RESUMO
Retrospective lineage reconstruction of humans predicts that dramatic clonal imbalances in the body can be traced to the 2-cell stage embryo. However, whether and how such clonal asymmetries arise in the embryo is unclear. Here, we performed prospective lineage tracing of human embryos using live imaging, non-invasive cell labeling, and computational predictions to determine the contribution of each 2-cell stage blastomere to the epiblast (body), hypoblast (yolk sac), and trophectoderm (placenta). We show that the majority of epiblast cells originate from only one blastomere of the 2-cell stage embryo. We observe that only one to three cells become internalized at the 8-to-16-cell stage transition. Moreover, these internalized cells are more frequently derived from the first cell to divide at the 2-cell stage. We propose that cell division dynamics and a cell internalization bottleneck in the early embryo establish asymmetry in the clonal composition of the future human body.
Assuntos
Blastômeros , Linhagem da Célula , Embrião de Mamíferos , Feminino , Humanos , Blastômeros/citologia , Blastômeros/metabolismo , Divisão Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Masculino , Animais , CamundongosRESUMO
Stem cell-based mammalian embryo models facilitate the discovery of developmental mechanisms because they are more amenable to genetic and epigenetic perturbations than natural embryos. Here, we highlight exciting recent advances that have yielded a plethora of models of embryonic development. Imperfections in these models highlight gaps in our current understanding and outline future research directions, ushering in an exciting new era for embryology.
Assuntos
Embrião de Mamíferos , Desenvolvimento Embrionário , Animais , Feminino , Gravidez , Desenvolvimento Embrionário/genética , Organogênese , Células-Tronco , MamíferosRESUMO
These protocols investigate the interaction of cytonemes with localized Wnt. Cell-niche signaling between naive or primed mouse embryonic stem cells (ESCs) and either Wnt-secreting trophoblast stem cells (TSCs) or Wnt signals tethered to microbeads can be scrutinized in vitro. This approach analyzes cytoneme reactivity during Wnt-interaction initiation, Ca2+ transients at Wnt-contacting cytonemes, and subsequent pairing between ESCs and Wnt-sources. This pairing interaction is crucial to synthetic embryogenesis; hence this protocol is effective for in vitro studies of developmental biology. For complete details on the use and execution of this protocol, please refer to Junyent et al. (2020, 2021a, 2021b).
Assuntos
Bioengenharia/métodos , Células-Tronco Embrionárias Murinas , Pseudópodes , Via de Sinalização Wnt/fisiologia , Animais , Técnicas de Cultura de Células , Células Cultivadas , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Pseudópodes/metabolismo , Pseudópodes/fisiologia , Trofoblastos/citologiaRESUMO
The Wnt-pathway is part of a signalling network that regulates many aspects of cell biology. Recently, we discovered crosstalk between AMPA/Kainate-type ionotropic glutamate receptors (iGluRs) and the Wnt-pathway during the initial Wnt3a-interaction at the cytonemes of mouse embryonic stem cells (ESCs). Here, we demonstrate that this crosstalk persists throughout the Wnt3a-response in ESCs. Both AMPA and Kainate receptors regulate early Wnt3a-recruitment, dynamics on the cell membrane, and orientation of the spindle towards a Wnt3a-source at mitosis. AMPA receptors specifically are required for segregating cell fate components during Wnt3a-mediated asymmetric cell division (ACD). Using Wnt-pathway component knockout lines, we determine that Wnt co-receptor Lrp6 has particular functionality over Lrp5 in cytoneme formation, and in facilitating ACD. Both Lrp5 and 6, alongside pathway effector ß-catenin act in concert to mediate the positioning of the dynamic interaction with, and spindle orientation to, a localised Wnt3a-source. Wnt-iGluR crosstalk may prove pervasive throughout embryonic and adult stem cell signalling.
Assuntos
Divisão Celular , Células-Tronco Embrionárias Murinas/metabolismo , Receptores de AMPA/metabolismo , Receptores de Ácido Caínico/metabolismo , Via de Sinalização Wnt , Proteína Wnt3A/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Microscopia de Vídeo , Receptor Cross-Talk , Receptores de AMPA/genética , Receptores de Ácido Caínico/genética , Fatores de Tempo , Proteína Wnt3A/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
To coordinate cell fate with changes in spatial organization, stem cells (SCs) require specific and adaptable systems of signal exchange and cell-to-cell communication. Pluripotent embryonic stem cells (ESCs) use cytonemes to pair with trophoblast stem cells (TSCs) and form synthetic embryonic structures in a Wnt-dependent manner. How these interactions vary with pluripotency states remains elusive. Here we show that ESC transition to an early primed ESC (pESC) state reduces their pairing with TSCs and impairs synthetic embryogenesis. pESCs can activate the Wnt/ß-catenin pathway in response to soluble Wnt ligands, but their cytonemes form unspecific and unstable interactions with localized Wnt sources. This is due to an impaired crosstalk between Wnt and glutamate receptor activity and reduced generation of Ca2+ transients on the cytonemes upon Wnt source contact. Induced iGluR activation can partially restore cytoneme function in pESCs, while transient overexpression of E-cadherin improves pESC-TSC pairing. Our results illustrate how changes in pluripotency state alter the mechanisms SCs use to self-organize.
Assuntos
Comunicação Celular , Desenvolvimento Embrionário , Células-Tronco Embrionárias Murinas/metabolismo , Trofoblastos/metabolismo , Via de Sinalização Wnt , Animais , Linhagem Celular , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Trofoblastos/citologiaRESUMO
The maintenance of human skeletal stem cells (hSSCs) and their progeny in bone defects is a major challenge. Here, we report on a transplantable bandage containing a three-dimensional Wnt-induced osteogenic tissue model (WIOTM). This bandage facilitates the long-term viability of hSSCs (8 weeks) and their progeny, and enables bone repair in an in vivo mouse model of critical-sized calvarial defects. The newly forming bone is structurally comparable to mature cortical bone and consists of human and murine cells. Furthermore, we show that the mechanism of WIOTM formation is governed by Wnt-mediated asymmetric cell division of hSSCs. Covalently immobilizing Wnts onto synthetic materials can polarize single dividing hSSCs, orient the spindle and simultaneously generate a Wnt-proximal hSSC and a differentiation-prone Wnt-distal cell. Our results provide insight into the regulation of human osteogenesis and represent a promising approach to deliver human osteogenic constructs that can survive in vivo and contribute to bone repair.
Assuntos
Osso e Ossos/citologia , Divisão Celular , Osteogênese , Crânio/citologia , Células-Tronco/citologia , Engenharia Tecidual/métodos , Proteínas Wnt/metabolismo , Animais , Humanos , Camundongos , Crânio/fisiologiaRESUMO
Spatial cellular organization is fundamental for embryogenesis. Remarkably, coculturing embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) recapitulates this process, forming embryo-like structures. However, mechanisms driving ESC-TSC interaction remain elusive. We describe specialized ESC-generated cytonemes that react to TSC-secreted Wnts. Cytoneme formation and length are controlled by actin, intracellular calcium stores, and components of the Wnt pathway. ESC cytonemes select self-renewal-promoting Wnts via crosstalk between Wnt receptors, activation of ionotropic glutamate receptors (iGluRs), and localized calcium transients. This crosstalk orchestrates Wnt signaling, ESC polarization, ESC-TSC pairing, and consequently synthetic embryogenesis. Our results uncover ESC-TSC contact-mediated signaling, reminiscent of the glutamatergic neuronal synapse, inducing spatial self-organization and embryonic cell specification.
Assuntos
Comunicação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Pseudópodes/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Drosophila , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/fisiologia , Camundongos , Trofoblastos/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologiaRESUMO
Wnt signaling is crucial during embryonic development and for the maintenance of adult tissues. Depending on the tissue type, the Wnt pathway can promote stem cell self-renewal and/or direct lineage commitment. Wnt proteins are subject to lipid modification, often restricting them to act in a localized manner on responsive cells. Most methods for inducing Wnt signaling in stem cell cultures do not control the spatial presentation of the protein. To recreate the local presentation of Wnt proteins often seen in vivo, we previously developed a method to immobilize the protein onto synthetic surfaces. Here we describe a detailed protocol based on covalent binding of nucleophilic groups on Wnt proteins to activated carboxylic acid (COOH) or glutaraldehyde (COH) groups functionalized on synthetic surfaces. As an example, we describe how this method can be used to covalently immobilize Wnt3a proteins on microbeads or a glass surface. This procedure requires â¼3 h and allows for the hydrophobic protein to be stored in the absence of detergent. The immobilization efficiency of active Wnt proteins can be assessed using different T-cell factor (TCF) reporter assays as a readout for Wnt/ß-catenin-dependent transcription. Immobilization efficiency can be measured 12-18 h after seeding the cells and takes 2-4 h. The covalent immobilization of Wnt proteins can also be used for single-cell analysis using Wnt-coated microbeads (12-18 h of live imaging) and to create a Wnt platform on a glass surface for stem cell maintenance and cell population analysis (3 d). The simple chemistry used for Wnt immobilization allows for adaptation to new materials and other developmental signals. Therefore, this method can also be incorporated into tissue engineering platforms in which depletion of the stem cell pool restricts the complexity and maturity of the tissue developed.
Assuntos
Técnicas Citológicas/métodos , Proteínas Imobilizadas/metabolismo , Proteínas Wnt/metabolismo , Vidro , Microesferas , Ligação Proteica , Análise de Célula Única/métodos , Fatores de TempoRESUMO
Single-nucleotide polymorphism studies have linked the chromosome 17q12-q21 region, where the human orosomucoid-like (ORMDL)3 gene is localized, to the risk of asthma and several other inflammatory diseases. Although mast cells are involved in the development of these diseases, the contribution of ORMDL3 to the mast cell physiology is unknown. In this study, we examined the role of ORMDL3 in antigen-induced activation of murine mast cells with reduced or enhanced ORMDL3 expression. Our data show that in antigen-activated mast cells, reduced expression of the ORMDL3 protein had no effect on degranulation and calcium response, but significantly enhanced phosphorylation of AKT kinase at Ser 473 followed by enhanced phosphorylation and degradation of IκBα and translocation of the NF-κB p65 subunit into the nucleus. These events were associated with an increased expression of proinflammatory cytokines (TNF-α, IL-6, and IL-13), chemokines (CCL3 and CCL4), and cyclooxygenase-2 dependent synthesis of prostaglandin D2. Antigen-mediated chemotaxis was also enhanced in ORMDL3-deficient cells, whereas spreading on fibronectin was decreased. On the other hand, increased expression of ORMDL3 had no significant effect on the studied signaling events, except for reduced antigen-mediated chemotaxis. These data were corroborated by increased IgE-antigen-dependent passive cutaneous anaphylaxis in mice with locally silenced ORMDL3 using short interfering RNAs. Our data also show that antigen triggers suppression of ORMDL3 expression in the mast cells. In summary, we provide evidence that downregulation of ORMDL3 expression in mast cells enhances AKT and NF-κB-directed signaling pathways and chemotaxis and contributes to the development of mast cell-mediated local inflammation in vivo.