High fidelity lineage tracing in mouse pre-implantation embryos using primed conversion of photoconvertible proteins (original) (raw)
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eLife
Accurate lineage reconstruction of mammalian pre-implantation development is essential for inferring the earliest cell fate decisions. Lineage tracing using global fluorescence labeling techniques is complicated by increasing cell density and rapid embryo rotation, which hampers automatic alignment and accurate cell tracking of obtained four-dimensional imaging data sets. Here, we exploit the advantageous properties of primed convertible fluorescent proteins (pr-pcFPs) to simultaneously visualize the global green and the photoconverted red population in order to minimize tracking uncertainties over prolonged time windows. Confined primed conversion of H2B-pr-mEosFP-labeled nuclei combined with light-sheet imaging greatly facilitates segmentation, classification, and tracking of individual nuclei from the 4-cell stage up to the blastocyst. Using green and red labels as fiducial markers, we computationally correct for rotational and translational drift, reduce overall data size, and a...
Live imaging of mouse embryos during pre-implantation and peri-implantation development
Live visualisation of embryo development is a powerful tool for scientists to understand how morphogenetic events shape the embryo. Here, we report on a culturing technique that allows live imaging of pre- and peri-implantation mouse embryos throughout the process of blastocyst to egg cylinder transition, enabling single cell tracking and delineation of the tissue dynamics accompanying this morphogenetic stage. At the same time, this protocol can be used for pharmacological manipulations of mouse embryos.
Toward high-content/high-throughput imaging and analysis of embryonic morphogenesis
Genesis (New York, N.Y. : 2000), 2011
In vivo study of embryonic morphogenesis tremendously benefits from recent advances in live microscopy and computational analyses. Quantitative and automated investigation of morphogenetic processes opens the field to high-content and high-throughput strategies. Following experimental workflow currently developed in cell biology, we identify the key challenges for applying such strategies in developmental biology. We review the recent progress in embryo preparation and manipulation, live imaging, data registration, image segmentation, feature computation, and data mining dedicated to the study of embryonic morphogenesis. We discuss a selection of pioneering studies that tackled the current methodological bottlenecks and illustrated the investigation of morphogenetic processes in vivo using quantitative and automated imaging and analysis of hundreds or thousands of cells simultaneously, paving the way for high-content/high-throughput strategies and systems analysis of embryonic morphogenesis. genesis 49:555–569, 2011. © 2011 Wiley-Liss, Inc.
Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual Conference, 2009
We present a simple and parameter-free nuclei tracking method for reconstructing cell dynamics in fluorescence 3D+t images of embryogenesis. The strategy is based on the use of the mathematical morphology operators directly in the 4D image. The morphological reconstruction of a marker -manually or automatically selected- in an initial spatio-temporal position generates a connected path over the time representing the cell migration. Thus, the processing provides a coherent spatiotemporal estimation of cell movement. The algorithm has been validated on in vivo images of early zebrafish and sea urchin embryogenesis acquired with two-photon laser scanning microscopy providing mean tracking rates above 98% per time step.
Embryonic lineage analysis using three-dimensional, time-lapse in-vivo fluorescent microscopy
Bioimaging and Two-Dimensional Spectroscopy, 1990
Drosophila melanogaster has become one of the most extensively studied organisms because of its amenability to genetic analysis. Unfortunately, the biochemistry and cell biology ofDrosophila has lagged behind. To this end we have been microinjecting fluorescently labelled proteins into the living embryo and observing the behavior of these proteins to determine their role in the cell cycle and development. Imaging of these fluorescent probes is an extremely important element to this form of analysis. We have taken advantage of the sensitivity and well behaved characteristics of the charge coupled device (CCD) camera in conjunction with digital image enhancement schemes to produce highly accurate images of these fluorescent probes in vivo. One of our major goals is to produce a detailed map of cell fate so that we can understand how fate is determined and maintained. In order produce such a detailed map, protocols for following the movements and mitotic behavior of a large number of cells in three dimensions over relatively long periods of time were developed. We will present our results using fluorescently labelled histone proteins as a marker for nuclear location1. In addition, we will also present our initial results using a photoactivatable analog of fluorescein to mark single cells so that their long range fate can be unambiguously determined.
PLoS ONE, 2011
The extraction of fluorescence time course data is a major bottleneck in high-throughput live-cell microscopy. Here we present an extendible framework based on the open-source image analysis software ImageJ, which aims in particular at analyzing the expression of fluorescent reporters through cell divisions. The ability to track individual cell lineages is essential for the analysis of gene regulatory factors involved in the control of cell fate and identity decisions. In our approach, cell nuclei are identified using Hoechst, and a characteristic drop in Hoechst fluorescence helps to detect dividing cells. We first compare the efficiency and accuracy of different segmentation methods and then present a statistical scoring algorithm for cell tracking, which draws on the combination of various features, such as nuclear intensity, area or shape, and importantly, dynamic changes thereof. Principal component analysis is used to determine the most significant features, and a global parameter search is performed to determine the weighting of individual features. Our algorithm has been optimized to cope with large cell movements, and we were able to semi-automatically extract cell trajectories across three cell generations. Based on the MTrackJ plugin for ImageJ, we have developed tools to efficiently validate tracks and manually correct them by connecting broken trajectories and reassigning falsely connected cell positions. A gold standard consisting of two time-series with 15,000 validated positions will be released as a valuable resource for benchmarking. We demonstrate how our method can be applied to analyze fluorescence distributions generated from mouse stem cells transfected with reporter constructs containing transcriptional control elements of the Msx1 gene, a regulator of pluripotency, in mother and daughter cells. Furthermore, we show by tracking zebrafish PAC2 cells expressing FUCCI cell cycle markers, our framework can be easily adapted to different cell types and fluorescent markers.
Methods in Molecular Biology, 2010
A common problem in research laboratories that study the mammalian embryo is the limited supply of live material. For this reason, new methods are always developed and existing methods for cells in culture are adapted to suit this peculiar experimental model. Three-Dimensional fluorescent in situ hybridisation (3D-FISH) is an important tool to study how genomic sequences are positionned in nuclei without altering their 3D organisation. This provides vital information about the distribution of specific sequences in relation to nuclear substructures such as chromocenters and nucleoli. In this manuscript, we will present a detailed description on how to carry out 3D-FISH in the early preimplantation mouse embryo.
Reconstructing embryonic development
genesis, 2011
Novel approaches to bio-imaging and automated computational image processing allow the design of truly quantitative studies in developmental biology. Cell behavior, cell fate decisions, cell interactions during tissue morphogenesis, and gene expression dynamics can be analyzed in vivo for entire complex organisms and throughout embryonic development. We review state-of-the-art technology for live imaging, focusing on fluorescence light microscopy techniques for system-level investigations of animal development, and discuss computational approaches to image segmentation, cell tracking, automated data annotation, and biophysical modeling. We argue that the substantial increase in data complexity and size requires sophisticated new strategies to data analysis to exploit the enormous potential of these new resources. genesis 49:488-513, 2011. V V C 2010 Wiley-Liss, Inc.
Scientific Reports, 2016
Embryonic stem cells can spontaneously differentiate into cell types of all germ layers within embryoid bodies (EBs) in a highly variable manner. Whether there exists an intrinsic differentiation program common to all EBs is unknown. Here, we present a novel combination of high-throughput live twophoton imaging and gene expression profiling to study early differentiation dynamics spontaneously occurring within developing EBs. Onset timing of Brachyury-GFP was highly variable across EBs, while the spatial patterns as well as the dynamics of mesendodermal progression following onset were remarkably similar. We therefore defined a 'developmental clock' using the Brachyury-GFP signal onset timing. Mapping snapshot gene expression measurements to this clock revealed their temporal trends, indicating that loss of pluripotency, formation of primitive streak and mesodermal lineage progression are synchronized in EBs. Exogenous activation of Wnt or BMP signaling accelerated the intrinsic clock. CHIR down-regulated Wnt3, allowing insights into dependency mechanisms between canonical Wnt signaling and multiple genes. Our findings reveal a developmental clock characteristic of an early differentiation program common to all EBs, further establishing them as an in vitro developmental model. The coordinated progression of different cell lineages is essential for the formation of functional tissues and organs. Recent efforts have been focused on developing and optimizing in vitro platforms to study the mechanisms underlying stem cell differentiation as well as multi-lineage progression, using two-and three-dimensional cultures 1-4. Better definition of the constraints on multi-lineage differentiation processes in these systems would enhance their use in the study of development and developmental defects. Embryonic stem cells can be aggregated into embryoid bodies (EBs), which have the potential to differentiate to a diverse population of adult specialized cells 2,5-8. Even though EBs differentiate in a less strictly defined fashion than embryos, they display embryogenesis-like processes such as germ layer formation, ECM secretion, and primitive streak formation 2,6-8. The timing and pattern of these processes are influenced by a profusion of variables such as medium composition, growth surfaces and physical constraints 2. For example, initial EB size affects the extent of mesodermal (and subsequently cardiac) vs. ectodermal differentiation 9. The differentiation of EBs into cells of the three germ layers, even in the absence of externally added directive signals, indicates that the required signals for these processes can autonomously build up within each EB. BMP, Wnt, FGF and activin signaling pathways have all been shown to play important roles in inducing these transitions, both in vivo and in vitro 10-12. FGF signaling was shown to be essential for the onset of neural ectoderm markers 13,14. Wnt and BMP signaling components are essential for the establishment of primitive streak and mesendoderm formation and, in EBs, influence mesendoderm differentiation and axis formation 15-17. How these signals interact to coordinate the growth and relative composition of multiple lineages is not fully characterized. EBs can serve as a valuable tool for studying cell to cell signaling mediated development, and are particularly compatible with high-throughput analysis of post-implantation differentiation processes.
Using migrating cells as probes to illuminate features in live embryonic tissues
Science Advances, 2020
The biophysical and biochemical properties of live tissues are important in the context of development and disease. Methods for evaluating these properties typically involve destroying the tissue or require specialized technology and complicated analyses. Here, we present a novel, noninvasive methodology for determining the spatial distribution of tissue features within embryos, making use of nondirectionally migrating cells and software we termed “Landscape,” which performs automatized high-throughput three-dimensional image registration. Using the live migrating cells as bioprobes, we identified structures within the zebrafish embryo that affect the distribution of the cells and studied one such structure constituting a physical barrier, which, in turn, influences amoeboid cell polarity. Overall, this work provides a unique approach for detecting tissue properties without interfering with animal’s development. In addition, Landscape allows for integrating data from multiple sample...