RESUMO
Embryo implantation into the uterus marks a key transition in mammalian development. In mice, implantation is mediated by the trophoblast and is accompanied by a morphological transition from the blastocyst to the egg cylinder. However, the roles of trophoblast-uterine interactions in embryo morphogenesis during implantation are poorly understood due to inaccessibility in utero and the remaining challenges to recapitulate it ex vivo from the blastocyst. Here, we engineer a uterus-like microenvironment to recapitulate peri-implantation development of the whole mouse embryo ex vivo and reveal essential roles of the physical embryo-uterine interaction. We demonstrate that adhesion between the trophoblast and the uterine matrix is required for in utero-like transition of the blastocyst to the egg cylinder. Modeling the implanting embryo as a wetting droplet links embryo shape dynamics to the underlying changes in trophoblast adhesion and suggests that the adhesion-mediated tension release facilitates egg cylinder formation. Light-sheet live imaging and the experimental control of the engineered uterine geometry and trophoblast velocity uncovers the coordination between trophoblast motility and embryo growth, where the trophoblast delineates space for embryo morphogenesis.
Assuntos
Blastocisto , Implantação do Embrião , Feminino , Camundongos , Animais , Trofoblastos , Útero , Desenvolvimento Embrionário , MamíferosRESUMO
Biological systems are highly complex, yet notably ordered structures can emerge. During syncytial stage development of the Drosophila melanogaster embryo, nuclei synchronously divide for nine cycles within a single cell, after which most of the nuclei reach the cell cortex. The arrival of nuclei at the cortex occurs with remarkable positional order, which is important for subsequent cellularisation and morphological transformations. Yet, the mechanical principles underlying this lattice-like positional order of nuclei remain untested. Here, using quantification of nuclei position and division orientation together with embryo explants, we show that short-ranged repulsive interactions between microtubule asters ensure the regular distribution and maintenance of nuclear positions in the embryo. Such ordered nuclear positioning still occurs with the loss of actin caps and even the loss of the nuclei themselves; the asters can self-organise with similar distribution to nuclei in the wild-type embryo. The explant assay enabled us to deduce the nature of the mechanical interaction between pairs of nuclei. We used this to predict how the nuclear division axis orientation changes upon nucleus removal from the embryo cortex, which we confirmed in vivo with laser ablation. Overall, we show that short-ranged microtubule-mediated repulsive interactions between asters are important for ordering in the early Drosophila embryo and minimising positional irregularity.
Assuntos
Blastoderma/metabolismo , Divisão do Núcleo Celular , Células Gigantes/metabolismo , Animais , Blastoderma/citologia , Núcleo Celular/metabolismo , Drosophila melanogaster , Células Gigantes/citologia , Microtúbulos/metabolismo , Estresse MecânicoRESUMO
Upon implantation, mammalian embryos undergo major morphogenesis and key developmental processes such as body axis specification and gastrulation. However, limited accessibility obscures the study of these crucial processes. Here, we develop an ex vivo Matrigel-collagen-based culture to recapitulate mouse development from E4.5 to E6.0. Our system not only recapitulates embryonic growth, axis initiation, and overall 3D architecture in 49% of the cases, but its compatibility with light-sheet microscopy also enables the study of cellular dynamics through automatic cell segmentation. We find that, upon implantation, release of the increasing tension in the polar trophectoderm is necessary for its constriction and invagination. The resulting extra-embryonic ectoderm plays a key role in growth, morphogenesis, and patterning of the neighboring epiblast, which subsequently gives rise to all embryonic tissues. This 3D ex vivo system thus offers unprecedented access to peri-implantation development for in toto monitoring, measurement, and spatiotemporally controlled perturbation, revealing a mechano-chemical interplay between extra-embryonic and embryonic tissues.
Assuntos
Implantação do Embrião , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Animais , Padronização Corporal , Ectoderma/citologia , Aprendizado de Máquina , Camundongos Endogâmicos C57BL , Microcirurgia , Morfogênese , Trofoblastos/citologiaRESUMO
Visualizing dynamic processes over large, three-dimensional fields of view at high speed is essential for many applications in the life sciences. Light-field microscopy (LFM) has emerged as a tool for fast volumetric image acquisition, but its effective throughput and widespread use in biology has been hampered by a computationally demanding and artifact-prone image reconstruction process. Here, we present a framework for artificial intelligence-enhanced microscopy, integrating a hybrid light-field light-sheet microscope and deep learning-based volume reconstruction. In our approach, concomitantly acquired, high-resolution two-dimensional light-sheet images continuously serve as training data and validation for the convolutional neural network reconstructing the raw LFM data during extended volumetric time-lapse imaging experiments. Our network delivers high-quality three-dimensional reconstructions at video-rate throughput, which can be further refined based on the high-resolution light-sheet images. We demonstrate the capabilities of our approach by imaging medaka heart dynamics and zebrafish neural activity with volumetric imaging rates up to 100 Hz.
Assuntos
Aprendizado Profundo , Coração/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Animais , Fenômenos Biomecânicos , Cálcio/química , Larva/fisiologia , Oryzias/fisiologia , Reprodutibilidade dos Testes , Peixe-Zebra/fisiologiaRESUMO
Marine invertebrate ascidians display embryonic reproducibility: Their early embryonic cell lineages are considered invariant and are conserved between distantly related species, despite rapid genomic divergence. Here, we address the drivers of this reproducibility. We used light-sheet imaging and automated cell segmentation and tracking procedures to systematically quantify the behavior of individual cells every 2 minutes during Phallusia mammillata embryogenesis. Interindividual reproducibility was observed down to the area of individual cell contacts. We found tight links between the reproducibility of embryonic geometries and asymmetric cell divisions, controlled by differential sister cell inductions. We combined modeling and experimental manipulations to show that the area of contact between signaling and responding cells is a key determinant of cell communication. Our work establishes the geometric control of embryonic inductions as an alternative to classical morphogen gradients and suggests that the range of cell signaling sets the scale at which embryonic reproducibility is observed.
Assuntos
Urocordados/citologia , Urocordados/embriologia , Animais , Comunicação Celular , Divisão Celular , Rastreamento de Células , ReproduçãoRESUMO
Single-cell RNA sequencing (scRNA-seq) provides a leap forward in resolving cellular diversity and developmental trajectories but fails to comprehensively delineate the spatial organization and precise cellular makeup of individual embryos. Here, we reconstruct from scRNA-seq and light sheet imaging data a canonical digital embryo that captures the genome-wide gene expression trajectory of every single cell at every cell division in the 18 lineages up to gastrulation in the ascidian Phallusia mammillata. By using high-coverage scRNA-seq, we devise a computational framework that stratifies single cells of individual embryos into cell types without prior knowledge. Unbiased transcriptome data analysis mapped each cell's physical position and lineage history, yielding the complete history of gene expression at the genome-wide level for every single cell in a developing embryo. A comparison of individual embryos reveals both extensive reproducibility between symmetric embryo sides and a large inter-embryonic variability due to small differences in embryogenesis timing.
Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Linhagem da Célula/genética , Cordados/genética , Biologia Computacional/métodos , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Reprodutibilidade dos Testes , Transcriptoma/genética , Urocordados/genéticaRESUMO
Three-dimensional live imaging has become an indispensable technique in the fields of cell, developmental and neural biology. Precise spatio-temporal manipulation of biological entities is often required for a deeper functional understanding of the underlying biological process. Here we present a home-built integrated framework and optical design that combines three-dimensional light-sheet imaging over time with precise spatio-temporal optical manipulations induced by short infrared laser pulses. We demonstrate their potential for sub-cellular ablation of neurons and nuclei, tissue cauterization and optogenetics by using the Drosophila melanogaster and zebrafish model systems.
Assuntos
Microscopia , Animais , Drosophila melanogaster/fisiologia , Imageamento Tridimensional/métodos , Raios Infravermelhos , Lasers , Peixe-Zebra/fisiologiaRESUMO
To capture highly dynamic biological processes at cellular resolution is a recurring challenge in biology. Here we show that combining selective-volume illumination with simultaneous acquisition of orthogonal light fields yields three-dimensional images with high, isotropic spatial resolution and a significant reduction of reconstruction artefacts, thereby overcoming current limitations of light-field microscopy implementations. We demonstrate medaka heart and blood flow imaging at single-cell resolution and free of motion artefacts at volume rates of up to 200 Hz.
Assuntos
Coração/diagnóstico por imagem , Coração/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Análise de Célula Única/métodos , Algoritmos , Animais , Animais Geneticamente Modificados , Artefatos , Velocidade do Fluxo Sanguíneo , Humanos , Imageamento Tridimensional/métodos , OryziasRESUMO
Extraembryonic tissues contribute to animal development, which often entails spreading over embryo or yolk. Apart from changes in cell shape, the requirements for this tissue spreading are not well understood. Here, we analyze spreading of the extraembryonic serosa in the scuttle fly Megaselia abdita. The serosa forms from a columnar blastoderm anlage, becomes a squamous epithelium, and eventually spreads over the embryo proper. We describe the dynamics of this process in long-term, whole-embryo time-lapse recordings, demonstrating that free serosa spreading is preceded by a prolonged pause in tissue expansion. Closer examination of this pause reveals mechanical coupling to the underlying yolk sac, which is later released. We find mechanical coupling prolonged and serosa spreading impaired after knockdown of M. abdita Matrix metalloprotease 1. We conclude that tissue-tissue interactions provide a critical functional element to constrain spreading epithelia.
Assuntos
Dípteros/embriologia , Embrião não Mamífero/metabolismo , Membranas Extraembrionárias/metabolismo , Saco Vitelino/embriologia , Âmnio/citologia , Âmnio/embriologia , Animais , Blastoderma/citologia , Forma Celular , Dípteros/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Membrana Serosa/citologia , Membrana Serosa/embriologia , Imagem com Lapso de TempoRESUMO
Embryogenesis relies on instructions provided by spatially organized signaling molecules known as morphogens. Understanding the principles behind morphogen distribution and how cells interpret locally this information remains a major challenge in developmental biology. Here, we introduce morphogen-age measurements as a novel approach to test models of morphogen gradient formation. Using a tandem fluorescent timer as a protein age sensor, we find a gradient of increasing age of Bicoid along the anterior-posterior axis in the early Drosophila embryo. Quantitative analysis of the protein age distribution across the embryo reveals that the synthesis-diffusion-degradation model is the most likely model underlying Bicoid gradient formation, and rules out other hypotheses for gradient formation. Moreover, we show that the timer can detect transitions in the dynamics associated with syncytial cellularization. Our results provide new insight into Bicoid gradient formation and demonstrate how morphogen-age information can complement knowledge about movement, abundance, and distribution, which should be widely applicable to other systems.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Imunofluorescência/métodos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Imagem Óptica/métodos , Transativadores/genética , Animais , Padronização Corporal/genética , Proteínas de Drosophila/biossíntese , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/diagnóstico por imagem , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/biossíntese , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Estabilidade Proteica , Transporte Proteico , Proteólise , Transdução de Sinais , Transativadores/biossíntese , Proteína Vermelha FluorescenteRESUMO
At the beginning of mammalian life, the genetic material from each parent meets when the fertilized egg divides. It was previously thought that a single microtubule spindle is responsible for spatially combining the two genomes and then segregating them to create the two-cell embryo. We used light-sheet microscopy to show that two bipolar spindles form in the zygote and then independently congress the maternal and paternal genomes. These two spindles aligned their poles before anaphase but kept the parental genomes apart during the first cleavage. This spindle assembly mechanism provides a potential rationale for erroneous divisions into more than two blastomeric nuclei observed in mammalian zygotes and reveals the mechanism behind the observation that parental genomes occupy separate nuclear compartments in the two-cell embryo.
Assuntos
Segregação de Cromossomos , Embrião de Mamíferos/embriologia , Herança Materna/genética , Herança Paterna/genética , Polos do Fuso/metabolismo , Zigoto/metabolismo , Anáfase , Animais , Blastômeros/citologia , Núcleo Celular/metabolismo , Feminino , Genoma , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismoRESUMO
Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.
Assuntos
Caenorhabditis elegans/metabolismo , Corrente Citoplasmática , Retículo Endoplasmático/metabolismo , Microtúbulos/metabolismo , Animais , Grânulos Citoplasmáticos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Hidrodinâmica , Microscopia Confocal , Interferência de RNA , Imagem com Lapso de Tempo , Xenopus laevis , Zigoto/metabolismoRESUMO
Cellular structures are hydrodynamically interconnected, such that force generation in one location can move distal structures. One example of this phenomenon is cytoplasmic streaming, whereby active forces at the cell cortex induce streaming of the entire cytoplasm. However, it is not known how the spatial distribution and magnitude of these forces move distant objects within the cell. To address this issue, we developed a computational method that used cytoplasm hydrodynamics to infer the spatial distribution of shear stress at the cell cortex induced by active force generators from experimentally obtained flow field of cytoplasmic streaming. By applying this method, we determined the shear-stress distribution that quantitatively reproduces in vivo flow fields in Caenorhabditis elegans embryos and mouse oocytes during meiosis II. Shear stress in mouse oocytes were predicted to localize to a narrower cortical region than that with a high cortical flow velocity and corresponded with the localization of the cortical actin cap. The predicted patterns of pressure gradient in both species were consistent with species-specific cytoplasmic streaming functions. The shear-stress distribution inferred by our method can contribute to the characterization of active force generation driving biological streaming.
Assuntos
Caenorhabditis elegans/embriologia , Corrente Citoplasmática , Oócitos/metabolismo , Animais , Teorema de Bayes , Hidrodinâmica , Funções Verossimilhança , Camundongos , Modelos Biológicos , Estresse MecânicoRESUMO
Tackling modern cell and developmental biology questions requires fast 3D imaging with sub-cellular resolution over extended periods of time. Fluorescence microscopy has emerged as a powerful tool to image biological samples with high spatial and temporal resolution with molecular specificity. In particular, the highly efficient illumination and detection scheme of light-sheet fluorescence microscopy is starting to revolutionize the way we can monitor cellular and developmental processes in vivo. Here we summarize the state-of-the art of light-sheet imaging with a focus on mammalian development - from instrumentation, mounting and sample handling to data processing.
Assuntos
Mamíferos/embriologia , Microscopia de Fluorescência/métodos , Envelhecimento/fisiologia , Animais , Desenvolvimento Embrionário , Imageamento TridimensionalRESUMO
We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5-12-fold compared with their conventional diffraction-limited LS analogs.
RESUMO
Animal development is marked by the repeated reorganization of cells and cell populations, which ultimately determine form and shape of the growing organism. One of the central questions in developmental biology is to understand precisely how cells reorganize, as well as how and to what extent this reorganization is coordinated. While modern microscopes can record video data for every cell during animal development in 3D+t, analyzing these videos remains a major challenge: reconstruction of comprehensive cell tracks turned out to be very demanding especially with decreasing data quality and increasing cell densities. In this paper, we present an analysis pipeline for coordinated cellular motions in developing embryos based on the optical flow of a series of 3D images. We use numerical integration to reconstruct cellular long-term motions in the optical flow of the video, we take care of data validation, and we derive a LIC-based, dense flow visualization for the resulting pathlines. This approach allows us to handle low video quality such as noisy data or poorly separated cells, and it allows the biologists to get a comprehensive understanding of their data by capturing dynamic growth processes in stills. We validate our methods using three videos of growing fruit fly embryos.
Assuntos
Movimento Celular/fisiologia , Gráficos por Computador , Imageamento Tridimensional/métodos , Microscopia/métodos , Algoritmos , Animais , Drosophila/embriologia , Embrião não MamíferoRESUMO
Despite its importance for understanding human infertility and congenital diseases, early mammalian development has remained inaccessible to in toto imaging. We developed an inverted light-sheet microscope that enabled us to image mouse embryos from zygote to blastocyst, computationally track all cells and reconstruct a complete lineage tree of mouse pre-implantation development. We used this unique data set to show that the first cell fate specification occurs at the 16-cell stage.
Assuntos
Blastocisto/citologia , Microscopia/instrumentação , Microscopia/métodos , Animais , Imageamento Tridimensional/instrumentação , Imageamento Tridimensional/métodos , Camundongos , Imagem com Lapso de Tempo/instrumentação , Imagem com Lapso de Tempo/métodosRESUMO
Selective-plane illumination microscopy has proven to be a powerful imaging technique due to its unsurpassed acquisition speed and gentle optical sectioning. However, even in the case of multiview imaging techniques that illuminate and image the sample from multiple directions, light scattering inside tissues often severely impairs image contrast. Here we combine multiview light-sheet imaging with electronic confocal slit detection implemented on modern camera sensors. In addition to improved imaging quality, the electronic confocal slit detection doubles the acquisition speed in multiview setups with two opposing illumination directions allowing simultaneous dual-sided illumination. Confocal multiview light-sheet microscopy eliminates the need for specimen-specific data fusion algorithms, streamlines image post-processing, easing data handling and storage.
RESUMO
Morphogenesis of an organism requires the development of its parts to be coordinated in time and space. While past studies concentrated on defined cell populations, a synthetic view of the coordination of these events in a whole organism is needed for a full understanding. Drosophila gastrulation begins with the embryo forming a ventral furrow, which is eventually internalized. It is not understood how the rest of the embryo participates in this process. Here we use multiview selective plane illumination microscopy coupled with infrared laser manipulation and mutant analysis to dissect embryo-scale cell interactions during early gastrulation. Lateral cells have a denser medial-apical actomyosin network and shift ventrally as a compact cohort, whereas dorsal cells become stretched. We show that the behaviour of these cells affects furrow internalization. A computational model predicts different mechanical properties associated with tissue behaviour: lateral cells are stiff, whereas dorsal cells are soft. Experimental analysis confirms these properties in vivo.