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author:("Bao, zhikong")
1.  De Novo Inference of Systems-Level Mechanistic Models of Development from Live-Imaging-Based Phenotype Analysis 
Cell  2014;156(0):359-372.
Elucidation of complex phenotypes for mechanistic insights presents a significant challenge in systems biology. We report a strategy to automatically infer mechanistic models of cell fate differentiation based on live-imaging data. We use cell lineage tracing and combinations of tissue-specific marker expression to assay progenitor cell fate and detect fate changes upon genetic perturbation. Based on the cellular phenotypes, we further construct a model for how fate differentiation progresses in progenitor cells and predict cell-specific gene modules and cell-to-cell signaling events that regulate the series of fate choices. We validate our approach in C. elegans embryogenesis by perturbing 20 genes in over 300 embryos. The result not only recapitulates current knowledge but also provides insights into gene function and regulated fate choice, including an unexpected self-renewal. Our study provides a powerful approach for automated and quantitative interpretation of complex in vivo information.
PMCID: PMC3998820  PMID: 24439388
2.  Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy 
Nature biotechnology  2013;31(11):1032-1038.
Optimal four-dimensional imaging requires high spatial resolution in all dimensions, high speed and minimal photobleaching and damage. We developed a dual-view, plane illumination microscope with improved spatiotemporal resolution by switching illumination and detection between two perpendicular objectives in an alternating duty cycle. Computationally fusing the resulting volumetric views provides an isotropic resolution of 330 nm. As the sample is stationary and only two views are required, we achieve an imaging speed of 200 images/s (i.e., 0.5 s for a 50-plane volume). Unlike spinning-disk confocal or Bessel beam methods, which illuminate the sample outside the focal plane, we maintain high spatiotemporal resolution over hundreds of volumes with negligible photobleaching. To illustrate the ability of our method to study biological systems that require high-speed volumetric visualization and/or low photobleaching, we describe microtubule tracking in live cells, nuclear imaging over 14 h during nematode embryogenesis and imaging of neural wiring during Caenorhabditis elegans brain development over 5 h.
PMCID: PMC4105320  PMID: 24108093
3.  A semi-local neighborhood-based framework for probabilistic cell lineage tracing 
BMC Bioinformatics  2014;15:217.
Advances in fluorescence labeling and imaging have made it possible to acquire in vivo records of complex biological processes. Analysis has lagged behind acquisition in part because of the difficulty and computational expense of accurate cell tracking. In vivo analysis requires, at minimum, tracking hundreds of cells over hundreds of time points in complex three dimensional environments. We address this challenge with a computational framework capable of efficiently and accurately tracing entire cell lineages.
The bulk of the tracking problem—tracking cells during interphase—is straightforward and can be executed with simple and fast methods. Difficult cases originate from detection errors and relatively rare large motions. Therefore, our method focuses computational effort on difficult cases identified by local increases in cell number. We force these cases into tentative cell track bifurcations, which define natural semi-local neighborhoods that permit Bayesian judgment about the underlying cell behavior. The bifurcation judgment process not only correctly tracks through cell divisions and large movements, but also offers corrections to detection errors. We demonstrate that this method enables large scale analysis of Caenorhabditis elegans development, an ideal validation platform because of an invariant cell lineage.
The high accuracy achieved by our method suggests that a bifurcation-based semi-local neighborhood provides sufficient information to recognize dependencies between nearby tracking choices, and to interpret difficult tracking cases without reverting to global optimization. Our method makes large amounts of lineage data accessible and opens the door to new types of statistical analysis of complex in vivo processes.
PMCID: PMC4085468  PMID: 24964866
Image processing; Microscopy; In vivo; Cell tracking; Developmental biology
4.  Irises 
Worm  2014;3:e29041.
The DNA content of nuclei is a valuable measure of cell cycle status. Irises is a software tool to facilitate systematic in situ determination of DNA content for cell cycle analysis at single-nucleus resolution within complex tissues. We demonstrate the utility of the tool with analysis of DNA content in germline nuclei of C. elegans. Compared with results obtained by manual analysis, we find the tool greatly facilitates analysis by improving speed at least 5-fold while maintaining accuracy. The source code and instruction manual (including installation for both Mac and PC) are provided.
PMCID: PMC4165541  PMID: 25254149
C. elegans; germline; ploidy; cell cycle; image analysis
5.  Actomyosin-based Self-organization of cell internalization during C. elegans gastrulation 
BMC Biology  2012;10:94.
Gastrulation is a key transition in embryogenesis; it requires self-organized cellular coordination, which has to be both robust to allow efficient development and plastic to provide adaptability. Despite the conservation of gastrulation as a key event in Metazoan embryogenesis, the morphogenetic mechanisms of self-organization (how global order or coordination can arise from local interactions) are poorly understood.
We report a modular structure of cell internalization in Caenorhabditis elegans gastrulation that reveals mechanisms of self-organization. Cells that internalize during gastrulation show apical contractile flows, which are correlated with centripetal extensions from surrounding cells. These extensions converge to seal over the internalizing cells in the form of rosettes. This process represents a distinct mode of monolayer remodeling, with gradual extrusion of the internalizing cells and simultaneous tissue closure without an actin purse-string. We further report that this self-organizing module can adapt to severe topological alterations, providing evidence of scalability and plasticity of actomyosin-based patterning. Finally, we show that globally, the surface cell layer undergoes coplanar division to thin out and spread over the internalizing mass, which resembles epiboly.
The combination of coplanar division-based spreading and recurrent local modules for piecemeal internalization constitutes a system-level solution of gradual volume rearrangement under spatial constraint. Our results suggest that the mode of C. elegans gastrulation can be unified with the general notions of monolayer remodeling and with distinct cellular mechanisms of actomyosin-based morphogenesis.
PMCID: PMC3583717  PMID: 23198792
C. elegans; gastrulation; actomyosin; cellular rosette
6.  Chiral forces organize left-right patterning in C. elegans by uncoupling midline and anteroposterior axis 
Developmental cell  2010;19(3):402-412.
Left-right (LR) patterning is an intriguing but poorly understood process aspect of bilaterian embryogenesis. We report a mechanism for LR patterning in C. elegans in which the embryo uncouples its midline from the anteroposterior (AP) axis. Specifically, the eight-cell embryo establishes a midline that is tilted 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, involves 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 cytokinetic furrow of a neighbor of the sister pair, providing a developmental clock and an unexpected signaling interaction between the contractile ring and adjacent cells.
PMCID: PMC2952354  PMID: 20833362
7.  A hybrid blob-slice model for accurate and efficient detection of fluorescence labeled nuclei in 3D 
BMC Bioinformatics  2010;11:580.
To exploit the flood of data from advances in high throughput imaging of optically sectioned nuclei, image analysis methods need to correctly detect thousands of nuclei, ideally in real time. Variability in nuclear appearance and undersampled volumetric data make this a challenge.
We present a novel 3D nuclear identification method, which subdivides the problem, first segmenting nuclear slices within each 2D image plane, then using a shape model to assemble these slices into 3D nuclei. This hybrid 2D/3D approach allows accurate accounting for nuclear shape but exploits the clear 2D nuclear boundaries that are present in sectional slices to avoid the computational burden of fitting a complex shape model to volume data. When tested over C. elegans, Drosophila, zebrafish and mouse data, our method yielded 0 to 3.7% error, up to six times more accurate as well as being 30 times faster than published performances. We demonstrate our method's potential by reconstructing the morphogenesis of the C. elegans pharynx. This is an important and much studied developmental process that could not previously be followed at this single cell level of detail.
Because our approach is specialized for the characteristics of optically sectioned nuclear images, it can achieve superior accuracy in significantly less time than other approaches. Both of these characteristics are necessary for practical analysis of overwhelmingly large data sets where processing must be scalable to hundreds of thousands of cells and where the time cost of manual error correction makes it impossible to use data with high error rates. Our approach is fast, accurate, available as open source software and its learned shape model is easy to retrain. As our pharynx development example shows, these characteristics make single cell analysis relatively easy and will enable novel experimental methods utilizing complex data sets.
PMCID: PMC3008706  PMID: 21114815
8.  Comparative Analysis of Embryonic Cell Lineage between Caenorhabditis briggsae and C. elegans 
Developmental biology  2007;314(1):93-99.
Comparative genomic analysis of important signaling pathways in C. briggase and C. elegans reveals both conserved features and also differences. To build a framework to address the significance of these features we determined the C. briggsae embryonic cell lineage, using the tools StarryNite and AceTree. We traced both cell divisions and cell positions for all cells through all but the last round of cell division and for selected cells through the final round. We found the lineage to be remarkably similar to that of C. elegans. Not only did the founder cells give rise to similar numbers of progeny, the relative cell division timing and positions were largely maintained. These lineage similarities appear to give rise to similar cell fates as judged both by the positions of lineally-equivalent cells and by the patterns of cell deaths in both species. However, some reproducible differences were seen, e.g., the P4 cell cycle length is more than 40% longer in C. briggsae than that in C. elegans (p < 0.01). The extensive conservation of embryonic development between such divergent species suggests that substantial evolutionary distance between these two species has not altered these early developmental cellular events, although the developmental defects of transpecies hybrids suggest that the details of the underlying molecular pathways have diverged sufficiently so as to not be interchangeable.
PMCID: PMC2696483  PMID: 18164284
C. briggsae; C. elegans; embryo; cell lineage; signaling pathway
9.  Control of Cell Cycle Timing during C. elegans Embryogenesis 
Developmental biology  2008;318(1):65-72.
As a fundamental process of development, cell proliferation must be coordinated with other processes such as fate differentiation. Through statistical analysis of individual cell cycle lengths of the first eight out of ten rounds of embryonic cell division in C. elegans, we identified synchronous and invariantly ordered divisions that are tightly associated with fate differentiation. Our results suggest a three-tier model for fate control of cell cycle pace: the primary control of cell cycle pace is established by lineage and the founder cell fate, then fine-tuned by tissue and organ differentiation within each lineage, then further modified by individualization of cells as they acquire unique morphological and physiological roles in the variant body plan. We then set out to identify the pace-setting mechanisms in different fates. Our results suggest that ubiquitin-mediated degradation of CDC-25.1 is a rate-determining step for the E (gut) and P3 (muscle and germline) lineages but not others, even though CDC-25.1 and its apparent decay have been detected in all lineages. Our results demonstrate the power of C. elegans embryogenesis as a model to dissect the interaction between differentiation and proliferation, and an effective approach combining genetic and statistical analysis at single-cell resolution.
PMCID: PMC2442716  PMID: 18430415
statistics; single cell; fate differentiation; cdc25; Skp1-related
10.  Automated analysis of embryonic gene expression with cellular resolution in C. elegans 
Nature methods  2008;5(8):703-709.
We describe a system that permits the automated analysis of reporter gene expression in Caenorhabditis elegans with cellular resolution continuously during embryogenesis and demonstrate its utility by defining the expression patterns of reporters for several embryonically expressed transcription factors. The invariant cell lineage permits the automated alignment of multiple expression profiles, allowing the direct comparison of the expression of different genes' reporters. We have also used the system to monitor perturbations to normal development involving changes both in cell division timing and in cell fate. Systematic application could reveal the gene activity of each cell throughout development.
PMCID: PMC2553703  PMID: 18587405
11.  AceTree: a tool for visual analysis of Caenorhabditis elegans embryogenesis 
BMC Bioinformatics  2006;7:275.
The invariant lineage of the nematode Caenorhabditis elegans has potential as a powerful tool for the description of mutant phenotypes and gene expression patterns. We previously described procedures for the imaging and automatic extraction of the cell lineage from C. elegans embryos. That method uses time-lapse confocal imaging of a strain expressing histone-GFP fusions and a software package, StarryNite, processes the thousands of images and produces output files that describe the location and lineage relationship of each nucleus at each time point.
We have developed a companion software package, AceTree, which links the images and the annotations using tree representations of the lineage. This facilitates curation and editing of the lineage. AceTree also contains powerful visualization and interpretive tools, such as space filling models and tree-based expression patterning, that can be used to extract biological significance from the data.
By pairing a fast lineaging program written in C with a user interface program written in Java we have produced a powerful software suite for exploring embryonic development.
PMCID: PMC1501046  PMID: 16740163
12.  The Genome Sequence of Caenorhabditis briggsae: A Platform for Comparative Genomics 
PLoS Biology  2003;1(2):e45.
The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.
With the Caenorhabditis briggsae genome now in hand, C. elegans biologists have a powerful new research tool to refine their knowledge of gene function in C. elegans and to study the path of genome evolution
PMCID: PMC261899  PMID: 14624247

Results 1-12 (12)