Self-Organizing Properties of Mouse Pluripotent Cells Initiate Morphogenesis upon Implantation

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’.

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Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells

INTRODUCTION Early mammalian development requires the formation of embryonic and extraembryonic tissues and a highly coordinated partnership between them. This close partnership is a prerequisite for successful construction of embryo architecture, with the embryonic tissue generating cells of the embryo proper and the extraembryonic tissues, trophectoderm, and primitive endoderm forming the placenta and the yolk sac. Each of these components and the interactions between them are critical for embryonic development to birth. Embryonic stem cells (ESCs) in culture have the potential to participate in development when introduced into the early embryo. However, when cultured in vitro on their own, they do not recapitulate the spatial and temporal events of early embryogenesis. RATIONALE We hypothesized that in order to faithfully model with stem cells the morphogenetic steps involved in mammalian embryogenesis, we would need to establish a developmental dialogue between ESCs and extraembryonic trophoblast stem cells (TSCs) in a three-dimensional (3D) extracellular matrix (ECM) scaffold, to potentially substitute for the basement membrane normally provided by the primitive endoderm. RESULTS We combined embryonic and extraembryonic stem cells in vitro on such a 3D matrix and found that these cells were capable of self-assembly into a structure whose development and architecture were similar to that of the natural embryo, leading us to name them in vitro ESC and TSC stem cell–embryos (ETS-embryos). By building ETS-embryos from genetically modified stem cells and using specific inhibitors, we identify morphogenetic events and signaling pathways involved in these early developmental stages. Furthermore, we show that in vitro stem cell embryogenesis can be broken down into a sequence of key steps from implantation stage to germ layer specification. First is the self-organization of ESCs, which leads to polarization and lumenogenesis of ESC-derived embryonic compartment, followed by cavitation in the TSC-derived extraembryonic compartment. Second is the unification of embryonic and extraembryonic cavities into the equivalent of the embryo’s proamniotic cavity. Third is the requirement for a dialogue between embryonic and extraembryonic compartments, involving Nodal signaling, that builds characteristic embryo architecture. Fourth is the patterning of the embryonic compartment, revealed by the localized expression of mesoderm markers at the boundary between embryonic and extraembryonic compartments that, as in the natural embryo, is preceded by and dependent upon Wnt signaling. Fifth is the specification of a small cluster of PGC-like cells at the embryonic and extraembryonic boundary in a bone morphogenetic protein (BMP) signaling–dependent manner. Remarkably, such events in in vitro stem cell embryogenesis occur with very similar spatial and temporal dynamics to those taking place in natural embryogenesis. CONCLUSION In this work, we demonstrate that establishing a close cross-talk between embryonic and extraembryonic stem cells in a 3D ECM scaffold is necessary and sufficient for the self-assembly of a structure that recapitulates many of the key spatial and temporal steps of early mammalian embryogenesis. This in vitro ETS-embryo models embryonic architecture and patterning more accurately than has been possible before using ESCs alone. They also provide a simplified platform to dissect the physical, cellular, and molecular mechanisms mediating the critical cross-talk between embryonic and extraembryonic cells during development, and hence are a powerful tool for the future study of mammalian embryogenesis.

/pdf/assembly-of-embryonic-and-extraembryonic-stem-cells-to-mimic-2plgmupm9f.pdf

Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro

A crucial question in mammalian development is how cells of the early embryo differentiate into distinct cell types. The first decision is taken when cells undertake waves of asymmetric division that generate one daughter on the inside and one on the outside of the embryo. After this division, some cells on the inside remain pluripotent and give rise to the epiblast, and hence the future body, whereas others develop into the primitive endoderm, an extraembryonic tissue. How the fate of these inside cells is decided is unknown: Is the process random, or is it related to their developmental origins? To address this question, we traced all cells by live-cell imaging in intact, unmanipulated embryos until the epiblast and primitive endoderm became distinct. This analysis revealed that inner cell mass (ICM) cells have unrestricted developmental potential. However, cells internalized by the first wave of asymmetric divisions are biased toward forming pluripotent epiblast, whereas cells internalized in the next two waves of divisions are strongly biased toward forming primitive endoderm. Moreover, we show that cells internalized by the second wave up-regulate expression of Gata6 and Sox17, and changing the expression of these genes determines whether the cells become primitive endoderm. Finally, with our ability to determine the origin of cells, we find that inside cells that are mispositioned when they are born can sort into the correct layer. In conclusion, we propose a model in which the timing of cell internalization, cell position, and cell sorting combine to determine distinct lineages of the preimplantation mouse embryo.

https://www.pnas.org/content/pnas/107/14/6364.full.pdf

Origin and formation of the first two distinct cell types of the inner cell mass in the mouse embryo

In the mammalian brain, neurogenesis continues only in few regions of the forebrain. The molecular signals governing neurogenesis in these unique neurogenic niches, however, are still ill defined. Here, we show that bone morphogenic protein (BMP)-mediated signaling is active in adult neural stem cells and is crucial to initiate the neurogenic lineage in the adult mouse subependymal zone. Conditional deletion of Smad4 in adult neural stem cells severely impairs neurogenesis, and this is phenocopied by infusion of Noggin, an extracellular antagonist of BMP. Smad4 deletion in stem, but not progenitor cells, as well as Noggin infusion lead to an increased number of Olig2-expressing progeny that migrate to the corpus callosum and differentiate into oligodendrocytes. Transplantation experiments further verified the cell-autonomous nature of this phenotype. Thus, BMP-mediated signaling via Smad4 is required to initiate neurogenesis from adult neural stem cells and suppress the alternative fate of oligodendrogliogenesis.

https://www.jneurosci.org/content/jneuro/28/2/434.full.pdf

Adult neurogenesis requires Smad4-mediated bone morphogenic protein signaling in stem cells.

The visualization of live cell behaviors operating in situ combined with the power of mouse genetics represents a major step toward understanding the mechanisms regulating embryonic development, homeostasis, and disease progression in mammals. The availability of genetically encoded fluorescent protein reporters, combined with improved optical imaging modalities, have led to advances in our ability to examine cells in vivo. We developed a series of lipid-modified fluorescent protein fusions that are targeted to and label the secretory pathway and the plasma membrane, and that are amenable for use in mice. Here we report the generation of two strains of mice, each expressing a spectrally distinct lipid-modified GFP-variant fluorescent protein fusion. The CAG::GFP-GPI strain exhibited widespread expression of a glycosylphosphatidylinositol-tagged green fluorescent protein (GFP) fusion, while the CAG::myr-Venus strain exhibited widespread expression of a myristoyl-Venus yellow fluorescent protein fusion. Imaging of live transgenic embryonic stem (ES) cells, either live or fixed embryos and postnatal tissues demonstrated that glycosylphosphatidyl inositol- and myristoyl-tagged GFP-variant fusion proteins are targeted to and serve as markers of the plasma membrane. Moreover, our data suggest that these two lipid-modified protein fusions are dynamically targeted both to overlapping as well as distinct lipid-enriched compartments within cells. These transgenic strains not only represent high-contrast reporters of cell morphology and plasma membrane dynamics, but also may be used as in vivo sensors of lipid localization. Furthermore, combining these reporters with the study of mouse mutants will be a step forward in understanding the inter- and intracellular behaviors underlying morphogenesis in both normal and mutant contexts.

In vivo imaging and differential localization of lipid-modified GFP-variant fusions in embryonic stem cells and mice.

The ascidian Ciona intestinalis is one of the model organisms of choice for comparative investigations of chordate development and for unraveling the molecular mechanisms underlying morphogenesis and cell fate specification. Taking advantage of the availability of various genetically encoded fluorescent proteins and of defined cis-regulatory elements, we combined transient transgenesis with laser scanning confocal imaging to acquire and quantitate 3D time-lapse data from living Ciona embryos. We used Ciona tissue-specific enhancers to drive expression of spectrally distinct fluorescent protein reporters to label and simultaneously visualize axially and paraxially positioned mesodermal derivatives, as well as neural precursors in individual embryos. We observed morphogenetic movements, without perturbing development, from the early gastrula throughout the larval stage, including gastrulation, neurulation, convergent extension of the presumptive notochord, and tail elongation. These multidimensional data allowed us to establish a reference system of metrics to quantify key developmental events including blastopore closure and muscle extension. The approach we describe can be used to document morphogenetic cell and tissue rearrangements in living embryos and paves the way for a live digitized anatomical atlas of Ciona. genesis 43:136–147, 2005. © 2005 Wiley-Liss, Inc.

Jerry M. Rhee

Papers

In vivo imaging and differential localization of lipid-modified GFP-variant fusions in embryonic stem cells and mice.

Live imaging and morphometric analysis of embryonic development in the ascidian Ciona intestinalis