Tissue-intrinsic beta-catenin signals antagonize Nodal-driven anterior visceral endoderm differentiation.

The anterior-posterior axis of the mammalian embryo is laid down by the anterior visceral endoderm (AVE), an extraembryonic signaling center that is specified within the visceral endoderm. Current models posit that AVE differentiation is promoted globally by epiblast-derived Nodal signals, and spatially restricted by a BMP gradient established by the extraembryonic ectoderm. Here, we report spatially restricted AVE differentiation in bilayered embryo-like aggregates made from mouse embryonic stem cells that lack an extraembryonic ectoderm. Notably, clusters of AVE cells also form in pure visceral endoderm cultures upon activation of Nodal signaling, indicating that tissue-intrinsic factors can restrict AVE differentiation. We identify ß-catenin activity as a tissue-intrinsic factor that antagonizes AVE-inducing Nodal signals. Together, our results show how an AVE-like population can arise through interactions between epiblast and visceral endoderm alone. This mechanism may be a flexible solution for axis patterning in a wide range of embryo geometries, and provide robustness to axis patterning when coupled with signal gradients.

Highly cooperative chimeric super-SOX induces naive pluripotency across species.

Our understanding of pluripotency remains limited: iPSC generation has only been established for a few model species, pluripotent stem cell lines exhibit inconsistent developmental potential, and germline transmission has only been demonstrated for mice and rats. By swapping structural elements between Sox2 and Sox17, we built a chimeric super-SOX factor, Sox2-17, that enhanced iPSC generation in five tested species: mouse, human, cynomolgus monkey, cow, and pig. A swap of alanine to valine at the interface between Sox2 and Oct4 delivered a gain of function by stabilizing Sox2/Oct4 dimerization on DNA, enabling generation of high-quality OSKM iPSCs capable of supporting the development of healthy all-iPSC mice. Sox2/Oct4 dimerization emerged as the core driver of naive pluripotency with its levels diminished upon priming. Transient overexpression of the SK cocktail (Sox+Klf4) restored the dimerization and boosted the developmental potential of pluripotent stem cells across species, providing a universal method for naive reset in mammals.

3D biomimetic environment enabling ex utero trophoblast invasion and co-culture of embryos and somatic cells.

The first direct contact between the embryo and the mother is established during implantation. This process is inaccessible for direct studies as the implanting embryo is concealed by the maternal tissues. Here, we present a protocol for establishing a 3D biomimetic environment based on synthetic hydrogels which harbor key biomechanical properties of the uterine stroma. We describe steps for isolating and culturing embryos in PEG/DexMA hydrogel. We then detail the co-culture of embryos and endothelial cells in a microfluidic device. For complete details on the use and execution of this protocol, please refer to Govindasamy et al. (2021)1 and Ozguldez et al. (2023).2.

Polarity inversion reorganizes the stem cell compartment of the trophoblast lineage.

The extra-embryonic tissues that form the placenta originate from a small population of trophectoderm cells with stem cell properties, positioned at the embryonic pole of the mouse blastocyst. During the implantation stages, the polar trophectoderm rapidly proliferates and transforms into extra-embryonic ectoderm. The current model of trophoblast morphogenesis suggests that tissue folding reshapes the trophoblast during the blastocyst to egg cylinder transition. Instead of through folding, here we found that the tissue scale architecture of the stem cell compartment of the trophoblast lineage is reorganized via inversion of the epithelial polarity axis. Our findings show the developmental significance of polarity inversion and provide a framework for the morphogenetic transitions in the peri-implantation trophoblast.

Early developmental plasticity enables the induction of an intermediate extraembryonic cell state.

Two fundamental elements of pre-implantation embryogenesis are cells' intrinsic self-organization program and their developmental plasticity, which allows embryos to compensate for alterations in cell position and number; yet, these elements are still poorly understood. To be able to decipher these features, we established culture conditions that enable the two fates of blastocysts' extraembryonic lineages-the primitive endoderm and the trophectoderm-to coexist. This plasticity emerges following the mechanisms of the first lineage segregation in the mouse embryo, and it manifests as an extended potential for extraembryonic chimerism during the pre-implantation embryogenesis. Moreover, this shared state enables robust assembly into higher-order blastocyst-like structures, thus combining both the cell fate plasticity and self-organization features of the early extraembryonic lineages.

Rap1 controls epiblast morphogenesis in sync with the pluripotency states transition.

The complex architecture of the murine fetus originates from a simple ball of pluripotent epiblast cells, which initiate morphogenesis upon implantation. In turn, this establishes an intermediate state of tissue-scale organization of the embryonic lineage in the form of an epithelial monolayer, where patterning signals delineate the body plan. However, how this major morphogenetic process is orchestrated on a cellular level and synchronized with the developmental progression of the epiblast is still obscure. Here, we identified that the small GTPase Rap1 plays a critical role in reshaping the pluripotent lineage. We found that Rap1 activity is controlled via Oct4/Esrrb input and is required for the transmission of polarization cues, which enables the de novo epithelialization and formation of tricellular junctions in the epiblast. Thus, Rap1 acts as a molecular switch that coordinates the morphogenetic program in the embryonic lineage, in sync with the cellular states of pluripotency.

Mechanisms of formation and functions of the early embryonic cavities.

As the early mouse embryo develops, fundamental steps include the sequential formation of the first lumens in the murine conceptus. The first cavity established in the pre-implantation embryo is the blastocoel, followed by the emergence of the proamniotic cavity during the peri-implantation stages. The mouse embryo is a dynamic system which switches its modes of lumenogenesis before and after implantation. The blastocoel emerges in between the basolateral membranes, whereas the proamniotic cavity is formed on the apical interface. Defects in the sculpting of these luminal spaces are associated with developmental abnormalities and embryonic lethality. Here, we review the mechanisms by which these early embryonic cavities are formed and discuss the cavities in terms of their common and stage-specific principles of lumenogenesis and their functions.

Tissue clearing may alter emission and absorption properties of common fluorophores.

In recent years, 3D cell culture has been gaining a more widespread following across many fields of biology. Tissue clearing enables optical analysis of intact 3D samples and investigation of molecular and structural mechanisms by homogenizing the refractive indices of tissues to make them nearly transparent. Here, we describe and quantify that common clearing solutions including benzyl alcohol/benzyl benzoate (BABB), PEG-associated solvent system (PEGASOS), immunolabeling-enabled imaging of solvent-cleared organs (iDISCO), clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC), and ScaleS4 alter the emission spectra of Alexa Fluor fluorophores and fluorescent dyes. Clearing modifies not only the emitted light intensity but also alters the absorption and emission peaks, at times to several tens of nanometers. The resulting shifts depend on the interplay of solvent, fluorophore, and the presence of cells. For biological applications, this increases the risk for unexpected channel crosstalk, as filter sets are usually not optimized for altered fluorophore emission spectra in clearing solutions. This becomes especially problematic in high throughput/high content campaigns, which often rely on multiband excitation to increase acquisition speed. Consequently, researchers relying on clearing in quantitative multiband excitation experiments should crosscheck their fluorescent signal after clearing in order to inform the proper selection of filter sets and fluorophores for analysis.

Lima1 mediates the pluripotency control of membrane dynamics and cellular metabolism.

Lima1 is an extensively studied prognostic marker of malignancy and is also considered to be a tumour suppressor, but its role in a developmental context of non-transformed cells is poorly understood. Here, we characterise the expression pattern and examined the function of Lima1 in mouse embryos and pluripotent stem cell lines. We identify that Lima1 expression is controlled by the naïve pluripotency circuit and is required for the suppression of membrane blebbing, as well as for proper mitochondrial energetics in embryonic stem cells. Moreover, forcing Lima1 expression enables primed mouse and human pluripotent stem cells to be incorporated into murine pre-implantation embryos. Thus, Lima1 is a key effector molecule that mediates the pluripotency control of membrane dynamics and cellular metabolism.

Induction of osteogenesis by bone-targeted Notch activation.

Declining bone mass is associated with aging and osteoporosis, a disease characterized by progressive weakening of the skeleton and increased fracture incidence. Growth and lifelong homeostasis of bone rely on interactions between different cell types including vascular cells and mesenchymal stromal cells (MSCs). As these interactions involve Notch signaling, we have explored whether treatment with secreted Notch ligand proteins can enhance osteogenesis in adult mice. We show that a bone-targeting, high affinity version of the ligand Delta-like 4, termed Dll4(E12), induces bone formation in male mice without causing adverse effects in other organs, which are known to rely on intact Notch signaling. Due to lower bone surface and thereby reduced retention of Dll4(E12), the same approach failed to promote osteogenesis in female and ovariectomized mice but strongly enhanced trabecular bone formation in combination with parathyroid hormone. Single cell analysis of stromal cells indicates that Dll4(E12) primarily acts on MSCs and has comparably minor effects on osteoblasts, endothelial cells, or chondrocytes. We propose that activation of Notch signaling by bone-targeted fusion proteins might be therapeutically useful and can avoid detrimental effects in Notch-dependent processes in other organs.

A balanced Oct4 interactome is crucial for maintaining pluripotency.

Oct4 collaborates primarily with other transcriptional factors or coregulators to maintain pluripotency. However, how Oct4 exerts its function is still unclear. Here, we show that the Oct4 linker interface mediates competing yet balanced Oct4 protein interactions that are crucial for maintaining pluripotency. Oct4 linker mutant embryonic stem cells (ESCs) show decreased expression of self-renewal genes and increased expression of differentiation genes, resulting in impaired ESC self-renewal and early embryonic development. The linker mutation interrupts the balanced Oct4 interactome. In mutant ESCs, the interaction between Oct4 and Klf5 is decreased. In contrast, interactions between Oct4 and Cbx1, Ctr9, and Cdc73 are increased, disrupting the epigenetic state of ESCs. Control of the expression level of Klf5, Cbx1, or Cdc73 rebalances the Oct4 interactome and rescues the pluripotency of linker mutant ESCs, indicating that such factors interact with Oct4 competitively. Thus, we provide previously unidentified molecular insights into how Oct4 maintains pluripotency.

3D biomimetic platform reveals the first interactions of the embryo and the maternal blood vessels.

The process of implantation and the cellular interactions at the embryo-maternal interface are intrinsically difficult to analyze, as the implanting embryo is concealed by the uterine tissues. Therefore, the mechanisms mediating the interconnection of the embryo and the mother are poorly understood. Here, we established a 3D biomimetic culture environment that harbors the key features of the murine implantation niche. This culture system enabled direct analysis of trophoblast invasion and revealed the first embryonic interactions with the maternal vasculature. We found that implantation is mediated by the collective migration of penetrating strands of trophoblast giant cells, which acquire the expression of vascular receptors, ligands, and adhesion molecules, assembling a network for communication with the maternal blood vessels. In particular, Pdgf signaling cues promote the establishment of the heterologous contacts. Together, the biomimetic platform and our findings thereof elucidate the hidden dynamics of the early interactions at the implantation site.

Ronin governs the metabolic capacity of the embryonic lineage for post-implantation development.

During implantation, the murine embryo transitions from a "quiet" into an active metabolic/proliferative state, which kick-starts the growth and morphogenesis of the post-implantation conceptus. Such transition is also required for embryonic stem cells to be established from mouse blastocysts, but the factors regulating this process are poorly understood. Here, we show that Ronin plays a critical role in the process by enabling active energy production, and the loss of Ronin results in the establishment of a reversible quiescent state in which naïve pluripotency is promoted. In addition, Ronin fine-tunes the expression of genes that encode ribosomal proteins and is required for proper tissue-scale organisation of the pluripotent lineage during the transition from blastocyst to egg cylinder stage. Thus, Ronin function is essential for governing the metabolic capacity so that it can support the pluripotent lineage's high-energy demands for cell proliferation and morphogenesis.

Deciphering epiblast lumenogenesis reveals proamniotic cavity control of embryo growth and patterning.

During the peri-implantation stages, the mouse embryo radically changes its appearance, transforming from a hollow-shaped blastocyst to an egg cylinder. At the same time, the epiblast gets reorganized from a simple ball of cells to a cup-shaped epithelial monolayer enclosing the proamniotic cavity. However, the cavity's function and mechanism of formation have so far been obscure. Through investigating the cavity formation, we found that in the epiblast, the process of lumenogenesis is driven by reorganization of intercellular adhesion, vectoral fluid transport, and mitotic paracellular water influx from the blastocoel into the emerging proamniotic cavity. By experimentally blocking lumenogenesis, we found that the proamniotic cavity functions as a hub for communication between the early lineages, enabling proper growth and patterning of the postimplantation embryo.

In Vitro Culture of Mouse Blastocysts to the Egg Cylinder Stage via Mural Trophectoderm Excision.

The developmental transition from the blastocyst to the egg cylinder stage is associated with stark changes in the overall shape of the embryo, as well as with reorganization of the transcriptional network and epigenetic landscape in the pluripotent and the supportive extraembryonic lineages. To directly analyze this pre- to postimplantation switch, culture conditions are needed that can support mouse embryogenesis beyond the blastocyst stage without maternal input. Here we provide a step-by-step protocol describing an experimental pipeline for isolating late blastocysts, excising (manually or via laser assistance) the mural trophectoderm, and, finally, culturing the embryo to the egg cylinder stage.

Wnt/Beta-catenin/Esrrb signalling controls the tissue-scale reorganization and maintenance of the pluripotent lineage during murine embryonic diapause.

The epiblast, which provides the foundation of the future body, is actively reshaped during early embryogenesis, but the reshaping mechanisms are poorly understood. Here, using a 3D in vitro model of early epiblast development, we identify the canonical Wnt/ß-catenin pathway and its central downstream factor Esrrb as the key signalling cascade regulating the tissue-scale organization of the murine pluripotent lineage. Although in vivo the Wnt/ß-catenin/Esrrb circuit is dispensable for embryonic development before implantation, autocrine Wnt activity controls the morphogenesis and long-term maintenance of the epiblast when development is put on hold during diapause. During this phase, the progressive changes in the epiblast architecture and Wnt signalling response show that diapause is not a stasis but instead is a dynamic process with underlying mechanisms that can appear redundant during transient embryogenesis.

Placental gene editing via trophectoderm-specific Tat-Cre/loxP recombination.

The ways in which placental defects affect embryonic development are largely overlooked because of the lack of a trophoblast-specific approach for conditional gene ablation. To tackle this, we have established a simple, fast and efficient method for trophectodermal Tat-Cre/loxP recombination. We used the natural permeability barrier in mouse blastocysts in combination with off-the-shelf Tat-Cre recombinase to achieve editing of conditional alleles in the trophoblast lineage. This direct approach enables gene function analysis during implantation and placentation in mice, thereby crucially helping to broaden our understanding of human reproduction and development.

Isolation and Culture of Periimplantation and Early Postimplantation Mouse Embryos.

Key developmental processes of cell fate decisions and morphogenetic transformations take place during the periimplantation and early postimplantation stages of mouse embryogenesis. However analysing these fundamental events relies on direct observations of cultured embryos, which are challenging to obtain. To address this challenge, here we provide a detailed protocol describing a workflow for isolating early implanted embryos, removing of redundant extraembryonic tissues and describing the culture conditions that support further embryo development in vitro.

Test-tube embryos - mouse and human development in vitro to blastocyst stage and beyond.

Mammalian embryogenesis is intrauterine and depends on support from the maternal environment. Therefore, in order to directly study and manipulate early mouse and human embryos, fine-tuned culture conditions have to be provided to maintain embryo growth in vitro. Over time, the establishment and implementation of embryo culture methods have come a long way, initially enabling the development of few pre-implantation stages, expanding later to support in vitro embryogenesis from fertilization until blastocyst and even ex utero development beyond the implantation stages. Designing culture conditions that enable near physiological development of early embryos without maternal input, especially during the peri- and post-implantation stages, requires overcoming numerous experimental challenges, and it is still far from optimal. Nevertheless, embryo culture methods are an essential cornerstone of both assisted reproductive technologies and basic research, and these methods provide a platform to understand life's greatest miracle - the development of a new organism.