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[
International Worm Meeting,
2013]
WormGUIDES (Global Understanding in Dynamic Embryonic Systems) is a novel resource that aims to create and share the first 4D atlas with single cell resolution of embryogenesis and neurodevelopment for any animal. The first goal of WormGUIDES is production of an interactive atlas of nuclear positions from zygote until hatching. A simple navigation program for computers or hand-held devices facilitates the use of WormGUIDES as a reference tool in cell identification, quantification of developmental processes and visualization of nascent patterns and symmetries in the embryo. This program and data will be demonstrated at the meeting. WormGUIDES will also incorporate a complementary atlas of neurodevelopmental processes-neurite outgrowth and synaptogenesis-that will facilitate a dynamic understanding of the connectome emerging throughout development. Building on the C. elegans community's open-sharing of systems-level knowledge and resources, WormGUIDES will further enhance the value of C. elegans as a model organism.
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[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
Left-right (LR) patterning is an intriguing but poorly understood process of bilaterian embryogenesis. We report a novel mechanism to break LR symmetry, whereby the embryo uncouples its midline from the anteroposterior (AP) axis. Specifically, the eight-cell C. elegans embryo establishes a midline that tilts rightward from the AP axis and positions more cells on the left, allowing subsequent differential LR fate inductions. To establish the tilted midline, cells exhibit LR asymmetric protrusions and a handed collective movement. This process, termed chiral morphogenesis, is based on differential regulation of cortical contractility between a pair of sister cells that are bilateral counterparts fate-wise, and is activated by non-canonical Wnt signaling. Chiral morphogenesis is timed by the division furrow of a neighbor of the sister pair, suggesting a nov el developmental clock and a novel signaling mechanism from the contractile ring to adjacent cells.
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[
International Worm Meeting,
2005]
The invariant cell lineage of C. elegans provides a powerful scheme to understand how the information stored in an organisms genome directs its development. We are developing methods that will exploit the lineage to define gene expression patterns throughout development at the single-cell level with high temporal resolution. Conceptually, the system is based on 3D, time-lapse images of developing embryos expressing histone-GFP fusions in each cell. We will track each nucleus through movement, division and death by automated image analysis, thus automatically construct the lineage and determine the identity of each cell. Simultaneously, we will detect a second color reporter expressed under the control of a candidate transcriptional regulatory region and map the resulting temporal and spatial expression patterns onto the embryonic lineage.We have obtained a universally and brightly nuclear-labeled worm strain. Using a confocal microscope, we are able to image the whole embryogenesis at one-minute interval and 30 z-planes (1 um apart) per time point without apparent disturbance of development. We have also developed image analysis algorithms to trace the lineage. For all but the final embryonic stages, our software correctly finds >99.5% of the cells, traces >99.5% of non-dividing cells across time points and correctly assigns >90% of newborn daughter cells to the mother. Thus, we can automatically construct the lineage to the 300-cell stage with 20 to 30 errors. We estimate that it would take about 30 minutes of human effort per lineage to correct these errors with the aid of a proper graphic interface. A prototype of the interface has been developed and is being optimized. Furthermore, because we do not use the known lineage to guide the image analysis, it can also be applied to lineage mutants. The software package will be freely available to the research community upon publication. One of our long-term goals is to use this system to assay the expression pattern, function and regulatory relationship of the ~600 transcription factors in C. elegans using native promoters, promoter bashing, RNAi treatment and mutant lineaging.
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[
International Worm Meeting,
2009]
Reliable, automated cell detection is a critical component of automated biological image analysis. Accuracy is particularly important for efficient, high-throughput cell lineaging. When tracing a C. elegans lineage overall detection error rates as low as one or two percent result in lineages that can require hours of hand editing to correct. To achieve more reliable automation we are investigating image processing approaches tailored to the appearance of nuclei in confocal fluorescence images. Our approach uses a Difference of Gaussians blob detector to guide an efficient extraction of the nuclear boundary as a set of disks. Preliminary results show a significant improvement in detection rates over Starrynite, our existing detection and lineaging system. Total detection error during the 9th stage of cell division drops from 4.5 to 1.1 %, overall error through the 9th round is reduced from 1.5 to .78%. The method is both accurate and fast enough to be used in real time imaging applications. Results suggest that with further development on extraction and tracking, editing will cease to be a bottleneck. This will enable automated lineaging through the previously daunting 10th and final round of cell divisions and make possible uniquely detailed, large-scale studies of embryonic development.
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[
International Worm Meeting,
2011]
The study of embryonic phenotypes at high detail and large scale requires accurate and fast assembly of detected nuclei into a cell lineage. Accuracy is essential. Every error limits analysis, requiring either manual correction or a retreat from the goal of single cell tracking. In addition, high throughput work limits the effort that can be spent on correction and quality control of results. The time it would take to confirm, unguided, the correctness of even a perfectly accurate cell lineage may be unsupportable. As such, a measure of the local reliability of results is just as important as low error. Existing methods do not to provide this combination of features. Common, simple methods such as nearest neighbor association across time are error prone and lack a model that can provide confidence in results. More sophisticated general tracking methods such as particle filters lack an explicit model of cell division, a key source of ambiguity and error. These requirements lead to an approach that scores lineage configurations against a learned model. The key design decision is what aspects of the lineage, as revealed via imaging and cell detection, to include in the model used to judge alternative tree configurations. Our desire is for a general method applicable to any embryo capable of normal cell division. As such, we model key local behaviors of nuclei, firstly their individual change over time (in appearance and position), and secondly the correlation in these measures expected between daughter cells at division. We also model two key aspects of the detection method that provides the raw nuclear positions for lineage assembly, firstly the relationship between nuclear appearance and the probability of a detection being a false positive and secondly the probability of a hypothesized series of detection failures. This model, which can be learned from a set of corrected lineages, is relatively simple but sufficient to produce accurate results and to highlight ambiguous areas for human inspection.
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[
International Worm Meeting,
2017]
Morphogenesis is the fundamental process in biology that describes the formation of a tissue or organism. It encompasses multiple layers of controlling upon molecular basis (Turing, 1952) and cellular basis. Taking advantage of the invariant cell lineage in C. elegans (Sulston, 1983), we asked how cells move to correct destinations. Previous studies proposed cell movement is guided by local cell-cell interaction (Schnabel, 2006), which were supported by evidences in early-stage embryos and limited types of mutants. Here, we hypothesized that, upon cell-cell interaction, differential affinities between neighboring cells drive their next moves. With our collection of all cell tracks up to 350-cell stage in hundreds of wild-type and perturbed embryos (Du, 2015), we devised a computational model to optimize cellular configuration over time and infer cell-cell affinities for individual embryos. The comparison between wild-type and perturbed embryos demonstrated those affinities were specified by cell fate rather than cell lineage. This study provided a basis to systematically elucidate how affinity network can serve as a new paradigm to positional information and orchestration in complex tissues.
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[
International Worm Meeting,
2021]
Advances in Electron Microscopy (EM) bring about the possibility of temporal analysis using staged samples over time as well as offering opportunities to gain insights by synthesizing EM with other imaging modalities. We present a pseudo time series of C. elegans embryonic development, four volumes covering around 2 hours particularly rich in development between 320 and 475 minutes post first cleavage. This period encompasses neurulation, neural organogenesis and neuropil formation key events that build most of the major structures of the nervous system as well as critical events for many other organ systems. We correlate this EM data with florescence data spanning the first eight hours of embryogenesis. Every cell in the florescence data is identifiable via lineaging. To correlate these data sets we develop a novel computational method for alignment of identities between data sets in the challenging presence of spatial and temporal variation. This approach involves co-optimization of spatial alignment and the structure of labeled data based on a model of dynamic anatomy in the form of an adjacency graph with expected variation. This model captures both variable elements and consistent spatial proximity relationships. We identify every cell in three of the four time points. Identity results are accurate, ranging from 72 to 78 percent correct when assessed against a large set of manual annotations based on position and morphology. This is better than any previously reported results for identifying all cells in an organism based on position alone. The resulting single cell level annotation allows efficient navigation of this large EM data set. We use the sequence to probe the interactions over time between different components within the nerve ring elucidating the relationship between the temporal and spatial location of initial outgrowths into the ring and ultimate structure. We also examine the interaction between cells during the formation of the amphid dendrite structure providing insight into the timing and succession of events at the inter and intra cellular level. Our observations only scratch the surface of the details available in the data set. We will provide the EM data with single-cell level annotation as a resource for the community.
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[
International Worm Meeting,
2015]
While in toto imaging and image analysis methods have advanced the study of multicellular phenomena in development at single-cell resolutions, not much progress has been made in the design of tools to perturb embryos with comparable spatial and temporal resolution. Both classical techniques such as laser ablation and new technologies built around light-activated proteins offer significant promise in filling this need. Their use to-date, however, remains limited by a need for cell-specific promoters or completely manual operation. We have developed a platform for the real-time segmentation and tracking of cells in the C. elegans embryo to enable more reproducible perturbations at higher throughput and without a need for cell-specific markers. The platform consists of three components: 1) Automated cell detection and tracking. 2) An interface for curating detection and tracking results. 3) Laser control for carrying out a pre-defined perturbation protocol when a target cell identity is detected (ie. cell killing by ablation with a pulse laser).The performance of the cell detection pipeline exhibits little dependence on the number of cells present in the embryo; segmentation requires only 3 s per volume on a high-performance workstation. The time required to track detected cells at each time-point, however, is strongly dependent on the number of cells present; currently matching an average of 7.6 cells / s. For an imaging period of 75 s, the on-line segmentation pipeline executes faster than the imaging rate through the 500-cell stage and thus potentially up until twitching begins. More heavily parallelizable strategies for cell tracking are also being pursued to enable perturbations later and in larger model systems. This platform should prove valuable for performing single-cell ablations in the late embryo (ie. for the ablation of individual neurons following their terminal division) and could be easily adapted for other perturbations such as the induction of photoactivatable signaling proteins or even to use in other model systems.
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[
International Worm Meeting,
2009]
It is generally believed that the reproducible cell positions and contacts in the early C. elegans embryo are set up through fine regulation of spindle orientation, and no active migration is involved in adjusting cell positions until gastrulation. We have observed dynamic cell shape changes and active migration in as early as the 6-cell stage embryo. Specifically, one of the six cells, ABpl, ruffles and migrates circumferentially toward the ventral side. Consequently, ABpl detaches from its sister, ABpr, and new contact forms between ABar and C. Disrupting the migration prevents the contact between ABar and C, and subsequently, the ABar spindle fails to rotate, a process known to require a Wnt signal from C. Interestingly, ABpr does not ruffle or migrate, even though it is the bilateral equivalent of ABpl. The earliest left-right asymmetry can be observed as the embryo proceeds from the 4-cell to the 6-cell stage, during which the initially left-right aligned spindles of ABa and ABp skew so that the right daughters are more posterior than their sisters. Temporally, the asymmetric ruffling follows immediately. We are now investigating if the asymmetric ruffling is due to the different cell contacts of ABpl and ABpr resulted from the skewed division of ABp, or that certain factors are distributed asymmetrically during the division. Dynamic ruffling behavior continues as the embryo develops. For example, in the next generation of cells, ABplp, ABarp and Ca, threes cells that are adjacent to each other, all ruffle. Thus, our results suggest that the early embryo is more dynamic than previously thought.
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[
International Worm Meeting,
2011]
Breaking left-right (LR) symmetry is a fascinating process in bilaterian embryogenesis as the asymmetry arises without apparent outside spatial cue. In C. elegans, LR symmetry is broken at the 4 to 6 cell division. As ABa and ABp divide synchronously, their spindles are initially aligned to the LR axis, but skew in such a manner that by the end of the division, the left side daughters are situated anterior to the right side daughters. This positional bias between the left and right side AB cells is sufficient to specify the handedness of the animal. Using micromanipulation to force the right AB daughters more anterior to the left is enough to reverse all normal L/R asymmetries of the worm. However, the actual molecular mechanism underlying this initial AB spindle skew is largely unknown. We used high-resolution time lapse fluorescent microscopy to delineate the cellular level events of the spindle skew. We found that this skew appears to be fundamentally different from spindle repositioning events found at the one- and two-cell stages, where the spindle rotates within the cell and determines the cleavage plane before cytokinesis starts. Instead, the ABa/p spindle skew occurs as the cytokinesis furrow forms and the contractile ring begins to ingress. This observation indicates that the skew is a whole cell movement rather than the spindle rotating within the cell. In previous experiments, we showed that in the events directly after the ABa/p skew, GSK-3, the C. elegans glycogen synthase kinase ortholog, is required in the global rearrangement of cells to translate the initial LR asymmetry into a LR body plan. Here we show that in about 9% of
gsk-3(
nr2047) embryos, ABa/p skew in the reversed direction, suggesting that
gsk-3 plays a role in the initial LR symmetry breaking cue. In an enhancer screen we found that the reversal rate can be increased to 30-40%. Currently, we are using quantitative analysis to uncover the molecular mechanism of these reversal cases.