RESUMO
Inducing cellular senescence in mouse embryonic fibroblasts (MEFs) is a robust tool to study the molecular mechanisms underlying senescence establishment and their heterogeneity. This protocol provides a detailed guide to generate MEFs and routinely induce senescence in MEFs using several DNA damage-dependent and DNA damage-independent induction methods.
Assuntos
Senescência Celular , Dano ao DNA , Fibroblastos , Animais , Fibroblastos/citologia , Fibroblastos/metabolismo , Senescência Celular/genética , Camundongos , Embrião de Mamíferos/citologia , Técnicas de Cultura de Células/métodos , Células CultivadasRESUMO
Pluripotent mouse embryonic stem cells (ESCs) can differentiate to all germ layers and serve as an in vitro model of embryonic development. To better understand the differentiation paths traversed by ESCs committing to different lineages, we track individual differentiating ESCs by timelapse imaging followed by multiplexed high-dimensional Imaging Mass Cytometry (IMC) protein quantification. This links continuous live single-cell molecular NANOG and cellular dynamics quantification over 5-6 generations to protein expression of 37 different molecular regulators in the same single cells at the observation endpoints. Using this unique data set including kinship history and live lineage marker detection, we show that NANOG downregulation occurs generations prior to, but is not sufficient for neuroectoderm marker Sox1 upregulation. We identify a developmental cell type co-expressing both the canonical Sox1 neuroectoderm and FoxA2 endoderm markers in vitro and confirm the presence of such a population in the post-implantation embryo. RNASeq reveals cells co-expressing SOX1 and FOXA2 to have a unique cell state characterized by expression of both endoderm as well as neuroectoderm genes suggesting lineage potential towards both germ layers.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Fator 3-beta Nuclear de Hepatócito , Células-Tronco Embrionárias Murinas , Fatores de Transcrição SOXB1 , Animais , Camundongos , Fator 3-beta Nuclear de Hepatócito/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Rastreamento de Células/métodos , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Linhagem da Célula , Endoderma/metabolismo , Endoderma/citologia , Análise de Célula Única/métodos , Desenvolvimento Embrionário/genética , Placa Neural/metabolismo , Placa Neural/embriologia , Placa Neural/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologiaRESUMO
The lymphatic system is formed during embryonic development by the commitment of specialized lymphatic endothelial cells (LECs) and their subsequent assembly in primary lymphatic vessels. Although lymphatic cells are in continuous contact with mesenchymal cells during development and in adult tissues, the role of mesenchymal cells in lymphatic vasculature development remains poorly characterized. Here, we show that a subpopulation of mesenchymal cells expressing the transcription factor Osr1 are in close association with migrating LECs and established lymphatic vessels in mice. Lineage tracing experiments revealed that Osr1+ cells precede LEC arrival during lymphatic vasculature assembly in the back of the embryo. Using Osr1-deficient embryos and functional in vitro assays, we show that Osr1 acts in a non-cell-autonomous manner controlling proliferation and early migration of LECs to peripheral tissues. Thereby, mesenchymal Osr1+ cells control, in a bimodal manner, the production of extracellular matrix scaffold components and signal ligands crucial for lymphatic vessel formation.
Assuntos
Células Endoteliais , Linfangiogênese , Vasos Linfáticos , Fatores de Transcrição , Animais , Vasos Linfáticos/embriologia , Vasos Linfáticos/metabolismo , Vasos Linfáticos/citologia , Camundongos , Linfangiogênese/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células Endoteliais/metabolismo , Células Endoteliais/citologia , Movimento Celular/genética , Proliferação de Células , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Mesoderma/metabolismo , Mesoderma/citologia , Regulação da Expressão Gênica no Desenvolvimento , Linhagem da CélulaRESUMO
During homeostasis, the endoplasmic reticulum (ER) maintains productive transmembrane and secretory protein folding that is vital for proper cellular function. The ER-resident HSP70 chaperone, binding immunoglobulin protein (BiP), plays a pivotal role in sensing ER stress to activate the unfolded protein response (UPR). BiP function is regulated by the bifunctional enzyme filamentation induced by cyclic-AMP domain protein (FicD) that mediates AMPylation and deAMPylation of BiP in response to changes in ER stress. AMPylated BiP acts as a molecular rheostat to regulate UPR signaling, yet little is known about the molecular consequences of FicD loss. In this study, we investigate the role of FicD in mouse embryonic fibroblast (MEF) response to pharmacologically and metabolically induced ER stress. We find differential BiP AMPylation signatures when comparing robust chemical ER stress inducers to physiological glucose starvation stress and recovery. Wildtype MEFs respond to pharmacological ER stress by down-regulating BiP AMPylation. Conversely, BiP AMPylation in wildtype MEFs increases upon metabolic stress induced by glucose starvation. Deletion of FicD results in widespread gene expression changes under baseline growth conditions. In addition, FicD null MEFs exhibit dampened UPR signaling, altered cell stress recovery response, and unconstrained protein secretion. Taken together, our findings indicate that FicD is important for tampering UPR signaling, stress recovery, and the maintenance of secretory protein homeostasis.
Assuntos
Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Fibroblastos , Glucose , Resposta a Proteínas não Dobradas , Animais , Camundongos , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologia , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Glucose/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Camundongos Knockout , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Transdução de SinaisRESUMO
Primordial germ cells (PGCs) are the precursors of gametes and the sole mechanism by which animals transmit genetic information across generations. In the mouse embryo, the transcriptional and epigenetic regulation of PGC specification has been extensively characterized. However, the initial event that triggers the soma-germline segregation remains poorly understood. Here, we uncover a critical role for the basement membrane in regulating germline entry. We show that PGCs arise in a region of the mouse embryo that lacks contact with the basement membrane, and the addition of exogenous extracellular matrix (ECM) inhibits both PGC and PGC-like cell (PGCLC) specification in mouse embryos and stem cell models, respectively. Mechanistically, we demonstrate that the engagement of ß1 integrin with laminin blocks PGCLC specification by preventing the Wnt signaling-dependent down-regulation of the PGC transcriptional repressor, Otx2. In this way, the physical segregation of cells away from the basement membrane acts as a morphogenetic fate switch that controls the soma-germline bifurcation.
Assuntos
Células Germinativas , Células-Tronco Pluripotentes , Animais , Camundongos , Células Germinativas/metabolismo , Células Germinativas/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Transdução de Sinais , Integrinas/metabolismo , Integrinas/genética , Membrana Basal/metabolismo , Via de Sinalização Wnt , Diferenciação Celular , Matriz Extracelular/metabolismo , Laminina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Integrina beta1/metabolismo , Integrina beta1/genética , Fatores de Transcrição Otx/metabolismo , Fatores de Transcrição Otx/genética , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologiaRESUMO
High quality label-free imaging of oocytes and early embryos is essential for accurate assessment of their developmental potential, a key element of assisted reproduction procedures. To achieve this goal, we propose full-field optical coherence microscopy (FF-OCM), constructed as a compact module fully integrated with a commercial wide-field fluorescence microscope. Our system achieves optical sectioning in wide-field, high in-plane resolution of 0.5 µm, and high sensitivity to backscattered light. To demonstrate its imaging capabilities, we study live mouse oocytes and embryos at all important stages of meiotic maturation and early embryogenesis. Our system enables visualization of intracellular structures, which are not visible in common bright-field microscopy, i.e., internal structure of nuclear apparatus, cytoskeletal filaments, cellular cortex, cytoplasmic protrusions, or zona pellucida features. Additionally, we visualize and quantify intracellular dynamics like cytoplasmic stirring motion, nuclear envelope fluctuations and nucleolus mobility. Altogether, we demonstrate that FF-OCM is a powerful tool for research in developmental biology that also holds great potential for non-invasive time-lapse monitoring of oocyte and embryo quality in assisted reproduction.
Assuntos
Oócitos , Animais , Oócitos/citologia , Camundongos , Feminino , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Tomografia de Coerência Óptica/métodosRESUMO
Faithful DNA replication is essential for genome integrity1-4. Under-replicated DNA leads to defects in chromosome segregation, which are common during embryogenesis5-8. However, the regulation of DNA replication remains poorly understood in early mammalian embryos. Here we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and identified an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing program, and the entire genome replicated gradually and uniformly using extremely slow-moving replication forks. In 4-cell embryos, a somatic-cell-like replication timing program commenced abruptly. However, the fork speed was still slow, S phase was extended, and markers of replication stress, DNA damage and repair increased. This was followed by an increase in break-type chromosome segregation errors specifically during the 4-to-8-cell division with breakpoints enriched in late-replicating regions. These errors were rescued by nucleoside supplementation, which accelerated fork speed and reduced the replication stress. By the 8-cell stage, forks gained speed, S phase was no longer extended and chromosome aberrations decreased. Thus, a transient period of genomic instability exists during normal mouse development, preceded by an S phase lacking coordination between replisome-level regulation and megabase-scale replication timing regulation, implicating a link between their coordination and genome stability.
Assuntos
Período de Replicação do DNA , Embrião de Mamíferos , Desenvolvimento Embrionário , Instabilidade Genômica , Animais , Feminino , Masculino , Camundongos , Blastocisto/citologia , Blastocisto/metabolismo , Aberrações Cromossômicas/efeitos dos fármacos , Segregação de Cromossomos , Dano ao DNA/efeitos dos fármacos , Reparo do DNA , Período de Replicação do DNA/efeitos dos fármacos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/embriologia , Desenvolvimento Embrionário/genética , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/genética , Fase S/efeitos dos fármacos , Fase S/genética , Análise de Célula Única , Pontos de Quebra do Cromossomo , Divisão Celular , Nucleosídeos/metabolismo , Nucleosídeos/farmacologia , DNA Polimerase Dirigida por DNA/metabolismo , Complexos Multienzimáticos/metabolismoRESUMO
Oogenesis is the central process required to produce viable oocytes in female mammals. It is initiated during embryonic development, and it involves the specification of primordial germ cells (PGCs) and progresses through the activation of the meiotic program, reaching a crucial phase in prophase I before pausing at diplotene around the time of birth. The significance of meiosis, particularly the prophase I stage, cannot be overstated, as it plays a pivotal role in ensuring the formation of healthy gametes, a prerequisite for successful reproduction. While research has explored meiosis across various organisms, understanding how environmental factors, including radiation, drugs, endocrine disruptors, reproductive age, or diet, influence this complex developmental process remains incomplete. In this chapter, we describe an ex vivo culture method to investigate meiotic prophase I and beyond and the disruption of oogenesis by external factors. Using this methodology, it is possible to evaluate the effects of individual xenobiotics by administering chemicals at specific points during oogenesis. This culture technique was optimized to study the effects of two selected endocrine disruptors (vinclozolin and MEHP), demonstrating that vinclozolin exposure delayed meiotic differentiation and MEHP exposure reduced follicle size. This approach also opens avenues for future applications, involving the exploration of established or novel pharmaceutical substances and their influence on essential events during prophase I, such as homologous recombination and chromosome segregation. These processes collectively dictate the ultimate fitness of oocytes, with potential implications for factors relevant to the reproductive age and fertility.
Assuntos
Meiose , Ovário , Animais , Feminino , Camundongos , Ovário/citologia , Meiose/efeitos dos fármacos , Oogênese/efeitos dos fármacos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Prófase Meiótica I/efeitos dos fármacos , Disruptores Endócrinos/farmacologia , Oxazóis/farmacologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacosRESUMO
Morphogen gradients are critical regulators of embryonic development. In this issue, Liu et al.1 introduce a microfluidic system that externally applies morphogen gradients to an in vitro model of human embryo segmentation. It enables the investigation of signaling gradients during this developmental process at unprecedented levels of precision.
Assuntos
Desenvolvimento Embrionário , Humanos , Embrião de Mamíferos/citologia , Padronização Corporal , Microfluídica , AnimaisRESUMO
During the epithelial-mesenchymal transition driving mouse embryo gastrulation, cells divide more frequently at the primitive streak, and half of those divisions happen away from the apical pole. These observations suggest that non-apical mitoses might play a role in cell delamination. We aim to uncover and challenge the molecular determinants of mitosis position in different regions of the epiblast through computational modeling and pharmacological treatments of embryos and stem cell-based epiblast spheroids. Blocking basement membrane degradation at the streak has no impact on the asymmetry in mitosis frequency and position. By contrast, disturbance of the actomyosin cytoskeleton or cell cycle dynamics elicits ectopic non-apical mitosis and shows that the streak region is characterized by local relaxation of the actomyosin cytoskeleton and less stringent regulation of cell division. These factors are essential for normal dynamics at the streak and favor cell delamination from the epiblast.
Assuntos
Actomiosina , Gastrulação , Camadas Germinativas , Mitose , Animais , Camundongos , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Actomiosina/metabolismo , Transição Epitelial-Mesenquimal , Embrião de Mamíferos/citologia , Linha Primitiva/citologia , Linha Primitiva/metabolismo , Feminino , Membrana Basal/metabolismo , Citoesqueleto/metabolismo , Ciclo CelularRESUMO
Regenerative therapy is considered a novel option for treating various diseases, whereas a developing embryo is a prime source of molecules that help repair diseased tissue and organs. Organoid culture studies also confirmed the inherent biological functions of several embryonic factors. However, the in vivo safety and efficacy of embryonic protein fraction (EPF) were not validated. In this study, we investigated the effectiveness of EPF on healthy adult rats. We obtained embryos from Sprague-Dawley (SD) female rats of E14, E16, and E19 embryonic days and collected protein lysate. This lysate was administered intravenously into adult SD rats on sequential days. We collected blood and performed hematological and biochemical parameters of rats that received EPF. C-reactive protein levels, interleukin-6, blood glucose levels, serum creatinine, blood urea, total leucocyte counts, and % of neutrophils and lymphocytes were comparable between rats receiving EPF and saline. Histological examination of rats' tissues administered with EPF is devoid of abnormalities. Our study revealed that intravenous administration of EPF to healthy adult rats showed that EPF is non-immunogenic, non-inflammatory, non-tumorigenic, and safe for in vivo applications. Our analysis suggests that EPF or its components could be recommended for validating its therapeutic abilities in organ regenerative therapy.
Assuntos
Medicina Regenerativa , Animais , Ratos , Feminino , Medicina Regenerativa/métodos , Ratos Sprague-Dawley , Embrião de Mamíferos/citologiaRESUMO
Thymic epithelial cells (TECs) are crucial to the ability of the thymus to generate T cells for the adaptive immune system in vertebrates. However, no in vitro system for studying TEC function exists. Overexpressing the transcription factor FOXN1 initiates transdifferentiation of fibroblasts into TEC-like cells (iTECs) that support T-cell differentiation in culture or after transplant. In this study, we have characterized iTEC programming at the cellular and molecular level in mouse to determine how it proceeds, and have identified mechanisms that can be targeted for improving this process. These data show that iTEC programming consists of discrete gene expression changes that differ early and late in the process, and that iTECs upregulate markers of both cortical and medullary TEC (cTEC and mTEC) lineages. We demonstrate that promoting proliferation enhances iTEC generation, and that Notch inhibition allows the induction of mTEC differentiation. Finally, we show that MHCII expression is the major difference between iTECs and fetal TECs. MHCII expression was improved by co-culturing iTECs with fetal double-positive T-cells. This study supports future efforts to improve iTEC generation for both research and translational uses.
Assuntos
Diferenciação Celular , Células Epiteliais , Fibroblastos , Fatores de Transcrição Forkhead , Timo , Animais , Células Epiteliais/metabolismo , Células Epiteliais/citologia , Timo/citologia , Timo/metabolismo , Timo/embriologia , Fibroblastos/metabolismo , Fibroblastos/citologia , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição Forkhead/genética , Camundongos , Proliferação de Células , Transdiferenciação Celular , Linfócitos T/citologia , Linfócitos T/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Técnicas de Cocultura , Receptores Notch/metabolismoRESUMO
Red blood cells (RBCs) comprise a critical component of the cardiovascular network, which constitutes the first functional organ system of the developing mammalian embryo. Examination of circulating blood cells in mammalian embryos revealed two distinct types of erythroid cells: large, nucleated "primitive" erythroblasts followed by smaller, enucleated "definitive" erythrocytes. This review describes the current understanding of primitive and definitive erythropoiesis gleaned from studies of mouse and human embryos and induced pluripotent stem cells (iPSCs). Primitive erythropoiesis in the mouse embryo comprises a transient wave of committed primitive erythroid progenitors (primitive erythroid colony-forming cells, EryP-CFC) in the early yolk sac that generates a robust cohort of precursors that mature in the bloodstream and enucleate. In contrast, definitive erythropoiesis has two distinct developmental origins. The first comprises a transient wave of definitive erythroid progenitors (burst-forming units erythroid, BFU-E) that emerge in the yolk sac and seed the fetal liver where they terminally mature to provide the first definitive RBCs. The second comprises hematopoietic stem cell (HSC)-derived BFU-E that terminally mature at sites colonized by HSCs particularly the fetal liver and subsequently the bone marrow. Primitive and definitive erythropoiesis are derived from endothelial identity precursors with distinct developmental origins. Although they share prototypical transcriptional regulation, primitive and definitive erythropoiesis are also characterized by distinct lineage-specific factors. The exquisitely timed, sequential production of primitive and definitive erythroid cells is necessary for the survival and growth of the mammalian embryo.
Assuntos
Embrião de Mamíferos , Eritropoese , Eritropoese/fisiologia , Animais , Humanos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Camundongos , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Saco Vitelino/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismoRESUMO
Mouse lung branching morphogenesis creates epithelial tree structures required for respiration. Here, we present a protocol for studying mouse lung developmental branching using lung explant cultures. We describe steps for isolating lungs with a video at embryonic day 12.5 (E12.5) and culturing as an explant for 2 days. We also detail procedures for microscopic imaging on days 0-2 and analysis of peripheral lung buds. This technique has the potential to investigate lung development in various conditions. For complete details on the use and execution of this protocol, please refer to Talvi et al.1.
Assuntos
Pulmão , Animais , Camundongos , Pulmão/embriologia , Pulmão/citologia , Embrião de Mamíferos/citologia , Técnicas de Cultura de Órgãos/métodos , Morfogênese , FemininoAssuntos
Apoptose , Animais , Camundongos , Humanos , Morte Celular , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologiaRESUMO
Cellular Potts models are broadly applied across developmental biology and cancer research. We overcome limitations of the traditional approach, which reinterprets a modified Metropolis sampling as ad hoc dynamics, by introducing a physical timescale through Poissonian kinetics and by applying principles of stochastic thermodynamics to separate thermal and relaxation effects from athermal noise and nonconservative forces. Our method accurately describes cell-sorting dynamics in mouse-embryo development and identifies the distinct contributions of nonequilibrium processes, e.g., cell growth and active fluctuations.
Assuntos
Modelos Biológicos , Processos Estocásticos , Animais , Camundongos , Cinética , Termodinâmica , Desenvolvimento Embrionário/fisiologia , Embrião de Mamíferos/citologiaRESUMO
Hematopoietic stem cells (HSCs) develop from hemogenic endothelial cells (HECs) in vivo during mouse embryogenesis. When cultured in vitro, cells from the embryo phenotypically defined as pre-HSC-I and pre-HSC-II have the potential to differentiate into HSCs. However, minimal factors required for HSC induction from HECs have not yet been determined. In this study, we demonstrated that stem cell factor (SCF) and thrombopoietin (TPO) induced engrafting HSCs from embryonic day (E) 11.5 pre-HSC-I in a serum-free and feeder-free culture condition. In contrast, E10.5 pre-HSC-I and HECs required an endothelial cell layer in addition to SCF and TPO to differentiate into HSCs. A single-cell RNA sequencing analysis of E10.5 to 11.5 dorsal aortae with surrounding tissues and fetal livers detected TPO expression confined in hepatoblasts, while SCF was expressed in various tissues, including endothelial cells and hepatoblasts. Our results suggest a transition of signal requirement during HSC development from HECs. The differentiation of E10.5 HECs to E11.5 pre-HSC-I in the aorta-gonad-mesonephros region depends on SCF and endothelial cell-derived factors. Subsequently, SCF and TPO drive the differentiation of E11.5 pre-HSC-I to pre-HSC-II/HSCs in the fetal liver. The culture system established in this study provides a beneficial tool for exploring the molecular mechanisms underlying the development of HSCs from HECs.
Assuntos
Diferenciação Celular , Hemangioblastos , Células-Tronco Hematopoéticas , Fator de Células-Tronco , Trombopoetina , Animais , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Camundongos , Trombopoetina/metabolismo , Fator de Células-Tronco/metabolismo , Hemangioblastos/metabolismo , Hemangioblastos/citologia , Células Endoteliais/metabolismo , Células Endoteliais/citologia , Transdução de Sinais , Hematopoese/fisiologia , Desenvolvimento Embrionário , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/citologia , Fígado/embriologia , Fígado/metabolismo , Fígado/citologiaRESUMO
The first embryonic division represents a starting point for the development of a new individual. In many species, tight control over the first embryonic division ensures its accuracy. However, the first division in humans is often erroneous and can impair embryo development. To delineate the spatiotemporal organization of the first mitotic division typical for normal human embryo development, we systematically analyzed a unique timelapse dataset of 300 IVF embryos that developed into healthy newborns. The zygotic division pattern of these best-quality embryos was compared to their siblings that failed to implant or arrested during cleavage stage. We show that division at the right angle to the juxtaposed pronuclei is preferential and supports faithful zygotic division. Alternative configurations of the first mitosis are associated with reduced clustering of nucleoli and multinucleation at the 2-cell stage, which are more common in women of advanced age. Collectively, these data imply that orientation of the first division predisposes human embryos to genetic (in)stability and may contribute to aneuploidy and age-related infertility.