RESUMO
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.
Assuntos
Blastocisto/citologia , Implantação do Embrião , Embrião de Mamíferos/citologia , Camadas Germinativas/citologia , Animais , Apoptose , Blastocisto/metabolismo , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Matriz Extracelular/metabolismo , Feminino , Integrinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Morfogênese , Transdução de SinaisRESUMO
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.
Assuntos
Blastocisto , Endoderma , Animais , Implantação do Embrião , Embrião de Mamíferos , Desenvolvimento Embrionário , CamundongosRESUMO
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.
Assuntos
Blastocisto/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Animais , Blastocisto/citologia , Feminino , Edição de Genes , Humanos , Integrases/genética , Integrases/metabolismo , Camundongos , Gravidez , Trofoblastos/citologiaRESUMO
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.
Assuntos
Desenvolvimento Embrionário , Células-Tronco Embrionárias , Animais , Blastocisto/metabolismo , Implantação do Embrião/fisiologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/metabolismo , CamundongosRESUMO
Shortly after implantation the embryonic lineage transforms from a coherent ball of 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.
Assuntos
Camadas Germinativas/citologia , Morfogênese/fisiologia , Animais , Apoptose/fisiologia , Blastocisto/citologia , Implantação do Embrião/fisiologia , Desenvolvimento Embrionário/fisiologia , Feminino , Camadas Germinativas/fisiologia , Camundongos , GravidezRESUMO
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.
Assuntos
Blastocisto , Caderinas , Desenvolvimento Embrionário/genética , Receptor IGF Tipo 1 , Transdução de Sinais/genética , Animais , Apoptose , Blastocisto/citologia , Blastocisto/metabolismo , Caderinas/genética , Caderinas/metabolismo , Adesão Celular/genética , Comunicação Celular/genética , Sobrevivência Celular , Células-Tronco Embrionárias , Regulação da Expressão Gênica no Desenvolvimento , Homozigoto , Ligantes , Camundongos , Mutação , Estrutura Terciária de Proteína/genética , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismoRESUMO
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.
Assuntos
Padronização Corporal , Diferenciação Celular , Endoderma , Proteína Nodal , Transdução de Sinais , beta Catenina , Animais , Endoderma/citologia , Endoderma/metabolismo , Endoderma/embriologia , beta Catenina/metabolismo , Camundongos , Proteína Nodal/metabolismo , Proteína Nodal/genética , Camadas Germinativas/metabolismo , Camadas Germinativas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Regulação da Expressão Gênica no Desenvolvimento , Embrião de Mamíferos/citologiaRESUMO
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.
RESUMO
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.
Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Humanos , Camundongos , Ratos , Animais , Suínos , Macaca fascicularis/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Reprogramação Celular , Fatores de Transcrição SOXB1/metabolismo , Diferenciação Celular , Mamíferos/metabolismoRESUMO
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.
Assuntos
Biomimética , Células Endoteliais , Técnicas de Cocultura , Embrião de Mamíferos , TrofoblastosRESUMO
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.
Assuntos
Blastocisto , Trofoblastos , Gravidez , Feminino , Camundongos , Animais , Células-Tronco , Implantação do Embrião , Placenta , Linhagem da Célula , Diferenciação CelularRESUMO
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.
Assuntos
Implantação do Embrião , Camadas Germinativas , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , MorfogêneseRESUMO
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.
Assuntos
Corantes Fluorescentes , Imageamento Tridimensional , Encéfalo/diagnóstico por imagem , Corantes Fluorescentes/química , Imageamento Tridimensional/métodos , Ionóforos , SolventesRESUMO
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.
RESUMO
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.
Assuntos
Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Blastocisto , Proliferação de Células , Desenvolvimento Embrionário/fisiologia , Células-Tronco Embrionárias/citologia , Feminino , Masculino , Camundongos , Células-Tronco Pluripotentes/citologiaRESUMO
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.
RESUMO
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.
Assuntos
Osteogênese , Osteoporose/metabolismo , Receptores Notch/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Osso e Ossos/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Condrócitos/metabolismo , Células Endoteliais/metabolismo , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Alelos , Proteínas de Ciclo Celular , Técnicas de Introdução de Genes , Fenótipo , Placentação/fisiologia , Animais , Apoptose , Blastocisto/fisiologia , Proteínas Cdh1 , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Teste de Complementação Genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese , Placenta/anormalidades , Placenta/fisiologia , GravidezRESUMO
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.
Assuntos
Blastocisto/fisiologia , Técnicas de Cultura Embrionária/métodos , Camundongos/embriologia , Óvulo/fisiologia , Animais , Blastocisto/citologia , Blastocisto/ultraestrutura , Desenvolvimento Embrionário , Feminino , Óvulo/citologia , Óvulo/ultraestruturaRESUMO
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.