic cells. The identity of these extra-embryonic cells has been controversial, and we use RNA sequencing (RNA-seq) to obtain their transcriptomes and show that they closely resemble human trophoblast cells in vivo. The domain of BMP signaling is identical to the domain of differentiation of these trophoblast-like cells; however, neither WNT nor NODAL forms a spatial pattern that maps directly to the mesodermal region, suggesting that mesoderm differentiation is controlled dynamically by the combinatorial effect of multiple signals. We synthesize our data into a mathematical model that accurately recapitulates signaling dynamics and predicts cell fate patterning upon chemical and physical perturbations. Taken together, our study shows that the dynamics of signaling events in the BMP, WNT, and NODAL cascade in the absence of a stable signaling gradient control fate patterning of human gastruloids.

Quantifying cell transitions in C. elegans with data-fitted landscape models.

Increasing interest has emerged in new mathematical approaches that simplify the study of complex differentiation processes by formalizing Waddington's landscape metaphor. However, a rational method to build these landscape models remains an open problem. Here we study vulval development in C. elegans by developing a framework based on Catastrophe Theory (CT) and approximate Bayesian computation (ABC) to build data-fitted landscape models. We first identify the candidate qualitative landscapes, and then use CT to build the simplest model consistent with the data, which we quantitatively fit using ABC. The resulting model suggests that the underlying mechanism is a quantifiable two-step decision controlled by EGF and Notch-Delta signals, where a non-vulval/vulval decision is followed by a bistable transition to the two vulval states. This new model fits a broad set of data and makes several novel predictions.

Synergy with TGFß ligands switches WNT pathway dynamics from transient to sustained during human pluripotent cell differentiation.

WNT/ß-catenin signaling is crucial to all stages of life. It controls early morphogenetic events in embryos, maintains stem cell niches in adults, and is dysregulated in many types of cancer. Despite its ubiquity, little is known about the dynamics of signal transduction or whether it varies across contexts. Here we probe the dynamics of signaling by monitoring nuclear accumulation of ß-catenin, the primary transducer of canonical WNT signals, using quantitative live cell imaging. We show that ß-catenin signaling responds adaptively to constant WNT signaling in pluripotent stem cells, and that these dynamics become sustained on differentiation. Varying dynamics were also observed in the response to WNT in commonly used mammalian cell lines. Signal attenuation in pluripotent cells is observed even at saturating doses, where ligand stability does not affect the dynamics. TGFß superfamily ligands Activin and BMP, which coordinate with WNT signaling to pattern the gastrula, increase the ß-catenin response in a manner independent of their ability to induce new WNT ligand production. Our results reveal how variables external to the pathway, including differentiation status and cross-talk with other pathways, dramatically alter WNT/ß-catenin dynamics.

Roadmap for the multiscale coupling of biochemical and mechanical signals during development.

The way in which interactions between mechanics and biochemistry lead to the emergence of complex cell and tissue organization is an old question that has recently attracted renewed interest from biologists, physicists, mathematicians and computer scientists. Rapid advances in optical physics, microscopy and computational image analysis have greatly enhanced our ability to observe and quantify spatiotemporal patterns of signalling, force generation, deformation, and flow in living cells and tissues. Powerful new tools for genetic, biophysical and optogenetic manipulation are allowing us to perturb the underlying machinery that generates these patterns in increasingly sophisticated ways. Rapid advances in theory and computing have made it possible to construct predictive models that describe how cell and tissue organization and dynamics emerge from the local coupling of biochemistry and mechanics. Together, these advances have opened up a wealth of new opportunities to explore how mechanochemical patterning shapes organismal development. In this roadmap, we present a series of forward-looking case studies on mechanochemical patterning in development, written by scientists working at the interface between the physical and biological sciences, and covering a wide range of spatial and temporal scales, organisms, and modes of development. Together, these contributions highlight the many ways in which the dynamic coupling of mechanics and biochemistry shapes biological dynamics: from mechanoenzymes that sense force to tune their activity and motor output, to collectives of cells in tissues that flow and redistribute biochemical signals during development.

Conservation of Epithelial-to-Mesenchymal Transition Process in Neural Crest Cells and Metastatic Cancer.

Epithelial to mesenchymal transition (EMT) is a highly conserved cellular process in several species, from worms to humans. EMT plays a fundamental role in early embryogenesis, wound healing, and cancer metastasis. For neural crest cell (NCC) development, EMT typically results in forming a migratory and potent cell population that generates a wide variety of cell and tissue, including cartilage, bone, connective tissue, endocrine cells, neurons, and glia amongst many others. The degree of conservation between the signaling pathways that regulate EMT during development and metastatic cancer (MC) has not been fully established, despite ample studies. This systematic review and meta-analysis dissects the major signaling pathways involved in EMT of NCC development and MC to unravel the similarities and differences. While the FGF, TGFß/BMP, SHH, and NOTCH pathways have been rigorously investigated in both systems, the EGF, IGF, HIPPO, Factor Receptor Superfamily, and their intracellular signaling cascades need to be the focus of future NCC studies. In general, meta-analyses of the associated signaling pathways show a significant number of overlapping genes (particularly ligands, transcription regulators, and targeted cadherins) involved in each signaling pathway of both systems without stratification by body segments and cancer type. Lack of stratification makes it difficult to meaningfully evaluate the intracellular downstream effectors of each signaling pathway. Finally, pediatric neuroblastoma and melanoma are NCC-derived malignancies, which emphasize the importance of uncovering the EMT events that convert NCC into treatment-resistant malignant cells.

Reaction-diffusion models for morphological patterning of hESCs.

In this paper we consider mathematical modeling of the dynamics of self-organized patterning of spatially confined human embryonic stem cells (hESCs) treated with BMP4 (gastruloids) described in recent experimental works (Warmflash in Nat Methods 11:847-854, 2014; Chhabra in PloS Biol 17: 3000498, 2019). In the first part of the paper we use the activator-inhibitor equations of Gierer and Meinhardt to identify 3 reaction-diffusion regimes for each of the three morphogenic proteins, BMP4, Wnt and Nodal, based on the characteristic features of the dynamic patterning. We identify appropriate boundary conditions which correspond to the experimental setup and perform numerical simulations of the reaction-diffusion (RD) systems, using the finite element approximation, to confirm that the RD systems in these regimes produce realistic dynamics of the protein concentrations. In the second part of the paper we use analytic tools to address the questions of the existence and stability of non-homogeneous steady states for the reaction-diffusion systems of the type considered in the first part of the paper.

Morphogen and community effects determine cell fates in response to BMP4 signaling in human embryonic stem cells.

Paracrine signals maintain developmental states and create cell fate patterns in vivo and influence differentiation outcomes in human embryonic stem cells (hESCs) in vitro Systematic investigation of morphogen signaling is hampered by the difficulty of disentangling endogenous signaling from experimentally applied ligands. Here, we grow hESCs in micropatterned colonies of 1-8 cells ('µColonies') to quantitatively investigate paracrine signaling and the response to external stimuli. We examine BMP4-mediated differentiation in µColonies and standard culture conditions and find that in µColonies, above a threshold concentration, BMP4 gives rise to only a single cell fate, contrary to its role as a morphogen in other developmental systems. Under standard culture conditions BMP4 acts as a morphogen but this requires secondary signals and particular cell densities. We find that a 'community effect' enforces a common fate within µColonies, both in the state of pluripotency and when cells are differentiated, and that this effect allows a more precise response to external signals. Using live cell imaging to correlate signaling histories with cell fates, we demonstrate that interactions between neighbors result in sustained, homogenous signaling necessary for differentiation.

Coco is a dual activity modulator of TGFß signaling.

The TGFß signaling pathway is a crucial regulator of developmental processes and disease. The activity of TGFß ligands is modulated by various families of soluble inhibitors that interfere with the interactions between ligands and receptors. In an unbiased, genome-wide RNAi screen to identify genes involved in ligand-dependent signaling, we unexpectedly identified the BMP/Activin/Nodal inhibitor Coco as an enhancer of TGFß1 signaling. Coco synergizes with TGFß1 in both cell culture and Xenopus explants. Molecularly, Coco binds to TGFß1 and enhances TGFß1 binding to its receptor Alk5. Thus, Coco acts as both an inhibitor and an enhancer of signaling depending on the ligand it binds. This finding raises the need for a global reconsideration of the molecular mechanisms regulating TGFß signaling.

A method to recapitulate early embryonic spatial patterning in human embryonic stem cells.

Embryos allocate cells to the three germ layers in a spatially ordered sequence. Human embryonic stem cells (hESCs) can generate the three germ layers in culture; however, differentiation is typically heterogeneous and spatially disordered. We show that geometric confinement is sufficient to trigger self-organized patterning in hESCs. In response to BMP4, colonies reproducibly differentiated to an outer trophectoderm-like ring, an inner ectodermal circle and a ring of mesendoderm expressing primitive-streak markers in between. Fates were defined relative to the boundary with a fixed length scale: small colonies corresponded to the outer layers of larger ones. Inhibitory signals limited the range of BMP4 signaling to the colony edge and induced a gradient of Activin-Nodal signaling that patterned mesendodermal fates. These results demonstrate that the intrinsic tendency of stem cells to make patterns can be harnessed by controlling colony geometries and provide a quantitative assay for studying paracrine signaling in early development.

Pluripotent stem cells as a model for embryonic patterning: From signaling dynamics to spatial organization in a dish.

In vivo studies have identified the signaling pathways and transcription factors involved in patterning the vertebrate embryo, but much remains unknown about how these are organized in space and time to orchestrate embryogenesis. Recently, embryonic stem cells have been established as a platform for studying spatial pattern formation and differentiation dynamics in the early mammalian embryo. The ease of observing and manipulating stem cell systems promises to fill gaps in our understanding of developmental dynamics and identify aspects that are uniquely human. Developmental Dynamics 245:976-990, 2016. © 2016 Wiley Periodicals, Inc.

Dynamics of TGF-ß signaling reveal adaptive and pulsatile behaviors reflected in the nuclear localization of transcription factor Smad4.

The TGF-ß pathway plays a vital role in development and disease and regulates transcription through a complex composed of receptor-regulated Smads (R-Smads) and Smad4. Extensive biochemical and genetic studies argue that the pathway is activated through R-Smad phosphorylation; however, the dynamics of signaling remain largely unexplored. We monitored signaling and transcriptional dynamics and found that although R-Smads stably translocate to the nucleus under continuous pathway stimulation, transcription of direct targets is transient. Surprisingly, Smad4 nuclear localization is confined to short pulses that coincide with transcriptional activity. Upon perturbation, the dynamics of transcription correlate with Smad4 nuclear localization rather than with R-Smad activity. In Xenopus embryos, Smad4 shows stereotyped, uncorrelated bursts of nuclear localization, but activated R-Smads are uniform. Thus, R-Smads relay graded information about ligand levels that is integrated with intrinsic temporal control reflected in Smad4 into the active signaling complex.

SMAD7 directly converts human embryonic stem cells to telencephalic fate by a default mechanism.

Human embryonic stem cells (hESCs) provide a valuable window into the dissection of the molecular circuitry underlying the early formation of the human forebrain. However, dissection of signaling events in forebrain development using current protocols is complicated by non-neural contamination and fluctuation of extrinsic influences. Here, we show that SMAD7, a cell-intrinsic inhibitor of transforming growth factor-ß (TGFß) signaling, is sufficient to directly convert pluripotent hESCs to an anterior neural fate. Time course gene expression revealed downregulation of MAPK components, and combining MEK1/2 inhibition with SMAD7-mediated TGFß inhibition promoted telencephalic conversion. Fibroblast growth factor-MEK and TGFß-SMAD signaling maintain hESCs by promoting pluripotency genes and repressing neural genes. Our findings suggest that in the absence of these cues, pluripotent cells simply revert to a program of neural conversion. Hence, the "primed" state of hESCs requires inhibition of the "default" state of neural fate acquisition. This has parallels in amphibians, suggesting an evolutionarily conserved mechanism.

Endogenous Nodal switches Wnt interpretation from posteriorization to germ layer differentiation in geometrically constrained human pluripotent cells.

The Wnt pathway is essential for inducing the primitive streak, the precursor of the mesendoderm, as well as setting anterior-posterior coordinates. How Wnt coordinates these diverse activities remains incompletely understood. Here, we show that in Wnt-treated human pluripotent cells, endogenous Nodal signaling is a crucial switch between posteriorizing and primitive streak-including activities. While treatment with Wnt posteriorizes cells in standard culture, in micropatterned colonies, higher levels of endogenously induced Nodal signaling combine with exogenous Wnt to drive endoderm differentiation. Inhibition of Nodal signaling restores dose-dependent posteriorization by Wnt. In the absence of Nodal inhibition, micropatterned colonies undergo spontaneous, elaborate morphogenesis concomitant with endoderm differentiation even in the absence of added extracellular matrix proteins like Matrigel. Our study shows how Wnt and Nodal combinatorially coordinate germ layer differentiation with AP patterning and establishes a system to study a natural self-organizing morphogenetic event in in vitro culture.

Combinatorial interpretation of BMP and WNT controls the decision between primitive streak and extraembryonic fates.

BMP signaling is essential for mammalian gastrulation, as it initiates a cascade of signals that control self-organized patterning. As development is highly dynamic, it is crucial to understand how time-dependent combinatorial signaling affects cellular differentiation. Here, we show that BMP signaling duration is a crucial control parameter that determines cell fates upon the exit from pluripotency through its interplay with the induced secondary signal WNT. BMP signaling directly converts cells from pluripotent to extraembryonic fates while simultaneously upregulating Wnt signaling, which promotes primitive streak and mesodermal specification. Using live-cell imaging of signaling and cell fate reporters together with a simple mathematical model, we show that this circuit produces a temporal morphogen effect where, once BMP signal duration is above a threshold for differentiation, intermediate and long pulses of BMP signaling produce specification of mesoderm and extraembryonic fates, respectively. Our results provide a systems-level picture of how these signaling pathways control the landscape of early human development.

An incoherent regulatory network architecture that orchestrates B cell diversification in response to antigen signaling.

The B-lymphocyte lineage is a leading system for analyzing gene regulatory networks (GRNs) that orchestrate distinct cell fate transitions. Upon antigen recognition, B cells can diversify their immunoglobulin (Ig) repertoire via somatic hypermutation (SHM) and/or class switch DNA recombination (CSR) before differentiating into antibody-secreting plasma cells. We construct a mathematical model for a GRN underlying this developmental dynamic. The intensity of signaling through the Ig receptor is shown to control the bimodal expression of a pivotal transcription factor, IRF-4, which dictates B cell fate outcomes. Computational modeling coupled with experimental analysis supports a model of 'kinetic control', in which B cell developmental trajectories pass through an obligate transient state of variable duration that promotes diversification of the antibody repertoire by SHM/CSR in direct response to antigens. More generally, this network motif could be used to translate a morphogen gradient into developmental inductive events of varying time, thereby enabling the specification of distinct cell fates.