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PUBLICATIONS
2026
Chen F, Bruder JM, Govindasamy N, Wu J, Chen R, Prit D, Drexler HCA, Leidel SA, Schöler HR, Bedzhov I
Science Advances 2026
https://doi.org/10.1126/sciadv.adq7469
During the peri-implantation phase of murine embryogenesis, the epiblast proliferates rapidly and undergoes epithelialization. At the same time, the preimplantation pluripotent state transforms into a more developmentally advanced, pregastrulation state. While extensive research has elucidated cell-extrinsic signals that direct the developmental progression, such as the Fgf/Mek/Erk pathway, the potential interplay of intrinsic cellular cues remains largely unexplored. To address this, we conducted a comprehensive phenotypic screen using an in vitro model of epiblast development. We identified aurora kinase A as a cell-intrinsic factor contributing to Erk activation and transcriptional response. Consequently, suppressing aurora kinase A activity delayed exit from naïve pluripotency. Moreover, our results show that upon entry into mitosis, Erk relocates to the cell division machinery. We found that in dividing cells, a fraction of Erk, with yet elusive functions, localizes on the centrosomes, where its phosphorylation depends on polo-like kinase 1.
2025
Chandramohan D, Yan R, Kruse K, Brinkmann H, Jeong H-W, Hou Y, Adachi K, Schöler HR, Bedzhov I
Developmental Cell 2025
https://doi.org/10.1016/j.devcel.2025.11.003
The fundamental processes of cell fate specification, differentiation, and morphogenesis must be finely synchronized to enable proper developmental progression, yet the molecular factors coordinating these processes are not well understood. A key driver of embryonic morphogenesis is the establishment of epithelial polarity, which organizes and structures the early cell layers. Here, we investigated factors controlling the epithelialization in epiblast cells and implemented sequential loss-of-function approaches in mouse embryos to define the timing of developmental significance. We found that the expression wave of the core pluripotency factors Oct4 and Sox2 following the 8-cell stage plays a critical role in this process. In this context, one of the key shared functions of these factors is to prevent premature activation of the epithelial program until the completion of the second lineage segregation. Thus, Oct4 and Sox2 simultaneously govern developmental capacity and regulate the developmental timing of tissue morphogenesis of the embryonic lineage.
Chen R, Bedzhov I
Development 2025
https://doi.org/10.1242/dev.205121
Typically, embryonic development is a continuous process. However, in some species, embryos can halt their development and enter a dormant state known as diapause. During this period, the embryo retains its viability and developmental capacity to resume transient embryogenesis. While the diapausing embryo appears to be in a state of suspended animation, recent studies have revealed a more dynamic picture of modulated signalling responses, metabolic rewiring and slow-paced but active tissue morphogenesis. In this Spotlight, we discuss the emerging concepts of the molecular and cellular mechanisms that govern mammalian embryonic diapause, focusing on the mouse as a model system.
Chandramohan D, Chen F, Chen R, Bedzhov I
Current Topics in Developmental Biology 2025
https://doi.org/10.1016/bs.ctdb.2025.10.001
The mammalian embryo establishes the first direct contact with the maternal tissues during implantation. As the blastocyst attaches to the uterine wall, it initiates transformation into a post-implantation conceptus, laying the foundations for placentation and foetal development. The peri-implantation embryogenesis relies on the coordinated growth, morphogenesis, and cell fate transitions of the embryonic and extra-embryonic tissues, entailing changes in cell shape and tissue architecture. In some species, including mice, this process is suspended in a non-receptive uterine environment, where embryonic development arrests at the blastocyst stage and the embryo enters a dormant state known as diapause. However, despite being dormant, relatively slow morphogenetic changes continue to reshape the embryonic cells. Here, we will briefly review the process of murine blastocyst formation before focusing primarily on cell shape and tissue dynamics during peri-implantation embryogenesis. Finally, we will synthesise the current knowledge on tissue morphogenesis during embryo dormancy.
Riedel NC, Walter C, de Faria FW, Altendorf L, Aust P, Göbel C, Verma A, Ballast A, Bedzhov I, Roy R, Münter D, Schüftan E, Albert TK, Rössig C, Johann P, von Zezschwitz B, Sandmann S, Varghese J, Thomas C, Schüller U, Bruder JM and Kerl K.
Cancer Communications 2025
https://doi.org/10.1002/cac2.70074
No abstract available.
Chen R, Bedzhov I
Biospektrum 2025
https://doi.org/10.1007/s12268-025-2502-0
Embryonic dormancy (diapause) is a reproductive adaptation that allows some mammalian species to halt the otherwise continuous pace of embryonic development. While the dormant embryo remains in a state of suspended animation, molecular analysis at single-cell resolution has revealed extensive signalling, transcriptional and morpho genetic changes, indicating that diapause is not a stasis but a dynamic process.
de Faria FW, Riedel NC, Münter D, Interlandi M, Göbel C, Altendorf L, Richter M, Melcher V, Thomas C, Roy R, Schoof M, Bedzhov I, Moreno N, Graf M, Hotfilder M, Holdhof D, Hartmann W, Bruns AK, Brentrup A, Liesche-Starnecker F, Maerkl B, Sandmann S, Varghese J, Dugas M, Pinto PH, Balbach ST, Lu IN, Rossig C, Soehnlein O, Canak A, Ebinger M, Schuhmann M, Schittenhelm J, Frühwald MF, Schüller U, Albert TK, Walter C, Bruder JM, Kerl K.
Nature Communications 2025
https://doi.org/10.1038/s41467-025-60442-9
Embryonal tumor with multilayered rosettes (ETMR) is a lethal embryonal brain tumor entity. To investigate the intratumoral heterogeneity and cellular communication in the tumor microenvironment (TME), we analyze in this work single-cell RNA sequencing of about 250,000 cells of primary human and murine ETMR, in vitro cultures, and a 3D forebrain organoid model of ETMR, supporting the main findings with immunohistochemistry and spatial transcriptomics of human tumors. We characterize three distinct malignant ETMR subpopulations – RG-like, NProg-like and NB-like – positioned within a putative neurodevelopmental hierarchy. We reveal PDGFRβ+ pericytes as key communication partners in the TME, contributing to stem cell signaling through extracellular matrix-mediated interactions with tumor cells. PDGF signaling is upregulated in chemoresistant RG-like cells in vivo and plays a role in recruiting pericytes to ETMR TME by finalizing a signaling cascade which promotes the differentiation of non-malignant radial glia cells, derived from our 3D model, into pericyte-like cells. Selective PDGFR-inhibition blocked the lineage differentiation into pericytes in vitro and reduced the tumor cell population in vivo. Targeting ETMR-pericyte interactions in the TME presents a promising therapeutic approach.
Prit D, Jaitly S, Govindasamy N, Ranga A, Bedzhov I.
Methods Mol Biol. 2025
https://doi.org/10.1007/7651_2025_646
The cellular dynamics during peri-implantation embryogenesis and the concurrent interactions at the embryo-maternal interface are inherently difficult to study due to intrauterine development in mammals. To model certain aspects of these processes in vitro, we have generated a biomimetic environment resembling the mechanical properties of the murine uterine stroma. Here we describe a step-by-step methodology for 3D culture of mouse embryos and ectoplacental cone explants in synthetic hydrogels that allow ex utero trophoblast invasion.
Chen R, Fan R, Bedzhov I.
STAR Protocols 2025
https://doi.org/10.1016/j.xpro.2025.103813
Embryonic dormancy (diapause) is a reproductive adaptation that allows some mammalian species to prolong pregnancy and delay birth by temporarily suspending embryonic development just before implantation. Here, we present a step-by-step protocol for inducing and maintaining embryonic diapause in mice by tamoxifen administration or ovariectomy. We describe steps for setting up mouse matings, the administration of pharmacological compounds, the surgical procedure for the removal of the ovaries, postoperative care, and the isolation of dormant embryos. We then describe procedures for triggering exit from diapause by administration of β-estradiol and the subsequent isolation of reactivated embryos.
Hou Y, Nie Z, Jiang Q, Velychko S, Heising S, Bedzhov I, Wu G, Adachi K, Scholer HR.
Elife 2025
https://doi.org/10.7554/elife.100735
During the first lineage segregation, mammalian embryos generate the inner cell mass (ICM) and trophectoderm (TE). ICM gives rise to the epiblast (EPI) that forms all cell types of the body, an ability referred to as pluripotency. The molecular mechanisms that induce pluripotency in embryos remain incompletely elucidated. Using knockout (KO) mouse models in conjunction with low-input ATAC-seq and RNA-seq, we found that Oct4 and Sox2 gradually come into play in the early ICM, coinciding with the initiation of Sox2 expression. Oct4 and Sox2 activate the pluripotency-related genes through the putative OCT-SOX enhancers in the early ICM. Furthermore, we observed a substantial reorganization of chromatin landscape and transcriptome from the morula to the early ICM stages, which was partially driven by Oct4 and Sox2, highlighting their pivotal role in promoting the developmental trajectory toward the ICM. Our study provides new insights into the establishment of the pluripotency network in mouse preimplantation embryos.
2024
Chen R, Fan R, Chen F, Govindasamy N, Brinkmann H, Stehling M, Adams RH, Jeong HW, Bedzhov I.
Cell Stem Cell 2024
https://doi.org/10.1016/j.stem.2024.06.015
Embryonic diapause is a reproductive adaptation that enables some mammalian species to halt the otherwise continuous pace of embryonic development. In this dormant state, the embryo exploits poorly understood regulatory mechanisms to preserve its developmental potential for prolonged periods of time. Here, using mouse embryos and single-cell RNA sequencing, we molecularly defined embryonic diapause at single-cell resolution, revealing transcriptional dynamics while the embryo seemingly resides in a state of suspended animation. Additionally, we found that the dormant pluripotent cells rely on integrin receptors to sense their microenvironment and preserve their viability via Yap/Taz mediated prosurvival signaling.
Schumacher S, Fernkorn M, Marten M, Chen R, Kim YS, Bedzhov I, Schröter C.
Nature Communications 2024
https://doi.org/10.1038/s41467-024-49380-0
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 β-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.
MacCarthy CM, Wu G, Malik V, Menuchin-Lasowski Y, Velychko T, Keshet G, Fan R, Bedzhov I, Church GM, Jauch R, Cojocaru V, Schöler HR, Velychko S.
Cell Stem Cell 2024
https://doi.org/10.1016/j.stem.2023.11.010
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.
2023
Govindasamy N, Long H, Ranga A, Trappmann B, Bedzhov I.
STAR Protocols 2023
https://doi.org/10.1016/j.xpro.2023.102456
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.
Ozguldez HO, Govindasamy N, Fan R, Long H, Mildner K, Zeuschner D, Trappmann B, Ranga A, Bedzhov I.
Cell Reports 2023
https://doi.org/10.1016/j.celrep.2023.112313
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.
2022
Sathyanarayanan A, Ing-Simmons E, Chen R, Jeong HW, Ozguldez HO, Fan R, Duethorn B, Kim KP, Kim YS, Stehling M, Brinkmann H, Schöler HR, Adams RH, Vaquerizas JM, Bedzhov I.
Science Advances 2022
https://doi.org/10.1126/sciadv.abl9583
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.
Kim YS, Fan R, Lith SC, Dicke AK, Drexler HCA, Kremer L, Kuempel-Rink N, Hekking L, Stehling M, Bedzhov I.
Developmental Cell 2022
https://doi.org/10.1016/j.devcel.2022.07.011
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.
Kim YS, Bedzhov I.
Seminars in Cell & Developmental Biology 2023
https://doi.org/10.1016/j.semcdb.2022.04.020
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.
Eliat F, Sohn R, Renner H, Kagermeier T, Volkery S, Brinkmann H, Kirschnick N, Kiefer F, Grabos M, Becker K, Bedzhov I, Schöler HR, Bruder JM.
Scientific Reports 2022
https://doi.org/10.1038/s41598-022-09303-9
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.
Han D, Wu G, Chen R, Drexler HCA, MacCarthy CM, Kim KP, Adachi K, Gerovska D, Mavrommatis L, Bedzhov I, Araúzo-Bravo MJ, Schöler HR.
Science Advances 2022
https://doi.org/10.1126/sciadv.abe4375
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.
Xu C, Dinh VV, Kruse K, Jeong HW, Watson EC, Adams S, Berkenfeld F, Stehling M, Rasouli SJ, Fan R, Chen R, Bedzhov I, Chen Q, Kato K, Pitulescu ME, Adams RH.
eLife 2022
https://doi.org/10.7554/elife.60183
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.
Duethorn B, Groll F, Rieger B, Drexler HCA, Brinkmann H, Kremer L, Stehling
M, Borowski MT, Mildner K, Zeuschner D, Zernicka-Goetz M, Stemmler MP, Busch KB, Vaquerizas JM, Bedzhov I.
Nature Communications 2022
https://doi.org/10.1038/s41467-022-28139-5
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.
2021
Govindasamy N, Long H, Jeong HW, Raman R, Özcifci B, Probst S, Arnold SJ, Riehemann K, Ranga A, Adams RH, Trappmann B, Bedzhov I.
Developmental Cell 2021
https://doi.org/10.1016/j.devcel.2021.10.014
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.
Salewskij K, Gross-Thebing T, Ing-Simmons E, Duethorn B, Rieger B, Fan R, Chen R, Govindasamy N, Brinkmann H, Kremer L, Kuempel-Rink N, Mildner K, Zeuschner D, Stehling M, Dejosez M, Zwaka TP, Schöler HR, Busch KB, Vaquerizas JM, Bedzhov I.
EMBO Reports 2022
https://doi.org/10.15252/embr.202153048
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.
Kim YS, Fan R, Kremer L, Kuempel-Rink N, Mildner K, Zeuschner D, Hekking L, Stehling M, Bedzhov I.
Science Advances 2022
https://doi.org/10.1126/sciadv.abe1640
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.
2020
Fan R, Kim YS, Wu J, Chen R, Zeuschner D, Mildner K, Adachi K, Wu G, Galatidou S, Li J, Schöler HR, Leidel SA, Bedzhov I.
Nature Communications 2020
https://doi.org/10.1038/s41467-020-19353-0
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/β-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/β-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.
Ozguldez HO, Bedzhov I.
Methods Mol Biol. 2020
https://doi.org/10.1007/978-1-0716-0958-3_3
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.
Ozguldez HO, Fan R, Bedzhov I.
Development 2020
https://doi.org/10.1242/dev.190371
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.
2019
Agrawal M, Schwarz P, Giaimo BD, Bedzhov I, Corbacioglu A, Weber D, Gaidzik VI, Jahn N, Rücker FG, Schroeder T, Kindler T, Wattad M, Götze K, Lübbert M, Salwender H, Ringhoffer M, Lange E, Koller E, Thol F, Heuser M, Ganser A, Bullinger L, Paschka P, Döhner H, Geiger H, Borggrefe T, Döhner K, Oswald F.
Leukemia 2019
https://doi.org/10.1038/s41375-019-0551-4
No abstract available.
Govindasamy N, Bedzhov I.
Methods Mol Biol. 2019
https://doi.org/10.1007/978-1-4939-9566-0_25
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.
Govindasamy N, Duethorn B, Oezgueldez HO, Kim YS, Bedzhov I.
Int J Dev Biol. 2019
https://doi.org/10.1387/ijdb.180379ib
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.
2015
Bedzhov I, Bialecka M, Zielinska A, Kosalka J, Antonica F, Thompson AJ, Franze K, Zernicka-Goetz M.
Cell Research 2015
https://doi.org/10.1038/cr.2015.104
No abstract available.
Bedzhov I, Zernicka-Goetz M.
Bioessays 2015
https://doi.org/10.1002/bies.201400147
Shortly after implantation the embryonic lineage transforms from a coherent ballbof cells into polarized cup shaped epithelium. Recently we elucidated a previously unknown apoptosis-independent morphogenic event that reorganizes the pluripotent lineage. Polarization cues from the surrounding basement membrane rearrange the epiblast into a polarized rosette-like structure, where subsequently a central lumen is established. Thus, we provided a new model revising the current concept of apoptosis-dependent epiblast morphogenesis. Cell death however has to be tightly regulated during embryogenesis to ensure developmental success. Here, we follow the stages of early mouse development and take a glimpse at the critical signaling and morphogenic events that determine cells destiny and reshape the embryonic lineage.
Bedzhov I, Graham SJ, Yan Leung C, Zernicka-Goetz M.
Philos Trans R Soc Lond B Biol Sci 2015
https://doi.org/10.1098/rstb.2014.0339
Erratum for Philos Trans R Soc Lond B Biol Sci. 2014 Dec 5;369(1657):20130538. doi:10.1098/rstb.2013.0538.
2014
Bedzhov I, Leung CY, Bialecka M, Zernicka-Goetz M.
Nature Protocols 2014
https://doi.org/10.1038/nprot.2014.186
The implanting mouse blastocyst invades the uterine stroma and undergoes a dramatic transformation into an egg cylinder. The morphogenetic and signaling events during this transition are largely unexplored, as the uterine tissues engulf the embryo. Here we describe a protocol supporting the development of the mouse embryo beyond the blastocyst stage in vitro. We established two types of medium to be applied sequentially, and we used a substrate permitting high-resolution imaging of the transition from blastocyst to egg cylinder. We developed two variants of this protocol: the first starts with intact early blastocysts that upon zona removal can attach to the substrate and develop into egg cylinders after 5 d, and the second starts with late blastocysts that upon dissection of the mural trophectoderm form egg cylinders in only 3 d. This method allows observation of a previously hidden period of development, and it provides a platform for novel research into peri-implantation embryogenesis and beyond.
Bedzhov I, Graham SJ, Leung CY, Zernicka-Goetz M.
Philos Trans R Soc Lond B Biol Sci 2014
https://doi.org/10.1098/rstb.2013.0538
A critical point in mammalian development is when the early embryo implants into its mother’s uterus. This event has historically been difficult to study due to the fact that it occurs within the maternal tissue and therefore is hidden from view. In this review, we discuss how the mouse embryo is prepared for implantation and the molecular mechanisms involved in directing and coordinating this crucial event. Prior to implantation, the cells of the embryo are specified as precursors of future embryonic and extra-embryonic lineages. These preimplantation cell fate decisions rely on a combination of factors including cell polarity, position and cell-cell signalling and are influenced by the heterogeneity between early embryo cells. At the point of implantation, signalling events between the embryo and mother, and between the embryonic and extraembryonic compartments of the embryo itself, orchestrate a total reorganization of the embryo, coupled with a burst of cell proliferation. New developments in embryo culture and imaging techniques have recently revealed the growth and morphogenesis of the embryo at the time of implantation, leading to a new model for the blastocyst to egg cylinder transition. In this model, pluripotent cells that will give rise to the fetus self-organize into a polarized three-dimensional rosette-like structure that initiates egg cylinder formation.
Erratum in Philos Trans R Soc Lond B Biol Sci. 2015 Feb 5;370(1661):20140339. doi: 10.1098/rstb.2014.0339.
Bedzhov I, Stemmler MP.
Methods Mol Biol. 2014
https://doi.org/10.1007/978-1-4939-1789-1_6
Analysis of protein-protein interactions in mouse preimplantation embryos is hindered by the low cell number of the embryo. Here we describe the use of the proximity ligation assay to overcome these limitations and outline how protein-protein interactions can be visualized in situ. The method is based on a normal immunofluorescence labeling protocol of preimplantation embryos. Events of binding of the two primary antibodies directed against two individual proteins close to each other are visualized. If the two primary antibodies and the corresponding oligo-linked secondary antibodies bind in close proximity a cascade of events is initiated. This includes oligo ligation, DNA amplification, and hybridization with a fluorescent probe that allows visualization of this close proximity. In contrast to normal immunofluorescence labeling, here detection of red fluorescent dots reflects protein-protein interaction.
Bedzhov I, Zernicka-Goetz M.
Cell 2014
https://doi.org/10.1016/j.cell.2014.01.023
Transformation of pluripotent epiblast cells into a cup-shaped epithelium as the mouse blastocyst implants is a poorly understood and yet key developmental step. Studies of morphogenesis in embryoid bodies led to the current belief that it is programmed cell death that shapes the epiblast. However, by following embryos developing in vivo and in vitro, we demonstrate that not cell death but a previously unknown morphogenetic event transforms the amorphous epiblast into a rosette of polarized cells. This transformation requires basal membrane-stimulated integrin signaling that coordinates polarization of epiblast cells and their apical constriction, a prerequisite for lumenogenesis. We show that basal membrane function can be substituted in vitro by extracellular matrix (ECM) proteins and that ES cells can be induced to form similar polarized rosettes that initiate lumenogenesis. Together, these findings lead to a completely revised model for peri-implantation morphogenesis in which ECM triggers the self-organization of the embryo’s stem cells.
2013
Bedzhov I, Alotaibi H, Basilicata MF, Ahlborn K, Liszewska E, Brabletz T, Stemmler MP.
Stem Cell Research 2013
https://doi.org/10.1016/j.scr.2013.08.009
Embryonic stem (ES) cell pluripotency and induced pluripotent stem (iPS) cell generation is dependent on a core transcriptional network and proper cell-cell adhesion mediated by E-cadherin (E-cad). Whereas E-cad is associated with pluripotency, N-cadherin (N-cad) expression is correlated with differentiation into mesodermal and neuroectodermal lineages. We investigated whether E-cad harbors unique molecular features in establishing or maintaining pluripotency. By using a gene replacement knock-in (ki) approach to express N-cadherin (N-cad) or E-cad/N-cad chimeric cadherins under the control of the E-cad locus, we show that all E-cad-depleted ki/ki ES cells are maintained in an undifferentiated state. Surprisingly, these cells retained key features of pluripotency, such as Nanog expression and full differentiation capacity in vitro and in vivo, whereas E-cad knockout (ko) ES cells irreversibly lost most of these features. Moreover, our results indicate that E-cad mediated adhesion is essential for iPS cell generation, since E-cad depleted fibroblasts were not reprogrammed. In contrast, N-cad efficiently supports somatic reprogramming similar to E-cad, and permits initiation of the crucial initial step of mesenchymal-epithelial transition. Thus, we show that cell adhesion and a robust pluripotent phenotype are ultimately connected. Since N-cad properly compensates for loss of E-cad, no specific ‘cadherin code’ is required.
2012
Bedzhov I, Liszewska E, Kanzler B, Stemmler MP.
PLOS Genetics 2012
https://doi.org/10.1371/journal.pgen.1002609
Insulin-like growth factor I receptor (Igf1r) signaling controls proliferation, differentiation, growth, and cell survival in many tissues; and its deregulated activity is involved in tumorigenesis. Although important during fetal growth and postnatal life, a function for the Igf pathway during preimplantation development has not been described. We show that abrogating Igf1r signaling with specific inhibitors blocks trophectoderm formation and compromises embryo survival during murine blastocyst formation. In normal embryos total Igf1r is present throughout the membrane, whereas the activated form is found exclusively at cell contact sites, colocalizing with E-cadherin. Using genetic domain switching, we show a requirement for E-cadherin to maintain proper activation of Igf1r. Embryos expressing exclusively a cadherin chimera with N-cadherin extracellular and E-cadherin intracellular domains (NcEc) fail to form a trophectoderm and cells die by apoptosis. In contrast, homozygous mutant embryos expressing a reverse-structured chimera (EcNc) show trophectoderm survival and blastocoel cavitation, indicating a crucial and non-substitutable role of the E-cadherin ectodomain for these processes. Strikingly, blastocyst formation can be rescued in homozygous NcEc embryos by restoring Igf1r signaling, which enhances cell survival. Hence, perturbation of E-cadherin extracellular integrity, independent of its cell-adhesion function, blocked Igf1r signaling and induced cell death in the trophectoderm. Our results reveal an important and yet undiscovered function of Igf1r during preimplantation development mediated by a unique physical interaction between Igf1r and E-cadherin indispensable for proper receptor activation and anti-apoptotic signaling. We provide novel insights into how ligand-dependent Igf1r activity is additionally gated to sense developmental potential in utero and into a bifunctional role of adhesion molecules in contact formation and signaling.
2010
Stemmler MP, Bedzhov I.
Developmental Dynamics 2010
https://doi.org/10.1002/dvdy.22375
The specific roles of classical cadherins at key morphogenetic events during development are still not fully understood. As part of a project to study cadherin function during early mammalian development, we generated mice carrying an HA-epitope tagged Cdh1 (E-cadherin) cDNA knocked into the Cdh1 locus, similar to the previously described mouse mutants in which we forced Cdh2 (N-cadherin) expression in the Cdh1 expression domain. As expected and in contrast to Cdh1(Cdh2/Cdh2) and Cdh1(-/-), our Cdh1(HA/HA) mutant embryos form proper trophectoderm, implant and undergo both gastrulation and neurulation. However, Cdh1(HA/HA) mice display an unexpected phenotype at embryonic day 10.5. Cdh1(HA/HA) embryos are smaller, paler and suffer from an insufficient nutrient supply. We detected a reduced expression of Cdh1(HA) specifically in the extraembryonic ectoderm and in the labyrinth layer, whereas expression in the embryo proper was normal. With this approach, we show for the first time that Cdh1 is essential for the correct formation of the placenta. Placentas without Cdh1 expression are impaired and incapable of establishing a proper connection between the embryonic and the maternal blood vessels for efficient nutrient and oxygen transport.
2005
Borissova, T. Hvarleva, I. Bedzhov, V. Kondakova, A. Atanassov, I. Atanassov.
Biotechnology & Biotechnological Equipment 2005
https://doi.org/10.1080/13102818.2005.10817156
A genetic transformation protocol, based on co-cultivation of Rosa hybrida L. secondary embryos with Agrobacterium was established. Transgenic phosphinothricin (ppt) – resistant plants were obtained through application of interrupted selection procedure. Following the Agrobacterium inoculation step, the somatic embryos were initially cultivated on ppt—free media containing cefatoxim for elimination of the bacteria and later transferred on media containing the selective agent. The ppt—resistant secondary somatic embryos were matured and regenerated on ppt—free media and the putative transgenic plants were subsequently selected on media contained ppt and chlorphenol red. The performed PCR—and PAT protein—assays demonstrated presence and expression of bar gene encoding the phosphinothricin acetyltransferase (PAT) in part of the selected plants. The possibilities for application of the established transformation protocol are discussed.
PATENTS
Sathyanarayanan A, Bedzhov I.
WO2021259909A1
2021
The present invention relates to a method for preparing trophectoderm-like cells comprising culturing a blastocyst, extraembryonic endoderm stem (XEN) cells and/or XEN-like cells in a medium comprising a glycogen synthase kinase 3 (GSK-3) inhibitor.
Zernicka-Goetz M, Bedzhov I.
WO2015022541A1
2014
Media, kits and methods for culturing embryos and stem cells in vitro are provided. The media comprise an insulin receptor agonist, an oestrogen receptor agonist, and a progesterone receptor agonist. The media enable reliable and reproducible in vitro culture of embryos from a pre-implantation stage of development to a post- implantation stage of development.
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