AceTree: a tool for visual analysis of Caenorhabditis elegans embryogenesis

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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

Computer Vision Approaches for Mapping Gene Expression onto Lineage Trees

This project concerns studying the early development of living organisms. This period is accompanied by dynamic morphogenetic events. There is an increase in the number of cells, changes in the shape of cells and specification of cell fate during this time. Typically, in order to capture the dynamic morphological changes, one can employ a form of microscopy imaging such as Selective Plane Illumination Microscopy (SPIM) which offers a single-cell resolution across time, and hence allows observing the positions, velocities and trajectories of most cells in a developing embryo. Unfortunately, the dynamic genetic activity which underlies these morphological changes and influences cellular fate decision, is captured only as static snapshots and often requires processing (sequencing or imaging) multiple distinct individuals. In order to set the stage for characterizing the factors which influence cellular fate, one must bring the data arising from the above-mentioned static snapshots of multiple individuals and the data arising from SPIM imaging of other distinct individual(s) which characterizes the changes in morphology, into the same frame of reference. In this project, a computational pipeline is established, which achieves the aforementioned goal of mapping data from these various imaging modalities and specimens to a canonical frame of reference. This pipeline relies on the three core building blocks of Instance Segmentation, Tracking and Registration. In this dissertation work, I introduce EmbedSeg which is my solution to performing instance segmentation of 2D and 3D (volume) image data. Next, I introduce LineageTracer which is my solution to performing tracking of a time-lapse (2d+t, 3d+t) recording. Finally, I introduce PlatyMatch which is my solution to performing registration of volumes. Errors from the application of these building blocks accumulate which produces a noisy observation estimate of gene expression for the digitized cells in the canonical frame of reference. These noisy estimates are processed to infer the underlying hidden state by using a Hidden Markov Model (HMM) formulation. Lastly, for wider dissemination of these methods, one requires an effective visualization strategy. A few details about the employed approach are also discussed in the dissertation work. The pipeline was designed keeping imaging volume data in mind, but can easily be extended to incorporate other data modalities, if available, such as single cell RNA Sequencing (scRNA-Seq) (more details are provided in the Discussion chapter). The methods elucidated in this dissertation would provide a fertile playground for several experiments and analyses in the future. Some of such potential experiments and current weaknesses of the computational pipeline are also discussed additionally in the Discussion Chapter

Simulation of Early C. elegans Embryogenesis

Simulations are powerful tools that can be utilized to understand complex mechanisms within a system. Our project focuses on simulating the first several cell divisions of C. elegans embryogenesis. C. elegans is used as biological model for development, aging, and cell biology and is an ideal simulation candidate. Our four-dimensional simulation contains known information and hypotheses about molecular interactions within cells. Using rules to represent biological functions, our project visually and computationally shows the effects of several different mutations

Transcription profiling of fertilization and early seed development events in a solanaceous species using a 7.7 K cDNA microarray from Solanum chacoense ovules

<p>Abstract</p> <p>Background</p> <p>To provide a broad analysis of gene expression changes in developing embryos from a solanaceous species, we produced amplicon-derived microarrays with 7741 ESTs isolated from <it>Solanum chacoense </it>ovules bearing embryos from all developmental stages. Our aims were to: 1) identify genes expressed in a tissue-specific and temporal-specific manner; 2) define clusters of genes showing similar patterns of spatial and temporal expression; and 3) identify stage-specific or transition-specific candidate genes for further functional genomic analyses.</p> <p>Results</p> <p>We analyzed gene expression during <it>S. chacoense </it>embryogenesis in a series of experiments with probes derived from ovules isolated before and after fertilization (from 0 to 22 days after pollination), and from leaves, anthers, and styles. From the 6374 unigenes present in our array, 1024 genes were differentially expressed (≥ ± 2 fold change, p value ≤ 0.01) in fertilized ovules compared to unfertilized ovules and only limited expression overlap was observed between these genes and the genes expressed in the other tissues tested, with the vast majority of the fertilization-regulated genes specifically or predominantly expressed in ovules (955 genes). During embryogenesis three major expression profiles corresponding to early, middle and late stages of embryo development were identified. From the early and middle stages, a large number of genes corresponding to cell cycle, DNA processing, signal transduction, and transcriptional regulation were found. Defense and stress response-related genes were found in all stages of embryo development. Protein biosynthesis genes, genes coding for ribosomal proteins and other components of the translation machinery were highly expressed in embryos during the early stage. Genes for protein degradation were overrepresented later in the middle and late stages of embryo development. As expected, storage protein transcripts accumulated predominantly in the late stage of embryo development.</p> <p>Conclusion</p> <p>Our analysis provides the first study in a solanaceous species of the transcriptional program that takes place during the early phases of plant reproductive development, including all embryogenesis steps during a comprehensive time-course. Our comparative expression profiling strategy between fertilized and unfertilized ovules identified a subset of genes specifically or predominantly expressed in ovules while a closer analysis between each consecutive time point allowed the identification of a subset of stage-specific and transition-specific genes.</p

Distribution of lipid transfer protein 1 (LTP1) epitopes associated with morphogenic events during somatic embryogenesis of Arabidopsis thaliana

Using immunocytochemical methods, at both the light and electron microscopic level, we have investigated the spatial and temporal distribution of lipid transfer protein 1 (LTP1) epitopes during the induction of somatic embryogenesis in explants of Arabidopsis thaliana. Immunofluorescence labelling demonstrated the presence of high levels of LTP1 epitopes within the proximal regions of the cotyledons (embryogenic regions) associated with particular morphogenetic events, including intense cell division activity, cotyledon swelling, cell loosening and callus formation. Precise analysis of the signal localization in protodermal and subprotodermal cells indicated that cells exhibiting features typical of embryogenic cells were strongly labelled, both in walls and the cytoplasm, while in the majority of meristematic-like cells no signal was observed. Staining with lipophilic dyes revealed a correlation between the distribution of LTP1 epitopes and lipid substances within the cell wall. Differences in label abundance and distribution between embryogenic and non-embryogenic regions of explants were studied in detail with the use of immunogold electron microscopy. The labelling was strongest in both the outer periclinal and anticlinal walls of the adaxial, protodermal cells of the proximal region of the cotyledon. The putative role(s) of lipid transfer proteins in the formation of lipid lamellae and in cell differentiation are discussed

Bioimage informatics in the context of drosophila research

Modern biological research relies heavily on microscopic imaging. The advanced genetic toolkit of drosophila makes it possible to label molecular and cellular components with unprecedented level of specificity necessitating the application of the most sophisticated imaging technologies. Imaging in drosophila spans all scales from single molecules to the entire populations of adult organisms, from electron microscopy to live imaging of developmental processes. As the imaging approaches become more complex and ambitious, there is an increasing need for quantitative, computer-mediated image processing and analysis to make sense of the imagery. Bioimage informatics is an emerging research field that covers all aspects of biological image analysis from data handling, through processing, to quantitative measurements, analysis and data presentation. Some of the most advanced, large scale projects, combining cutting edge imaging with complex bioimage informatics pipelines, are realized in the drosophila research community. In this review, we discuss the current research in biological image analysis specifically relevant to the type of systems level image datasets that are uniquely available for the drosophila model system. We focus on how state-of-the-art computer vision algorithms are impacting the ability of drosophila researchers to analyze biological systems in space and time. We pay particular attention to how these algorithmic advances from computer science are made usable to practicing biologists through open source platforms and how biologists can themselves participate in their further development

Targeted sequencing supports morphology and embryo features in resolving the classification of Cyperaceae tribe Fuireneae s.l.

Molecular phylogenetic studies based on Sanger sequences have shown that Cyperaceae tribe Fuireneae s.l. is paraphyletic. However, taxonomic sampling in these studies has been poor, topologies have been inconsistent, and support for the backbone of trees has been weak. Moreover, uncertainty still surrounds the morphological limits of Schoenoplectiella, a genus of mainly small, amphicarpic annuals that was recently segregated from Schoenoplectus. Consequently, despite ample evidence from molecular analyses that Fuireneae s.l. might consist of two to four tribal lineages, no taxonomic changes have yet been made. Here, we use the Angiosperms353 enrichment panel for targeted sequencing in order to: (1) clarify the relationships of Fuireneae s.l. with the related tribes Abildgaardieae, Eleocharideae and Cypereae; (2) define the limits of Fuireneae s.s., and (3) test the monophyly of Fuireneae s.l. genera with emphasis on Schoenoplectus and Schoenoplectiella. Using more than a third of Fuireneae s.l. diversity, our phylogenomic analyses strongly support six genera and four major Fuireneae s.l. clades that we recognise as tribes: Bolboschoeneae stat.nov., Fuireneae s.s., Schoenoplecteae, and Pseudoschoeneae tr.nov. These results are consistent with morphological, micromorphological (nutlet epidermal cell shape), and embryo differences detected for each tribe. At the generic level, most sub‐Saharan African perennials currently treated in Schoenoplectus are transferred to Schoenoplectiella. Our targeted sequencing results show that these species are nested in Schoenoplectiella, and their treatment here is consistent with micromorphological and embryo characters shared by all Schoenoplectiella species. Keys to recognised tribes and genera are provided

Interactive Similarity Analysis for 3D+t Cell Trajectory Data

Recent data acquisition techniques permit an improved analysis of living organisms. These techniques produce 3D+t information of cell developments in unprecedentedly high resolution. Biologists have a strong desire to analyze these cell evolutions in order to find similarities in their migration and division behaviors. The exploration of such patterns helps them in understanding how cells and hence organisms are able to ensure a regular shape development. However, the enormous size of the time-dependent data with several tens of thousands of cells and the need to analyze it in 3D hinder an interactive analysis. Visualizing the data to identify and extract relevant features provides a solution to this problem. For this, new visualization approaches are required that reduce the complexity of the data to detect important features in the visual analysis. In this thesis, novel visual similarity analysis methods are presented to interactively process very large 3D+t data of cell developments. Three main methods are developed that allow different visual analysis strategies. The usefulness of them is demonstrated by applications to cells from zebrafish embryos and Arabidopsis thaliana plants. Both data sets feature a high regularity in the shape formation of the organs and domain experts seek to research similar cell behaviors that are responsible for this development. For example, the identification of 3D division behaviors in plants is still an unresolved issue. The first method is a novel visualization approach that can automatically classify cell division types in plant data sets with high memory and time efficiency. The visualization is based on the generation of newly introduced cell isosurfaces that allow a quantitative and spatial comparison of cell division behaviors among individual plants. The method is applied to cells of the lateral root of Arabidopsis plants and reveals similar division schemes with respect to their temporal order. The second method enables a new visual similarity analysis for arbitrary 3D trajectory data in order to extract similar movement behaviors. The algorithm performs a grouping of thousands of trajectories with an optional level of detail modification. The clustering is based on a newly weighted combination of geometry and migratory features for which the weights are used to emphasize feature combinations. As a result, similar collective cell movements in zebrafish as well as a hitherto unknown correlation between division types and subsequent nuclei migrations in the Arabidopsis plants are detected. The third method is a novel visualization technique called the structure map. It permits a compact and interactive similarity analysis of thousands of binary tree structures. Unique trees are pre-ordered in the map based on spectral similarities and substructures are highlighted according to user-selected tree descriptors. Applied to cell developments from zebrafish depicted as trees, the map achieves compression rates up to 95% according to spectral analysis and facilitates an immediate identification of biologically implausible events and outliers. Additionally, similar quantities of feature appearances are detected in the center of the lateral root of several Arabidopsis plants

Differential Expressions of Nodal-Signal Transducers and Global Transcriptional Repression Commit Vegetal Cells in Eleutherodactylus Coqui to Form Nutritional Endoderm

The vegetal core cells of a Xenopus laevis embryo commit to mesendoderm via the Nodal-signaling pathway. In Eleutherodactylus coqui, a direct developing frog, mesendoderm is specified at the marginal zone of the early gastrulae and vegetal core cells transform into nutritional endoderm. It is a novel tissue consisting of transient, yolk rich cells that provide nutrition, but do not differentiate into adult tissues. We hypothesized that a disruption of Nodal-signaling is responsible for committing vegetal core cells to nutritional endoderm. I report a dual regulation involved in the generation of nutritional endoderm. First, differential expression of Nodal-signaling components like Smad2 and Smad4 was observed during early gastrulation between cells in the marginal zone and in the vegetal core. Although EcSmad2 RNA, as well as both native and the active forms of EcSmad2, were detected in the vegetal core by qPCR and western blot respectively, western analysis revealed that Smad4 isoforms were expressed at a low level during early gastrulation. Immunostaining showed that only 12% and 50% of vegetal core cells were positive for nuclear Smad2 and Smad4 signals, respectively, compared to 100% in marginal zone cells. These differential expressions may indicate a signaling blockade in vegetal core cells. Second, I found global transcription repression in vegetal core cells by immunostaining. At late blastula, both the marginal zone and vegetal core cell were transcriptionally silent. At the onset of gastrulation, marginal zone cells, but not vegetal core ones, became transcriptionally active. This indicates the occurrence of a mid-blastula transition in the marginal zone by early gastrulation. Global transcriptional repression prevails in the vegetal core through development. A combination of differential Nodal-signaling and global transcriptional repression in vegetal core cells may account for its lack of differentiation