RESUMO
During development, pseudostratified epithelia undergo large scale morphogenetic events associated with increased mechanical stress. Using a variety of genetic and imaging approaches, we uncover that in the mouse E6.5 epiblast, where apical tension is highest, ASPP2 safeguards tissue integrity. It achieves this by preventing the most apical daughter cells from delaminating apically following division events. In this context, ASPP2 maintains the integrity and organisation of the filamentous actin cytoskeleton at apical junctions. ASPP2 is also essential during gastrulation in the primitive streak, in somites and in the head fold region, suggesting that it is required across a wide range of pseudostratified epithelia during morphogenetic events that are accompanied by intense tissue remodelling. Finally, our study also suggests that the interaction between ASPP2 and PP1 is essential to the tumour suppressor function of ASPP2, which may be particularly relevant in the context of tissues that are subject to increased mechanical stress.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Epitélio/crescimento & desenvolvimento , Morfogênese , Proteínas Supressoras de Tumor/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Células CACO-2 , Polaridade Celular , Cães , Técnicas de Cultura Embrionária , Embrião de Mamíferos , Epitélio/metabolismo , Feminino , Gastrulação , Camadas Germinativas , Humanos , Células Madin Darby de Rim Canino , Camundongos , Camundongos Transgênicos , Mutação , Linha Primitiva , Receptores de Neuropeptídeo Y/metabolismo , Estresse Mecânico , Junções Íntimas/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Droplet microfluidics technology provides a powerful approach to isolate and process millions of single cells simultaneously. Despite many exciting applications that have emerged based on this technology, workflows based on multi-step operations, including molecular biology and cell-based phenotypic screening assays, cannot be easily adapted to droplet format. Here, we present a microfluidics-based technique to isolate single cells, or biological samples, into semi-permeable hydrogel capsules and perform multi-step biological workflows on thousands to millions of individual cells simultaneously. The biochemical reactions are performed by changing the aqueous buffer surrounding the capsules, without needing sophisticated equipment. The semi-permeable nature of the capsules' shell retains large encapsulated biomolecules (such as genome) while allowing smaller molecules (such as proteins) to passively diffuse. In contrast to conventional hydrogel bead assays, the approach presented here improves bacterial cell retention during multi-step procedures as well as the efficiency of biochemical reactions. We showcase two examples of capsule use for single genome amplification of bacteria, and expansion of individual clones into isogenic microcolonies for later screening for biodegradable plastic production.
Assuntos
Hidrogéis , Microfluídica , Bactérias , Cápsulas , Biologia MolecularRESUMO
Precise patterning within the three-dimensional context of tissues, organs and embryos implies that cells can sense their relative position. During preimplantation development, outside and inside cells rely on apicobasal polarity and the Hippo pathway to choose their fate. Despite recent findings suggesting that mechanosensing might be central to this process, the relationship between blastomere geometry (i.e. shape and position) and the Hippo pathway effector YAP remains unknown. We used a highly quantitative approach to analyse information on the geometry and YAP localisation of individual blastomeres of mouse and human embryos. We identified the proportion of exposed cell surface area as most closely correlating with the nuclear localisation of YAP. To test this relationship, we developed several hydrogel-based approaches to alter blastomere geometry in cultured embryos. Unbiased clustering analyses of blastomeres from such embryos revealed that this relationship emerged during compaction. Our results therefore pinpoint the time during early embryogenesis when cells acquire the ability to sense changes in geometry and provide a new framework for how cells might integrate signals from different membrane domains to assess their relative position within the embryo.
Assuntos
Blastocisto/citologia , Blastocisto/metabolismo , Blastômeros/metabolismo , Animais , Blastômeros/citologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Advances in fluorescence microscopy approaches have made it relatively easy to generate multi-dimensional image volumes and have highlighted the need for flexible image analysis tools for the extraction of quantitative information from such data. Here we demonstrate that by focusing on simplified feature-based nuclear segmentation and probabilistic cytoplasmic detection we can create a tool that is able to extract geometry-based information from diverse mammalian tissue images. Our open-source image analysis platform, called 'SilentMark', can cope with three-dimensional noisy images and with crowded fields of cells to quantify signal intensity in different cellular compartments. Additionally, it provides tissue geometry related information, which allows one to quantify protein distribution with respect to marked regions of interest. The lightweight SilentMark algorithms have the advantage of not requiring multiple processors, graphics cards or training datasets and can be run even with just several hundred megabytes of memory. This makes it possible to use the method as a Web application, effectively eliminating setup hurdles and compatibility issues with operating systems. We test this platform on mouse pre-implantation embryos, embryonic stem cell-derived embryoid bodies and mouse embryonic heart, and relate protein localization to tissue geometry. This article is part of a discussion meeting issue 'Contemporary morphogenesis'.
Assuntos
Embrião de Mamíferos/embriologia , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/instrumentação , Microscopia de Fluorescência/métodos , Proteínas/análise , Animais , Núcleo Celular/fisiologia , CamundongosRESUMO
A long cylindrical cavity through a soft solid forms a soft microfluidic channel, or models a vascular capillary. We observe experimentally that, when such a channel bears a pressurized fluid, it first dilates homogeneously, but then becomes unstable to a peristaltic elastic instability. We combine theory and numerics to fully characterize the instability in a channel with initial radius a through an incompressible bulk neo-Hookean solid with shear modulus µ. We show instability occurs supercritically with wavelength 12.278 a when the cavity pressure exceeds 2.052 µ. In finite solids, the wavelength for peristalsis lengthens, with peristalsis ultimately being replaced by a long-wavelength bulging instability in thin-walled cylinders. Peristalsis persists in Gent strain-stiffening materials, provided the material can sustain extension by more than a factor of 6. Although naively a pressure driven failure mode of soft channels, the instability also offers a route to fabricate periodically undulating channels, producing, e.g., waveguides with photonic or phononic stop bands.
RESUMO
Biological samples are inherently heterogeneous and complex. Tackling this complexity requires innovative technological and analytical solutions. Recent advances in high-throughput single-cell isolation and nucleic acid barcoding methods are rapidly changing the technological landscape of biological sciences and now make it possible to measure the (epi)genomic, transcriptomic, or proteomic state of individual cells. In addition, few experimental approaches enable multi-omics measurements of the same cell. However, merging-omics data collected from different experiments remains a considerable challenge. Although several strategies for merging transcriptomics datasets have recently been introduced, cell-to-cell variability and heterogeneity remains one of the confounding factors limiting data fusion and integration. Here, we focus our discussion on the latest single-cell technological and analytical solutions to achieve high data dimensionality and resolution. Obtaining datasets with a wealth of multi-omics information will undoubtedly provide new avenues for researchers to unravel the complexity of biological samples encountered in modern biological research and molecular diagnostics.
Assuntos
Análise de Dados , Genômica/métodos , Análise de Célula Única/métodos , Animais , Bases de Dados como Assunto , Epigênese Genética , Humanos , Transcriptoma/genéticaRESUMO
Mammalian embryos are surrounded by an acellular shell, the zona pellucida. Hatching out of the zona is crucial for implantation and continued development of the embryo. Clinically, problems in hatching can contribute to failure in assisted reproductive intervention. Although hatching is fundamentally a mechanical process, due to limitations in methodology most studies focus on its biochemical properties. To understand the role of mechanical forces in hatching, we developed a hydrogel deformation-based method and analytical approach for measuring pressure in cyst-like tissues. Using this approach, we found that, in cultured blastocysts, pressure increased linearly, with intermittent falls. Inhibition of Na/K-ATPase led to a dosage-dependent reduction in blastocyst cavity pressure, consistent with its requirement for cavity formation. Reducing blastocyst pressure reduced the probability of hatching, highlighting the importance of mechanical forces in hatching. These measurements allowed us to infer details of microphysiology such as osmolarity, ion and water transport kinetics across the trophectoderm, and zona stiffness, allowing us to model the embryo as a thin-shell pressure vessel. We applied this technique to test whether cryopreservation, a process commonly used in assisted reproductive technology (ART), leads to alteration of the embryo and found that thawed embryos generated significantly lower pressure than fresh embryos, a previously unknown effect of cryopreservation. We show that reduced pressure is linked to delayed hatching. Our approach can be used to optimize in vitro fertilization (IVF) using precise measurement of embryo microphysiology. It is also applicable to other biological systems involving cavity formation, providing an approach for measuring forces in diverse contexts.
Assuntos
Blastocisto/fisiologia , Embrião de Mamíferos/fisiologia , Desenvolvimento Embrionário/fisiologia , Fertilização in vitro , Hidrogéis/química , Zona Pelúcida/fisiologia , Animais , Blastocisto/citologia , Células Cultivadas , Criopreservação , Transferência Embrionária , Embrião de Mamíferos/citologia , Feminino , Camundongos , GravidezRESUMO
Elongation of the body axis is a key aspect of body plan development. Bipotential neuromesoderm progenitors (NMPs) ensure axial growth of embryos by contributing both to the spinal cord and mesoderm. The current model for the mechanism controlling NMP deployment invokes Tbx6, a T-box factor, to drive mesoderm differentiation of NMPs. Here, we identify a new population of Tbx6+ cells in a subdomain of the NMP niche in mouse embryos. Based on co-expression of a progenitor marker, Sox2, we identify this population as representing a transient cell state in the mesoderm-fated NMP lineage. Genetic lineage tracing confirms the presence of the Tbx6+ NMP cell state. Furthermore, we report a novel aspect of the documented Tbx6 mutant phenotype, namely an increase from two to four ectopic neural tubes, corresponding to the switch in NMP niche, thus highlighting the importance of Tbx6 function in NMP fate decision. This study emphasizes the function of Tbx6 as a bistable switch that turns mesoderm fate 'on' and progenitor state 'off', and thus has implications for the molecular mechanism driving NMP fate choice.
Assuntos
Células-Tronco Embrionárias/citologia , Mesoderma/citologia , Tubo Neural/embriologia , Fatores de Transcrição SOXB1/biossíntese , Medula Espinal/embriologia , Fatores de Transcrição/biossíntese , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Diferenciação Celular , Linhagem da Célula , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Transgênicos , Tubo Neural/citologia , Fatores de Transcrição SOXB1/genética , Proteínas com Domínio T , Fatores de Transcrição/genéticaRESUMO
A very simple, environmentally friendly, one-step oxidative polymerization route to fabricate polypyrrole (Ppy) nanoparticles of fixed size and morphology was developed and investigated. The herein proposed method is based on the application of sodium dodecyl sulfate and hydrogen peroxide, both easily degradable and cheap materials. The polymerization reaction is performed on 24 h time scale under standard conditions. We monitored a polaronic peak at 465 nm and estimated nanoparticle concentration during various stages of the reaction. Using this data we proposed a mechanism for Ppy nanoparticle formation in accordance with earlier emulsion polymerization mechanisms. Rates of various steps in the polymerization mechanism were accounted for and the resulting particles identified using atomic force microscopy. Application of Ppy nanoparticles prepared by the route presented here seems very promising for biomedical applications where biocompatibility is paramount. In addition, this kind of synthesis could be suitable for the development of solar cells, where very pure and low-cost conducting polymers are required.