11 research outputs found
The Role and Control of WNT Signalling in an HESC Model of Human Primitive Streak
In amniotes, gastrulation is marked by the creation of the primitive streak (PS) and is largely controlled by WNT, BMP, and ACTIVIN/NODAL signalling. Despite detailed characterization in model organisms, the human PS and the role these pathways play in its formation and patterning remains a mystery. In this work I focused on understanding the role and control of the WNT pathway in human PS development. Due to the ethical limitations of working with human embryos, I used an in vitro human embryonic stem cell (hESC) micropatterned âgastruloidâ system. I first showed that in the human PS there is a conserved BMP â WNTâ NODAL signalling initiation hierarchy, and that WNT is necessary and sufficient for PS formation. Next, I found that structured subpopulations of endoderm and mesoderm emerge and self-organize depending on different BMP, WNT, and ACTIVIN/NODAL levels, and that by comparison to the mouse embryo I could arrange these subpopulations along an anterior-posterior axis. With the development of a new cell tracking technique, I was also able to identify and characterize robust cell migrations from the PS region of each gastruloid that depended on which fates the cells would ultimately adopt. Putting these pieces together, I was able to derive a rudimentary first fate map of the human PS, as well as a rough picture of the BMP, WNT, and ACTIVIN/NODAL signalling gradients that determine it. One interesting and unforeseen result from this fate map was the hint of a human âorganizerâ cell fate that emerged under joint WNT and ACTIVIN/NODAL stimulation. To characterize and functionally prove this organizerâs existence, I devised an ex ovo cross-species transplantation strategy grafting treated gastruloids into chick embryos. The assay demonstrated that the human cells induce and contribute autonomously to a secondary axis while inducing neural fate in the host, thus fulfilling the most stringent criteria for an organizer. This work adds an important milestone to the research program begun in 1924 with the first famous organizer experiment of Hilde Mangold and Hans Spemann, and the methods I developed have opened a door to new functional explorations and tests of early human development. Having learned more about the role of WNT in determining cell fates in the gastruloid model, I next endeavoured to understand how the spatial extent and duration of the WNT signal itself was controlled. With the use of various CRISPR/Cas9 knockout lines, I discovered that DKK1 and E-CADHERIN were the two dominant factors, with E-CADHERIN transducing boundary forces to focus WNT signalling to colony border at early times, and DKK1 controlling the late WNT pattern via cell non-autonomous negative feedback. With the help of time-lapse imaging of a fluorescent reporter line and mathematical modelling, I showed that these two factors mediate a wave of WNT signalling that spreads across the tissue to be patterned, and that this wave is a generic property of a bistable system and thus likely generalizable to other instances in development. While limited by the use of hESCs, taken together my findings provide a first glimpse into the role and control of WNT signalling early on in our own, human development
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Pluripotent stem cell models of early mammalian development.
Pluripotent stem cells derived from the early mammalian embryo offer a convenient model system for studying cell fate decisions in embryogenesis. The last 10 years have seen a boom in the popularity of two-dimensional micropatterns and three-dimensional stem cell culture systems as a way to recreate the architecture and interactions of particular cell populations during development. These methods enable the controlled exploration of cellular organization and patterning during development, using cell lines instead of embryos. They have established a new class of in vitro model system for pre-implantation and peri-implantation embryogenesis, ranging from models of the blastocyst stage, through gastrulation and toward early organogenesis. This review aims to set these systems in context and to highlight the strengths and suitability of each approach in modelling early mammalian development.This work was supported by the European Research Council (Advanced Grant to A.M.A., number 834580) and the Leverhulme Trust (RPG-2018-356). N.M. holds the Constance Work Junior Research Fellowship at Newnham College, Cambridge
High-resolution transcriptional and morphogenetic profiling of cells from micropatterned human ESC gastruloid cultures
During mammalian gastrulation, germ layers arise and are shaped into the body plan while extraembryonic layers sustain the embryo. Human embryonic stem cells, cultured with BMP4 on extracellular matrix micro-discs, reproducibly differentiate into gastruloids, expressing markers of germ layers and extraembryonic cells in radial arrangement. Using single-cell RNA sequencing and cross-species comparisons with mouse, cynomolgus monkey gastrulae, and post-implantation human embryos, we reveal that gastruloids contain cells transcriptionally similar to epiblast, ectoderm, mesoderm, endoderm, primordial germ cells, trophectoderm, and amnion. Upon gastruloid dissociation, single cells reseeded onto micro-discs were motile and aggregated with the same but segregated from distinct cell types. Ectodermal cells segregated from endodermal and extraembryonic but mixed with mesodermal cells. Our work demonstrates that the gastruloid system models primate-specific features of embryogenesis, and that gastruloid cells exhibit evolutionarily conserved sorting behaviors. This work generates a resource for transcriptomes of human extraembryonic and embryonic germ layers differentiated in a stereotyped arrangement
Reassembling gastrulation
During development, a single cell is transformed into a highly complex organism through progressive cell division, specification and rearrangement. An important prerequisite for the emergence of patterns within the developing organism is to establish asymmetries at various scales, ranging from individual cells to the entire embryo, eventually giving rise to the different body structures. This becomes especially apparent during gastrulation, when the earliest major lineage restriction events lead to the formation of the different germ layers. Traditionally, the unfolding of the developmental program from symmetry breaking to germ layer formation has been studied by dissecting the contributions of different signaling pathways and cellular rearrangements in the in vivo context of intact embryos. Recent efforts, using the intrinsic capacity of embryonic stem cells to self-assemble and generate embryo-like structures de novo, have opened new avenues for understanding the many ways by which an embryo can be built and the influence of extrinsic factors therein. Here, we discuss and compare divergent and conserved strategies leading to germ layer formation in embryos as compared to in vitro systems, their upstream molecular cascades and the role of extrinsic factors in this process
Quantitative developmental biology in vitro using micropatterning
Micropatterning encompasses a set of methods aimed at precisely controlling the spatial distribution of molecules onto the surface of materials. Biologists have borrowed the idea and adapted these methods, originally developed for electronics, to impose physical constraints on biological systems with the aim of addressing fundamental questions across biological scales from molecules to multicellular systems. Here, I approach this topic from a developmental biologist's perspective focusing specifically on how and why micropatterning has gained in popularity within the developmental biology community in recent years. Overall, this Primer provides a concise overview of how micropatterns are used to study developmental processes and emphasises how micropatterns are a useful addition to the developmental biologistâs toolbox
3D gastruloids: a novel frontier in stem cell-based in vitro modeling of mammalian gastrulation
3D gastruloids, aggregates of embryonic stem cells that recapitulate key aspects of gastrula-stage embryos, have emerged as a powerful tool to study the early stages of mammalian post-implantation development in vitro. Owing to their tractable nature and the relative ease by which they can be generated in large numbers, 3D gastruloids provide an unparalleled opportunity to study normal and pathological embryogenesis from a bottom-up perspective and in a high-throughput manner. Here, we review how gastruloid models can be exploited to deepen our understanding of mammalian development. In addition, we discuss current limitations, potential clinical applications, and ethical implications of this emerging model system
Differentiating Human Embryonic Stem Cells in Micropatterns to Study Cell Fate Specification and Morphogenetic Events During Gastrulation
During mammalian embryogenesis, the first major lineage segregation occurs when embryonic epiblast, and extraembryonic trophectoderm and hypoblast arise in the blastocyst. In the next fundamental and conserved phase of animal embryogenesis known as gastrulation, extraembryonic cells provide signals to epiblast to instruct embryonic patterning, and epiblast gives rise to germ layers ectoderm, mesoderm, and endoderm, that will establish all embryonic tissues. Proper specification and morphogenesis of germ layers during gastrulation is vital for correct embryonic development. Due to ethical and legal restrictions limiting human embryo studies, human gastrulation is poorly understood. Our knowledge of human gastrulation has largely been derived from studies in model organisms, including mouse and more recently, cynomolgus monkey. However, interspecies differences underscore the need for alternative human gastrulation models. In this regard, human and mouse embryonic stem cells have been shown to recapitulate aspects of in vivo gastrulation including germ layer specification, and internalization and elongation morphogenesis. These in vitro systems represent powerful models of gastrulation due to the ease of genetic manipulations and the ability to finely control experimental factors. Human embryonic stem cells, treated with BMP4 for 44 hours in spatially confined micro-discs of extracellular matrix, have been shown to differentiate into 2D micro-colonies termed gastruloids. These gastruloids display highly reproducible differentiation of germ layers and extraembryonic cell types in a radial arrangement. We used combinatorial single-cell RNA sequencing and immunofluorescence imaging to characterize these BMP4-treated 2D gastruloids, and showed the formation in gastruloids of seven cell types, including epiblast, prospective ectoderm, two populations of mesoderm, and endoderm, as well as previously undescribed cell types in 2D gastruloids, primordial germ cell-like cells, and extraembryonic cells that are transcriptionally similar to trophectoderm and amnion. Comparative transcriptomic analyses with human, mouse, and cynomolgus monkey gastrulae support the notion that 2D gastruloid differentiation recapitulates formation of cell types relevant to and models an early-mid stage of in vivo gastrulation. Time course scRNA-seq and immunofluorescence analyses of 2D gastruloid differentiation after 12, 24, and 44 hours of BMP4 treatment showed that germ layer emergence in gastruloids follows the temporal sequence of in vivo gastrulation, with epiblast and ectoderm precursors forming at 12 hour, mesendoderm precursors arising from epiblast at 24 hour to give rise to nascent mesoderm and endoderm at 44 hour, when primordial germ cell-like cells also form. Comparison with human gastrula also showed similarity in transcriptomes and differentiation trajectories of gastruloid cells to their in vivo counterparts. Dynamic changes in transcripts encoding components of key signaling pathways support a BMP, WNT and Nodal hierarchy underlying germ layer specification conserved across mammals, with FGF and HIPPO signaling being active throughout the time course of 2D micropattern gastruloid differentiation. To probe morphogenetic properties of gastruloid cells, differentiated gastruloids treated with BMP4 for 44 hours were dissociated and re-seeded onto extracellular matrix micro-discs. The reseeded cells were highly motile and tended to form aggregates with the same but segregate from or mix with distinct cell types, supporting that 2D gastruloid system exhibits evolutionarily conserved sorting behaviors. In particular, ectodermal cells segregated from endodermal and extraembryonic cells but mixed with mesodermal cells. These results demonstrate that 2D gastruloid system models specification of germ layers and extraembryonic cell types, temporal order and differentiation trajectories of germ layer emergence, and signaling interactions found in early-mid in vivo gastrulation. Dissociated and reseeded gastruloid cells also exhibit conserved cell sorting behaviors. Lastly, this work provides a resource for mining genes and pathways expressed in a stereotyped 2D gastruloid model, common with other species or unique to human gastrulation
SINGLE-CELL DYNAMICS OF CORE PLURIPOTENCY FACTORS IN HUMAN PLURIPOTENT STEM CELLS
Human pluripotent stem cell therapy is a novel approach to cellular therapeutics that can treat and model multiple diseases, accelerate regenerative medicine, and progress drug discovery. To induce pluripotency in somatic cells through the process of reprogramming, core pluripotency TFs are upregulated to force a somatic cell to adopt a pluripotent cell fate. From this hiPSC state, these cells can be differentiated to any cell type and used for downstream research and clinical applications. These pluripotency transcription factors (TFs) are OCT4, SOX2, and NANOG, which form a core signaling network critical for maintaining stem cell pluripotency and self-renewal. Currently, the spatiotemporal expression dynamics of these pluripotency TFs throughout differentiation and reprogramming is unclear, limiting our understanding of stem cell fate decisions. We investigated the temporal dynamics of pluripotency TFs underlying stem cell pluripotency and elucidated these dynamics during differentiation and reprogramming while potentially improving reprogramming strategies for clinical applications. We initially combined CRISPR/Cas9-mediated gene editing with microraft array technology to generate human embryonic stem cell lines with endogenously tagged fluorophores for the pluripotent TFs. We used time-lapse imaging on a SOX2/NANOG reporter to reveal that pluripotent stem cells show gastrulation-like patterning without direct chemical or spatial induction. Directed differentiation to the three primary germ layersâendoderm, mesoderm, and ectodermârevealed distinct spatiotemporal patterns of SOX2 and NANOG expression in single cells. Finally, we captured dynamic changes in cell morphology during ectoderm differentiation corresponding to the formation of neural rosettes.To elucidate pluripotent TF reactivation during reprogramming, the SOX2/NANOG reporter was differentiated to various cell types and then reprogrammed back into a pluripotent state. We found that the cells initially underwent morphological changes, then had subsequent inverse pluripotent TF reactivation, showing pluripotency recovery happens in reverse order from which it was lost. We then utilized the differentiated reporter cells to perform a screen of chemical combinations that can reactivate the pluripotency TFs. The results from this work provide insight into pluripotency TF dynamics during differentiation and reprogramming as well as knowledge for generating live cell reporters and a potential reprogramming alternative for future stem cell applications.Doctor of Philosoph