Towards a digital model of zebrafish embryogenesis. Integration of Cell Tracking and Gene Expression Quantification

We elaborate on a general framework composed of a set of computational tools to accurately quantificate cellular position and gene expression levels throughout early zebrafish embryogenesis captured over a time-lapse series of in vivo 3D images. Our modeling strategy involves nuclei detection, cell geometries extraction, automatic gene levels quantification and cell tracking to reconstruct cell trajectories and lineage tree which describe the animal development. Each cell in the embryo is then precisely described at each given time t by a vector composed of the cell 3D spatial coordinates (x; y; z) along with its gene expression level g. This comprehensive description of the embryo development is used to assess the general connection between genetic expression and cell movement. We also investigate genetic expression propagation between a cell and its progeny in the lineage tree. More to the point, this paper focuses on the evolution of the expression pattern of transcriptional factor goosecoid (gsc) through the gastrulation process between 6 and 9 hours post fertilization (hpf

Digital Three-Dimensional Atlas of Quail Development Using High-Resolution MRI

We present an archetypal set of three-dimensional digital atlases of the quail embryo based on microscopic magnetic resonance imaging (µMRI). The atlases are composed of three modules: (1) images of fixed ex ovo quail, ranging in age from embryonic day 5 to 10 (e05 to e10); (2) a coarsely delineated anatomical atlas of the µMRI data; and (3) an organ system–based hierarchical graph linked to the anatomical delineations. The atlas is designed to be accessed using SHIVA, a free Java application. The atlas is extensible and can contain other types of information including anatomical, physiological, and functional descriptors. It can also be linked to online resources and references. This digital atlas provides a framework to place various data types, such as gene expression and cell migration data, within the normal three-dimensional anatomy of the developing quail embryo. This provides a method for the analysis and examination of the spatial relationships among the different types of information within the context of the entire embryo

3D + t Morphological Processing: Applications to Embryogenesis Image Analysis

We propose to directly process 3D + t image sequences with mathematical morphology operators, using a new classi?cation of the 3D+t structuring elements. Several methods (?ltering, tracking, segmentation) dedicated to the analysis of 3D + t datasets of zebra?sh embryogenesis are introduced and validated through a synthetic dataset. Then, we illustrate the application of these methods to the analysis of datasets of zebra?sh early development acquired with various microscopy techniques. This processing paradigm produces spatio-temporal coherent results as it bene?ts from the intrinsic redundancy of the temporal dimension, and minimizes the needs for human intervention in semi-automatic algorithms

Green Fluorescent Protein in the sea urchin: new experimental approaches to transcriptional regulatory analysis in embryos and larvae

The use of Green Fluorescent Protein (GFP) as a reporter for expression transgenes opens the way to several new experimental strategies for the study of gene regulation in sea urchin development. A GFP coding sequence was associated with three different previously studied cis-regulatory systems, viz those of the SM50 gene, expressed in skeletogenic mesenchyme, the CyIIa gene, expressed in archenteron, skeletogenic and secondary mesenchyme, and the Endo16 gene, expressed in vegetal plate, archenteron and midgut. We demonstrate that the sensitivity with which expression can be detected is equal to or greater than that of whole-mount in situ hybridization applied to detection of CAT mRNA synthesized under the control of the same cis-regulatory systems. However, in addition to the important feature that it can be visualized nondestructively in living embryos, GFP has other advantages. First, it freely diffuses even within fine cytoplasmic cables, and thus reveals connections between cells, which in sea urchin embryos is particularly useful for observations on regulatory systems that operate in the syncytial skeletogenic mesenchyme. Second, GFP expression can be dramatically visualized in postembryonic larval tissues. This brings postembryonic larval developmental processes for the first time within the easy range of gene transfer analyses. Third, GFP permits identification and segregation of embryos in which the clonal incorporation of injected DNA has occurred in any particular desired region of the embryo. Thus, we show explicitly that, as expected, GFP transgenes are incorporated in the same nuclei together with other transgenes with which they are co-injected

Automated processing of zebrafish imaging data: a survey

Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines

Assembling models of embryo development: Image analysis and the construction of digital atlases

Digital atlases of animal development provide a quantitative description of morphogenesis, opening the path toward processes modeling. Prototypic atlases offer a data integration framework where to gather information from cohorts of individuals with phenotypic variability. Relevant information for further theoretical reconstruction includes measurements in time and space for cell behaviors and gene expression. The latter as well as data integration in a prototypic model, rely on image processing strategies. Developing the tools to integrate and analyze biological multidimensional data are highly relevant for assessing chemical toxicity or performing drugs preclinical testing. This article surveys some of the most prominent efforts to assemble these prototypes, categorizes them according to salient criteria and discusses the key questions in the field and the future challenges toward the reconstruction of multiscale dynamics in model organisms

IR Laser-Induced Gene Expression for Tracking Development of Single Embryonic Neurons and Glia in C. Elegans

The assembly of neural circuits requires a complex choreography of developmental events: neurons must be generated, extend neurites at the correct time and location, and then integrate extracellular information, like long-range guidance cues or cellular contacts, with an internal developmental program to make correct wiring decisions. Visualizing neural-circuit assembly in vivo can provide insight into how these events are coordinated. The C. elegans embryo, which contains only 222 neurons and 56 glia, is an attractive setting to study nervous system development comprehensively in an intact, living organism. However, methods to label and track optically-resolvable neurites or manipulate single neurons through gene expression do not exist, as most embryonic reporters are broadly expressed. Here, I present a method for expressing fluorescent reporters or any gene of interest in specific C. elegans embryonic neurons, glia, or other cell types, without cell specific drivers. Our method is based on a previous setup (Kamei et al., 2009), and uses an infrared (IR) laser to localize heat to the volume of a single precursor cell in the embryo. This induces gene expression in the progeny of that cell (1-4 cells/embryo) through heat-shock-response regulatory elements. I perform significant optimizations to adapt this strategy to cells in the C. elegans embryo, which are highly sensitive to heat toxicity. Direct temperature measurements of IR heating in the embryo reveal that cells are heated to physiological temperatures (320C) for 5 minute durations using our modified irradiation protocol. These conditions lead to high rates of gene induction (\u3e60%) with no signs of damage. First, I use our system to label and track single neurons during early nervous system assembly. These studies reveal a retrograde extension mechanism for axon growth in specific interneurons. I also study the etiology of axon-guidance defects in sax-3/Robo and vab-1/EphR mutants; these studies suggest that a timing/competence mechanism controls axon-outgrowth dynamics in the nerve ring. Next, I demonstrate the versatility of IR irradiation by performing cell-specific rescues, determining DAF-6/Patched-related site of action during sensory-organ development. Finally, I demonstrate that IR cell irradiation can be used to perform simultaneous ablation and labeling of cells in the same embryo. I use this system to uncover a role for the amphid sheath glia in dendrite extension. As IR induction can be used for targeted labeling, gene expression, and ablation without the need for cell-specific drivers, this tool opens to door to high resolution systematic analyses of C. elegans morphogenesis